Anti-chemokin like receptor 1 humanized antibodies and their therapeutic applications

Abstract

To provide molecules having the capability to take part in initiating or enhancing the resolution stage of the inflammatory response like pro-resolving factors.SOLUTION: The present invention provides humanized anti-CMKLR1 compounds having an agonist capability on the interaction between Resolvin E1 and CMKLR1, and their uses for treating or preventing a disease, in particular wherein the resolution of inflammation is delayed or disrupted.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the field of immunotherapy. The present invention provides novel humanized anti-chemerin receptor antibodies with resolvin E1-like agonistic activity against chemokine-like receptor-1 (CMKLR1). The present invention also provides the use of such antibodies in therapy, particularly for treating autoimmune and chronic inflammatory diseases, infectious diseases, cancer, and any condition in which the resolution phase of inflammation is disrupted or delayed. [Background technology]

[0002] The important role of the inflammatory process in health and disease has long been recognized. The detailed molecular mechanisms and biological events that regulate the progression and resolution of inflammation remain of great interest. Recent studies have provided strong evidence that the resolution of inflammation is not a passive process, as previously believed. Rather, it is a biosynthetically active process regulated by biochemical mediators and receptor signaling pathways. Resolution is therefore driven by specific inflammation-resolving mediators. Inflammation is a spontaneous mechanism that occurs during infection, injury, or traumatic insult. Inflammation is unavoidable and usually beneficial, and the response is regulated by a delicate balance between positive and negative feedback loops. Inflammation is typically divided into three steps: initiation, amplification, and resolution.

[0003] The resolution process that allows the end of the inflammatory response is a complex process that involves the sequential and sequential involvement of cellular (e.g., granulocytes or macrophages) and chemical (e.g., cytokines or specific inflammation-resolving mediators or factors) effectors.

[0004] Chemokine-like receptor 1 (CMKLR1) and chemokine receptor-like 2 (CCRL2), also known as ChemR23, are seven-transmembrane receptors identified by their homology to known G protein-coupled receptors (AJ Kennedy and AP Davenport, 2018). Chemokine-like receptor 1 (CMKLR1; also named Dez in mice) is an orphan G protein-coupled receptor related to GPR-1 (38% overall amino acid identity), C3a receptor (38%), C5a anaphylatoxin receptor (36%), and formyl Met-Leu-Phe receptor (35%). ChemR23 is more distantly related to the chemokine receptor subfamily (Samson et al., 1998). CMKLR1 is expressed on monocytes, macrophages, dendritic cells, and NK cells, as well as adipocytes and endothelial cells. Recent studies have identified ligands for these receptors and begun to clarify their functions. Thus, the first plasma protein-derived chemoattractant, designated chemerin, was identified as a ligand for CMKLR1.

[0005] The second ligand of CMKLR1 is resolvin E1 (RvE1), a lipid mediator belonging to the resolvin family. Resolvin E1, an anti-inflammatory lipid mediator, inhibits leukocyte infiltration and inflammatory gene expression.

[0006] Initially, interest in the chemerin system (i.e., the signaling pathway activated or not by chemerin receptors and their ligands, such as chemerins and resolvins) focused on its role in inflammation and immune cell chemotaxis after its discovery in psoriatic disease. More recently, its potential role in cardiovascular function, as well as in reproductive biology, has been considered in connection with its role in inflammation, obesity, and metabolic syndrome. Thus, the chemerin system has attracted considerable interest for its role in inflammatory processes, particularly in the resolution of inflammation. Several diseases are associated with a delay or disruption of the resolution process. Many of the currently known specific inflammation-resolving factor mediators are derived from polyunsaturated fatty acids, including lipoxins, the resolvin family, including E-series and D-series resolvins, protectins, and maresins. Nevertheless, inflammation-resolving molecules are difficult to synthesize due to their lipid nature. For example, producing inflammation-resolving molecules in sufficient quantities for clinical trials is burdensome, and very few SPMs have been efficiently produced. Additionally, antibodies that specifically target G protein-coupled receptors are difficult to produce. Therefore, there is a need for molecules that have the ability to participate in initiating or enhancing the resolution phase of the inflammatory response, particularly inflammation-resolving factors. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] WO2008068637 [Patent Document 2] EP1270725B1 [Patent Document 3] U.S. Patent No. 8,536,307 [Non-patent literature]

[0008] [Non-Patent Document 1] Krehenbrink et al., J. Mol. Biol., 383:5, 2008 [Non-patent document 2] Skerra, Febs J., 275:11, 2008 [Non-patent document 3] Schlehuber and Skerra, Biophys. Chem., 96:2-3, 2002 [Non-patent document 4] Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998) [Non-Patent Document 5] Colligan et al., Current Protocols in Immunology, Green Publishing Assoc., NY (1992;1993) [Non-patent document 6] Muller, Meth. Enzym, 92:589~601 (1983) Summary of the Invention

[0009] In a first aspect, the present invention relates to a humanized anti-CMKLR1 antibody or an antigen-binding fragment thereof or an antigen-binding antibody mimetic or modified antibody.

[0010] The present inventors sought to obtain improved humanized antibodies compared to wild-type anti-CMKLR1 antibodies known in the prior art. It is known that some residues in the variable domain CDR and framework (FR) sequences, including residues in the Vernier zone, canonical residues, and residues at the interface between the variable heavy and variable light chains, are important for antibody structure and should not be mutated to maintain the antibody's biochemical and biological activity. However, the present inventors surprisingly discovered that mutations in the heavy and / or light chain variable domain sequences of wild-type antibodies, including some of these important residues, can increase the content of human residues (up to 99% humanization) while maintaining functional characteristics associated with these humanized antibodies that are not shared by the wild-type anti-CMKLR1 to be improved, and at the same time, can improve the production of these humanized antibodies compared to less humanized or clearly humanized anti-CMKLR1 antibodies. These humanized antibodies provide compounds that can be produced on a large scale for candidate drug development and exhibit functional characteristics useful in the treatment of diseases, including those involving inflammation. The combination of these two features results in the provision of anti-CMKLR1 antibodies that exhibit improved potency compared to prior art antibodies.

[0011] Starting from a non-humanized anti-CMKLR1 antibody and selecting human germline sequences, the inventors designed specific humanized heavy and light chain variable domains. Humanized heavy and light chain variable domains derived from non-humanized antibodies allow for the production of functional antibodies in different cell lines, such as, but not limited to, Chinese hamster ovary (CHO) cell lines, transformed African green monkey kidney fibroblast (COS-7) cell lines, and human embryonic kidney cell lines (HEK293), under conditions that allow for the recovery of significant yields of antibodies that bind to their targets with avidity that can even be increased relative to the binding activity of the wild-type antibody. Some of the resulting humanized heavy and light chain variable domains were further humanized, particularly with respect to the initial humanization step performed on the framework regions of the heavy and light chain variable domains, resulting in the provision of antibodies suitable for high-yield production in different cell lines with reduced immunogenicity and at least functional characteristics of their parent antibodies. Those skilled in the art would not have expected that the above mutations at such positions in the CDR domains and framework regions of the heavy and / or light chain variable domains, in particular, would result in the provision of anti-CMKLR1 antibodies with retained binding ability, in particular affinity, retained stability, and a reduced immunogenicity index, while improving the production scale of these antibodies comprising the heavy and light chain variable domains according to the present invention compared to less humanized or differently humanized antibodies. In particular, it could not have been predicted that the introduction of the disclosed mutations into CDR2 of the heavy chain would result in improved production while still retaining the binding properties and, advantageously, other functional characteristics of prior art anti-CMKLR1 antibodies. Anti-CMKLR1 antibodies exhibiting the described characteristics (binding ability to CMKLR1, particularly to the specific third extra loop of CMKLR1, in particular affinity, low immunogenicity, good production scale and resolvin-like agonist ability to CMKLR1) are useful for the potential treatment of several diseases, including diseases involving inflammation, more particularly inflammatory diseases.

[0012] Furthermore, as will be explained later in this application, several highly advantageous biological effects have been achieved, notably related to a reduction in neutrophil apoptosis and neutrophil and / or macrophage migration and / or transmigration, which results in a potent beneficial effect in the resolution of inflammation. The new compounds are thus able to combine productivity with biological activity.

[0013] Therefore, in a first aspect of the present invention, the antibody or antigen-binding fragment thereof that binds to chemokine-like receptor 1 (CMKLR1), particularly human CMKLR1, comprises: a) an antibody heavy chain variable (VH) domain comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, - VHCDR1 is selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7; - VHCDR2 is selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:61; - an antibody heavy chain variable (VH) domain, wherein VHCDR3 is selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16; b) an antibody light chain variable (VL) domain comprising three CDRs, VLCDR1, VLCDR2 and VLCDR3, - VLCDR1 is selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23; - VLCDR2 is selected from the group consisting of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:33; - an antibody light chain variable (VL) domain, wherein VLCDR3 is selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36 Includes:

[0014] In certain embodiments, the antibody or antigen-binding fragment thereof that binds to chemokine-like receptor 1 (CMKLR1), particularly human CMKLR1, is a) an antibody heavy chain variable (VH) domain comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, - VHCDR1 is selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7; - VHCDR2 is selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 61; or VHCDR2 corresponds to the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 63, with the proviso that VHCDR1 is not SEQ ID NO: 3 or SEQ ID NO: 4; - an antibody heavy chain variable (VH) domain, wherein VHCDR3 is selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16; b) an antibody light chain variable (VL) domain comprising three CDRs, VLCDR1, VLCDR2 and VLCDR3, - VLCDR1 is selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23; - VLCDR2 is selected from the group consisting of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:33; - an antibody light chain variable (VL) domain, wherein VLCDR3 is selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36 Includes:

[0015] In another particular embodiment, the antibody or antigen-binding fragment thereof that binds to chemokine-like receptor 1 (CMKLR1), particularly human CMKLR1, is a) an antibody heavy chain variable (VH) domain comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, - VHCDR1 is selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7; - VHCDR2 is selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 61; if VHCDR1 is SEQ ID NO: 3 or SEQ ID NO: 4 and VHCDR2 corresponds to the amino acid sequence of SEQ ID NO: 12, preferably said heavy chain variable (VH) domain does not comprise a framework VHFR3 of SEQ ID NO: 70, provided that said heavy chain variable (VH) domain comprises a framework FR3 of SEQ ID NO: 69; - an antibody heavy chain variable (VH) domain, wherein VHCDR3 is selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16; b) an antibody light chain variable (VL) domain comprising three CDRs, VLCDR1, VLCDR2 and VLCDR3, - VLCDR1 is selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23; - VLCDR2 is selected from the group consisting of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:33; - an antibody light chain variable (VL) domain, wherein VLCDR3 is selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36 Includes:

[0016] Particularly advantageously, the antibody or antigen-binding fragment thereof specifically binds to the third extracellular loop (EL3) of CMKLR1, in particular to an epitope located within the third extracellular loop (EL3) of CMKLR1; more particularly, the antibody or antigen-binding fragment thereof specifically binds to a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 59, or to an epitope located within the amino acid sequence of SEQ ID NO: 60.

[0017] The antibody or antigen-binding fragment thereof according to this embodiment is suitable for production in various cell lines, including, but not limited to, mammalian cell lines, at production yields suitable for drug candidate development purposes, while retaining its ability to specifically bind to the extracellular loop 3 of CMKLR1 and its Resolvin E1-like agonist activity against CMKLR1. The humanized antibody disclosed herein can therefore be produced efficiently and share the functional capabilities of its parent antibody.

[0018] A particular aspect of the present invention, which is distinguished from the definition of an antibody of the present invention only by its CDR domains, but which can be combined with such definition in certain embodiments, provides an antibody or antigen-binding fragment thereof that binds to chemokine-like receptor 1 (CMKLR1), in particular human CMKLR1, suitable for production in mammalian cells such as COS or CHO or HEK cells, in particular in a yield of more than 0.1 mg / ml, in particular in a yield of more than 1 mg / ml, more particularly in a yield of at least 10 mg / ml, and even more particularly in a yield of more than 100 mg / ml, a) the variable heavy (VH) domain comprises the amino acid sequences of frameworks (FR1, FR2, FR3 and FR4) of a heavy chain variable domain, each of which has a sequence identity with the framework of the same rank in the sequence of SEQ ID NO: 41 of 100% for FR1, at least 60% for FR2, at least 78% for FR3 and at least 80% for FR4, respectively; more specifically, 100% for FR1, at least 80% for FR2, at least 85% for FR3 and at least 90% for FR4; b) the variable light (VL) domain comprises the amino acid sequence of the framework of the light chain variable domain (FR1, FR2, FR3 and FR4), each of which has a sequence identity with the framework of the same rank in the sequence of SEQ ID NO: 50 of 60% for FR1, at least 70% for FR2, at least 75% for FR3 and at least 80% for FR4, respectively; more specifically, 100% for FR1, at least 90% for FR2, at least 90% for FR3 and 100% for FR4, Antibodies or antigen-binding fragments thereof are disclosed.

[0019] In particular, the humanized anti-CMKLR1 antibody or its antigen-binding fragment specifically binds to the third extracellular loop (EL3) of CMKLR1, and in particular, the antibody or its antigen-binding fragment specifically binds to a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 59 or an epitope located within the amino acid sequence of SEQ ID NO: 60.

[0020] Starting from the anti-CMKLR1 antibody 2G1, the present inventors synthesized various heavy and light chain variable domains. The humanized heavy and light chain variable domains of SEQ ID NO: 41 and SEQ ID NO: 50 were particularly suitable for producing humanized anti-CMKLR1 antibodies in cells or cell lines, including mammalian cells such as CHO cells, COS cells, or HEK cells. Once specific human germline sequences have been selected for the design of the heavy and light chain variable domains, humanized antibodies with the identities defined herein in the framework regions can also be produced in sufficient quantities in cells or cell lines for the purpose of developing antibodies that can generate drug candidates. There is considerable interest in providing humanized antibodies that can be produced in large quantities in cells or cell lines, particularly mammalian cells or cell lines, for the development of therapeutic antibodies. The particular anti-CMKLR1 antibody provided herein shares a highly similar structure with its parent antibody, which comprises a heavy chain variable domain of SEQ ID NO: 41 and a light chain variable domain of SEQ ID NO: 50, allowing for the precise production, conformation and secretion of the anti-CMKLR1 antibody, thereby enabling the provision of an antibody that specifically binds to a particular epitope of Chemerin-like receptor 1 and has resolvin E1-like agonistic activity against this receptor in sufficient quantities for therapeutic purposes.

[0021] In particular embodiments of the present invention, there is provided a humanized anti-chemerin-like receptor 1 (CMKLR1) antibody or antigen-binding fragment thereof suitable for production in mammalian cells, such as COS or CHO cells, particularly in a yield of greater than 0.1 mg / ml, more particularly greater than 1 mg / ml, more particularly greater than 10 mg / ml, and even more particularly greater than 100 mg / ml, a) the variable heavy (VH) domain comprises the amino acid sequences of heavy chain variable domain frameworks (FR1, FR2, FR3 and FR4), each of which has a sequence identity with the framework of the same rank in the sequence of SEQ ID NO: 41 of at least 90% for FR1, at least 70% for FR2, at least 80% for FR3, and at least 80% for FR4, respectively; b) Antibodies are provided in which the variable heavy (VL) domain comprises the amino acid sequence of the framework (FR1, FR2, FR3 and FR4) of the light chain variable domain, each framework having a sequence identity with the framework of the same rank in the sequence of SEQ ID NO: 50 of at least 60% for FR1, at least 80% for FR2, at least 75% for FR3 and at least 70% for FR4, respectively.

[0022] In particular, the humanized anti-CMKLR1 antibody or its antigen-binding fragment specifically binds to the third extracellular loop (EL3) of CMKLR1, and in particular, the antibody or its antigen-binding fragment specifically binds to a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 59, or located within the amino acid sequence of SEQ ID NO: 60.

[0023] In certain embodiments of the humanized antibodies or antigen-binding fragments thereof defined above, such humanized antibodies or antigen-binding fragments thereof comprise: a) an antibody heavy chain variable (VH) domain comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, - VHCDR1 is selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7; - VHCDR2 is selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:61; - an antibody heavy chain variable (VH) domain, wherein VHCDR3 is selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16; and b) an antibody light chain variable (VL) domain comprising three CDRs, VLCDR1, VLCDR2 and VLCDR3, - an antibody light chain variable (VL) domain, wherein VLCDR1 is selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23 Includes:

[0024] In certain embodiments of the humanized antibodies or antigen-binding fragments thereof defined above, such humanized antibodies or antigen-binding fragments thereof comprise: a) an antibody heavy chain variable (VH) domain comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, - VHCDR1 is selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7; - VHCDR2 is selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:61; or VHCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:12 or SEQ ID NO:63, with the proviso that VHCDR1 is not SEQ ID NO:3 or SEQ ID NO:4; - an antibody heavy chain variable (VH) domain, wherein VHCDR3 is selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16; and b) an antibody light chain variable (VL) domain comprising three CDRs, VLCDR1, VLCDR2 and VLCDR3, - VLCDR1 is selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23; - VLCDR2 is selected from the group consisting of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:33; - an antibody light chain variable (VL) domain, wherein VLCDR3 is selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36 Includes:

[0025] In another particular embodiment of the humanized antibody or antigen-binding fragment thereof defined above, such humanized antibody or antigen-binding fragment thereof comprises: a) an antibody heavy chain variable (VH) domain comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, - VHCDR1 is selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7; - VHCDR2 is selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 61; When VHCDR1 is SEQ ID NO: 3 or SEQ ID NO: 4 and VHCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 12, preferably said heavy chain variable (VH) domain does not comprise the framework VHFR3 of SEQ ID NO: 70, provided that said heavy chain variable (VH) domain comprises the VHFR3 of SEQ ID NO: 69; - an antibody heavy chain variable (VH) domain, wherein VHCDR3 is selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16; and b) an antibody light chain variable (VL) domain comprising three CDRs, VLCDR1, VLCDR2 and VLCDR3, - VLCDR1 is selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23; - VLCDR2 is selected from the group consisting of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:33; - VLCDR3 comprises an antibody light chain variable (VL) domain selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36.

[0026] Such antibodies offer the same advantageous characteristics as the antibodies defined according to the first and second aspects of the present invention, namely high yield production, specific binding to an epitope located within the third extracellular loop of Chemerin-like receptor 1, and resolvin E1-like agonist ability.

[0027] The disclosed antibodies are suitable for use in treating conditions in which resolution of inflammation is delayed or disrupted. All of the antibodies described herein are suitable for inducing resolution of inflammation and / or enhancing resolution of inflammation and / or initiating resolution of inflammation.

[0028] In the following disclosure, a humanized anti-CMKLR1 compound is considered to be either a humanized anti-CMKLR1 antibody, an antigen-binding fragment thereof, an antigen-binding antibody mimic, or a modified antibody. In a specific embodiment of the present invention, the compound is defined by the sequences of its CDRs. In a more specific embodiment of the present invention, the anti-CMKLR1 compound is an antibody defined by the sequences of its CDRs and its framework regions (FRs). An anti-CMKLR1 compound is a compound that specifically binds to chemokine-like receptor 1 (CMKLR1). In the following disclosure, the terms chemokine-like receptor 1, CMKLR1, and ChemR23 are used interchangeably and all refer to receptors encoded by the CMKLR1 gene in humans or the cmklr1 gene in non-human animals. In a specific embodiment of the present invention, an anti-CMKLR1 compound specifically binds to human CMKLR1, or in other words, the present invention relates to anti-human CMKLR1 compounds. As used herein, the term "CMKLR1" refers to a chemokine-like receptor 1 protein (also designated chemR23), a member of the G protein-coupled receptor family derived from mammalian species, preferably human CMKLR1. The reference sequence of the human CMKLR1 protein used in the examples of this application corresponds to the sequence associated with Uniprot accession number Q99788 (SEQ ID NO: 1).

[0029] In a specific aspect, the present invention relates to an anti-CMKLR1 antibody or an antigen-binding fragment, an antigen-binding antibody mimic, or a modified antibody thereof defined by at least one functional characteristic. In a preferred embodiment, the anti-CMKLR1 compound is defined by its ability to inhibit the secretion of pro-inflammatory cytokines, particularly IL12, and / or its ability to enhance the secretion of anti-inflammatory cytokines, particularly IL10. In a more specific embodiment, the anti-CMKLR1 compound inhibits or enhances cytokine secretion by macrophages, particularly by pro-inflammatory macrophages and / or the inflammation-resolving properties of inflammatory macrophages. In a specific embodiment, the anti-CMKLR1 compound of the present invention enhances the polarization of macrophages into anti-inflammatory macrophages, particularly inflammation-resolving macrophages. In a specific embodiment, the anti-CMKLR1 compound of the present invention enhances neutrophil apoptosis compared to a control antibody.

[0030] In a particular aspect, the present invention relates to an anti-CMKLR1 antibody or its antigen-binding fragment or antigen-binding antibody mimic that has agonistic properties against resolvin E1 (RvE1) / CMKLR1 interaction, thereby mimicking at least one of the effects induced by the binding of RvE1 to CMKLR1 on CMKLR1-positive cells. "Agonistic properties against RvE1-CMKLR1 interaction" means that the antibody or its antigen-binding fragment or antigen-binding antibody mimic or modified antibody of the present invention targeting CMKLR1 has an effect of mimicking at least one of the effects provided by the binding of RvE1 to CMKLR1, thereby activating receptor signaling pathways normally activated by RvE1, particularly the binding of human RvE1 to human CMKLR1 on dendritic cells, neutrophils, monocytes, and macrophages. As a result of receptor binding and activation resulting in a biological response, the compounds of the present invention activate G protein signaling pathways, particularly Gα, without activating the β-arrestin pathway. iThe compound of the present invention can activate the signal transduction pathway and / or Gα0. In particular, the compound of the present invention can inhibit the β-arrestin pathway. In particular, the binding of the compound according to the present invention induces the activation of Akt and / or Erk proteins in vitro and / or in vivo. In certain embodiments, if the G protein signal transduction pathway is activated in CMKLR1-positive cells stimulated with the compound of the present invention, or more particularly, if the B-arrestin pathway is not activated or, in certain conditions, if the B-arrestin pathway is inhibited, the compound can be considered an anti-CMKLR1 agonist with resolvin E1-like activity. In other words, a resolvin E1-like agonist antibody can be defined as an antibody that can bind to CMKLR1 and thereby induce phosphorylation of Akt and / or Erk proteins compared to a control antibody. The control antibody may also be an antibody that does not specifically bind to CMKLR1. Protein phosphorylation can be determined according to methods well known to those skilled in the art, for example, the methods disclosed in the Examples herein. In certain embodiments, the compounds of the present invention enhance the activation of the G protein pathway induced by CMKLR1. In another embodiment, the compounds of the present invention do not induce the activation of the β-arrestin pathway induced by CMKLR1. In another embodiment, the compounds of the present invention inhibit the β-arrestin pathway induced by CMKLR1. In another embodiment, the compounds of the present invention are inflammation-resolving factors or inflammation-resolving mediators because they induce at least one agonistic effect of RvE1 binding to CMKLR1, and RvE1 is an inflammation-resolving factor or inflammation-resolving mediator. For example, an inflammation-resolving factor can be defined as a compound that inhibits the β-arrestin pathway induced by CMKLR1 and / or enhances the activation of the G protein pathway induced by CMKLR1 in CMKLR1-positive cells, compared to a control compound known not to specifically interact with CMKLR1. The activation / inhibition of these pathways can be evaluated according to the methods disclosed in the examples of the present invention. In certain embodiments, the effects of agonist compounds are evaluated in human cells.

