Heterodimeric Fc-CLEC-1 fusion molecules and uses thereof

Abstract

The present invention relates to a heterodimeric Fc-CLEC1 fusion molecule 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-CLEC1 fusion molecules having improved capabilities in terms of toxicity, half-life, and bioavailability compared to fusion molecules binding to CLEC-1 in the prior art. The invention also relates to the use of said fusion molecules in therapy.

Description

Technical Field

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

[0002] One problem in cancer treatment is to destroy tumor cells without damaging healthy tissues. In the past decade, anti-tumor immunotherapy has gained increasing interest because they redirect the patient's immune system to destroy cancer cells. It has been clearly demonstrated that tumor cells use immune checkpoints to escape immune defense. The key role of these inhibitory checkpoints in blocking anti-tumor immunity has been illustrated by the remarkable success of antibody-driven T cell immune checkpoint blockade. However, usually after the failure of classical chemotherapy, a considerable proportion of patients do not respond to or develop resistance to these checkpoint inhibitors (CKI) therapies. These patients need effective new therapies, and myeloid cells represent promising therapeutic targets. In fact, myeloid cells are the most abundant tumor-infiltrating immune cells. C-type lectins (CLECs) expressed on immune cells are sensors of environmental and immune response regulators. CLEC-1 represents a potential therapeutic target for enhancing myeloid cells and anti-tumor responses. In order to better explain the function of CLEC-1 and develop compounds intended to regulate its activity, it is necessary to mimic the molecules of CLEC-1 action, which may have an improved ability to enhance or stimulate signaling pathways and / or cells expressing CLEC-1.

[0003] CLEC-1 (also known as CLEC-1A, or CLEC1, or CLEC1A) is a C-type lectin-like receptor, i.e., C-type lectin-like receptor-1, which is particularly expressed 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 capable of recognizing a variety of glycans on pathogens or on self-proteins. For these receptors, glycan recognition is dependent on Ca 2+ Many of the related -CLRs are still able to recognize carbohydrates, but with Ca 2+ Unrelated; These receptors refer to C-type lectin-like receptors (CTLRs), which cover the receptor family of CLEC-1. These receptors are of particular interest due to their role in coupling innate and adaptive immunity. CTLRs are mainly expressed by myeloid cells such as monocytes, macrophages, dendritic cells (DCs) and neutrophils. CTLRs not only act as antigen uptake receptors for internalization and presentation to T cells, but also trigger a variety of signal transduction pathways that lead to NF-κB, type I interferon (IFN) and / or inflammasome activation. Through their ability to present antigens and ensure the balance between cell activation and inhibition, CTLRs have become challenging pharmacological targets for the treatment of a variety of diseases (including cancer, autoimmune diseases or allergies). CTLR regulation seems to represent a promising strategy for disease management, although attempts to identify ligands and efforts to clarify their role in immunity are still incomplete.

[0004] CLEC-1 is known to be expressed on bone marrow and endothelial cells. CLEC-1 has been described as a receptor that can be up-regulated by immunomodulatory mediators and can regulate 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 inventors show that CLEC-1 is expressed on the cell surface by conventional DC (cDC) and small subtypes of monocytes and DC in human blood, and is enhanced by immunosuppressive cytokine TGFβ (see International Application No. WO 2018073440). They demonstrated that in rodents and humans, CLEC-1 acts as an inhibitory receptor in myeloid cells and prevents 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 activationand downstream Th17 responses Blood Adv. 2017; Mar 22; 1(9)557-568).

[0005] These properties provide interest in considering CLEC-1 in the design of additional means for treating health conditions involving the patient's immune response, in particular the development of new therapies against cancer. In particular, these results require additional research to provide compounds capable of binding to and / or capturing at least one ligand of CLEC-1 with enhanced properties, such as better half-life, or enhanced functions, such as complement dependent cytotoxicity (CDC), antibody dependent cellular cytotoxicity (ADCC), and antibody dependent cellular phagocytosis (ADCP). Summary of the invention

[0006] The present invention relies on the association within a fusion molecule of a binding moiety comprising or consisting of at least a portion of the extracellular domain of CLEC-1, which may or may not be mutant compared to wild-type CLEC-1, and a heterodimeric Fc compound comprising at least two different Fc polypeptide chains.

[0007] As will be described in further detail in this application, the inventors provide evidence that the fusion molecules of the invention notably result in molecules having at least one of the following properties:

[0008] - heterodimeric Fc-CLEC-1 fusion molecules having reduced in vivo toxicity compared to prior art anti-CLEC-1 compounds, in particular prior art Fc-CLEC-1 fusion proteins;

[0009] - heterodimeric Fc-CLEC-1 fusion molecules that do not form aggregates (e.g. accumulations of fusion molecules that aggregate together and form a body or mass); aggregates are considered when more than two fusion proteins accumulate and aggregate together - dimers of fusion proteins are not considered aggregates in the context of the present invention).

[0010] - heterodimeric Fc-CLEC-1 fusion molecules having a prolonged half-life, in particular compared to prior art anti-CLEC-1 compounds, in particular prior art Fc-CLEC-1 fusion proteins;

[0011] - Heterodimeric Fc-CLEC-1 fusion molecules having enhanced bioavailability compared to prior art anti-CLEC-1 compounds, in particular prior art Fc-CLEC-1 fusion proteins;

[0012] - A heterodimeric Fc-CLEC-1 fusion molecule that does not lead to an increase in the secretion of proinflammatory cytokines, in particular IL-6.

[0013] As shown in the examples of the present invention, the heterodimeric Fc-CLEC-1 fusion molecules of the present invention have lower toxicity than the Fc-CLEC-1 fusions of the prior art, thereby reducing the risks associated with administering the compound to patients in need (e.g., side effects, tolerance), and / or allowing the administration of higher doses of Fc-CLEC-1 fusion proteins to enhance their in vivo therapeutic effects. In addition, the therapeutic ability of the heterodimeric Fc-CLEC-1 fusion molecules of the present invention was demonstrated in cancer models, thereby demonstrating that these fusion molecules have a powerful therapeutic effect in vivo. Data are also provided to demonstrate that the heterodimeric Fc-CLEC-1 fusion molecules of the present invention can bind to the ligand of CLEC-1, referred to as CLEC-1-ligand or CLEC-1L, with the same affinity as the Fc-CLEC-1 fusion proteins or CLEC-1-HIS proteins of the prior art. In addition, the heterodimeric Fc-CLEC-1 fusion molecules of the present invention can bind to tumor cells expressing the ligand of CLEC-1.

[0014] Compared with the Fc-CLEC-1 fusion protein of the prior art, the heterodimeric Fc-CLEC-1 fusion molecule of the present invention has enhanced in vivo bioavailability. As shown in the examples of the present invention, the in vivo concentration of the heterodimeric Fc-CLEC-1 fusion molecule is detectable over a longer period of time than the Fc-CLEC-1 fusion protein of the prior art; when the fusion protein of the prior art reaches the lower limit of quantification (LLOQ), the concentration of the heterodimeric Fc-CLEC-1 fusion molecule of the present invention remains close to its initial concentration.

[0015] The heterodimeric Fc-CLEC-1 fusion molecules of the present invention do not form aggregates, thereby reducing their immunogenicity. Although the Fc-CLEC-1 fusion proteins of the prior art may form aggregates in some cases, thereby triggering an immune response against the aggregates, the heterodimeric Fc-CLEC-1 fusion molecules of the present invention do not form aggregates when administered and do not cause increased secretion of proinflammatory cytokines (particularly IL-6).

[0016] In a first aspect, the present invention relates to a fusion molecule comprising:

[0017] (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

[0018] (ii) a heterodimeric immunoglobulin or a fragment thereof, comprising:

[0019] a. a first Fc polypeptide chain comprising a first CH3 domain (CH3B chain or hole chain), and

[0020] b. a second Fc polypeptide chain comprising a second CH3 domain (CH3A chain or knob chain),

[0021] wherein the first CH3 domain and the second CH3 domain are different,

[0022] One of the at least one binding moieties is fused to the C-terminus or N-terminus of the first Fc polypeptide, 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.

[0023] In another aspect, the present invention relates to a fusion molecule comprising:

[0024] (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

[0025] (ii) a heterodimeric immunoglobulin or a fragment thereof, comprising:

[0026] 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 (CH3B chain or hole chain) and a linker, and

[0027] 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 (CH3B chain or hole chain),

[0028] The 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.

[0029] In another aspect, the present invention relates to a fusion molecule comprising:

[0030] (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

[0031] (ii) a heterodimeric immunoglobulin or a fragment thereof, comprising:

[0032] 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), and

[0033] 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 (CH3B chain or hole chain) and a linker;

[0034] The 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.

[0035] In another aspect, the present invention relates to a fusion molecule comprising:

[0036] (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

[0037] (ii) a heterodimeric immunoglobulin or a fragment thereof, comprising:

[0038] a. a first Fc polypeptide chain comprising a first CH3 domain (CH3B chain or hole chain), and

[0039] b. a second Fc polypeptide chain comprising a second CH3 domain (CH3A chain or knob chain),

[0040] wherein the first CH3 domain and the second CH3 domain are different,

[0041] One of the at least one binding moieties is fused to the C-terminus or N-terminus of the first Fc polypeptide, 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.

[0042] For use as a medicament, in particular for the treatment of a subject, in particular a human subject, suffering from a disease selected from the group consisting of cancer, or an infectious disease, or sepsis, an autoimmune disease, or an inflammatory disease, including acute or chronic inflammatory diseases, in particular the cancers listed in the present description, in particular liquid cancers, solid cancers, cancers expressing at least one ligand of human CLEC-1, cancers with CLEC-1 ligand-positive tumors (also referred to as cancers with CLEC-1L-positive tumors), cancers with CLEC-1L-positive tumor cells, cancers with Cancer of 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, nasopharyngeal carcinoma, adenocarcinoma, pancreatic cancer, or selected from the following group: chronic infection, sepsis, infection, viral infection (especially coxsackievirus or encephalitis virus, more particularly coxsackievirus B3 or Japanese encephalitis virus), fungal infection, cardiovascular disease, autoimmune disease (especially Sjogren's syndrome or systemic lupus erythematosus or systemic sclerosis), inflammatory disease.

[0043] In a specific embodiment of the present invention, at least one binding portion of the fusion molecule of the present invention comprises or consists of a portion of the extracellular domain of human CLEC-1, said portion comprising at least 50 amino acid residues, particularly at least 100 amino acid residues, more particularly at least 200 amino acid residues, and sharing at least 70% identity, particularly at least 80% identity, more particularly at least 90% identity, even more particularly at least 95% identity with the extracellular domain of human CLEC-1 of SEQ ID NO:2.

[0044] In a specific embodiment of the present invention, the extracellular domain of human CLEC-1 comprises at least the amino acid sequence shown in SEQ ID NO:2.

[0045] In a specific embodiment of the present invention, the CH3 domains of the first Fc polypeptide chain and the second Fc polypeptide are selected from the reference KiH, KiH S-S , HA-TF, ZW1, 7.8.90, DD-KK, EW-RVT, SEED and the CH3 domain of A107.

[0046] In a specific aspect of the present invention, a fusion molecule is provided, comprising:

[0047] (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

[0048] (ii) a heterodimeric immunoglobulin or a fragment thereof, comprising:

[0049] a. a first Fc polypeptide chain comprising a first CH2 domain (CH2B chain or hole chain), and

[0050] b. a second Fc polypeptide chain comprising a second CH2 domain (CH2A chain or knob chain),

[0051] wherein the first CH2 domain and the second CH2 domain are different,

[0052] One of the at least one binding moieties is fused to the C-terminus or N-terminus of the first Fc polypeptide, and / or one of the at least one binding moieties is fused to the C-terminus or N-terminus of the second Fc polypeptide chain. 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), the CH3 domains being different or identical, in particular different. The Fc polypeptide chain may further comprise a hinge region and / or a linker region as disclosed herein.

