Anti-ccr1 humanized antibodies and therapeutic uses thereof

Abstract

This invention provides humanized antibodies against chemokine receptor 1 and their therapeutic applications. This invention also provides humanized anti-CMKLR1 compounds that have agonistic activity against the interaction between regressor E1 and CMKLR1, and their use in the treatment or prevention of diseases, particularly those in which the resolution of inflammation is delayed or interrupted.

Description

[0001] This application is a divisional application of the invention application filed on October 9, 2020, with Chinese national application number 202080084501.6, entitled "Humanized Anti-Chemokines Receptor 1 Antibody and Its Therapeutic Application". Technical Field

[0002] This invention relates to the field of immunotherapy. Specifically, it provides novel humanized anti-chemerin receptor antibodies that exhibit resolvin E1 agonist activity against chemokine-like receptor-1 (CMKLR1). Furthermore, this invention provides the use of such antibodies in treatment, particularly for the treatment of autoimmune diseases and chronic inflammatory diseases, infectious diseases, cancer, and any condition in which the inflammatory resolution phase is interrupted or delayed. Background Technology

[0003] The crucial role of inflammation in health and disease is well-established. The detailed molecular mechanisms and biological events regulating the progression and resolution of inflammation remain of great significance. Recent research provides strong evidence that the resolution of inflammation is not a passive process as previously thought. Rather, it is a biosynthetically active process regulated by biochemical mediators and receptor signaling pathways. Thus, resolution is driven by specific pro-resolution mediators. Inflammation is a spontaneous mechanism that occurs during infection, injury, or trauma. Inflammation is inevitable and often beneficial, with the inflammatory response coordinated by a delicate balance between positive and negative feedback loops. Inflammation is generally divided into three phases: initiation, amplification, and resolution.

[0004] The process of remission that ends an inflammatory response is a complex one that involves the sequential and chronological engagement of cellular (e.g., granulocytes or macrophages) and chemical (e.g., cytokines or specific pro-remission mediators or factors) effectors.

[0005] Chemkokine receptor 1 (CMKLR1), also known as ChemR23, and chemokine receptor 2 (CCRL2) are seven-transmembrane receptors identified by their homology with known G protein-coupled receptors (AJ Kennedy and AP Davenport, 2018). Chemkokine receptor 1 (CMKLR1; also known as Dez in murine animals) is an orphan G protein-coupled receptor associated with GPR-1 (38% total 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 the ligands for these receptors and revealed their functions. Therefore, the first plasma protein-derived chemical attractant named chemerin has been identified as a ligand for CMKLR1.

[0006] The second ligand of CMKLR1 is the lipid mediator Resolvine E1 (RvE1), which belongs to the resolvine family. The anti-inflammatory lipid mediator Resolvine E1 inhibits leukocyte infiltration and pro-inflammatory gene expression.

[0007] Following its discovery in psoriasis, initial interest in the chemokine system (i.e., signaling pathways activated or inactive by chemokine receptors through their ligands, such as chemokines and regressors) focused on its role in the chemotaxis of inflammatory and immune cells. More recently, its potential role in cardiovascular function, as well as its role in reproductive biology, has been considered in relation to inflammation, obesity, and metabolic syndrome. Therefore, the role of the chemokine system in inflammatory processes, particularly in the resolution of inflammation, has attracted considerable attention. Many diseases are associated with delays or interruptions in the process of inflammatory resolution. Most known specific pro-regression factor mediators are derived from polyunsaturated fatty acids, including lipoxygenin, the regression family (including E-series and D-series regressors), protectins, and maresin. Nevertheless, pro-regression molecules are difficult to synthesize due to their lipid nature. Producing sufficient quantities of pro-regression molecules (e.g., for clinical trials) is a burden, and few SPMs have undergone efficient production. Furthermore, it is difficult to generate antibodies that specifically target G protein-coupled receptors. Therefore, molecules capable of participating in, and especially initiating or enhancing, the resolution phase of the inflammatory response, such as resolution-promoting factors, are needed. Summary of the Invention

[0008] In a first aspect, the present invention relates to humanized anti-CMKLR1 antibodies or antigen-binding fragments thereof or antigen-binding antibody mimics or modified antibodies.

[0009] Compared to existing wild-type anti-CMKLR1 antibodies, the inventors sought to obtain improved humanized antibodies. While it is known that certain residues in the variable domain (CDR) and backbone (FR) sequences (including residues in the vernier zone, canonical residues, residues at the variable heavy chain and variable light chain interfaces, etc.) are crucial to the antibody structure and should not be mutated to maintain the antibody's biochemical and biological activity, the inventors unexpectedly discovered that mutations in certain heavy chain variable domain and / or light chain variable domain sequences of wild-type antibodies (including some of the aforementioned key residues) allow for increased human residue content (up to 99% humanization), and functional characteristics associated with these humanized antibodies are not present in wild-type anti-CMKLR1, particularly improved wild-type anti-CMKLR1. Furthermore, the production of these humanized antibodies can be improved compared to anti-CMKLR1 antibodies with fewer or different humanizations. These humanized antibodies enable the provision of compounds exhibiting functional characteristics suitable for treating diseases, including those involving inflammation, and these compounds can be mass-produced for drug candidate development. The combination of these two characteristics allows the provided anti-CMKLR1 antibodies to demonstrate improved capabilities compared to antibodies of the prior art.

[0010] Starting with a dehumanized anti-CMKLR1 antibody and selecting human lineage sequences, the inventors designed specific humanized heavy chain variable domains and light chain variable domains. These humanized heavy chain and light chain variable domains derived from the dehumanized antibody allow for the production of functional antibodies in various cell lines (e.g., but not limited to Chinese hamster ovary (CHO) cell lines, transformed African green monkey kidney fibroblast (COS-7) cell lines, and human embryonic kidney cell line (HEK 293)). Under conditions where significant antibody yields can be recovered, the binding activity of these antibodies to their targets may even be increased relative to the binding activity of wild-type antibodies. Further humanization of the obtained humanized heavy chain and light chain variable domains, particularly targeting the backbone regions of the heavy chain and light chain variable domains, through a first humanization step, provides antibodies suitable for high production in various cell lines, exhibiting low immunogenicity and possessing at least the functional characteristics of their parent antibody. Those skilled in the art would not anticipate that the aforementioned mutations, particularly at these positions in the CDR domain and in the backbone regions of the heavy and / or light chain variable domains, would enable the provision of anti-CMKLR1 antibodies with preserved binding capacity (especially preserved affinity and stability), preservation of the CMKLR1 regressor agonist activity, a low immunogenicity index, and improved production scale of these antibodies comprising the heavy and light variable domains according to the invention compared to fewer or different humanized antibodies. Unexpectedly, the introduction of the disclosed mutations, particularly those in the CDR2 of the heavy chain, would increase yield while retaining the binding characteristics of prior art anti-CMKLR1 antibodies and advantageously preserving other functional characteristics. Anti-CMKLR1 antibodies exhibiting the aforementioned characteristics (binding ability (especially affinity) to CMKLR1 (particularly to a specific third outer ring of CMKLR1), low immunogenicity, good production scale, and ability to act as a regressor agonist against CMKLR1) are useful for the effective treatment of several diseases, including those involving inflammation, and more particularly inflammatory diseases.

[0011] Furthermore, as will be explained below in this application, several highly advantageous biological effects have been achieved, most notably a potent beneficial effect on inflammation resolution associated with neutrophil apoptosis and a reduction in neutrophil and / or macrophage migration and / or transmigration. Thus, the new compound allows for the combination of production capacity and biological activity.

[0012] Therefore, in a first aspect of the invention, an antibody or antigen-binding fragment thereof that binds to chemokine receptor 1 (CMKLR1), particularly to human CMKLR1, is disclosed, the antibody or antigen-binding fragment thereof comprising: a) Antibody heavy chain variable (VH) domain, which contains 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) Antibody light chain variable (VL) domain, which contains 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.

[0013] In one particular embodiment, the antibody or its antigen-binding fragment that binds to chemokine receptor 1 (CMKLR1), particularly human CMKLR1, comprises: a) Antibody heavy chain variable (VH) domain, which contains 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 corresponds to the amino acid sequence 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; b) Antibody light chain variable (VL) domain, which contains 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.

[0014] In another specific embodiment, the antibody or its antigen-binding fragment that binds to chemokine receptor 1 (CMKLR1), particularly human CMKLR1, comprises: a) Antibody heavy chain variable (VH) domain, which contains 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 corresponds to the amino sequence of SEQ ID NO: 12, the heavy chain variable (VH) domain does not contain the framework region VHFR3 of SEQ ID No. 70, preferably, the heavy chain variable (VH) domain contains the framework region FR3 of SEQ ID NO: 69. - 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) Antibody light chain variable (VL) domain, which contains 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.

[0015] Particularly advantageously, the antibody or its antigen-binding fragment specifically binds to the third extracellular loop (EL3) of CMKLR1, especially 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 containing the amino acid sequence SEQ ID No: 2 or SEQ ID No. 59 or to an epitope located within the amino acid sequence SEQ ID No. 60.

[0016] The antibodies or antigen-binding fragments thereof according to this embodiment are suitable for production in various cell lines (including but not limited to mammalian cell lines) at yields suitable for drug candidate development, while retaining their ability to specifically bind to the extracellular third ring of CMKLR1 and their resolvin E1-like agonist activity against CMKLR1. Therefore, the humanized antibodies disclosed herein can be efficiently produced and possess the functional capabilities of their parent antibodies.

[0017] In a particular aspect of the invention (which may be distinguished from the definition of antibodies of the invention solely by their CDR domains, but may be associated with the definition in certain embodiments), an antibody or antigen-binding fragment thereof is disclosed that binds to chemokine receptor 1 (CMKLR1), particularly human CMKLR1, and is suitable for production in mammalian cells (e.g., COS, CHO, or HEK cells), particularly in yields exceeding 0.1 mg / ml, particularly exceeding 1 mg / ml, more particularly at least 10 mg / ml, and further more particularly exceeding 100 mg / ml, wherein: a) The variable weight (VH) domain comprises the amino acid sequences of the backbone regions (FR1, FR2, FR3, and FR4) of the heavy chain variable domain, wherein each backbone region has sequence identity with the backbone region of the same order in sequence SEQ ID No. 41: FR1 is 100%, FR2 is at least 60%, FR3 is at least 78%, and FR4 is at least 80%; more specifically, FR1 is 100%, FR2 is at least 80%, FR3 is at least 85%, and FR4 is at least 90%. b) The variable light (VL) domain comprises the amino acid sequence of the backbone regions (FR1, FR2, FR3, and FR4) of the light chain variable domain, wherein each backbone region has sequence identity with the backbone region of the same order in sequence SEQ ID No. 50: at least 60% for FR1, at least 70% for FR2, at least 75% for FR3, and at least 80% for FR4, and more particularly, at least 100% for FR1, at least 90% for FR2, at least 90% for FR3, and at least 100% for FR4.

[0018] Specifically, the humanized anti-CMKLR1 antibody or its antigen-binding fragment specifically binds to the third extracellular loop (EL3) of CMKLR1. In particular, the antibody or its antigen-binding fragment specifically binds to a polypeptide containing the amino acid sequence SEQ ID No: 2 or SEQ ID No. 59 or to an epitope located in the amino acid sequence SEQ ID No. 60.

[0019] Starting with 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 are particularly suitable for generating humanized anti-CMKLR1 antibodies in cells or cell lines, including mammalian cells such as CHO cells, COS cells, or HEK cells. After selecting specific human lineage sequences for designing the heavy chain and light chain variable domains, humanized antibodies with backbone region identity as defined herein can also be generated in appropriate amounts in cells or cell lines for the purpose of developing antibodies that can generate drug candidates. Providing humanized antibodies that can be generated in large quantities in cells or cell lines, especially mammalian cells or cell lines, for the development of therapeutic antibodies has received considerable attention. The specific anti-CMKLR1 antibody described herein has a highly similar structure to its parent antibody, which contains the heavy chain variable domain of SEQ ID No. 41 and the light chain variable domain of SEQ ID No. 50, allowing for the proper production, conformation, and secretion of the anti-CMKLR1 antibody. This allows for the provision of an antibody that binds to a specific epitope of chemokine receptor 1 and has sufficient amount of regressor E1 agonist activity against that receptor for therapeutic purposes.

[0020] In a particular embodiment of the invention, an antibody is provided that is a humanized anti-chemokine receptor 1 (CMKLR1) antibody or antigen-binding fragment thereof suitable for production in mammalian cells (e.g., COS or CHO cells), particularly in a yield exceeding 0.1 mg / ml, more particularly in a yield exceeding 1 mg / ml, even more particularly in a yield exceeding 10 mg / ml, and further, even more particularly in a yield exceeding 100 mg / ml, wherein: a) The variable heavy (VH) domain contains the amino acid sequences of the backbone regions (FR1, FR2, FR3, and FR4) of the heavy chain variable domain, wherein each backbone region has sequence identity with the backbone region of the same order in sequence SEQ ID No. 41: FR1 at least 90%, FR2 at least 70%, FR3 at least 80%, and FR4 at least 80%; b) The variable light (VL) domain contains the amino acid sequences of the backbone regions (FR1, FR2, FR3 and FR4) of the light chain variable domain, wherein each backbone region has sequence identity with the backbone region of the same order in the sequence SEQ ID No. 50: at least 60% for FR1, at least 80% for FR2, at least 75% for FR3 and at least 70% for FR4.

[0021] Specifically, the humanized anti-CMKLR1 antibody or its antigen-binding fragment specifically binds to the third extracellular loop (EL3) of CMKLR1. In particular, the antibody or its antigen-binding fragment specifically binds to a polypeptide containing the amino acid sequence SEQ ID No: 2 or SEQ ID No. 59 or located within the amino acid sequence SEQ ID No. 60.

[0022] In a particular embodiment of the humanized antibody or its antigen-binding fragment as defined above, the humanized antibody or its antigen-binding fragment comprises a) Antibody heavy chain variable (VH) domain, which contains three CDRs, namely 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; and - 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; and - 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) Antibody light chain variable (VL) domain, which contains 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; and In a particular embodiment of the humanized antibody or its antigen-binding fragment as defined above, the humanized antibody or its antigen-binding fragment comprises: a) Antibody heavy chain variable (VH) domain, which contains three CDRs, namely 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; and - 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 contains or is composed of the amino acid sequence SEQ ID No. 12 or SEQ ID No. 63, provided that VHCDR1 is not SEQ ID No. 3 or SEQ ID No. 4; and - 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) Antibody light chain variable (VL) domain, which contains 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; and - VLCDR2 is selected from 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 - VLCDR3 is selected from SEQ ID No.34, SEQ ID No.35 and SEQ ID No.36.

[0023] In another specific embodiment of the humanized antibody or its antigen-binding fragment as defined above, the humanized antibody or its antigen-binding fragment comprises: a) Antibody heavy chain variable (VH) domain, which contains three CDRs, namely 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; and - 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 is composed of the amino acid sequence SEQ ID No. 12, the heavy chain variable (VH) domain does not contain the backbone region VHFR3 of SEQ ID No. 70, preferably, the heavy chain variable (VH) domain contains VHFR3 of SEQ ID No. 69; and - 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) Antibody light chain variable (VL) domain, which contains 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; and - VLCDR2 is selected from 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 - VLCDR3 is selected from SEQ ID No.34, SEQ ID No.35 and SEQ ID No.36.

[0024] The antibody provides the same advantageous characteristics as antibodies defined according to the first and second aspects of the invention; namely, high yield, specific binding to epitopes located within the third outer ring of chemokine receptor 1, and having effusive E1 agonist activity.

[0025] The disclosed antibodies are suitable for treating conditions in which inflammatory resolution is delayed or interrupted. All antibodies described herein are suitable for inducing, and / or enhancing, and / or triggering inflammatory resolution.

[0026] In the following disclosure, humanized anti-CMKLR1 compounds are considered to be humanized anti-CMKLR1 antibodies or their antigen-binding fragments or antigen-binding antibody mimics or modified antibodies. In one particular embodiment of the invention, the compound is defined by its CDR sequence. In a more particular embodiment of the invention, the anti-CMKLR1 compound is an antibody defined by the sequence of its CDR and its backbone region (FR). Anti-CMKLR1 compounds are compounds that specifically bind to chemokine receptor 1 (CMKLR1). In the following disclosure, the terms chemokine receptor 1, CMKLR1, and ChemR23 are used interchangeably and all refer to human-derived anti-CMKLR1 antibodies. CMKLR1 Genes or non-human animals cmklr1 Genetically encoded receptor. In a particular embodiment of the invention, the anti-CMKLR1 compound specifically binds to human CMKLR1, or in other words, the present invention relates to an anti-human CMKLR1 compound. As used herein, the term "CMKLR1" refers to a chemokine receptor 1 protein (also known as chemR23), a member of the mammalian G protein-coupled receptor family, preferably human CMKLR1. The reference sequence of the human CMKLR1 protein used in the embodiments of this application corresponds to the sequence associated with Uniprot accession number Q99788 (SEQ ID No: 1).