[0031] In certain embodiments, the compounds of the present invention do not inhibit the binding of chemerin to CMKLR1. Chemerin is one of the natural ligands of CMKLR1. In other words, the compounds of the present invention are not agonists and / or antagonists of the interaction between chemerin and CMKLR1. The absence of such agonist and / or antagonist ability can be assessed according to the Examples of the present invention, which disclose a competition assay for measuring chemerin-dependent beta-arrestin recruitment by the CMKLR1 receptor in the presence of an anti-CMKLR1 antibody of the present invention. In a preferred embodiment, the anti-CMKLR1 compounds of the present invention do not compete with chemerin for binding to CMKLR1. Absence of competition between the anti-CMKLR1 compounds of the present invention and chemerin can be determined when the binding of chemerin to CMKLR1 in the presence of the CMKLR1 compounds of the present invention is at least 50%, more preferably at least 80%, even more preferably at least 90%, and most preferably similar to the binding of chemerin to CMKLR1 in the absence of the anti-CMKLR1 compounds of the present invention under the same experimental conditions. Alternatively, absence of competition between the anti-CMKLR1 compounds of the present invention and chemerin can be determined according to the method exemplified in Example 9.

[0032] In certain embodiments, the anti-CMKLR1 compound has the in vitro and / or in vivo ability to activate at least one of the Akt signaling pathway proteins (known as the PI3K-Akt pathway) and / or the Erk signaling pathway proteins, preferably the Akt protein and / or the Erk protein, preferably both the Akt and the Erk protein. Pathway activation can be assessed according to methods known in the art, in particular using the methods disclosed in the examples of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0033] The present invention relates to an agonist of CMKLR1 having resolvin E1-like capabilities for use in the therapeutic treatment of inflammatory conditions in patients, particularly chronic inflammation, especially inflammatory conditions in which the resolution phase of inflammation is delayed or disrupted, in particular said agonist being selected from the group consisting of an antibody or antigen-binding fragment thereof, a peptide, a polypeptide and a protein.

[0034] In the following description, unless otherwise stated, an agonist of CMKLR1 having resolvin E1-like ability can be defined as "agonist"; both terms include antibodies or antigen-binding fragments thereof (also referred to as anti-CMKLR1 antibodies), proteins, peptides, or polypeptides; the term compound or anti-CMKLR1 compound can also be used in the following description as a synonym for "agonist" (of the present invention), thereby including antibodies or antigen-binding fragments thereof (also referred to as anti-CMKLR1 antibodies), proteins, peptides, or polypeptides. Among the effects provided by the use of such agonists, the following specific effects have been demonstrated: Agonists of CMKLR1 with resolvin E1-like ability induce apoptosis of polymorphonuclear neutrophils (also referred to herein simply as neutrophils, PMNs or PMNs) and / or reduce or inhibit the migratory ability of these cells, particularly by inhibiting their ability to migrate through the endothelium towards sites of inflammation, as described later in this application. - agonists of CMKLR1 with resolvin E1-like capabilities induce the internalization of different receptors expressed on the cell surface on various myeloid cells, notably macrophages and / or dendritic cells, thereby enhancing the processes that induce or perpetuate the resolution of inflammation, as described later in this application; - in particular, agonists of CMKLR1 with resolvin E1-like capabilities induce the internalization of various receptors CMKLR1, as well as CXCR4 and / or CCR7, expressed on the cell surface of macrophages and / or dendritic cells, and such internalization leads to much lower targeting and recognition of CXCR4, CCR7 receptors by cytokines known to induce migration of cells towards inflammatory sites, resulting in a reduction or inhibition of migration of macrophages and / or dendritic cells from the inflammatory site to secondary lymphoid organs and / or towards the inflammatory site; - Agonists of CMKLR1 with resolvin E1-like ability reduce or inhibit the ability of neutrophils and macrophages and / or dendritic cells to migrate; in particular, they reduce or inhibit the ability of neutrophils to migrate through inflammatory sites by reducing the rolling ability of these cells and their ability to migrate through the endothelium due to the internalization of CD62L and / or a decrease in its cell surface expression; In a particular embodiment of the present invention, the inventors have demonstrated that agonists of CMKLR1 with resolvin E1-like activity, which contain a domain suitable for interaction with Fc receptors, such as an IgG constant domain, in particular an IgG1 constant domain, are particularly efficient. Fc receptors on macrophages or neutrophils particularly recognize anti-CMKLR1 IgG constant domains or Fc fragments of IgG1 constant domains very efficiently, resulting in or contributing to apoptosis of neutrophils recognized by anti-CMKLR1 IgG1 antibodies; - The inventors now demonstrate that myeloid cells that are involved in inflammatory processes (i.e., that perpetuate inflammation) and that express CMKLR1, as well as CXCR4 and / or CCR7, are particularly suitable targets for agonists of CMKLR1 with Resolvin E1-like capabilities for the treatment of pathological inflammatory processes.

[0035] The present invention particularly relates to an anti-Chemerin-like receptor 1 (CMKLR1) agonist with resolvin E1-like ability for use in the treatment of patients suffering from inflammatory conditions, particularly inflammatory conditions in which resolution of inflammation is delayed or disrupted, wherein the CMKLR1 agonist with resolvin E1-like ability is selected from the group consisting of an antibody or antigen-binding fragment thereof, a peptide, a polypeptide and a protein; - Inducing apoptosis or activating neutrophils at the site of inflammation, and / or - inhibiting or reducing the ability of neutrophils to migrate through the endothelium towards the site of inflammation, and / or - relates to agonists that inhibit the migration of macrophages and / or dendritic cells from and / or towards the site of inflammation to secondary lymphoid organs.

[0036] The present invention relates to the use of agonists of CMKLR1 with resolvin E1-like ability, such as antibodies, particularly humanized antibodies, or antigen-binding fragments thereof, to induce, enhance, or activate neutrophil apoptosis in the treatment of inflammatory conditions, particularly inflammatory conditions in which resolution of inflammation is delayed or disrupted.

[0037] In certain embodiments, the present invention relates to a humanized anti-CMKLR1 antibody or an antigen-binding fragment thereof or an antigen-binding antibody mimetic or modified antibody.

[0038] As used herein, the term "antibody" includes polyclonal, monoclonal, or recombinant antibodies. As used herein, "monoclonal antibody" is intended to refer to the preparation of antibody molecules to obtain antibodies with a common heavy chain and a common light chain amino acid sequence, as opposed to "polyclonal" antibody preparations which contain a mixture of antibodies with different amino acid sequences. Monoclonal antibodies can be produced by several known techniques, such as phage, bacterial, yeast, or ribosome display, as well as by classical methods exemplified by hybridoma-derived antibodies. They can also be synthesized using the disclosed amino acid sequence as a reference. Thus, the term "monoclonal" is used to refer to all antibodies derived from a single nucleic acid clone.

[0039] As used herein, the term "antibody" further includes antibodies that have been modified relative to wild-type antibodies, and encompasses chimeric antibodies, humanized antibodies, modified antibodies, and antigen-binding antibody mimetics. A particular wild-type antibody of reference in the context of the present invention is antibody 2G1.

[0040] Antibodies of the present invention include recombinant antibodies. As used herein, the term "recombinant antibody" refers to antibodies that are produced, expressed, generated, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell; antibodies isolated from a recombinant combinatorial antibody library; antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes; or antibodies that are produced, expressed, generated, or isolated by any other method that brings together particular immunoglobulin gene sequences (such as human immunoglobulin gene sequences) with other DNA sequences. Recombinant antibodies include, for example, chimeric and humanized antibodies.

[0041] As used herein, "chimeric antibody" refers to an antibody in which variable domain sequences derived from the germline of a mammalian species, such as a mouse, have been grafted onto constant domain sequences derived from the germline of another mammalian species, such as a human.

[0042] As used herein, "humanized antibody" refers, in a first embodiment, to an antibody in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human or, in particular, humanized framework sequences. In a further embodiment, "humanized antibody" refers to an antibody in which at least one CDR and framework sequence has been humanized in whole or in part.

[0043] As used herein, "antigen-binding fragment of an antibody" refers to a molecule corresponding to a portion of the structure of the antibody of the present invention, which, optionally in its native form, exhibits antigen-binding ability to CMKLR1; such a fragment exhibits the same or substantially the same antigen-binding specificity for the antigen, in particular compared to the antigen-binding specificity of the corresponding four-chain antibody. Advantageously, the antigen-binding fragment has a binding affinity similar to that of the corresponding four-chain antibody. However, antigen-binding fragments with reduced antigen-binding affinity relative to the corresponding four-chain antibody are also encompassed by the present invention. The antigen-binding ability can be determined by measuring the affinity of the antibody to the target fragment. These antigen-binding fragments can also be designated as "functional fragments" of antibodies.

[0044] An antigen-binding fragment of an antibody is a fragment that includes the recognition site for the antigen, i.e., the extracellular domain of CMKLR1, particularly its hypervariable domain designated as CDR (complementarity-determining region), or a portion thereof, which encompasses the third loop (designated EL3) of the extracellular domain of CMKLR1, thereby defining the antigen recognition specificity. EL3 is located between amino acid residue 283 and amino acid residue 300 of SEQ ID NO: 1. The amino acid sequence of the EL3 domain is that of SEQ ID NO: 2. EL3 is also contained within the polypeptide of SEQ ID NO: 52. Each light and heavy chain variable domain (VL and VH, respectively) of a four-chain immunoglobulin has three CDRs designated VLCDR1, VLCDR2, and VLCDR3 for the light chain variable domain; and VHCDR1, VHCDR2, and VHCDR3 for the heavy chain variable domain. Each light and heavy chain variable domain of a four-chain immunoglobulin has four framework regions (FRs), designated LFR1, LFR2, LFR3, and LFR4 for the light chain variable domain; and HFR1, HFR2, HFR3, and HFR4 for the heavy chain variable domain. The nomenclature system used to define CDR and framework domains is the Kabat system.

[0045] Those skilled in the art can determine the location of the various regions / domains of an antibody by reference to the standard definitions described herein, including the reference numbering system, by reference to the KABAT numbering system, or by applying the IMGT "collier de perle" algorithm. In this regard, it is noted that for the purposes of the definition of the sequences of the present invention, the limits of a region / domain may differ from one reference system to another. Thus, a region / domain as defined in the present invention encompasses sequences that exhibit a variation of about + / - 10% in length or position of the corresponding sequence within the full-length sequence of an antibody variable domain.

[0046] Thus, based on the structure of four-chain immunoglobulins, keeping in mind that the positions of framework and constant domains are well defined for various classes of antibodies, in particular IgG, and especially mammalian IgG, antigen-binding fragments can be defined by comparison with sequences of antibodies in available databases and prior art, in particular by comparison of the positions of functional domains in these sequences. Such comparison also includes data on the three-dimensional structure of the antibody.

[0047] To illustrate specific embodiments of the present invention, antigen-binding fragments of antibodies containing variable domains comprising the antibody CDRs include Fv, dsFv, scFv, Fab, Fab', and F(ab')2. Fv fragments consist of the VL and VH domains of an antibody bound together by hydrophobic interactions; in dsFv fragments, the VH:VL heterodimer is stabilized by disulfide bonds; in scFv fragments, the VL and VH domains are connected to each other by a flexible peptide linker, thus forming a single-chain protein. Fab fragments are monomeric fragments obtained by papain digestion of antibodies; they contain the entire L chain and the VH-CH1 fragment of the H chain, bound together by disulfide bonds. F(ab')2 fragments can be produced by pepsin digestion of antibodies below the hinge disulfide; they contain two Fab' fragments and also a portion of the hinge region of an immunoglobulin molecule. Fab' fragments can be obtained from F(ab')2 fragments by cleaving the disulfide bond in the hinge region. F(ab')2 fragments are bivalent, i.e., they contain two antigen-binding sites, like natural immunoglobulin molecules; on the other hand, Fv (a VHVL dimer constituting the variable portion of Fab), dsFv, scFv, Fab, and Fab' fragments are monovalent, i.e., they contain one antigen-binding site. These basic antigen-binding fragments of the present invention can be combined together to obtain multivalent antigen-binding fragments, such as diabodies, tribodies, or tetrabodies. These multivalent antigen-binding fragments are also part of the present invention.

[0048] As used herein, the term engineered antibody includes "bispecific" antibodies and refers to antibodies that recognize two different antigens by virtue of having at least one region specific for a first antigen (e.g., derived from the variable region of a first antibody) and at least a second region specific for a second antigen (e.g., derived from the variable region of a second antibody). Bispecific antibodies specifically bind to two target antigens and are thus a type of multispecific antibody. Multispecific antibodies that recognize two or more different antigens can be produced by recombinant DNA methods or include, but are not limited to, antibodies produced chemically by any convenient method. Bispecific antibodies include any antibody or conjugate of antibodies, or multimeric forms of antibodies, that are capable of recognizing two different antigens. Bispecific antibodies include antibodies that have been reduced and reshaped to retain their bivalent properties, and antibodies that have been chemically coupled to each other so that they have several antigen recognition sites for each antigen, such as BiME (bispecific macrophage-enhancing antibody), BiTE (bispecific T-cell engager), DART (dual affinity retargeting); DNL (dock-and-lock), DVD-Ig (dual variable domain immunoglobulin), HAS (human serum albumin), and kih (knobs-into-holes).

[0049] Antigen-binding antibody mimetics are organic compounds that specifically bind to antigens but are structurally unrelated to antibodies. They are typically artificial peptides or small proteins with molecular weights of approximately 3-20 kDa. Nucleic acids and small molecules are also sometimes considered antibody mimetics, but not artificial antibodies, antibody fragments, or fusion proteins composed of these. Their general advantages over antibodies include better solubility, tissue penetration, heat and enzyme stability, and relatively low production costs. Antibody mimetics are being developed as therapeutic and diagnostic agents. Antigen-binding antibody mimetics can also be selected from the group including affibodies, affilins, affimers, affitins, DARPins, and monobodies.

[0050] The antigen-binding antibody mimic is more preferably selected from the group consisting of affitins and anticalins. Affitins are artificial proteins capable of selectively binding to antigens. They are structurally derived from the DNA-binding protein Sac7d, found in Sulfolobus acidocaldarius, a microorganism belonging to the archaea domain. Affitin libraries can be generated by randomizing the amino acids on the binding surface of Sac7d, for example, by generating variants corresponding to random substitutions of 11 residues in the binding interface of Sac7d. The resulting protein library can then be subjected to rounds of ribosome display to target affinities for various targets, such as peptides, proteins, viruses, and bacteria. Affitins are antibody mimics that have been developed as tools in biotechnology. They have also been used as specific inhibitors of various enzymes (Krehenbrink et al., J. Mol. Biol., 383:5, 2008). Those skilled in the art can easily develop affitins with the required binding properties using methods known in the art, in particular the generation of phage display and / or ribosome display libraries and screening them using the antigens disclosed herein, as disclosed in patent application WO2008068637 and the publications cited above. Anticalins are artificial proteins that can bind to either antigens, proteins, or small molecules. They are antibody mimics derived from human lipocalins, a family of natural binding proteins. Anticalins are approximately eight times smaller, with a size of approximately 180 amino acids and a mass of approximately 20 kDa (Skerra, Febs J., 275:11, 2008). In particular, anticalin phage display libraries have been generated that allow the screening and selection of anticalins with specific binding properties.Those skilled in the art can readily develop anticalins with the required binding properties using methods known in the art, such as those disclosed in European Patent EP 1270725 B1, U.S. Patent No. 8,536,307, Schlehuber and Skerra, Biophys. Chem., 96:2-3, 2002, and the publications cited above, particularly the generation of phage display and / or ribosome display libraries and screening them using the antigens disclosed herein. Both anticalins and affitins can be produced in several expression systems, including bacterial expression systems. Thus, the present invention encompasses the use of affitins, anticalins, and other similar antibody mimetics having the characteristics of the antibodies described herein, particularly with respect to their ability to bind to CMKLR1, their agonistic ability to bind to the binding between RvE1 and CMKLR1, their ability to induce or inhibit the secretion of specific cytokines described herein, and their use in the treatment or prevention of diseases described herein, all of which are contemplated as mimetics of the present invention.

[0051] As used herein, "modified antibody" refers to an antibody whose amino acid sequence has been altered by mutation of at least one amino acid residue. Thus, "modified antibody" encompasses chimeric or humanized antibodies as defined herein, and may also refer to a molecule comprising an antibody or an antigen-binding fragment thereof, in which said monoclonal antibody or functional fragment thereof binds to a functionally distinct molecule. Modified antibodies of the present invention may also be fusion chimeric proteins or conjugates resulting from any suitable form of conjugation, including covalent bonding, grafting, chemical bonding with chemical or biological groups, or with molecules such as PEG polymers or other protecting groups, or molecules suitable for protection against protease cleavage in vivo, to improve the stability and / or half-life of the antibody or functional fragment.

[0052] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (Fv, Fab, Fab', F(ab')2, or other target-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. Generally, humanized antibodies will comprise substantially all of at least one, and typically two, variable domains, in which all CDR regions correspond to those of the non-human immunoglobulin and / or humanized versions thereof; and all or substantially all of the FR regions are those of the human immunoglobulin template sequence, or with substitutions of non-human residues (such as rodent residues) for amino acid residues present in the human immunoglobulin template sequence at corresponding positions. Humanized antibodies may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of the human immunoglobulin template selected. In certain embodiments, the invention relates to an antibody comprising a heavy chain variable region disclosed herein and a light chain variable region disclosed herein, wherein the heavy chain variable region and / or the light chain variable region further comprises a constant region, in particular an Fc region.

[0053] The terms "specifically bind" and "specifically bind to" mean that an antibody, antigen-binding fragment thereof, antigen-binding antibody mimetic, or modified antibody according to the present invention binds to at least 1 x 10 -6 M, 1 x 10 -7 M, 1 x 10 -8 M, 1 x 10 -9 M, 1 x 10 -10 M, 1 x 10 -11 M, 1 x 10 -12 It refers to the ability to bind to CMKLR1 with an affinity of M or greater and / or with an affinity at least two-fold higher than its affinity for a non-specific target (e.g., a protein other than CMKLR1). Affinity can be assessed according to various methods well known to those skilled in the art. These methods include, but are not limited to, biosensors such as Biacore analysis, Blitz analysis, and Scatchard plots.

[0054] The term "therapeutically effective amount" is used to refer to that amount of any given compound, as defined herein, sufficient to at least improve the clinical or physiological condition of the patient being treated. The therapeutically effective amount of an antibody, antigen-binding fragment thereof, antigen-binding antibody mimetic, or modified antibody according to the invention to be administered will be influenced by considerations such as the disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the schedule of administration, and other factors known to a physician.

[0055] All embodiments disclosed herein relating to antibodies or antigen-binding fragments thereof may be substituted mutatis mutandis for the macromolecules of the invention, in particular antigen-binding antibody mimetics and engineered antibodies.

[0056] In a specific embodiment of the invention, the CMKLR1 is human CMKLR1, corresponding to NCBI protein accession number Q99788.2.

[0057] Both the heavy and light chain variable domains comprise three CDRs (CDR1, CDR2, and CDR3, respectively, from the 5' to the 3' end) and four framework regions (FR1, FR2, FR3, and FR4, respectively, from the 5' to the 3' end). Humanization of a murine antibody may consist of humanizing at least one framework region in the light chain variable region and / or the heavy chain variable region, or both. In certain embodiments, some framework regions may be humanized, particularly within the heavy and light chain variable regions. Wild-type CDRs may be preserved, or CDRs may be replaced by CDRs described herein. Thus, the anti-CMKLR1 compounds according to the present invention may comprise at least one, or at least two, or at least three, or at least four, or at least five, or at least six wild-type CDRs when the framework regions are humanized. In other words, the anti-CMKLR1 compound is a humanized version of the parent chimeric antibody 2G1 (comprising a heavy chain variable domain of SEQ ID NO: 37 and a light chain variable domain of SEQ ID NO: 49) in which at least one framework region has been humanized, particularly in which at least one framework region and at least one CDR have been humanized. In a specific embodiment of the present invention, the variable region of the antibody may be combined with an antibody constant region, such as an IgG1, IgG2, IgG3, or IgG4 constant region, particularly an IgG1 constant region. These constant regions can be further mutated or modified by methods known in the art to alter their binding ability to Fc receptors.In a particular embodiment, the antibody or antigen-binding fragment thereof according to the invention is a humanized monoclonal antibody, in particular wherein the antibody light chain constant domain is derived from a human kappa light chain constant domain, in particular wherein the light chain constant domain comprises or consists of the sequence of SEQ ID NO: 79, and wherein the antibody heavy chain constant domain is derived from a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant domain, in particular an IgG1 heavy chain constant domain, in particular wherein the antibody heavy chain constant domain comprises or consists of the amino acid sequence of SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83 or SEQ ID NO: 84, in particular derived from a human IgG1 heavy chain constant domain, in particular wherein the antibody heavy chain constant domain comprises or consists of the amino acid sequence of SEQ ID NO: 80 or SEQ ID NO: 83.

[0058] Binding to a polypeptide comprising or consisting of the amino acid residues located within the sequence of SEQ ID NO: 2 and / or SEQ ID NO: 59 and / or the amino acid sequence of SEQ ID NO: 60, and / or binding to the third extracellular loop of CMKLR1, can be assessed by binding affinity analysis using an ELISA assay, as described in the examples of the present invention. To determine whether a test antibody can compete for binding to the same antigen, third loop, or epitope bound by the 2G1 antibody (or an antigen-binding fragment comprising a heavy chain variable domain corresponding to SEQ ID NO: 37 and a light chain domain corresponding to SEQ ID NO: 49), a cross-blocking assay (e.g., a competitive ELISA assay) can be performed. In an exemplary competitive ELISA assay, a polypeptide comprising or consisting of the epitope or third loop is coated on the wells of a microtiter plate and pre-incubated with or without a candidate competing antibody, followed by the addition of a biotin-labeled 2G1 antibody of the present invention. The amount of labeled anti-2G1 antibody bound to the polypeptide comprising or consisting of a polypeptide located within the amino acid sequence of SEQ ID NO: 2 and / or SEQ ID NO: 59, and / or SEQ ID NO: 60, and / or the third loop of CMKLR1 in the well is measured using an avidin-peroxidase conjugate and an appropriate substrate. The antibody can be labeled with a radioactive label, a fluorescent label, or some other detectable and measurable label. The amount of labeled anti-2G1 antibody bound to the polypeptide located within the amino acid sequence of SEQ ID NO: 2 and / or SEQ ID NO: 59, and / or SEQ ID NO: 60, and / or the third loop will indirectly correlate with the ability of a candidate competing antibody (test antibody) to compete for binding to the same epitope or the same loop; i.e., the higher the affinity of the test antibody for the same epitope, the less labeled 2G1 antibody will bind to the well coated with antigen. A candidate competing antibody is considered to be a candidate for antibody 2G1 antibody if the candidate antibody is able to block binding of the 2G1 antibody by at least 20%, preferably at least 20-50%, and even more preferably at least 50%, compared to a control run simultaneously in the absence of the candidate competing antibody (but which may also be in the presence of a known non-competing antibody). If the antibody is a 2G1 antibody of the present invention, it will be considered to be an antibody that competes for binding to the same polypeptide or third loop as the 2G1 antibody of the present invention. It will be understood that variations of this assay can be performed to arrive at the same quantitative value.