[0053] In a specific embodiment of the invention, there is a single binding moiety in the fusion protein, which is fused to the C-terminus or N-terminus of the first Fc polypeptide chain, particularly to the C-terminus of the first Fc polypeptide chain.

[0054] In a specific embodiment of the invention, the binding moiety is fused to the C-terminus or N-terminus of the first Fc polypeptide chain, in particular the C-terminus of the first Fc polypeptide chain, and wherein 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 the second Fc polypeptide chain. A "binding molecule" can be a protein (for example, but not limited to, a portion of the extracellular domain of a target protein) or a functional equivalent of 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.

[0055] In a specific embodiment of the invention, the binding moiety is fused to the C-terminus or N-terminus of the first Fc polypeptide chain, in particular the C-terminus of the first Fc polypeptide chain, and the other binding moiety is fused to the C-terminus or N-terminus of the second Fc polypeptide chain, in particular the C-terminus of the second Fc polypeptide chain, and the binding moieties are identical. In this case, both 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.

[0056] In a specific embodiment of the invention, at least one binding moiety is fused to the Fc polypeptide chain via a linker peptide, more specifically via a linker peptide of SEQ ID NO:13.

[0057] In a specific embodiment of the invention, the first Fc polypeptide chain, or the second Fc polypeptide chain, or both the first and the second Fc polypeptide chain comprise a CH2 domain, particularly N-terminal to the CH3 domain.

[0058] In a specific embodiment of the invention, the first Fc polypeptide chain, or the second Fc polypeptide chain, or both the first and the second Fc polypeptide chain comprise a hinge region, particularly at the N-terminus of the Fc polypeptide chain, more particularly at the N-terminus of the CH2 domain when present.

[0059] In a specific embodiment of the present invention, the fusion molecule further comprises:

[0060] (iii) at least one antigen binding domain comprising or consisting of a variable domain of an antibody or antigen binding fragment thereof, said antigen binding domain being fused to a first Fc polypeptide chain or a second Fc polypeptide chain, or the first antigen binding domain being fused to a first Fc polypeptide chain and the second antigen binding domain being fused to a second Fc polypeptide chain. In a specific embodiment, 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 binds to an antigen or epitope selected from the group consisting of SIRPα, SIRPβ, SIRPγ, 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), SI GLEC, 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. In particular, when 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.

[0061] In a specific embodiment, 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, in particular to the N-terminus of the CH2 domain, or when the hinge region is present, to the hinge region.

[0062] In a specific embodiment, there are two antigen binding domains in the fusion molecule, each associated with a single Fc polypeptide chain, the first antigen binding domain and the second antigen binding domain binding to two different epitopes, in particular binding to two different antigens, or binding to the same epitope or the same antigen.

[0063] In a specific embodiment of the present invention, a dimeric molecule comprising two fusion molecules according to the present invention is provided.

[0064] In a specific embodiment of the present invention, a soluble fusion molecule according to the present invention is provided.

[0065] In a specific embodiment of the present invention, a soluble dimeric molecule is provided, which comprises two fusion molecules according to the present invention.

[0066] In one embodiment of the present invention, a bispecific fusion molecule is provided.

[0067] In one embodiment of the present invention, a monospecific fusion molecule is provided.

[0068] In a specific embodiment of the present invention, a fusion molecule is provided, which comprises or consists of the amino acid sequences shown in SEQ ID NO:10 and SEQ ID NO:12, or SEQ ID NO:3 and SEQ ID NO:5.

[0069] 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 use as a medicament, in particular for the treatment of a subject, in particular a human subject, suffering from a disease selected from the group consisting of: a cancer listed in the present specification, in particular 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, thyroid cancer, liver cancer, testicular cancer, kidney cancer, melanoma, colorectal cancer, adenocarcinoma, nasopharyngeal carcinoma, pancreatic cancer, chronic infection, sepsis, infection (in particular coxsackievirus or encephalitis virus, more particularly coxsackievirus B3 or Japanese encephalitis virus), cardiovascular disease, autoimmune disease (in particular Sjogren's syndrome or systemic lupus erythematosus or systemic sclerosis), inflammatory disease.

[0070] The structures of different fusion molecules according to the present invention are as follows Figure 1 shown.

[0071] ·definition

[0072] As used herein, the terms "CLEC-1" and "CLEC-1A" relate to a CLEC-1A protein from a mammalian species, preferably human CLEC-1 or CLEC-1A. The reference sequence of the human CLEC-1A receptor corresponds to the sequence associated with the accession number Q8NC01 Uniprot. Preferably, the term "human CLEC-1" refers to the protein encoded by the CLEC-1 gene with reference to the amino acid sequence of the Q8NC01 Uniprot accession number and the 51267 NCBI accession number. In this specification, the terms CLEC-1A, CLEC-1, CLECA, CLEC-1, Clec1, CLEC-1, CLEC-A1 and CLEC-1A are used interchangeably and all refer to the mammalian CLEC1 receptor corresponding to the human CLEC-1A receptor, characterized by the amino acid sequence associated with the accession number Q8NC01 Uniprot, or its orthologous protein, or its homologous protein.

[0073] Preferably, the term "human CLEC-1" refers to the protein encoded by the CLEC-1 gene with reference to the amino acid sequence of the Q8NC01 Uniprot accession number and the 51267 NCBI accession number. CLEC-1 is characterized by the amino acid sequence shown in SEQ ID NO: 1. In a specific embodiment, the amino acid sequence of the extracellular domain of human wild-type CLEC-1 comprises or consists of the sequence of QYYQLSNTGQDTISQMEERLGNTSQELQSLQVQNIKLAGSLQHVAEKLCRELYNKAGAHRCSPCTEQWKWHGDNCYQFYKDSKSWEDCKYFCLSENSTMLKINKQEDLEFAASQSYSEFFYSYWTGLLRPDSGKAWLWMDGTPFTSELFHIIIDVTSPRSRDCVAILNGMIFSKDCKELKRCVCERRAGMVKPESLHVPPETLGEGD (SEQ ID NO: 2).

[0074] A fusion molecule is a "fusion protein" comprising all or part of a functional equivalent of CLEC-1 (usually biologically active) operably linked to at least one heterologous polypeptide (i.e., a polypeptide other than the same polypeptide) produced from, derived from, or selected from an Fc polypeptide. In the context of a fusion protein, the term "fused to" means that the functional equivalent of CLEC-1 and the heterologous polypeptide are fused in frame to each other. The heterologous polypeptide may be fused to the N-terminus or the 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, since the first and second Fc polypeptide chains are bound by subnanomolar affinity and, when present, by disulfide bonds in the hinge region.

[0075] According to a specific embodiment of the present invention, a fusion molecule is provided, characterized in particular in that it is a functional equivalent of CLEC-1 through at least a part of the extracellular domain of CLEC-1. Thus, at least one fragment of the extracellular domain of CLEC-1 is fused to the constant domain of a human immunoglobulin or a fragment thereof comprising at least two Fc polypeptide chains (referred to as "heterodimeric Fc polypeptide" or "Fc polypeptide chain" or "Fc fragment"); the combination of the fragment of the immunoglobulin and the extracellular domain of CLEC-1 forms a fusion molecule that can have many valuable chemical and biological properties of human antibodies. The Fc polypeptide chain exhibits a spontaneous pairing interaction between the first Fc polypeptide chain and the second Fc polypeptide chain, and the first and second Fc polypeptides are different at least in their CH3 domain and / or CH2 domain, in particular at least in their CH3 domain, in particular only in their CH3 domain, more particularly in their CH2 and CH3 domains. Thus, the first and second Fc polypeptide chains of the fusion molecule form a heterodimer by their heterodimerization.

[0076] In a specific embodiment of the fusion molecule of the present invention, the Fc fragment of the human immunoglobulin contained in the Fc-CLEC-1 fusion molecule is the Fc fragment of human IgG1 or IgG4, and the Fc fragment is fused to the N-terminus or C-terminus of the extracellular domain of the mammalian CLEC-1 domain, particularly the human CLEC-1 domain.

[0077] As used herein, the term "polypeptide" refers to an amino acid polymer having amino acid residues of each or any length. Therefore, peptides, oligopeptides and proteins are included in the definition of "polypeptide", and these terms are used interchangeably throughout the specification and claims. The term "polypeptide" does not exclude post-translational modifications, including but not limited to phosphorylation, acetylation, glycosylation, etc.

[0078] Heterodimeric Fc polypeptide chain or part thereof

[0079] The fragment crystallizable region (Fc region or Fc) is the tail region of the antibody that interacts with the cell surface receptors called Fc receptors and some proteins of the complement system.Usually, the Fc region of an antibody isotype is composed of two identical protein fragments derived from the constant domains of the two heavy chains of the antibody (with reference to CH2 and CH3, and CH4 when present).IgA, IgD and IgG Fc regions contain 2 heavy chain constant regions, while IgM and IgE Fc regions contain 3 heavy chain constant regions in each polypeptide chain.With regard to IgA, IgD and IgG antibodies, IgA, IgD and IgG isotypes each have three CH regions, two of which belong to the Fc region.As an example, " CH " domain in the IgG case is as follows: " CH1 " refers to the 118th-215th according to the EU index in Kabat. " Hinge " refers to the 216th-230th according to the EU index in 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. The Fc region of IgG consists of two paired CH3 domains, which, by contrast, are separated and do not interact with each other, but two oligosaccharide chains are inserted between them. Wild-type Fc homodimerization is mediated by a huge and tightly packed interface between two identical CH3 domains with subnanomolar affinity, followed by disulfide bonds in the hinge region. Heterodimeric Fc molecules are mainly engineered by replacing homodimer-favorable interactions with heterodimerization-favorable interactions at the CH3 domain interface. This is achieved by introducing asymmetric mutations in each CH3 domain of each Fc chain, which promotes the assembly of Fc chains from two different antibodies.

[0080] The functional equivalent of the extracellular domain of CLEC-1 (i.e., at least one binding moiety present in the fusion molecule of the present invention) is fused to an immunoglobulin constant domain (Fc region) to form an immunoadhesin. Immunoadhesin can have many valuable chemical and biological properties of human antibodies. Since immunoadhesin can be constructed by a human protein sequence with the desired specificity connected to an appropriate human immunoglobulin hinge region and constant region (Fc) sequence, the binding specificity of interest can be achieved using completely human components. This immunoadhesin has the lowest immunogenicity to the patient and is safe for long-term or repeated use. In some embodiments, the Fc polypeptide is from a native sequence Fc region. In some embodiments, the Fc polypeptide is a variant Fc region. In 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 a native sequence Fc region and a variant Fc region. Although the boundaries of the immunoglobulin heavy chain Fc region are variable, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at the Cys226 or Pro230 position to its carboxyl terminus. The adhesion part of immunoadhesin and immunoglobulin sequence part can be connected by the smallest joint.Immunoglobulin sequence is usually but not necessarily immunoglobulin constant region.The immunoglobulin part in the chimera of the present invention can be obtained from IgG1, IgG2, IgG3 or IgG4 subtype, IgA, IgE, IgD or IgM, but is usually IgG1 or IgG4.In some embodiments, the extracellular domain of CLEC-1 and the immunoglobulin sequence part of immunoadhesin are connected by the smallest joint.As used herein, the term "joint" refers to the sequence of at least one amino acid that connects the polypeptide of the present invention and the immunoglobulin sequence part.Such a joint can be used to prevent steric hindrance.In some embodiments, the joint has 4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30 amino acid residues. However, the upper limit is not critical, but is selected for reasons of convenience, such as biopharmaceutical production of these polypeptides. The linker sequence can be the linker (G4S)3 referenced in this specification. The linker sequence can be a naturally occurring sequence or a non-naturally occurring sequence. If used for therapeutic purposes, the linker is generally non-immunogenic in a subject to which the immunoadhesin is administered.