[0027] In one specific aspect, the present invention relates to an anti-CMKLR1 antibody or its antigen-binding fragment or antigen-binding antibody mimic or modified antibody defined by at least one functional characteristic. In a preferred embodiment, the anti-CMKLR1 compound is defined by its ability to inhibit the secretion of pro-inflammatory cytokines (especially IL12) and / or its ability to enhance the secretion of anti-inflammatory cytokines (especially IL10). In a more specific embodiment, the anti-CMKLR1 compound inhibits or enhances cytokine secretion by macrophages (especially pro-inflammatory macrophages and / or pro-regression macrophages). In a specific embodiment, the anti-CMKLR1 compound of the present invention enhances macrophage polarization into anti-inflammatory macrophages, especially pro-regression macrophages. In a specific embodiment, the anti-CMKLR1 compound of the present invention enhances neutrophil apoptosis compared to a control antibody.

[0028] In one particular aspect, the present invention relates to an anti-CMKLR1 antibody or an antigen-binding fragment thereof or an antigen-binding antibody mimic, which has agonist properties for the regressor E1 (RvE1) / CMKLR1 interaction, thereby mimicking at least one effect induced by the binding of RvE1 to CMKLR1 on CMKLR1-positive cells. Excitation of RvE1-CMKLR1 interaction Agent properties"This refers to the antibodies of the present invention targeting CMKLR1, or their antigen-binding fragments, antigen-binding antibody mimics, or modified antibodies, having the effect mimicking at least one effect provided by the binding of RvE1 to CMKLR1 (especially the binding of human RvE1 to human CMKLR1, particularly on dendritic cells, neutrophils, monocytes, and macrophages), thereby activating receptor signaling pathways normally activated by RvE1. Because receptor binding and activation produce a biological response, the compounds of the present invention can lead to the activation of G protein signaling pathways, particularly Gα." i Signaling pathways and / or Gα o Without activating the β-repressor protein pathway. Specifically, the compounds of the present invention can lead to the inhibition of the β-repressor protein pathway. In particular, binding of the compounds according to the present invention induces activation of Akt and / or Erk proteins in vitro or in vivo. In a particular embodiment, the compound can be considered an anti-CMKLR1 agonist with chemokine E1-like ability when activating the G protein signaling pathway in CMKLR1-positive cells stimulated with the compounds of the present invention, and in a more particular embodiment, this is also true when the β-repressor protein pathway is not activated and especially when the β-repressor protein pathway is inhibited. In other words, a chemokine E1-like agonist antibody can be defined as an antibody that, compared to a control antibody, can bind to CMKLR1, thereby inducing phosphorylation of Akt and / or Erk proteins. The control antibody can be an antibody that does not specifically bind to CMKLR1. Protein phosphorylation can be determined according to methods well known to those skilled in the art, such as those disclosed in the embodiments of this specification. In a particular embodiment, the compounds of the present invention enhance the activation of the G protein pathway induced by CMKLR1. In another embodiment, the compounds of the present invention do not induce the activation of the β-repressor protein pathway induced by CMKLR1. In another embodiment, the compounds of the present invention inhibit the CMKLR1-induced β-repressor protein pathway. In another embodiment, the compounds of the present invention are regressors or regressors because they induce at least one agonist effect of RvE1 binding to CMKLR1, and because RvE1 is a regressor or regressor mediator; for example, a regressor can be defined as a compound that inhibits the CMKLR1-induced β-repressor protein pathway and / or enhances the activation of the CMKLR1-induced G protein pathway in CMKLR1-positive cells, compared to control compounds known not to specifically interact with CMKLR1. The activation / inhibition of these pathways can be evaluated using the methods disclosed in the working embodiments of the present invention. In one particular embodiment, the effects of the agonist compounds are evaluated in human cells.

[0029] In one particular embodiment, the compounds of the present invention do not interfere with the binding of chemokines to CMKLR1. Chemokines are one of the natural ligands of CMKLR1. In other words, the compounds of the present invention are not agonists and / or antagonists of the interaction between chemokines and CMKLR1. The lack of said agonist and / or antagonist ability can be assessed according to embodiments of the present invention, wherein a competitive assay of the recruitment of the CMKLR1 receptor to chemokine-dependent beta-inhibitor protein is disclosed in the presence of the anti-CMKLR1 antibody of the present invention is disclosed. In a preferred embodiment, the anti-CMKLR1 compounds of the present invention do not compete with chemokines for binding to CMKLR1. When, in the presence of the CMKLR1 compound of the present invention, the binding of chemokines to CMKLR1 is at least 50%, more preferably at least 80%, still more preferably at least 90%, and most preferably similar to the binding of chemokines to CMKLR1 under the same experimental conditions but in the absence of the anti-CMKLR1 compound of the present invention, it can be determined that there is no competition between the anti-CMKLR1 compound of the present invention and chemokines. Alternatively, it can be determined, according to the method described in Example 9, that there is no competition between the anti-CMKLR1 compound of the present invention and the chemokine.

[0030] In one particular embodiment, the anti-CMKLR1 compound has the ability to activate at least one Akt signaling pathway protein (also known as the PI3K-Akt pathway) and / or an Erk signaling pathway protein, preferably Akt protein and / or Erk protein, preferably both Akt protein and Erk protein, in vitro or in vivo. The activation of the pathway can be evaluated according to methods known in the art, particularly using the methods disclosed in the embodiments of this invention. Attached Figure Description

[0031] Figure 1The effects of chimeric antibodies against CMKLR1 variants 1G1, 2G1, 3G1, and 4G1 on DC maturation and differentiation. Mouse dendritic cells were incubated at maturation (24 or 48 hours) followed by differentiation using excipients or RvE1 or anti-CMKLR1 antibody variants (mutations at positions 1 and 2 of SEQ ID No. 8, corresponding to VH-CDR3: 1G1 (LL), 2G1 (LI), 3G1 (IL), or 4G1 (II) or isotype controls (hIgG1 or mIgG)). Cells were then stained with cell marker antibodies for FACS analysis: A. CD80-PE; B. CD86-FITC; C. CD103-PerCPCy5.5; DI / Ab-APC. Fluorescence mean values ​​were measured under each condition. E and F represent cell viability measured by FACS under each condition at 24 or 48 hours after maturation using the Life Technologies LIVE / DEAD® kit.

[0032] Figure 2 Effect of anti-CMKLR1 antibody on a DSS-induced mouse model of acute inflammatory colitis. Six days after DSS induction, mice were injected with isotype control hIgG1 (10 µg per mouse) (x), RvE1 (1 µg per mouse) daily (●), or 2G1 antibody (10 µg per mouse) three times daily (x). (Continued for 5 days. Use ▼ to represent untreated wild-type mice.) A. Animal weight loss. B. Animal fecal score. C. Colon length. D. Resolution index.

[0033] Figure 3 Effect of anti-CMKLR1 antibody on a TNBS-induced mouse model of acute inflammatory colitis. Mice received 200 µL of 5% haptenant TNBS in 50% ethanol on day 0, followed by injection of isotype control hIgG1 (10 µg per mouse) (x), daily injection of RvE1 (1 µg per mouse) (●), or injection of 2G1 antibody (10 µg per mouse) three times. (▲) Continue for 5 days, or without treatment (wild-type animals). Mice were sacrificed and colon length was measured in each case.

[0034] Figure 4 Effect of anti-CMKLR1 antibody on a chronic inflammatory colitis model in IL10 KO mice. IL10 KO mice developed spontaneous inflammatory colitis. Anti-CMKLR1 antibody (2G1) was used... IL10 KO mice were treated with intraperitoneal injection (25 µg / injection, 3 weeks) or isotype control (hIgG1) (x). A. Weight loss of animals during and after treatment. B. Animal fecal score.

[0035] Figure 5 Effects of anti-CMKLR1 antibody on a mouse model of type 1 non-obese diabetes. Mice developed spontaneous diabetes. When blood glucose levels were between 180 mg / dL and 234 mg / dL, anti-CMKLR1 antibody (A and B) was administered. (■ in C) or isotype control antibody (x in A and B; in C) Mice were treated with intraperitoneal injection of 20 µg / injection, three times a week for two weeks. A. Survival percentage; B. Individual blood glucose concentration in mg / dL; C. Summary blood glucose concentration in mg / dL.

[0036] Figure 6 Effects of anti-CMKLR1 agonists on autoimmune diseases, such as mouse models of psoriasis. Aldara-treated mice were intraperitoneally injected for 4 to 6 consecutive days with either 2G1 (●) or the isotype control antibody (x). A. Thickness and B. Weight.

[0037] Figure 7 Effect of anti-CMKLR1 antibody on a mouse peritonitis model. Prior to Zymosan A injection (1 mg per mouse, in 1 mL), a prophylactic injection of anti-CMKLR1 was administered: RvE1 (1 µg per mouse) (●), and 2G1 antibody (10 µg per mouse) (●). A. PMN counts within the first 50 hours after Zymosan A injection. B. Macrophage counts within the first 50 hours after Zymosan A injection. C. Regression index.

[0038] Figure 8 Effect of anti-CMKLR1 antibody on a 4T1 mammary tumor model. 4T1 cells (250,000 cells) were seeded into the mammary glands of mice. Then, anti-CMKLR1 antibody (2G1) was injected twice (10 μg / injection) on days 4 and 7. B. (●) or anti-41BB antibody (3H3) (●) or both antibodies (▲), or a control antibody (IgG1 isotype antibody clone 3G8) (x in A. and B.) injected 3 times a week for 3 weeks. A. Tumor area was measured 8 days after tumor injection, and then every 2 days thereafter. B. Tumor lung metastasis was measured by bioluminescence imaging (BLI) in animals treated with isotype control and anti-CMKLR1 antibody (n=4).

[0039] Figure 9 The effects of anti-CMKLR1 antibody on two different mouse models of colon cancer. Two mouse models were investigated, and animals received either the isotype control antibody (3G8) (x) or anti-CMKLR1 (2G1) (x). ) Intraperitoneal injection, 20 µg / injection, for 3 weeks. AC: Results in the CT26 colon cancer model, comparing monotherapy with p84 (A) and 2G1 (B) with combination therapy of the two antibodies (C). D: Results in the MC38 colon cancer model.

[0040] Figure 10 Expression of CMKLR1 in biopsies of patients with ulcerative colitis (UC) or Crohn's disease before and after anti-TNFα treatment. x represents control; The symbol represents CMKLR1 expression in patients who responded to corticosteroid therapy and / or immunosuppressive therapy prior to infliximab treatment; the symbol ■ represents CMKLR1 expression in patients who did not respond to corticosteroid therapy and / or immunosuppressive therapy prior to infliximab treatment. The figures show CMKLR1 expression in patients who responded to corticosteroid and / or immunosuppressive therapy after infliximab treatment; ▲ represents CMKLR1 expression in patients who did not respond to corticosteroid and / or immunosuppressive therapy after infliximab treatment. A. Expression of CMKLR1 transcripts in patients with ulcerative colitis before and after infliximab treatment. B. Expression of CMKLR1 transcripts in inflamed colonic biopsies from patients with Crohn's disease before and after infliximab treatment.

[0041] Figure 11 CMKLR1 expression in biopsies of UC patients before and after anti-α4β7 (VDZ) treatment. A represents CMKLR1 transcript expression in inflamed colon biopsies of ulcerative colitis patients before and after vedoolizumab treatment. R corresponds to patients who responded to VDZ treatment, and NR corresponds to patients who did not respond to VDZ treatment.

[0042] Figure 12 The binding of anti-CMKLR1 antibody to the CMKLR1 peptide was analyzed by ELISA. The OD at 450 nm was measured by ELISA, and the 2G1 antibody on the CMKLR1 EL3 loop (SEQ ID No: 18) was analyzed. The binding of ) was compared with that of the isotype control antibody (3G8) (x).

[0043] Figure 13The expression of CMKLR1 in different cell lines was investigated using FACS and Western blotting. A. CMKLR1 protein expression on the cell surface was measured using different concentrations (ng / ml) of 2G1 antibody in two different human T cell lines, Thrp1 and U937. B. CMKLR1 protein expression was tested using Western blotting in T cell lines (Thp1 and U937), fibroblast cell line MRC5, NK cell line NKL, and CMKLR1-negative and transduced CHO cells.

[0044] Figure 14 CMKLR1 expression in mouse bone marrow cells was tested using FACS. After differentiation, CMKLR1 expression on the cell surface of mouse bone marrow lineage cells was analyzed. A. Expression on macrophages / monocytes (M0). B and C. Expression on macrophages (pro-inflammatory mM1 and pro-regressive mM2 macrophages). D and E. Expression on dendritic cells (mDC and iDC).

[0045] Figure 15 CMKLR1 expression stimulated by pro-inflammatory stimuli in human monocytes and mouse bone marrow cells.

[0046] The expression of CMKLR1 (ChemR23) was measured by FACS 16 or 48 hours after cells were stimulated with LPS, TNFα, or IL6. A. Results from human blood mononuclear cells; B. and C. Results from bone marrow cells and neutrophils in mouse bone marrow.

[0047] Figure 16 A study of markers of bone marrow cell activation following CMKLR1 pathway activation. Dendritic cells were incubated in excipients, RvE1, 2G1, or isotype control (hIgG1) and then stained for FACS analysis using marker antibodies: A. CD80-PE. B. CD86-FITC. C. CD103-PerCPCy5.5. D. CD40-PeCy7. EI / Ab-APC. The mean fluorescence value was measured under each condition.

[0048] Figure 17 Studies on CMKLR1 pathway activation: Akt and Erk phosphorylation. Western blot analysis was performed on the ERK and AKT activation pathways after incubating mouse pro-inflammatory (M1) macrophages with 2G1 or RvE1 for 5, 10, and 30 minutes. Phosphorylated Akt or Erk proteins were evaluated using p-Akt or p-Erk antibodies (p44 / 42). A. Activation of Erk and Akt using RvE1. B. Activation of Erk and Akt using 2G1.

[0049] Figure 18A competitive study on the interaction between chemokine-CMLKR1 and anti-CMKLR1 antibody.

[0050] A. Tested DiscoverX from two different vendors ( The inhibitory effect of chemokines from R&D Systems (▲) on cAMP, or used alone in combination with anti-CMKLR1 antibody (2G1) ), or with DiscoverX ( It is used in combination with chemokines.

[0051] B. Different concentrations of anti-CMKLR1 antibody from 1 µM to 1 nM and 2 nM ( ) or 6 nM ( Beta-inhibiting protein activation in the presence of chemokines.

[0052] Figure 19 Anti-CMKLR1 antibody on CD45Rb 高 Role of T-cell metastasis in a mouse model of chronic colitis. A. Tracking weight changes in treated animals for up to sixty days. Animals were treated with isotype control hIgG1 (x) or anti-CMKLR1 antibody (■). B. Histological staining of colon tissue from mice treated with anti-CMKLR1 (right) compared to the control (left). C. Anatomical pathological scores (inflammation score, vasculitis score, colon thickness, and fibrotic colonic wall) used to calculate symptom and inflammation severity. D. CD3 and Ly6G infiltration.

[0053] Figure 20 The effect of anti-CMKLR1 antibody on a mouse model of hepatocellular carcinoma (HCC). The antitumor effect of anti-CMKLR1 antibody (2G1, 0.8 mg / kg) was assessed by intraperitoneal administration three times a week for two weeks, followed by two injections of anti-PD1 mAb on days 4 and 8. RMP1-14 Cloning, 8 mg / kg) in combination (▲) or without combination ( ), or in combination with a single injection (twice a week) of anti-PD-1 antibody ( In an orthogonal model of hepatocellular carcinoma in mice, mice were treated for 2 weeks (2.5 × 10^6 Hepatocellular carcinoma 1.6 cells were injected via the portal vein on day 0). Isotype control antibodies were administered at 0.8 mg / kg three times a week for 2 weeks. A partial response (PR) was defined as mice surviving for several days to one month after treatment cessation; a complete response (CR) was defined as mice surviving for more than one month or being cured when their survival time was three times longer than that required for staining in all control mice.

[0054] Figure 21 Anti-CMKLR1 antibodies were generated in different cell lines.

[0055] The CDR of the 2G1 heavy chain was grafted into three human germline backbones and named IGHV3-23*04, IGHV1-46*01, and IGHV7-4-1 (IMGT nomenclature). The CDR of the 2G1 light chain was grafted into three human germline backbones and named IGKV1-13*02, IGKV6-21*01, and IGKV3-11*01 (IMGT nomenclature). Each sequence was fused with a constant fragment of human immunoglobulin and co-transfected in mammalian cells to generate humanized antibodies in COS and CHO cells. Yields were evaluated in the supernatant. VH WT and VL WT correspond to the heavy and light chains of the 2G1 antibody, respectively.