[0059] The anti-CMKLR1 antibody or antigen-binding fragment thereof has the effect of an inflammation-resolving factor, particularly by interacting with myeloid lineage cells.

[0060] In certain embodiments of the invention, the presence of certain VHCDR2s in antibodies that may exhibit reduced immunogenicity in humans allows for the provision of antibodies that may be more potent for therapeutic purposes, as antibodies with reduced immunogenicity are expected to have fewer side effects when administered to patients.

[0061] In a particular aspect of the present invention, there is provided an anti-CMKLR1 antibody or an antigen-binding fragment thereof that binds to chemokine-like receptor 1 (CMKLR1), particularly human CMKLR1, comprising: a. an antibody heavy chain variable (VH) domain comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3; - VHCDR1 is selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7; - VHCDR2 is selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:61; - an antibody heavy chain variable (VH) domain, wherein VHCDR3 is selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16; b. An antibody light chain variable (VL) domain comprising three CDRs, VLCDR1, VLCDR2 and VLCDR3, - VLCDR1 is selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23; - VLCDR2 is selected from the group consisting of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:33; - an antibody light chain variable (VL) domain, wherein VLCDR3 is selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36 The antibody or antigen-binding fragment thereof is disclosed, comprising:

[0062] In particular, the antibody or antigen-binding fragment thereof specifically binds to an epitope located within the third extracellular loop (EL3) of CMKLR1, and more particularly, the antibody or antigen-binding fragment thereof specifically binds to a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 59, or an epitope located within the amino acid sequence of SEQ ID NO: 60.

[0063] In another specific embodiment of the present invention, there is provided an anti-CMKLR1 antibody or an antigen-binding fragment thereof that binds to chemokine-like receptor 1 (CMKLR1), particularly human CMKLR1, comprising: a. an antibody heavy chain variable (VH) domain comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3; - VHCDR1 is selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7; - VHCDR2 is selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:61; or VHCDR2 corresponds to the amino acid residues of SEQ ID NO:12 or SEQ ID NO:63, with the proviso that VHCDR1 is not SEQ ID NO:3 or SEQ ID NO:4; - an antibody heavy chain variable (VH) domain, wherein VHCDR3 is selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16; b. An antibody light chain variable (VL) domain comprising three CDRs, VLCDR1, VLCDR2 and VLCDR3, - VLCDR1 is selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23; - VLCDR2 is selected from the group consisting of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:33; - an antibody light chain variable (VL) domain, wherein VLCDR3 is selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36 The antibody or antigen-binding fragment thereof is disclosed, comprising:

[0064] In particular, the antibody or antigen-binding fragment thereof specifically binds to an epitope located within the third extracellular loop (EL3) of CMKLR1, and more particularly, the antibody or antigen-binding fragment thereof specifically binds to a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 59, or an epitope located within the amino acid sequence of SEQ ID NO: 60.

[0065] In another specific embodiment of the present invention, there is provided an anti-CMKLR1 antibody or an antigen-binding fragment thereof that binds to chemokine-like receptor 1 (CMKLR1), particularly human CMKLR1, comprising: a. an antibody heavy chain variable (VH) domain comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3; - VHCDR1 is selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7; - VHCDR2 is selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:61; when VHCDR1 is SEQ ID NO: 3 or SEQ ID NO: 4 and VHCDR2 corresponds to the amino acid sequence of SEQ ID NO: 12, preferably said heavy chain variable (VH) domain does not comprise a framework VHFR3 of SEQ ID NO: 70, provided that said heavy chain variable (VH) domain comprises a framework FR3 of SEQ ID NO: 69; - an antibody heavy chain variable (VH) domain, wherein VHCDR3 is selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16; b. An antibody light chain variable (VL) domain comprising three CDRs, VLCDR1, VLCDR2 and VLCDR3, - VLCDR1 is selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23; - VLCDR2 is selected from the group consisting of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:33; - an antibody light chain variable (VL) domain, wherein VLCDR3 is selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36 The antibody or antigen-binding fragment thereof is disclosed, comprising:

[0066] In particular, the antibody or antigen-binding fragment thereof specifically binds to an epitope located within the third extracellular loop (EL3) of CMKLR1, and more particularly, the antibody or antigen-binding fragment thereof specifically binds to a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 59, or an epitope located within the amino acid sequence of SEQ ID NO: 60.

[0067] In particular, if an antibody or antigen-binding fragment thereof specifically binds to an epitope located within a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 59, or the amino acid sequence of SEQ ID NO: 60, binding to an epitope located within the third extracellular loop (EL3) of CMKLR1 can be assessed according to the methods disclosed herein above. The specific selection of a VHCDR2 can provide an antibody with reduced immunogenicity compared to an antibody having a different VHCDR2.

[0068] Such antibodies are agonists of CMKLR1 that mimic the effect of binding of resolvin E1 to CMKLR1, i.e., have resolvin E1-like ability as defined hereinabove. Anti-CMKLR1 antibodies or antigen-binding fragments thereof have the effect of an inflammation-resolving factor, particularly by interacting with myeloid cell lineages.

[0069] In a specific embodiment of the present invention, the anti-CMKLR1 antibody or antigen-binding fragment thereof - VHCDR1 selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 4 Includes:

[0070] In a specific embodiment of the present invention, the anti-CMKLR1 antibody or antigen-binding fragment thereof - a VHCDR2 selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 61 Includes:

[0071] In a specific embodiment of the present invention, the anti-CMKLR1 antibody or antigen-binding fragment thereof - a VHCDR3 selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15 Includes:

[0072] In a specific embodiment of the present invention, the anti-CMKLR1 antibody or antigen-binding fragment thereof - VLCDR1 selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 23 Includes:

[0073] In a specific embodiment of the present invention, the anti-CMKLR1 antibody or antigen-binding fragment thereof - VLCDR2 selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 33 Includes:

[0074] In a specific embodiment of the present invention, the anti-CMKLR1 antibody or antigen-binding fragment thereof - a VLCDR3 selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36 Includes:

[0075] In a specific embodiment of the present invention, the anti-CMKLR1 antibody or antigen-binding fragment thereof - a VHCDR1 selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 4; and / or - a VHCDR2 selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 61; and / or - a VHCDR3 selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15; - a VLCDR1 selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 23, and / or - a VLCDR2 selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 33; and / or - a VLCDR3 selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36 Includes:

[0076] In certain embodiments, the anti-CMKLR1 antibody or antigen-binding fragment thereof comprises the following CDRs: VHCDR1 of SEQ ID NO: 4, VHCDR2 of SEQ ID NO: 12, VHCDR3 of SEQ ID NO: 13, VLCDR1 of SEQ ID NO: 19, VLCDR2 of SEQ ID NO: 26, and VLCDR3 of SEQ ID NO: 35.

[0077] In certain embodiments of the present invention, the anti-CMKLR1 antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 62, SEQ ID NO: 89, SEQ ID NO: 90 and SEQ ID NO: 91.

[0078] In a more specific embodiment, the anti-CMKLR1 antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO:91.

[0079] In certain embodiments of the present invention, the anti-CMKLR1 antibody or antigen-binding fragment thereof comprises a light chain variable domain comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:92 and SEQ ID NO:93.

[0080] In a more specific embodiment, the anti-CMKLR1 antibody or antigen-binding fragment thereof comprises a light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO:93.

[0081] In a specific embodiment of the present invention, the anti-CMKLR1 antibody or antigen-binding fragment thereof - a heavy chain variable domain comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 62, SEQ ID NO: 89, SEQ ID NO: 90 and SEQ ID NO: 91; and a light chain variable domain comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 92 and SEQ ID NO: 93. Includes:

[0082] Any combination of a particular heavy chain variable domain and light chain variable domain disclosed herein is encompassed by the present disclosure.

[0083] In a more specific embodiment, the anti-CMKLR1 antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain variable domain comprising or consisting of amino acid residues selected from the group consisting of SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 62, and a light chain variable domain comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56.

[0084] In a more specific embodiment of the present invention, the anti-CMKLR1 antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO:38 and a light chain variable domain of SEQ ID NO:49.

[0085] In a more specific embodiment of the present invention, the anti-CMKLR1 antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 91 and a light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 93.

[0086] Furthermore, said antibody is advantageously characterized in that its Fc fragment is characteristic of IgG1.

[0087] In a second aspect of the present invention, there is provided a compound selected from the group consisting of antibodies, antigen-binding fragments thereof, or chimeric, modified, or humanized antibodies, which specifically bind to CMKLR1, in particular human CMKLR1, and which comprises an antibody heavy chain variable domain comprising (i) a VHCDR2 comprising or consisting of the amino acid sequences set forth in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:61, and (ii) a VHCDR3 comprising or consisting of the amino acid sequences set forth in SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16, or a mutant sequence thereof in which amino acid residues are substituted, with the proviso that the amino acid residues at positions 1 and 2 of the mutant sequence are L and I, respectively; The anti-CMKLR1 compound specifically binds to an epitope located within the third extracellular loop (EL3) of CMKLR1, in particular, the compound specifically binds to an epitope located within a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 59, or the amino acid sequence of SEQ ID NO: 60; The compound competes with an antibody comprising a heavy chain variable domain corresponding to SEQ ID NO: 37 and a light chain variable domain corresponding to SEQ ID NO: 49 for binding to a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 59 or SEQ ID NO: 60, or to a polypeptide comprising or consisting of the third loop (EL3) of the extracellular domain of CMKLR1. is disclosed.

[0088] The combination of the heavy chain variable domain of SEQ ID NO: 37 and the light chain variable domain of SEQ ID NO: 49 corresponds to the parent antibody 2G1. The binding of a compound to a polypeptide comprising or consisting of the sequence of SEQ ID NO: 2 or SEQ ID NO: 59, or an amino acid sequence located within SEQ ID NO: 60, or a polypeptide comprising or consisting of the third loop (EL3) of the extracellular domain of CMKLR1 can be assessed according to the methods disclosed herein above and illustrated in the examples of the present invention.

[0089] In a more specific embodiment, the anti-CMKLR1 antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO:91.

[0090] In a more specific embodiment, the anti-CMKLR1 antibody or antigen-binding fragment thereof comprises a light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO:93.

[0091] In a more specific embodiment of the present invention, the anti-CMKLR1 antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 91 and a light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 93.

[0092] Furthermore, said antibody is advantageously characterized in that its Fc fragment is characteristic of IgG1.

[0093] The present invention relates to diseases in which the resolution of inflammation is delayed or disrupted, and / or inflammatory diseases, in particular chronic inflammatory diseases such as acute inflammatory diseases, chronic inflammatory lung diseases (e.g., asthma), keratoconjunctivitis, periodontal disease, eczema, inflammatory bowel diseases, in particular Crohn's disease or colitis, in particular ulcerative colitis or spontaneous colitis, cystic fibrosis, skin inflammation; autoimmune diseases such as diabetes, NASH, in particular type 1 diabetes, psoriasis, lupus, rheumatoid arthritis, multiple sclerosis, Sjogren's syndrome, celiac disease, vasculitis, myasthenia gravis; severe inflammatory conditions such as sepsis, coronaviruses (e.g., COVID-19), etc. the compound of the present invention as defined above for use in the prevention and / or treatment of a disease selected from the group consisting of severe viral symptoms accompanied by steroids, infectious diseases such as peritonitis; degenerative diseases; wound healing disorders, dry eye syndrome; cancer diseases, in particular solid and liquid cancers, metastatic cancers, in particular carcinomas, in particular breast cancer or colon cancer, or colorectal cancer or lung cancer or mesothelioma, or myeloid cancers, in particular leukemias, in particular cancers in which the cancer cells express CMKLR1 or the tumor microenvironment is infiltrated by cells that express or overexpress CMKLR1.

[0094] Of particular interest are fibrosis (particularly pulmonary and hepatic fibrosis), ANCA (anti-neutrophil cytoplasmic autoantibody) pathologies (vasculitis), and pathologies resulting from neuronal apoptosis associated with ChermR23.

[0095] In a particular embodiment, the invention relates to any compound of the invention as defined above for use in the prevention and / or treatment of diseases in which the resolution of inflammation is delayed or disrupted, and / or inflammatory diseases, in particular chronic inflammatory diseases such as acute inflammatory diseases, asthma, keratoconjunctivitis, periodontal disease, eczema, inflammatory bowel disease, in particular Crohn's disease or colitis, in particular ulcerative colitis or spontaneous colitis, cystic fibrosis; autoimmune diseases such as diabetes, in particular type 1 diabetes, psoriasis, lupus, rheumatoid arthritis, multiple sclerosis, Sjögren's syndrome, celiac disease, vasculitis, myasthenia gravis; infectious diseases such as sepsis, peritonitis, severe viral conditions accompanied by a severe inflammatory state, such as coronaviruses (e.g. COVID-19); degenerative diseases; wound healing disorders, NASH (non-alcoholic steatohepatitis), scleroderma, and dry eye syndrome. In another particular embodiment of the present invention, the present invention relates to any of the compounds of the present invention as defined above for use in the prevention and / or treatment of cancer, in particular solid and liquid cancers, metastatic cancers, in particular carcinomas, in particular breast cancer or colon cancer, or colorectal cancer or lung cancer or myeloid cancer, in particular leukemia, in particular cancers in which the cancer cells express CMKLR1 or the tumor microenvironment is infiltrated by cells that express or overexpress CMKLR1.

[0096] In certain embodiments, the antibodies of the invention elicit at least one of the following effects that favor resolution in vitro and / or in vivo: - Increases apoptosis of polymorphonuclear neutrophils, referred to herein under the acronym PMN or PMN, at inflammation sites; such effects are illustrated in the examples of the present invention. Regulation of neutrophil apoptosis is important for therapeutic intervention in inflammation-related diseases. As illustrated in the examples of the present invention, the use of a CMKLR1 agonist with a resolvin E1-like antibody against PMN induces potent apoptosis of neutrophils during inflammation induction compared to a control antibody. - Enhance caspase-3 expression in neutrophils, thereby resulting in caspase-3 dependent apoptosis; apoptosis may be considered to be enhanced or induced if the expression of caspase-3 exceeds 1 log, preferably 2 logs, most preferably 3 logs, 11 hours after treatment with an agonist of CMKLR1 having a resolvin E1-like ability antibody compared to a negative control; the method is disclosed in the examples of the present invention. - Reduces or inhibits the migration of PMNs (neutrophils) from the endothelium toward the site of inflammation; preventing them from relocating to the site of inflammation and exerting their pro-inflammatory effects. In a specific embodiment of the present invention, neutrophil migration and migration are prevented or reduced by administering an agonist of CMKLR1 having an IgG1 antibody with resolvin E1-like capabilities. Thus, in a specific embodiment, the agonist of CMKLR1 having resolvin E1-like capabilities is a humanized antibody with a human constant region derived from or generated from human IgG1. In a specific embodiment, the agonist of CMKLR1 having an antibody with resolvin E1-like capabilities is of the human IgG1 isotype, i.e., constant fragments of heavy and light chains derived from or generated from human IgG1 antibody constant heavy and light chain fragments. Thus, the agonist of CMKLR1 having an antibody with resolvin E1-like capabilities of the present invention comprises an Fc domain of the IgG1 isotype. Neutrophil migration can be evaluated by the method disclosed in the Examples of the present invention; if cell surface expression of CD62L is reduced by at least 1 log in a staining experiment compared to a negative control, the neutrophil can be considered to have reduced migration and / or migration ability. The present inventors have found that CMKLR1 agonists having resolvin E1-like ability humanized IgG1 antibodies of the present invention do not exhibit cytotoxicity in vivo; in other words, the use of CMKLR1 agonists does not show significant depletion of CMKLR1-positive cells in vivo. In certain embodiments, the use of the agonists of the present invention does not show cytotoxic activity against CMKLR1-positive cells. - Reduces cell surface expression of CMKLR1, CXCR4, and / or CCR7. In certain embodiments, the cell surface expression is considered to be on macrophages and / or dendritic cells. When a CMKLR1 agonist with resolvin E1-like activity binds to its target, CMKLR1, CXCR4, and / or CCR7 heterodimerize and are internalized. In a preferred embodiment, the antibody of the present invention induces the internalization and / or inhibits the expression of CMKLR1 and / or CXCR4 and / or CCR7 on the cell surface of CMKLR1-positive cells. Thus, the cell surface expression of CMKLR1, CXCR4, and / or CCR7 in cells incubated in the presence of the antibody is reduced or significantly reduced compared to the cell surface expression in cells incubated under otherwise identical conditions but in the absence of a CMKLR1 agonist with resolvin E1-like activity.

[0097] In a particular aspect, the present invention provides a humanized anti-chemerin-like receptor 1 (CMKLR1) antibody or antigen-binding fragment thereof suitable for production in mammalian cells, such as COS or CHO cells, at a yield of greater than 0.1 mg / ml, particularly greater than 1 mg / ml, particularly greater than 10 mg / ml, and more particularly greater than 100 mg / ml, a) the variable heavy (VH) domain comprises the amino acid sequences of frameworks (FR1, FR2, FR3 and FR4) of a heavy chain variable domain, each of which has a sequence identity with the framework of the same rank in the sequence of SEQ ID NO: 41 of 100% for FR1, at least 60% for FR2, at least 78% for FR3 and at least 80% for FR4, respectively; more specifically, 100% for FR1, at least 80% for FR2, at least 85% for FR3 and at least 90% for FR4; b) An antibody or antigen-binding fragment thereof, wherein the variable light (VL) domain comprises the amino acid sequence of the framework of the light chain variable domain (FR1, FR2, FR3 and FR4), and each framework has a sequence identity with the framework of the same rank in the sequence of SEQ ID NO: 50 of 60% for FR1, at least 70% for FR2, at least 75% for FR3 and at least 80% for FR4, respectively; more specifically, 100% for FR1, at least 90% for FR2, at least 90% for FR3 and 100% for FR4.

[0098] In certain embodiments, the humanized anti-CMKLR1 antibody or antigen-binding fragment thereof specifically binds to the third extracellular loop (EL3) of CMKLR1, and in particular, the antibody or antigen-binding fragment thereof specifically binds to a polypeptide comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:59, or located within the amino acid sequence of SEQ ID NO:60. This embodiment may facilitate in vitro production of the antibody (or antigen-binding fragment thereof). It should be noted that this definition of an antibody is independent of any other definition of the anti-CMKLR1 antibody of the present invention, including the definition of an antibody by its CDR domain. In certain aspects of the present invention, the definition of the antibody of the present invention by its FR sequence can be combined with the definition of its CDR domain and / or functional characteristics. To this end, antibodies defined by their FR domains and characterized by their CDR domains selected from a specific group are also encompassed by the present invention. To this end, such antibodies, which may or may not exhibit identity to the framework domains disclosed herein, may comprise the following CDRs: - a VHCDR1 selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7; and / or - a VHCDR2 selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:61, SEQ ID NO:63 and SEQ ID NO:64; and / or - a VHCDR3 selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16; and / or The variable light (VL) domain comprises: - a VLCDR1 selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23; and / or - a VLCDR2 selected from the group consisting of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:33; and / or - a VLCDR3 selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36 It includes at least one, especially six, of the following:

[0099] In a more particular embodiment, at least one of the framework domains HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4 is selected from the group: SEQ ID NO: 65 for VHFR1; for VHFR2, SEQ ID NO: 66, SEQ ID NO: 67 or SEQ ID NO: 68; for VHFR3, SEQ ID NO: 69 or SEQ ID NO: 70; SEQ ID NO: 71 for VHFR4; SEQ ID NO: 72 for VLFR1; for VLFR2, SEQ ID NO: 73 or SEQ ID NO: 74; for VLFR3, SEQ ID NO: 75 or SEQ ID NO: 76; For VLFR4, SEQ ID NO: 77 is selected from.

[0100] It should be understood that multiple framework domains, or all, may be selected from the group set forth herein above.

[0101] In more specific embodiments, the antibody or antigen-binding fragment thereof described in the two paragraphs above has an amino acid sequence of a heavy chain variable domain selected from the group of SEQ ID NO: 41, SEQ ID NO: 38, SEQ ID NO: 42, SEQ ID NO: 43, and an amino acid sequence of a light chain variable domain that is the sequence of SEQ ID NO: 50.

[0102] In a more particular embodiment, the antibody or antigen-binding fragment thereof comprises the following framework domains: - VHFR1 of SEQ ID NO: 65, - VHFR2 of SEQ ID NO: 67, - VHFR3 of SEQ ID NO: 69, - VHFR4 of SEQ ID NO: 71, - VLFR1 of SEQ ID NO: 72, - VLFR2 of SEQ ID NO: 73, - VLFR3 of SEQ ID NO: 76, and VLFR4 of SEQ ID NO: 77 Includes:

[0103] In a more particular embodiment, the antibody comprises the following framework domain: - VHFR1 of SEQ ID NO: 65, - VHFR2 of SEQ ID NO: 67, - VHFR3 of SEQ ID NO: 69, - VHFR4 of SEQ ID NO: 71, - VLFR1 of SEQ ID NO: 72, - VLFR2 of SEQ ID NO: 73, - VLFR3 of SEQ ID NO: 76, VLFR4 of SEQ ID NO: 77 Including, The following CDRs: - a VHCDR1 selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7; and / or - a VHCDR2 selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:61, SEQ ID NO:63 and SEQ ID NO:64; and / or - a VHCDR3 selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16; and / or The variable light (VL) domain comprises: - a VLCDR1 selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23; and / or - a VLCDR2 selected from the group consisting of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:33; and / or - a VLCDR3 selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36 Includes:

[0104] This embodiment may facilitate the production of antibodies (or antigen-binding fragments thereof) in vitro.

[0105] In particular, the present invention relates to a humanized anti-CMKLR1 antibody or an antigen-binding fragment thereof, which has been optimized to reduce immunogenicity while maintaining high binding activity, stability, and biological function, and which has the following CDRs: - VHCDR1 of SEQ ID NO: 4, - VHCDR2 of SEQ ID NO: 12, - VHCDR3 of SEQ ID NO: 13, - VLCDR1 of SEQ ID NO: 19, - VLCDR2 of SEQ ID NO: 26, and VLCDR3 of SEQ ID NO: 35 The present invention relates to an antibody or an antigen-binding fragment thereof comprising:

[0106] In certain embodiments, the antibodies or antigen-binding fragments thereof having CDR sequences of SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:19, SEQ ID NO:26, and SEQ ID NO:35 contain the following framework domains: - VHFR1 of SEQ ID NO: 65, - VHFR2 of SEQ ID NO: 67, - VHFR3 of SEQ ID NO: 69, - VHFR4 of SEQ ID NO: 71, - VLFR1 of SEQ ID NO: 72, - VLFR2 of SEQ ID NO: 73, - VLFR3 of SEQ ID NO: 76, VLFR4 of SEQ ID NO: 77 Includes:

[0107] In particular, the present disclosure relates to an anti-CMKLR1 antibody or antigen-binding fragment thereof that is optimized to reduce immunogenicity while maintaining high binding activity, stability, and biological function, which are suitable for in vitro production. The anti-CMKLR1 antibody or antigen-binding fragment thereof described in the preceding paragraph has an amino acid sequence of a heavy chain variable domain set forth in SEQ ID NO: 91 and an amino acid sequence of a light chain variable domain set forth in SEQ ID NO: 93.

