Humanized antibody against chemokine-like receptor 1 and its therapeutic applications

Abstract

The present invention provides humanized anti-CMKLR1 compounds having agonistic activity against the interaction between Resolvin E1 and CMKLR1, and their use for treating or preventing diseases, particularly those in which the resolution of inflammation is delayed or disrupted.

Description

Technical Field

[0001] The present invention relates to the field of immunotherapy. The present invention provides novel humanized anti-chemerin receptor antibodies having resolvin E1-like agonist activity against chemokine-like receptor-1 (CMKLR1). The present invention also provides the use of such antibodies in therapy, in particular 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 Art

[0002] The important role of inflammatory processes in health and disease has long been recognized. The detailed molecular mechanisms and biological events that regulate the progression and resolution of inflammation remain highly interesting. Recent investigations have provided strong evidence that the resolution of inflammation is not a passive process as previously believed. Instead, the resolution of inflammation is a biosynthetically active process that is regulated by biochemical mediators and receptor signaling pathways. Resolution is thus driven by specific pro-resolving mediators. Inflammation is an innate mechanism that occurs during infection, injury or traumatic insult. Inflammation is inevitable and is usually beneficial, and its response is regulated by a delicate balance between positive and negative feedback loops. Inflammation is typically divided into three stages: initiation, amplification and resolution.

[0003] The resolution process that enables the termination of the inflammatory response is a complex process involving the sequential and time-dependent involvement of cellular (e.g., granulocytes or macrophages) and chemical (e.g., cytokines or specific pro-resolving mediators or factors) effectors.

[0004] Chemokine-like receptor 1 (CMKLR1), also known as ChemR23, and chemokine receptor-like 2 (CCRL2) 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 murine animals) 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, named chemerin, was identified as a ligand for CMKLR1.

[0005] The second ligand for 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 chemokine system (i.e., signal transduction pathways activated or not activated by its ligands such as chemokines and resolvins by chemokine receptors) focused on its role in inflammation and the chemotaxis of immune cells after its discovery in psoriatic diseases. More recently, its potential role related to cardiovascular function, as well as its role in reproductive biology, has been considered in relation to its role in inflammation, obesity, and metabolic syndrome. Thus, the chemokine system is of great interest regarding its role in the inflammatory process, particularly its role 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-resolution factor mediators include polyunsaturated fatty acids containing lipoxins, the resolvin family containing E-series resolvins and D-series resolvins, and protectins, as well as maresins. Nevertheless, due to their lipid nature, it is difficult to synthesize inflammation-resolution molecules. For example, the production of inflammation-resolution molecules in amounts sufficient for clinical trials is burdensome, and very few SPMs have been efficiently produced. In addition, antibodies specifically targeting G protein-coupled receptors are difficult to produce. Therefore, there is a need for molecules, particularly those with the ability to initiate or enhance the resolution phase of the inflammatory response, such as inflammation-resolution factors.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0008]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

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 mimic or a modified antibody.

[0010] The inventors sought to obtain improved humanized antibodies when compared to wild-type anti-CMKLR1 antibodies known in the prior art. Some residues in the variable domain CDR and framework (FR) sequences, including residues in the Vernier zone, canonical residues, residues at the interface between the variable heavy and variable light chains, etc., are important in the structure of the antibody and are known not to be mutated in order to retain the biochemical and biological activities of the antibody. However, the inventors surprisingly found that some mutations within the heavy chain variable domain and / or light chain variable domain sequences of the wild-type antibody, including some of such important residues, are associated with these humanized antibodies with functional characteristics that are not shared, in particular, by the wild-type anti-CMKLR1 that is improved, enabling an increase in the human residue content (up to 99% humanized), and at the same time, the production of these humanized antibodies can be improved as compared to less humanized or clearly humanized anti-CMKLR1 antibodies. These humanized antibodies provide compounds that exhibit functional characteristics useful in the treatment of diseases including those involving inflammation, which can be produced on a large scale for candidate drug development. The combination of these two features results in the provision of anti-CMKLR1 antibodies that exhibit an improved ability as compared to antibodies of the prior art.

[0011] Starting from a non-humanized anti-CMKLR1 antibody and selecting human germline sequences, the inventors designed specific humanized heavy chain variable domains and light chain variable domains. The humanized heavy chain variable domains and light chain variable domains derived from the non-humanized antibody are not limited, but under conditions that enable the recovery of a significant yield of antibodies that bind to their target with an activity that can also increase with respect to the binding activity of the wild-type antibody, such as Chinese hamster ovary (CHO) cell lines, transformed African green monkey kidney fibroblast (COS-7) cell lines, and human embryonic kidney cell lines (HEK293), enable the production of functional antibodies in different cell lines. When some of the obtained humanized heavy chain variable domains and light chain variable domains were further humanized, particularly with respect to the first humanization step performed on the framework regions of the heavy chain and light chain variable domains, it resulted in a decrease in immunogenicity and provided antibodies suitable for high-yield production in different cell lines that have at least the functional characteristics of their parental antibody. Those skilled in the art can improve the production scale of these antibodies containing the heavy chain variable domain and light chain variable domain according to the present invention, compared with antibodies that are less humanized or humanized differently, particularly where the mutations at such positions in the CDR domains as well as in the framework regions of the heavy chain and / or light chain variable domains maintain the binding ability, particularly the affinity, maintain the stability, and provide an anti-CMKLR1 antibody with a reduced immunogenicity index, and it was not expected to result in the retention of the resolvin-like agonist ability of these antibodies on CMKLR1. In particular, it was not predictable that the introduction of the disclosed mutations into CDR2 of the heavy chain would result in improved production while still retaining the binding characteristics, advantageously other functional characteristics of the prior art anti-CMKLR1 antibodies. Anti-CMKLR1 antibodies exhibiting the described characteristics (binding ability, particularly affinity, low immunogenicity, good production scale, and resolvin-like agonist ability for CMKLR1, particularly for a specific third extra loop of CMKLR1) are useful for the potent treatment of several diseases, more specifically diseases involving inflammation, including inflammatory diseases.

[0012] Furthermore, as will be described later in the present application, notably, several highly advantageous biological effects have been achieved, which are particularly related to a strong beneficial effect in the dissipation of inflammation, specifically, a decrease in neutrophil apoptosis and in the migration and / or movement of neutrophils and / or macrophages. The new compounds can thus combine production capacity and biological activity.

[0013] Accordingly, in a first aspect of the present invention, said antibody or antigen-binding fragment thereof that binds to chemokine-like receptor 1 (CMKLR1), particularly human CMKLR1, a) an antibody heavy chain variable (VH) domain comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, wherein - 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; - 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, 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; - VLCDR3 is selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36; and comprises.

[0014] In certain embodiments, said antibody or antigen-binding fragment thereof that binds to chemokine-like receptor 1 (CMKLR1), particularly human CMKLR1, a) An antibody heavy chain variable (VH) domain comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, wherein - 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 is identical to the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 63, provided that VHCDR1 is not SEQ ID NO: 3 or SEQ ID NO: 4; - 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, an antibody heavy chain variable (VH) domain; b) An antibody light chain variable (VL) domain comprising three CDRs, VLCDR1, VLCDR2 and VLCDR3, 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; - VLCDR3 is selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36, an antibody light chain variable (VL) domain comprising.

[0015] In another specific 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, wherein - 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 matches the amino acid sequence of SEQ ID NO: 12, preferably, under the condition that the heavy chain variable (VH) domain contains the framework FR3 of SEQ ID NO: 69, the heavy chain variable (VH) domain does not contain the framework VHFR3 of SEQ ID NO: 70. - 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 is included.

[0016] Particularly preferably, the antibody or its antigen-binding fragment specifically binds to the third extracellular loop (EL3) of CMKLR1, particularly to an epitope located within the third extracellular loop (EL3) of CMKLR1; more specifically, 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 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 retains the ability to specifically bind to the extracellular loop 3 of CMKLR1 and its resolvin E1-like agonist ability for CMKLR1, while having a production yield suitable for the purpose of drug candidate development, although not limited to, it is suitable for production in various cell lines including mammalian cell lines. The humanized antibody disclosed herein can thus be produced efficiently and share the functional ability of its parental antibody.

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

[0019] In particular, the humanized anti-CMKLR1 antibody or the antigen-binding fragment thereof specifically binds to the third extracellular loop (EL3) of CMKLR1, and in particular, the antibody or the 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.

[0020] Starting from the anti-CMKLR1 antibody 2G1, the inventors synthesized various heavy chain variable domains and light chain variable domains. The humanized heavy chain variable domain of SEQ ID NO: 41 and the humanized light chain variable domain of SEQ ID NO: 50 were particularly suitable for the production of humanized anti-CMKLR1 antibodies in cells or cell lines, including mammalian cells such as CHO cells or COS cells or HEK cells. Once specific human germline sequences were selected for the design of the heavy chain variable domain and the light chain variable domain, humanized antibodies having the identity defined herein in the framework region could also be produced in sufficient amounts in cells or cell lines for the purpose of developing antibodies that could give rise to drug candidates. There is great 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 specific anti-CMKLR1 antibodies provided herein share a highly similar structure with their parental antibodies containing the heavy chain variable domain of SEQ ID NO: 41 and the light chain variable domain of SEQ ID NO: 50, enabling the accurate production, conformation, and secretion of anti-CMKLR1 antibodies, thereby enabling the provision of antibodies that specifically bind to a specific epitope of chemokine-like receptor 1 in an amount sufficient for therapeutic purposes and have a resolvin E1-like agonist ability against this receptor.

[0021] In certain embodiments of the invention, in particular, a humanized anti-chemokine-like receptor 1 (CMKLR1) antibody or an 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, more specifically greater than 1 mg / ml, more specifically greater than 10 mg / ml, and even more specifically greater than 100 mg / ml, a) the variable heavy (VH) domain comprises the amino acid sequences of the frameworks (FR1, FR2, FR3, and FR4) of the heavy chain variable domain, and each framework has sequence identity with the same-rank framework in the sequence of SEQ ID NO: 41 that is at least 90% for FR1, at least 70% for FR2, at least 80% for FR3, and at least 80% for FR4; b) The variable heavy chain (VL) domain comprises the amino acid sequences of the frameworks (FR1, FR2, FR3, and FR4) of the light chain variable domain, and each framework has sequence identity with the framework of the same rank in the sequence of SEQ ID NO: 50 that is at least 60% for FR1, at least 80% for FR2, at least 75% for FR3, and at least 70% for FR4, respectively. An antibody is provided.

[0022] In particular, the humanized anti-CMKLR1 antibody or the antigen-binding fragment thereof specifically binds to the third extracellular loop (EL3) of CMKLR1. In particular, the antibody or the 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.

[0023] In a particular embodiment of the humanized antibody or the antigen-binding fragment thereof defined above, such a humanized antibody or the antigen-binding fragment thereof a) An antibody heavy chain variable (VH) domain comprising three CDRs, VHCDR1, VHCDR2, and VHCDR3, wherein - 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; - 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, 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, comprising.

[0024] In certain embodiments of the humanized antibody or antigen-binding fragment thereof defined above, such humanized antibody or antigen-binding fragment thereof is a) an antibody heavy chain variable (VH) domain comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, wherein - 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, provided that VHCDR1 is not SEQ ID NO: 3 or SEQ ID NO: 4; - 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, an antibody heavy chain variable (VH) domain; and b) an antibody light chain variable (VL) domain comprising three CDRs, VLCDR1, VLCDR2 and VLCDR3, 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; - VLCDR3 is selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36, an antibody light chain variable (VL) domain comprising.

[0025] In another specific embodiment of the humanized antibody or antigen-binding fragment thereof defined above, such humanized antibody or antigen-binding fragment thereof is a) an antibody heavy chain variable (VH) domain comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, wherein - 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; - The 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 contains or consists of the amino acid sequence of SEQ ID NO: 12, preferably, under the condition that the heavy chain variable (VH) domain contains VHFR3 of SEQ ID NO: 69, the heavy chain variable (VH) domain does not contain the framework VHFR3 of SEQ ID NO: 70; - The 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, an antibody heavy chain variable (VH) domain; and b) An antibody light chain variable (VL) domain comprising three CDRs, VLCDR1, VLCDR2, and VLCDR3, - The 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; - The 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; - The VLCDR3 is selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36, an antibody light chain variable (VL) domain.

[0026] Such antibodies provide the same advantageous features 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 the chemmerin-like receptor 1, and resolvin E1-like agonist ability.

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

[0028] In the following disclosure, the humanized anti-CMKLR1 compound is considered to be either a humanized anti-CMKLR1 antibody or its antigen-binding fragment or antigen-binding antibody mimetic or modified antibody. In certain embodiments of the invention, said compound is defined by the sequence of its CDRs. In more specific embodiments of the 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 the receptor encoded by the CMKLR1 gene in humans or the cmklr1 gene in non-human animals. In certain embodiments of the invention, the 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 the chemokine-like receptor 1 protein (also designated as ChemR23), which is 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 the present application corresponds to the sequence associated with Uniprot accession number Q99788 (SEQ ID NO: 1).