[0056] Figure 22 The generation of humanized antibodies derived from 2G1.

[0057] The concentrations and yields of different combinations of heavy and light variable-strand domains in CHO cells. VHWT and VLWT correspond to the variable domains of the 2G1 antibody. Germplasm lines were generated from humanized forms of the variable domains of 2G1 and are disclosed in the examples below.

[0058] Figure 23 Humanized anti-CMKLR1 derived from 2G1 binds to and recognizes the C7 peptide. The binding of C7biot in the supernatant of CHO cells transfected with different combinations of heavy and light chains (VHWT+VLWT: combination of SEQ ID No. 37 and SEQ ID No. 49; HCLC: combination of SEQ ID No. 42 and SEQ ID No. 52; HDLC: combination of SEQ ID No. 43 and SEQ ID No. 52; HCLD: combination of SEQ ID No. 42 and SEQ ID No. 53; HDLD: combination of SEQ ID No. 43 and SEQ ID No. 53).

[0059] Figure 24ED50 of anti-CMKLR1 derived from 2G1 antibody. 2G1wt: HALA: combination of SEQ ID No. 41 and SEQ ID No. 50; combination of SEQ ID No. 37 and SEQ ID No. 49; HCLC: combination of SEQ ID No. 42 and SEQ ID No. 52; HDLC: combination of SEQ ID No. 43 and SEQ ID No. 52; HCLD: combination of SEQ ID No. 42 and SEQ ID No. 53; HDLD: combination of SEQ ID No. 43 and SEQ ID No. 53.

[0060] Figure 25 CCR7 expression was inhibited on M1 macrophages coated with anti-ChemR23 antibody in vitro. 2G1 macrophages and all humanized 2G1 variants (HALA, HCLC, HCLD, HDLC, and HDLD) were immobilized on plates. Isotype controls were added as a reference. Two isotypes that inhibit FcRγ binding were also added: 2G1-N297A (2G1wt mutated in N297A to reduce FcγR binding) and 2G4 (wild-type, where isotype IgG4 is mutated in S228P to stabilize the hinge region). Pro-inflammatory macrophages M1 were added to the coated plates for 48 hours, and CCR7 expression on the macrophage surface was measured by flow cytometry.

[0061] Figure 26 Neutrophil apoptosis. A) Neutrophil survival / death B) Caspase-3 expression C) ROS production.

[0062] Neutrophils were isolated from the blood of healthy volunteers and cultured for 24 hours (mortality analysis) or 4, 6, and 11 hours (Caspase-3 assay) or 5 hours (ROS assay) on coated isotype controls (crosses), chimeric anti-ChemR23 2G1 (squares), or different humanized forms of 2G1 (diamonds). PMN mortality was analyzed by incubating PMNs with specific markers of death and viability, followed by counting via image analysis. Caspase-3 was visualized by Western blotting, and signal intensity was determined using software. ROS generation was visualized using specific markers and analyzed on images.

[0063] Figure 27 -In vivo neutrophil apoptosis-A) Experimental procedure-B) Expression of ChemR23-C) Neutrophil frequency in exudate-D) Macrophage frequency in exudate-E) Neutrophil mortality rate-F) Dead / surviving neutrophil ratio.

[0064] Sterile air was injected twice on day 3 (d3) and day 6 (d6), and carrageenan was injected to induce inflammation. Exudate was collected at different times and stained for flow cytometry analysis.

[0065] Figure 28 - Neutrophil migration rate - A) PMN migration rate in healthy patients - B) PMN ratio in patients with ANCA-associated inflammation Endothelial cells were coated with 100 U / mL TNFα and activated overnight (+ / -). PMNs were then added or not added with 100 U / mL TNFα and antibody (Ab) for 2 hours. Migrating PMNs were collected and analyzed by flow cytometry.

[0066] Figure 29 - CD62L expression. PMNs from healthy volunteers were incubated for different times in medium containing 10 µg / mL coated antibody and collected for CD62L staining by flow cytometry analysis (left panel). Soluble form of CD62L released through detachment was detected by ELISA in the PMN supernatant incubated with the coated antibody (right panel).

[0067] Figure 30 - Survival rate of mesothelioma models Starting on day 4, AK-7 cells (3M) were injected into the pleural cavity, and mice received 1 mg / kg of isotype control or 2G1 three times a week for three weeks.

[0068] Figure 31 - CRC Models A) CRC Tumor Cell Inoculation Model: Tumor Development and Survival B) Chemical and Inflammation-Induced CRC.

[0069] Subcutaneous tumor inoculation was performed using 0.5M MC38 CRC cell line. Starting on day 4 post-tumor induction, mice were injected three times weekly with 1 mg / kg of 2G1 or control antibody b for 3 weeks. Cyclophosphamide was administered intraperitoneally at 100 mg / kg once a week. Tumor development was assessed three times weekly, and tumors >1000 mm in mice were monitored. 3 Establish survival rate curves in time.

[0070] In the AOM-DSS model, mice received an intraperitoneal injection of 7.5 mg / kg azomethane. Five days later, three cycles of 5-day-DSS and 14-day-water therapy were initiated. After the first cycle, either a treatment agent or a placebo was administered twice weekly until the end of the cycle. Mice were weighed and their feces analyzed twice weekly. Colon and tumors were measured and counted 80 days after the chemical injection.

[0071] Figure 32- An experimental model of autoimmune encephalomyelitis. – A) Changes in body weight over time; B) Disease score. Central nervous system injury was induced by injection of an adjuvant-conjugated immunogenic MOG peptide. When the animal's clinical score was equal to 2, indicating that the central nervous system had been affected by T cell activity, the treatment agent (2G1) or isotype control was administered at 1 mg / kg until the end of the experiment.

[0072] Figure 33 - The binding of purified antibody to human ChemR23 peptide was determined by ELISA (A), ED50 concentration (ng / ml) (B). For the activity ELISA assay, Fc-specific donkey anti-human IgG (Jackson Immunoresearch; USA; Reference No. 709-005-098) was immobilized on a plastic at 1.3 µg / ml in borate buffer (pH 9), and purified antibody was added to measure binding in 1% BSA buffer (compared to wild-type 2G1). After incubation and washing, a biotinylated antigen-specific peptide (Biot-C7 peptide) was added, followed by peroxidase-streptomycin (Jackson Immunoresearch; USA; Reference No. 016-030-084) and visualized using standard methods.

[0073] Figure 34 - A. ELISA assay of the binding of purified antibodies in human ChemR23 after incubation at 4°C or 37°C for 7 days. B. SEC analysis of purified antibodies incubated at 37°C for 7 days by gel filtration chromatography. 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 purified antibodies was analyzed by ELISA, and aggregate formation was analyzed by gel filtration (Superdex 200 10 / 300GL, GeHealthcare).

[0074] Figure 35 - Inhibition of CCR7 expression on in vitro M1 macrophages coated with anti-ChemR23 antibody. Humanized 2G1 variant HEF-LD-T52S was immobilized on plates. An isotype control was added as a control. Pro-inflammatory M1 macrophages were added to the coated plates, and CCR7 expression on the macrophage surface was measured by flow cytometry.

[0075] Figure 36- Increase in in vitro dead PMN neutrophils coated with anti-ChemR23 antibody. PMNs from healthy volunteers were incubated for 24 hours in medium containing 10 µg / mL of HEF-LDT52S, HEF-LEF, and HDLD antibody variants and stained with a death / survival assay kit (LIVE / DEAD, Invitrogen). The percentage of positive cells was obtained by analyzing the images using Fiji software. An isotype control was added as a control. A mutant HEF-LDT52S antibody (HEF-LDT52S N297A) that does not bind to the Fc receptor (FcR) was also added. Detailed Implementation

[0076] This invention relates to a CMKLR1 agonist with regressor-like E1 ability, which is used to treat inflammatory conditions in patients, particularly inflammatory conditions in which the inflammatory resolution phase is delayed or interrupted, especially in chronic inflammation, and particularly the agonist is selected from the group consisting of antibodies or their antigen-binding fragments, peptides, polypeptides and proteins.

[0077] In the following description, unless otherwise stated, a CMKLR1 agonist with regressor E1-like ability may be referred to as the "agonist" or "an agonist"; both terms include antibodies or antigen-binding fragments thereof (also known as anti-CMKLR1 antibodies), proteins, peptides, or polypeptides; the terms "compound" or "anti-CMKLR1 compound" may also be used in the following description as a synonym for "agonist" (of the present invention), thereby including antibodies or antigen-binding fragments thereof (also known as anti-CMKLR1 antibodies), proteins, peptides, or polypeptides. Among the effects provided by the use of such an agonist, the following specific effects have been demonstrated: - CMKLR1 agonists with regression-like E1 capabilities induce apoptosis of polymorphonuclear neutrophils (also referred to herein as neutrophils, PMNs, or PMNs) and / or reduce or inhibit the migration ability of these cells, particularly by inhibiting their ability to migrate through the endothelium to sites of inflammation, as described below in this application; - CMKLR1 agonists with relapse-like E1 capabilities induce the internalization of different receptors expressed on the cell surface of various bone marrow cells, especially macrophages and / or dendritic cells, thereby enhancing the induction or maintenance of inflammatory resolution processes, as described below in this application; - In particular, CMKLR1 agonists with insufficiency-like E1 capabilities induce the internalization of different receptors CMKLR1, as well as CXCR4 and / or CCR7, expressed on the cell surface of macrophages and / or dendritic cells. This internalization results in much lower targeting and recognition of CXCR4 and CCR7 receptors by cytokines known to induce cell migration to sites of inflammation. As a result, it leads to reduced or inhibited migration of macrophages and / or dendritic cells from sites of inflammation to secondary lymphoid organs and / or toward sites of inflammation. - CMKLR1 agonists with regression-like E1 capabilities reduce or inhibit the ability of neutrophils and macrophages and / or dendritic cells to migrate; in particular, they reduce or inhibit the ability of neutrophils to migrate through sites of inflammation by reducing the rolling ability of these cells (due to internalization of CD62L and / or reduced expression on the cell surface), thereby reducing their ability to migrate through the endothelium. - In one particular aspect of the invention, the inventors have demonstrated that CMKLR1 agonists with regressive E1-like capabilities, including domains suitable for interaction with Fc receptors (such as the IgG constant domain), particularly the IgG1 constant domain, are particularly effective. Specifically, Fc receptors on macrophages or neutrophils very effectively recognize Fc fragments of the anti-CMKLR1 IgG constant domain or the IgG1 constant domain, thereby leading to or facilitating neutrophil apoptosis recognized by anti-CMKLR1 IgG1 antibodies.

[0078] - The inventors here demonstrate that bone marrow cells that participate in the inflammatory process (i.e., that maintain inflammation) and express CMKLR1 and CXCR4 and / or CCR7 are particularly suitable targets for treating pathological inflammatory processes by CMKLR1 agonists with regression-like E1 capabilities.

[0079] This invention particularly relates to an anti-chemokine receptor 1 (CMKLR1) agonist with regressor-like E1 capability, for the treatment of patients with inflammatory conditions, especially those in which the resolution of inflammation is delayed or interrupted. The CMKLR1 agonist with regressor-like E1 capability is selected from the group consisting of antibodies or their antigen-binding fragments, peptides, polypeptides, and proteins, and wherein the agonist: - Induces or activates neutrophil apoptosis at sites of inflammation, and / or - Inhibit or reduce the ability of neutrophils to migrate through the endothelium to sites of inflammation, and / or - Inhibits the migration of macrophages and / or dendritic cells from the site of inflammation to secondary lymphoid organs and / or to the site of inflammation.

[0080] - This invention relates to the use of CMKLR1 agonists (such as antibodies, especially humanized antibodies or their antigen-binding fragments) with regression-like E1 capabilities in inducing or enhancing or activating neutrophil apoptosis in the treatment of inflammatory conditions, particularly those in which the resolution of inflammation is delayed or interrupted.

[0081] In a particular embodiment, the present invention relates to a humanized anti-CMKLR1 antibody or its antigen-binding fragment or antigen-binding antibody mimic or modified antibody.

[0082] As used herein, the term "antibody" includes polyclonal antibodies, monoclonal antibodies, or recombinant antibodies. As used herein, "monoclonal antibody" refers to an antibody molecule formulation used to obtain antibodies that share a common heavy chain and a common light chain amino acid sequence, in contrast to "polyclonal" antibody formulations, which are mixtures of antibodies containing different amino acid sequences. Monoclonal antibodies can be produced using several known techniques, such as phage, bacterial, yeast, or ribosome display, as well as classical methods such as hybridoma-derived antibodies. They can also be synthesized using a published amino acid sequence as a reference. Therefore, the term "monoclonal" is used to refer to all antibodies derived from a single nucleic acid clone.

[0083] As used herein, the term "antibody" further includes antibodies that have been modified compared to wild-type antibodies; and includes chimeric antibodies, humanized antibodies, modified antibodies, and antigen-binding antibody mimics. The specific wild-type reference antibody in the context of this invention is antibody 2G1.

[0084] The antibodies of this invention include recombinant antibodies. As used herein, the term "recombinant antibody" means an antibody produced, expressed, generated, or isolated in a recombinant manner, such as an antibody expressed using a recombinant expression vector transfected into a host cell; an antibody isolated from a library of recombinant antibody combinations; 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 in any other manner in which a specific immunoglobulin gene sequence (e.g., a human immunoglobulin gene sequence) is assembled with other DNA sequences. Recombinant antibodies include, for example, chimeric antibodies and humanized antibodies.

[0085] As used herein, a "chimeric antibody" refers to an antibody in which a sequence of a variable domain derived from a lineage of one mammal species (e.g., mouse) has been transplanted onto a sequence of a constant domain derived from a lineage of another mammal species (e.g., human).

[0086] As used herein, in a first embodiment, a “humanized antibody” refers to an antibody in which a CDR sequence derived from another mammalian species (e.g., mouse) has been transplanted onto a human or, in particular, humanized backbone region sequence. In a further embodiment, a “humanized antibody” refers to an antibody in which at least one CDR and all or part of the backbone region sequence has been humanized.

[0087] As used herein, "antigen-binding fragment of an antibody" means a portion of an antibody, i.e., a molecule corresponding to a portion of the antibody structure of the present invention, which exhibits antigen-binding ability to CMKLR1, and may be in its native form; the fragment exhibits the same or substantially the same antigen-binding specificity to the antigen as the corresponding four-chain antibody. Advantageously, the antigen-binding fragment has a binding affinity similar to that of the corresponding four-chain antibody. However, antigen-binding fragments with reduced antigen-binding affinity to the corresponding four-chain antibody are also included in the present invention. Antigen-binding ability can be determined by measuring the affinity between the antibody and the target fragment. These antigen-binding fragments may also be referred to as "functional fragments" of the antibody.

[0088] The antigen-binding fragment of an antibody is a segment containing a hypervariable domain called a CDR (complementarity-determining region) or a portion thereof containing the antigen recognition site, namely the extracellular domain of CMKLR1, particularly the third loop of the extracellular domain of CMKLR1 (called EL3), thus defining antigen recognition specificity. EL3 is located between amino acid residues 283 and 300 of SEQ ID No: 1. The amino acid sequence of the EL3 domain is the amino acid sequence SEQ ID No: 2. EL3 is also contained in the polypeptide of SEQ ID No: 52. Each light chain variable domain and heavy chain variable domain (VL and VH, respectively) of a four-chain immunoglobulin has three CDRs: the CDRs of the light chain variable domain are called VLCDR1, VLCDR2, and VLCDR3; the CDRs of the heavy chain variable domain are called VHCDR1, VHCDR2, and VHCDR3. Each light and heavy chain variable domain of a four-chain immunoglobulin has four framework regions (FRs). The FRs of the light chain variable domains are called LFR1, LFR2, LFR3, and LFR4; the FRs of the heavy chain variable domains are called HFR1, HFR2, HFR3, and HFR4. The nomenclature system used to define the CDR domains and framework domains is the Kabat system.

[0089] Technicians can determine the location of each region / domain of the antibody by referring to standard definitions in this regard (including reference numbering systems, reference KABAT numbering systems) or by applying the IMGT "collier de perle" algorithm. In this regard, it is worth noting that the delimitation of regions / domains can vary depending on the reference system, for the definition of the sequence of this invention. Therefore, regions / domains as defined in this invention include sequences that show a change in the length or location of the relevant sequence (approximately + / - 10%) within the full-length sequence of the antibody's variable domain.

[0090] Based on the structure of four-stranded immunoglobulins, antigen-binding fragments can thus be defined by comparing them with antibody sequences in existing databases and prior art, particularly by comparing the positions of functional domains within these sequences. Note that for various types of antibodies, especially IgG, and especially mammalian IgG, the positions of the backbone and constant domains are well-defined. This comparison also involves data related to the 3D structure of the antibody.