[0108] Furthermore, said antibody is advantageously characterized in that its Fc fragment is characteristic of IgG1.

[0109] In certain embodiments, the antibody of the present invention is characterized by the amino acid sequence of the framework domain disclosed above and may further be characterized by the amino acid sequence of at least one of its CDR domains. In particular, the humanized anti-CMKLR1 antibody or antigen-binding fragment thereof according to this embodiment has a heavy chain variable domain VH CDR2 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:61, SEQ ID NO:63, and SEQ ID NO:64.

[0110] In certain embodiments of the present invention, - inflammatory diseases, in particular acute inflammatory diseases, chronic inflammatory lung diseases (e.g. asthma), keratoconjunctivitis, periodontal disease, eczema, inflammatory bowel diseases, in particular Crohn's disease or colitis, in particular ulcerative colitis or spontaneous colitis, chronic inflammatory diseases such as cystic fibrosis, NASH (non-alcoholic steatohepatitis), liver fibrosis, pulmonary fibrosis, antineutrophil cytoplasmic antibody-associated diseases (ANCA), vasculitis, in particular ANCA-mediated vasculitis, scleroderma, in particular in which administration of the treatment results in enhanced resolution of inflammation; - autoimmune diseases such as diabetes, especially type 1 diabetes, psoriasis, lupus, rheumatoid arthritis, multiple sclerosis, Sjögren's syndrome, celiac disease, vasculitis, myasthenia gravis, or severe viral conditions accompanied by severe inflammatory conditions such as infections such as sepsis, peritonitis, degenerative diseases, wound healing disorders or dry eye syndrome, coronaviruses (e.g., COVID-19), in particular where administration of the treatment results in enhanced resolution of inflammation; In particular, cancers, in particular metastatic cancers, solid or liquid cancers, such as carcinomas, more particularly liver cancer, in particular breast cancer or colon cancer, or lung cancer or myeloid cancers such as leukemia, in which administration of the treatment results in enhanced resolution of inflammation, in particular cancers in which the cancer cells express CMKLR1 or the tumor microenvironment is infiltrated by cells that express or overexpress CMKLR1. For the prophylactic or therapeutic treatment of a) an antibody heavy chain variable (VH) domain comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, - VHCDR1 is selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7; - VHCDR2 is selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 61; or VHCDR2 corresponds to the amino acid residues of SEQ ID NO: 12, with the proviso that VHCDR1 is not SEQ ID NO: 3 or SEQ ID NO: 4; - an antibody heavy chain variable (VH) domain, wherein VHCDR3 is selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16; b) an antibody light chain variable (VL) domain comprising three CDRs, VLCDR1, VLCDR2 and VLCDR3, - an antibody light chain variable (VL) domain, wherein VLCDR1 is selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23; - VLCDR2 is selected from the group consisting of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:33; - an antibody light chain variable (VL) domain, wherein VLCDR3 is selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36 The present invention provides an antibody or antigen-binding fragment thereof comprising:

[0111] In another particular embodiment of the present invention, for the prophylactic or therapeutic treatment of the diseases described above, a) an antibody heavy chain variable (VH) domain comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, - VHCDR1 is selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7; - VHCDR2 is selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 61; when VHCDR1 is SEQ ID NO: 3 or SEQ ID NO: 4 and VHCDR2 corresponds to the amino acid sequence of SEQ ID NO: 12, preferably the heavy chain variable (VH) domain does not comprise the framework VHFR3 of SEQ ID NO: 70, provided that said heavy chain variable (VH) domain comprises the framework FR3 of SEQ ID NO: 69; - an antibody heavy chain variable (VH) domain, wherein VHCDR3 is selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16; b) an antibody light chain variable (VL) domain comprising three CDRs, VLCDR1, VLCDR2 and VLCDR3, - VLCDR1 is selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23; - VLCDR2 is selected from the group consisting of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:33; - an antibody light chain variable (VL) domain, wherein VLCDR3 is selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36 The present invention provides an antibody or antigen-binding fragment thereof comprising:

[0112] In certain embodiments of the invention, the humanized antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or engineered antibody comprises: - inflammatory diseases, in particular acute inflammatory diseases, chronic inflammatory lung diseases (e.g. asthma), keratoconjunctivitis, periodontal disease, eczema, inflammatory bowel diseases, in particular Crohn's disease or colitis, in particular ulcerative colitis or spontaneous colitis, chronic inflammatory diseases such as cystic fibrosis, NASH (non-alcoholic steatohepatitis), liver fibrosis, pulmonary fibrosis, antineutrophil cytoplasmic antibody-associated diseases (ANCA), vasculitis, in particular ANCA-mediated vasculitis, scleroderma, in particular in which administration of the treatment results in enhanced resolution of inflammation; - autoimmune diseases such as diabetes, especially type 1 diabetes, psoriasis, lupus, rheumatoid arthritis, multiple sclerosis, Sjögren's syndrome, celiac disease, vasculitis, myasthenia gravis, or infections such as sepsis, peritonitis, degenerative diseases, wound healing disorders, severe viral conditions accompanied by severe inflammatory conditions such as coronaviruses (e.g., COVID-19), or dry eye syndrome, in particular in which administration of the treatment results in enhanced resolution of inflammation; In particular, cancers, in particular metastatic cancers, solid or liquid cancers, such as carcinomas, more particularly liver cancer, in particular breast cancer or colon cancer, or lung cancer or myeloid cancers such as leukemia, in which administration of the treatment results in enhanced resolution of inflammation, in particular cancers in which the cancer cells express CMKLR1 or the tumor microenvironment is infiltrated by cells that express or overexpress CMKLR1. For the prophylactic or therapeutic treatment of a) a variable heavy chain (VH) domain comprising the amino acid sequences of the frameworks (FR1, FR2, FR3 and FR4) of a heavy chain variable domain, each framework having a sequence identity with the framework of the same rank in the sequence of SEQ ID NO: 41 of 100% for FR1, at least 60% for FR2, at least 78% for FR3 and at least 80% for FR4, respectively; more specifically, 100% for FR1, at least 80% for FR2, at least 85% for FR3 and at least 90% for FR4; b) a variable light chain (VL) domain comprising the amino acid sequences of the frameworks (FR1, FR2, FR3 and FR4) of the light chain variable domain, each of which has a sequence identity with the framework of the same rank in the sequence of SEQ ID NO: 50 of 60% for FR1, at least 70% for FR2, at least 75% for FR3 and at least 80% for FR4, respectively; more specifically, 100% for FR1, at least 90% for FR2, at least 90% for FR3 and 100% for FR4. Includes:

[0113] In more particular embodiments, the antibody comprises the following framework domains: - VHFR1 of SEQ ID NO: 65, - VHFR2 of SEQ ID NO: 67, - VHFR3 of SEQ ID NO: 69, - VHFR4 of SEQ ID NO: 71, - VLFR1 of SEQ ID NO: 72, - VLFR2 of SEQ ID NO: 73, - VLFR3 of SEQ ID NO: 76, VLFR4 of SEQ ID NO: 77 Including, The following CDRs: - a VHCDR1 selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7; and / or - a VHCDR2 selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 61; - a VHCDR3 selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16; The variable light (VL) domain comprises: - a VLCDR1 selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23; and / or - a VLCDR2 selected from the group consisting of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:33; and / or - a VLCDR3 selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36 Including, - inflammatory diseases, in particular acute inflammatory diseases, chronic inflammatory lung diseases (e.g. asthma), keratoconjunctivitis, periodontal disease, eczema, inflammatory bowel diseases, in particular Crohn's disease or colitis, in particular ulcerative colitis or spontaneous colitis, chronic inflammatory diseases such as cystic fibrosis, NASH (non-alcoholic steatohepatitis), liver fibrosis, pulmonary fibrosis, antineutrophil cytoplasmic antibody-associated diseases (ANCA), vasculitis, in particular ANCA-mediated vasculitis, scleroderma, in particular in which administration of the treatment results in enhanced resolution of inflammation; - autoimmune diseases such as diabetes, especially type 1 diabetes, psoriasis, lupus, rheumatoid arthritis, multiple sclerosis, Sjögren's syndrome, celiac disease, vasculitis, myasthenia gravis, or infections such as sepsis, peritonitis, degenerative diseases, wound healing disorders, severe viral conditions accompanied by severe inflammatory conditions such as coronaviruses (e.g., COVID-19), or dry eye syndrome, in particular in which administration of the treatment results in enhanced resolution of inflammation; - In particular for use in the prophylactic or therapeutic treatment of cancer, in particular metastatic cancer, solid or liquid cancer, e.g. carcinoma, more particularly liver cancer, in particular breast or colon cancer, or lung cancer or myeloid cancer such as leukemia, where administration of the treatment results in enhanced resolution of inflammation, in particular cancers in which the cancer cells express CMKLR1 or the tumor microenvironment is infiltrated by cells expressing or overexpressing CMKLR1.

[0114] Such a particular antibody or antigen-binding fragment thereof for the prophylactic or therapeutic treatment of one of the above-disclosed conditions is in particular characterized in that it comprises the following CDRs: VHCDR1 of SEQ ID NO: 4, VHCDR2 of SEQ ID NO: 12, VHCDR3 of SEQ ID NO: 13 and VLCDR1 of SEQ ID NO: 19, VLCDR2 of SEQ ID NO: 26, VLCDR3 of SEQ ID NO: 35, and the following frameworks: VHFR1 of SEQ ID NO: 65, VHFR2 of SEQ ID NO: 67, VHFR3 of SEQ ID NO: 69, VHFR4 of SEQ ID NO: 71, VLFR1 of SEQ ID NO: 72, VLFR2 of SEQ ID NO: 73, VLFR3 of SEQ ID NO: 76, VLFR4 of SEQ ID NO: 77. In particular, for the above-disclosed prophylactic or therapeutic uses of an antibody or antigen-binding fragment thereof, the amino acid sequence of the heavy chain variable domain of the antibody comprises or consists of SEQ ID NO: 91, and the amino acid sequence of the light chain variable domain of the antibody comprises or consists of SEQ ID NO: 93.

[0115] In another aspect, the present invention relates to a composition comprising an anti-CMKLR1 compound described herein, in particular a pharmaceutical composition comprising an anti-CMKLR1 compound according to the present invention and a further therapeutic agent or a pharmaceutically acceptable carrier. In a particular embodiment, the present invention relates to a composition comprising an anti-CMKLR1 compound according to the present invention and a therapeutic agent selected from the group consisting of an immunomodulator, an immune checkpoint blocker, an immune checkpoint activator, an antibody, or an anti-SIRPα antibody (P84 - anti-mouse SIRPa from Merck Millipore).

[0116] In another aspect, the present invention relates to a combination of compounds comprising the anti-CMKLR1 compounds described herein, in particular a pharmaceutical composition comprising an anti-CMKLR1 compound according to the present invention and an anti-PD1 or anti-PDL1 compound, in particular an anti-PD1 compound; such compounds are in particular selected from the group consisting of small molecules such as antibodies, antigen-binding antibody fragments, antigen-binding antibody mimics, aptamers or peptides, and modified antibodies such as, but not limited to, humanized or chimeric antibodies, which are capable of binding to PD1 or PDL1.

[0117] In another aspect, the present invention relates to a combination of compounds comprising the anti-CMKLR1 compounds described herein, in particular a pharmaceutical composition comprising an anti-CMKLR1 compound according to the present invention and an anti-SIRPa compound; such compounds are in particular selected from the group consisting of small molecules such as antibodies, antigen-binding antibody fragments, antigen-binding antibody mimics, aptamers or peptides, and modified antibodies such as, but not limited to, humanized or chimeric antibodies, capable of binding to SIRPa, in particular human SIRPa.

[0118] The present invention also relates to combinations, eg, for treating fibrosis, that include therapeutic compounds that stimulate inflammation-resolving macrophages, whether or not they are cytokines.

[0119] In another aspect, the present invention relates to the therapeutic use of the anti-CMKLR1 compounds of the present invention, in particular for inducing and / or enhancing the resolution of inflammation, particularly when said resolution is delayed or disrupted, in view of treating diseases in which prolonged inflammation or the duration of resolution of inflammation is pathological.

[0120] In certain embodiments of the present invention, the anti-CMKLR1 compound binds to CMKLR1 with an affinity (KD value) of at least 10E-8M, more preferably with an affinity of at least 10E-9M. Specific binding of the antibody, or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody of the present invention to CMKLR1 (or a region of CMKLR1 comprising the third extracellular loop comprising the amino acid sequences set forth in SEQ ID NO:2 and SEQ ID NO:59, or located within the amino acid sequence of SEQ ID NO:60) means that the antibody exhibits an applicable affinity for CMKLR1. "Applicable affinity" refers to an affinity of at least about 10 -8 M(KD) or stronger. Preferably, the binding affinity is 10 -8 M~10 -12 M, 10 as needed -9 M~10 -10 M, especially at least 10 -9If the binding is M, the binding is considered specific. Whether a binding domain specifically reacts with or binds to a target can be easily tested, particularly by comparing the reaction of the binding domain with the target protein or antigen and the reaction of the binding domain with proteins or antigens other than the target protein. Such antibodies of the present invention specifically bind to CMKLR1 and have an agonistic effect on the interaction between RvE1 and CMKLR1. Methods for determining antibody specificity and affinity by competitive inhibition are known in the art (see, e.g., Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); Colligan et al., Current Protocols in Immunology, Green Publishing Assoc., NY (1992; 1993); Muller, Meth. Enzym, 92:589-601 (1983)). These methods include, but are not limited to, Biacore analysis, Blitz analysis, flow cytometry, and ELISA assays.

[0121] In certain embodiments of the present invention, the anti-CMKLR1 compound specifically binds to an epitope located within the third extracellular loop of CMKLR1, particularly an epitope located within the amino acid residue sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 59 or SEQ ID NO: 60, particularly SEQ ID NO: 2. Anti-CMKLR1 compounds that bind within this specific region of CMKLR1 have agonistic properties for CMKLR1, thereby mimicking the binding of RvE1 to CMKLR1.

[0122] In another aspect, the present invention relates to an anti-CMKLR1 antibody or an antigen-binding fragment thereof or an antigen-binding antibody mimetic or modified antibody as defined herein above, having agonistic activity against the interaction between RvE1 and CMKLR1, for use as a pharmaceutical.

[0123] In another aspect, the present invention relates to an anti-CMKLR1 antibody, or an antigen-binding fragment thereof, or an antigen-binding antibody mimic, or a modified antibody as defined herein above, which has the ability to induce activation of Akt and / or Erk proteins in vitro and / or in vivo. Activation of these proteins can be evaluated by the methods described in the Examples of the present invention. In particular, the anti-CMKLR1 antibody, or an antigen-binding fragment thereof, or an antigen-binding antibody mimic, or a modified antibody has the ability to activate either or both of Akt and / or Erk proteins in macrophages, particularly human macrophages.

[0124] The present invention also relates to a method of treatment in a subject in need thereof, comprising administering to the subject an effective amount of an anti-CMKLR1 antibody, or an antigen-binding fragment thereof, or an antigen-binding antibody mimic as defined above, which has agonistic ability for the interaction between RvE1 and CMKLR1, or in other words, is an RvE1 agonist-like factor or modulator.

[0125] Altering macrophage polarization in favor of anti-inflammatory cells can be useful in several pathologies or situations. As noted above, this alteration is particularly useful in the context of diseases selected from the group of inflammatory diseases, including, but not limited to, acute and chronic inflammatory diseases, inflammatory bowel disease, Crohn's disease, asthma, keratoconjunctivitis, periodontal disease, eczema, colitis, particularly ulcerative or spontaneous colitis, cystic fibrosis; diabetes, particularly type 1 diabetes, peritonitis, psoriasis, carcinoma, particularly breast or colon cancer, cancer, metastatic cancer, lung cancer, degenerative diseases, infectious diseases, particularly sepsis, autoimmune diseases, NASH, scleroderma, colitis or Crohn's disease in subjects refractory to corticosteroid and / or immunosuppressive treatment.

[0126] The present invention also relates to the use of an anti-CMKLR1 antibody or an antigen-binding fragment thereof or an antigen-binding antibody mimetic as defined above, which has resolvin E1-like agonist ability, in the manufacture of a medicament.

[0127] In another aspect, the present invention relates to an anti-CMKLR1 antibody or an antigen-binding fragment thereof or an antigen-binding antibody mimetic or modified antibody, such as, but not limited to, a humanized or chimeric antibody, as defined herein above, for use in the treatment of chronic inflammatory diseases, in particular for treating chronic colitis.

[0128] In another aspect, the present invention relates to an anti-CMKLR1 antibody or an antigen-binding fragment thereof or an antigen-binding antibody mimetic as defined above, having agonistic activity against CMKLR1 interaction, for use in the treatment of inflammatory conditions, particularly inflammatory diseases whose resolution is delayed or disrupted, and / or in the treatment or prevention of a disease selected from the group of inflammatory diseases including, but not limited to, acute and chronic inflammatory diseases, inflammatory bowel disease, Crohn's disease, asthma, keratoconjunctivitis, periodontal disease, eczema, colitis, particularly ulcerative colitis or spontaneous colitis, cystic fibrosis, diabetes, particularly type 1 diabetes, peritonitis, psoriasis, carcinoma, particularly breast cancer or colon cancer, cancer, degenerative diseases, infectious diseases, particularly sepsis, autoimmune diseases.

[0129] As defined herein, "delayed or impaired resolution of an inflammatory state" occurs when resolution of inflammation is delayed or impaired compared to normal resolution (i.e., resolution that occurs in patients who experience physiological resolution after an inflammatory event). Delayed or impaired resolution can result in increased infiltration of granulocytes at the site of inflammation. Therefore, delayed or impaired resolution can be assessed by quantifying granulocytes at the site of inflammation. For example, granulocyte populations can be measured by histology, blood count, or indirect biochemical techniques, such as elastase quantification by enzyme immunoassay or molecular quantification by PCR of granulocyte receptor 1. Delayed or impaired resolution can also be assessed by determining delayed apoptosis of granulocytes, measured, for example, by blood count using a specific antibody against annexin 5. Defective or delayed resolution of inflammation can also be assessed by quantifying the synthesis of pro-inflammatory cytokines, such as TNF-alpha, IL8, or IL12, and anti-inflammatory cytokines, such as IL-10. Cytokine secretion can be assessed by enzyme immunoassay or PCR. Defective or delayed resolution of inflammation can also be determined by assessing the activation of transcription factors involved in the synthesis of proinflammatory cytokines, such as NF-kappaB, which can be measured, for example, by nuclear translocation or by Western blot and / or quantification of IkappaB degradation levels. Defective or delayed resolution of inflammation can also be determined by quantifying specific inflammation-resolving mediators (such as lipoxins, resolvins, protectins, or maresins) or their precursors (17-HDOHE or 14-HDOHE) by mass spectrometry or enzyme immunoassay. Defective or delayed resolution then results from a defect in the synthesis of one or more of these mediators. Defective or delayed resolution can also be determined when the expression of receptors for resolution molecules is reduced. These receptors can be selected from the group including ALX, CMK1R1, GPR32, or GPR18. Alternatively, or complementary, the internalization and processing of these receptors into the cytoplasm can be assessed. Alternatively, or complementary, the expression of some receptors for inflammatory cytokines or lipids can be assessed, with overexpression compared to normal indicating a significant delay in the resolution of inflammation. or defective resolution. These conditions can be measured by histology, cytology, or PCR. Defective resolution can also result in a reduced or inhibited switch to inflammatory, inflammation-resolving macrophages, and impaired phagocytosis or efferocytosis of the same cells. Therefore, delayed or defective resolution can be assessed by analyzing the switch to inflammatory, inflammation-resolving macrophages in a particular condition compared to a normal condition, as exemplified in the examples of the present invention.

[0130] According to certain embodiments, anti-CMKLR1 compounds can be used to treat individuals with cancers selected from the group consisting of breast cancer, particularly breast cancer, melanoma, colon cancer, particularly colon cancer, and leukemia, particularly acute myeloid leukemia, particularly when the cancer cells overexpress CMKLR1.

[0131] In one embodiment, the present invention relates to an anti-human CMKLR1 antibody or its antigen-binding fragment or antigen-binding antibody mimic or modified antibody as defined above for the use as defined above, wherein the anti-human CMKLR1 antibody or its antigen-binding fragment or antigen-binding antibody mimic or modified antibody of the present invention is administered to a patient presenting with a CMKLR1-positive tumor.

[0132] The antibody or antigen-binding fragment thereof of the present invention can be administered to a subject by various suitable routes, for example, intravenously (IV), subcutaneously (SC), or intramuscularly (IM). The anti-CMKLR1 compound can be administered alone or together with another therapeutic agent, for example, a second human monoclonal antibody or its antigen-binding fragment. In another example, the antibody is administered together with another drug, for example, an immunosuppressant or an erythropoiesis-stimulating agent (ESA), together with a therapeutic cell composition, etc. In one embodiment, the present invention relates to an anti-CMKLR1 compound or its antigen-binding fragment or antigen-binding antibody mimic for use as defined above, in which the anti-CMKLR1 antibody or its antigen-binding fragment is combined with a second therapeutic agent.

[0133] The administration of the second therapeutic agent may or may not be simultaneous with the administration of the anti-CMKLR1 compound. Depending on the nature of the second agent, simultaneous administration can be prepared in the form of a combination drug (product), also known as a "combo." A combo is a fixed-dose combination containing two or more active pharmaceutical ingredients mixed in a single dosage form, which is manufactured and distributed in a fixed dose. However, the dosage regimen and / or route of administration may be different.

[0134] In preferred embodiments, the second therapeutic agent is selected from the group consisting of a chemotherapeutic agent, a radiotherapeutic agent, an immunotherapeutic agent, a cellular therapy agent (CAR-T cells), an antibiotic, and a probiotic.

[0135] In particular, immunotherapeutic agents useful in the context of the present invention are selected from the group consisting of therapeutic vaccines (DNA, RNA or peptide vaccines), immune checkpoint blockers or activators, in particular adaptive immune cells (T or B lymphocytes) or immunoconjugates such as antibody-drug conjugates.

[0136] As used herein, the term "immunotherapeutic agent" refers to agents that can translate interesting biological phenomena into effective therapeutic agents for cancer vaccines, including, inter alia, T cell growth factors that increase the number and repertoire of naive T cells, growth factors that increase the number of dendritic cells (DCs), agonists that activate DCs and other antigen-presenting cells (APCs), adjuvants that enable and increase cancer vaccines, agonists that activate and stimulate T cells, inhibitors of T cell checkpoint blockade, T cell growth factors that increase the proliferation and survival of immune T cells, agents that inhibit, block, or neutralize cancer cells, and immune cell-derived immunosuppressive cytokines.

[0137] Several immune checkpoint blockers or activators are known in the art. In the context of the present invention, examples of immune checkpoint blockers or activators of adaptive immune cells (B or T lymphocytes) that may be useful include anti-PDL1, anti-PD1, anti-CTLA4, anti-SIRPa, anti-CD137, anti-CD2, anti-CD28, anti-CD40, anti-HVEM, anti-BTLA, anti-CD160, anti-TIGIT, anti-TIM-1 / 3, anti-LAG-3, anti-2B4, and anti-OX40, anti-CD40 agonists, CD40-L, TLR agonists, anti-ICOS, ICOS-L, and B cell receptor agonists, particularly anti-CD137 and anti-SIRPa. In certain embodiments of the present invention, the second therapeutic agent is an anti-PDL1 or anti-PD1 compound, particularly an anti-PD1 compound, more particularly an anti-PD1 antibody. In certain embodiments of the present invention, the second therapeutic agent is an anti-SIRPa compound, particularly an anti-SIRPa antibody.