[0081] In the present invention, a fusion molecule is provided, which has at least two different protein fragments derived from the constant region of the heavy chain of an antibody. The two different protein fragments are the first Fc polypeptide chain and the second Fc polypeptide chain of reference, respectively. In particular, the first and second Fc polypeptide chains each contain a CH3 domain and / or a CH2 domain, respectively referred to as the first CH3 (or CH2) domain or the second CH3 (or CH2) domain. In particular, the first CH3 (or CH2) domain and the second CH3 (or CH2) domain are different, that is, they do not share the same amino acid residue sequence (e.g., they do not have 100% identity). Such CH3 (or CH2) domains can differ by a single amino acid residue, or by 2 amino acid residues, or by 3 amino acid residues, or by 4 amino acid residues, or by 5 or more amino acid residues. In a preferred embodiment of the present invention, the first CH3 (or CH2) domain and the second CH3 (or CH2) domain can interact with each other, allowing the first and second Fc polypeptide chains to pair.

[0082] In a specific embodiment of the invention, the two Fc polypeptide chains are produced or derived from or selected from human immunoglobulin heavy chains, in particular IgG1 immunoglobulin heavy chains, more particularly the Fc region of IgG1, IgG2, IgG3, IgG4 immunoglobulin heavy chains. More particularly, the Fc polypeptide chains are produced, derived from or selected from IgG4 Fc-regions. More particularly, the Fc polypeptide chains are produced, derived from or selected from IgG4 Fc-regions having the substitution S228P that allows for enhanced stability of the Fc region.

[0083] In one embodiment of the invention, each Fc polypeptide chain comprises a CH3 domain.

[0084] The CH3 domain may be derived from, generated from or selected from an immunoglobulin heavy chain, particularly a human immunoglobulin heavy chain, more particularly an IgG heavy chain, such as an IgG1, IgG2, IgG3, IgG4 heavy chain.

[0085] In one embodiment of the invention, each Fc polypeptide chain comprises a truncated Fc region or an Fc region fragment comprising a CH3 domain.

[0086] In one embodiment of the present invention, the heterodimeric Fc domain comprises a first Fc chain and a complementary second Fc chain based on the "knobs and holes" technology. For example, the first Fc chain is a "knob" or K chain, meaning that it comprises substitutions that characterize a knob chain, and the second Fc chain is a "hole chain" or H chain, meaning that it comprises substitutions that characterize a hole chain. Vice versa, the first Fc chain is a "hole" or H chain, meaning that it comprises substitutions that characterize a hole chain, and the second Fc chain is a "knob" or K chain, meaning that it comprises substitutions that characterize a knob chain. 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.

[0087] 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 following table:

[0088]

[0089]

[0090] Table 1: List of complementary Fc polypeptide chains that can bind to the heterodimeric immunoglobulin of the fusion protein.

[0091] In a preferred embodiment, the "Hole" Fc polypeptide chain comprises the following substitutions: T366S, L368A, Y407V and Y349C. The Hole Fc chain may further comprise additional substitutions.

[0092] 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.

[0093] 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. Both Fc chains may also comprise additional substitutions.

[0094] In another embodiment, each Fc polypeptide chain comprises a CH3 domain as disclosed above, and at least a portion of a CH2 domain, more particularly a complete CH2 domain. The CH2 domain may be derived from, generated from or selected from an immunoglobulin heavy chain, particularly a human immunoglobulin heavy chain, more particularly an IgG heavy chain, such as an IgG1, IgG2, IgG3, IgG4 heavy chain.

[0095] In another embodiment, each Fc polypeptide chain comprises a CH3 domain and a hinge region as disclosed above. The hinge region is a short sequence of an antibody heavy chain that connects the Fab (fragment antigen binding) region to the Fc region. The hinge region can be derived from, produced from, or selected from an immunoglobulin heavy chain, particularly a human immunoglobulin heavy chain, more particularly an IgG heavy chain, such as an IgG1, IgG2, IgG3, IgG4 heavy chain. One group of useful hinge sequences is derived from the hinge region of a heavy chain antibody as described in WO96 / 34103 and WO94 / 04678. Other examples are polyalanine linker sequences. The hinge region can correspond to the amino acid sequence shown in SEQ ID NO:20.

[0096] In another embodiment, each Fc polypeptide chain comprises a constant domain produced or derived from an IgG1 isotype or an IgG4 isotype.

[0097] In one embodiment, the fusion molecule of the present invention comprises the amino acid residues shown in SEQ ID NO: 3, which is a KIH-Fc-G1 Knob chain.

[0098] In one embodiment, the fusion molecule of the present invention comprises the amino acid residues shown in SEQ ID NO: 4, which is a KIH-Fc-G1 Hole chain.

[0099] In one embodiment, the fusion molecule of the invention comprises the amino acid residues shown in SEQ ID NO:3 and the amino acid residues shown in SEQ ID NO:4.

[0100] In one embodiment, the fusion molecule of the present invention comprises the amino acid residues shown in SEQ ID NO: 5, which is a KIH-Fc-G1-CLEC-1 Hole chain.

[0101] In one embodiment, the fusion molecule of the present invention comprises the amino acid residues shown in SEQ ID NO:5 and the amino acid residues shown in SEQ ID NO:3.

[0102] In one embodiment, the fusion molecule of the invention comprises an Fc polypeptide chain produced or derived from the amino acid residues shown in SEQ ID NO: 6, which is an Fc-G1 chain. The mutations disclosed herein (see Table 1) may be present in this chain.

[0103] In one embodiment, the fusion molecule of the invention comprises an Fc polypeptide chain produced or derived from the amino acid residues shown in SEQ ID NO: 7, which is an Fc-G1e3 chain. The mutations disclosed herein (see Table 1) may be present in this chain.

[0104] In one embodiment, the fusion molecule of the invention comprises an Fc polypeptide chain produced or derived from the amino acid residues shown in SEQ ID NO: 8, which is an FcG1-N297A chain. The mutations disclosed herein (see Table 1) may be present in this chain.

[0105] In one embodiment, the fusion molecule of the invention comprises an Fc polypeptide chain produced or derived from the amino acid residues shown in SEQ ID NO: 9, which is an FcG4-S228P chain. The mutations disclosed herein (see Table 1) may be present in this chain.

[0106] In one embodiment, the fusion molecule of the invention comprises an Fc polypeptide chain produced or derived from the amino acid residues shown in SEQ ID NO: 10, which is a KIH-FcG4 Knob chain.

[0107] In one embodiment, the fusion molecule of the invention comprises an Fc polypeptide chain produced or derived from the amino acid residues shown in SEQ ID NO: 11, which is a KIH-FcG4 Hole chain.

[0108] In one embodiment, the fusion molecule of the invention comprises an Fc polypeptide chain produced or derived from the amino acid residues shown in SEQ ID NO:10 and the amino acid residues shown in SEQ ID NO:11.

[0109] In one embodiment, the fusion molecule of the invention comprises an Fc polypeptide chain produced or derived from the amino acid residues shown in SEQ ID NO: 12, which is a KIH-FcG4-CLEC-1 Hole chain.

[0110] In one embodiment, the fusion molecule of the invention comprises or consists of an Fc polypeptide chain produced or derived from the amino acid residues shown in SEQ ID NO:10 and the amino acid residues shown in SEQ ID NO:12.

[0111] In one embodiment, the fusion molecule comprises:

[0112] (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 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

[0113] (ii) a heterodimeric immunoglobulin or a fragment thereof, comprising

[0114] a. a first Fc polypeptide chain comprising a first CH3 domain (CH3B chain or hole chain), and

[0115] b. a second Fc polypeptide chain comprising a second CH3 domain (CH3A chain or knob chain),

[0116] wherein the first and second CH3 domains are different,

[0117] The binding moiety is fused to the C-terminus of the first Fc polypeptide or the second Fc polypeptide chain, and

[0118] (iii) an antigen binding domain comprising or consisting of a variable domain of an antibody or antigen binding fragment thereof, in particular which binds to PD-1, said antigen binding domain being fused to the N-terminus of an Fc polypeptide chain to which the binding moiety is fused.

[0119] In a specific embodiment, both the binding moiety and the antigen binding domain are fused to a Hole Fc polypeptide chain.

[0120] CLEC-1 binding part

[0121] The term "functional equivalent fragment" as used herein may refer to any fragment or assembly of fragments of CLEC-1. Therefore, the present invention provides a polypeptide capable of reducing / reducing or inhibiting the binding of CLEC-1 to at least one of its ligands, in particular a functional equivalent, the polypeptide comprising consecutive amino acids having a sequence that is a sequence of at least a portion of the extracellular domain of CLEC-1 of a functional equivalent to CLEC-1, in a preferred embodiment, the functional equivalent of CLEC-1 is a portion that binds to at least one ligand of CLEC-1, in particular a portion that binds to human CLEC-1L.

[0122] In a specific embodiment of the present 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 of SEQ ID NO: 2. Alternatively, the extracellular domain of CLEC-1 is a peptide or polypeptide comprising at least 20 amino acid residues, particularly at least 25 amino acid residues, particularly at least 30 amino acid residues, particularly at least 40 amino acid residues, particularly 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 having at least 70% identity, particularly at least 80% identity, more particularly at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% and even more particularly at least 99% identity with the extracellular domain of CLEC-1 of SEQ ID NO:2, or sharing 100% identity. The extracellular domain of CLEC-1 may be a polypeptide fused to a linker sequence, which 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%, even more particularly at least 99% or sharing 100% identity with the extracellular domain of CLEC-1 of SEQ ID NO:2.

[0123] The percent identity with a reference in the description of the present invention is determined based on a global alignment of the sequences to be compared, that is to say based on an alignment of the sequences over their entire length, using for example the algorithm of Needleman and Wunsch 1970. This sequence comparison can be performed, for example, using the needle software by using the parameter "Gapopen" equal to 10.0, the parameter "GapExtend" equal to 0.5 and the matrix "BLOSUM62". Software (e.g. needle) is available under the name "needle" at the global website ebi.ac.uk.

[0124] As used herein, a "functional equivalent" of CLEC-1 is a compound that is able to bind 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 mutant proteins of CLEC-1. Thus, the term "functional equivalent" includes any equivalent of CLEC-1 obtained by changing the amino acid sequence, for example by deletion, substitution or addition of one or more amino acids, such that the protein analog retains the ability to bind to its ligand. For example, amino acid substitutions can be made by point mutations of the DNA encoding the amino acid sequence.

[0125] 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 to form a molecule capable of binding to at least one ligand of CLEC-1, in particular CLEC-1L (in particular to form a soluble receptor capable of capturing at least one ligand of CLEC-1). Functional equivalents include soluble forms of CLEC-1. Suitable soluble forms of these proteins or their functional equivalents may include, for example, mutant, in particular truncated forms of the protein from which the transmembrane domain has been removed by chemical, proteolytic or recombinant methods. In particular, functional equivalents consist of an amino acid sequence that is at least 70%, in particular at least 80% identical, 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%, even more particularly at least 99% identical to the corresponding protein over its entire length. The term "corresponding protein" as used herein refers to a protein having a similar function to a functional equivalent of the present invention. Based on a global alignment of the sequences to be compared, that is, based on an alignment of the sequences over their entire length, the percent identity with the reference in the description of the present invention is determined using, for example, the algorithm of Needleman and Wunsch 1970. The sequence comparison can be performed, for example, using the needle software by using the parameter "Gapopen" equal to 10.0, the parameter "Gap Extend" equal to 0.5 and the matrix "BLOSUM62". Software (e.g. needle) is available under the name "needle" on the global website ebi.ac.uk.