[0029] In certain embodiments, the present invention relates to an anti-CMKLR1 antibody or an antigen-binding fragment or antigen-binding antibody mimetic or modified antibody thereof defined by at least one functional characteristic. In a preferred embodiment, said anti-CMKLR1 compound is defined by its ability to inhibit the secretion of inflammatory cytokines, particularly IL12, and / or its ability to enhance the secretion of anti-inflammatory cytokines, particularly IL10. In a more particular embodiment, the anti-CMKLR1 compound inhibits or enhances cytokine secretion by macrophages, particularly by inflammatory macrophages and / or resolution-phase macrophages. In certain embodiments, the anti-CMKLR1 compound of the present invention enhances the polarization of macrophages into anti-inflammatory macrophages, particularly resolution-phase macrophages. In certain embodiments, the anti-CMKLR1 compound of the present invention enhances neutrophil apoptosis as compared to a control antibody.

[0030] In certain embodiments, the present invention relates to an antigen-binding fragment or antigen-binding antibody mimetic of an anti-CMKLR1 antibody that mimics at least one of the effects induced by the binding of RvE1 to CMKLR1 on CMKLR1-positive cells by having agonist properties with respect to the RvE1 / CMKLR1 interaction. "Agonist properties with respect to the RvE1-CMKLR1 interaction" means that the antibody or antigen-binding fragment or antigen-binding antibody mimetic or modified antibody of the present invention that targets CMKLR1 has the effect of mimicking at least one of the effects provided by the binding of RvE1 to CMKLR1, thereby activating receptor signaling pathways that are normally activated by RvE1, particularly on dendritic cells, neutrophils, monocytes, and macrophages, particularly the binding of human RvE1 to human CMKLR1. As a result of the binding and activation of the receptor that leads to a biological response, the compounds of the present invention activate G protein signaling pathways, particularly Gα, without activating the β-arrestin pathway. iIt can bring about signal transduction pathways and / or activation of Gα0. In particular, the compounds of the present invention can bring about inhibition of the β-arrestin pathway. In particular, the binding of the compounds according to the present invention induces activation of Akt and / or Erk proteins in vitro and / or in vivo. In certain embodiments, when the G protein signal transduction pathway is activated in CMKLR1-positive cells stimulated with the compounds of the present invention, in more specific embodiments, even when the B-arrestin pathway is not activated, and in certain conditions, when the B-arrestin pathway is inhibited, the compound can be considered an anti-CMKLR1 agonist having a resolvin R1-like ability. 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 as compared to a control antibody. The control antibody may be an antibody that does not specifically bind to CMKLR1. Phosphorylation of proteins can be determined by methods well known to those skilled in the art, such as the methods disclosed in the examples of this specification. 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 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, since the compounds of the present invention induce at least one agonist effect of the binding of RvE1 to CMKLR1, and since RvE1 is a proresolving factor or a proresolving mediator, the compounds of the present invention are proresolving factors or proresolving mediators; for example, a proresolving factor can inhibit the β-arrestin pathway induced by CMKLR1 and / or enhance the activation of the G protein pathway induced by CMKLR1 in CMKLR1-positive cells as compared to a control compound known not to specifically interact with CMKLR1. 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 invention do not inhibit the binding of chemerin to CMKLR1. Chemerin is one of the natural ligands of CMKLR1. In other words, the compounds according to the invention are not agonists and / or antagonists of the interaction between chemerin and CMKLR1. The absence of such agonist and / or antagonist capabilities can be evaluated according to the examples of the invention, in which a competitive assay is disclosed for measuring chemerin-dependent beta-arrestin recruitment by the CMKLR1 receptor in the presence of the anti-CMKLR1 antibody of the invention. In a preferred embodiment, the anti-CMKLR1 compounds of the invention do not compete with chemerin for binding to CMKLR1. The binding of chemerin to CMKLR1 in the presence of the CMKLR1 compound of the invention is at least 50%, more preferably at least 80%, even more preferably at least 90%, and most preferably, when similar, the absence of competition between the anti-CMKLR1 compound of the invention and chemerin can be determined compared to the binding of chemerin to CMKLR1 under the same experimental conditions but in the absence of the anti-CMKLR1 of the invention. Alternatively, the absence of competition between the anti-CMKLR1 compound of the invention and chemerin can be determined according to the method exemplified in Example 9.

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

Mode for Carrying Out the Invention

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

[0034] In the following description, where there is no conflicting description, an agonist of CMKLR1 having a resolvin E1-like ability can be defined as an "agonist"; both terms include an antibody or an antigen-binding fragment thereof (also referred to as an anti-CMKLR1 antibody), a protein, a peptide, or a polypeptide; 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 an antibody or an antigen-binding fragment thereof (also referred to as an anti-CMKLR1 antibody), a protein, a peptide, or a polypeptide. Among the effects provided by the use of such an agonist, the following specific effects have been demonstrated: - An agonist of CMKLR1 having a resolvin E1-like ability induces a decrease or inhibition of the migratory ability of these cells by apoptosis of polymorphonuclear neutrophils (also simply referred to as neutrophils, PMNs or PMNs herein) and / or, particularly, inhibition of its ability to migrate towards the inflammatory site through the endothelium, as described later in the present application. - An agonist of CMKLR1 having a resolvin E1-like ability induces the internalization of different receptors expressed on the cell surface on various myeloid cells, notably macrophages and / or dendritic cells, thereby enhancing the process of inducing or sustaining the resolution of inflammation, as described later in the present application. - In particular, agonists of CMKLR1 having resolvin E1-like ability 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 results in much lower targeting and recognition of CXCR4 and CCR7 receptors by cytokines known to induce the migration of cells towards the site of inflammation, and as a result, leads to a decrease or inhibition of the migration of macrophages and / or dendritic cells from the site of inflammation to secondary lymphoid organs and / or towards the site of inflammation; - Agonists of CMKLR1 having resolvin E1-like ability decrease or inhibit the ability of neutrophils and macrophages and / or dendritic cells to migrate; in particular, it decreases the rolling ability of these cells and reduces its ability to move through the endothelium, thereby decreasing or inhibiting the ability of neutrophils to move through the site of inflammation, due to the internalization of CD62L and / or a decrease in its cell surface expression; - In certain embodiments of the invention, the inventors have demonstrated that agonists of CMKLR1 having resolvin E1-like ability, which contain domains suitable for interaction with Fc receptors, such as IgG constant domains, particularly IgG1 constant domains, are particularly efficient. Fc receptors on macrophages or neutrophils recognize very efficiently, in particular, the Fc fragment of the anti-CMKLR1 IgG constant domain or IgG1 constant domain, and result in or contribute to the apoptosis of neutrophils recognized by anti-CMKLR1 IgG1 antibodies; - The inventors have now demonstrated that myeloid cells expressing CMKLR1, as well as CXCR4 and / or CCR7, which are involved in the inflammatory process (i.e., sustain inflammation), are particularly suitable targets for agonists of CMKLR1 having resolvin E1-like ability for the treatment of pathological inflammatory processes.

[0035] The present invention relates in particular to an agonist of chemokine-like receptor 1 (CMKLR1) having resolvin E1-like ability for use in the treatment of patients suffering from inflammatory conditions, in particular inflammatory conditions in which the resolution of inflammation is delayed or disrupted, wherein the agonist of CMKLR1 having resolvin E1-like ability is selected from the group consisting of antibodies or antigen-binding fragments thereof, peptides, polypeptides and proteins, - inducing or activating apoptosis of 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 - inhibiting the migration of macrophages and / or dendritic cells from the site of inflammation to secondary lymphoid organs and / or towards the site of inflammation, relating to the agonist.

[0036] The present invention relates to the use of an agonist of CMKLR1 having resolvin E1-like ability, such as an antibody, in particular a humanized antibody, or an antigen-binding fragment thereof, for inducing, enhancing or activating apoptosis of neutrophils in the treatment of inflammatory conditions, in particular inflammatory conditions in which the 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 a modified antibody.

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

[0039] As used herein, the term "antibody" further includes antibodies modified as compared to wild-type antibodies, and encompasses chimeric antibodies, humanized antibodies, modified antibodies, and antigen-binding antibody mimetics. The specific wild-type antibody for reference in the context of the present invention is antibody 2G1.

[0040] The antibodies of the present invention include recombinant antibodies. As used herein, the term "recombinant antibody" refers to an antibody expressed using a recombinant expression vector transfected into a host cell; an antibody isolated from a recombinant combinatorial antibody library; an antibody produced, expressed, generated or isolated by recombinant means such as an antibody isolated from an animal (e.g., a mouse) transgenic due to a human immunoglobulin gene; or an antibody produced, expressed, generated or isolated by any other method of assembling a specific immunoglobulin gene sequence (such as a human immunoglobulin gene sequence) 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 the sequence of a variable domain derived from the germline of a mammalian species such as a mouse is transplanted onto the sequence of a constant domain 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, are grafted onto a human or, in particular, a humanized framework sequence. In a further embodiment, "humanized antibody" refers to an antibody in which all or part of at least one CDR and framework sequence has been humanized.

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

[0044] An antigen-binding fragment of an antibody includes a recognition site for an antigen, i.e., the extracellular domain of CMKLR1, particularly the third loop of the extracellular domain of CMKLR1 (designated as EL3), and thereby includes its hypervariable domain or a fragment thereof designated as CDR (complementary determining region) that defines 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 the amino acid sequence of SEQ ID NO: 2. EL3 is also included within the polypeptide of SEQ ID NO: 52. Each light and heavy chain variable domain of a four-chain immunoglobulin (VL and VH, respectively) has three CDRs designated as VLCDR1, VLCDR2, and VLCDR3 for the light chain variable domain; and VHCDR1, VHCDR2, 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 as 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 the CDR domains and framework domains is the Kabat system.

[0045] One of ordinary skill in the art can determine the positions of the various regions / domains of an antibody by reference to standard definitions described herein, including a reference numbering system, reference to the Kabat numbering system, or application of the IMGT "collier de perle" algorithm. In this regard, it is known that the limits of the regions / domains may vary for each reference system for the definition of the sequences of the present invention. Accordingly, the regions / domains defined in the present invention include sequences that show a change in the length or position of the corresponding sequence within the full-length sequence of the variable domain of the antibody of about + / - 10%.

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

[0047] To illustrate specific embodiments of the invention, antigen-binding fragments of antibodies containing variable domains that include the CDRs of said antibodies include Fv, dsFv, scFv, Fab, Fab', F(ab')2. The Fv fragment consists of the VL and VH domains of the antibody that are bound together by hydrophobic interactions; in the dsFv fragment, the VH:VL heterodimer is stabilized by a disulfide bond; in the scFv fragment, the VL and VH domains are connected to each other by a flexible peptide linker, thus forming a single-chain protein. The Fab fragment is a monomeric fragment obtained by papain digestion of the antibody; they contain the entire L chain and the VH-CH1 fragment of the H chain, bound together by a disulfide bond. The F(ab')2 fragment can be produced by pepsin digestion of the antibody under hinge disulfides; it contains two Fab' fragments and, further, a part of the hinge region of the immunoglobulin molecule. The Fab' fragment is obtained from the F(ab')2 fragment by cleaving the disulfide bond in the hinge region. The F(ab')2 fragments are bivalent, i.e., they contain two antigen-binding sites, like the natural immunoglobulin molecule; on the other hand, the Fv (VHVL dimer that constitutes the variable part 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 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 invention.

[0048] As used herein, the term "modified antibody" includes "bispecific" antibodies and refers to antibodies that recognize two different antigens in that they have 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 one type of multispecific antibody. Multispecific antibodies that recognize more than two different antigens can be produced by recombinant DNA methods or include antibodies chemically produced by any convenient method, including but not limited to. Bispecific antibodies include all antibodies or antibody conjugates, or multimeric forms of antibodies, that can recognize two different antigens. Bispecific antibodies include antibodies and 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), kih (knob into hole), etc. that have been chemically coupled to have several antigen recognition sites for their respective antigens and that retain their bivalent nature upon reduction and reformation.

[0049] Antigen-binding antibody mimetics are organic compounds that specifically bind to an antigen but are not structurally related to antibodies. They are typically artificial peptides or small proteins with a molecular weight of about 3 - 20 kDa. Nucleic acids and small molecules may also be considered antibody mimetics, but are not artificial antibodies, antibody fragments, or fusion proteins composed thereof. General advantages over antibodies are better solubility, tissue penetration, stability to heat and enzymes, and relatively low production costs. Antigen-binding antibody mimetics can also be selected from the group including affibodies, affilins, affimers, affitins, DARPins, and monobodies.