[0138] The immunotherapeutic agent may also be an antibody that targets a tumor antigen, particularly selected from the group consisting of anti-Her2, anti-EGFR, anti-CD20, anti-CD19, anti-CD52.

[0139] Antibodies can be provided at an effective dose of about 1 ng / kg body weight to about 30 mg / kg body weight or more, in certain embodiments, dosages can range from 1 μg / kg to about 20 mg / kg, or, as appropriate, 10 μg / kg to 10 mg / kg or 100 μg / kg to 5 mg / kg.

[0140] The term "effective dose" or "effective dosage" or "effective amount" is defined as an amount sufficient to achieve, or at least partially achieve, the desired effect. The term "effective dose" is meant to encompass an amount sufficient to cure or at least partially halt the disease and its complications, or to alleviate symptoms of the disease in a patient already suffering from the disease. The amount or dose effective for this use will depend on the condition being treated, the antibody construct being delivered, the context and purpose of the treatment, the severity of the disease, previous therapies, the patient's clinical history and response to the therapeutic agent, the route of administration, the patient's size (weight, body surface area, or organ size) and / or condition (age and general health), and the general state of the patient's own immune system. The appropriate dose can be adjusted so that it can be administered to the patient once or over a series of administrations, and to obtain the optimal therapeutic effect.

[0141] Dosages for such purposes can be repeated as needed, for example, daily, twice weekly, weekly, twice monthly, monthly, or as needed during recurrence.

[0142] In another aspect, the present invention relates to a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as defined above and a pharmaceutically acceptable carrier.

[0143] As used herein, "pharmaceutical composition" is meant to encompass compositions suitable for administration to a subject or patient, such as a mammal, particularly a human. Generally, a "pharmaceutical composition" is sterile and usually free of contaminants that may elicit an undesirable response in the subject (e.g., the compounds in the pharmaceutical composition are of pharmaceutical grade). Pharmaceutical compositions can be designed for administration to a subject or patient in need thereof by several different routes of administration, such as oral, buccal, rectal, parenteral, intraperitoneal, intradermal, intratracheal, etc.

[0144] As used herein, "pharmaceutically acceptable carrier" is meant to encompass excipients, diluents, carriers, and adjuvants that are generally safe, non-toxic, and not biologically or otherwise undesirable and are useful in preparing pharmaceutical compositions, including excipients, diluents, carriers, and adjuvants that are acceptable for veterinary use as well as for human pharmaceutical use. As used herein, "pharmaceutically acceptable" includes both one and more than one such excipient, diluent, carrier, and adjuvant.

[0145] In particular, the present invention relates to a pharmaceutical composition comprising, as an active ingredient, an antibody or an antigen-binding fragment thereof as defined above, and a pharmaceutically acceptable carrier.

[0146] In another aspect, the present invention relates to a therapeutic means, in particular a combination product means, comprising as active ingredients an anti-SIRPα antibody or antigen-binding fragment or antigen-binding antibody mimetic thereof as defined above and a second therapeutic agent, wherein said active ingredients are formulated for individual, sequential or combination therapy, in particular for combined or sequential use.

[0147] In particular, the present invention relates to a combination product comprising an anti-CMKLR1 agent as defined above and a second therapeutic agent for simultaneous, separate or sequential pharmaceutical use.

[0148] In one embodiment, the invention relates to a combination product as defined above, wherein the second therapeutic agent is selected from the group consisting of a chemotherapeutic agent, a radiotherapeutic agent, a cellular therapeutic agent, an immunotherapeutic agent, an antibiotic, and a probiotic.

[0149] In one embodiment, the present invention relates to a combination product as defined above, wherein said immunotherapeutic agent is selected from the group consisting of therapeutic vaccines, in particular immune checkpoint blockers or activators of adaptive immune cells (T and B lymphocytes), and antibody-drug conjugates.

[0150] In one embodiment, the present invention relates to a combination product as defined above, wherein the immune checkpoint blocker or activator of adaptive immune cells (T and B lymphocytes) is selected from the group consisting of anti-PDL1, anti-PD1, anti-SIRPA, anti-CTLA4, anti-CD137, anti-CD2, anti-CD28, anti-CD40, anti-HVEM, anti-BTLA, anti-CD160, anti-TIGIT, anti-TIM-1 / 3, anti-LAG-3, anti-2B4, and anti-OX40, anti-CD40 agonists, CD40-L, TLR agonists, anti-ICOS, ICOS-L, and B cell receptor agonists, in particular, selected from the group consisting of anti-PDL1, anti-PD1, and anti-CD137. In a particular embodiment of the present invention, the second therapeutic agent is an anti-PDL1 or anti-PD1 compound, in particular an anti-PD1 compound, more particularly an anti-PD1 antibody. In a particular embodiment of the present invention, the second therapeutic agent is an anti-SIRPa compound, in particular an anti-SIRPa antibody.

[0151] In one embodiment, the immunotherapeutic agent is also an antibody that specifically targets a tumor antigen selected from the group consisting of anti-Her2, anti-EGFR, anti-CD20, anti-CD19, anti-CD52.

[0152] In one aspect, the present invention relates to a combination product as defined above for simultaneous, separate or sequential use in the treatment of any condition susceptible to amelioration or prevention by altering macrophage polarization towards inflammation-resolving macrophages.

[0153] In one embodiment, the present invention relates to a method for the treatment of any condition susceptible to amelioration or prevention by altering macrophage polarization towards inflammation-resolving macrophages in a subject in need thereof, comprising the step of simultaneously, separately or sequentially administering to said subject an effective amount of a combination product as defined above.

[0154] In one embodiment, the present invention relates to the use of a combination product as defined above in the manufacture of a medicament for the treatment of any condition susceptible to the induction of inflammation-resolving pro-inflammatory macrophages.

[0155] In one aspect, the invention relates to a combination product as defined above for simultaneous, separate or sequential use in the treatment of a pathology selected from the group consisting of inflammatory diseases, including but not limited to acute and chronic inflammatory diseases, inflammatory bowel disease, Crohn's disease, NASH, scleroderma, asthma, keratoconjunctivitis, periodontal disease, eczema, colitis, in particular ulcerative colitis or spontaneous colitis, cystic fibrosis, diabetes, in particular type I diabetes, peritonitis, psoriasis, carcinoma, in particular breast or colon cancer, cancer, metastatic cancer, lung cancer, degenerative diseases, infectious diseases, in particular sepsis, autoimmune diseases, or for use in vaccination.

[0156] In one embodiment, the present invention relates to a method for the treatment of a pathology selected from the group consisting of inflammatory diseases, including but not limited to acute and chronic inflammatory diseases, inflammatory bowel disease, Crohn's disease, NASH, scleroderma, asthma, keratoconjunctivitis, periodontal disease, eczema, colitis, in particular ulcerative colitis or spontaneous colitis, cystic fibrosis, diabetes, in particular type I diabetes, peritonitis, psoriasis, carcinoma, in particular breast or colon cancer, cancer, metastatic cancer, lung cancer, degenerative diseases, infectious diseases, in particular sepsis, autoimmune diseases, in a subject in need thereof, comprising the simultaneous, separate or sequential administration to said subject of an effective amount of a combination product as defined above.

[0157] The present invention also relates to polynucleotides encoding the anti-CMKLR1 compounds defined herein. To this end, the present invention also relates to a nucleic acid molecule or a group of nucleic acid molecules, more particularly isolated and / or recombinant nucleic acid molecules, encoding any of the anti-CMKLR1 compounds according to the present disclosure, more particularly encoding a heavy chain variable domain comprising or consisting of the amino acid residues of the sequences set forth in SEQ ID NOs: 38, 39, 40, and 62, and a light chain variable domain comprising or consisting of the amino acid residues of the sequences set forth in SEQ ID NOs: 49, 50, 51, 52, 53, 54, 55, 56, 57, and 58. In a specific embodiment, the present invention relates to a nucleic acid molecule or a group of nucleic acid molecules, more particularly isolated and / or recombinant nucleic acid molecules, encoding the amino acid sequences of the heavy chain variable domain of an antibody consisting of SEQ ID NO: 91 and the light chain variable domain of an antibody consisting of SEQ ID NO: 93.

[0158] The nucleic acid molecule may further comprise regulatory sequences such as, but not limited to, enhancers, silencers, promoters, particularly expression promoters, signal peptides, for the transcription and expression of the encoded heavy chain variable domain and / or light chain variable domain.

[0159] The present invention also relates to vectors comprising the polynucleotides disclosed herein or comprising the nucleic acid molecules disclosed herein. As used herein, a vector is a nucleic acid molecule used as a vehicle for introducing genetic material into cells, and in preferred embodiments, allows for the expression of a polynucleotide inserted into the vector. The term vector encompasses plasmids, viruses, cosmids, and artificial chromosomes. A vector generally contains an origin of replication, a multiple cloning site, and a selectable marker. The vector itself is generally a nucleotide sequence, typically a DNA sequence, that contains an insert (transgene) and a larger sequence that serves as the "backbone" of the vector. In addition to the transgene insert and backbone, modern vectors may also contain additional features: promoters, genetic markers, antibiotic resistance, reporter genes, targeting sequences, and protein purification tags. Specifically, vectors called expression vectors (expression constructs) are designed for the expression of a transgene in target cells and generally contain regulatory sequences.

[0160] In another aspect, the present invention relates to a cell, an isolated cell, a host cell, an isolated host cell, or a cell line comprising the vector defined above. As used herein, these terms with respect to cells are intended to include any individual cell or cell culture that may be or has been a recipient of vectors encoding the antibody constructs of the present invention, exogenous nucleic acid molecules, and polynucleotides, and / or the antibody construct itself. Introduction of the respective materials into a cell can be performed by transformation, transfection, etc. These terms are also intended to include the progeny or potential progeny of a single cell. Suitable host cells include prokaryotic or eukaryotic cells, including, but not limited to, bacteria, yeast cells, fungal cells, plant cells, and animal cells such as insect cells and mammalian cells, e.g., mouse, rat, rabbit, macaque, or human cells.

[0161] In particular embodiments of the invention, the cell or cell line is selected from the group consisting of CHO, COS and HEK cells, which when genetically engineered with a vector, exogenous nucleic acid molecule, and vector comprising a polynucleotide encoding an antibody construct of the invention, produces at least 0.1 mg / ml, particularly at least 1 mg / ml of antibody, particularly at least 10 mg / ml, more particularly at least 100 mg / ml; and / or is a recipient of the antibody construct itself.

[0162] The following figures and examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the experiments below are all or the only experiments performed. While the invention has been described with reference to specific embodiments thereof, those skilled in the art will recognize that various modifications can be made and equivalents can be substituted without departing from the true spirit and scope of the invention. In addition, many modifications can be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

[0163] In certain embodiments, the present invention relates to the use of an antibody or antigen-binding fragment thereof as defined in any one of the embodiments disclosed herein for the manufacture of a medicament. In certain embodiments, the present invention relates to the use of an antibody or antigen-binding fragment thereof as defined in any one of the embodiments disclosed herein for the manufacture of a medicament useful for treating a condition involving inflammation. In certain embodiments, the present invention relates to the use of an antibody or antigen-binding fragment thereof as defined in any one of the embodiments disclosed herein for the manufacture of a medicament useful for treating a condition involving inflammation. - inflammatory diseases, in particular acute inflammatory diseases, chronic inflammatory lung diseases (e.g. asthma), keratoconjunctivitis, periodontal disease, eczema, inflammatory bowel diseases, in particular Crohn's disease or colitis, in particular ulcerative colitis or spontaneous colitis, chronic inflammatory diseases such as cystic fibrosis, NASH (non-alcoholic steatohepatitis), scleroderma, antineutrophil cytoplasmic antibody-associated diseases (ANCA-associated diseases), in particular in which the administration of the treatment results in enhanced resolution of inflammation; - autoimmune diseases such as diabetes, especially type 1 diabetes, psoriasis, lupus, rheumatoid arthritis, multiple sclerosis, Sjögren's syndrome, celiac disease, vasculitis, myasthenia gravis, or infections such as sepsis, peritonitis, degenerative diseases, wound healing disorders, severe viral conditions accompanied by severe inflammatory conditions such as coronaviruses (e.g., COVID-19), or dry eye syndrome, in particular in which administration of the treatment results in enhanced resolution of inflammation; - cancers, in particular metastatic cancers, solid or liquid cancers, such as carcinomas, more particularly liver cancers, in particular breast cancer or colon cancer, colorectal cancer or lung cancer or myeloid cancers such as mesothelioma or leukemia, in particular cancers in which the administration of the treatment results in enhanced resolution of inflammation, in particular cancers in which the cancer cells express CMKLR1 or the tumor microenvironment is infiltrated by cells that express or overexpress CMKLR1; - NASH (non-alcoholic steatohepatitis), scleroderma, cystic fibrosis or antineutrophil cytoplasmic antibody-associated disease (ANCA) The present invention relates to the use of an antibody or antigen-binding fragment thereof as defined in any one of the embodiments disclosed herein for the manufacture of a medicament useful for the prophylactic or therapeutic treatment of [Brief explanation of the drawings]