[0126] In one embodiment of the present invention, the portion of the extracellular domain of human CLEC-1 corresponds to a truncated portion of the extracellular domain of CLEC-1, wherein the N-terminus of the extracellular domain of CLEC-1, in particular the N-terminus of SEQ ID NO:2 is deleted. The N-terminus may correspond to the first 10, or first 11, or first 13, or first 14, or first 15, or first 16, or first 17, or first 18, or first 19, or first 20, or first 21, or first 22, or first 23, or first 24, or 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 to up to 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 amino acid residue sequence as shown in SEQ ID NO:19.

[0127] Linker peptide and hinge domain

[0128] In a specific embodiment of the present invention, any binding moiety contained in the fusion molecule can be fused to the Fc polypeptide chain via a linker peptide. The linker peptide generally ensures that the linked binding moiety is linked to the Fc polypeptide chain in a conformation in which each performs its function.

[0129] In a specific embodiment, each binding moiety is preferably connected to the Fc polypeptide chain by a linker peptide. In a specific embodiment, when the binding moiety is fused to the C-terminus of the Fc polypeptide chain, the binding moiety can be fused to the Fc polypeptide chain by a linker peptide. As used herein, the term "linker peptide" refers to a sequence of at least one amino acid residue. The joint is usually 1-44 amino acid residues in length. Preferably, the joint has 3-30 amino acid residues. In a specific embodiment, the linker peptide has 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.

[0130] The linker peptide can be a naturally occurring sequence or a non-naturally occurring sequence. In a specific embodiment, the linker peptide is non-immunogenic in a subject to which the fusion molecule is administered. Other examples are polyalanine linker sequences. Other preferred examples of linker sequences are Gly / Ser linkers of varying lengths, including (Gly4Ser)4, (Gly4Ser)3, (Gly4Ser)2, Gly4Ser, Gly3Ser, Gly3, Gly2Ser and (Gly3Ser2)3, particularly (Gly4Ser)3. Preferably, the linker is selected from (Gly4Ser)4, (Gly4Ser)3 and (Gly3Ser2)3. Even more preferably, the linker is (GGGGS)3. In a specific embodiment, the linker peptide contained in the fusion molecule is selected from (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 (Gly4Ser)4, (Gly4Ser)3 and (Gly3Ser2)3. In a specific embodiment, the linker peptide is (Gly4Ser)3 of SEQ ID NO: 13. In a specific embodiment, the binding portion can be fused to the Fc chain through a hinge sequence naturally found in immunoglobulin heavy chains (commonly referred to as a "hinge"), which is used to connect the VH domain, in particular, to connect the CH1 domain to the CH2 domain. In a specific embodiment, when the binding moiety is fused to the N-terminus of the Fc polypeptide chain, the binding moiety can be fused to the Fc polypeptide chain via a hinge domain. A useful set of hinge domains is derived from the hinge region of a heavy chain antibody as described in WO96 / 34103 and WO94 / 04678.

[0131] In one embodiment of the invention, the fusion molecule comprises at least one binding moiety, in particular a single binding moiety, fused to the C-terminus of the first Fc polypeptide chain. In a preferred embodiment, at least one binding moiety or a single binding moiety is fused to the C-terminus of the first Fc polypeptide via a linker peptide, in particular 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, in particular a truncated portion of the extracellular domain of CLEC-1, more particularly having the amino acid residue sequence as shown in SEQ ID NO: 19. As shown in the examples of the present invention, such constructs are less likely to aggregate.

[0132] In one embodiment of the invention, the fusion molecule comprises at least one binding moiety, in particular a single binding moiety, fused to the C-terminus of the second Fc polypeptide chain. In a preferred embodiment, at least one binding moiety or a single binding moiety is fused to the C-terminus of the first Fc polypeptide via a linker peptide, in particular 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, in particular a truncated portion of the extracellular domain of CLEC-1, more particularly having the amino acid residue sequence as shown in SEQ ID NO: 19. As shown in the examples of the present invention, such constructs are less likely to aggregate.

[0133] In one embodiment of the present 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.

[0134]

[0135] Table 2: Various Fc KIH regions, linkers, and ligands that may be present in the heterodimeric immunoglobulins of the present invention. Sequences of the head and CLEC-1 domains.

[0136] SEQ ID NO: 3 corresponds to Fc-KIH (Knob) shown in the Examples of the present invention, which specifically has an IgG1 domain with T366W and S354C.

[0137] SEQ ID NO: 4 corresponds to Fc-KIH (Hole) shown in the Examples of the present invention, which specifically has an IgG1 domain with T366S, L368A, Y407V and Y349C.

[0138] SEQ ID NO: 10 corresponds to Fc-KIH (Hole). It specifically has an IgG4 domain with S228P, T366W and S354C.

[0139] SEQ ID NO: 11 corresponds to Fc-KIH (Knob), which specifically has an IgG4 domain with S228P, T366S, L368A, Y407V and Y349C.

[0140] SEQ ID NO: 20 corresponds to the hinge domain found in, for example, Fc-KIH (Knob or Hole) as shown in the Examples of the present invention.

[0141] SEQ ID NO: 21 corresponds to the CH2 domain found in, for example, Fc-KIH (Knob or Hole) as shown in the Examples of the present invention.

[0142] SEQ ID NO: 22 corresponds to the CH3 domain found in, for example, Fc-KIH (Hole) as shown in the Examples of the present invention.

[0143] Linkers are those detailed above.

[0144] The CLEC-1 domain of SEQ ID NO: 2 corresponds to the extracellular domain of CLEC-1.

[0145] The CLEC-1 domain of SEQ ID NO: 19 corresponds to the truncated extracellular domain of CLEC-1 as described in detail above.

[0146] In one embodiment of the invention, the fusion molecule comprises a heterodimeric immunoglobulin comprising a second Fc polypeptide chain comprising or consisting of the following domains:

[0147] - the Fc-KIH domain of SEQ ID NO: 3; and

[0148] - a linker of SEQ ID NO: 13; and

[0149] - the CLEC-1 domain of SEQ ID NO: 2; and optionally in a preferred embodiment, the first Fc polypeptide chain comprises or consists of the Fc KIH domain of SEQ ID NO: 4.

[0150] In one embodiment of the invention, the fusion molecule comprises a heterodimeric immunoglobulin comprising a second Fc polypeptide chain comprising or consisting of the following domains:

[0151] - the Fc-KIH domain of SEQ ID NO: 3; and

[0152] - a linker of SEQ ID NO: 13; and

[0153] - the CLEC-1 domain of SEQ ID NO: 19; and optionally in a preferred embodiment the first Fc polypeptide chain comprises or consists of the Fc KIH domain of SEQ ID NO:4.

[0154] In one embodiment of the invention, the fusion molecule comprises a heterodimeric immunoglobulin comprising a first Fc polypeptide chain comprising or consisting of the following domains:

[0155] - the Fc-KIH domain of SEQ ID NO: 4; and

[0156] - a linker of SEQ ID NO: 13; and

[0157] - the CLEC-1 domain of SEQ ID NO: 2, and optionally in a preferred embodiment the second Fc polypeptide chain comprises or consists of the Fc KIH domain of SEQ ID NO: 3.

[0158] In one embodiment of the invention, the fusion molecule comprises a heterodimeric immunoglobulin comprising a first Fc polypeptide chain comprising or consisting of the following domains:

[0159] - the Fc-KIH domain of SEQ ID NO: 4; and

[0160] - a linker of SEQ ID NO: 13; and

[0161] - the CLEC-1 domain of SEQ ID NO: 19; optionally in a preferred embodiment the second Fc polypeptide chain comprises or consists of the Fc KIH domain of SEQ ID NO:3.

[0162] In one embodiment of the invention, the fusion molecule comprises a heterodimeric immunoglobulin comprising a first Fc polypeptide chain comprising or consisting of the following domains:

[0163] - the hinge domain of SEQ ID NO: 20; and

[0164] - the CH2 domain of SEQ ID NO: 21; and

[0165] - the CH3 domain of SEQ ID NO: 22; and

[0166] - a linker of SEQ ID NO: 13; and

[0167] - the CLEC-1 domain of SEQ ID NO: 2; and optionally in a preferred embodiment the second Fc polypeptide chain comprises or consists of the Fc KIH domain of SEQ ID NO: 3.

[0168] In one embodiment of the invention, the fusion molecule comprises a heterodimeric immunoglobulin comprising a first Fc polypeptide chain comprising or consisting of the following domains:

[0169] - the hinge domain of SEQ ID NO: 20; and

[0170] - the CH2 domain of SEQ ID NO: 21; and

[0171] - the CH3 domain of SEQ ID NO: 22; and

[0172] - a linker of SEQ ID NO: 13; and

[0173] - the CLEC-1 domain of SEQ ID NO: 19; and optionally in a preferred embodiment the second Fc polypeptide chain comprises or consists of the Fc KIH domain of SEQ ID NO:3.

[0174] In one embodiment of the invention, the fusion protein comprises a first Fc polypeptide chain and a second Fc polypeptide chain, the first Fc polypeptide chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 4 and the second Fc polypeptide chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 3, and at least the binding part comprises or consists of the amino acid sequence shown in SEQ ID NO: 2. In particular, at least one binding part 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.

[0175] In one embodiment of the invention, the fusion protein comprises a first Fc polypeptide chain and a second Fc polypeptide chain, the first Fc polypeptide chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 4 and the second Fc polypeptide chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 3, and at least the binding part comprises or consists of the amino acid sequence shown in SEQ ID NO: 19. In particular, at least one binding part 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.

[0176] In one embodiment of the present invention, the fusion protein comprises a first Fc polypeptide chain and a second Fc polypeptide chain, wherein the first Fc polypeptide chain comprises or consists of the amino acid sequence as shown in SEQ ID NO:5 and the second Fc polypeptide chain comprises or consists of the amino acid sequence as shown in SEQ ID NO:3, and the binding portion of SEQ ID NO:2 is present in the first Fc polypeptide chain.

[0177] In one embodiment of the present invention, the fusion protein comprises a first Fc polypeptide chain and a second Fc polypeptide chain, wherein the first Fc polypeptide chain comprises or consists of the amino acid sequence as shown in SEQ ID NO:4 and the second Fc polypeptide chain comprises or consists of the amino acid sequence as shown in SEQ ID NO:23, and the binding portion of SEQ ID NO:23 is present in the second Fc polypeptide chain.

[0178] Other binding molecules (antigen binding domain and other binding molecules that are different from at least one binding moiety of the fusion protein)

[0179] In a specific embodiment of the invention, the fusion molecule further comprises at least one antigen binding domain or another binding moiety that is different from 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.

[0180] The fusion molecule may comprise an additional binding moiety that is different from 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 an extracellular domain of a protein or a portion thereof, in particular an extracellular domain of a protein known to be involved in an immune response, such as, but not limited to, PD1, PDL1, CTLA4, 4-1BBL, CD80, CD137. The binding moiety should have the binding ability of its native protein against at least one of its ligands.

[0181] When the fusion molecule comprises an antigen binding domain, the domain comprises or consists of the variable domain of an antibody or antigen binding fragment thereof, which domain is fused to the first Fc polypeptide chain or the 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.