[0050] Antigen-binding antibody mimetics are more preferably selected from the group consisting of affitins and anticalins. Affitins are artificial proteins having the ability to selectively bind to an antigen. Structurally, they are derived from the DNA-binding protein Sac7d found in Sulfolobus acidocaldarius, a microorganism belonging to the archaebacterial domain. By randomizing the amino acids on the binding surface of Sac7d, for example, by generating variants corresponding to the random substitution of 11 residues at the binding interface of Sac7d, an affitin library can be generated, and the resulting protein library can be subjected to rounds of ribosome display to direct affinity towards various targets such as peptides, proteins, viruses, and bacteria. Affitins are antibody mimetics and have been developed as tools in biotechnology. They have also been used as specific inhibitors for various enzymes (Krehenbrink et al., J. mol. Biol., 383:5, 2008). A person skilled in the art can easily develop an affitin having the required binding properties using methods known in the art, in particular, phage display and / or ribosome display library generation and their screening using the antigens disclosed herein, especially as disclosed in patent application WO2008068637 and the publications cited above. Anticalins are artificial proteins that can bind to either an antigen, a protein, or a small molecule. They are antibody mimetics derived from the family of natural binding proteins, human lipocalins. Anticalins are approximately one-eighth the size, having a size of approximately 180 amino acids and a mass of approximately 20 kDa (Skerra, Febs J., 275:11, 2008). In particular, an anticalin phage display library has been generated that enables the screening and selection of anticalins with specific binding properties.One skilled in the art can readily develop anti-kallins with the required binding properties, in particular using methods known in the art such as those disclosed in European Patent EP1270725B1, US Patent No. 8,536,307, Schlehuber and Skerra, Biophys. Chem., 96:2-3, 2002 and the publications cited above, in particular the generation of phage display and / or ribosome display libraries and their screening using the antigens disclosed herein. Both anti-kallins and affitins can be produced in several expression systems, including bacterial expression systems. Thus, the present invention relates to the use of affitins, anti-kallins and other similar antibody mimetics having the characteristics of the antibodies described herein, in particular with respect to their binding ability to CMKLR1, their agonist ability with respect to the binding between RvE1 and CMKLR1, their ability to induce or inhibit the secretion of the specific cytokines described herein, and their use in the treatment or prevention of the 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 modified by the mutation of at least one amino acid residue. Thus, "modified antibody" encompasses chimeric or humanized antibodies as defined herein, and "modified antibody" may also coincide with a molecule comprising an antibody or an antigen-binding fragment thereof, wherein the monoclonal antibody or a functional fragment thereof binds to a molecule that is functionally different. The modified antibodies of the present invention may be fusion chimeric proteins or conjugates obtained from any suitable form of binding, including covalent binding, grafting, chemical binding with a chemical or biological group, or a molecule suitable for protection against proteolytic cleavage in vivo with a molecule such as a PEG polymer or another protecting group, for the purpose of improving the stability and / or half-life of the antibody or functional fragment.

[0052] A "humanized" form of a non-human (e.g., mouse) antibody is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (Fv, Fab, Fab', F(ab')2, or other target-binding subsequence of an antibody) that contains minimal sequences derived from non-human immunoglobulins. Generally, a humanized antibody has all CDR regions identical to those of the non-human immunoglobulin and / or their humanized versions; all or substantially all of the FR regions are those of a human immunoglobulin template sequence or have substitutions of non-human residues (such as rodent residues) at corresponding positions to amino acid residues present in the human immunoglobulin template sequence, and will typically include substantially all of at least one, typically two variable domains. A humanized antibody may also include at least a portion of the immunoglobulin constant region (Fc), typically that of a selected human immunoglobulin template. 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, particularly an Fc region.

[0053] The terms "specifically binds" and "specifically binds to" mean that an antibody, antigen-binding fragment thereof, antigen-binding antibody mimetic, or modified antibody according to the invention binds to CMKLR1 with an affinity of at least 1×10 -6 M, 1×10 -7 M, 1×10 -8 M, 1×10 -9 M, 1×10 -10 M, 1×10 -11 M, 1×10 -12 M or greater and / or binds to CMKLR1 with an affinity that is at least 2-fold higher than its affinity for a non-specific target (e.g., a protein other than CMKLR1). Affinity can be evaluated 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 an amount of any given compound as defined herein that is sufficient for at least the amelioration of the clinical or physiological state of a 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 affected by considerations such as the disorder being treated, the particular mammal being treated, the clinical state 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 the physician.

[0055] All embodiments disclosed herein regarding an antibody or antigen-binding fragment thereof are replaced, where appropriate, with polymers of the invention, in particular, antigen-binding antibody mimetics and modified antibodies.

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

[0057] Both the heavy chain variable domain and the light chain variable domain each contain three CDRs (CDR1, CDR2, and CDR3, respectively, from the 5' end to the 3' end) and four framework regions (FR1, FR2, FR3, and FR4, respectively, from the 5' end to the 3' end). Humanization of a mouse antibody may consist of humanizing at least one framework region within the light chain variable region and / or within the heavy chain variable region or both. In certain embodiments, some framework regions can be humanized, particularly within the heavy chain variable region and within the light chain variable region. The wild-type CDRs may be preserved, or the CDRs can be replaced with the CDRs described herein. Thus, an anti-CMKLR1 compound according to the present invention may contain 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 parental chimeric antibody 2G1 (comprising the heavy chain variable domain of SEQ ID NO: 37 and the light chain variable domain of SEQ ID NO: 49) in which at least one framework region is humanized, particularly at least one framework region and at least one CDR are humanized. In certain embodiments of the present invention, the variable region of the antibody may be linked to 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 certain embodiments, the antibody or antigen-binding fragment thereof according to the invention is such that, in particular, the antibody light chain constant domain is derived from the human kappa light chain constant domain, and in particular, the light chain constant domain comprises or consists of the sequence of SEQ ID NO: 79, and the antibody heavy chain constant domain is derived from the human IgG1, IgG2, IgG3, or IgG4 heavy chain constant domain, in particular, the IgG1 heavy chain constant domain, and in particular, 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, which is particularly derived from the human IgG1 heavy chain constant domain, and in particular, the antibody heavy chain constant domain comprises or consists of the amino acid sequence of SEQ ID NO: 80 or SEQ ID NO: 83, and is a humanized monoclonal antibody.

[0058] For the binding to a polypeptide comprising or consisting of an amino acid residue located within the sequence of SEQ ID NO: 2 and / or SEQ ID NO: 59 and / or within the amino acid sequence of SEQ ID NO: 60, and / or for the binding to the third extracellular loop of CMKLR1, it can be evaluated by the binding affinity analysis by ELISA assay according to the examples disclosed in the embodiments of the present invention. To determine whether a test antibody can compete for binding to the same antigen or the third loop or the same epitope that is bound by the 2G1 antibody (or an antigen-binding fragment comprising the heavy chain variable domain corresponding to SEQ ID NO: 37 and the 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 an epitope or the third loop is coated on the wells of a microtiter plate, pre-incubated with or without a candidate competing antibody, and then the biotinylated 2G1 antibody of the present invention is added. The amount of the labeled anti-2G1 antibody bound to the polypeptide comprising or consisting of the polypeptide located within the amino acid sequence of SEQ ID NO: 2 and / or SEQ ID NO: 59 and / or within the amino acid sequence of SEQ ID NO: 60, and / or to 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 or a fluorescent label or some other detectable and measurable label. The amount of the 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 within the amino acid sequence of SEQ ID NO: 60, and / or to the third loop will have an indirect correlation 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 antigen-coated well. A candidate competing antibody is one where the candidate antibody can block the binding of the 2G1 antibody by at least 20%, preferably at least 20 - 50%, more preferably at least 50% compared to a control performed simultaneously in the absence of the candidate competing antibody (however, it may also be in the presence of a known non-competing antibody). It is contemplated that antibodies that bind to the same polypeptide as the 2G1 antibody of the present invention or compete for binding to the third loop. It will be understood that variations of this assay can be performed to reach the same quantitative values.

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

[0060] In certain embodiments of the present invention, certain VHCDRs are present in antibodies that can exhibit reduced immunogenicity in humans, such that when an antibody with reduced immunogenicity is administered to a patient, there are fewer expected side effects, enabling the provision of an antibody that can be more potent for therapeutic purposes.

[0061] In certain aspects of the present invention, an anti-CMKLR1 antibody or antigen-binding fragment thereof that binds to chemokine-like receptor 1 (CMKLR1), particularly human CMKLR1, a. An antibody heavy chain variable (VH) domain comprising three CDRs, VHCDR1, VHCDR2, and VHCDR3, wherein - 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; - 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, 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; - VLCDR3 is a variable light (VL) domain of an antibody, 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 as described above is disclosed.

[0062] In particular, the antibody or antigen-binding fragment thereof specifically binds to an epitope located within the third extracellular loop (EL3) of CMKLR1. More specifically, 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.

[0063] In another specific embodiment of the present invention, an anti-CMKLR1 antibody or antigen-binding fragment thereof that binds to chemokine-like receptor 1 (CMKLR1), particularly human CMKLR1, wherein a. A variable heavy (VH) domain of an antibody comprising three CDRs, VHCDR1, VHCDR2, and VHCDR3, wherein - 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 is identical to the amino acid residue of SEQ ID NO: 12 or SEQ ID NO: 63, provided that VHCDR1 is not SEQ ID NO: 3 or SEQ ID NO: 4; - 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, a variable heavy (VH) domain of an antibody; b. A variable light (VL) domain of an antibody comprising three CDRs, VLCDR1, VLCDR2, and VLCDR3, 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; - VLCDR3 is selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36, an antibody light chain variable (VL) domain is disclosed, said antibody or antigen-binding fragment thereof.

[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 specifically, 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.

[0065] In another specific embodiment of the present invention, an anti-CMKLR1 antibody or antigen-binding fragment thereof that binds to chemokine-like receptor 1 (CMKLR1), particularly human CMKLR1, wherein a. an antibody heavy chain variable (VH) domain comprising three CDRs, VHCDR1, VHCDR2, and VHCDR3, wherein - 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 matches the amino acid sequence of SEQ ID NO: 12, preferably, said heavy chain variable (VH) domain does not contain the framework FR3 of SEQ ID NO: 69, provided that said heavy chain variable (VH) domain contains the framework VHFR3 of SEQ ID NO: 70; - 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, an antibody heavy chain variable (VH) domain; b. An antibody light chain variable (VL) domain comprising three CDRs, VLCDR1, VLCDR2 and VLCDR3, 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; - VLCDR3 is selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36, the antibody light chain variable (VL) domain Including, said antibody or antigen-binding fragment thereof is disclosed.

[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 specifically, the antibody or antigen-binding fragment thereof is a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 59, or specifically binds to an epitope located within the amino acid sequence of SEQ ID NO: 60.

[0067] In particular, when 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, the binding to the epitope located within the third extracellular loop (EL3) of CMKLR1 can be evaluated according to the method disclosed above in this specification. The specific selection of VHCDR2 enables the provision of an antibody with reduced immunogenicity compared to antibodies having different VHCDR2s.

[0068] Such an antibody is an agonist of CMKLR1 that mimics the effect of the binding of resolvin E1 to CMKLR1, i.e., has the resolvin E1-like ability as defined above in this specification. The anti-CMKLR1 antibody or antigen-binding fragment thereof has the effect of a dissipative factor of inflammation, in particular, has such an effect by interacting with the myeloid cell lineage.

[0069] In certain embodiments of the present invention, the anti-CMKLR1 antibody or antigen-binding fragment thereof is - VHCDR1 selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 4 and comprises.

[0070] In certain embodiments of the present invention, the anti-CMKLR1 antibody or antigen-binding fragment thereof is - 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 comprises.

[0071] In certain embodiments of the present invention, the anti-CMKLR1 antibody or antigen-binding fragment thereof is - VHCDR3 selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15 and comprises.

[0072] In certain embodiments of the present invention, the anti-CMKLR1 antibody or antigen-binding fragment thereof is - 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 comprises.

[0073] In certain embodiments of the present invention, the anti-CMKLR1 antibody or antigen-binding fragment thereof is - 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 comprises.

[0074] In certain embodiments of the present invention, the anti-CMKLR1 antibody or antigen-binding fragment thereof is - VLCDR3 selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36 and comprises.

[0075] In certain embodiments of the present invention, the anti-CMKLR1 antibody or antigen-binding fragment thereof is - VHCDR1 selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 4; and / or - 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 - VHCDR3 selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15 - 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 - 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 - VLCDR3 selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36 comprising.

[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 invention, the anti-CMKLR1 antibody or antigen-binding fragment thereof comprises 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, or consists of a heavy chain variable domain.

[0078] In a more particular 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 an antigen-binding fragment thereof comprises, or consists of, a light chain variable domain comprising 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 an antigen-binding fragment thereof comprises, or consists of, a light chain variable domain comprising the amino acid sequence of SEQ ID NO:93.

[0081] In certain embodiments of the present invention, the anti-CMKLR1 antibody or an antigen-binding fragment thereof - comprises, or consists of, a heavy chain variable domain comprising 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 - comprises, or consists of, a light chain variable domain comprising 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 and is included.

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

[0083] In a more specific embodiment, the anti-CMKLR1 antibody or an antigen-binding fragment thereof according to the present invention comprises, or consists of, a heavy chain variable domain comprising an amino acid residue 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 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 an 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 an 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, the antibody is preferably characterized in that its Fc fragment is characteristic of IgG1.

[0087] In a second aspect of the present invention, a compound selected from the group of antibodies, antigen-binding fragments or chimeric, modified or humanized antibodies that specifically bind to CMKLR1, particularly human CMKLR1, (i) comprises or consists 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 VHCDR2, and (ii) comprises or consists 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 VHCDR3, provided that the amino acid residues at positions 1 and 2 of the mutant sequence are L and I, respectively, and includes its mutant sequence in which the amino acid residues are substituted. The anti-CMKLR1 compound specifically binds to an epitope located within the third extracellular loop (EL3) of CMKLR1, particularly, the compound specifically binds to a polypeptide comprising or consisting of 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. A compound that 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 is identical to the parental antibody 2G1. Binding of a compound to a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 59 or located within SEQ ID NO: 60, or to a polypeptide comprising or consisting of the third loop (EL3) of the extracellular domain of CMKLR1 can be evaluated according to the methods disclosed above in this specification and exemplified in the examples of the present invention.