[0091] For the purpose of illustrating specific embodiments of the present invention, antigen-binding fragments containing variable domains of the antibody including the CDR of the antibody include Fv, dsFv, scFv, Fab, Fab', and F(ab')2. The Fv fragment consists of the VL and VH domains of the antibody linked together by hydrophobic interactions; in the dsFv fragment, the VH:VL heterodimer is stabilized by disulfide bonds; in the scFv fragment, the VL and VH domains are linked together by flexible peptide linkers to form a single-chain protein. Fab fragments are monomeric fragments obtainable by papain digestion; they contain a VH-CH1 fragment of the entire light L and H chains linked together by disulfide bonds. The F(ab')2 fragment can be produced by pepsin digestion of the antibody below the hinge disulfide bond; it contains two Fab' fragments, as well as a portion of the hinge region of an immunoglobulin molecule. The Fab' fragment can be obtained from the F(ab')2 fragment by cleaving the disulfide bonds in the hinge region. The F(ab')2 fragments are bivalent, meaning they contain two antigen-binding sites, like innate immunoglobulin molecules; on the other hand, the Fv (the VHVL dimer constituting the variable part of Fab), dsFv, scFv, Fab, and Fab' fragments are monovalent, meaning they contain a single antigen-binding site. These basic antigen-binding fragments of the present invention can be combined to obtain multivalent antigen-binding fragments, such as dimeric, trimeric, or tetrameric ones. These multivalent antigen-binding fragments are also part of the present invention.

[0092] As used herein, the term "antibody" includes "bispecific" antibody, and refers to an antibody that recognizes two different antigens by having at least one region specific to a first antigen (e.g., a variable region derived from a first antibody) and at least one second region specific to a second antigen (e.g., a variable region derived from a second antibody). A bispecific antibody specifically binds to two target antigens and is therefore a multispecific antibody. Multispecific antibodies that recognize two or more different antigens can be produced by recombinant DNA methods or can be, but are not limited to, antibodies produced chemically by any convenient method. Bispecific antibodies include all antibodies or antibody conjugates, or polymeric forms of antibodies, capable of recognizing two different antigens. Bispecific antibodies include antibodies that have been reduced and recombined to retain their bivalent properties, as well as antibodies that have been chemically conjugated so that they can have multiple antigen recognition sites for each of the following antigens: BiME (Bispecific Macrophage Enhancing antibodies), BiTE (Bispecific T cell engager), DART (Dual affinity retargeting); DNL (dock-and-lock), DVD-Ig (dual variable domain immunoglobulins), HAS (human serum albumin), and kih (knobs-into-holes).

[0093] Antigen-binding antibody mimics are organic compounds that specifically bind to antigens but are structurally independent of antibodies. They are typically artificial peptides or small proteins with a molar mass of about 3–20 kDa. Nucleic acids and small molecules are sometimes considered antibody mimics, but they are not artificial antibodies, antibody fragments, or fusion proteins composed of these. Their shared advantages with antibodies are better solubility, tissue penetration, stability to heat and enzymes, and relatively low production costs. Antigen-binding antibody mimics are being developed as therapeutic and diagnostic agents. Antigen-binding antibody mimics can also be selected from the group including affibody, affilin, affimier, affitin, designed ankyrin repeat proteins (DARPin), and monobody.

[0094] Antigen-binding antibody mimics are more preferably selected from the group including affitin and anti-carrier protein (anticalin). Affitin is an artificial protein with the ability to selectively bind antigens. They are structurally derived from the DNA-binding protein Sac7d, which is found in *Saccharomyces cerevisiae* (acidophilic thermosulfuric leaf mold). Sulfolobus acidocaldariusAffitins are microorganisms belonging to the archaeal domain. Affitin libraries can be generated by randomizing the amino acids on the Sac7d binding surface (e.g., by generating variants with random substitutions of 11 residues corresponding to the Sac7d binding interface), and the resulting protein libraries can be subjected to multiple rounds of ribosome display, with affinity targeting a variety of targets, such as peptides, proteins, viruses, and bacteria. Affitins are antibody mimics and are being developed as tools in biotechnology. They are also used as specific inhibitors of various enzymes (Krehenbrink et al., Journal of Molecular Biology (J. mol. Biol.), 383:5, 2008). Those skilled in the art can readily develop affitins with desired binding properties using methods known in the art, particularly those disclosed in patent application WO2008068637 and the publications cited above, especially using the antigen generation and screening of phage display and / or ribosome display libraries disclosed herein. Anticarrier proteins are artificial proteins capable of binding to antigens, whether proteins or small molecules. These are antibody mimics derived from human lipid transport proteins, a naturally bound family of proteins. The anti-transport proteins are approximately eight times smaller, with a size of about 180 amino acids and a mass of about 20 kDa (Skerra, Journal of the Federation of European Biochemical Societies (Febs J.), 275:11, 2008). Phage display libraries of anti-transport proteins, particularly those with specific binding properties, have been generated to allow for screening and selection. Those skilled in the art can readily develop anti-transport proteins with desired binding properties using methods known in the art, particularly those disclosed in European Patent EP1270725 B1, US Patent US8536307 B2, Schlehuber and Skerra, Journal of Biophysical Chemistry (Biophys. Chem.), 96:2-3, 2002, and the publications cited above, especially using the antigen generation and screening of phage display and / or ribosome display libraries disclosed herein. Both anticarrier proteins and affitin can be produced in many expression systems that include bacterial expression systems. Therefore, this invention includes the use of affitin, anticarrier proteins, and other similar antibody mimics having the characteristics of the antibodies described herein, particularly regarding their binding affinity to CMKLR1, their agonistic ability to bind between RvE1 and CMKLR1, their ability to induce or inhibit the secretion of specific cytokines as described herein, and their use in the treatment or prevention of diseases as described herein; all such mimics are considered mimics according to the invention.

[0095] As used herein, “modified antibody” refers to an antibody whose amino acid sequence has been modified by mutation of at least one amino acid residue. Therefore, “modified antibody” encompasses chimeric antibodies or humanized antibodies as defined herein, and can also correspond to a molecule containing an antibody or an antigen-binding fragment thereof, wherein the monoclonal antibody or its functional fragment is associated with a molecule with a different function. The modified antibodies of this invention can be fusion chimeric proteins or conjugates generated by any suitable attachment method, including covalent attachment, grafting, chemical binding to a chemical or biological group or molecule (such as a PEG polymer) or another protective group or molecule suitable for preventing cleavage by proteases in vivo, for improving the stability and / or half-life of the antibody or functional fragment.

[0096] "Humanized" non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other target-binding sequences of the antibody) containing a minimal sequence derived from a non-human immunoglobulin. Typically, humanized antibodies comprise substantially all of at least one, typically two, variable domains, wherein all CDR regions correspond to the CDR regions of non-human immunoglobulins and / or their humanized forms; and all or substantially all FR regions are FR regions of a human immunoglobulin template sequence, or contain substitutions of amino acid residues present in the human immunoglobulin template sequence with non-human residues (e.g., rodent residues) at corresponding positions. Humanized antibodies may also comprise at least a portion of an immunoglobulin constant region (Fc), typically a constant region of a selected human immunoglobulin template. In one particular embodiment, the present invention relates to an antibody comprising a heavy chain variable region as disclosed herein and a light chain variable region as disclosed herein, the heavy chain variable region and / or the light chain variable region further comprising a constant region, particularly an Fc region.

[0097] The terms "specifically binding" and "specifically bind to" refer to antibodies, their antigen-binding fragments, antigen-binding antibody mimics, or modified antibodies according to the present invention, with a binding affinity of at least 1 x 10⁻⁶. -6 M, 1 x 10 -7 M, 1 x 10 -8 M, 1 x 10 -9 M, 1 x 10 -10 M, 1 x 10 -11 M, 1 X10 -12The ability to bind to CMKLR1 with an affinity of M or higher, and / or to CMKLR1 with an affinity at least twice that to nonspecific targets (e.g., another protein besides CMKLR1). Affinity can be assessed using a variety of 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 plot.

[0098] The term "therapeutic effective amount" is used to refer to the amount of any given compound as defined herein that is sufficient to at least improve the clinical or physiological condition of the patient being treated. The therapeutic effective amount of the antibody, its antigen-binding fragment, antigen-binding antibody mimic, or modified antibody to be administered according to the invention is affected by factors such as the disease being treated, the specific mammal being treated, the individual patient's clinical symptoms, the cause of the disease, the site of delivery of the agent, the method of administration, the timing of administration, and other factors known to a medical practitioner.

[0099] All embodiments of the antibodies or antigen-binding fragments disclosed herein may be transformed into the macromolecules of the present invention, particularly antigen-binding antibody mimics and modified antibodies, with necessary modifications.

[0100] In one specific embodiment of the present invention, CMKLR1 is human CMKLR1, which corresponds to the NCBI protein with accession number Q99788.2.

[0101] Both the heavy chain variable domain and the light chain variable domain comprise three CDRs (CDR1, CDR2, and CDR3 from the 5' to the 3' end, respectively) and four backbone regions (FR1, FR2, FR3, and FR4 from the 5' to the 3' end, respectively). Humanization of the mouse antibody may include humanizing at least one backbone region of the light chain variable region and / or the heavy chain variable region, or both. In a particular embodiment, several backbone regions may be humanized, particularly within the heavy chain variable region and within the light chain variable region. Wild-type CDRs may be conserved, but these CDRs may also be replaced by the CDRs described herein. Thus, when the backbone regions are humanized, the anti-CMKLR1 compound according to the invention may comprise at least one, at least two, at least three, at least four, at least five, or six wild-type CDRs. In other words, the anti-CMKLR1 compound is a humanized form of the parental chimeric antibody 2G1 (which contains the heavy chain variable domain of SEQ ID No. 37 and the light chain variable domain of SEQ ID No. 49), wherein at least one backbone region is humanized, and in particular, at least one backbone region and at least one CDR are humanized. In a particular embodiment of the invention, the variable region of the antibody may be associated with an antibody constant region (e.g., the constant region of IgG1, IgG2, IgG3, or IgG4, especially the constant region of IgG1). These constant regions can be further mutated or modified by methods known in the art to alter their binding affinity to the Fc receptor. In one particular embodiment, the antibody or antigen-binding fragment according to the invention is a humanized monoclonal antibody, particularly wherein the antibody light chain constant domain is derived from the human kappa light chain constant domain, particularly wherein the light chain constant domain comprises or consists of the sequence SEQ ID No: 79, and wherein the antibody heavy chain constant domain is derived from the human IgG1, IgG2, IgG3, or IgG4 heavy chain constant domain, particularly the IgG1 heavy chain constant domain, particularly wherein the antibody heavy chain constant domain comprises or consists of the amino acid sequences SEQ ID No. 80, SEQ ID No. 81, SEQ ID No. 82, SEQ ID No. 83, or SEQ ID No. 84, particularly from the human IgG1 heavy chain constant domain, particularly wherein the antibody heavy chain constant domain comprises or consists of the amino acid sequences SEQ ID No. 80, SEQ ID No. 81, SEQ ID No. 82, SEQ ID No. 83, or SEQ ID No. 84, particularly from the human IgG1 heavy chain constant domain, particularly wherein the antibody heavy chain constant domain comprises or consists of the amino acid sequences SEQ ID No. 80 or SEQ ID No. 83, or SEQ ID No. 84. Composed of IDNo.83.

[0102] The binding affinity assay performed by ELISA, as disclosed in the embodiments of the present invention, can be used to evaluate the binding affinity of peptides comprising amino acid residue sequences of SEQ ID No: 2 and / or SEQ ID No: 59, or consisting of amino acid residue sequences of SEQ ID No: 2 and / or SEQ ID No: 59, and / or located within amino acid sequence SEQ ID No: 60, and / or for binding the third outer ring of CMKLR1. To determine whether a test antibody can competitively bind the same antigen or third ring, or the same epitope bound by the 2G1 antibody (or an antigen-binding fragment comprising the heavy 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 peptide comprising an epitope or third ring, or consisting thereof, can be coated onto the wells of a microtiter plate and pre-incubated with or without a candidate competitive antibody, followed by the addition of the biotin-labeled 2G1 antibody of the present invention. The amount of labeled anti-2G1 antibody bound to the peptides containing or composed of the peptides of SEQ ID No: 2 and / or SEQ ID No: 59 and / or located within the third ring of the amino acid sequence SEQ ID No: 60 and / or CMKLR1 in the wells is measured using an avidin-peroxidase conjugate and a suitable substrate. The antibodies can be labeled with radioactive or fluorescent labels or some other detectable and measurable labels. The amount of labeled anti-2G1 antibody bound to the peptides of SEQ ID No: 2 and / or SEQ ID No: 59 and / or located within the amino acid sequence SEQ ID No: 60 and / or bound to the third ring is indirectly related to the ability of candidate competitive antibodies (test antibodies) to compete for binding to the same epitope or the same ring; that is, the greater the affinity of the test antibody for the same epitope, the less labeled 2G1 antibody will bind to the antigen-coated wells. Compared to a parallel control performed in the absence of a candidate competitive antibody (but the presence of known non-competitive antibodies), a candidate competitive antibody is considered to be an antibody that competes with the 2G1 antibody of the present invention for binding to the same polypeptide or third ring when the candidate antibody can block the binding of at least 20%, preferably at least 20%-50%, or even more preferably at least 50% of the 2G1 antibody. It should be understood that variations of this assay can be performed to obtain the same quantitative value.

[0103] Anti-CMKLR1 antibodies or their antigen-binding fragments have the effect of pro-inflammatory remission factors, especially through their interaction with myeloid cell lineage.

[0104] In one particular embodiment of the invention, a specific VHCDR2 is present in an antibody that can exhibit reduced immunogenicity in the human body, thereby allowing the provision of an antibody that can be more effectively used for therapeutic purposes, since fewer side effects are desired when the reduced immunogenic antibody is administered to a patient.

[0105] In one particular aspect of the invention, antibodies against chemokine-like receptor 1 (CMKLR1) or antigen-binding fragments thereof that bind to CMKLR1, particularly human CMKLR1, are disclosed, said antibodies or antigen-binding fragments comprising: a. Antibody heavy chain variable (VH) domain, which contains 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. Antibody light chain variable (VL) domain, which contains 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.

[0106] Specifically, the antibody or its antigen-binding fragment specifically binds to an epitope located in the third extracellular loop (EL3) of CMKLR1. More specifically, the antibody or its antigen-binding fragment specifically binds to a polypeptide containing the amino acid sequence SEQ ID No: 2 or SEQ ID No. 59 or to an epitope located in the amino acid sequence SEQ ID No. 60.

[0107] In another specific aspect of the invention, antibodies against chemokine receptor 1 (CMKLR1) or antigen-binding fragments thereof that bind to CMKLR1, particularly human CMKLR1, are disclosed, said antibodies or antigen-binding fragments comprising: a. Antibody heavy chain variable (VH) domain, which contains 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 corresponds to an 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; b. Antibody light chain variable (VL) domain, which contains 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.

[0108] Specifically, the antibody or its antigen-binding fragment specifically binds to an epitope located in the third extracellular loop (EL3) of CMKLR1. More specifically, the antibody or its antigen-binding fragment specifically binds to a polypeptide containing the amino acid sequence SEQ ID No: 2 or SEQ ID No. 59 or to an epitope located in the amino acid sequence SEQ ID No. 60.

[0109] In another specific aspect of the invention, antibodies against chemokine receptor 1 (CMKLR1) or antigen-binding fragments thereof that bind to CMKLR1, particularly human CMKLR1, are disclosed, said antibodies or antigen-binding fragments comprising: a. Antibody heavy chain variable (VH) domain, which contains 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 corresponds to the amino sequence of SEQ ID NO: 12, the heavy chain variable (VH) domain does not contain the backbone VHFR3 of SEQ ID No. 70, preferably, the heavy chain variable (VH) domain contains the backbone FR3 of SEQ ID NO: 69. ;as well as - 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. Antibody light chain variable (VL) domain, which contains 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.

[0110] Specifically, the antibody or its antigen-binding fragment specifically binds to an epitope located in the third extracellular loop (EL3) of CMKLR1. More specifically, the antibody or its antigen-binding fragment specifically binds to a polypeptide containing the amino acid sequence SEQ ID No: 2 or SEQ ID No. 59 or to an epitope located in the amino acid sequence SEQ ID No. 60.

[0111] Binding to epitopes located within the third extracellular loop (EL3) of CMKLR1 can be assessed using the methods disclosed above, particularly where the antibody or its antigen-binding fragment specifically binds to a polypeptide containing the amino acid sequence SEQ ID No: 2 or SEQ ID No. 59, or specifically binds to an epitope located within the amino acid sequence SEQ ID No. 60. Specific selection of VHCDR2 compared to antibodies with different VHCDR2s may allow for the provision of antibodies with reduced immunogenicity.