[0182] In specific embodiments, when the antigen binding domain is present on an Fc polypeptide chain that is associated with a binding moiety at one end, then the antigen binding fragment or binding molecule is associated at the opposite end of the Fc polypeptide chain. For example, when the binding moiety is associated with the C-terminus of the Fc polypeptide chain, then the antigen binding domain or binding molecule is associated with the N-terminus of the Fc polypeptide chain. When the binding moiety is associated with the N-terminus of the Fc polypeptide chain, then the antigen binding domain or binding molecule is associated with the C-terminus of the Fc polypeptide chain.

[0183] As used herein, an antigen binding domain means a portion or fragment of an antibody, i.e., a molecule corresponding to a portion of the antibody structure, which may exhibit antigen binding ability to its target in its native form; such a fragment particularly exhibits the same or substantially the same antigen binding specificity to the antigen 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 4-chain antibody. However, antigen binding fragments with reduced antigen binding affinity relative to the corresponding 4-chain antibody are also included in the present invention. Antigen binding ability can be determined by measuring the affinity between the antibody and the antibody target fragment. These antigen binding fragments may also be referred to as "functional fragments" of antibodies.

[0184] The antigen-binding fragment of an antibody is a fragment comprising a hypervariable domain, which is designated as the CDR (complementarity determining region) of a reference antibody or a portion thereof, which comprises the recognition site of the antigen. Antigen-binding fragments of antibodies containing variable domains comprising the CDRs of the antibody include Fv, dsFv, scFv, Fab, Fab', F(ab')2. In a specific embodiment of the invention, the antigen-binding domain is a Fab domain.

[0185] These basic antigen-binding fragments for use according to the invention can be combined together to obtain multivalent antigen-binding fragments, such as diabodies, tribodies or tetrabodies. When the Fab domain is combined with a fusion molecule, these multivalent antigen-binding fragments are also part of the invention.

[0186] Antigen-binding antibody mimics are organic compounds that specifically bind antigens but are structurally unrelated to antibodies. They are usually artificial peptides or small proteins with a molar mass of about 3-20kDa. Nucleic acids and small molecules are sometimes also considered to be antibody mimics, but are not artificial antibodies, antibody fragments and fusion proteins composed of them. The common advantages relative to antibodies are better solubility, tissue permeability, stability to heat and enzymes, and relatively low production costs. Antibody mimics are developed as therapeutic agents and diagnostic agents. Antigen-binding antibody mimics can also be selected from affibodies, affilins, aptamers, anticalins, affimers, affitins, DARPins and monomeric antibodies.

[0187] In a specific embodiment of the invention, the fusion molecule comprises two antigen binding domains, each associated with a single Fc polypeptide chain, the first antigen binding domain and the second antigen binding domain bind to the same epitope, or the same antigen, or two different epitopes, or two different antigens. The epitope is a part of the antigen molecule that the antibody itself binds to. Therefore, when the fusion molecule comprises two antigen binding domains, the two antigen binding domains can bind to the same epitope or can bind to two different epitopes located in the same antigen, or can bind to two different epitopes located in two different antigens. In other words, when the fusion molecule comprises two antigen binding domains, they can bind to the same antigen or bind to two different antigens.

[0188] In a particular embodiment 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.

[0189] In a specific embodiment of the present invention, at least one antigen binding domain specifically binds to an antigen selected from the following list: SIRPα, SIRPβ, SIRPγ, 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, 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, EMR 2. 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.

[0190] In a specific embodiment of the invention, the fusion molecule comprises two antigen binding domains, each associated with a single Fc polypeptide chain, and the two antigen binding domains bind to the same antigen selected from the following list: SIRPα, SIRPβ, SIRPγ, 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, 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.

[0191] In a specific embodiment of the invention, the fusion molecule comprises two antigen binding domains, each binding antigen, and the two antigens recognized by each antigen binding domain are independently selected from the following list: SIRPα, SIRPβ, SIRPγ, 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), lactophilus Lipoprotein-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, EMR 1. 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.

[0192] In specific embodiments, the antigen binding domain is at the N-terminus of the first Fc polypeptide chain or the second Fc polypeptide chain.

[0193] In a specific embodiment, the antigen binding domain is at the N-terminus of a first Fc polypeptide chain and the second antigen binding domain is fused to a second Fc polypeptide chain.

[0194] In specific embodiments, the antigen binding domain is at the N-terminus of the CH2 domain, or in the hinge region when a hinge region is present.

[0195] Uses of fusion molecules

[0196] Fusion molecules used as drugs are provided. In particular, fusion molecules for treating diseases in subjects (i.e., patients, particularly human patients), particularly those suffering from cancer, or infectious diseases, or sepsis, or autoimmune diseases, or inflammatory diseases (including acute or chronic inflammatory diseases) are provided. In another embodiment, fusion molecules for preventing diseases in subjects can be provided. As used herein, "treatment" or "treating" is a method for obtaining beneficial or desired results. 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 caused by the disease, curing the disease, reducing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the deterioration of the disease), preventing or delaying the spread of the disease, preventing or delaying the recurrence of the disease, delaying or slowing down the progress of the disease, improving the disease state, providing relief (partial or complete) of the disease, reducing the dosage of one or more other drugs required for treating the disease, delaying the progress of the disease, improving the quality of life and / or prolonging survival. According to one embodiment, the term "treatment" relates to preventive treatment. As used herein, the term "prevention" refers to reducing the risk of obtaining or developing a given condition.

[0197] In a specific embodiment of the invention, a fusion molecule for treating a patient is provided, wherein the phagocytosis of target cells, in particular cells expressing at least one ligand of CLEC-1, in particular cells expressing CLEC-1L, more particularly CLEC-1L positive tumor cells by myeloid cells, in particular dendritic cells and / or macrophages is regulated to improve the outcome of the disease. Thus, in a specific embodiment, a fusion molecule is provided to increase the phagocytosis of cells, in particular CLEC1 ligand positive cells, in particular 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, in particular dendritic cells and / or macrophages. Thus, the fusion molecule of the invention can target CLEC-1L positive tumors (i.e. tumors comprising at least one cell expressing, in particular aberrantly expressing, CLEC-1L). Indeed, it has been shown that antagonists of the CLEC-1L-CLEC1 signaling pathway (e.g., compounds that inhibit the binding between CLEC-1 and CLEC-1L, compounds that bind to CLEC-1L or CLEC1, or functional equivalents of CLEC-1L or CLEC1, or compounds that reduce the expression of CLEC-1L or functional equivalents of CLEC1, or compounds that reduce or inhibit the signaling pathway induced by the binding between CLEC-1L and CLEC-1) are able to modulate, in particular enhance, the phagocytosis of target cells (e.g., tumor cells expressing CLEC-1L) by myeloid cells, in particular dendritic cells and / or macrophages, leading to their use in therapy or treatment of diseases, wherein modulating the phagocytosis of target cells (e.g., tumor cells or cells expressing CLEC-1L) by myeloid cells, in particular dendritic cells and / or macrophages improves the health of the patient. Thus, the use of the fusion molecules of the invention that 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) can be used to modulate the phagocytosis of cells (e.g., tumor cells) by myeloid cells, in particular dendritic cells and / or macrophages. When myeloid cells, in particular macrophages or dendritic cells, expressing CLEC-1A interact with cells expressing CLEC-1L, the phagocytosis of these macrophages or dendritic cells can be inhibited or reduced. For example, it has been shown that tumor cells expressing CLEC-1Lescape (i.e., Raji cells) escape the phagocytosis exerted by macrophages. By using an antagonist compound of the invention that modulates the interaction between myeloid cells expressing CLEC-1 and cells expressing CLEC-1L, in particular CLEC-1L-positive tumor cells, the reduction or inhibition of the phagocytosis of cells expressing CLEC-1L by myeloid cells, in particular dendritic cells or macrophages, is reduced.Thus, phagocytosis of cells expressing CLEC-1L (eg, tumor cells) is enhanced in the presence of the fusion molecules of the present invention.

[0198] More specifically, the present invention relates to the use of the fusion molecule in treating a disorder or disease in a patient, wherein the disorder or disease is or involves a cancer listed in the present specification, in particular cancer with CLEC1 ligand-positive tumor cells, in particular CLEC-1L-positive tumor cells, cancer 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, infection (in particular coxsackievirus or encephalitis virus, more particularly coxsackievirus B3 or Japanese encephalitis virus), cardiovascular disease, autoimmune disease (in particular Sjogren's syndrome or systemic lupus erythematosus or systemic sclerosis), inflammatory disease.

[0199] The term "cancer" has its ordinary meaning in the art and refers to a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. The term "cancer" also encompasses primary and metastatic cancers. Examples of cancers that can be treated by the methods and compositions of the present invention include, but are not limited to, liquid cancers, solid cancers, cancers expressing at least one human CLEC-1 ligand, cancers with CLEC-1 ligand-positive tumors, cancers with CLEC-1L-positive tumor cells, cancers with CLEC-1L-positive tumors, cancers from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gums, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testicles, tongue, or uterus. In addition, cancer can be specifically of the following histological types, but is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; trichomonuclear carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; bronchioloalveolar adenocarcinoma; papillary Adenocarcinoma; Chromophobe cell carcinoma; Oncocytic carcinoma; Oncocytic adenocarcinoma; Basophilic cell carcinoma; Clear cell adenocarcinoma; Granular cell carcinoma; Follicular adenocarcinoma; Papillary and follicular adenocarcinoma; Nonencapsulated sclerosing carcinoma; Adrenocortical carcinoma; Endometrioid carcinoma; Carcinoma of skin appendages; Apocrine carcinoma; Sebaceous gland carcinoma; Cereus; Adenocarcinoma; Mucoepidermoid carcinoma; Cystadenoma; Papillary cystadenocarcinoma; Papillary serous cystadenocarcinoma; Mucinous cystadenocarcinoma; Mucinous adenocarcinoma; Signet ring cell carcinoma; Infiltrating ductal carcinoma; Medullary carcinoma; Lobular carcinoma; Inflammatory carcinoma; Paget's disease, breast; Acinar cell carcinoma; Adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; lymphoma, malignant; granulosa cell tumor, malignant; and retinoblastoma, malignant; Sertoli cell tumor, malignant; Leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extramammary paraganglioma, malignant; pheochromocytoma; angiosarcoma; melanoma, malignant; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma ; fibrosarcoma; fibrohistiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; Wilms' tumor; hepatoblastoma; carcinosarcoma; mesenchymal tumor, malignant; Brenner's tumor, malignant; cystosarcoma phyllodes, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; goiter, malignant; choriocarcinoma; mesonephroma, malignant; angiosarcoma;Hemangioendothelioma, malignant; Kaposi's sarcoma; Hemangiopericytoma, malignant; Lymphangiosarcoma; Osteosarcoma; Juxtacortical osteosarcoma; Chondrosarcoma; Chondroblastoma, malignant; Mesenchymal chondrosarcoma; Giant cell tumor of bone; Ewing's sarcoma; Odontogenic tumors, malignant; Odontogenic ameloblastic cementumoma; Ameloblastoma, malignant; Ameloblastic fibrosarcoma; Pinealoma, malignant; Chordoma; Glioma, malignant; Ependymoma; Astrocytoma; Protoplasmic astrocytoma; Fibrous astrocytoma; Astroblastoma; Glioblastoma; Oligodendroglioma; Oligodendroglioma; Primitive neuroectodermal; Cerebellar sarcoma; Ganglioneuroblastoma; Neuroblastoma; Retinoblastoma; Olfactory neurogenic tumors; 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 lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemias; lymphoid leukemias; plasma cell leukemias; erythroleukemias; lymphosarcoma cell leukemias; myeloid leukemias; basophilic leukemias; eosinophilic leukemias; monocytic leukemias; mast cell leukemias; megakaryoblastic leukemias; myeloid sarcoma; and hairy cell leukemias.