[0089] In a more specific embodiment, the anti-CMKLR1 antibody or an 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 an 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 an 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, the antibody is preferably characterized in that its Fc fragment is characteristic of IgG1.

[0093] The present invention relates to the use of a compound of the present invention as defined above for the prevention and / or treatment of diseases in which the resolution of inflammation is delayed or disrupted, and / or 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, cystic fibrosis, chronic inflammatory diseases such as skin inflammation; autoimmune diseases such as diabetes, NASH, in particular, type I diabetes, psoriasis, lupus, rheumatoid arthritis, multiple sclerosis, Sjögren's syndrome, celiac disease, vasculitis, myasthenia gravis; infectious diseases with severe inflammatory conditions such as sepsis, coronavirus (e.g., COVID-19), 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, leukemia, in particular, cancers selected from the group of cancers in which cancer cells express CMKLR1, or the tumor microenvironment expresses CMKLR1, or is infiltrated by cells that overexpress it.

[0094] Fibrosis (especially pulmonary and hepatic fibrosis), ANCA (antineutrophil cytoplasmic autoantibody) pathology (vasculitis), and pathologies resulting from apoptosis of neurons associated with ChemR23 are of particular interest.

[0095] In certain embodiments, the present invention relates to the use of the compounds of the present invention as defined above for the prevention and / or treatment of diseases in which the resolution of inflammation is delayed or disrupted, and / or inflammatory diseases, in particular, 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, chronic inflammatory diseases such as cystic fibrosis; autoimmune diseases such as diabetes, in particular, type I diabetes, psoriasis, lupus, rheumatoid arthritis, multiple sclerosis, Sjögren's syndrome, celiac disease, vasculitis, myasthenia gravis; infectious diseases such as sepsis, peritonitis, severe viral symptoms with severe inflammatory conditions such as coronavirus (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 the use of the compounds of the present invention as defined above for 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 cancer cells express CMKLR1, or the tumor microenvironment expresses CMKLR1, or is infiltrated by cells that overexpress CMKLR1.

[0096] In certain embodiments, the antibodies of the present invention elicit at least one of the following effects that are advantageous for dissipation in vitro and / or in vivo: - Increasing the apoptosis of polymorphonuclear neutrophils, referred to herein under the acronym PMN or PMN, on the inflammatory site; such an effect is exemplified in the examples of the present invention. The regulation of neutrophil apoptosis is important for therapeutic intervention in inflammation-related diseases. As exemplified in the examples of the present invention, the use of an agonist of CMKLR1 having a resolvin E1-like ability antibody against PMN induces strong apoptosis of neutrophils during inflammation induction as compared to control antibodies. - Enhance caspase-3 expression in neutrophils, thereby leading to caspase-3-dependent apoptosis; apoptosis may be considered to be enhanced or induced when the expression of caspase 3 exceeds 1 log, preferably 2 log, most preferably 3 log, 11 hours after treatment with an agonist of CMKLR1 having a resolvin E1-like ability antibody, compared to the negative control; the method is disclosed in the examples of the present invention. - Decrease or inhibit the migration of PMNs (neutrophils) from the endothelium towards the site of inflammation; prevent them from being repositioned to the site of inflammation and exerting their pro-inflammatory effect. In certain embodiments of the present invention, the migration and movement of neutrophils are prevented or reduced by administration of an agonist of CMKLR1 having a resolvin E1-like ability IgG1 antibody. Thus, in certain embodiments, the agonist of CMKLR1 having a resolvin E1-like ability is a humanized antibody having a human constant region derived from or resulting from human IgG1. In certain embodiments, the agonist of CMKLR1 having a resolvin E1-like ability antibody is of the human IgG1 isotype, i.e., a constant fragment of the heavy and light chains derived from or resulting from the human IgG1 antibody constant heavy and light chain fragments. Thus, the agonist of CMKLR1 having a resolvin E1-like ability antibody of the present invention comprises the Fc domain of the IgG1 isotype. The migration of neutrophils can be evaluated by the method disclosed in the examples of the present invention; when the cell surface expression of CD62L is reduced by at least 1 log in a staining experiment compared to the negative control, the neutrophils can be considered to have reduced migration and / or movement ability. The inventors have found that the agonist of CMKLR1 having a resolvin E1-like ability humanized IgG1 antibody of the present invention does not exhibit cytotoxicity in vivo; in other words, the use of the agonist of CMKLR1 does not show significant depletion of CMKLR1-positive cells in vivo. In certain embodiments, the use of the agonist of the present invention does not show cytotoxic activity against CMKLR1-positive cells. - It reduces the cell surface expression of CMKLR1, as well as CXCR4 and / or CCR7. In certain embodiments, the cell surface expression is believed to be on macrophages and / or dendritic cells. When an agonist of CMKLR1 having the ability of resolvin E1 binds to its target, the CMKLR1, as well as the CXCR4 and / or CCR7 moieties, heterodimerize and internalize. In a preferred embodiment, the antibody of the present invention induces the internalization of CMKLR1 and / or CXCR4 and / or CCR7 on the cell surface of CMKLR1-positive cells and / or inhibits their expression. Thus, the cell surface expression of CMKLR1, as well as CXCR4 and / or CCR7, in cells incubated in the presence of the antibody is decreased or significantly decreased compared to the cell surface expression in cells incubated under the same conditions but in the absence of an agonist of CMKLR1 having a resolvin E1-like ability.

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

[0098] In certain embodiments, the humanized anti-CMKLR1 antibody or an antigen-binding fragment thereof specifically binds to the third extracellular loop (EL3) of CMKLR1, and in particular, the antibody or an 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 the production of the antibody (or an antigen-binding fragment thereof) in vitro. It should be noted that this definition of the antibody is independent of any other definition of the anti-CMKLR1 antibody of the present invention, including the definition of the antibody by the CDR domain. In certain aspects of the present invention, the definition of the antibody of the present invention by the FR sequence can be combined with the CDR domain and / or functional characteristics by definition. For this purpose, antibodies defined by their FR domains selected from a particular group and characterized by their CDR domains are also encompassed by the present invention. For this purpose, such antibodies, which may or may not show identity to the framework domains disclosed herein, have the following CDRs: - 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 - 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 chain (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 Of which at least one, in particular including 6.

[0099] In a more specific embodiment, at least one of the framework domains HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4 is from the following group: For VHFR1, SEQ ID NO: 65; 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; For VHFR4, SEQ ID NO: 71; For VLFR1, SEQ ID NO: 72; 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 Selected from.

[0100] It should be understood that multiple framework domains, or all, can be selected from the groups described above in this specification.

[0101] In a more specific embodiment, the antibody or antigen-binding fragment thereof described in the above two paragraphs has an amino acid sequence of a heavy chain variable domain selected from the group consisting 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 which is the sequence of SEQ ID NO: 50.

[0102] In a more specific embodiment, the antibody or antigen-binding fragment thereof has 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 and includes.

[0103] In a more specific embodiment, the antibody has 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 and includes, the following CDRs: - 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 - 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 chain (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 comprises.

[0104] This embodiment can facilitate the production of an antibody (or its antigen-binding fragment) in vitro.

[0105] The present invention particularly relates to a humanized anti-CMKLR1 antibody or its antigen-binding fragment optimized to reduce immunogenicity while maintaining high binding activity, stability and biological function, and having 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 relates to an antibody or its antigen-binding fragment comprising.

[0106] In certain embodiments, said antibody or its antigen-binding fragment wherein the CDR sequences are 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 has 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 is included.

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

[0108] Furthermore, the antibody is preferably 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 an antigen-binding fragment thereof according to this embodiment has a heavy chain variable domain VHCDR2 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, - In particular, as a result of the administration of treatment, enhanced resolution of inflammation, inflammatory diseases, particularly acute inflammatory diseases, chronic inflammatory lung diseases (e.g., asthma), keratoconjunctivitis, periodontal disease, eczema, inflammatory bowel diseases, particularly Crohn's disease or colitis, particularly ulcerative colitis or spontaneous colitis, chronic inflammatory diseases such as cystic fibrosis, NASH (non-alcoholic steatohepatitis), liver fibrosis, pulmonary fibrosis, anti-neutrophil cytoplasmic antibody-related diseases (ANCA), vasculitis, particularly ANCA-mediated vasculitis, scleroderma; - In particular, as a result of the administration of the treatment, enhanced dissipation of inflammation, diabetes, especially type I diabetes, psoriasis, lupus, rheumatoid arthritis, multiple sclerosis, Sjögren's syndrome, celiac disease, vasculitis, autoimmune diseases such as myasthenia gravis, or infectious diseases such as sepsis, peritonitis, degenerative diseases, wound healing disorders or dry eye syndrome, severe viral symptoms with severe inflammatory conditions such as coronavirus (e.g., COVID-19); - In particular, as a result of the administration of the treatment, enhanced dissipation of inflammation, cancer, especially metastatic cancer, solid or liquid cancer, e.g., carcinoma, more specifically, liver cancer, especially breast cancer or colon cancer, or myeloid cancer such as lung cancer or leukemia, especially cancer in which cancer cells express CMKLR1, or the tumor microenvironment expresses CMKLR1, or is infiltrated by cells that overexpress it for prophylactic or therapeutic treatment of a) An antibody heavy chain variable (VH) domain comprising three CDRs, VHCDR1, VHCDR2, and VHCDR3, wherein - 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 is identical to the amino acid residues of SEQ ID NO: 12 on the condition that VHCDR1 is not SEQ ID NO: 3 or SEQ ID NO: 4; - 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, an antibody heavy chain variable (VH) domain; b) An antibody light chain variable (VL) domain comprising three CDRs, VLCDR1, VLCDR2, and VLCDR3, 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, an antibody light chain variable (VL) domain; - 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; - The 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 An antibody or an antigen-binding fragment thereof is provided, which comprises the same.

[0111] In another specific 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, wherein - 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 matches the amino acid sequence of SEQ ID NO: 12, preferably, the heavy chain variable (VH) domain does not contain the framework FR3 of SEQ ID NO: 69, provided that the heavy chain variable (VH) domain contains the framework VHFR3 of SEQ ID NO: 70 - 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, 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; - VLCDR3 is selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36 An antibody or an antigen-binding fragment thereof is provided, which comprises the same.

[0112] In certain embodiments of the present invention, a humanized antibody or antigen-binding fragment thereof, or an antigen-binding antibody mimetic or engineered antibody is - In particular, as a result of administration of the treatment, enhanced resolution of 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), liver fibrosis, pulmonary fibrosis, anti-neutrophil cytoplasmic antibody-related diseases (ANCA), vasculitis, in particular, ANCA-mediated vasculitis, scleroderma; - In particular, as a result of administration of the treatment, enhanced resolution of inflammation, diabetes, in particular, type I diabetes, psoriasis, lupus, rheumatoid arthritis, multiple sclerosis, Sjogren's syndrome, celiac disease, vasculitis, autoimmune diseases such as myasthenia gravis, or infectious diseases such as sepsis, peritonitis, degenerative diseases, impaired wound healing, severe viral symptoms with severe inflammatory conditions such as coronavirus (e.g., COVID-19), or dry eye syndrome; - In particular, as a result of administration of the treatment, enhanced resolution of inflammation, cancer, in particular, metastatic cancer, solid or liquid cancer, e.g., carcinoma, more specifically, liver cancer, in particular, breast cancer or colon cancer, or myeloid cancer such as lung cancer or leukemia, in particular, cancer in which cancer cells express CMKLR1, or the tumor microenvironment expresses CMKLR1, or is infiltrated by cells that overexpress CMKLR1 for prophylactic or therapeutic treatment of a variable heavy (VH) domain having sequence identity with the framework of the same rank in the sequence of SEQ ID NO: 41, comprising the amino acid sequences of the frameworks (FR1, FR2, FR3 and FR4) of the heavy chain variable domain, each framework being, respectively, 100% for FR1, at least 60% for FR2, at least 78% for FR3 and at least 80% for FR4; 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 chain (VL) domain includes the amino acid sequences of the frameworks (FR1, FR2, FR3, and FR4) of the light chain variable domain, with each framework having, respectively, 60% for FR1, at least 70% for FR2, at least 75% for FR3, and at least 80% for FR4; more specifically, 100% for FR1, at least 90% for FR2, at least 90% for FR3, and 100% for FR4, and having sequence identity with the frameworks of the same rank in the sequence of SEQ ID NO: 50. and includes.

[0113] In a more specific embodiment, the antibody has 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 and includes the following CDRs: - 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 - 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; - 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 chain (VL) domain includes the following: - 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 - 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 - VLCDR3 selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36 comprising - In particular, as a result of administration of the treatment, enhanced resolution of inflammation, inflammatory diseases, in particular, acute inflammatory diseases, chronic inflammatory lung diseases (e.g., asthma), keratoconjunctivitis, periodontal diseases, 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, anti-neutrophil cytoplasmic antibody-related diseases (ANCA), vasculitis, in particular, ANCA-mediated vasculitis, scleroderma; - In particular, as a result of administration of the treatment, enhanced resolution of inflammation, diabetes, in particular, type I diabetes, psoriasis, lupus, rheumatoid arthritis, multiple sclerosis, Sjögren's syndrome, celiac disease, vasculitis, autoimmune diseases such as myasthenia gravis, or infectious diseases such as sepsis, peritonitis, degenerative diseases, wound healing disorders, severe viral symptoms with severe inflammatory conditions such as coronavirus (e.g., COVID-19), or dry eye syndrome; - In particular, as a result of administration of the treatment, enhanced resolution of inflammation, cancer, in particular, metastatic cancer, solid or liquid cancer, e.g., carcinoma, more specifically, liver cancer, in particular, breast cancer or colon cancer, or myeloid cancer such as lung cancer or leukemia, in particular, for use in prophylactic or therapeutic treatment of cancer in which cancer cells express CMKLR1, or the tumor microenvironment expresses CMKLR1, or is infiltrated by cells that overexpress it.