[0112] Such antibodies are CMKLR1 agonists that mimic the effect of regression E1 binding to CMKLR1, i.e., they possess the regression E1-like ability defined above. Anti-CMKLR1 antibodies or their antigen-binding fragments have pro-inflammatory regressor effects, particularly through interaction with bone marrow cell lineages.

[0113] In a specific embodiment of the present invention, the anti-CMKLR1 antibody or its antigen-binding fragment comprises: - VHCDR1 is selected from the group consisting of SEQ ID No. 3 and SEQ ID No. 4.

[0114] In a specific embodiment of the present invention, the anti-CMKLR1 antibody or its antigen-binding fragment comprises: - 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.

[0115] In a specific embodiment of the present invention, the anti-CMKLR1 antibody or its antigen-binding fragment comprises: - VHCDR3 is selected from the group consisting of SEQ ID No. 13, SEQ ID No. 14 and SEQ ID No. 15.

[0116] In a specific embodiment of the present invention, the anti-CMKLR1 antibody or its antigen-binding fragment comprises: - VLCDR1 is selected from the group consisting of SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19 and SEQ ID No. 23.

[0117] In a specific embodiment of the present invention, the anti-CMKLR1 antibody or its antigen-binding fragment comprises: - VLCDR2 is 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.

[0118] In a specific embodiment of the present invention, the anti-CMKLR1 antibody or its antigen-binding fragment comprises: - VLCDR3 is selected from the group consisting of SEQ ID No. 34, SEQ ID No. 35 and SEQ ID No. 36.

[0119] In a specific embodiment of the present invention, the anti-CMKLR1 antibody or its antigen-binding fragment comprises: - VHCDR1 is selected from the group consisting of SEQ ID No. 3 and SEQ ID No. 4; and / or - 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; and / or - VHCDR3 is selected from the group consisting of SEQ ID No. 13, SEQ ID No. 14 and SEQ ID No. 15.

[0120] - VLCDR1 is 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 is 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 is selected from the group consisting of SEQ ID No. 34, SEQ ID No. 35 and SEQ ID No. 36.

[0121] In one particular embodiment, the anti-CMKLR1 antibody or its antigen-binding fragment 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.

[0122] In one specific embodiment of the invention, the anti-CMKLR1 antibody or its antigen-binding fragment comprises a heavy chain variable domain comprising or consisting of an amino acid sequence selected from the group consisting of: SEQ ID No. 38, SEQ ID No. 39, SEQ ID No. 40, SEQ ID No. 62, SEQ ID No. 89, SEQ ID No. 90 and SEQ ID No. 91.

[0123] In a more specific embodiment, the anti-CMKLR1 antibody or its antigen-binding fragment comprises a heavy chain variable domain comprising or consisting of the amino acid sequence SEQ ID No. 91.

[0124] In one specific embodiment of the invention, the anti-CMKLR1 antibody or its antigen-binding fragment comprises a light chain variable domain comprising or consisting of an amino acid sequence selected from the group consisting of: SEQ ID No. 49, SEQ ID No. 50, SEQ ID No. 51, SEQ ID No. 52, SEQ ID No. 53, SEQ ID No. 54, SEQ ID No. 55, SEQ ID No. 56, SEQ ID No. 57, SEQ ID No. 58, SEQ ID No. 92, and SEQ ID No. 93.

[0125] In a more specific embodiment of the invention, the anti-CMKLR1 antibody or its antigen-binding fragment comprises a light chain variable domain comprising or consisting of the amino acid sequence SEQ ID No. 93.

[0126] In a specific embodiment of the present invention, the anti-CMKLR1 antibody or its antigen-binding fragment comprises: - A heavy chain variable domain comprising or consisting of an amino acid sequence selected from or composed of the following: SEQ ID No. 38, SEQ ID No. 39, SEQ ID No. 40, SEQ ID No. 62, SEQ ID No. 89, SEQ ID No. 90, and SEQ ID No. 91; and - A light chain variable domain comprising or consisting of an amino acid sequence selected from or composed of the following groups: 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.

[0127] Any combination of the specific heavy chain variable structural domains and light chain variable structural domains disclosed herein is included in this disclosure.

[0128] In a more specific embodiment, the anti-CMKLR1 antibody or its antigen-binding fragment according to the invention comprises a heavy chain variable domain comprising or consisting of amino acid residues selected from the group consisting of: SEQ ID No. 38, SEQ ID No. 39, SEQ ID No. 40 and SEQ ID No. 62; and a light chain variable domain comprising or consisting of an amino acid sequence selected from the group consisting of: SEQ ID No. 54, SEQ ID No. 55 and SEQ ID No. 56.

[0129] In a more specific embodiment of the invention, the anti-CMKLR1 antibody or its antigen-binding fragment comprises a heavy chain variable domain comprising or consisting of the amino acid sequence SEQ ID No. 38, and a light chain variable domain SEQ ID No. 49.

[0130] In a more specific embodiment of the invention, the anti-CMKLR1 antibody or its antigen-binding fragment comprises a heavy chain variable domain comprising or consisting of the amino acid sequence SEQ ID No. 91, and a light chain variable domain comprising or consisting of the amino acid sequence SEQ ID No. 93.

[0131] Furthermore, an advantageous feature of the antibody is that its Fc fragment is characteristic of IgG1.

[0132] In a second aspect of the invention, a compound selected from antibodies, their antigen-binding fragments, or chimeric, modified, or humanized antibodies is disclosed, which specifically binds to CMKLR1, particularly human CMKLR1, said compound comprising an antibody heavy chain variable domain comprising (i) VHCDR2, comprising the amino acid sequences SEQ ID No: 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, and SEQ ID No. 61 or consisting of the amino acid sequences SEQ ID No: 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, and SEQ ID No. 61, and (ii) VHCDR3, comprising the amino acid sequences SEQ ID No: 13, SEQ ID No. 14, SEQ ID No. 15, and SEQ ID No. 16 or mutant sequences thereof or consisting of the amino acid sequences SEQ ID No: 13, SEQ ID No. 14, SEQ ID No. 15, and SEQ ID No. 61, and (ii) VHCDR3, comprising the amino acid sequences SEQ ID No: 13, SEQ ID No. 14, SEQ ID No. 15, and SEQ ID No. 16, or mutant sequences thereof, or consisting of the amino acid sequences SEQ ID No: 13, SEQ ID No. 14, SEQ ID No. 15, and SEQ ID No. 61, and (ii) VHCDR3, comprising the amino acid sequences SEQ ID No: 13, SEQ ID No. 14, SEQ ID No. 15, and SEQ ID No. 16, and (ii) VHCDR3, comprising the amino acid sequences SEQ ID No: 13, SEQ ID No. 14, SEQ ID No. 15, and (ii) VHCDR3, comprising the amino acid sequences SEQ ID No: 13, SEQ ID No. 14, SEQ ID No. 15, and (ii) VHCDR3, IDNo. ​​16 or its mutated sequence, wherein amino acid residues are substituted, provided that amino acid residues at positions 1 and 2 of the mutated sequence are L and I, respectively; and The anti-CMKLR1 compound specifically binds to an epitope located within the third extracellular loop (EL3) of CMKLR1, particularly a polypeptide comprising or consisting of the amino acid sequence SEQ ID No:2 or SEQ ID No.59, or specifically binds to an epitope located within the amino acid sequence SEQ ID No.60; and The compound thereon competes with antibodies containing the heavy chain variable domain corresponding to SEQ ID No: 37 and the light chain variable domain corresponding to SEQ ID No: 49 for binding to the following peptides: peptides containing the amino acid sequence SEQ ID No: 2 or SEQ ID No. 59 or SEQ ID No. 60 or composed of the amino acid sequence SEQ ID No: 2 or SEQ ID No. 59 or SEQ ID No. 60, or peptides containing the third loop (EL3) of the extracellular domain of CMKLR1 or composed of peptides thereof.

[0133] The combination of the heavy chain variable domain of SEQ ID No. 37 and the light chain variable domain of SEQ ID No. 49 corresponds to the parental antibody 2G1. The binding of the compound to a polypeptide comprising or consisting of the amino acid sequence SEQ ID No. 2 or SEQ ID No. 59, or a polypeptide located within SEQ ID No. 60, or to a third loop (EL3) comprising or consisting of the extracellular domain of CMKLR1, can be evaluated according to the methods disclosed above and is illustrated in the embodiments of the present invention.

[0134] In a more specific embodiment, the anti-CMKLR1 antibody or its antigen-binding fragment comprises a heavy chain variable domain comprising or consisting of the amino acid sequence SEQ ID No. 91.

[0135] In a more specific embodiment of the invention, the anti-CMKLR1 antibody or its antigen-binding fragment comprises a light chain variable domain comprising or consisting of the amino acid sequence SEQ ID No. 93.

[0136] In a more specific embodiment of the invention, the anti-CMKLR1 antibody or its antigen-binding fragment comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises or is composed of the amino acid sequence SEQ ID No. 91, and the light chain variable domain comprises or is composed of the amino acid sequence SEQ ID No. 93.

[0137] Furthermore, an advantageous feature of the antibody is that its Fc fragment is characteristic of IgG1.

[0138] This invention relates to any of the compounds of the invention as defined above, for the prevention and / or treatment of diseases in which the resolution of inflammation is delayed or interrupted, and / or diseases selected from: inflammatory diseases, especially acute inflammatory diseases, chronic inflammatory diseases such as chronic inflammatory lung disease (e.g., asthma), keratoconjunctivitis, periodontitis, eczema, inflammatory bowel disease (especially Crohn's disease or colitis, especially ulcerative colitis or spontaneous colitis), cystic fibrosis, skin inflammation ( cutaneous inflammation Autoimmune diseases, such as diabetes and non-alcoholic steatohepatitis (NAH). NASH( ), especially type I diabetes, psoriasis, lupus, rheumatoid arthritis, multiple sclerosis, Sjögren's syndrome, celiac disease, vasculitis, myasthenia gravis; infectious diseases, such as sepsis, severe viral indications with severe inflammatory symptoms, such as coronaviruses (e.g., COVID-19), peritonitis; degenerative diseases; wound healing disorders, dry eye syndrome; cancers, especially solid cancer or liquid cancer, metastatic cancer, especially carcinoma, especially breast cancer or colon cancer or colorectal cancer or lung cancer or mesothelioma or myeloma, especially leukemia, especially cancers in which cancer cells express CMKLR1 or the tumor microenvironment is invaded by cells that express or overexpress CMKLR1.

[0139] Fibrosis (especially pulmonary and hepatic fibrosis), ANCA (anti-neutrophil cytoplasmic autoantibody) symptoms (vasculitis), and symptoms caused by neuronal apoptosis associated with ChermR23 have received particular attention.

[0140] In one particular embodiment, the present invention relates to any of the compounds defined above for the prevention and / or treatment of diseases in which the resolution of inflammation is delayed or interrupted, and / or diseases selected from: inflammatory diseases, especially acute inflammatory diseases; chronic inflammatory diseases such as asthma, keratoconjunctivitis, periodontitis, eczema, inflammatory bowel disease (especially Crohn's disease or colitis, especially ulcerative colitis or spontaneous colitis); cystic fibrosis; autoimmune diseases such as diabetes, especially type 1 diabetes, psoriasis, lupus, rheumatoid arthritis, multiple sclerosis, Sjögren's syndrome, celiac disease, vasculitis, myasthenia gravis; infectious diseases such as sepsis, peritonitis; severe viral indications with severe inflammatory symptoms, such as coronaviruses (e.g., COVID-19); degenerative diseases; wound healing disorders, NASH (non-alcoholic steatohepatitis), scleroderma, and dry eye syndrome. In another specific embodiment of the invention, the invention relates to any of the compounds of the invention as defined above, for the prevention and / or treatment of cancer, particularly solid or liquid cancer, metastatic cancer, particularly carcinoma, particularly breast cancer or colon cancer or colorectal cancer or lung cancer or bone marrow cancer, particularly leukemia, particularly cancer in which cancer cells express CMKLR1 or the tumor microenvironment is invaded by cells expressing or overexpressing CMKLR1.

[0141] In one particular embodiment, the antibody of the present invention induces at least one of the following effects that promote regression in vitro and / or in vivo: - Increases apoptosis of polymorphonuclear neutrophils (PMNs or acronym) at sites of inflammation; this effect is illustrated in the embodiments of the invention. Regulation of neutrophil apoptosis is crucial for therapeutic interventions in inflammation-related diseases. As illustrated in the embodiments of the invention, during inflammation induction, the use of a CMKLR1 agonist with an anti-inflammation E1-like antibody on PMNs induces strong apoptosis of neutrophils, compared to a control antibody.

[0142] - Enhanced expression of caspase-3 in neutrophils, leading to caspase-3-dependent apoptosis; compared to the negative control, after treatment with a CMKLR1 agonist with an antibody possessing anti-regression E1 ability for 11 hours, when caspase-3 expression exceeds 1-log, more preferably 2-log, and most preferably 3-log, apoptosis can be considered enhanced or induced; one method is disclosed in the embodiments of the present invention; - Reduces or inhibits the migration of neutrophils from the PMN (neutrophil) endothelium to the site of inflammation; prevents them from migrating to the site of inflammation and exerting their pro-inflammatory effects. In a particular embodiment of the invention, neutrophil migration and migration are prevented or reduced by administering a CMKLR1 agonist with an IgG1 antibody possessing regressive E1-like capabilities. Thus, in a particular embodiment, the CMKLR1 agonist with regressive E1-like capabilities is a humanized antibody having a human constant region derived from or generated from human IgG1. In a particular embodiment, the CMKLR1 agonist with an antibody possessing regressive E1-like capabilities is a human IgG1 isotype, i.e., constant fragments of the heavy and light chains are derived from or generated from constant heavy and light chain fragments of human IgG1 antibodies. Therefore, the CMKLR1 agonist with an antibody possessing regressive E1-like capabilities of the present invention comprises an Fc domain of an IgG1 isotype. Neutrophil migration can be assessed using the methods disclosed in the embodiments of this invention; compared to the negative control, a decrease of at least 1-log in the cell surface expression of CD62L in the staining experiment indicates that neutrophils have reduced migration and / or migratory capacity. The inventors have found that the CMKLR1 agonist of the present invention, which is a humanized IgG1 antibody with regressor-like E1 activity, does not exhibit cytotoxicity in vivo; in other words, the use of the CMKLR1 agonist does not show significant depletion of CMKLR1-positive cells in vivo. In one specific embodiment, the use of the agonist of the present invention does not exhibit cytotoxic activity against CMKLR1-positive cells; - Reduces cell surface expression of CMKLR1 and CXCR4 and / or CCR7. In a particular embodiment, cell surface expression is considered to occur on macrophages and / or dendritic cells. When a CMKLR1 agonist with regressor-E1 activity binds to its target, CMKLR1 and CXCR4 and / or CCR7 are heterodimerized and internalized. In a preferred embodiment, the antibody of the present invention does induce the internalization of CMKLR1 and / or CXCR4 and / or CCR7 on the cell surface of CMKLR1-positive cells, and / or inhibit the expression of CMKLR1 and / or CXCR4 and / or CCR7. Therefore, cell surface expression of CMKLR1 and CXCR4 and / or CCR7 in cells incubated in the presence of the antibody is reduced or significantly reduced compared to cell surface expression in cells incubated under other identical conditions but in the absence of a CMKLR1 agonist with regressor-E1 activity.

[0143] In one particular aspect, the present invention relates to a humanized anti-chemokine receptor 1 (CMKLR1) antibody or an antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof being adapted to be produced in mammalian cells (such as COS or CHO cells) at a yield of more than 0.1 mg / ml, particularly more than 1 mg / ml, particularly more than 10 mg / ml, and more particularly more than 100 mg / ml, wherein: a) The variable weight (VH) domain comprises the amino acid sequences of the backbone regions (FR1, FR2, FR3, and FR4) of the heavy chain variable domain, wherein each backbone region has sequence identity with the backbone region of the same order in sequence SEQ ID No. 41: FR1 is 100%, FR2 is at least 60%, FR3 is at least 78%, and FR4 is at least 80%; more specifically, FR1 is 100%, FR2 is at least 80%, FR3 is at least 85%, and FR4 is at least 90%. b) The variable light (VL) domain comprises the amino acid sequences of the backbone regions (FR1, FR2, FR3, and FR4) of the light chain variable domain, wherein each backbone region has sequence identity with the backbone region of the same order in sequence SEQ ID No. 50: at least 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 at least 100% for FR4.