[0200] In a specific embodiment, the subject has a cancer selected from the group consisting of bile duct cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, Castleman's disease, cervical cancer, colorectal cancer, adenocarcinoma, endometrial cancer, esophageal cancer, gallbladder cancer, gastrointestinal carcinoid tumors, Hodgkin's disease, non-Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancer, mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cancer and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, stomach cancer, testicular cancer, thymic cancer, thyroid cancer, vaginal cancer, vulvar cancer, and uterine cancer.

[0201] CLEC-1A is also known to be involved in various pathological conditions, including sepsis, infection, allergic inflammation. Tone et al. (Frontiers in Immunology, 2021), Gao et al. (iSciences, 2020), or Zhu et al. (Invest. Ophthalmo. Vis. Scis., 2021) emphasize that blocking CLEC-1A signaling is associated with the regulation of immune responses (CLEC-1 is an immunosuppressant of myeloid cells), which can enhance patient outcomes.

[0202] The present invention also relates to the use of heterodimeric fusion molecules in treating infectious diseases.

[0203] The present invention also relates to the use of the heterodimeric fusion molecule in treating sepsis.

[0204] The present invention also relates to the use of heterodimeric fusion molecules in treating autoimmune diseases.

[0205] The present invention also relates to the use of heterodimeric fusion molecules in treating inflammatory diseases, especially acute or chronic inflammatory diseases.

[0206] The present invention also relates to the use of heterodimeric fusion molecules in the treatment (including prophylactic treatment) of adverse conditions or diseases, in particular where dendritic cells and / or T cells are involved, and where the proliferation of T cells and / or the stimulation of phagocytosis by myeloid cells (in particular dendritic cells and / or macrophages) can improve or treat the condition or disease. In a specific embodiment, the disease or condition is selected from cancer, in particular the cancers listed above, more particularly cancers with CLEC-1L positive tumor cells, liquid cancers, solid cancers, lymphomas, colorectal cancers, adenocarcinomas, mesothelioma or liver cancer.

[0207] Pharmaceutical composition

[0208] The present invention also relates to a pharmaceutical composition for treating a patient suffering from a disease, comprising as a therapeutic agent a fusion molecule as defined herein (alone or in combination with a second therapeutic agent) and a pharmaceutically suitable vehicle, the vehicle being pharmaceutically acceptable for a formulation that can be administered to a patient in need thereof. These formulations may in particular be isotonic, sterile saline solutions (monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, etc. or mixtures of these salts), or dried, in particular lyophilized compositions, which, after addition of sterile water or physiological saline, as the case may be, allow the preparation of injectable solutions.

[0209] Nucleic acid molecules

[0210] The present invention also relates to a nucleic acid molecule or a combination of nucleic acid molecules encoding at least a portion of a fusion protein described herein.

[0211] To this end, the present invention also relates to a nucleic acid molecule or a combination of nucleic acid molecules encoding a fusion protein according to any definition disclosed herein. In particular, the nucleic acid molecule encodes at least the first Fc polypeptide chain disclosed herein, and / or the second Fc polypeptide chain, and / or at least one binding moiety. Preferably, the nucleic acid molecule encodes the first Fc polypeptide chain, the second Fc polypeptide chain and at least one binding moiety. In particular, when a hinge portion, a linker and / or CH2 are present, the nucleic acid molecule can encode these elements.

[0212] The present invention also relates 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 wherein the second nucleic acid molecule encodes at least a second Fc polypeptide chain.

[0213] Any nucleic acid molecule according to the present invention may be inserted into an expression vector, such as a plasmid suitable for expressing the coding sequence in a host cell.

[0214] Combination of compounds

[0215] The present invention also relates to combinations of compounds comprising a first therapeutic agent and at least one additional therapeutic agent.

[0216] A combination of compounds is a preparation comprising at least two different products or agents or compounds, which may be packaged together or separately and prepared for simultaneous, co-administered or coordinated administration (or sequential administration). In particular, the amount of each compound in the combination may be 1 μg to 100 mg per kg of patient body weight.

[0217] The first therapeutic agent is a fusion molecule as defined in any embodiment of the invention disclosed herein (ie 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).

[0218] At least one additional therapeutic agent is selected from the following list: a tumor-targeting antibody or an antigen-binding fragment thereof, in particular a tumor-targeting monoclonal antibody or an antigen-binding fragment thereof, more particularly a tumor-targeting monoclonal antibody or an antigen-binding fragment thereof that activates and / or enhances phagocytosis of tumor cells or cells expressing CLEC-1L by myeloid cells, in particular dendritic cells and / or macrophages, and further more particularly a monoclonal antibody selected from the group consisting of alemtuzumab, atezolizumab, bevacizumab, cetuximab, trastuzumab, panitumumab, rituximab, trastuzumab, anti-PDL-1 antibodies and anti-CD47 antibodies, or another antibody or monoclonal antibody selected from the group consisting of anti-PD1 antibodies and anti-SIRPα antibodies; and / or a chemotherapeutic agent, in particular a cytotoxic agent having an anti-proliferative, pro-apoptotic, cell cycle arrest and / or differentiation-inducing effect, more particularly a cytotoxic agent selected from the group consisting of a cytotoxic antibody, an alkylating drug, an anthracycline, an antimetabolite, an antimicrotubule agent, a topoisomerase inhibitor, an alkaloid, bleomycin, an anti-tumor drug, cyclophosphamide. Tumor targeting antibodies can be defined as therapeutic monoclonal antibodies that recognize tumor-specific membrane proteins, block cell signaling, and induce tumor killing through Fc-driven innate immune responses. Chemotherapeutic agents can be conventional cytotoxic agents, i.e. compounds that induce irreversible lethal damage by interfering with DNA replication, mitosis, etc. after exposure. These agents can have anti-proliferation, pro-apoptosis, cell cycle arrest, and differentiation induction effects. These agents are preferably selected from the following group: alkylating drugs (cisplatin, chlorambucil, procarbazine, carmustine), anthracyclines and other cytotoxic antibiotics, antimetabolites (i.e. methotrexate, cytarabine, gemcitabine), anti-microtubule agents (i.e. vinblastine, paclitaxel, docetaxel), topoisomerase inhibitors (i.e. etoposide, doxorubicin), alkaloids (i.e. vincristine, vinblastine, vinorelbine, camptothecin) or bleomycin (inhibits thymidine incorporation into DNA chains). The combination may include more than one second therapeutic agent selected from the list. The combination may also further comprise additional therapeutic agents, and / or components not listed in the table, such as but not limited to pharmaceutical excipients or administration vehicles.

[0219] In a specific embodiment, in the treatment of disease, particularly in the treatment of cancer, the therapeutic agents may be administered simultaneously, separately or sequentially.

[0220] In a specific embodiment, a fusion molecule as defined in any embodiment of the invention disclosed herein is provided for use in the treatment of liquid cancers, in particular leukemias, lymphomas or myelomas, or for the treatment of solid cancers, in particular for the treatment of colorectal cancer or adenocarcinoma.

[0221] In a specific embodiment, there is provided a combination of a fusion molecule as defined in any of the embodiments of the invention disclosed herein and cyclophosphamide.

[0222] In a specific embodiment, there is provided a combination of a fusion molecule as defined in any embodiment of the invention disclosed herein and cyclophosphamide for use in the treatment of cancer, in particular a liquid cancer, more particularly a leukemia, lymphoma or myeloma, or in particular a solid cancer, more particularly for the treatment of colorectal cancer.

[0223] In a specific embodiment of the present invention, a fusion molecule as defined in any embodiment of the present invention disclosed herein is provided, which inhibits the C-type lectin-like receptor-1 (CLEC-1) signaling pathway, wherein the compound binds to CLEC1 or reduces the expression of functional CLEC1 or is a functional equivalent of CLEC1, in particular binds to CLEC1, more particularly binds to human CLEC1, an antagonist fusion protein of the binding between CLEC-1 and CLEC-1L, for treating a subject, in particular a human subject, having a cancer with CLEC-1L-positive tumor cells or CLEC-1-positive tumor cells, preferably the fusion protein increases the phagocytic capacity of myeloid cells, in particular dendritic cells and / or macrophages, in particular increases the phagocytosis of CLEC-1L-positive cells, more particularly CLEC-1L-positive tumor cells and / or secondary necrotic cells by dendritic cells and / or macrophages, in particular increases the phagocytosis of tumor cells and / or secondary necrotic cells by dendritic cells and / or macrophages.

[0224] In a specific embodiment of the present invention, a fusion molecule is provided, which enhances the phagocytic ability of myeloid cells, particularly dendritic cells and / or macrophages, particularly 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, more particularly tumor cells and / or secondary necrotic cells by dendritic cells and / or macrophages,

[0225] For treating patients suffering from cancer, or infectious diseases, or sepsis, autoimmune diseases or inflammatory diseases (including acute or chronic inflammatory diseases), more preferably cancer, in particular liquid cancer or solid cancer, in particular cancer with CLEC-1L positive tumor cells and / or CLEC-1 positive tumor cells, cancer 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, infection (in particular coxsackievirus or encephalitis virus, more particularly coxsackievirus B3 or Japanese encephalitis virus), cardiovascular disease, autoimmune disease (in particular Sjogren's syndrome or systemic lupus erythematosus or systemic sclerosis), inflammatory disease.

[0226] Methods for treating patients

[0227] Therefore, in another aspect, the present invention relates to a method for treating a human patient diagnosed with cancer, or an infectious disease, or sepsis, an autoimmune disease, or an inflammatory disease (including acute or chronic inflammatory diseases), more preferably cancer, particularly a liquid cancer or a solid cancer, the method comprising administering to the subject a therapeutically effective amount of a fusion molecule according to any embodiment disclosed herein. The fusion molecule can be administered in combination with conventional treatments such as cyclophosphamide.

[0228] Thus, in another aspect, the invention relates to a method for treating a human patient diagnosed with cancer, the method comprising:

[0229] - assessing whether the patient has tumor cells expressing CLEC-1L and / or CLEC-1;

[0230] - administering the fusion molecule according to any embodiment disclosed herein when the patient has CLEC-1L-positive tumor cells and / or CLEC-1-positive tumor cells.

[0231] Thus, in another aspect, the invention relates to a method of 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.

[0232] As used herein, the term "standard or conventional treatment" refers to any cancer treatment (drugs, radiotherapy, etc.) that is typically administered to a subject with cancer. In particular, the fusion molecules of the present invention are used in combination with chemotherapeutic agents, radiotherapeutic agents, immunotherapeutic agents (e.g., tumor-targeted monoclonal antibodies), cell therapeutic agents (e.g., CAR-T cells), immunosuppressants, pro-apoptotic agents, antibiotics, targeted cancer therapies, and / or probiotics.

[0233] In another aspect, a fusion molecule according to any embodiment disclosed herein is provided for use in treating a patient having CLEC-1 -ligand-positive tumor cells (ie, cells expressing a ligand for CLEC-1).

[0234] In another aspect, a fusion molecule according to any embodiment disclosed herein is provided for use in treating a patient having tumor cells recognized by CLEC-1, particularly by an anti-CLEC-1 antibody or Fc-CLEC-1, more particularly by an Fc-CLEC-1 fusion molecule according to the invention.

[0235] In another aspect, a fusion molecule according to any embodiment disclosed herein is provided for use in treating a patient who is being or has been treated with conventional treatments for cancer.

[0236] In another aspect, a fusion molecule according to any embodiment disclosed herein is provided for use in treating a patient having cancer and being or having been treated with an agent selected from the group consisting of a chemotherapeutic agent, a targeted cancer therapy, an immunotherapeutic agent, or a radiotherapeutic agent.