[0164] [Figure 1-1]Effect of anti-CMKLR1 variant 1G1, 2G1, 3G1, and 4G1 chimeric antibodies on DC maturation and differentiation. Mouse dendritic cells were incubated with vehicle or RvE1 or anti-CMKLR1 antibody variants (mutations at positions 1 and 2 of SEQ ID NO: 8, corresponding to VH-CDR3: 1G1(LL), 2G1(LI), 3G1(IL), or 4G1(II) or isotype control (hIgG1 or mIgG)) during the maturation phase (24 or 48 hours), followed by incubation during the differentiation phase. The cells were then stained with cell marker antibodies: A. CD80-PE; B. CD86-FITC; C. CD103-PerCPCy5.5; DI / Ab-APC for FACS analysis. The average fluorescence was determined for each condition. E and F represent cell viability measured by FACS using the LIVE / DEAD® kit from Life Technologies in each condition after 24 or 48 hours of maturation, respectively. [Figure 1-2] Effect of anti-CMKLR1 variant 1G1, 2G1, 3G1, and 4G1 chimeric antibodies on DC maturation and differentiation. Mouse dendritic cells were incubated with vehicle or RvE1 or anti-CMKLR1 antibody variants (mutations at positions 1 and 2 of SEQ ID NO: 8, corresponding to VH-CDR3: 1G1(LL), 2G1(LI), 3G1(IL), or 4G1(II) or isotype control (hIgG1 or mIgG)) during the maturation phase (24 or 48 hours), followed by incubation during the differentiation phase. The cells were then stained with cell marker antibodies: A. CD80-PE; B. CD86-FITC; C. CD103-PerCPCy5.5; DI / Ab-APC for FACS analysis. The average fluorescence was determined for each condition. E and F represent cell viability measured by FACS using the LIVE / DEAD® kit from Life Technologies in each condition after 24 or 48 hours of maturation, respectively. [Figure 2-1]Effect of anti-CMKLR1 antibody on a mouse model of acute inflammatory colitis induced by DSS. Six days after DSS induction, mice were injected with isotype control hIgG1 (10 μg per mouse) (x), daily RvE1 (1 μg per mouse) (●), or three injections of 2G1 antibody (10 μg per mouse) (□) for 5 days. Untreated, wild-type mice are represented by ▼. A. Animal weight loss. B. Animal fecal score. C. Colon length. D. Resolution index. [Figure 2-2] Effect of anti-CMKLR1 antibody on a mouse model of acute inflammatory colitis induced by DSS. Six days after DSS induction, mice were injected with isotype control hIgG1 (10 μg per mouse) (x), daily RvE1 (1 μg per mouse) (●), or three injections of 2G1 antibody (10 μg per mouse) (□) for 5 days. Untreated, wild-type mice are represented by ▼. A. Animal weight loss. B. Animal fecal score. C. Colon length. D. Resolution index. [Figure 3] Effect of anti-CMKLR1 antibodies on a murine model of acute inflammatory colitis induced by TNBS. Mice received 200 μL of 5% haptenizing agent TNBS in 50% ethanol on day 0, followed by 5 days of injections of isotype control hIgG1 (10 μg per mouse) (x), daily RvE1 (1 μg per mouse) (●), or three injections of 2G1 antibody (10 μg per mouse) (□), or no treatment (wild-type animals) (▲). Mice were sacrificed, and colon length was measured for each condition. [Figure 4] Effect of anti-CMKLR1 antibody on IL10 KO mouse chronic inflammatory colitis model. Mice KO for IL10 develop spontaneous inflammatory colitis. They were treated intraperitoneally with anti-CMKLR1 antibody (2G1) (□) or isotype control (hIgG1) (x) (25 μg / injection, 3 times a week). A. Weight loss of animals during and after treatment. B. Fecal score of animals. [Figure 5]Effect of anti-CMKLR1 antibody on a murine type 1 non-obese diabetes model. Mice developed spontaneous diabetes. When blood glucose was 180-234 mg / dL, they were treated with anti-CMKLR1 antibody (□ in A and B, ■ in C) or isotype control antibody (x in A and B, □ in C) intraperitoneally at 20 μg / injection, three times a week for two weeks. A. Percentage of survival. B. Individual blood glucose levels in mg / dL. C. Pooled blood glucose levels in mg / dL. [Figure 6] Effect of anti-CMKLR1 agonists on autoimmune diseases, including mouse psoriasis models. Mice treated with Aldara for 4-6 consecutive days were injected intraperitoneally with 2G1 (●) or isotype control antibody (x). A. Thickness and B. Body weight. [Figure 7-1] Effect of anti-CMKLR1 antibody on a mouse peritonitis model. Prophylactic injection of anti-CMKLR1 was performed before Zymosan A injection (1 mg per mouse in 1 mL): RvE1 (1 μg per mouse) (●), 2G1 antibody (10 μg per mouse) (□), or isotype control antibody (x). A. PMNs counted during the first 50 hours after Zymosan A injection. B. Macrophages counted during the first 50 hours after Zymosan A injection. C. Resolution index. [Figure 7-2] Effect of anti-CMKLR1 antibody on a mouse peritonitis model. Prophylactic injection of anti-CMKLR1 was performed before Zymosan A injection (1 mg per mouse in 1 mL): RvE1 (1 μg per mouse) (●), 2G1 antibody (10 μg per mouse) (□), or isotype control antibody (x). A. PMNs counted during the first 50 hours after Zymosan A injection. B. Macrophages counted during the first 50 hours after Zymosan A injection. C. Resolution index. [Figure 8]Effect of anti-CMKLR1 antibody on the 4T1 breast tumor model. Mice were inoculated with 250,000 4T1 cells into the mammary gland. Anti-CMKLR1 antibody (2G1) (A.□, B.●) or anti-41BB antibody (3H3) (●), or both antibodies (▲), were then injected twice (10 μg / injection) on days 4 and 7, or a control antibody (IgG1 isotype antibody clone 3G8) (x in A and B) was injected three times a week for three weeks. A. Tumor area was measured for 8 days after tumor injection, and then every two days. B. Tumor lung metastasis was measured by bioluminescence imaging (BLI) in animals treated with the isotype control and anti-CMKLR1 antibody (n = 4). [Figure 9] Effect of anti-CMKLR1 antibodies on two different mouse models of colon cancer. Two mouse models were tested, with animals receiving either an isotype control antibody (3G8) (x) or anti-CMKLR1 (2G1) (□) at 20 μg / injection intraperitoneally for 3 weeks. A-C. Results in the CT26 colon cancer model comparing single treatments with p84 (A) and 2G1 (B) with combination therapy using both antibodies (C). D: Results in the MC38 colon cancer model. [Figure 10] Expression of CMKLR1 on biopsies from patients with ulcerative colitis (UC) or Crohn's disease before and after anti-TNFα treatment. x represents control; □ represents CMKLR1 expression in patients responding to corticosteroid treatment and / or immunosuppressive treatment before infliximab treatment; ■ represents CMKLR1 expression in patients not responding to corticosteroid treatment and / or immunosuppressive treatment before infliximab treatment; △ represents CMKLR1 expression in patients responding to corticosteroid treatment and / or immunosuppressive treatment after infliximab treatment; ▲ represents CMKLR1 expression in patients not responding to corticosteroid treatment and / or immunosuppressive treatment after infliximab treatment. A. Expression of CMKLR1 transcripts in patients with ulcerative colitis before and after infliximab treatment. B. Expression of CMKLR1 transcripts in patients with Crohn's disease before and after infliximab treatment in biopsies of inflamed colon. [Figure 11]Expression of CMKLR1 on UC patient biopsies before and after anti-α4β7 (VDZ) treatment. A. Expression of CMKLR1 (CMKLTR1) transcripts in inflamed colon biopsies in ulcerative colitis patients before and after vedolizumab treatment. R corresponds to patients who respond to VDZ treatment, and NR corresponds to patients who do not respond to VDZ treatment. [Figure 12] Binding analysis of anti-CMKLR1 antibodies to CMKLR1 peptides by ELISA. Binding of 2G1 antibody (□) to the CMKLR1 EL3 loop (SEQ ID NO: 18) was compared with that of an isotype control antibody (3G8) (x) by measuring the OD at 450 nm by ELISA. [Figure 13] Testing CMKLR1 expression on different cell lines by FACS and Western blot. A. Cell surface protein expression of CMKLR1 was determined on two different human T cell lines, Thp1 and U937, using different concentrations (ng / ml) of 2G1 antibody. B. CMKLR1 protein expression was tested by Western blot on T cell lines (Thp1 and U937), the fibroblast cell line MRC5, the NK cell line NKL, and CMKLR1-negative, transduced CHO cells. [Figure 14] Expression of CMKLR1 on mouse myeloid cells by FACS. After differentiation, mouse myeloid lineage cells were analyzed for their CMKLR1 expression on the cell surface. A. Expression on macrophage monocytes (M0). B and C. Expression on macrophages (inflammatory mM1 and inflammation-resolving mM2 macrophages). D and E. Expression on dendritic cells (mDC and iDC). [Figure 15] Expression of CMKLR1 on human monocytes and mouse bone marrow cells stimulated by inflammatory stimuli. 16 or 48 hours after cell stimulation with LPS, TNFα, or IL6, CMKLR1 (ChemR23) expression was measured by FACS. A. Results on human blood monocytes. B. and C. Results on mouse bone marrow-derived myeloid cells and neutrophils. [Figure 16]Examination of myeloid cell activation markers after CMKLR1 pathway activation. Dendritic cells were incubated in vehicle, RvE1, 2G1, or isotype control (hIgG1) and then stained with marker antibodies for FACS analysis: A. CD80-PE. B. CD86-FITC. C. CD103-PerCPCy5.5. D. CD40-PeCy7. E. I / Ab-APC. The mean fluorescence was determined for each condition. [Figure 17] Testing CMKLR1 pathway activation: Akt and Erk phosphorylation. Western blot analysis of ERK and AKT pathway activation after 5, 10, and 30 minutes of incubation of murine inflammatory (M1) macrophages with 2G1 or RvE1. Phosphorylated proteins Akt or Erk were assessed using P-Akt antibody or P-Erk antibody (p44 / 42). A. Activation of Erk and Akt with RvE1. B. Activation of Erk and Akt with 2G1. [Figure 18] Competition study of chemerin-CMLKR1 interaction with anti-CMKLR1 antibodies. A. Inhibition of cAMP by chemerin from two different suppliers, DiscoverX (●) or R&D Systems (▲), or by chemerin combined with anti-CMKLR1 antibody (2G1) alone (◇), or by chemerin combined with chemerin from DiscoverX (□) was tested. B. Activation of beta-arrestin in the presence of various concentrations of anti-CMKLR1 antibodies ranging from 1 μM to 1 nM and 2 nM (◇) or 6 nM (●) chemerin. [Figure 19-1] Effect of anti-CMKLR1 antibody in a mouse model of chronic colitis transferred with CD45Rbhigh T cells. A. Weight changes in treated animals were followed for up to 60 days. Animals were treated with isotype control hIgG1(x) or anti-CMKLR1 antibody (■). B. Histological staining of colon tissue in mice treated with anti-CMKLR1 (right) compared to control (left). C. Anatomical pathology scores (inflammation score, vasculitis score, colon thickness, and fibrous colon wall) used to calculate the severity of pathology and inflammation. D. CD3 and Ly6G infiltration. [Figure 19-2]Effect of anti-CMKLR1 antibody in a mouse model of chronic colitis transferred with CD45Rbhigh T cells. A. Weight changes in treated animals were followed for up to 60 days. Animals were treated with isotype control hIgG1(x) or anti-CMKLR1 antibody (■). B. Histological staining of colon tissue in mice treated with anti-CMKLR1 (right) compared to control (left). C. Anatomical pathology scores (inflammation score, vasculitis score, colon thickness, and fibrous colon wall) used to calculate the severity of pathology and inflammation. D. CD3 and Ly6G infiltration. [Figure 19-3] Effect of anti-CMKLR1 antibody in a mouse model of chronic colitis transferred with CD45Rbhigh T cells. A. Weight changes in treated animals were followed for up to 60 days. Animals were treated with isotype control hIgG1(x) or anti-CMKLR1 antibody (■). B. Histological staining of colon tissue in mice treated with anti-CMKLR1 (right) compared to control (left). C. Anatomical pathology scores (inflammation score, vasculitis score, colon thickness, and fibrous colon wall) used to calculate the severity of pathology and inflammation. D. CD3 and Ly6G infiltration. [Figure 20] Effect of anti-CMKLR1 antibody on a mouse model of liver cancer (HCC model). Antitumor effect of ip administration of anti-CMKLR1 antibody (2G1, 0.8 mg / kg) three times a week for two weeks, combined with (▲) or not combined with (●) two injections of anti-PD1 mAb (RMP1-14 clone, 8 mg / kg) on ​​days 4 and 8, or with injections of anti-PD-1 antibody alone (twice a week) (△). Mice were treated for two weeks in an orthotopic model of mouse liver cancer (2.5 × 10 Hepa 1.6 cells were injected via the portal vein on day 0). An isotype control antibody was used at 0.8 mg / kg three times a week for two weeks. Mice were considered to have a partial response (PR) if they survived for several days to one month after treatment was stopped, or a complete response (CR) if they survived for more than one month, or cured if they survived three times longer than the time required for all control mice to die. [Figure 21]Anti-CMKLR1 antibody production in different cell lines. The CDRs of the 2G1 heavy chain were grafted into three human germline frameworks, designated IGHV3-23*04, IGHV1-46*01, and IGHV7-4-1 (IMGT nomenclature). The CDRs of the 2G1 light chain were grafted into three human germline frameworks, designated IGKV1-13*02, IGKV6-21*01, and IGKV3-11*01 (IMGT nomenclature). Each sequence was fused to a human immunoglobulin constant fragment and cotransfected into mammalian cells to produce humanized antibodies in COS and CHO cells. Production was assessed in the supernatant. VH WT and VL WT correspond to the heavy and light chains of the 2G1 antibody, respectively. [Figure 22] Production of humanized antibodies derived from 2G1. Production and yield of different combinations of heavy and light chain variable domains in CHO cells. VH WT and VL WT correspond to the variable domains of the 2G1 antibody. Germline sequences are derived from humanized versions of the variable domains of 2G1 and are disclosed in the Examples herein. [Figure 23] Binding recognition of C7 peptide by humanized anti-CMKLR1 derived from 2G1. C7biot binding in the supernatant of CHO cells transfected with various combinations of heavy and light chains (VHWT+VLWT: combination of SEQ ID NO: 37 and SEQ ID NO: 49; HCLC: combination of SEQ ID NO: 42 and SEQ ID NO: 52; HDLC: combination of SEQ ID NO: 43 and SEQ ID NO: 52; HCLD: combination of SEQ ID NO: 42 and SEQ ID NO: 53; HDLD: combination of SEQ ID NO: 43 and SEQ ID NO: 53). [Figure 24] Anti-CMKLR1 ED50 derived from 2G1 antibody. 2G1wt: HALA: combination of SEQ ID NO: 41 and SEQ ID NO: 50; combination of SEQ ID NO: 37 and SEQ ID NO: 49; HCLC: combination of SEQ ID NO: 42 and SEQ ID NO: 52; HDLC: combination of SEQ ID NO: 43 and SEQ ID NO: 52; HCLD: combination of SEQ ID NO: 42 and SEQ ID NO: 53; HDLD: combination of SEQ ID NO: 43 and SEQ ID NO: 53. [Figure 25]In vitro inhibition of CCR7 expression on M1 macrophage cells using coated anti-ChemR23 antibodies. 2G1 and all humanized 2G1 variants (HALA, HCLC, HCLD, HDLC, and HDLD) were immobilized on plates. Isotype controls were added as controls. Two isotypes that prevent FcRγ binding, namely, 2G1-N297A (2G1 wt mutated at N297A to reduce FcγR binding) and 2G4 (wt with isotype IgG4 mutated at S228P to stabilize the hinge region), were also added. Inflamed M1 macrophages were added to the coated plates for 48 hours, and CCR7 expression on the surface of the macrophages was measured by flow cytometry. [Figure 26-1] Neutrophil apoptosis: A) Neutrophil survival / death, B) Caspase-3 expression, C) ROS production. Neutrophils were isolated from the blood of healthy volunteers and cultured on coated Iso ctrl (x), chimeric anti-ChemR23 2G1 (square), or different humanized versions of 2G1 (diamond) for 24 hours (death assay), or for 4, 6, 11, or 5 hours (caspase-3 assay). PMN death was analyzed by incubating them with specific markers for death and survival, followed by counting by photographic analysis. Caspase-3 was visualized by Western blot, and signal intensity was determined using software. ROS production was visualized using specific markers and analyzed photographically. [Figure 26-2]Neutrophil apoptosis: A) Neutrophil survival / death, B) Caspase-3 expression, C) ROS production. Neutrophils were isolated from the blood of healthy volunteers and cultured on coated Iso ctrl (x), chimeric anti-ChemR23 2G1 (square), or different humanized versions of 2G1 (diamond) for 24 hours (death assay), or for 4, 6, 11, or 5 hours (caspase-3 assay). PMN death was analyzed by incubating them with specific markers for death and survival, followed by counting by photographic analysis. Caspase-3 was visualized by Western blot, and signal intensity was determined using software. ROS production was visualized using specific markers and analyzed photographically. [Figure 27] Neutrophil apoptosis in vitro: A) Experimental procedure, B) ChemR23 expression, C) Neutrophil frequency in exudates, D) Macrophage frequency in exudates, E) Neutrophil death, F) Dead / live neutrophil ratio. Sterile air was injected twice on days 3 and 6, and inflammation was initiated by injection of carrageenan. Exudates were collected at various time points and stained for flow cytometry analysis. [Figure 28] Neutrophil migration ratio: A) PMN migration ratio in healthy patients, B) PMN migration ratio in inflammatory patients with ANCA. Endothelial cells were coated and activated with 100 U / mL TNFα overnight. PMN were then added with or without 100 U / mL TNFα and Ab for 2 hours. Migrated PMN were collected and analyzed by flow cytometry. [Figure 29] CD62L expression. PMNs from healthy volunteers were incubated with 10 μg / mL of coated Ab in culture medium for various times, collected for CD62L staining, and analyzed by flow cytometry (left panel). Soluble CD62L released by shedding is detected by ELISA in the supernatant of PMNs incubated with coated Ab (right panel). [Figure 30]Mesothelioma Model Survival. AK-7 cells (3M) were injected into the thoracic cavity and mice received Iso ctrl or 2G1 at 1 mg / kg, 3 times a week for 3 weeks, starting on day 4. [Figure 31] CRC Model. A) Tumor cell inoculation model of CRC: tumor progression and survival; B) Chemo-induced and inflammation-induced CRC. Tumor inoculation was performed subcutaneously using 0.5M MC38 CRC cell line. Mice were injected with 1 mg / kg 2G1 or Ctrl Ab three times a week for three weeks, starting on day 4 after tumor induction. Cyclophosphamide was injected IP once at 100 mg / kg. Tumor progression was assessed three times a week, and survival curves were established when mice developed tumors exceeding 1000 mm3. In the AOM-DSS model, mice received an IP injection of 7.5 mg / kg azoxymethane and, five days later, began three cycles of 5-day DSS and 14-day water. Treatment or placebo was administered after the first cycle and continued twice a week until completion. Mice were weighed and feces were analyzed twice a week. Colons and tumors were measured and counted for 80 days after chemical injection. [Figure 32] Experimental autoimmune encephalomyelitis model. A) Weight change over time, B) Disease score. Lesions in the central nervous system were induced by injection of immunogenic MOG peptide combined with adjuvant. If, by the end of the experiment, the animals had a clinical score equal to 2, meaning that the central nervous system was already affected by T cell activity, treatment (2G1) or isotype control was administered at 1 mg / kg. [Figure 33]Binding of purified antibodies to human ChemR23 peptides by ELISA assay (A) and ED50 concentration (ng / ml) (B). For the activity ELISA assay, donkey anti-human IgG, Fc-specific (Jackson Immunoresearch, USA; ref. no. 709-005-098) was immobilized on plastic at 1.3 μg / ml in borate buffer (pH 9), and purified antibodies were added to measure binding in 1% BSA buffer relative to wild-type 2G1. After incubation and washing, biotinylated antigen-specific peptide (Biot-C7 peptide) was added, followed by peroxidase-streptavidin (Jackson Immunoresearch, USA; ref. no. 016-030-084), and revealed by conventional methods. [Figure 34] A. Binding of purified antibodies incubated at 4°C or 37°C for 7 days on a human ChemR23 ELISA assay. B. SEC profile by gel filtration chromatography of purified antibodies incubated at 37°C for 7 days. Each purified humanized anti-ChemR23 antibody (HALA, HDLD, HD-LDT52S, HEF-LDT52S, HEF-LEF) was incubated at 4°C or 37°C for 7 days. After 7 days, binding of the purified antibodies was analyzed by ELISA assay, and aggregate formation was analyzed by gel filtration (Superdex 200 10 / 300GL, GeHealthcare). [Figure 35] In vitro inhibition of CCR7 expression on M1 macrophage cells using coated anti-ChemR23 antibody. Humanized 2G1 variant HEF-LD-T52S was immobilized on the plate. An isotype control was added as a control. Inflammatory M1 macrophages were added to the coated plate, and CCR7 expression on the surface of the macrophages was measured by flow cytometry. [Figure 36]In vitro expansion of killed PMN neutrophils using coated anti-ChemR23 antibodies. PMNs from healthy volunteers were incubated with 10 μg / mL of coated HEF-LDT52S, HEF-LEF, and HDLD antibody variants in culture medium for 24 hours and stained with one of the dead / live kits (LIVE / DEAD, Invitrogen). The percentage of positive cells was obtained by analyzing the photographs using Fiji software. An isotype control was added as a control. A mutant version of the HEF-LDT52S antibody (HEF-LDT52S N297A), which does not bind to Fc receptors (FcR), was also added. [Example]

[0165] Production and selection of anti-CMKLR1 antibodies Several antibodies with different CDR sequences within the heavy and light chain variable domains were synthesized. The different antibodies were tested for their ability to induce maturation and differentiation of dendritic cells toward pro-inflammatory or anti-inflammatory pathways. We selected antibody 2G1 (SEQ ID NO: 37 and SEQ ID NO: 49) to evaluate its properties in resolving inflammatory conditions, at least in the resolution phase, and three germline generations to evaluate antibody production in vitro.

[0166] As shown in Figure 1, the detection level of CD103- and IAb-expressing DCs treated with 2G1 was lower than that of DC cells treated with C7 antibody, indicating that 2G1 and 1G1 (another synthetic antibody) antibodies were able to inhibit DC activation and / or maturation in a more potent manner than other synthetic antibody (3G1 and 4G1) cells toward the inflammatory pathway (the method used in this assay is described in Example 10.2). As shown in Figure 1E and Figure 1F, cell viability is enhanced after treatment with 1G1 or 2G1 antibody compared to cells treated with other antibodies, including C7 or resolvin E1.

[0167] 2G1 was humanized using in silico CDR grafting, a method of humanization. The resulting humanized sequences derived from the CDR and FR regions and from the variable heavy and light chains are listed in the table below.

[0168] [Table 1]

[0169] [Table 2]

[0170] Examples of the therapeutic efficacy of anti-CMKLR1 antibody treatment on preclinical models of autoimmune and inflammatory diseases Example 1 Induction of colitis by DSS Colitis was induced in 8-10 week-old C57Bl / 6 male mice by adding 2% (wt / vol) DSS to ad libitum sterile drinking water for 6 days. Treatments were administered intraperitoneally: isotype control hIgG1 (10 μg per mouse), daily RvE1 (1 μg per mouse), or 2G1 antibody (10 μg per mouse) administered three times over 5 days. Colitis was monitored daily, consisting of body weight and fecal score (0: normal feces; 4: bloody feces). At the time of euthanasia, colon length, representing the severity of pathology, was measured. Resolution index was determined under various conditions as described by Bannenberg et al. (2005).

[0171] Results: The DSS animal model presented in Figure 2 is an acute inflammation model. Figure 1 shows a better overall condition of animals treated with anti-CMKLR1 antibody than that of animals receiving control antibody or resolvin RvE1. Mice treated with anti-CMKLR1 lost significantly less weight (Figure 2A), and fecal scores (Figure 2B) were significantly better. With regard to colon length and resolution index (Figures 2C and 2D), animals receiving anti-CMKLR1 or RvE1 exhibited similar results.

[0172] Example 2 Induction of colitis by TNBS Colitis was induced in 8-10 week-old C57Bl / 6 male mice by intrarectal injection of 200 μL of 5% haptenizing agent TNBS in 50% ethanol on day 0. Treatments were administered intraperitoneally: RvE1 (1 μg per mouse) daily for 3 days, or 2G1 antibody (10 μg per mouse) twice for 3 days. Colitis was monitored daily, consisting of body weight and fecal score (0: normal feces; 4: bloody feces) parameters (data not shown). At the time of euthanasia, colon length, which represents the severity of pathology, was measured.

[0173] Results: TNBS-induced colitis is another model of acute inflammation. Figure 3 shows that animals treated with anti-CMKLR1 or RvE1 had the same colon length as normal animals (wt). However, animals treated with the isotype control exhibited a shorter colon length. These results confirmed the therapeutic potential of anti-CMKLR1 antibodies that act like RvE1 in this mouse model of acute inflammation.

[0174] Example 3 IL10-KO model - spontaneous colitis model IL-10KO mice develop spontaneous colitis from 20 weeks of age, largely due to the absence of regulatory T cell function through IL-10 secretion in the intestine. IL-10KO mice were monitored for weight loss and fecal consistency, clinical hallmarks of this pathology, three times a week from 18 weeks of age. If weight loss was 5% higher and fecal scores were high or equal to 1 over a two-week period, anti-CMKLR1 antibody (2G1) or isotype control (hIgG1) was injected intraperitoneally (25 μg / injection, three times a week).

[0175] Results: A chronic inflammation model was used to test the efficacy of anti-CMKLR1 antibody treatment. Figure 4 shows an analysis of the percentage of weight loss (Figure 4A) and fecal scores (Figure 4B) when animals were treated with an isotype control or an anti-CMKLR1 antibody. The results show that animals treated with anti-CMKLR1 antibody lost less weight and had better fecal scores than animals receiving the isotype control. Anti-CMKLR1 antibodies are therefore believed to exhibit therapeutic potential for chronic inflammatory diseases.

[0176] Example 4 Preclinical model of type 1 diabetes: the mouse NOD model Eight-week-old NOD female mice were obtained from Charles River Laboratory. These mice develop spontaneous type 1 diabetes between 12 and 20 weeks of age. The onset of diabetes can be measured by hyperglycemia. Anti-CMKLR1 and isotype control were administered intraperitoneally at 20 μg / injection three times a week for 2 weeks when blood glucose levels were between 180 and 234 mg / dL. Mice were euthanized when blood glucose levels exceeded 600 mg / dL, corresponding to irreversible diabetes.

[0177] Results: This type 1 diabetes model is also considered a mouse autoimmune disease model. The results presented in Figures 5A-5C show that animals treated with anti-CMKLR1 antibody exhibited a better survival rate and near-normoglycemia, as indicated by blood glucose concentration measurements. This recovery was stable over time and is believed to indicate the possibility of full recovery in previously ill animals. The anti-CMKLR1 antibody restored glucose tolerance.

[0178] Example 5 Imiquimod-induced psoriasis-like skin inflammation Aldara® Cream, known to induce psoriasis in mice, was used on male C57B1 / 6 mice (8-10 weeks old). Mice received a daily topical dose of Aldara. Treatments (anti-CMKLR1 agonist or control compound) were injected intraperitoneally.

[0179] Results: The anti-CMKLR1 agonist (2G1) reduced skin thickness after Aldara administration (FIG. 6A) (see, e.g., day 15), but the animal's weight was not affected by agonist administration (FIG. 6B). These results suggest the application of anti-CMKLR1 agonist compound therapy to a mouse model of psoriasis, which exhibits autoimmune disease.

[0180] Example 6 Preclinical model of sepsis: Therapeutic efficacy of anti-CNKLR1 antibody in a murine peritonitis model General peritonitis was induced by intraperitoneal injection of Zymosan A® (1 mg per mouse in 1 mL). Prophylactic injections of anti-CMKLR1 antibodies were administered 5 minutes prior to Zymosan A injection: RvE1 (1 μg per mouse), 2G1 antibody (10 μg per mouse). Mouse peritoneal polymorphonuclear neutrophils (PMNs) and macrophages were collected 2, 4, 8, 16, 24, and 48 hours after Zymosan A injection and counted by flow cytometry analysis to determine the resolution index (Bannenberg et al., 2005).

[0181] Results: The results presented in Figure 7 regarding the number of PMNs (Figure 7A) and macrophages (Figure 7B) and the resolution index (Figure 7C) show that animals treated with RvE1 or anti-CMKLR1 antibody exhibited slightly fewer PMNs and macrophage cells, with identical results and a better resolution index compared to the isotype control. In sepsis, even small differences can be important for therapy. Therefore, these results are very positive for the applicability of anti-CMKLR1 antibodies in sepsis.

[0182] Example 7 Therapeutic efficacy of anti-CMKLR1 antibody treatment in preclinical cancer models: Example 7.1 Effect of anti-CMKLR1 antibody on primary tumor growth and its pulmonary metastasis development in an orthotopic breast cancer model Mice were anesthetized with 3% isoflurane. The abdominal hair of the mice was shaved, and 4T1 cells (250,000 cells) were injected into the mammary gland in 50 μL of PBS using an insulin syringe (30 gauge). Anti-CMKLR1 antibody (2G1) or anti-41BB antibody (3H3), or both antibodies, were injected twice on days 4 and 7 (10 μg / injection); control antibody was injected intraperitoneally in PBS three times a week for three weeks (100 μg / injection). In a second study to measure lung metastasis after breast cancer development, animals were treated with 0.8 mg / kg of anti-CMKLR1 antibody or control antibody (100 μg / injection) three times a week for three weeks.

[0183] Results: As shown in Figure 8A, animals treated with a single compound (2G1 or 3H3) did not show an improvement in tumor growth compared to animals receiving an isotype control antibody. However, animals treated with a combination of anti-CMKLR1 and anti-41BB antibodies showed a significant (p<0.01) reduction in tumor growth in the breast cancer model. This result is considered positive because this model of breast cancer is a highly aggressive model. As shown in Figure 8B, which shows the effect of anti-CMKLR1 compounds on lung metastasis by bioluminescence imaging, it can be seen that anti-CMKLR1 treatment reduced lung metastasis compared to animals treated with a control antibody. Analysis of lymph node metastasis showed that animals treated with anti-CMKLR1 compounds did not have metastases, while two control animals did (data not shown). These results indicate that anti-CMKLR1 antibodies with agonistic activity that mimics RvE1 have an anti-metastatic effect. In this model, the antibodies of the present invention did not exhibit significant efficacy against primary tumor development. However, the results show improvement when animals were treated with a combination of anti-CMKLR1 and anti-41BB antibodies.

[0184] Example 7.2 Therapeutic effects on tumor growth in a colon cancer model Eight-week-old C57bl / 6J male mice were anesthetized with 3% isoflurane. The flanks of the mice were shaved and transfected with MC38 cells (0.5 × 10 6The cell line (1 × 10 cells / mouse) was subcutaneously injected in 50 μL of PBS using an insulin syringe (30 gauge). Another model was also used in which 8-week-old Balb / c male mice were anesthetized with 3% isoflurane. The flanks of the mice were shaved and CT26 cells (1 × 10 6 Cells / mouse) were injected subcutaneously in 50 μL of PBS using an insulin syringe (30 gauge).

[0185] An agonistic anti-CMKLR1 antibody (2G1) or an anti-SIRPα antibody (p84-anti-mouse SIRPa from Merck Millipore) (SIRPα is a novel checkpoint inhibitor) was injected intraperitoneally once a week (20 μg / injection) for 3 weeks, alone or in combination, starting on day 4 after tumor inoculation.

[0186] Results: As shown in Figures 9A-9C, in the CT26 cancer model, anti-CMKLR1 antibody alone (Figure 9B) showed no clinical effect on tumor progression compared to the control group, nor did anti-SIRPa alone (Figure 9A). Surprisingly, however, the combination of both compounds enabled timely tumor growth inhibition (Figure 9C). In another mouse colon cancer model, shown in Figure 9D, anti-CMKLR1 demonstrated efficacy in inhibiting tumor growth compared to the isotype control. Collectively, these results for two different colon cancer models indicate that anti-CMKLR1 antibody agonists, alone or in combination with another therapeutic agent, can prevent tumor progression.

[0187] Example 8 Meta-analysis of CMKLR1 expression on biopsies from human patients with UC or CD treated with anti-TNFα or anti-α4β7 antibody therapy The signaling networks that perpetuate chronic gastrointestinal inflammation in two major forms of inflammatory bowel disease (IBD), Crohn's disease (CD) and ulcerative colitis (UC), remain unclear in humans. Through analysis of nearly 500 patients with IBD and 100 controls, we report here that CMKLR1 transcripts accumulate in the inflamed colonic tissue of severe IBD patients who have failed to respond to immunosuppressants / corticosteroids and immunotherapies such as anti-TNFα (infliximab) or anti-α4β7 integrin (vedolizumab) therapy.