[0144] In one particular embodiment, a humanized anti-CMKLR1 antibody or its antigen-binding fragment specifically binds to the third extracellular loop (EL3) of CMKLR1, particularly wherein the antibody or its antigen-binding fragment specifically binds to a polypeptide comprising the amino acid sequence SEQ ID No: 2 or SEQ ID No. 59 or located within the amino acid sequence SEQ ID No. 60. This embodiment can result in readily in vitro production of the antibody (or its antigen-binding fragment). It should be noted that the definition of this 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 one particular aspect of the invention, the definition of the antibody of the present invention by the FR sequence can be combined with the definition of the CDR domain and / or functional characteristics. For this purpose, the invention also includes antibodies defined by their FR domain and characterized by their CDR domain selected from a particular group. For this purpose, antibodies that may or may not be identical with the backbone domains disclosed herein comprise at least one, especially six, of 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 - 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 Furthermore, the variable light (VL) structural domain includes: - 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, which is selected from the group consisting of SEQ ID No. 34, SEQ ID No. 35 and SEQ ID No. 36.

[0145] In a more specific aspect, at least one of the skeleton structural domains HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4 is selected from the group consisting of: SEQ ID No. 65 for VHFR1; For VHFR2, SEQ ID No. 66, SEQ ID No. 67 or SEQ ID No. 68; For VHFR3, SEQ ID No. 69 or SEQ ID No. 70; SEQ ID No: 71 for VHFR4; SEQ ID No. 72 for VLFR1; For VLFR2, SEQ ID No. 73 or SEQ ID No. 74; For VLFR3, SEQ ID No. 75 or SEQ ID No. 76; SEQ ID No. 77 for VLFR4.

[0146] It should be understood that multiple or all of the skeleton structural domains may be selected from the groups described above.

[0147] In a more specific embodiment, the antibody or its antigen-binding fragment according to the first two paragraphs has an amino acid sequence of a heavy chain variable domain selected from SEQ ID No:41, SEQ ID No:38, SEQ ID No:42, SEQ ID No:43, and the amino acid sequence of the light chain variable domain is sequence SEQ ID No:50.

[0148] In a more specific embodiment, the antibody or its antigen-binding fragment comprises the following backbone domains: - VHFR1 SEQ ID NO: 65 - VHFR2 SEQ ID NO: 67, - VHFR3 SEQ ID NO: 69 - VHFR4 SEQ ID NO: 71, - VLFR1 SEQ ID NO: 72, - VLFR2 SEQ ID NO: 73 - VLFR3 SEQ ID NO: 76, and - VLFR4 SEQ ID NO: 77. In a more specific embodiment, the antibody comprises the following backbone domains: - VHFR1 SEQ ID NO: 65 - VHFR2 SEQ ID NO: 67, - VHFR3 SEQ ID NO: 69 - VHFR4 SEQ ID NO: 71, - VLFR1 SEQ ID NO: 72, - VLFR2 SEQ ID NO: 73 - VLFR3 SEQ ID NO: 76, - VLFR4 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 - 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 Furthermore, the variable light (VL) structural domain includes: - 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, which is selected from the group consisting of SEQ ID No. 34, SEQ ID No. 35 and SEQ ID No. 36.

[0149] This implementation method can lead to the easy generation of antibodies (or their antigen-binding fragments) in vitro.

[0150] This invention particularly relates to humanized anti-CMKLR1 antibodies or antigen-binding fragments thereof optimized to reduce immunogenicity while maintaining high binding activity and stability as well as biological function, said antibodies or antigen-binding fragments thereof comprising the following CDRs: - VHCDR1 SEQ ID NO: 4, - VHCDR2 SEQ ID NO: 12, - VHCDR3 SEQ ID NO: 13, - VLCDR1 SEQ ID NO: 19 - VLCDR2's SEQ ID NO: 26, and - VLCDR3 SEQ ID NO: 35.

[0151] In one particular embodiment, the antibody or its antigen-binding fragment with CDR sequences 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 comprises the following backbone domains: - VHFR1 SEQ ID NO: 65 - VHFR2 SEQ ID NO: 67, - VHFR3 SEQ ID NO: 69 - VHFR4 SEQ ID NO: 71, - VLFR1 SEQ ID NO: 72, - VLFR2 SEQ ID NO: 73 - VLFR3 SEQ ID NO: 76, - VLFR4 SEQ ID NO: 77.

[0152] This disclosure specifically relates to an anti-CMKLR1 antibody or its antigen-binding fragment thereof, which is optimized to reduce immunogenicity while maintaining high binding activity and stability suitable for in vitro production and biological function. The anti-CMKLR1 antibody or antigen-binding antibody described in the preceding paragraph has the amino acid sequence SEQ ID No: 91 of the heavy chain variable domain and the amino acid sequence SEQ ID No: 93 of the light chain variable domain.

[0153] Furthermore, an advantageous feature of the antibody is that its Fc fragment is characteristic of IgG1.

[0154] In one particular embodiment, the antibodies of the present invention are characterized by the amino acid sequences of the backbone domains disclosed above, and are further characterized by the amino acid sequences of at least one CDR domain thereof. In particular, the humanized anti-CMKLR1 antibody or its antigen-binding fragment according to this embodiment has a heavy chain variable domain VHCDR2, which comprises or consists 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.

[0155] In a specific embodiment of the present invention, an antibody or an antigen-binding fragment thereof is provided, comprising: a) Antibody heavy chain variable (VH) domain, which contains 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 corresponds to the amino acid residue of SEQ ID No. 12, provided that VHCDR1 is not 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; b) Antibody light chain variable (VL) domain, which contains 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; For prevention or treatment: - Inflammatory diseases, especially acute inflammatory diseases, chronic inflammatory diseases such as chronic inflammatory lung diseases (e.g., asthma), keratoconjunctivitis, periodontitis, eczema, inflammatory bowel disease (especially Crohn's disease or colitis (especially ulcerative colitis or spontaneous colitis)), cystic fibrosis, NASH (non-alcoholic steatohepatitis), liver fibrosis, pulmonary fibrosis, antineutrophil cytoplasmic antibody (ANCA) related diseases, vasculitis (especially ANCA-mediated vasculitis), scleroderma, especially in cases where inflammation is reduced due to treatment; - Autoimmune diseases, such as diabetes, especially type 1 diabetes, psoriasis, lupus, rheumatoid arthritis, multiple sclerosis, Sjögren's syndrome, celiac disease, vasculitis, myasthenia gravis, or infectious diseases, such as sepsis, peritonitis, degenerative diseases, impaired wound healing, or dry eye syndrome, severe viral indications with severe inflammatory symptoms, such as coronaviruses (e.g., COVID-19), especially where the inflammation is reduced due to treatment; - Cancer, especially metastatic cancer, whether solid or liquid, such as carcinoma, especially liver cancer, especially breast or colon cancer or lung cancer or bone marrow cancer, such as leukemia, especially cancer in which cancer cells express CMKLR1 or the tumor microenvironment is invaded by cells that express or overexpress CMKLR1, especially in cases where inflammation is enhanced due to the administration of treatment.

[0156] In another specific embodiment of the present invention, an antibody or antigen-binding fragment thereof is provided, the antibody or antigen-binding fragment thereof comprising: a) Antibody heavy chain variable (VH) domain, which contains 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 corresponds to the amino sequence SEQ ID NO: 12, the heavy chain variable (VH) domain does not contain the backbone VHFR3 of SEQ ID No. 70, preferably, the heavy chain variable (VH) domain contains the backbone FR3 of SEQ ID NO: 69. - 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) Antibody light chain variable (VL) domain, which contains 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; it is used for the preventive or therapeutic treatment of the above-mentioned diseases.

[0157] In a specific embodiment of the present invention, the humanized antibody or its antigen-binding fragment, antigen-binding antibody mimic, or modified antibody comprises: a) The variable weight (VH) domain comprises the amino acid sequences of the backbone regions (FR1, FR2, FR3, and FR4) of the heavy chain variable domain, wherein each backbone region has sequence identity with the backbone region of the same order in sequence SEQ ID No. 41: FR1 is 100%, FR2 is at least 60%, FR3 is at least 78%, and FR4 is at least 80%; more specifically, FR1 is 100%, FR2 is at least 80%, FR3 is at least 85%, and FR4 is at least 90%. b) The variable light (VL) domain comprises the amino acid sequences of the backbone regions (FR1, FR2, FR3, and FR4) of the light chain variable domain, wherein each backbone region has sequence identity with the backbone region of the same order in sequence SEQ ID No. 50: FR1 at least 60%, FR2 at least 70%, FR3 at least 75%, and FR4 at least 80%; more specifically, FR1 at 100%, FR2 at least 90%, FR3 at least 90%, and FR4 at least 100%. For prevention or treatment: - Inflammatory diseases, especially acute inflammatory diseases, chronic inflammatory diseases such as chronic inflammatory lung diseases (e.g., asthma), keratoconjunctivitis, periodontitis, eczema, inflammatory bowel disease (especially Crohn's disease or colitis (especially ulcerative colitis or spontaneous colitis)), cystic fibrosis, NASH (non-alcoholic steatohepatitis), liver fibrosis, pulmonary fibrosis, antineutrophil cytoplasmic antibody (ANCA) related diseases, vasculitis (especially ANCA-mediated vasculitis), scleroderma, especially in cases where inflammation is reduced due to treatment; - Autoimmune diseases, such as diabetes, especially type 1 diabetes, psoriasis, lupus, rheumatoid arthritis, multiple sclerosis, Sjögren's syndrome, celiac disease, vasculitis, myasthenia gravis, or infectious diseases such as sepsis, peritonitis, degenerative diseases, impaired wound healing, severe viral indications with severe inflammatory symptoms, such as coronaviruses (e.g., COVID-19), or dry eye syndrome, especially where the inflammation is reduced due to treatment. - Cancer, especially metastatic cancer, whether solid or liquid, such as carcinoma, especially liver cancer, especially breast cancer, colon cancer, lung cancer, or bone marrow cancer such as leukemia, especially cancer in which cancer cells express CMKLR1 or the tumor microenvironment is invaded by cells that express or overexpress CMKLR1, especially in cases where inflammation is enhanced due to the administration of treatment.

[0158] In one particular implementation, the antibody comprises the following backbone domains. - VHFR1 SEQ ID NO: 65 - VHFR2 SEQ UD NO: 67, - VHFR3 SEQ ID NO: 69 - VHFR4 SEQ ID NO: 71, - VLFR1 SEQ ID NO: 72, - VLFR2 SEQ ID NO: 73 - VLFR3 SEQ ID NO: 76, - VLFR4 SEQ ID NO: 77, And includes the following CDRs: - 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; and / or - 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; Furthermore, the variable light (VL) structural domain includes: - 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, and used for prevention or treatment: - Inflammatory diseases, especially acute inflammatory diseases, chronic inflammatory diseases such as chronic inflammatory lung diseases (e.g., asthma), keratoconjunctivitis, periodontitis, eczema, inflammatory bowel disease (especially Crohn's disease or colitis (especially ulcerative colitis or spontaneous colitis)), cystic fibrosis, NASH (non-alcoholic steatohepatitis), liver fibrosis, pulmonary fibrosis, antineutrophil cytoplasmic antibody (ANCA) related diseases, vasculitis (especially ANCA-mediated vasculitis), scleroderma, especially in cases where inflammation is reduced due to treatment; - Autoimmune diseases, such as diabetes, especially type 1 diabetes, psoriasis, lupus, rheumatoid arthritis, multiple sclerosis, Sjögren's syndrome, celiac disease, vasculitis, myasthenia gravis, or infectious diseases such as sepsis, peritonitis, degenerative diseases, impaired wound healing, severe viral indications with severe inflammatory symptoms, such as coronaviruses (e.g., COVID-19), or dry eye syndrome, especially where the inflammation is reduced due to treatment. - Cancer, especially metastatic cancer, whether solid or liquid, such as carcinoma, especially liver cancer, especially breast cancer, colon cancer, lung cancer, or bone marrow cancer such as leukemia, especially cancer in which cancer cells express CMKLR1 or the tumor microenvironment is invaded by cells that express or overexpress CMKLR1, especially in cases where inflammation is enhanced due to the administration of treatment.

[0159] Such specific antibodies or their antigen-binding fragments for the preventive or therapeutic treatment of one of the conditions disclosed above are characterized in particular by comprising the following CDRs: SEQ ID NO:4 of VHCDR1, SEQ ID NO:12 of VHCDR2, SEQ ID NO:13 of VHCDR3, and SEQ ID NO:19 of VLCDR1, SEQ ID NO:26 of VLCDR2, and SEQ ID NO:35 of VLCDR3; and the following backbones: SEQ ID NO:65 of VHFR1, SEQ ID NO:67 of VHFR2, SEQ ID NO:69 of VHFR3, SEQ ID NO:71 of VHFR4, SEQ ID NO:72 of VLFR1, SEQ ID NO:73 of VLFR2, SEQ ID NO:76 of VLFR3, and SEQ ID NO:77 of VLFR4. In particular, for the preventive or therapeutic use of the disclosed antibody or its antigen-binding fragment, the amino acid sequence of the heavy chain variable domain of the antibody contains or is composed of SEQ ID NO:91, and the amino acid sequence of the light chain variable domain of the antibody contains or is composed of SEQ ID NO:93.

[0160] In another aspect, the present invention relates to a composition comprising the anti-CMKLR1 compound described herein, and more particularly to a pharmaceutical composition comprising the anti-CMKLR1 compound according to the invention and an additional therapeutic agent or pharmaceutically acceptable carrier. In one particular embodiment, the present invention relates to a composition comprising the anti-CMKLR1 compound according to the invention and a therapeutic agent selected from the group consisting of: immunomodulators, immune checkpoint blockers, immune checkpoint activators, antibodies, or anti-SIRPa antibodies (P84 - anti-mouse SIRPa from Merck Millipore).

[0161] In another aspect, the present invention relates to a combination of compounds comprising the anti-CMKLR1 compound described herein, and in particular to a pharmaceutical composition comprising the anti-CMKLR1 compound according to the invention and an anti-PD1 or anti-PDL1 compound (especially an anti-PD1 compound); such compounds are particularly selected from antibodies capable of binding to PD1 or PDL1, antigen-binding antibody fragments, antigen-binding antibody mimics, small molecules (such as aptamers or peptides), and modified antibodies (such as, but not limited to, humanized antibodies or chimeric antibodies).

[0162] In another aspect, the present invention relates to a combination of compounds comprising the anti-CMKLR1 compound described herein, and in particular to a pharmaceutical composition comprising the anti-CMKLR1 compound and the anti-SIRPa compound according to the present invention; such compounds are particularly selected from antibodies capable of binding to SIRPa, especially human SIRPa, antigen-binding antibody fragments, antigen-binding antibody mimics, small molecules (such as aptamers or peptides), and modified antibodies (such as, but not limited to, humanized antibodies or chimeric antibodies).

[0163] The present invention also relates to a combination, for example for the treatment of fibrosis, comprising a therapeutic compound, which may or may not be a cytokine, and stimulating pro-regression macrophages.

[0164] In another aspect, the present invention relates to the therapeutic use of the anti-CMKLR1 compound of the present invention, particularly for inducing and / or enhancing inflammatory resolution, especially in the treatment of diseases in which the spread of inflammation is pathological or where the duration of inflammatory resolution is pathological, for inducing and / or enhancing inflammatory resolution when said resolution is delayed or interrupted.

[0165] In a particular embodiment of the invention, the anti-CMKLR1 compound binds to CMKLR1 with an affinity of at least 10E-8 M (KD value), more preferably at least 10E-9 M. The specific binding between the antibody of the present invention or its antigen-binding fragment or antigen-binding antibody mimic or modified antibody and CMKLR1 (or the region of CMKLR1 containing the third extracellular loop, including the amino acid sequence shown in SEQ ID No: 2 and SEQ ID No: 59 or located within the amino acid sequence SEQ ID No. 60) demonstrates that the antibody exhibits significant affinity for CMKLR1. "Significant affinity" includes an affinity of about 10... -8 M(KD) or stronger affinity binding. Preferably, when the binding affinity is 10... -8 M and 10 -12 Between M, optionally within 10 -9 M and 10 -10 Between M, especially for at least 10 -9 When M is present, binding is considered specific. Whether the binding domain specifically reacts with or binds to the target can be readily tested, especially by comparing the reaction between the binding domain and the target protein or antigen with the reaction between the binding domain and a protein or antigen other than the target protein. The antibody of this invention specifically binds to CMKLR1 and acts as an agonist for 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, New York (1998); Colligan et al., Current Protocols in Immunology, Greene Press, New York (1992; 1993); Muller, Meth. Enzym, 92:589-601 (1983)). These methods include, but are not limited to, Biacore assays, Blitz assays, flow cytometry, and ELISA assays.