[0237] In another aspect, a fusion molecule according to any embodiment disclosed herein is provided for use in treating a patient having cancer and being or having been treated with an agent selected from the group consisting of a cytotoxic agent, an anti-angiogenic agent, an anti-cancer agent, a cell cycle / apoptosis control regulator, an anti-cancer antibody, and a hormone regulator.

[0238] In another aspect, a fusion molecule according to any embodiment disclosed herein is provided for the preparation of a medicament for treating a patient suffering from cancer, or an infectious disease, or sepsis, an autoimmune disease, or an inflammatory disease (including acute or chronic inflammatory diseases), in particular a liquid cancer (more particularly a leukemia, lymphoma or myeloma), or a solid cancer (more particularly colorectal cancer or adenocarcinoma). In another aspect, a fusion molecule according to any embodiment disclosed herein is provided for the preparation of a medicament for treating cancer, in particular a liquid cancer, more particularly a leukemia, lymphoma or myeloma, or a solid cancer, more particularly colorectal cancer or adenocarcinoma.

[0239] Other aspects and features of the present invention will be found in the following examples and figures.

[0240] The following figures and examples are given to provide a complete disclosure and description of how to make and use the present invention to those of ordinary skill in the art, and are not intended to limit the scope of what the inventors consider to be their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Although the present invention has been described with reference to specific embodiments of the present invention, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the present invention. In addition, many modifications may be made to adapt specific circumstances, materials, compositions of matter, processes, process steps to the purpose, spirit and scope of the present invention. All of these modifications are within the scope of the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0241] Figure 1 ：Schematic representation of different Fc-CLEC-1 fusion molecules according to the invention. A represents a prior art Fc-CLEC comprising 2 identical Fc chains (blue rectangles) and 2 binding moieties comprising the extracellular domain of CLEC-1 (green spheres). B represents a heterodimeric Fc-CLEC-1 of the invention comprising 2 different Fc chains (blue rectangles with red triangles) and a single binding moiety comprising the extracellular domain of CLEC-1 (green spheres). C represents a heterodimeric Fc-CLEC-1 of the invention comprising 2 different Fc chains (blue rectangles with red triangles), a single binding moiety comprising the extracellular domain of CLEC-1 (green spheres), and another binding moiety corresponding to the extracellular domain of another protein different from CLEC-1 (blue spheres). D represents a heterodimeric Fc-CLEC-1 of the invention comprising two different Fc chains (blue rectangles with red triangles), a single binding moiety comprising the extracellular domain of CLEC-1 (green spheres) associated with the C-terminus of one Fc chain, and an antigen binding domain associated with the N-terminal domain of the Fc chain (blue and cyan rectangles). E represents a heterodimeric Fc-CLEC-1 of the invention comprising two different Fc chains (blue rectangles with red triangles), a single binding moiety comprising the extracellular domain of CLEC-1 (green spheres), another binding moiety corresponding to the extracellular domain of another protein different from CLEC-1 (blue spheres), and a single antigen binding domain associated with a single N-terminal domain of the Fc chain (blue and cyan rectangles). F represents a heterodimeric Fc-CLEC-1 of the invention comprising two different Fc chains (blue rectangles with red triangles), a single binding portion comprising the extracellular domain of CLEC-1 (green sphere), and two antigen binding domains, each associated with a single N-terminal domain of the Fc chain (blue and cyan rectangles). G represents a heterodimeric Fc-CLEC-1 of the invention comprising two different Fc chains (blue rectangles with red triangles), a single binding portion comprising the extracellular domain of CLEC-1 (green sphere), another binding portion corresponding to the extracellular domain of another protein different from CLEC-1 (blue sphere), each associated with the C-terminus of the Fc chain, and two antigen binding domains, each associated with a single N-terminal domain of the Fc chain (blue and cyan rectangles). H represents the heterodimeric Fc-CLEC- of the invention, comprising two different Fc chains (blue rectangles 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 different from CLEC-1 (blue sphere), each of which is associated with the C-terminus of the Fc chain, and two different antigen binding domains, each of which is associated with a single N-terminal domain of the Fc chain (blue, cyan and red rectangles).

[0242] Figure 2 ： Antitumor effect of Fc CLEC-1 treatment. A. Tumor volume and B. survival of MC38 colon adenocarcinoma-transplanted mice after combined treatment with cyclophosphamide (CPA) and Fc control or Fc CLEC-1 fusion protein.

[0243] Figure 3 ： Toxicity scores after intravenous injection of different Fc CLEC-1 formats. A. Toxicity scores were evaluated over time after intravenous injection of human Fc CLEC-1 (mouse protein), mouse Fc CLEC-1 (human protein) and two different Fc KIH CLEC-1 of the present invention (Fc-KIH G1(Hole)-(G4S)3-CLEC1 and Fc-KIH-G1(knob)-(G4S)3-CLEC1) (human protein), according to the table shown in B.

[0244] Figure 4 ： Analytical SEC profiles (evaluation of monomeric, dimeric and aggregate forms) of different Fc CLEC-1 or control recombinant proteins (A) and different preparations (B).

[0245] 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.

[0246] Figure 6 ： According to the pharmacokinetic curves of Fc-CLEC-1 and three Fc-KIH-CLEC1 recombinant proteins of the present invention in mice.

[0247] Figure 7 ： ELISA binding assay of anti-CLEC-1A antibodies to three different Fc KIH CLEC-1 constructs of the invention. Binding of anti-CLEC-1 antibodies to immobilized Fc KIH CLEC-1 and isotype controls.

[0248] Figure 8 ： Inflammatory cytokine secretion. Pro-inflammatory / pro-tumor mouse IL-6 secretion after intravenous injection of Fc-CLEC-1 and Fc-KIH-CLEC1 recombinant proteins, as measured in mouse serum by ELISA.

[0249] Fig. 9 ： Example of binding of the Fc KIH CLEC-1 construct and control Fc KIH to tumor cells (here MC38 murine colon adenocarcinoma cells).

[0250] Fig.10 ：The binding of Fc-CLEC-1 to different tumor cells was detected by A. UV-C light (CL-1000 UV Crosslinker, Analytik Jena) (150 mJ / cm 2 ), B. X-ray (Faxitron CP 160 (FaxitronX-rayCorp., Wheeling)) (10Gy) or chemotherapy, C. cisplatin (20μM Merck), D. staurosporine (1μM Sigma-Aldrich) treatment to kill human non-small cell lung cancer (NSCLC) (A549), triple negative breast cancer (SK-BR3), colorectal cancer (CRC) (DLD-1, HT-29), ovary (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), AML (THP-1, U937), and cultured at 37°C for 18 hours. Then, cells were attached and stained using 100 nM Fixable Viability Dye (eBioscience) and Fc-CLEC-1 (or Fc-Ctrl) for 30 minutes at 4° C. The percentage of Fc-CLEC1 positive cells was determined after reading on a hemacytometer. DETAILED DESCRIPTION

[0251] Materials and methods

[0252] Tumor models and treatments

[0253] MC38 colon adenocarcinoma cells (ATCC, Manassas, Virginia, USA) were cultured in 1640RPMI medium (Life Technologies) supplemented with 10% endotoxin-free fetal bovine serum (Thermo Fisher), 2 mM L-glutamine (Sigma-Aldrich), 100 U / ml penicillin, 100 μg / ml streptavidin (Life Technologies, Carlsbad, California, USA) at 37°C and 5% CO according to the manufacturer's instructions. One million cells / mouse were injected subcutaneously in the left flank of C57 / B16 mice (Janvier labs, Le Genest-Saint-Isle, France). When the tumor reached 65 mm in diameter, the cells were inoculated with 100 μg / ml of 1 ... 3At 7-12 days, mice were injected with cyclophosphamide (CPA) (150 mg / kg, Sigma-Adrich, St. Louis, Missouri, USA) and co-injected with mouse Fc control or Fc CLEC-1 fusion protein (5 mg / kg, twice a week for 3 weeks). Tumor growth was measured blindly with a caliper and expressed as an area based on two perpendicular diameters ((length × width) 1.5) × π / 6. When the tumor size reached the allowed size, the mice were euthanized.

[0254] Pharmacokinetics and in vivo toxicity analysis of Fc CLEC-1 recombinant protein:

[0255] The FcCLEC-1 recombinant protein was injected intravenously into the tail vein of naive Balb-c mice at 10 mg / kg. Figure 7 Blood was collected at various time points after injection as listed. Figure 3 Scoring method detailed in B, toxicity signs were closely monitored over time. Pharmacokinetic analysis was performed using sandwich ELISA assays. For ELISA assays, goat anti-human Fc (Jackson immunoresearch reference 109-005-098) was fixed on plastic at 1 μg / ml, and serum or standard recombinant protein was added to quantify the concentration in serum. After incubation and washing, peroxidase-labeled donkey anti-human IgG (Jackson immunoresearch reference 709-035-149) was added and displayed by conventional methods.

[0256] Analytical SEC profile of Fc CLEC-1 recombinant protein:

[0257] SEC profiles were performed by gel filtration chromatography (GeHealthcare Superdex200 10 / 300GL column) using an AktaPüre system. Antibodies were injected into loops (100 μl) with PBS buffer and analyzed by AktaPüre software.

[0258] ELISA IL6 assay:

[0259] Detection of murine IL-6 in mouse serum was performed using a mouse IL-6 ELISA kit (BD Biosciences, Franklin Lakes, New Jersey, USA, OptEIA-555240) according to the manufacturer's instructions.

[0260] ELISA combined with CLEC-1L-CLEC-1 assay:

[0261] Binding of different Fc CLEC-1 recombinant proteins or Fc control to human CLEC-1L recombinant protein was performed using CLEC-1L binding ELISA. For activity ELISA assays, recombinant hCLEC-1L-His (Biotechne; reference 18010-H07B) was immobilized on plastic in P96 polysorp plates at 2 μg / ml and purified recombinant protein was added to measure binding. After incubation and washing, peroxidase-labeled donkey anti-human IgG ((Jackson immunoresearch reference 709-035-149) was added and revealed by conventional methods.

[0262] ELISA binding assay of anti-CLEC-1A with coated Fc KIH CLEC-1A

[0263] For binding ELISA assays, human Fc KIH CLEC-1A molecules were immobilized on plastic at 1 μg / ml and 5 μg / ml of mouse anti-CLEC-1A monoclonal antibody (OSE Immunotherapeutics) was added to measure binding. After incubation and washing, peroxidase-labeled donkey anti-mouse IgG (Jackson immunoresearch reference 715-036-151) was added and revealed by conventional methods.

[0264] Cell binding assay:

[0265] Using A30009 Alexa Fluor TM Fc KIH or Fc KIH CLEC-1 constructs were coupled to AF647 using the AF647 Micro Protein Labeling Kit (FISHER, Hampton, New Hampshire, USA). Tumor cells were stained with PBS 1% BSA 0.02% azide solution at pH 6 with 200 nM of the fusion protein for 45 minutes each on ice.

[0266] Example 1: Anti-tumor effect of Fc-CLEC-1 fusion molecule.

[0267] Figure 2 The results obtained in the treatment of cancer models highly relevant to oncology were demonstrated. As shown, the Fc-CLEC-1 fusion molecule enhances the antitumor effect of chemotherapy. For example, in the MC38 colon adenocarcinoma syngeneic mouse model, Fc CLEC-1 treatment, but not control Fc recombinant treatment, enhanced the tumor shrinkage ( Figure 2 A) and mouse survival ( Figure 2 B) positive role.

[0268] Example 2: In vivo toxicity by adverse reaction analysis.