[0114] Such a specific antibody or antigen-binding fragment thereof for one of the prophylactic or therapeutic treatments in the state disclosed above, in particular, it contains 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, and is characterized by this. In particular, for the prophylactic or therapeutic use disclosed above of the antibody or antigen-binding fragment thereof, the amino acid sequence of the heavy chain variable domain of the antibody contains or consists of SEQ ID NO: 91, and the amino acid sequence of the light chain variable domain of the antibody contains 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 specific 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 immunomodulatory agent, an immune checkpoint blocker, an immune checkpoint activator, an antibody, or an anti-SIRPa antibody (anti-mouse SIRPa from P84-Merck Millipore).

[0116] In another aspect, the present invention relates to a combination of compounds comprising an anti-CMKLR1 compound 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 a compound is, in particular, selected from the group consisting of antibodies, antigen-binding antibody fragments, antigen-binding antibody mimetics, aptamers, or small molecules such as peptides, and modified antibodies such as humanized or chimeric antibodies that can bind to PD1 or PDL1, although not limited to these.

[0117] In another aspect, the present invention relates to a combination of compounds comprising an anti-CMKLR1 compound 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 small molecules such as antibodies, antigen-binding antibody fragments, antigen-binding antibody mimetics, aptamers or peptides that can bind to SIRPa, in particular human SIRPa, and are selected from the group consisting of modified antibodies such as humanized or chimeric antibodies, but are not limited thereto.

[0118] The present invention also relates to a combination for, for example, treating fibrosis, comprising a cytokine or a therapeutic compound that is not a cytokine and stimulates resolution macrophages.

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

[0120] In certain embodiments of the present invention, the anti-CMKLR1 compound binds to CMKLR1 with an affinity (KD value) of at least 10E-8 M, more preferably at least 10E-9 M. Specific binding of an antibody of the present invention, or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic or modified antibody, to CMKLR1 (or a region of CMKLR1 comprising the third extracellular loop containing 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" includes binding with an affinity of about 10 -8 M (KD) or stronger. Preferably, the binding affinity is 10 -8 M to 10 -12 M, and optionally 10 -9 M to 10 -10 M, particularly at least 10 -9When M, the binding is considered to be specific. Whether the binding domain specifically reacts with or binds to a target can be readily tested, in particular, by comparing the reaction of the binding domain with a target protein or antigen with the reaction of the binding domain with a protein or antigen other than the target protein. Such an antibody of the present invention specifically binds to CMKLR1 and has 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, for example, 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 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. An anti-CMKLR1 compound that binds within this specific region of CMKLR1 has agonistic properties for CMKLR1 and can thereby mimic the binding of RvE1 to CMKLR1.

[0122] In another aspect, the invention relates to an anti-CMKLR1 antibody or an antigen-binding fragment or antigen-binding antibody mimetic or modified antibody as defined hereinabove having agonistic ability for the interaction between RvE1 and CMKLR1 for use as a medicament.

[0123] In another aspect, the present invention relates to an anti-CMKLR1 antibody or an antigen-binding fragment or antigen-binding antibody mimetic or modified antibody as defined hereinabove having the ability to induce the activation of Akt and / or Erk protein in vitro and / or in vivo. The 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 or antigen-binding antibody mimetic or 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, the method comprising administering to the subject an effective amount of an anti-CMKLR1 antibody or an antigen-binding fragment or antigen-binding antibody mimetic as defined above, which has agonist ability for the interaction between RvE1 and CMKLR1, or in other words, a factor or modulator such as an RvE1 agonist.

[0125] Modulation of macrophage polarization favoring anti-inflammatory cells may be useful in several pathologies or situations. As described above, this modulation is useful, but not limited to, in the context of diseases selected from the group of inflammatory diseases including 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 I diabetes, peritonitis, psoriasis, cancer, particularly breast cancer or colon cancer, carcinoma, metastatic cancer, lung cancer, degenerative diseases, infectious diseases, particularly sepsis, autoimmune diseases, NASH, scleroderma, colitis or Crohn's disease in a subject 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 or antigen-binding antibody mimetic as defined above having 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 a modified antibody, such as a humanized or chimeric antibody as defined hereinabove, for use in the treatment of chronic inflammatory diseases, in particular for treating ulcerative 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 agonist activity against the interaction of CMKLR1, for use in the treatment or prevention of diseases selected from the group of inflammatory diseases, including inflammatory conditions, in particular those inflammatory diseases in which the dissipation is delayed or disrupted, and / or, without limitation, acute and chronic inflammatory diseases, inflammatory bowel diseases, Crohn's disease, asthma, keratoconjunctivitis, periodontal disease, eczema, colitis, in particular ulcerative colitis or spontaneous colitis, cystic fibrosis, diabetes, in particular type I diabetes, peritonitis, psoriasis, cancer, in particular breast cancer or colon cancer, tumors, degenerative diseases, infectious diseases, in particular sepsis, autoimmune diseases.

[0129] As defined herein, "delayed or disrupted resolution of the inflammatory state" occurs when the resolution of inflammation is delayed or disrupted compared to normal resolution (i.e., the resolution that occurs in patients who experience physiological resolution after an inflammatory event). Delayed or defective resolution can result in an increased infiltration of granulocytes at the site of inflammation. Thus, delayed or defective resolution can be evaluated by quantification of granulocytes at the site of inflammation. For example, the granulocyte population can be measured by indirect biochemical techniques such as histology, blood cell counting or quantification of elastase by enzyme immunoassay or molecular quantification of granulocyte receptor 1 by PCR. Delayed or defective resolution can also be evaluated by determination of delayed apoptosis of granulocytes, measured, for example, by blood cell counting using a specific antibody against annexin 5. Defects or delays in the details of inflammation can also be determined by evaluating the synthesis of inflammatory cytokines such as TNF-alpha, IL8 or IL12 and anti-inflammatory cytokines such as IL-10. Cytokine secretion can be evaluated by enzyme immunoassay or by PCR. Defects or delays in the resolution of inflammation can also be determined by evaluating the activation of transcription factors involved in the synthesis of inflammatory cytokines such as NF-kappaB, which can be measured, for example, by nuclear translocation or by quantification of the degradation level of Western blot and / or IkappaB. Defects or delays in the resolution of inflammation can also be determined by quantifying specific inflammatory resolving mediators (lipoxin, resolvin, protectin or maresin, etc.) or their precursors (17-HDOHE or 14-HDOHE) by mass spectrometry or enzyme immunoassay. Subsequently, defective or delayed resolution results in a defect in the synthesis of one or more of these mediators. Defects or delays in resolution can also be determined when the expression of the receptor for the resolving molecule is decreased. These receptors can be selected from the group comprising ALX, CMK1R1, GPR32 or GPR18. Alternatively, or complementarily, the internalization and processing of these receptors into the cytoplasm can also be evaluated. Alternatively, or complementarily, the expression of receptors for some inflammatory cytokines or lipids can also be evaluated, and overexpression compared to the normal state is a significant delay in the resolution of inflammation Or is a defect. These states can be measured by histology, cytology or PCR. Defects in resolution can also result in a decrease or inhibition of the switch to inflammatory resolution macrophages, or damage to the phagocytosis or efferocytosis of the same cells. Thus, a delay or defect in resolution can be evaluated by analyzing the switch to inflammatory resolution macrophages in a particular state compared to the normal state, as exemplified in the examples of the present invention.

[0130] According to certain embodiments, an anti-CMKLR1 compound can be used to treat an individual having a cancer selected from the group consisting of breast cancer, particularly breast carcinoma, melanoma, colon cancer, particularly colon carcinoma, leukemia, particularly acute myeloid leukemia, especially when the cancer cells overexpress CMKLR1.

[0131] In certain embodiments, the invention relates to the anti-human CMKLR1 antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic or modified antibody as defined above for use as defined above, which is administered to a patient presenting with a CMKLR1-positive tumor.

[0132] The antibody or antigen-binding fragment thereof of the 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 in combination with another therapeutic agent, such as a second human monoclonal antibody or antigen-binding fragment thereof. In another example, the antibody is administered together with another agent, such as an immunosuppressive agent, an erythropoiesis-stimulating agent (ESA), along with a therapeutic cell composition, etc. In certain embodiments, the invention relates to an anti-CMKLR1 compound or antigen-binding fragment thereof or antigen-binding antibody mimetic as defined above for its use as defined above, wherein the anti-CMKLR1 antibody or antigen-binding fragment thereof is combined with a second therapeutic agent.

[0133] 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, co-administration can be prepared in the form of a combination drug (product), also known as a "combo". A combo is a fixed-dose combination that contains two or more active pharmaceutical ingredients mixed in a single dosage form, which is manufactured and distributed at a fixed dose. However, the dosage regimen and / or route of administration may be different.

[0134] In a preferred embodiment, this second therapeutic agent is selected from the group consisting of chemotherapeutic agents, radiation therapy agents, immunotherapeutic agents, cell therapy agents (CAR-T cells), antibiotics, and probiotics.

[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" particularly refers to a T cell growth factor that increases the number and repertoire of naive T cells, a growth factor that increases the number of dendritic cells (DCs), an agonist that activates DCs and other antigen-presenting cells (APCs), an adjuvant that enables and increases cancer vaccines, an agonist that activates and stimulates T cells, an inhibitor of T cell checkpoint blockade, a T cell growth factor that increases the proliferation and survival of immune T cells, an agent that inhibits, blocks, or neutralizes cancer cells, and a drug that can take a cancer vaccine from interesting biological phenomena for effective therapeutic agents, including immunosuppressive cytokines derived from immune cells.

[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, in particular, anti-CD137 and anti-SIRPa. In certain embodiments of the present invention, the second therapeutic agent is an anti-PDL1 or anti-PD1 compound, in particular, an anti-PD1 compound, more specifically, an anti-PD1 antibody. In certain embodiments of the present invention, the second therapeutic agent is an anti-SIRPa compound, in particular, an anti-SIRPa antibody.

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

[0139] The antibody 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, the dosage may range from 1 μg / kg to about 20 mg / kg, optionally from 10 μg / kg to 10 mg / kg or 100 μg / kg to 5 mg / kg.

[0140] The terms "effective amount" or "effective dosage" or "effective dose" are defined as an amount sufficient to achieve, or at least partially achieve, the desired effect. The term "effective amount" is meant to include an amount sufficient to cure, or at least partially arrest, a disease and its complications, or to alleviate the symptoms of a disease in a patient already suffering therefrom. The amount 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 therapy, the patient's clinical history and response to the therapeutic agent, the route of administration, the size (weight, body surface area or organ size) and / or condition (age and general health) of the patient, and the general state of the patient's own immune system. Appropriate dosages can be adjusted to administer the compound to the patient either as a single dose or over a series of doses and to obtain the optimal therapeutic effect.

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

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

[0143] As used herein, "pharmaceutical composition" means a composition suitable for administration to a mammalian subject or patient, particularly a human. Generally, a "pharmaceutical composition" is sterile and usually free of contaminants that can cause undesirable responses within the subject (e.g., the compounds in the pharmaceutical composition are of pharmaceutical grade). The pharmaceutical composition 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" generally means excipients, diluents, carriers, and adjuvants that are useful in preparing pharmaceutical compositions that are generally safe, non-toxic, and neither biologically nor otherwise undesirable, and includes excipients, diluents, carriers, and adjuvants that are acceptable for veterinary use as well as pharmaceutical use in humans. As used herein, "pharmaceutically acceptable" includes both one and more than one such excipients, diluents, carriers, and adjuvants.

[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, particularly a combination product means, comprising, as an active ingredient, an anti-SIRPa antibody or an antigen-binding fragment thereof or an antigen-binding antibody mimetic as defined above and a second therapeutic agent, wherein the active ingredient is formulated for individual therapy, sequential therapy, or combination therapy, particularly for combined or sequential use.

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

[0148] In certain embodiments, the present invention relates to the combination product as defined above, wherein the second therapeutic agent is selected from the group consisting of chemotherapeutic agents, radiation therapy agents, cell therapy agents, immunotherapeutic agents, antibiotics, and probiotics.

[0149] In certain embodiments, the present invention relates to the combination product as defined above, wherein the immunotherapeutic agent is selected from the group consisting of therapeutic vaccines, particularly 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 the 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 agonist, CD40-L, TLR agonist, anti-ICOS, ICOSL and B cell receptor agonist, particularly 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, particularly an anti-PD1 compound, more specifically an anti-PD1 antibody. In a particular embodiment of the present invention, the second therapeutic agent is an anti-SIRPa compound, particularly an anti-SIRPa antibody.

[0151] In one embodiment, the immunotherapeutic agent is also an antibody targeting a tumor antigen, particularly 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 the combination product as defined above for simultaneous, separate or sequential use in the treatment of any condition that is amenable to improvement or prevention by modifying macrophage polarization to resolution-promoting macrophages.