[0188] We first analyzed mucosal CMKLR1 transcript expression by conducting a meta-analysis of publicly available transcriptional datasets from three cohorts of UC patients (GSE16879 (Arijs et al., 2009a), GSE12251 (Arijs et al., 2009b), and GSE73661) using colonic mucosal biopsies performed before (within 1 week of) anti-TNF treatment in patients refractory to corticosteroids and / or immunosuppression. In these three cohorts, anti-TNF response was defined as histological recovery analyzed 4–6 weeks after the first anti-TNF infusion (total: n = 18 non-IBD cohort, n = 41 UC non-responders, and n = 28 UC responders).

[0189] Results: Analysis showed that CMKLR1 transcript expression was significantly increased in colonic biopsies of primary UC non-responders before and after treatment with anti-TNF therapy compared with non-IBD controls or patients with UC who were pre-anti-TNF and would respond to anti-TNF therapy (Figure 10A). Mucosal CMKLR1 expression was also significantly increased in colonic or ileal biopsies of Crohn's disease patients (n = 24, non-IBD controls, n = 17, CD non-responders, and n = 20, CD responders; GSE16879 (Arijs et al., 2009a)) before and after anti-TNF therapy in patients who would not respond to anti-TNF therapy compared with non-IBD controls or future responders (Figure 10B). Finally, colonic mucosal gene expression analysis in a cohort of UC patients treated with anti-α4β7 (vedolizumab) therapy (GSE7366146) also confirmed that CMKLR1 expression was significantly increased in non-responders before and after treatment with vedolizumab (Figure 11). In summary, the meta-analysis shows that CMKLR1 is overexpressed in inflamed tissues of IBD patients, especially in patients who do not respond to current immunosuppressants or immunotherapies even before the start of treatment. Our meta-analysis provides evidence that CMKLR1 expression in the colon, or rather in the ileum for CD, from treatment-refractory UC or CD patients can, in contrast, be considered to be responsive to anti-CMKLR1 antibody agonist treatment, such as the antibody of the present invention.

[0190] Example 9 CMKLR1 expression and antibody binding assay - ELISA binding CMKLR1 (Figure 12) CMKLR1 peptide (273NH2-PYHTLNLLELHHTAMPGSVFSLGLPLATALAIA-COOH305) (SEQ ID NO: 60) (5 μg / ml) was coated overnight in borate buffer. Saturation was performed with PBS-Tween 0.1%-gelatin 0.25% for 2 hours at 37°C. 2G1 or hIgG1 antibody was then added at different concentrations for 2 hours at 37°C. Peroxidase-conjugated secondary antibody (0.8 μg / ml) was then added for 1 hour at 37°C and revealed with TMB substrate. The colorimetric reaction was read using a TECAN.

[0191] - CMKLR1 expression by FACS (Figure 13A) Cells were resuspended in PBS-FBS-EDTA and incubated with Fc block (1 / 50) on ice for 30 minutes. Staining on monocytes, macrophages, and dendritic cells was performed using A488-labeled 2G1 (5 μg) or A488-labeled hIgG1 (5 μg).

[0192] Western blot analysis of CMKLR1 (Figure 13B) After protein migration and transfer as previously described, 2G1 antibody (10 μg / membrane) was incubated overnight at 4 °C and revealed with a peroxidase-conjugated secondary antibody (1:2000). CMKLR1 expression was then detected using chemiluminescence and an image reader. Western blot images were quantified using MultiGauge software.

[0193] Results: The results shown in Figure 12 confirm that the anti-CMKLR1 antibody clone 2G1 can bind to the polypeptide that forms the loop EL3 of CMKLR1. Expression of CMKLR1 on various cell lines assessed by FACS and Western blot using the 2G1 antibody showed that the human tumor T cell lines Trp1 and U937 expressed CMKLR1, and CHO cells, the human lung fibroblast cell line MRC5, and the human NK cell line NKL expressed transduced CMKLR1 (Figure 13).

[0194] Example 10 Examination of CMKLR1 expression on myeloid lineages Example 10.1 Differentiation and polarization of human monocytes Monocytes were collected from PBMCs of healthy volunteers' buffy coats and isolated by magnetic separation or elutriation. They were then cultured with various cytokine cocktails to generate differentiated unpolarized or polarized macrophages. This protocol allowed for the generation of differentiated macrophages polarized to have either proinflammatory (M1) or proinflammatory-resolving (M2) properties in different wells. Monocytes were cultured at 0.5 × 10 in complete RPMI (RPMI containing 10% FBS, 1% glutamine, and 1% antibiotics). 6 Cells were seeded at 1000 cells / mL, and 500 μL of cell suspension was seeded per well in a 24-well plate. 100 ng / mL M-CSF was added to the medium for cell differentiation. Cells were incubated for 5 days, and on day 3, the medium was replaced with fresh medium supplemented with 100 ng / mL M-CSF. For the polarization phase, inflammatory macrophages were generated using an LPS-IFNg solution containing 100 ng / mL LPS and 20 ng / mL IFNg, supplemented with an isotype control (mIgG1 or hIgG4) (2 μg / mL), an anti-CMKLR1 antibody (2 μg / mL) (2G1 or 2G4, H6, BZ332, or 84939), C15 peptide (10 nM), or RvE1 (10 ng / mL) for 3 days. Proinflammatory-IFNg macrophages could also be generated by adding IFNg (20 ng / mL) alone to the culture medium. For inflammation-resolving macrophage polarization, cells were incubated with 20 ng / mL IL-4. After differentiation and / or polarization, phenotypic and functional cytokine / chemokine release were examined by FACS analysis, ELISA, and Western blot.

[0195] Example 10.2 Isolation and differentiation of mouse macrophages and DCs - Isolation of mouse bone marrow-derived macrophages Bone marrow cells were harvested and cultured for 5 days in RPMI medium supplemented with 10% FBS, glutamine, and antibiotics containing 100 ng / mL macrophage colony-stimulating factor (M-CSF) to induce macrophage differentiation. Macrophages were harvested and incubated with IFNg (20 ng / mL) and LPS (100 ng / mL) for 2 days to induce pro-inflammatory polarization, or with IL-4 (20 ng / mL) to induce inflammation-resolving polarization. Treatment was added at 2 μg / mL during macrophage polarization.

[0196] - Generation of bone marrow-derived dendritic cells Bone marrow cells were collected and cultured in RPMI medium supplemented with 10% FBS, glutamine, and antibiotics, and dendritic cell differentiation was induced with 20 ng / ml GM-CSF for 7 days. Immature dendritic cells (iDCs) were then collected and cultured with LPS (100 ng / ml) for 24 hours to induce iDC maturation into mDCs. Treatment was added at 2 μg / ml during differentiation and maturation.

[0197] After differentiation of mouse inflammatory or inflammation-resolving macrophages as described above, cells were incubated in the presence of medium and were tested with isotype controls, anti-CMKLR1 antibodies (clone H6 and BZ194), C15 peptide, and the target anti-CMKLR1 antibodies 2G1 or RvE1. Secretion of IL10, CCL17, and IL12p40 was then assessed by ELISA. Cytokine secretion was measured in the supernatant using ELISA kits from BD. Supernatants were diluted 1 / 10 for IL10 cytokine, 1 / 50 for CCL17 cytokine, and 1 / 100 for IL12p40 cytokine.

[0198] - Cytokine secretion test by ELISA Cytokine secretion was detected by ELISA according to the manufacturer's instructions. Briefly, the supernatant was diluted in the appropriate buffer and incubated for 2 hours after overnight coating and saturation with a capture antibody. The cytokine was then revealed using a biotin-conjugated detection antibody, and the signal was amplified using a biotin-streptavidin-conjugated peroxidase system. TMB, supplied by BD Bioscience, was used as the substrate, and the colorimetric reaction was read using a TECAN.

[0199] - Activated cell markers analyzed by FACS Dendritic cells were resuspended in PBS-FBS-EDTA and incubated with LIVE / DEAD Fixable Dead Cell Stains Yellow (Life Technologies) on ice for 30 minutes. Staining for CD11c-BV711, CD11b-APCCy7, I / Ab-APC, CD103-PerCPCy5.5, CCR7-V450, CD40-PeCy7, CD80-PE, and CD86-FITC (all supplied by BD Pharmingen) was performed.

[0200] - Western blot analysis of ERK / Akt Murine inflammatory macrophages (M1) were generated from bone marrow containing M-CSF and polarized with IFN-gamma (IFNg) and LPS. Briefly, bone marrow cells were collected by flushing the femur and cultured with 100 ng / mL M-CSF for 5 days, followed by polarization with 20 ng / mL IFNg and 100 ng / mL LPS for 24 hours. They were then deprived of FBS using RPMI FBS 2% medium for 24 hours. Finally, murine inflammatory macrophages were treated with 2 μg / mL 2G1 antibody for various times: 5, 10, and 30 minutes. Cells were collected in RIPA buffer. Protein concentration was measured using a BCA protein kit assay. Proteins were denatured by heating at 95°C for 5 minutes and diluted in DTT and Laemmli solution. After migration and transfer, nitrocellulose membranes were blocked with 5% BSA in TBS-T for 2 hours. Anti-phospho-ERK and anti-phospho-Akt antibodies (1:1000) were incubated with the membranes overnight at 4°C and revealed with peroxidase-conjugated secondary antibodies (1:2000). Western blot images were quantified using Multi Gauge software.

[0201] Example 10.3 CMLKR1 expression on monocytes in human blood and myeloid cells and neutrophils in mouse bone marrow after inflammatory stimulation Human monocytes were collected from the PBMCs of healthy volunteers and isolated by magnetic separation or elutriation. Monocytes (CD14+ cells) were then cultured in culture medium and treated with different inflammatory stimuli: LPS (100 ng / ml), TNFα (100 U / ml), or IL-6 (20 ng / ml) for 16 or 48 hours.

[0202] Mouse monocytes (CD11b+Ly6G-SSClow) and neutrophils (CD11b+Ly6G-SSClow) were obtained from bone marrow cells collected and cultured in RPMI medium supplemented with 10% FBS, glutamine, and antibiotics. The cells were then cultured in medium and treated with different inflammatory stimuli: LPS (100 ng / ml), TNFα (100 U / ml), or IL-6 (20 ng / ml) for 16 or 48 hours.

[0203] The expression of CMLKR1 was measured by FACS using commercially available anti-CMKR1 antibodies (human anti-ChemR23: clone 84939 and mouse anti-ChemR23: clone 477806).

[0204] Results: Analysis of CMKLR1 expression in mouse myeloid lineages, shown in Figure 14, demonstrated strong protein expression on monocytes and macrophages, as well as dendritic cells. Figure 15 shows CMKLR1 expression on human monocytes and myeloid cells and neutrophils from mouse bone marrow. This expression was clearly increased (at least 2-fold compared to controls after 48 hours) by inflammatory stimuli such as LPS, TNFα, or IL6, confirming that CMKLR1 expression on myeloid cell lineages and its overexpression during inflammation may be a therapeutic approach to downregulate and / or induce resolution of inflammation. DC activation markers were analyzed by FACS, and the results, shown in Figure 16, demonstrate a strong decrease in the expression of CD80, CD86, CD103, CD40, and IAb when cells were treated with RvE1 lipid or 2G1 antibody compared to vehicle or isotype control. These results indicate that the 2G1 antibody, like RVE1, is active against the CMKLR1 pathway on DCs. We then analyzed the CMKLR1 activation pathway in mouse macrophages by Western blot. Figure 17 shows that the anti-CMKLR1 antibody 2G1 was able to induce activation of both Akt and Erk proteins after 10 to 30 minutes of incubation. These results indicate that the 2G1 antibody, like the RvE1 lipid, exhibits agonistic properties for the CMKLR1 receptor.

[0205] Example 11 Competition assay for chemerin-induced CMKLR1 activation using anti-CMKLR1 antibody method: Competition assay to measure chemerin-dependent B-arrestin recruitment by the CMKLR1 receptor in the presence of anti-CMKLR1 antibodies: The day before the assay, CHO-K1 CMKLR1 cells (Discover'X Reference No. 93-0313E2) were seeded in prewarmed cell reagent (Discover'X Reference No. 15-103) and then seeded into 96-well plates at 100 μl / well of cells and incubated for 48 hours at 37°C in a 5% CO2 humidified incubator. Anti-CMLKR1 antibodies were diluted (22-fold with a seven-point series of 3-fold dilutions from 1 μM to 1 nM) and cells were incubated with the antibody for 30 minutes at 37°C. Cells were then stimulated with Chemerin (2 or 6 nM) for 90 minutes at 37°C according to the supplier's protocol (Discover'X Reference No. 92-1036). Luminescence was measured using a plate reader with a 0.5 s integration after adding the working dilution detection solution to the cells.

[0206] Measurement of competition between anti-CMKLR1 antibody and chemerin for AMPc production by the CMKLR1 receptor The day before the experiment, CHO-K1 CMKLR1 Gi cells (Discover'X reference number 95-0080C2) were seeded in pre-warmed cell reagent (Discover'X reference number 15-103) and then seeded into 96-well plates at 100 μl / well of cells and incubated at 37°C in a 5% CO2 humidified incubator for 24 hours.

[0207] Chemerin agonist (10 -7 μM to 10 -10A mixture of forskolin (40 μM) (6x in a 7-point series of 3-fold dilutions of 1 μM) (Discover'x reference number 92-1036 or 2324-CM-025 from R&D Systems) and forskolin (40 μM) (cAMP activator) (Discover'x reference number 92-0005) was added to the cells for 30 min at 37°C; or the cells were preincubated with anti-CMKLR1 antibody (serial dilutions: 6x in a 7-point series of 3-fold dilutions from 1 μM to 1 nM) for 30 min at 37°C. Then, a mixture of chemerin (2 nM) and forskolin (60 nM) was added to the cells for 30 min at 37°C. For cAMP detection, the antibody reagent and cAMP standard dilution detection solution were added to the plate for 1 h at room temperature, followed by the addition of cAMP solution A, and the cells were incubated for 3 h at room temperature in the dark. Bioluminescence was read using a plate reader with a 0.5 s integration.

[0208] Results: To test whether the antibodies of the present invention are antagonists of chemerin-induced CMKLR1 activation, two assays were performed, the results of which are presented in Figure 18. Chemerin-induced inhibition of forskolin-dependent cAMP production is shown in Figure 18A (filled circles or open squares); anti-CMKLR1 antibodies of the present invention were unable to reverse this inhibition of production (filled circles or open squares) compared to the control (gray diamonds). Chemerin-induced activation of beta-arrestin, presented in Figure 18B, shows that anti-CMKLR1 antibodies of the present invention do not significantly alter chemerin-dependent activation of beta-arrestin (open circles compared to black diamonds). Antibodies of the present invention do not have antagonist activity of the CMLKR1-chemerin interaction. Furthermore, the antibodies of the present invention are unable to induce the chemerin-induced CMKLR1 signaling pathway, confirming that these antibodies are not agonists of chemerin in the CMLKR1 pathway.

[0209] Example 12 CD45Rb high T cell-transferred chronic colitis mouse model Method: CD45Rb highCD4 T cells were isolated from the spleens of naive mice and sorted on an ARIA FACS after negative selection of CD4 T cells by magnetic sorting, followed by 0.5 × 10 cells in 100 μL of PBS. 6 The cells were injected intraperitoneally into 6-week-old female Rag1 knockout mice. Anti-CMKLR1 antibody (2G1) or isotype control was administered at 1 mg / kg three times a week for 3 weeks. high Dosing began on day 32 after CD4 T cell transfer. Body weight follow-up was assessed three times a week and weight fluctuations were determined relative to initial body weight. * p<0.05, ** p<0.01.

[0210] Results: Figure 19 shows the percentage weight change over time for animals treated with anti-CMLKR1 antibody or isotype control. Both groups showed the same initial weight development over the first 30 days when treated with anti-CMLKR1 antibody or isotype control. Mice treated with anti-CMLKR1 continued to gain weight, whereas control mice began to lose weight, indicating the expected development of chronic colitis in this control group (Figure 19A). A decrease in tissue thickness, corresponding to colonic repair, was observed in mice treated with anti-CMLKR1. A decrease in fibrotic tissue was also observed (Figure 19B). The different scores represent the anatomic pathology scores used to calculate the severity of pathology. Scores, including the inflammation score, were lower in mice treated with anti-CMLKR1 (Figure 19C). The inventors confirmed that the anti-CMLKR1 antibody of the present invention is interesting for treating chronic inflammatory and autoimmune diseases such as colitis in a third model of colitis, here a chronic inflammation model.

[0211] Example 13 Antitumor effect on overall survival in a mouse hepatocellular carcinoma tumor model Methods: Mice were anesthetized with a cocktail of xylazine / ketamine. After laparotomy, tumor Hepa 1.6 cells (2.5 × 10 cells / mL) were injected in PBS via the portal vein. 6Cells / 100 μL). Treatment began 4 days after tumor injection. Anti-CMKLR1 antibody (2G1 clone) and hIgG1 isotype control were injected at 0.8 mg / kg, three times a week for 2 weeks. Anti-PD1 monoclonal antibody was injected intraperitoneally in PBS twice a week for 2 weeks (8 mg / kg). The combination of anti-CMKLR1 and anti-PD1 antibodies was also tested (0.8 mg / kg and 8 mg / kg, respectively). Overall survival was followed for 60 days, and the percentage of survival for each condition is reported in Figure 22.

[0212] Results: As shown in Figure 20, animals treated with anti-CMKLR1 or anti-PD1 antibodies appeared to prolong their survival in only one of seven treated animals (15% of treated animals), indicating a partial response (PR). However, animals treated with a combination of anti-PD1 and anti-CMKLR1 antibodies significantly increased survival (from 15% to 45%), and the animals survived for 60 days after treatment, indicating a complete response (CR). This result demonstrates the unexpected efficacy of the therapeutic combination (anti-PD1 / anti-CMKLR1 antibodies) against HCC tumor models.

[0213] Example 14 Antibody production in different cell lines IGHV3-23 * 04 (corresponding to SEQ ID NO: 41), IGHV1-46 * The CDRs of the 2G1 heavy chain were grafted into three human germline frameworks, designated IGKV1-13, IGKV1-01, and IGHV7-4-1. * 02 (corresponding to SEQ ID NO: 50), IGKV6-21 * 01 and IGKV3-11 *The CDRs of the 2G1 light chain were grafted into three human germline frameworks, designated 01 (IMGT nomenclature). Each sequence was fused to a human immunoglobulin constant fragment and co-transfected into mammalian cells to produce a humanized antibody. More specifically, for construction of the heavy chain of the anti-ChemR23 antibody, the antibody variable domain VH sequence was synthesized and cloned into the pFUSE-CHIg-hG1 expression plasmid (pFUSE-CHIg-hG1 vector from Invivogen, Toulouse) containing the Fc of human IgG1 by EcoRV. For construction of the light chain of the anti-ChemR23 antibody, the variable domain VL was synthesized and cloned into the pFuse2CLIg-hk expression plasmid (pFuse2CLIg-hk from Invivogen, Toulouse) containing the human CL kappa by BsiWI. We co-transfected mammalian HEK or CHO cells with a plasmid containing VH-hFcG1 and a plasmid containing VL-CLkappa using the Lipofectamine method. After 3-7 days of incubation, the supernatant was collected and quantified by sandwich ELISA assay. The supernatant was purified by affinity chromatography on Protein A (HiTrap, GeHealthcare) using a 0.1 M citrate pH 3 elution buffer. The purified antibodies were dialyzed into PBS and concentrated. They were quantified by UV (A280 nm) and tested in an activity assay against a C7 antigen-specific peptide.

[0214] Results: As shown in Figure 21, human germline IGHV3-23 for the heavy chain * 04 was more efficient at producing antibodies in mammalian cells. Mutations in other frameworks did not result in highly productive chains. For the light chain, human germline IGKV1-13 * 02 was the best in producing humanized antibodies, while the other germlines reduced productivity by 1 log. * 04 and IGKV1-13 *The combination of both with 02 is suitable for the production of high-yield humanized anti-CMKLR1 antibodies. As shown in Figure 22, this combination allows the production of sufficient amounts of antibodies with satisfactory yields.

[0215] Therefore, germline IGHV3-23 * 04 and germline IGKV1-13 * 02 was selected for further antibody humanization.

[0216] Several mutations were added to the heavy or light chain. In the heavy chain, the mutations G33A (in CDR1), P60A (in CDR2), and R94K (in FR3) were substituted to increase humanization, and amino acid D61 (in CDR2) was replaced with amino acid E or A to reduce the risk of antibody deamination (sequence variant vB-vD). In the light chain, the mutations S24R, S27Q, M33L (in CDR1), T51A (in CDR2), and Y71F (in FR3) were substituted to increase humanization, and amino acid N92 was replaced with amino acid Q to reduce the risk of antibody glycosylation (sequence variant vB-vD). Each sequence was fused to a human immunoglobulin constant fragment and co-transfected into mammalian cells to produce humanized antibodies. The results showed that all combinations of heavy and light chains produced antibodies. Depending on the combination of heavy and light chains, productivity is affected differently in mammalian cells, but is always expected to be sufficient for efficient production, considering the therapeutic application of antibodies.

[0217] Example 15 Recognition ability of anti-CMKLR1 antibodies generated in vitro and derived from specific germline heavy and light chain variable domains For quantitative ELISA assays, donkey anti-human IgG, Fc specific (Jackson Immunoresearch, USA; ref. 709-005-098) was immobilized on plastic at 1.3 μg / ml in borate buffer (pH 9), and antibody-containing supernatant was added to measure binding compared with a standard antibody. After incubation and washing, mouse anti-human kappa antibody (Ose Immunotherapeutics, ref. NaM76-5F3) was added and detected with peroxidase-labeled donkey anti-mouse IgG antibody (Jackson Immunoresearch, USA; ref. 715-036-151). ELISA exposures were performed according to conventional methods.

[0218] For the activity ELISA assay, donkey anti-human IgG, Fc specific (Jackson Immunoresearch, USA; ref. 709-005-098) was immobilized on plastic at 1.3 μg / ml in borate buffer (pH 9), and purified antibody was added to measure binding in 1% BSA buffer compared to wild-type 2G1. After incubation and washing, a biotinylated antigen-specific peptide (Biot-C7 peptide: biotinylated NH2-PYHTLNLLELHHTAMPGSVFSLGLPLATALAIA-COOH, SEQ ID NO: 60, synthesized by synpeptide) was added, followed by peroxidase-streptavidin (Jackson Immunoresearch, USA; ref. 016-030-084) and revealed by conventional methods.

[0219] VHvAv3-23 * 04 (SEQ ID NO: 41 - 89.8% humanized) and VLvAv1-13 * The combination of heavy and light chains derived from wild-type antibody 2G1 (SEQ ID NO: 50-8, 2.1% humanized) produced a humanized antibody with good binding activity to an antigen-specific peptide (C7 peptide) like the wild-type antibody 2G1. As shown in Figure 23, the humanized antibody variable domain chain combination was derived from 2G1 (for the heavy chain variable domain, HA was VHvAv3-23 *04 and correspond to SEQ ID NO: 4; HC corresponds to SEQ ID NO: 42; HD corresponds to SEQ ID NO: 43; for the light chain variable domain, LA corresponds to SEQ ID NO: 50; LC corresponds to SEQ ID NO: 52; LD corresponds to SEQ ID NO: 53).