[0269] In order to select the most effective 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 (side effects) in animals treated with these molecules by intravenous administration (using mouse or human CLEC-1 protein), while the Fc KIH CLEC-1 format did not induce any signs of toxicity in vivo ( Figure 3 A), as monitored by careful scoring of overall mouse aspects ( Figure 3 B). 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. Therefore, when a reasonable dose is administered (side effects may occur when a prescribed drug is administered to a patient in need thereof), the administration of the prior art Fc-CLEC cannot be considered toxic, but the heterodimeric Fc-CLEC-1 fusion molecule of the present invention causes almost no side effects, which is a clear advantage over the prior art compounds that cause side effects, such as Figure 3 The Fc-CLEC-1 compound in A is shown.

[0270] Example 3: Aggregation of Fc-CLEC fusion molecules.

[0271] The analytical SEC profile of the prior art Fc CLEC-1 fusion protein showed the presence of large aggregates, whereas the analysis of the FcKIH CLEC-1 fusion protein of the present invention surprisingly showed that the molecule formed dimers without any aggregates. A summary of the different types of isoforms fused to the protein evaluated by analytical SEC is shown in Table Figure 4As shown, highlighting the fact that the Fc KIHCLEC-1 format of the present invention (grey) is the only Fc fusion format capable of the absence of aggregates. This aggregability of Fc CLEC-1 is specific for recombinant CLEC-1 protein, as other targets such as Fc-Dectin-1R do not induce as many dimers / aggregates. Furthermore, as shown, several different Fc KIH CLEC-1 according to the present invention (Fc-KIH-G1(hole)-(G4S)3-CLEC-1, comprising or consisting of chain (comprising or consisting of SEQ ID NO:5) and chain (comprising or consisting of SEQ ID NO:3); Fc-KIH-G1(knob)-(G4S)3-CLEC-1, comprising or consisting of chain (comprising or consisting of SEQ ID NO:23 (or SEQID NO:3+SEQ ID NO:13+SEQ ID NO:2)) and chain (comprising or consisting of SEQ ID NO:4); and Fc-KIH-G1(hole)-(G4S)3-mutatedCLEC-1, comprising chain (comprising or consisting of SEQ ID NO:4+SEQ ID NO:13+SEQID NO:19) and 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 replaces IL-15 in the fusion molecule) form aggregates (see Figure 4 =The lower row of the table). Therefore, the inability to form aggregates is achieved only by the heterodimeric Fc-CLEC-1 fusion molecules of the present invention. Different classical formulations were tested to prevent aggregation, such as Figure 4 However, any buffer may be effective in reducing aggregation in Fc CLEC-1. The lack of aggregation of the heterodimeric Fc-CLEC-1 fusion molecules of the present invention may underlie the lack of in vivo toxicity particularly observed after treatment with the molecules. Figure 4 As shown, a dimer of the heterodimeric Fc-KIH-CLEC fusion protein was obtained. As the prior art believes, the authors expected to obtain monomers based on the monomeric protein of heterodimeric Fc (see, for example, Ha JH, 2016, Front. Immunol 7: 394); in fact, as Figure 4As shown in A, when only one monomer is associated with the heterodimeric immunoglobulin, the native homodimeric protein (such as Dectin-1 or IL-15) results in monomers of the heterodimeric Fc-monomeric protein: Fc-dectin1R and Fab anti-PD1-KIH-Fc-IL15 fusion protein are almost only or only monomers. In the construction of the present invention shown in this example, the authors associated a single CLEC-1 domain with the heterodimeric immunoglobulin. Therefore, the CLEC-1 domain is only present on one Fc polypeptide chain of the construct of the present invention, but the dimers of the fusion protein of the present invention are dimers rather than obtained monomers. These dimers do not form large aggregates, which is contrary to other forms of Fc CLEC-1 or other Fc proteins: Fc CLEC-1, Fc-dectin1R and Fab anti-PD1-KIH-Fc-IL15 fusion protein aggregates. It is unexpected that the fusion protein of the present invention forms dimers and does not form large aggregates.

[0272] Example 4. Binding of the Fc-CLEC-1 fusion molecule according to the present invention to CLEC-1L, a known ligand of CLEC-1.

[0273] Human CLEC-1L is a ligand of human CLEC-1 (unpublished results). In order to verify 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 the binding of different Fc KIH CLEC-1 to the natural ligand of CLEC-1 (CLEC-1L) was conserved. It is important to emphasize the observation that this ability is independent of the binding of the CLEC binding moiety to the hole or knob chain and is independent of the presence of the complete extracellular domain of CLEC-1 or its mutant forms. Next, the pharmacokinetic profiles of the prior art Fc CLEC-1 and Fc KIH CLEC-1 forms were evaluated. After intravenous injection of 10 mg / kg, the Fc KIH CLEC-1 forms were cleared more slowly from mouse serum than Fc CLEC-1 ( Figure 6 ). The half-life of the Fc KIH CLEC-1 form of the invention is at least twice as long as the half-life of Fc-CLEC-1. Furthermore, the in vivo concentration of Fc-CLEC-1 decreases after 72 hours, but the Fc KIH CLEC-1 construct of the invention remains at high concentrations in the serum of treated mice for longer, even 6 days after injection, while being completely cleared at the same time point in its aggregated FcCLEC-1 form.

[0274] Example 5. Binding of anti-CLEC-1 antibodies to Fc KIH CLEC-1 constructs

[0275] In order to evaluate the conservation of CLEC-1 structure when fused to the Fc KIH domain, anti-CLEC-1 mouse antibodies have been used to target immobilized several different Fc KIH CLEC-1 proteins according to the present invention. Figure 7 As shown, the anti-CLEC-1 mouse antibody recognized CLEC-1 regardless of its binding to the hole chain or the knob chain. In addition, the anti-CLEC-1 mouse antibody also recognized mutant forms of CLEC-1, particularly truncated forms of CLEC-1 in which the N-terminus of the extracellular domain of CLEC-1 was deleted. These results demonstrate that CLEC-1 maintains its structural conformation when fused to Fc KIH, allowing for in vivo or in vitro interactions between the Fc KIH CLEC-1 of the present invention and the ligands of CLEC-1.

[0276] Example 6. There is no inflammatory effect induced by the heterodimeric Fc-CLEC-1 fusion protein of the present invention.

[0277] To evaluate the adverse 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 the aggregated Fc CLEC-1 fusion protein of the prior art induced IL-6 secretion in serum, but the heterodimeric Fc KIH CLEC-1 protein of the present invention did not induce IL-6 secretion ( Figure 8 ), thus confirming that this form is safer for oncology applications.

[0278] Example 7. Heterodimeric Fc-CLEC-1 fusion protein of the present invention binds to tumor cells expressing CLEC-1 ligand ability.

[0279] 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 ( Fig. 9 ), thus confirming that this format binds to CLEC-1 ligand-positive tumor cells.

[0280] Example 8. Binding of Fc-CLEC-1 fusion molecules

[0281] like Fig.10 As shown, Fc-CLEC-1 is able to bind to several different tumor cells treated by conventional methods against cancer (more than 21 tumor cell lines were tested). These results demonstrate the ability of Fc-CLEC-1 fusion molecules to bind to different tumor cells, thus supporting the broad use of the fusion molecules of the present invention in treating different types of cancer.

Claims

1. A fusion molecule 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 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 heterodimeric immunoglobulin or a fragment thereof, comprising: a. a first Fc polypeptide chain comprising a first CH3 domain (CH3B chain or hole chain), and b. a second Fc polypeptide chain comprising a second CH3 domain (CH3A chain or knob chain), wherein the first and second CH3 domains are different, One of the at least one binding moieties is fused to the C-terminus or N-terminus of the first Fc polypeptide, 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.

2. The fusion molecule of claim 1, wherein the CH3 domains of the first Fc polypeptide chain and the second Fc polypeptide are selected from reference KiH, KiH S-S , HA-TF, ZW1, 7.8.90, DD-KK, EW-RVT, SEED and A107, more specifically wherein the first Fc polypeptide chain has the amino acid residues shown in SEQ ID NO:3 and the second Fc polypeptide chain has the amino acid residues shown in SEQ ID NO:5, or wherein the first Fc polypeptide chain has the amino acid residues shown in SEQ ID NO:10 and the second Fc polypeptide chain has the amino acid residues shown in SEQ ID NO:

12.

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

4. The fusion molecule according to any one of claims 1 to 3, wherein the 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 wherein 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 molecules are at the opposite end of the Fc polypeptide chain. 5 . The fusion molecule according to claim 1 , wherein the at least one binding moiety is fused to the Fc polypeptide chain via a linker peptide, more specifically a linker peptide of SEQ ID NO:

13.

6. The fusion molecule according to any one of claims 1 to 5, wherein the first Fc polypeptide chain, or the second Fc polypeptide chain, or both the first and the second Fc polypeptide chain comprise a CH2 domain, particularly at the N-terminus of a CH3 domain, particularly wherein the CH2 domains are different.

7. The fusion molecule according to any one of claims 1 to 6, wherein the first Fc polypeptide chain, or the second Fc polypeptide chain, or both the first and the 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 the CH2 domain when the CH2 domain is present.

8. The fusion molecule according to any one of claims 1 to 7, wherein: 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 (CH3B chain or hole chain), and The second Fc polypeptide chain comprises, from its N-terminus to its C-terminus, a hinge region, a CH2 domain, a second CH3 domain (CH3B chain or hole chain), 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. The fusion molecule according to any one of claims 1 to 8, 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. The fusion molecule according to any one of claims 1 to 8, 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. The 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 SEQ ID NO:

2. 12 . The fusion molecule according to claim 11 , wherein the mutated extracellular domain of CLEC-1 has the amino acid residue sequence shown in SEQ ID NO:

19.

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

3.

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

3.

15. The fusion molecule according to any one of claims 1 to 13, comprising a first Fc polypeptide chain and a second Fc polypeptide chain, wherein the first Fc polypeptide chain comprises or consists of the amino acid sequence shown in SEQ ID NO:4, and the second Fc polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:

23.

16. The fusion molecule according to any one of claims 1 to 15, further comprising: (iii) at least one antigen binding domain comprising or consisting of a variable domain of an antibody or antigen binding fragment thereof, said antigen binding domain being fused to said first Fc polypeptide chain or said second Fc polypeptide chain, or the first antigen binding domain being fused to said first Fc polypeptide chain and the second antigen binding domain being fused to said second Fc polypeptide chain, in particular when said at least one binding moiety is present at one end of the same Fc polypeptide chain, said antigen binding domains are at opposite ends of said Fc polypeptide chain.

17. The fusion molecule according to claim 16, wherein the antigen binding domain specifically binds to an antigen expressed by macrophages and / or lymphocytes and / or tumor cells, in particular selected from SIRPα, SIRPβ, SIRPγ, 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), lactophilus The group consisted of 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.

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, in particular to the N-terminus of the CH2 domain, or to the hinge region when the hinge region is present.

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

20. A fusion molecule according to any one of claims 1 to 19 for use in treating a patient suffering from a disease selected from cancer, in particular solid cancer or liquid cancer, or an infectious disease, or sepsis, an autoimmune disease, or an inflammatory disease, including acute or chronic inflammatory diseases.

21. A fusion molecule according to any one of claims 1 to 20 for use in treating a patient suffering from cancer who is being or has been treated with conventional treatment for cancer, in particular with an agent selected from the group consisting of a chemotherapeutic agent, a targeted cancer therapy, an immunotherapeutic agent or a radiotherapeutic agent, more particularly with an agent selected from the group consisting of a cytotoxic agent, an anti-angiogenic agent, an anticancer agent, a cell cycle / apoptosis control regulator, an anticancer antibody and a hormone regulator, the fusion molecule and the agent being administered simultaneously, separately or sequentially.

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

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