[0153] In one embodiment, the present invention is a method for treating any condition that is amenable to improvement or prevention by modifying macrophage polarization to resolution-promoting macrophages in a subject in need thereof, the method comprising administering to the subject an effective amount of the combination product as defined above simultaneously, separately or sequentially.

[0154] In one embodiment, the present invention relates to the use of the combination product as defined above in the manufacture of a medicament for the treatment of any condition that is amenable to induction of resolution-promoting inflammatory macrophages.

[0155] In one aspect, the present invention relates to the combined 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 diseases, Crohn's disease, NASH, scleroderma, asthma, keratoconjunctivitis, periodontal disease, eczema, colitis, particularly ulcerative colitis or spontaneous colitis, cystic fibrosis, diabetes, particularly type I diabetes, peritonitis, psoriasis, cancer, particularly breast cancer or colon cancer, carcinoma, metastatic cancer, lung cancer, degenerative diseases, infectious diseases, particularly sepsis, and autoimmune diseases, or for use in vaccination.

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

[0157] The present invention also relates to polynucleotides encoding the anti-CMKLR1 compounds as defined herein. To this end, the invention also relates to nucleic acids encoding any anti-CMKLR1 compound according to the present disclosure, more specifically, a heavy chain variable domain comprising or consisting of the amino acid residues of the sequences set forth in SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 62, and a nucleic acid molecule, or group of nucleic acid molecules, encoding a light chain variable domain comprising or consisting of the amino acid residues of the sequences set forth in 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 and SEQ ID NO: 58, more specifically, an isolated nucleic acid molecule and / or a recombinant nucleic acid molecule. In certain embodiments, the invention relates to a nucleic acid molecule, or group of nucleic acid molecules, encoding the amino acid sequence of the heavy chain variable domain of the antibody consisting of SEQ ID NO: 91 and the light chain variable domain of the antibody consisting of SEQ ID NO: 93, more specifically, an isolated nucleic acid molecule and / or a recombinant nucleic acid molecule.

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

[0159] The present invention also relates to vectors comprising the polynucleotides disclosed herein or nucleic acid molecules comprising the nucleic acid molecules disclosed herein. As used herein, a vector is a nucleic acid molecule used as a vehicle to introduce genetic material into a cell and, in preferred embodiments, enables the expression of a polynucleotide inserted therein. The term vector includes plasmids, viruses, cosmids, and artificial chromosomes. Vectors generally contain an origin of replication, a multiple cloning site, and a selectable marker. The vector itself is generally a nucleotide sequence, typically a DNA sequence, containing an insert (transgene) and a larger sequence that serves as the "backbone" of the vector. Modern vectors may include additional features in addition to the transgene insert and backbone: promoters, gene markers, antibiotic resistance, reporter genes, targeting sequences, protein purification tags. Specifically, a vector called an expression vector (expression construct) is for the expression of a transgene in a target cell and generally has control 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 a vector as defined above. As used herein, these terms with respect to a cell may refer to a recipient of a vector encoding an antibody construct of the present invention, an exogenous nucleic acid molecule, and a polynucleotide, and / or a recipient of the antibody construct itself, or any individual cell or cell culture that was or may be. The introduction of each material into the cell can be carried out 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 and include, but are not limited to, bacterial, yeast, fungal, plant cells, and insect and mammalian cells, such as animal cells of mice, rats, rabbits, macaques, or humans.

[0161] In certain embodiments of the invention, the cell or cell line is selected from the group consisting of CHO, COS, and HEK cells, and when genetically engineered using a vector, exogenous nucleic acid molecule, and polynucleotide encoding the 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 specifically at least 100 mg / ml; and / or is a recipient of the antibody construct itself.

[0162] The following drawings and examples are set forth to provide a complete disclosure and description to those skilled in the art 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 show that the following experiments are all or the only experiments performed. The invention has been described with reference to specific embodiments, but those skilled in the art will understand that various changes can be made and equivalents can be substituted without departing from the true spirit and scope of the invention. Furthermore, many modifications can be made to adapt a particular situation, material, composition, process, process step or steps to the objective, spirit and scope of the invention. All such modifications are intended to be within the scope of the claims appended hereto.

[0163] In certain embodiments, the 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 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 invention - In particular, as a result of the administration of the treatment, the resolution of inflammation is enhanced in inflammatory diseases, especially acute inflammatory diseases, chronic inflammatory lung diseases (e.g., asthma), keratoconjunctivitis, periodontal diseases, eczema, inflammatory bowel diseases, especially Crohn's disease or colitis, especially ulcerative colitis or spontaneous colitis, chronic inflammatory diseases such as cystic fibrosis, NASH (non-alcoholic steatohepatitis), scleroderma, antineutrophil cytoplasmic antibody-related diseases ANCA-related diseases; - In particular, as a result of the administration of the treatment, the resolution of inflammation is enhanced in diabetes, especially type I diabetes, psoriasis, lupus, rheumatoid arthritis, multiple sclerosis, Sjogren's syndrome, celiac disease, vasculitis, autoimmune diseases such as myasthenia gravis, or infectious diseases such as sepsis, peritonitis, degenerative diseases, wound healing disorders, severe viral symptoms with severe inflammatory conditions such as coronavirus (e.g., COVID-19), or dry eye syndrome; - In particular, as a result of the administration of the treatment, the resolution of inflammation is enhanced in cancer, especially metastatic cancer, solid or liquid cancer, e.g., carcinoma, more specifically, liver cancer, especially breast cancer or colon cancer, colorectal cancer or lung cancer or mesothelioma or myeloid cancer such as leukemia, especially cancer in which cancer cells express CMKLR1, or the tumor microenvironment expresses CMKLR1, or cancer infiltrated by cells that overexpress it; - NASH (non-alcoholic steatohepatitis), scleroderma, cystic fibrosis or antineutrophil cytoplasmic antibody-related diseases (ANCA) Relates to the use of an antibody or an 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 Description of the Drawings

[0164]

Figure 1-1

Figure 1-2

Figure 2-1

Figure 2-2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7-1

Figure 7-2

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19-1

Figure 19-2

Figure 19-3

Figure 20

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26-1

Figure 26-2

Figure 27

Figure 28

Figure 29

Figure 30

Figure 31

Figure 32

Figure 33

Figure 34

Figure 35

Figure 36

Example

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

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

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

[0168] [Table 1]

[0169] [Table 2]

[0170] Examples of the Therapeutic Efficacy of Anti-CMKLR1 Antibody Treatment Against 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 autoclaved drinking water ad libitum for 6 days. Treatments were injected intraperitoneally: isotype control hIgG1 (10 μg per mouse), daily RvE1 (1 μg per mouse), or 2G1 antibody (10 μg per mouse) three times over 5 days. Colitis was monitored daily for body weight and fecal score (0: normal feces; 4: bloody feces) parameters. At the time of euthanasia of the mice, the length of the colon, representing the severity of the pathology, was measured. The resolution index was determined under various conditions described by Bannenberg et al., 2005.

[0171] Results: The DSS animal model presented in Figure 2 is an acute inflammation model. Figure 1 shows the better overall condition of the animals treated with the anti-CMKLR1 antibody compared to the condition of the animals receiving the control antibody or resolvin RvE1. The body weight of the mice treated with anti-CMKLR1 did not significantly decrease (Figure 2A), and the fecal score (Figure 2B) was significantly better. With respect to colon length and resolution index (Figures 2C and 2D), the animals receiving anti-CMKLR1 or RvE1 presented similar results.

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

[0173] Results: Colitis induced by TNBS is another model of acute inflammation. Figure 3 shows that animals treated with anti - CMKLR1 or RvE1 have the same colon length as normal animals (wt). However, animals treated with isotype control presented a shorter colon length. These results confirmed the therapeutic ability of anti - CMKLR1 antibodies acting like RvE1 against the acute - inflammation mouse model.

[0174] (Example 3) IL10 - KO model - Spontaneous colitis model IL - 10KO mice develop spontaneous colitis from 20 weeks of age because there is often no regulatory T - cell function due to IL - 10 secretion in the intestine. IL - 10KO mice were tracked three times a week from 18 weeks of age for weight loss and fecal consistency, which are the clinical characteristics of this pathology. When weight loss was 5% higher, fecal score was high or equal to 1 over a 2 - week period, anti - CMKLR1 antibody (2G1) or isotype control (hIgG1) was intraperitoneally injected (25 μg / injection, three times a week).

[0175] Results: Using a chronic inflammation model, the efficacy of anti-CMKLR1 antibody treatment was tested. Figure 4 shows the analysis of the percentage of weight loss (Figure 4A) and fecal score (Figure 4B) when animals were treated with an isotype control or anti-CMKLR1 antibody. The results indicate that animals did not lose much weight when treated with anti-CMKLR1 antibody and had a better fecal score than animals receiving the isotype control. Anti-CMKLR1 antibody is thus considered to exhibit therapeutic ability against chronic inflammatory diseases.

[0176] (Example 4) Preclinical model of type 1 diabetes: mouse NOD model 8-week-old female NOD mice were obtained from Charles River laboratory. These mice develop spontaneous type 1 diabetes at 12 - 20 weeks of age. The onset of diabetes can be measured by hyperglycemia. When the blood glucose was 180 - 234 mg / dL, anti-CMKLR1 and isotype control were administered intraperitoneally at 20 μg / injection three times a week for two weeks. When the blood glucose was higher than 600 mg / dL corresponding to irreversible diabetes, the mice were euthanized.

[0177] Results: This type 1 diabetes model is similarly 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 percentage and almost normal blood glucose as indicated by the measurement of blood glucose concentration. This recovery was stable over a long period and is considered to indicate the possibility of full recovery of previously diseased animals. Anti-CMKLR1 antibody restored the tolerance to glucose.

[0178] (Example 5) Imiquimod-induced psoriasiform skin inflammation Aldara (registered trademark) cream, known to induce psoriasis in mice, was used on male C57Bl / 6 mice (8 - 10 weeks old). The mice received a daily topical dose of Aldara. Treatment (anti-CMKLR1 agonist or control compound) was administered by intraperitoneal injection.

[0179] Results: The anti-CMKLR1 agonist (2G1) reduces the skin thickness after Aldara administration (Figure 6A) (see, for example, day 15), but the body weight of the animals is not affected by the administration of the agonist (Figure 6B). These results suggest the application of anti-CMKLR1 agonist compound therapy to a psoriasis mouse model that exhibits an autoimmune disease.

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

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

[0182] (Example 7) Therapeutic efficacy of anti-CMKLR1 antibody treatment against a preclinical model of cancer: (Example 7.1) Effect of anti-CMKLR1 antibody on the growth of primary tumors and the development of their lung metastases in a syngeneic breast cancer model The mice were anesthetized with 3% isoflurane. The hair on the abdomen of the mice was shaved, and 4T1 cells (250,000) were injected into the mammary gland using an insulin syringe (30 gauge) in 50 μL of PBS. 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 3 weeks (100 μg / injection). In a second test to measure lung metastasis after breast cancer development, the animals were treated three times a week for 3 weeks with 0.8 mg / kg of anti-CMKLR1 antibody or control antibody (100 μg / injection).

[0183] Results: As shown in Figure 8A, animals treated with a single compound (2G1 or 3H3) did not show improved tumor growth compared to animals receiving the isotype control antibody. However, animals treated with the combination of anti-CMKLR1 antibody and anti-41BB antibody showed a significant (p<0.01) decrease in tumor growth in the breast cancer model. Since this model of breast cancer is a fairly aggressive model, the results are considered positive. As shown in Figure 8B, which shows the effect of the anti-CMKLR1 compound on lung metastasis by bioluminescence imaging, it can be seen that anti-CMKLR1 treatment reduces lung metastasis compared to animals treated with the control antibody. Analysis of lymph node metastasis shows that animals treated with the anti-CMKLR1 compound have no metastases, while two control animals have metastases (data not shown). These results indicate that the anti-CMLKR1 antibody with agonist activity mimicking RvE1 has an anti-metastatic effect. In this model, the antibody of the present invention does not exhibit a significant efficacy against primary tumor development. However, the results show improvement when the animals are treated with the combination of anti-CMKLR1 and anti-41BB antibodies.

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

[0185] The agonistic anti-CMKLR1 antibody (2G1) or anti-SIRPa antibody (p84-anti-mouse SIRPa from Merck Millipore) (SIRPa is a new checkpoint inhibitor) was injected intraperitoneally once a week (20 μg / injection) alone or in combination starting at d4 after tumor inoculation for 3 weeks.

[0186] Results: As shown in FIGS. 9A - 9C, in the CT26 cancer model, the anti-CMKLR1 antibody alone (FIG. 9B) showed no clinical effect on tumor development compared to the control group, nor did anti-SIRPa alone (FIG. 9A). However, surprisingly, the combination of both compounds enabled timely inhibition of tumor growth (FIG. 9C). In another mouse colon cancer model presented in FIG. 9D, anti-CMKLR1 showed efficacy in inhibiting tumor growth compared to the isotype control. Overall, these results regarding two different colon cancer models indicate that the anti-CMKLR1 antibody agonist can prevent tumor development either alone or in combination with another therapeutic agent.