[0166] In one specific embodiment of the invention, the anti-CMKLR1 compound specifically binds to an epitope located within the third outer ring of CMKLR1, particularly an epitope located within the amino acid residue sequence shown in SEQ ID No:2, SEQ ID NO:59, or SEQ ID NO:60, especially SEQ ID No:2. The anti-CMKLR1 compound bound within this specific region of CMKLR1 can exhibit agonist properties towards CMKLR1, thereby mimicking the binding of RvE1 to CMKLR1.

[0167] In another aspect, the present invention relates to the use of anti-CMKLR1 antibodies or antigen-binding fragments thereof or antigen-binding antibody mimics or modified antibodies as defined above as pharmaceuticals, which have agonist ability for the interaction between RvE1 and CMKLR1.

[0168] In another aspect, the present invention relates to anti-CMKLR1 antibodies or antigen-binding fragments or antigen-binding antibody mimics or modified antibodies as defined above, which have the ability to induce activation of Akt and / or Erk proteins in vitro and / or in vivo. The activation of these proteins can be evaluated by the methods described in the embodiments of the invention. In particular, anti-CMKLR1 antibodies or antigen-binding fragments or antigen-binding antibody mimics or modified antibodies have the ability to activate Akt and / or Erk proteins, or both, in macrophages, particularly human macrophages.

[0169] The present invention also relates to a method of treating a subject in need, the method comprising administering to the subject an effective amount of an anti-CMKLR1 antibody or an antigen-binding fragment thereof or an antigen-binding antibody mimic as defined above, which has the ability to act as an agonist to the interaction between RvE1 and CMKLR1, or in other words, is an RvE1 agonist or modulator.

[0170] In many conditions or situations, altering macrophage polarization to favor anti-inflammatory cells can be useful. As mentioned above, this modification is particularly applicable to conditions involving diseases selected from the following group: inflammatory diseases, including but not limited to acute and chronic inflammatory diseases, inflammatory bowel disease, Crohn's disease, asthma, keratoconjunctivitis, periodontitis, eczema, colitis (especially ulcerative colitis or spontaneous colitis), cystic fibrosis; diabetes (especially type 1 diabetes), peritonitis, psoriasis, cancer (especially breast or colon cancer), cancer, metastatic cancer, lung cancer, degenerative diseases, infectious diseases, especially sepsis, autoimmune diseases, NASH, scleroderma, colitis, or Crohn's disease, in subjects in whom corticosteroids and / or immunosuppressive therapy are refractory.

[0171] The present invention also relates to the use of anti-CMKLR1 antibodies as defined above, having the ability to act as a regressor E1 agonist, or antigen-binding fragments thereof or antigen-binding antibody mimics thereof, in the preparation of pharmaceuticals.

[0172] In another aspect, the present invention relates to anti-CMKLR1 antibodies or antigen-binding fragments thereof or antigen-binding antibody mimics or modified antibodies (e.g., but not limited to humanized antibodies or chimeric antibodies as defined above) for the treatment of chronic inflammatory diseases, particularly for the treatment of chronic colitis.

[0173] In another aspect, the present invention relates to anti-CMKLR1 antibodies as defined above, or antigen-binding fragments thereof or antigen-binding antibody mimics thereof having agonist activity against CMKLR1 interaction, for the treatment of inflammatory conditions in which remission is delayed or interrupted (especially inflammatory diseases in which remission is delayed or interrupted) and / or for the treatment or prevention of diseases selected from the group consisting of: inflammatory diseases, including but not limited to acute and chronic inflammatory diseases, inflammatory bowel disease, Crohn's disease, asthma, keratoconjunctivitis, periodontitis, eczema, colitis (especially ulcerative colitis or spontaneous colitis), cystic fibrosis, diabetes (especially type I diabetes), peritonitis, psoriasis, cancer (especially breast cancer or colon cancer), cancer, degenerative diseases, infectious diseases (especially sepsis), and autoimmune diseases.

[0174] As defined in this article, “delayed or interrupted resolution of inflammatory symptoms” occurs when the resolution of inflammation is delayed or interrupted compared to normal resolution (i.e., resolution that occurs in patients who experience physiological resolution following an inflammatory event). Delayed or defective resolution can lead to increased granulocyte infiltration at the site of inflammation. Therefore, delayed or defective resolution can be assessed by quantifying granulocytes at the site of inflammation. Granulocyte populations can be measured using, for example, histological, cytological, or indirect biochemical techniques (e.g., elastase quantification via enzyme immunoassay or molecular quantification via PCR of granulocyte receptor 1). Delayed or defective resolution can also be assessed by measuring the delay in granulocyte apoptosis, for example, by measuring it cytologically using a specific antibody against annexin 5. Defects or delays in inflammatory resolution can also be identified by quantifying the synthesis of pro-inflammatory cytokines (such as TNF-α, IL-8, or IL-12) and anti-inflammatory cytokines (such as IL-10). Cytokine secretion can be assessed by enzyme immunoassay or by PCR. Defects or delays in inflammatory resolution can also be identified by assessing the activation of transcription factors involved in the synthesis of inflammatory cytokines (e.g., NF-kappaB), which can be measured, for example, by nuclear translocation or by Western blotting and / or by quantification of IkappaB degradation levels. Defects or delays in inflammatory resolution can also be determined by quantifying specific pro-remission mediators (e.g., lipoxygenin, remissionin, protectin, or maresin) or their precursors (e.g., 17-HDOHE or 14-HDOHE) using mass spectrometry or enzyme immunoassay. Defects or delays in resolution result in defects in the synthesis of one or more of these mediators. Defects or delays in resolution can also be identified when the expression of receptors for resolution molecules is reduced. These receptors can be selected from the group including ALX, CMK1R1, GPR32, or GPR18. Alternatively or complementaryly, the internalization and processing of those receptors that enter the cytoplasm can also be assessed. Alternatively or complementary, the expression of certain receptors for inflammatory cytokines or lipids, which are overexpressed and significantly contribute to delayed or deficient inflammatory resolution compared to normal conditions, can also be assessed. These conditions can be measured histologically, cytologically, or by PCR. Deficient resolution can also lead to a reduced or suppressed conversion of pro-inflammatory macrophages to pro-regressive macrophages, resulting in impaired phagocytosis or necrolysis of the same cells. Therefore, delayed or deficient resolution can be assessed by analyzing the conversion of pro-inflammatory macrophages to pro-regressive macrophages under specific conditions compared to normal conditions, as illustrated in the embodiments of the present invention.

[0175] According to one particular implementation, anti-CMKLR1 compounds can be used to treat individuals with cancers selected from the group consisting of: breast cancer, especially mammary carcinoma cancer, melanoma, colon cancer, especially colon carcinoma cancer, leukemia, especially more acute myeloid leukemia, particularly in cases where cancer cells overexpress CMKLR1.

[0176] In one embodiment, the present invention relates to an anti-human CMKLR1 antibody or its antigen-binding fragment or antigen-binding antibody mimic or modified antibody as defined above for its use as defined above, wherein the CMKLR1 antibody or its antigen-binding fragment or antigen-binding antibody mimic or modified antibody of the present invention is administered to a patient with a CMKLR1-positive tumor.

[0177] The antibodies or antigen-binding fragments thereof of the present invention can be administered to subjects via a variety of suitable routes, such as intravenous (IV), subcutaneous (SC), or intramuscular (IM). The anti-CMKLR1 compound can be administered alone or in combination with another therapeutic agent (e.g., a second human monoclonal antibody or its antigen-binding fragment). In another embodiment, the antibody is administered together with another agent (e.g., an immunosuppressant, an erythropoiesis-stimulating agent (ESA)), and a therapeutic cell composition. In one embodiment, the present invention relates to an anti-CMKLR1 compound or its antigen-binding fragment or antigen-binding antibody mimic for its use as defined above, wherein the anti-CMKLR1 antibody or antigen-binding fragment is combined with a second therapeutic agent.

[0178] The administration of the second therapeutic agent can be simultaneous with or separate from the administration of the anti-CMKLR1 compound. Depending on the nature of the second agent, a co-administration, also known as a "combo," can be prepared as a combination drug (product). A combo is a combination of two or more active pharmaceutical ingredients in fixed doses, prepared and dispensed in a fixed dosage form. However, the dosage regimen and / or route of administration can also differ.

[0179] In a preferred embodiment, the second therapeutic agent is selected from the group consisting of: chemotherapeutic agents, radiotherapy agents, immunotherapy agents, cell therapy agents (e.g., CAR-T cells), antibiotics, and probiotics.

[0180] In particular, the immunotherapeutic agents used in the context of this invention are selected from the group consisting of: therapeutic vaccines (DNA, RNA or peptide vaccines), immune checkpoint blockers or activators, especially adaptive immune cells (T or B lymphocytes) or immune conjugates (e.g., antibody-drug conjugates).

[0181] As used herein, the term "immunotherapy agent" specifically refers to agents that can transform cancer vaccines from a biological phenomenon of interest into effective therapeutic agents, including: T cell growth factors that increase the number and repertoire of naive T cells; growth factors that increase the number of dendritic cells (DCs); agonists that activate DCs and other antigen-presenting cells (APCs); adjuvants that allow and enhance cancer vaccines; agonists that activate and stimulate T cells; T cell checkpoint blockade inhibitors; T cell growth factors that increase the growth and survival of immune T cells; and agents that inhibit, block, or neutralize immunosuppressive cytokines derived from cancer cells and immune cells.

[0182] Various immune checkpoint inhibitors or activators are known in the art. In the context of this invention, examples of potentially useful immune checkpoint inhibitors or adaptive immune cell (B or T lymphocyte) activators 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, especially anti-CD137 and SIRPa. In one particular embodiment of the invention, the second therapeutic agent is an anti-PDL1 or anti-PD1 compound, especially an anti-PD1 compound, and more particularly an anti-PD1 antibody. In one particular embodiment of the invention, the second therapeutic agent is an anti-SIRPa compound, especially an anti-SIRPa antibody.

[0183] The immunotherapeutic agent may also be an antibody that targets a tumor antigen, especially an antibody selected from the group consisting of: anti-Her2, anti-EGFR, anti-CD20, anti-CD19, and anti-CD52.

[0184] The antibody can be delivered at an effective dose of about 1 ng / kg body weight to about 30 mg / kg body weight or higher. In one particular embodiment, the dose can be in the range of about 1 μg / kg to about 20 mg / kg, optionally from 10 μg / kg to a maximum of 10 mg / kg or from 100 μg / kg to a maximum of 5 mg / kg.

[0185] The terms "effective dose," "effective dosage," or "effective amount" are defined as an amount sufficient to achieve or at least partially achieve the desired effect. The term "effective dose" is intended to encompass an amount sufficient to cure or at least partially suppress a disease and its complications, or to alleviate the symptoms of a patient already suffering from the disease. The effective amount or dosage for this purpose will depend on the condition to be treated, the antibody construct delivered, the treatment context and objectives, the severity of the disease, prior treatment, the patient's clinical history and response to the therapeutic agent, the route of administration, the patient's body type (weight, body surface or organ size) and / or condition (age and general health status), and the general state of the patient's own immune system. Appropriate dosages can be adjusted so that they can be administered to the patient in a single dose or in a series of administrations to achieve the best therapeutic effect.

[0186] The dosage for such purposes may be repeated as needed, such as daily, every half week, weekly, every half month, monthly, or as needed during relapses.

[0187] In another aspect, the present invention relates to a pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as defined above, and a pharmaceutically acceptable carrier.

[0188] As used herein, "pharmaceutical composition" is intended to include compositions suitable for administration to subjects or patients (e.g., mammals, especially humans). Typically, "pharmaceutical compositions" are sterile and generally free of contaminants that could cause adverse reactions in subjects (e.g., the compounds in the pharmaceutical composition are pharmaceutical grade). Pharmaceutical compositions may be designed for administration to subjects or patients in need via a variety of different routes of administration, including oral, oral, rectal, parenteral, intraperitoneal, intradermal, and intratracheal administration.

[0189] As used herein, "pharmaceuticalally acceptable carrier" is intended to include excipients, diluents, carriers, and adjuvants that can be used in the preparation of pharmaceutical compositions, which are generally safe, non-toxic, and biologically or otherwise desirable, and include excipients, diluents, carriers, and adjuvants that can be used for veterinary and human pharmaceutical purposes. As used herein, "pharmaceuticalally acceptable carrier" includes one or more of the aforementioned excipients, diluents, carriers, and adjuvants.

[0190] In particular, the present invention relates to a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as defined above as an active ingredient, and a pharmaceutically acceptable carrier.

[0191] In another aspect, the present invention relates to a treatment method, particularly a combination product method, comprising an anti-SIRPa antibody as defined above or an antigen-binding fragment thereof or an antigen-binding antibody mimic as an active ingredient, and a second therapeutic agent, wherein said active ingredient is formulated for treatment alone, sequentially or in combination, particularly for combined or sequential use.

[0192] In particular, the present invention relates to a combination product comprising an anti-CMKLR1 compound as defined above and a second therapeutic agent for simultaneous, single or sequential use of the medicine.

[0193] In one embodiment, the present invention relates to a combination product as defined above, wherein the second therapeutic agent is selected from the group consisting of: chemotherapeutic agents, radiotherapy agents, cell therapy agents, immunotherapy agents, antibiotics, and probiotics.

[0194] In one embodiment, the present invention relates to a combination product as defined above, wherein the immunotherapeutic agent is selected from the group consisting of: therapeutic vaccines, immune checkpoint inhibitors or activators, particularly adaptive immune cells (T and B lymphocytes) and antibody-drug conjugates.

[0195] In one embodiment, the present invention relates to a combination product as defined above, wherein the immune checkpoint blocker or adaptive immune cell (T and B lymphocyte) activator is selected from the group consisting of: anti-PDL1, anti-PD1, anti-SIRPA, anti-CTLA4, anti-CD137, anti-CD2, anti-CD28, anti-CD40, anti-HVEM, anti-BTLA, anti-CD160, anti-TIGIT, anti-TIM-1 / 3, anti-LAG-3, anti-2B4 and anti-OX40, anti-CD40 agonists, CD40-L, TLR agonists, anti-ICOS, ICOS-L and B cell receptor agonists, particularly selected from anti-PDL1, anti-PD1 and anti-CD137. In a particular embodiment of the invention, the second therapeutic agent is an anti-PDL1 or anti-PD1 compound, particularly an anti-PD1 compound, and more particularly an anti-PD1 antibody. In a particular embodiment of the invention, the second therapeutic agent is an anti-SIRPA compound, particularly an anti-SIRPA antibody.

[0196] In one embodiment, the immunotherapeutic agent is an antibody that targets a tumor antigen, particularly an antibody selected from the group consisting of anti-Her2, anti-EGFR, anti-CD20, anti-CD19, and anti-CD52.

[0197] In one aspect, the present invention relates to a combination product as defined above for use simultaneously, alone or sequentially in treating any condition that is easily improved or prevented by altering macrophage polarization to pro-regression macrophages.

[0198] In one embodiment, the present invention relates to a method for treating any condition that is easily improved or prevented by changing macrophage polarization to pro-regression macrophages, the method comprising administering, simultaneously, individually or sequentially, an effective amount of the combination product as described above to the subject.

[0199] In one embodiment, the present invention relates to the use of a combination product as defined above in the preparation of a medicament for treating any condition that is prone to inducing regressive inflammatory macrophages.

[0200] In one aspect, the present invention relates to a combination product as defined above for use simultaneously, alone, or sequentially in treating conditions selected from the group consisting of: inflammatory diseases, including but not limited to acute and chronic inflammatory diseases, inflammatory bowel disease, Crohn's disease, NASH, scleroderma, asthma, keratoconjunctivitis, periodontitis, eczema, colitis (especially ulcerative colitis or spontaneous colitis), cystic fibrosis, diabetes (especially type I diabetes), peritonitis, psoriasis, cancer (especially breast cancer or colon cancer), cancer, metastatic cancer, lung cancer, degenerative diseases, infectious diseases (especially sepsis), autoimmune diseases, or for use in vaccination.

[0201] In one embodiment, the present invention relates to a method for treating a subject in need of a condition selected from the group consisting of inflammatory diseases, including but not limited to acute and chronic inflammatory diseases, inflammatory bowel disease, Crohn's disease, NASH, scleroderma, asthma, keratoconjunctivitis, periodontitis, eczema, colitis (especially ulcerative colitis or spontaneous colitis), cystic fibrosis, diabetes (especially type I diabetes), peritonitis, psoriasis, cancer (especially breast cancer or colon cancer), cancer, metastatic cancer, lung cancer, degenerative diseases, infectious diseases, especially sepsis, and autoimmune diseases, including administering, simultaneously, alone, or sequentially, effective amounts of a combination product as defined above to the subject.