[0220] All combinations bound to the antigen-specific peptide (C7 peptide) with at least the same activity as wild-type antibody 2G1. In some cases (combinations of HCLC, HCLD, HDLC, and HDLD), binding was even better than that of germline antibody HALA and wild-type antibody 2G1 (Figure 23). As shown in Figure 24, the ED50 (ng / ml) of the humanized antibodies was at least equivalent to, and in many cases better than, that of the 2G1 antibody.

[0221] Example 16 Biological effects on CCR7 internalization Materials and Methods. Macrophages were generated from monocytes of healthy volunteers using 100 ng / mL M-CSF for 5 days. The macrophages were then collected and incubated with 10 mg / mL of coated mAb in the presence of 20 ng / mL IFNγ to obtain M1 inflammatory macrophages. M1s were then phenotyped for CXCR4 and CCR7 by flow cytometry, and cytokines released into the supernatant were measured by ELISA. Dendritic cells were generated from monocytes of healthy volunteers using 50 nm / mL GM-CSF and 20 ng / mL IL-4 for 6 days. DCs were then phenotyped for CCR7 by flow cytometry.

[0222] Results: 2G1 and all humanized 2G1 variants (HALA, HCLC, HCLD, HDLC, and HDLD) were immobilized on plates. Isotype controls were added as controls. Two isotypes that prevent FcγR binding were also added: 2G1-N297A (2G1 wt mutated at N297A to reduce FcγR binding) and 2G4 (wt with isotype IgG4 mutated at S228P to stabilize the hinge region). Inflammatory macrophages M1 were added to the coated plates for 48 hours, and CCR7 expression on the surface of the macrophages was measured by flow cytometry.

[0223] As shown in Figure 25, 2G1 and all humanized 2G1 variants were able to reduce the expression of CCR7 on the surface of inflammatory macrophages (M1). However, no internalization of CCR7 was observed with isotypes IgG1-N297A or IgG4, which prevent FcRγ binding, indicating that isotype IgG1 was preferred for this biological activity.

[0224] Example 17 Neutrophil apoptosis and death Neutrophils are normally located at sites of inflammation, and their presence perpetuates the inflammatory process, thereby preventing resolution of inflammation from being initiated or actively sustained, which can lead to chronic inflammation. Neutrophil apoptosis prevents the release of the neutrophil's tissue toxic contents and exerts an anti-inflammatory effect.

[0225] As shown in Figure 26A, the neutrophil survival / death ratio was higher in cells treated with the antibody of the invention, thereby demonstrating the effect of the agonist of the invention on these cells.

[0226] Caspase-3 expression Materials and Methods: PMNs from healthy volunteers were incubated with 10 μg / mL of coated Ab in culture medium for various times, harvested for caspase-3 staining, and analyzed by Western blot. The intensity of caspase-3 expression was calculated on WB.

[0227] Results: As shown in Figure 26B, administration of anti-CMKLR1 agonists enhanced caspase-3 activity compared to cells treated with a control antibody. The antibody HALA showed a stronger effect on caspase-3 activity compared to the 2G1 antibody. 2G1 WT and HALA increased the cleavage of caspase-3, indicating that ChemR23 induction led to caspase-3-dependent apoptosis.

[0228] Percentage of dead PMNs and ROS test (Fig. 26C): PMNs from healthy volunteers were incubated with 10 μg / mL of coated Abs in culture medium for 24 or 5 hours and stained with either a dead / live kit (LIVE / DEAD (Invitrogen)) or a specific marker of reactive oxygen species (ROS), respectively. The percentage of positive cells was obtained by analyzing the photographs using ImageJ software.

[0229] Results: 2G1 and all humanized variants of 2G1 increase the killing of PMNs after 24 hours. At 48 hours, the percentage of dead cells incubated with the IgG1 control and the HALA variants was similar, indicating that the antibodies promote programmed cell death in PMNs by inducing CMKLR1 signaling alone. 2G1 and all humanized variants of 2G1 promote the killing of PMNs, thus the humanized variants retain inflammation-resolving properties. 2G1 and the HALA variant increase ROS production by PMNs after 5 hours.

[0230] As shown in Figure 27B, the percentage of ChemR23-positive cells (macrophages and neutrophils) increases when inflammation is induced. However, the percentage of neutrophils in the exudate is not significantly reduced in animals treated with anti-CMKLR1 antibody (Figure 27C; black squares), while the overall percentage of macrophages in the exudate is slightly enhanced (Figure 27D). This indicates that administration of a CMKLR1 agonist does not reduce the overall number of myeloid cells in the exudate, but has an effect primarily on neutrophil apoptosis at the inflammatory site. As shown in Figures 27E and 27F, the percentage of dead neutrophils increases when a CMKLR1 agonist is administered, as well as their time of death. Because the neutrophil population is affected at the inflammatory site, not in the exudate, these results indicate a positive effect of a CMKLR1 agonist for treating the delay in resolution of inflammation.

[0231] In conclusion, apart from inducing apoptosis of neutrophils at the site of inflammation, treatment with the antibodies of the invention does not result in apoptosis of all neutrophil populations, a feature that may be advantageous in reducing side effects.

[0232] Example 18 Neutrophil migration After recruitment, neutrophils migrate to the site of inflammation, thereby initiating, enhancing and / or perpetuating the inflammatory process.

[0233] material and method Human endothelial cells (HDMEC) were incubated in gelatin-coated transwells for 24 hours and activated overnight with 100 U / mL TNF-alpha or without inflammatory conditions. PMNs from healthy volunteers or ANCA patients were then incubated in the transwells containing the HDMEC monolayer for 4 hours. During the 4-hour migration assay, 10 μg / mL of antibodies (Iso Ctrl and 2G1) were added with 100 U / mL + / - TNF-alpha. The lower migrated portion of the transwell was collected, and the migrated PMNs were counted by flow cytometry using counting beads.

[0234] result Neutrophils treated with 2G1 have reduced migration capacity compared to cells treated with a control compound (Figure 28A). 2G1 prevents PMN migration through endothelial monolayers under inflammatory conditions, particularly when PMNs and endothelial cells are activated with TNFα in healthy volunteers and AIDS patients (Figure 28B).

[0235] The antibody of the present invention has the ability to reduce the migration ability of neutrophils.

[0236] Example 19 CD62L expression Materials and Methods: PMNs from healthy volunteers were incubated with 10 μg / mL of coated Ab in culture medium for various times, collected for CD62L staining, and analyzed by flow cytometry (Figure 29, left panel). Cell surface expression of CD62L in cells incubated with the antibody of the present invention is reduced compared to cells incubated in the absence of antibody. Soluble CD62L released by shedding is detected by ELISA in the supernatant of PMNs incubated with coated Ab. Treatment of PMNs with anti-ChemR23 antibody increases the concentration of soluble CD62L compared to isotype control conditions (Figure 29, right panel).

[0237] Example 20 Mesothelioma model survival Mice treated with a CMKLR1 agonist according to the methods illustrated in the brief description of the figures had a higher survival rate than mice treated with a control antibody, thereby demonstrating the positive effect of compounds according to the present invention for treating mesothelioma (Figure 30).

[0238] Example 21 CRC Model As shown in Figure 31A, tumor volume is reduced in animals treated with the anti-CMKLR1 antibody of the present invention compared to the control antibody. In some cases, complete remissions are also observed, thereby demonstrating the positive effect of the compounds according to the present invention for treating CRC.

[0239] Furthermore, as illustrated in Figure 31B, treatment with anti-CMKLR1 monoclonal antibody (OSE-230) results in a decrease in fecal scores and a decrease in tumor number.

[0240] Example 22 Experimental model of autoimmune encephalomyelitis In this model, therapeutic administration of the antibody of the present invention in the EAE model does not result in a decrease in body weight compared to animals treated with a control antibody (Figure 32A). However, when treatment is performed with an anti-CMKLR1 antibody, the disease score is significantly reduced (Figure 32B). Since the disease score is about 30% lower in animals treated with an agonist compound, the anti-CMKLR1 antibody produces the greatest improvement in the score as soon as 10 days after treatment compared to the control. Therefore, treatment with an anti-CMKLR1 compound is shown to be effective in treating autoimmune encephalomyelitis.

[0241] Example 23 Optimization of CDR amino acid residues Several mutations were added in the heavy or light chain of the HDLD variant to replace amino acids involved in immunogenicity predicted in silico using IEDB software and HLA-II prediction software (NetMHCpanII method).

[0242] It is further emphasized that, among the very large number of possible amino acid mutations, the inventors have tested and identified several highly advantageous ones that do not substantially and inappropriately affect the biological activity of the product. Expert knowledge guides the selections described hereafter.

[0243] In the heavy chain, amino acid substitutions of D61E (HD-61E, SEQ ID NO: 89) in CDR2, R52G (HD-R52G, SEQ ID NO: 90) in CDR2, or R52aG, A49S, N52S, and Y53S (HEF, SEQ ID NO: 91) in CDR2 were achieved to reduce immunogenicity. In the light chain, amino acid N92 in CDR3 was substituted with amino acid S (LD-N92S, SEQ ID NO: 92) or Q (LD-T52S, SEQ ID NO: 93) to prevent post-translational modification. T52S in CDR2 was also substituted to reduce immunogenicity in the LD-N92S variant. N92Q in CDR3 and T52S, T55E in CDR2, and W47L in framework 2 were substituted to reduce immunogenicity in the LEF variant (SEQ ID NO: 55). The sequences are shown in the table below.

[0244] [Table 3]

[0245] As detailed in the Examples below, among all possible combinations, antibodies containing the light chain LDT52S, particularly the HEF-LDT52S variant, are particularly advantageous: Compared to other antibodies tested, HEF-LDT52S, which has been optimized to reduce immunogenicity, exhibits high binding activity and stability while maintaining biological function.

[0246] Each sequence was fused to a human immunoglobulin constant fragment and co-transfected into mammalian cells to produce humanized antibodies. The results showed that all combinations of heavy and light chains produced antibodies. Depending on the combination of heavy and light chains, productivity in mammalian cells is differentially affected, but considering the therapeutic application of antibodies, it is believed that the amount is always suitable for efficient production.

[0247] Example 24 Recognition ability of anti-CMKLR1 antibodies produced in vitro For the activity ELISA assay, donkey anti-human IgG, Fc specific (Jackson Immunoresearch, USA; ref. 709-005-098) was immobilized on plastic at 1.3 μg / ml in borate buffer (pH 9), and purified antibody was added to measure binding in 1% BSA buffer compared to wild-type 2G1. After incubation and washing, a biotinylated antigen-specific peptide (Biot-C7 peptide: biotinylated NH2-PYHTLNLLELHHTAMPGSVFSLGLPLATALAIA-COOH, SEQ ID NO: 60, synthesized by synpeptide) was added, followed by peroxidase-streptavidin (Jackson Immunoresearch, USA; ref. 016-030-084) and revealed by conventional methods.

[0248] As shown in Figure 33, combinations of humanized antibody variable domain chains derived from 2G1 (for the heavy chain variable domain, HD corresponds to SEQ ID NO: 43, HEG corresponds to SEQ ID NO: 91; for the light chain variable domain, LD corresponds to SEQ ID NO: 53; LD-T52S corresponds to SEQ ID NO: 93, LEF corresponds to SEQ ID NO: 55) produced humanized antibodies with better binding activity than the wild-type antibody 2G1 to the germline antibody HALA and an antigen-specific peptide (C7 peptide).

[0249] Example 25 Stability assay Each purified humanized anti-ChemR23 antibody (HALA, HDLD, HD-LDT52S, HEF-LDT52S, HEF-LEF) was incubated at 4°C or 37°C for 7 days. After 7 days, the binding of the purified antibodies was analyzed by ELISA assay, and aggregate formation was analyzed by gel filtration (Superdex 200 10 / 300GL, GeHealthcare).

[0250] As shown in Figure 34A, all purified antibodies exhibited similar binding activity at 37°C, 4°C, or -80°C. As shown in Figure 34B, the percentage of aggregates did not change after 7 days at 37°C for HDLD and HEF-LDT52S.

[0251] Example 26 Biological effects on CCR7 internalization The humanized 2G1 variant HEF-LD-T52S was immobilized on the plate. An isotype control was added as a control. Inflammatory macrophages M1 were added to the coated plate for 48 hours, and CCR7 expression on the surface of the macrophages was measured by flow cytometry.

[0252] As shown in Figure 35, the humanized 2G1 variant HEF-LD-T52S was able to reduce the expression of CCR7 on the surface of inflammatory macrophages (M1) compared to the isotype control. The humanized 2G1 variant HEF-LD-T52S, which was optimized to reduce immunogenicity, preserved the functional properties of the 2G1 antibody.

[0253] Example 27 Biological effects on PMN survival PMNs from healthy volunteers were incubated with 10 μg / mL of coated HEF-LDT52S, HEF-LEF, and HDLD antibody variants in culture medium for 24 hours and stained using one of the LIVE / DEAD kits (Invitrogen). The percentage of positive cells was obtained by analyzing the photographs using Fiji software. An isotype control was added as a control. A mutant version of the HEF-LDT52S antibody (HEF-LDT52S N297A) that does not bind to Fc receptors (FcRs) was also added. As shown in Figure 36, 2G1 and all HEF-LDT52S, HEF-LEF, and HDLD humanized versions of 2G1 promoted PMN killing, and thus the humanized variants retain inflammation-resolving properties.

[0254] The humanized HEF-LDT52S variant was optimized to reduce immunogenicity, and this optimized variant maintains high binding activity and stability while retaining biological function.

[0255] [References] TIFF2025134864000004.tif236168TIFF2025134864000005.tif226168

Claims

1. An anti-CMKLR1 antibody or its antigen-binding fragment that binds to chemerin-like receptor 1 (CMKLR1), wherein the antibody or its antigen-binding fragment is a. Antibody heavy chain variable (VH) domains containing the CDRs of VHCDR1 of SEQ ID NO: 4, VHCDR2 of SEQ ID NO: 12, and VHCDR3 of SEQ ID NO: 13, and antibody light chain variable (VL) domains containing the CDRs of VLCDR1 of SEQ ID NO: 19, VLCDR2 of SEQ ID NO: 26, and VLCDR3 of SEQ ID NO: 35; b. The antibody VH domain containing the CDRs of VHCDR1 of SEQ ID NO: 4, VHCDR2 of SEQ ID NO: 63, and VHCDR3 of SEQ ID NO: 13, and the antibody VL domain containing the CDRs of VLCDR1 of SEQ ID NO: 19, VLCDR2 of SEQ ID NO: 25, and VLCDR3 of SEQ ID NO: 35; c. An antibody VH domain containing the CDRs of VHCDR1 of SEQ ID NO: 4, VHCDR2 of SEQ ID NO: 63, and VHCDR3 of SEQ ID NO: 13, and an antibody VL domain containing the CDRs of VLCDR1 of SEQ ID NO: 19, VLCDR2 of SEQ ID NO: 25, and VLCDR3 of SEQ ID NO: 34; d. The antibody VH domain containing the CDRs of VHCDR1 of SEQ ID NO: 4, VHCDR2 of SEQ ID NO: 63, and VHCDR3 of SEQ ID NO: 13, and the antibody VL domain containing the CDRs of VLCDR1 of SEQ ID NO: 19, VLCDR2 of SEQ ID NO: 25, and VLCDR3 of SEQ ID NO: 34; e. The antibody VH domain containing the CDRs of VHCDR1 of SEQ ID NO: 3, VHCDR2 of SEQ ID NO: 63, and VHCDR3 of SEQ ID NO: 13, and the antibody VL domain containing the CDRs of VLCDR1 of SEQ ID NO: 19, VLCDR2 of SEQ ID NO: 25, and VLCDR3 of SEQ ID NO: 34; f. The antibody VH domain containing the CDRs of VHCDR1 of SEQ ID NO: 3, VHCDR2 of SEQ ID NO: 63, and VHCDR3 of SEQ ID NO: 13, and the antibody VL domain containing the CDRs of VLCDR1 of SEQ ID NO: 19, VLCDR2 of SEQ ID NO: 25, and VLCDR3 of SEQ ID NO: 35; g. An antibody VH domain containing the CDRs of VHCDR1 of SEQ ID NO: 3, VHCDR2 of SEQ ID NO: 8, and VHCDR3 of SEQ ID NO: 13, and an antibody light chain variable (VL) domain containing the CDRs of VLCDR1 of SEQ ID NO: 17, VLCDR2 of SEQ ID NO: 24, and VLCDR3 of SEQ ID NO: 34; or h. The antibody VH domain containing the CDRs of VHCDR1 of SEQ ID NO: 4, VHCDR2 of SEQ ID NO: 12, and VHCDR3 of SEQ ID NO: 13, and the antibody VL domain containing the CDRs of VLCDR1 of SEQ ID NO: 19, VLCDR2 of SEQ ID NO: 27, and VLCDR3 of SEQ ID NO: 35 An anti-CMKLR1 antibody or its antigen-binding fragment, including the above.

2. The antibody or its antigen-binding fragment is a. A heavy chain variable domain containing SEQ ID NO: 91 and a light chain variable domain containing SEQ ID NO: 93; b. A heavy chain variable domain containing Sequence ID No. 43 and a light chain variable domain containing Sequence ID No. 53; c. A heavy chain variable domain containing Sequence ID No. 43 and a light chain variable domain containing Sequence ID No. 52; d. Heavy chain variable domain containing Sequence ID No. 43 and light chain variable domain containing Sequence ID No. 93; e. A heavy chain variable domain containing Sequence ID No. 42 and a light chain variable domain containing Sequence ID No. 52; f. A heavy chain variable domain containing Sequence ID No. 42 and a light chain variable domain containing Sequence ID No. 53; g. A heavy chain variable domain containing SEQ ID NO: 41 and a light chain variable domain containing SEQ ID NO: 50; or h. Heavy chain variable domain containing Sequence ID 91 and light chain variable domain containing Sequence ID 55 The anti-CMKLR1 antibody or antigen-binding fragment thereof according to claim 1, comprising:

3. An anti-CMKLR1 antibody or antigen-binding fragment thereof that binds to chemerin-like receptor 1 (CMKLR1), wherein the antibody or antigen-binding fragment comprises an antibody heavy chain variable (VH) domain containing the CDRs of VHCDR1 of SEQ ID NO: 4, VHCDR2 of SEQ ID NO: 12, and VHCDR3 of SEQ ID NO: 13, and an antibody light chain variable (VL) domain containing the CDRs of VLCDR1 of SEQ ID NO: 19, VLCDR2 of SEQ ID NO: 26, and VLCDR3 of SEQ ID NO:

35.

4. An anti-CMKLR1 antibody or antigen-binding fragment thereof that binds to chemerin-like receptor 1 (CMKLR1), wherein the antibody or antigen-binding fragment thereof comprises an antibody VH domain including the CDRs of VHCDR1 of SEQ ID NO: 4, VHCDR2 of SEQ ID NO: 12, and VHCDR3 of SEQ ID NO: 13, and the CDRs of VLCDR1 of SEQ ID NO: 19, VLCDR2 of SEQ ID NO: 27, and VLCDR3 of SEQ ID NO:

35.

5. The anti-chemerin-like receptor 1 (CMKLR1) antibody or antigen-binding fragment thereof according to claim 4, wherein the VH domain comprises the amino acid sequence of SEQ ID NO: 91 and the VL domain comprises the amino acid sequence of SEQ ID NO:

55.

6. An anti-CMKLR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, which specifically binds to the third extracellular loop (EL3) of CMKLR1.

7. The anti-CMKLR1 antibody or antigen-binding fragment thereof according to claim 6, which specifically binds to an epitope located in a polypeptide containing the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 59, or to an epitope located in the amino acid sequence of SEQ ID NO:

60.

8. An anti-CMKLR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, which is a resolvin E1-like agonist of CMKLR1.

9. An anti-CMKLR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, which does not activate the beta-arrestin signaling pathway in CMKLR1-positive cells in vitro and / or in vivo.

10. An anti-CMKLR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, which does not show significant depletion in CMKLR1-positive cells in vitro and / or in vivo.

11. An anti-CMKLR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, which does not compete with chemerin for binding to CMKLR1 and / or does not inhibit the binding of chemerin to CMKLR1.

12. The anti-CMKLR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, which is a humanized monoclonal antibody or an antigen-binding fragment thereof.

13. The anti-CMKLR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, wherein the antibody light chain constant domain is a humanized monoclonal antibody or an antigen-binding fragment thereof derived from a human kappa light chain constant domain.

14. An anti-CMKLR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, wherein the light chain constant domain comprises the sequence of SEQ ID NO:

79.

15. An anti-CMKLR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, wherein the antibody heavy chain constant domain is derived from a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant domain.

16. The anti-CMKLR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, wherein the antibody heavy chain constant domain comprises the amino acid sequence of SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, or SEQ ID NO:

84.

17. An anti-CMKLR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, wherein the antibody heavy chain constant domain comprises the amino acid sequence of SEQ ID NO: 80 or SEQ ID NO:

83.

18. An antibody or antigen-binding fragment thereof that specifically binds to CMKLR1, comprising (i) a VHCDR2 comprising or consisting of the amino acid sequence described in SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 61, and (ii) a mutant sequence in which amino acid residues are substituted such that the amino acid residues at positions 1 and 2 of the antibody heavy chain variable domain or mutant sequence comprising or consisting of the amino acid sequence described in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 are L and I, respectively. Anti-CMKLR1 compounds specifically bind to epitopes located within the third extracellular loop (EL3) of CMKLR1. An antibody or antigen-binding fragment thereof, which competes with an antibody containing a heavy chain variable domain corresponding to SEQ ID NO: 37 and a light chain variable domain corresponding to SEQ ID NO: 49 for binding to a polypeptide containing or comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 59 or SEQ ID NO: 60, or to a polypeptide containing or comprising the third loop (EL3) of the extracellular domain of CMKLR1.

19. The anti-CMKLR1 antibody or its antigen-binding fragment is VHCDR1 of sequence number 4; VHCDR2 of sequence number 12; VHCDR3 of sequence number 13; VLCDR1 of sequence number 19; VLCDR2 of Sequence ID No. 26; and VLCDR3 of Sequence ID 35 The anti-CMKLR1 antibody or antigen-binding fragment thereof according to claim 18, comprising the CDR.

20. VHFR1 of Sequence ID No. 65; VHFR2 of sequence number 67; VHFR3 of sequence number 69; VHFR4 of sequence number 71; VLFR1 of sequence number 72; VLFR2 of sequence number 73; VLFR3 of Sequence ID No. 76; and VLFR4 of Sequence ID 77 The anti-CMKLR1 antibody or antigen-binding fragment thereof according to claim 18, comprising the framework of the above.

21. The anti-CMKLR1 antibody or antigen-binding fragment thereof according to claim 20, comprising a heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 91 and a light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO:

93.

22. A pharmaceutical composition comprising an anti-CMKLR1 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 21, for use in the prophylactic or therapeutic treatment of inflammatory diseases, autoimmune diseases, or cancer.

23. A pharmaceutical composition comprising an anti-CMKLR1 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 21, for use in the prophylactic or therapeutic treatment of diabetes.

24. A pharmaceutical composition comprising an anti-CMKLR1 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 21, for use in the prophylactic or therapeutic treatment of chronic inflammatory diseases.

25. A pharmaceutical composition comprising an anti-CMKLR1 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 21, for use in the prophylactic or therapeutic treatment of NASH (non-alcoholic steatohepatitis), scleroderma, cystic fibrosis, or anti-neutrophil cytoplasmic antibody-associated disease (ANCA).

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