[0187] (Example 8) Meta-analysis of CMKLR1 expression on biopsies of UC or CD human patients treated with anti-TNFa or anti-α4β7 antibody therapy The signaling networks that perpetuate chronic gastrointestinal inflammation in Crohn's disease (CD) and ulcerative colitis (UC), the two major forms of inflammatory bowel disease (IBD), remain unknown in humans. By analyzing nearly 500 patients with IBD and 100 controls, we herein report that CMKLR1 transcripts accumulate in inflamed colonic tissue of severely ill IBD patients who did not respond to immunotherapies such as immunosuppressant / corticosteroid and anti-TNFα (infliximab) or anti-α4β7 integrin (vedolizumab) therapies.

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

[0189] Results: Analysis revealed that CMKLR1 transcript expression was significantly increased in colonic biopsies of primary UC non-responders before and after treatment with anti-TNF therapy compared to non-IBD controls or patients with UC who were anti-TNF naive and would respond to anti-TNF therapy (Figure 10A). Mucosal CMKLR1 expression was also significantly increased in colonic or ileal biopsies of patients with Crohn's disease (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 compared to non-IBD controls or future responders (Figure 10B). Finally, analysis of colonic mucosal gene expression in a cohort of UC patients (GSE7366146) treated with anti-α4β7 (vedolizumab) therapy also confirmed that CMKLR1 expression was significantly increased in non-responders before and after treatment with vedolizumab (Figure 11). In summary, the meta-analysis indicates that CMKLR1 is overexpressed in the inflamed tissues of IBD patients, particularly those who do not respond to current immunosuppressive agents or immunotherapies even before the start of treatment. Our meta-analysis provides evidence that CMKLR1 expression in the colon, or rather the ileum for CD, from treatment-refractory UC or CD patients, in contrast, responds to treatment with an anti-CMKLR1 antibody agonist such as the antibody of the present invention, allowing these patients to be considered as such.

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

[0191] - CMKLR1 expression by FACS (Figure 13A) Cells were resuspended in PBS-FBS-EDTA and incubated with Fc block (1 / 50) for 30 minutes on ice. 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 described previously, 2G1 antibody (10 μg / membrane) was incubated overnight at 4°C and visualized using peroxidase-conjugated secondary antibody (1:2000). Then, CMKLR1 expression was detected by using chemiluminescence and an image reader. Western blot images were quantified by Multi Gauge software.

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

[0194] (Example 10) Test of CMKLR1 expression on the myeloid lineage (Example 10.1) Differentiation and polarization of human monocytes Monocytes were collected from the PBMC of the pia mater of healthy volunteers and isolated by magnetic separation or sedimentation. The monocytes were then cultured with various cytokine cocktails to generate differentiated unpolarized macrophages or polarized macrophages. With this protocol, polarized and differentiated macrophages with inflammatory (M1) or inflammatory resolving (M2) properties of inflammatory macrophages could be generated in different wells. The monocytes were seeded at 0.5×10 6 cells / mL in complete RPMI (RPMI containing 10% FBS, 1% glutamine, 1% antibiotics), and 500 μL of cell suspension per well was seeded into a 24-well plate. 100 ng / mL of M-CSF was added with the medium for cell differentiation. The cells were incubated for 5 days, and on the 3rd day, the medium was replaced with fresh medium supplemented with 100 ng / mL of M-CSF. For the polarization period, for 3 days, an isotype control (mIgG1 or hIgG4) (2 μg / ml) or anti-CMKLR1 antibody (2 μg / ml) (2G1 or 2G4, H6, BZ332 or 84939) or C15 peptide (10 nM) or RvE1 (10 ng / ml) was supplemented to the LPS-IFNg solution of 100 ng / mL of LPS and 20 ng / mL of IFNg to generate inflammatory macrophages. Inflammatory-IFNg macrophages could also be generated by adding only IFNg (20 ng / mL) to the culture medium. For the polarization of inflammatory resolving macrophages, the cells were incubated with 20 ng / mL of IL-4. After differentiation and / or polarization, phenotypic and functional cytokine / chemokine release was tested 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 collected 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 collected and incubated with IFNγ (20 ng / ml) and LPS (100 ng / ml) for 2 days to induce inflammatory polarization, or incubated with IL-4 (20 ng / ml) to induce resolution of inflammation polarization. During macrophage polarization, treatment was added at 2 μg / ml.

[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 over 7 days. Then, immature dendritic cells (iDCs) were collected and cultured with LPS (100 ng / ml) for 24 hours to induce maturation from iDCs to mDCs. During differentiation and maturation, treatment was added at 2 μg / ml.

[0197] After differentiation of mouse inflammatory or resolution of inflammation macrophages described above, the cells were incubated in the presence of medium, and isotype control, anti-CMKLR1 antibodies: clone H6 and BZ194, C15 peptide, 2G1 or RvE1, the anti-CMKLR1 antibody of interest, were used. Then, the secretion of IL10, CCL17, and IL12p40 was evaluated by ELISA. ELISA kits from BD were used to measure cytokine secretion in the supernatant. The supernatant was 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 of BD. Briefly, the supernatant was diluted in an appropriate buffer, incubated for 2 hours after overnight coating and saturation with the capture antibody. Then, the cytokine was revealed using a biotin-conjugated antibody for detection, 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 on ice for 30 minutes with LIVE / DEAD (registered trademark) Fixable Dead Cell Stains Yellow - Life Technologies. Staining with CD11c-BV711, CD11b-APCCy7, I / Ab-APC, CD103-PerCPCy5.5, CCR7-V450, CD40-PeCy7, CD80-PE, CD86-FITC (all supplied by BD Pharmingen) was performed.

[0200] - Western blot analysis of ERK / Akt Mouse inflammatory macrophages (M1) were generated from bone marrow containing M-CSF and polarized using IFN-gamma (IFNg) and LPS. Briefly, bone marrow cells were collected by flushing the femurs and cultured for 5 days with 100 ng / mL of mM-CSF, then polarized for 24 hours using 20 ng / mL of IFNg and 100 ng / mL of LPS. They were then depleted of FBS for 24 hours using RPMI FBS2% medium. Finally, mouse inflammatory macrophages were treated with 2 μg / mL of 2G1 antibody for various times: 5, 10, and 30 minutes. Cells were collected in RIPA buffer. Protein concentration was measured by 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 antibody and anti-phospho-Akt antibody (1:1000) were incubated with the membranes overnight at 4 °C and visualized using peroxidase-conjugated secondary antibody (1:2000). Western blot images were quantified by Multi Gauge software.

[0201] (Example 10.3) CMLKR1 expression after inflammatory stimulation on human blood monocytes and murine bone marrow myeloid cells and neutrophils Human monocytes were collected from the pia mater PBMC of healthy volunteers and isolated by magnetic separation or sedimentation. The monocytes (CD14-positive cells) were then cultured in medium and treated for 16 hours or 48 hours with different inflammatory stimuli: LPS (100 ng / ml) or TNFa (100 U / ml) or IL6 (20 ng / ml).

[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) or TNFa (100 U / ml) or IL6 (20 ng / ml) for 16 hours or 48 hours.

[0203] The expression of CMKLR1 was measured by FACS using commercially available anti-CMKLR1 antibodies (human anti-ChemR23: clone 84939 and mouse anti-ChemR23: clone 477806).

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

[0205] (Example 11) Competition test against chemokine-induced CMKLR1 activation using an anti-CMKLR1 antibody Method: Competition assay for measuring chemokine-dependent β-arrestin recruitment by the CMKLR1 receptor in the presence of an anti-CMKLR1 antibody: One day before the assay, CHO-K1 CMKLR1 cells (reference number 93 - 0313E2 from Discover'X) were seeded in pre-warmed cell reagent and then seeded in a 96-well plate at 100 μl / well of cells (reference number 15 - 103 from Discover'X) and incubated at 37 °C for 48 hours in a 5% CO2 humidified incubator. The anti-CMLKR1 antibody was diluted (22-fold in a 7-point series of 3-fold dilutions from 1 μM to 1 nM) and the cells were incubated with the antibody at 37 °C for 30 minutes. The cells were then stimulated with chemokine (2 or 6 nM) at 37 °C for 90 minutes according to the supplier's protocol (reference number 92 - 1036 from Discover'X). Luminescence was measured using a plate reader with 0.5 s integration after adding the diluted standard detection solution to the cells.

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

[0207] Chemokine agonist (10 -7 μM~10 -10A mixture of a 6-fold dilution in a 7-point series of a 3-fold dilution of M (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 at 37 °C for 30 minutes; or the cells were pre-incubated at 37 °C for 30 minutes with an anti-CMKLR1 antibody (serial dilutions: 6-fold in a 7-point series of 3-fold dilutions from 1 μM to 1 nM). Then, a mixture of chemelin (2 nM) + forskolin (60 nM) was added to the cells at 37 °C for 30 minutes. For the detection of cAMP, antibody reagent and cAMP standard dilution detection solution were added to the plate at room temperature for 1 hour, then cAMP solution A was added, and the cells were incubated in the dark at room temperature for 3 hours. Bioluminescence was read using a plate reader with 0.5 s integration.

[0208] Results: To test whether the antibody of the present invention is an antagonist of chemelin-induced CMKLR1 activation, two assays were performed and the results are presented in FIG. 18. The chemelin-induced inhibition of forskolin-dependent cAMP production is shown in FIG. 18A (black circles or white squares); the anti-CMKLR1 antibody of the present invention was unable to reverse this inhibition of production (black circles or white squares) compared to the control (gray diamonds). The chemelin-induced activation of beta-arrestin presented in FIG. 18B indicates that the anti-CMKLR1 antibody of the present invention does not significantly modify the chemelin-dependent activation of beta-arrestin (white circles compared to black diamonds). The antibody of the present invention does not have antagonist activity for the CMLKR1-chemelin interaction. Furthermore, the antibody of the present invention was unable to induce the chemelin-induced CMKLR1 signaling pathway, confirming that these antibodies are not agonists of chemelin for the CMLKR1 pathway.

[0209] (Example 12) CD45Rb high T cell transfer chronic colitis mouse model Method: CD45Rb highCD4 T cells were isolated from the spleens of naive mice and sorted on ARIA FACS after negative selection of CD4 T cells by magnetic selection, and then 0.5×10 6 cells in 100 μL of PBS were intraperitoneally injected 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 starting on day 32 after CD45Rb high CD4 T cell transfer. Body weight was tracked three times a week, and body weight fluctuations were determined relative to the initial body weight. * p < 0.05, ** p < 0.01.

[0210] Results: Figure 19 presents the percentage of body weight fluctuations over time in animals treated with anti-CMLKR1 antibody or isotype control. Both groups that exhibited the same initial body weight development over the first 30 days were treated with anti-CMLKR1 antibody or isotype control. Mice treated with anti-CMKLR1 continued to gain weight, whereas in contrast, control mice began to lose weight, indicating the development of chronic colitis as predicted in this control group (Figure 19A). A decrease in the thickness of the tissue corresponding to colonic repair was observed in mice treated with anti-CMKLR1. A decrease in fibrous tissue was also observed (Figure 19B). Different scores represent the anatomic pathology scores used to calculate the severity of the pathology. Scores including the inflammation score were lower in mice treated with anti-CMKLR1 (Figure 19C). The inventors confirm that in a third model of colitis, here a chronic inflammation model, the anti-CMKLR1 antibody of the present invention is interesting for treating chronic inflammation and autoimmune diseases such as colitis.

[0211] (Example 13) Antitumor effect on overall survival in a mouse hepatocarcinoma tumor model Method: Mice were anesthetized with a cocktail of xylazine / ketamine. After laparotomy, tumor Hepa 1.6 cells were injected into the PBS via the portal vein in PBS (2.5×10 6Cells / 100 μL). Treatment was initiated 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 similarly (0.8 mg / kg and 8 mg / kg, respectively). Overall survival was followed for 60 days, and the percentage of survival under each condition was reported in Figure 22.

[0212] Results: As shown in Figure 20, animals treated with anti-CMKLR1 or anti-PD1 antibody had an extended survival rate for only 1 out of 7 treated animals (15% of the treated animals), which showed a partial remission (PR). However, animals treated with the combination of anti-PD1 and anti-CMKLR1 antibody enabled a significant increase in the survival rate (from 15% to 45%), and the animals survived for 60 days after treatment, which showed a complete remission (CR). This result indicates the unexpected efficiency of the therapeutic combination (anti-PD1 / anti-CMKLR1 antibody) against the HCC tumor model.

[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 transplanted into three human germline frameworks named 01 and IGHV7-4-1. IGKV1-13 * 02 (corresponding to SEQ ID NO: 50), IGKV6-21 * 01 and IGKV3-11 *The CDRs of the 2G1 light chain were transplanted into three human germline frameworks, named according to the IMGT nomenclature. Each sequence was fused to the constant fragment of human immunoglobulin and co-transfected into mammalian cells to produce humanized antibodies. More specifically, for the construction of the heavy chain of the anti-ChemR23 antibody, the antibody variable domain VH sequence was synthesized and cloned by EcoRV into the pFUSE-CHIg-hG1 expression plasmid (pFUSE-CHIg-hG1 vector from Invivogen, Toulouse) containing the Fc of human IgG1. For the construction of the light chain of the anti-ChemR23 antibody, the variable domain VL was synthesized and cloned by BsiWI into the pFuse2CLIg-hk expression plasmid (pFuse2CLIg-hk from Invivogen, Toulouse) containing human CL kappa. In mammalian HEK or CHO cells, the inventors co-transfected the plasmid containing VH-hFcG1 and the plasmid containing VL-CL kappa by the lipofectamine method. After incubation for 3 to 7 days, the supernatant was collected and quantified by sandwich ELISA assay. The supernatant could be purified by affinity on Protein A chromatography (HiTrap, GeHealthcare) using 0.1M citric acid pH3 elution buffer. The purified antibodies were dialyzed and concentrated in PBS. They were quantified by UV (A280nm) and tested in an activity assay against the C7 antigen-specific peptide.

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

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

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

[0217] (Example 15) Recognition ability of anti-CMKLR1 antibodies produced in vitro and derived from specific germlines of heavy chain variable domain and light chain variable domain For the quantitative ELISA assay, donkey anti-human IgG, Fc specific (Jackson Immunoresearch; USA; catalog number 709-005-098) was immobilized on plastic at 1.3 μg / ml in borate buffer (pH 9), and the supernatant containing the antibody was added to measure binding and compared with the standard antibody. After incubation and washing, mouse anti-human kappa antibody (Ose Immunotherapeutics, catalog number NaM76-5F3) was added and detected with peroxidase-labeled donkey anti-mouse IgG antibody (Jackson Immunoresearch; USA; catalog number 715-036-151). The ELISA exposure was performed by the conventional method.

[0218] For the activity ELISA assay, donkey anti-human IgG, Fc specific (Jackson Immunoresearch; USA; catalog number 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 and compared with wild-type 2G1. After incubation and washing, biotinylated antigen-specific peptide (Biot-C7 peptide synthesized by synpeptide: biotinylated NH2-PYHTLNLLELHHTAMPGSVFSLGLPLATALAIA-COOH, SEQ ID NO: 60), then peroxidase-streptavidin (Jackson Immunoresearch; USA; catalog number 016-030-084) was added and shown by the conventional method.

[0219] VHvAv3-23 * 04 (SEQ ID NOs: 41 - 89.8% humanized) and VLvAv1-13 * The combination of the heavy and light chains derived from 01 (SEQ ID NOs: 50 - 82.1% humanized) generated a humanized antibody with good binding activity to the antigen-specific peptide (C7 peptide) like the wild-type antibody 2G1. As shown in Figure 23, the combination of the humanized antibody variable domain chains was derived from 2G1 (for the heavy chain variable domain, HA is VHvAv3-23 *corresponding to SEQ ID NO: 4 and SEQ ID NO: 04; 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 the wild-type antibody 2G1. In some cases (combinations of HCLC, HCLD, HDLC, and HDLD), the binding was even better than that of the germline antibodies HALA and the wild-type antibody 2G1 (Figure 23). As shown in Figure 24, the ED50 (ng / ml) of the humanized antibodies was at least equivalent to that of the 2G1 antibody and, in many cases, better.

[0221] (Example 16) Biological effects on CCR7 internalization Materials and methods. Macrophages were generated from monocytes of healthy volunteers using 100 ng / mL of M-CSF over 5 days. The macrophages were then collected and incubated with 10 mg / mL of the coated mAb in the presence of 20 ng / mL of IFNγ to obtain M1 inflammatory macrophages. The M1 macrophages were then phenotyped for CXCR4 and CCR7 by flow cytometry, and the cytokines released into the supernatant were dosed by ELISA. Dendritic cells were generated from monocytes of healthy volunteers using 50 ng / mL of GM-CSF and 20 ng / mL of IL-4 over 6 days. The 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 FcRγ binding, namely, 2G1-N297A (2G1wt mutated at N297A to reduce FcγR binding), and 2G4 (wt with isotype IgG4 mutated at S228P to stabilize the hinge region) were also added. Inflammatory macrophages M1 were added onto the coated plates over 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, internalization of CCR7 was not observed for the isotypes IgG1-N297A or IgG4 that prevent FcRγ binding, indicating that isotype IgG1 was preferred for this biological activity to be obtained.

[0224] (Example 17) Neutrophil apoptosis and death Neutrophils are normally located at the site of inflammation, and their presence sustains the inflammatory process, thereby preventing the initiation or active continuation of the dissipation of inflammation and leading to chronic inflammation. Neutrophil apoptosis prevents the release of the tissue-toxic contents of neutrophils and exerts an anti-inflammatory effect.

[0225] As shown in Figure 26A, the survival-to-death ratio of neutrophils was higher in cells treated with the antibodies of the present invention, thereby indicating the effect of these agonists of the present invention on these cells.

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

[0227] Results: As shown in Fig. 26B, administration of the anti-CMKLR1 agonist enhances caspase-3 activity compared to cells treated with the control antibody. Antibody HALA shows a higher effect on caspase 3 activity compared to the 2G1 antibody. 2G1 WT and HALA increase the cleavage of caspase-3, which means that ChemR23 induction leads to caspase-3-dependent apoptosis.

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

[0229] Results: All humanized variants of 2G1 and 2G1 increase PMN death after 24 hours. At 48 hours, the percentage of dead cells incubated with the IgG1 control and HALA variants is similar, indicating that the antibody promotes programmed cell death in PMNs by inducing only CMKLR1 signaling. All humanized variants of 2G1 and 2G1 promote PMN death, and thus the humanized variants retain the inflammation resolution property. 2G1 and HALA variants increase ROS production by PMNs after 5 hours.

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

[0231] In conclusion, except for inducing neutrophil apoptosis at the inflammatory site, treatment with the antibodies of the present invention does not result in apoptosis of all neutrophil populations. This feature may be advantageous for reducing side effects.

[0232] (Example 18) Neutrophil migration Neutrophils migrate to the inflammatory site after mobilization, thereby initiating, enhancing and / or sustaining the inflammatory process.

[0233] Materials and methods Human endothelial cells (HDMEC) were incubated in gelatin-coated transwells for 24 hours and activated overnight with 100 U / mL of TNF-alpha or without an inflammatory condition. PMNs from healthy volunteers or ANCA patients were then incubated in transwells containing a monolayer of HDMEC for 4 hours. During the 4-hour migration assay, 10 μg / mL of antibodies (Iso Ctrl and 2G1) were added with 100 U / mL of + / - 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 a lower migratory capacity compared to cells treated with the control compound (Figure 28A). 2G1 avoids PMN migration through the endothelial monolayer, especially under inflammatory conditions, when PMN 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 migratory capacity of neutrophils.

[0236] (Example 19) Expression of CD62L Materials and methods: PMNs from healthy volunteers were incubated in culture medium with 10 μg / mL of coated Ab for various times, collected for CD62L staining, and analyzed by flow cytometry (Figure 29, left panel). The cell surface expression of CD62L in cells incubated with the antibody of the present invention is decreased compared to cells incubated in the absence of the 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) Survival in the mesothelioma model Mice treated according to the method exemplified in the brief description of the drawings with an agonist of CMKLR1 have a higher survival rate than mice treated with the control antibody, thereby demonstrating a positive effect of the compound according to the present invention for treating mesothelioma (Figure 30).

[0238] (Example 21) CRC model As shown in Fig. 31A, the tumor volume decreases in animals treated with the anti-CMKLR1 antibody of the present invention as compared to the control antibody. In some cases, complete remission is also observed, thereby indicating the positive effect of the compounds according to the present invention for treating CRC.

[0239] Furthermore, as illustrated in Fig. 31B, treatment with the anti-CMKLR1 monoclonal antibody (OSE-230) results in a decrease in fecal score and a decrease in the number of tumors.

[0240] (Example 22) Experimental model of autoimmune encephalomyelitis In this model, the curative administration of the antibody of the present invention in the EAE model does not result in a decrease in body weight as compared to animals treated with the control antibody (Fig. 32A). However, when treatment is carried out using the anti-CMKLR1 antibody, the disease score significantly decreases (Fig. 32B). Since the disease score is approximately 30% lower in animals treated with the agonist compound, the anti-CMKLR1 antibody brings about the greatest improvement in score immediately after 10 days of treatment as compared to the control. Therefore, treatment with the 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 to the heavy or light chain of the HDLD variant to substitute the 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 mutations of amino acids, the inventors have tested and identified several very advantageous ones that do not substantially and inappropriately affect the biological activity of the product. Expertise leads to the selections described hereinafter.

[0243] In the heavy chain, amino acid substitutions of D61E (HD-61E, SEQ ID NO: 89), R52G (HD-R52G, SEQ ID NO: 90), or R52aG, A49S, N52S, and Y53S (HEF, SEQ ID NO: 91) in CDR2 were achieved to reduce immunogenicity. In the light chain, the amino acid at 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, T52S, T55E in CDR2, and W47L in framework 2 were substituted to reduce immunogenicity in the LEF variant (SEQ ID NO: 55). These sequences are shown in the following table.

[0244]

Table 3

[0245] As detailed in the following examples, 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 optimized to reduce immunogenicity exhibits high binding activity and stability while maintaining biological function.

[0246] Each sequence was fused to the constant fragment of human immunoglobulin and co-transfected into mammalian cells to produce humanized antibodies. As a result, it was shown that all combinations of heavy and light chains produce antibodies. Depending on the combination of heavy and light chains, productivity is differentially affected in mammalian cells, but is considered to be in an amount suitable for efficient production considering the therapeutic application of the antibody.

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

[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) generated humanized antibodies with better binding activity to the germline antibody HALA and the antigen-specific peptide (C7 peptide) than the wild-type antibody 2G1.

[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 antibody was analyzed by ELISA assay and the formation of aggregates was analyzed by gel filtration (Superdex 200 10 / 300GL, GeHealthcare).

[0250] As shown in Fig. 34A, all purified antibodies exhibited similar binding activities at 37 °C, 4 °C, or -80 °C. As shown in Fig. 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 onto the coated plate over 48 hours, and the CCR7 expression on the surface of the macrophages was measured by flow cytometry.

[0252] As shown in Fig. 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 optimized to reduce immunogenicity retained the functional properties of the 2G1 antibody.

[0253] (Example 27) Biological effects on the survival of PMN PMNs from healthy volunteers were incubated in culture medium for 24 hours with 10 μg / mL of the coated HEF-LDT52S, HEF-LEF, and HDLD antibody variants and stained with either a death / survival kit (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 that does not bind to the Fc receptor (FcR) (HEF-LDT52S N297A) was also added. As shown in Fig. 36, 2G1 and all HEF-LDT52S, HEF-LEF, and HDLD humanized versions of 2G1 promoted the death of PMNs, and thus the humanized variants retain the inflammation resolution properties.

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

[0255] [References] TIFF0007701915000004.tif240170TIFF0007701915000005.tif240170

Claims

1. An anti-CMKLR1 antibody or an antigen-binding fragment thereof that binds to chemokine-like receptor 1 (CMKLR1), comprising a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 91 and a light chain variable domain containing the amino acid sequence of SEQ ID NO: 93, the antibody or antigen-binding fragment thereof.

2. The anti-CMKLR1 antibody or antigen-binding fragment thereof according to claim 1, which specifically binds to the third extracellular loop (EL3) of CMKLR1.

3. The anti-CMKLR1 antibody or antigen-binding fragment thereof according to claim 2, which specifically binds to an epitope located within a polypeptide containing 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.

4. The anti-CMKLR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, which is a resolvin E1-like agonist of CMKLR1.

5. The anti-CMKLR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, which does not activate the beta-arrestin signaling pathway in CMKLR1-positive cells in vitro and / or in vivo.

6. The anti-CMKLR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, which does not show significant depletion in CMKLR1-positive cells in vitro and / or in vivo.

7. The anti-CMKLR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, which does not compete with chemokine for binding to CMKLR1 and / or does not inhibit the binding of chemokine to CMKLR1.

8. The anti-CMKLR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, which is a humanized monoclonal antibody or antigen-binding fragment thereof.

9. The anti-CMKLR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, which is a humanized monoclonal antibody or antigen-binding fragment thereof, wherein the antibody light chain constant domain is derived from a human kappa light chain constant domain.

10. The anti-CMKLR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the light chain constant domain contains the sequence of SEQ ID NO:

79.

11. The anti-CMKLR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein the antibody heavy chain constant domain is derived from a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant domain.

12. The anti-CMKLR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, 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.

13. The anti-CMKLR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, wherein the antibody heavy chain constant domain comprises the amino acid sequence of SEQ ID NO: 80 or SEQ ID NO:

83.

14. The anti-CMKLR1 antibody or antigen-binding fragment thereof according to claim 13, wherein the antibody heavy chain constant domain comprises the amino acid sequence of SEQ ID NO:

80.

15. The anti-CMKLR1 antibody or antigen-binding fragment thereof according to claim 13, wherein the antibody heavy chain constant domain comprises the amino acid sequence of SEQ ID NO:

83.

16. A pharmaceutical composition comprising the anti-CMKLR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 15 for use in the prophylactic or therapeutic treatment of an inflammatory disease, an autoimmune disease, or cancer.

17. A pharmaceutical composition comprising the anti-CMKLR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 15 for use in the prophylactic or therapeutic treatment of diabetes.

18. A pharmaceutical composition comprising the anti-CMKLR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 15 for use in the prophylactic or therapeutic treatment of a chronic inflammatory disease.

19. A pharmaceutical composition comprising the anti-CMKLR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 15 for use in the prophylactic or therapeutic treatment of NASH (non-alcoholic steatohepatitis), scleroderma, cystic fibrosis, or antineutrophil cytoplasmic antibody-related diseases (ANCA).

Images