[0202] The present invention also relates to a polynucleotide encoding an anti-CMKLR1 compound as defined herein. Therefore, the present invention also relates to a nucleic acid molecule or a group of nucleic acid molecules, more particularly isolated nucleic acid molecules and / or recombinant nucleic acid molecules, encoding any anti-CMKLR1 compound disclosed herein, and more particularly encoding a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises or is composed of the amino acid residue sequences of SEQ ID No. 38, SEQ ID No. 39, SEQ ID No. 40, and SEQ ID No. 62, and the light chain variable domain comprises or is composed of the amino acid residue sequences 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, and SEQ ID No. 58, or the light chain variable domain comprises or is composed of the amino acid residue sequences of SEQ ID No. 49, SEQ ID No. 50, SEQ ID No. 51, and SEQ ID No. 58. The amino acid residue sequences of 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 are present. In one particular embodiment, the present invention relates to a nucleic acid molecule or a group of nucleic acid molecules, more particularly isolated nucleic acid molecules and / or recombinant nucleic acid molecules, encoding the amino acid sequences of the antibody heavy chain variable domain consisting of SEQ ID NO: 91 and the antibody light chain variable domain consisting of SEQ ID NO: 93.

[0203] Nucleic acid molecules may also contain regulatory sequences (such as, but not limited to, enhancers, silencers, promoters, especially expression promoters, signal peptides) for the transcription and expression of heavy chain variable domains and / or light chain variable domains.

[0204] This invention also relates to a vector comprising the polynucleotides disclosed herein or the nucleic acid molecules disclosed herein. As used herein, a vector is a nucleic acid molecule used as a vehicle to transfer genetic material into a cell, and in a preferred embodiment, it allows for the expression of polynucleotides inserted within the vector. The term "vector" includes plasmids, viruses, kinases, and artificial chromosomes. Vectors typically contain an origin of replication, a multiple cloning site, and selection markers. The vector itself is typically a nucleotide sequence, usually a DNA sequence, containing an insert (transgenic gene) and a larger sequence serving as the vector's "backbone." Modern vectors may include other features besides the transgenic insert and backbone: promoters, genetic markers, antibiotic resistance, reporter genes, target sequences, and protein purification tags. Vectors called expression vectors (expression constructs) are specifically designed to express transgenic genes in target cells and typically have control sequences.

[0205] In another aspect, the present invention relates to a cell, isolated cell, host cell, isolated host cell, or cell line comprising a mediator as defined above. As used herein, these cell-related terms are intended to include any single cell or cell culture that may be or is already a mediator, exogenous nucleic acid molecule, or acceptor of a polynucleotide encoding an antibody construct of the present invention; and / or acceptor of the antibody construct itself. The material may be introduced into the cell by transformation, transfection, or the like. 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 also include, but are not limited to, bacterial, yeast, fungal, and plant cells; and animal cells, such as insect cells and mammalian (e.g., mouse, rat, rabbit, macaque, or human) cells.

[0206] In a particular embodiment of the invention, the cells or cell lines are selected from CHO, COS, and HEK cells, and when genetically modified with a mediator comprising a mediator, a foreign nucleic acid molecule, and a polynucleotide encoding the antibody construct of the invention, the cells or cell lines are produced with an antibody at a concentration of at least 0.1 mg / ml, particularly at least 1 mg / ml, particularly at least 10 mg / ml, and more particularly at least 100 mg / ml; and / or the cells or cell lines are selected from acceptors of the antibody construct itself.

[0207] The following figures and embodiments are provided to give those skilled in the art a complete disclosure and description of how to prepare and use the invention, but are not intended to limit the scope of the invention as the inventors believe, nor are they intended to represent all or only the experiments conducted. Although the invention has been described with reference to specific embodiments thereof, those skilled in the art will understand that various changes and equivalent substitutions can be made without departing from the true spirit and scope of the invention. Furthermore, many modifications can be made to adapt particular circumstances, materials, combinations of materials, processes, process steps, or steps to the purpose, spirit, and scope of the invention. All such modifications are intended to fall within the scope of the appended claims.

[0208] In one particular embodiment, the present invention relates to the use of an antibody or antigen-binding fragment thereof as defined in any of the embodiments disclosed herein in the preparation of a medicament. In one particular embodiment, the present invention relates to the use of an antibody or antigen-binding fragment thereof as defined in any of the embodiments disclosed herein in the preparation of a medicament for treating a condition involving inflammation. In one particular embodiment, the present invention relates to the use of an antibody or antigen-binding fragment thereof as defined in any of the embodiments disclosed herein in the preparation of a medicament for preventing or treating the following diseases: - Inflammatory diseases, especially acute inflammatory diseases, chronic inflammatory diseases such as chronic inflammatory lung diseases (e.g., asthma), keratoconjunctivitis, periodontitis, eczema, inflammatory bowel disease (especially Crohn's disease or colitis, especially ulcerative colitis or spontaneous colitis), cystic fibrosis, NASH (non-alcoholic steatohepatitis), scleroderma, and antineutrophil cytoplasmic antibody-related diseases (ANCA-associated diseases), especially those in which inflammation is enhanced due to treatment; - Autoimmune diseases, such as diabetes (especially type 1 diabetes), psoriasis, lupus, rheumatoid arthritis, multiple sclerosis, Sjögren's syndrome, celiac disease, vasculitis, myasthenia gravis, or infectious diseases, such as sepsis, peritonitis, degenerative diseases, impaired wound healing, severe viral indications with severe inflammatory symptoms, such as coronaviruses (e.g., COVID-19), or dry eye syndrome, especially where the inflammation is reduced due to treatment; - Cancer, especially metastatic cancer, solid cancer or liquid cancer, such as carcinoma, especially liver cancer, especially breast cancer or colon cancer, colorectal cancer or lung cancer or mesothelioma or bone marrow cancer, such as leukemia, especially cancer in which cancer cells express CMKLR1 or in which the tumor microenvironment is invaded by cells that express or overexpress CMKLR1, especially in cases where inflammation is enhanced due to the administration of treatment; - NASH (non-alcoholic steatohepatitis), scleroderma, cystic fibrosis, or antineutrophil cytoplasmic antibody (ANCA) related diseases. Example

[0209] Production and selection of anti-CMKLR1 antibodies Several antibodies with different CDR sequences within their heavy chain and light chain variable domains have been synthesized. The ability of different antibodies to induce dendritic cells to mature and differentiate into pro-inflammatory or anti-inflammatory pathways was tested. Antibodies 2G1 (SEQ ID No: 37 and SEQ ID No: 49) were selected to assess their properties in resolving inflammatory states (at least affecting the states in the remission phase), and three germlines were generated to assess antibody production in vitro.

[0210] like Figure 1 As shown; 2G1 and 1G1 (another synthetic antibody) antibodies were able to more strongly inhibit the activation and / or maturation of DCs into pro-inflammatory pathways than cells treated with other synthetic antibodies (3G1 and 4G1), because the detection levels of DCs expressing CD103 and IAb treated with 2G1 were lower than those treated with C7 antibody (the method used in this assay is described in Example 10.2). Figure 1 E and Figure 1 As shown in F, the viability of cells treated with 1G1 or 2G1 antibodies was enhanced compared to cells treated with other antibodies (including C7 or desensitizing E1).

[0211] 2G1 is a computer ( in silico Humanization was performed using the CDR grafting method (a humanization method). The table below describes the resulting humanized sequences from the CDR and FR regions, as well as from the variable heavy chain and the variable light chain. Table 1. Phylogenetics of heavy chain variable structural domains Table 2. Light chain variable structural domain phylogenetics .

[0212] Examples of therapeutic effects of anti-CMKLR1 antibody therapy in preclinical models of autoimmune and inflammatory diseases. Example 1. DSS-induced colitis Colitis was induced in 8–10 week old male C57Bl / 6 mice by adding 2% (wt / vol) DSS to sterile drinking water for 6 days. Treatment included intraperitoneal injection of isotype control hIgG1 (10 µg per mouse), daily injection of RvE1 (1 µg per mouse), or three injections of 2G1 antibody (10 µg per mouse) for 5 days. Colitis was monitored daily, including body weight and fecal score (0: normal feces; 4: bloody feces). Colon length, representing disease severity, was measured when mice were euthanized. The regression index was determined under different conditions, as described by Bannenberg et al. (2005).

[0213] result: Figure 2 The DSS animal model shown in A-2D is an acute inflammation model. Figure 2 A-2D showed that the overall condition of animals treated with the anti-CMKLR1 antibody was better than that of animals receiving the control antibody or regressor RvE1. Mice treated with anti-CMKLR1 experienced significantly less weight loss. Figure 2 A), and stool score ( Figure 2 B) is significantly better. Regarding colon length and regression index ( Figure 2 C and Figure 2 Animals receiving anti-CMKLR1 or RvE1 (D) showed similar results.

[0214] Example 2. TNBS-induced colitis On day 0, colitis was induced in 8–10 week old male C57Bl / 6 mice by intrarectal injection of 200 µL of the hapten agent TNBS (5% in 50% ethanol). Treatment was administered via intraperitoneal injection; RvE1 (1 µg per mouse) was injected daily for 3 days, or 2G1 antibody (10 µg per mouse) was injected twice daily for 3 days. Colitis was monitored daily, including body weight and fecal score (0: normal feces; 4: bloody feces) parameters (data not shown). When mice were euthanized, colon length, representing the severity of pathology, was measured.

[0215] Results: TNBS-induced colitis is another acute inflammatory model. Figure 3 Animals treated with either anti-CMKLR1 or RvE1 showed the same colon length (wt) as normal animals. However, those treated with isotype controls had shortened colon length. These results confirm the therapeutic potential of anti-CMKLR1 antibodies in acute inflammatory mouse models, similar to that of RvE1.

[0216] Example 3. IL10-KO model – spontaneous colitis model IL-10KO mice develop spontaneous colitis starting at 20 weeks of age, primarily due to a lack of regulatory T cell function secreted via IL-10 in the gut. IL-10KO mice were followed up three times weekly from 18 weeks of age for weight loss and fecal consistency—clinical markers of this condition. When weight loss exceeded 5% and a fecal score greater than or equal to 1, intraperitoneal injection of anti-CMKLR1 antibody (2G1) or isotype control (hIgG1) was administered for 2 weeks (25 μg / injection, three times weekly).

[0217] Results: The efficacy of anti-CMKLR1 antibody therapy was studied using a chronic inflammation model. Figure 4 The percentage of body weight loss was shown when animals were treated with isotype control or anti-CMKLR1 antibody. Figure 4 A) and stool score ( Figure 4 Analysis of B). The results showed that animals treated with anti-CMKLR1 antibodies experienced less weight loss and had better fecal scores compared to animals receiving isotype controls. Therefore, anti-CMKLR1 antibodies appear to have therapeutic potential for chronic inflammatory diseases.

[0218] Example 4.1 Preclinical Model of Type 1 Diabetes: Mouse NOD Model Eight-week-old female NOD mice were obtained from the Charles River laboratory. These mice developed spontaneous type 1 diabetes between 12 and 20 weeks of age. Initial onset of diabetes was characterized by hyperglycemia. When blood glucose levels were between 180 mg / dL and 234 mg / dL, anti-CMKLR1 and isotype controls were administered intraperitoneally three times weekly for two weeks at 20 µg / injection. Mice were euthanized when blood glucose levels exceeded 600 mg / dL (corresponding to irreversible diabetes).

[0219] Results: This type 1 diabetes model is also considered a mouse model of autoimmune disease. Figure 5 Results presented by AC showed that animals treated with anti-CMKLR1 antibodies exhibited higher survival rates and near-normal blood glucose levels, as indicated by blood glucose concentration measurements. This recovery appeared to be stable over time, suggesting that previously ill animals may have fully recovered. The anti-CMKLR1 antibody restored glucose tolerance.

[0220] Example 5. Imiquimod-induced psoriasis-like skin inflammation Aldara® cream, known to induce psoriasis in mice, was used in male C57Bl / 6 mice (8-10 weeks old). Mice received topical Aldara daily. Intraperitoneal injection of the therapeutic agent (anti-CMKLR1 agonist or control compound): Results: Following Aldara administration (see, for example, day 15), the anti-CMKLR1 agonist (2G1) reduced skin thickness ( Figure 6 A), while the animal's weight was not affected by the administration of stimulants ( Figure 6 B). These results demonstrate the applicability of agonist anti-CMKLR1 compound therapy in a mouse model of psoriasis (an example of an autoimmune disease).

[0221] Example 6. Therapeutic effect of anti-CMKLR1 antibody on a preclinical model of sepsis: mouse peritonitis model Common peritonitis is induced by intraperitoneal injection of Zymosan A® (1 mg per mouse in 1 mL). Five minutes prior to Zymosan A injection, a prophylactic injection of anti-CMKLR1 was administered: RvE1 (1 µg per mouse) and 2G1 antibody (10 µg per mouse). Polymorphonuclear neutrophils (PMNs) and macrophages were collected from the mouse peritoneum at 2, 4, 8, 16, 24, and 48 hours after Zymosan A injection and counted by flow cytometry to determine the regression index (Bannenberg et al., 2005).

[0222] result: Figure 7 The results of PMN (7A) and macrophage (7B) counts, as well as the regression index (7C), shown indicate that animals treated with RvE1 or anti-CMKLR1 antibodies exhibited similar results, with slightly fewer PMNs and macrophages and a better regression index compared to isotype controls. Even slight differences can be significant for treatment in sepsis. Therefore, these results are very positive for the potential application of anti-CMKLR1 antibodies in sepsis.

[0223] Example 7. Therapeutic effect of anti-CMKLR1 antibody treatment on a preclinical cancer model: Example 7.1. The role of anti-CMKLR1 antibody in the growth of primary tumors and the development of lung metastases in an orthotopic breast cancer model.

[0224] Mice were anesthetized with 3% isoflurane. The abdomen of the mice was shaved, and 250,000 4T1 cells were injected into the mammary glands using an insulin syringe (30 Gauge) in 50 µL phosphate-buffered saline (PBS). On days 4 and 7, animals were injected twice (10 μg / injection) with either anti-CMKLR1 antibody (2G1) or anti-41BB antibody (3H3), or both; a control antibody (100 µg / injection) was injected intraperitoneally three times a week for three weeks. In a second study measuring lung metastasis after breast cancer development, animals were treated with 0.8 mg / kg of either anti-CMKLR1 antibody or the control antibody (100 μg / injection) three times a week for three weeks.

[0225] Result: As Figure 8 As shown in Figure A, animals treated with a single compound (2G1 or 3H3) showed no improvement in tumor growth compared to animals receiving isotype control antibodies. However, animals treated with a combination of anti-CMKLR1 and anti-41BB antibodies showed significantly reduced tumor growth in the breast cancer model (p<0.01). Given that this breast cancer model is a very aggressive one, the results are considered positive. Figure 8 As shown in Figure B, bioluminescence imaging illustrated the effect of the anti-CMKLR1 compound on lung metastasis, demonstrating that anti-CMKLR1 treatment reduced lung metastasis compared to animals treated with the control antibody. Analysis of lymph node metastasis showed that animals treated with the anti-CMKLR1 compound did not have metastasis, while the two control animals did (data not shown). These results indicate that the anti-CMLKR1 antibody, possessing RvE1-mimicking agonist activity, has anti-metastatic activity. In this model, the antibody of the present invention did not show any significant efficacy against primary tumor development. However, the results showed improvement when animals received combined treatment with anti-CMKLR1 and anti-41BB antibodies.

[0226] Example 7.2. Therapeutic effect on tumor growth in a colon cancer model Eight-week-old male C57bl / 6J mice were anesthetized with 3% isoflurane. The flanks of the mice were shaved, and MC38 cells (0 to 5.10^6 cells / mouse) were subcutaneously injected into 50 µL phosphate-buffered saline using an insulin syringe (outer diameter 30). An alternative model was used, in which eight-week-old male Balb / c mice were anesthetized with 3% isoflurane. The flanks of the mice were shaved, and CT26 cells (1.10^6 cells / mouse) were subcutaneously injected into 50 µL phosphate-buffered saline using an insulin syringe (outer diameter 30).

[0227] Following tumor inoculation alone or in combination, patients were given intraperitoneal injections (20 μg / injection) once a week for 3 weeks, starting from day 4, of either agonist anti-CMKLR1 antibody (2G1) or anti-SIRPa antibody (p84-anti-mouse SIRPa from Merck Millipore) (SIRPa is a novel checkpoint inhibitor).

[0228] Result: As Figure 9 As shown in AC, in the CT26 cancer model, compared with the control group, the anti-CMKLR1 antibody itself ( Figure 9 B) showed no clinical effect on tumor development, as did anti-SIRPa alone. Figure 9 A). However, surprisingly, the combination of these two compounds was able to inhibit tumor growth in a timely manner ( Figure 9 C). In Figure 9 In another mouse colon cancer model shown in Figure D, anti-CMKLR1 did indeed show inhibitory efficacy against tumor growth compared to isotype controls. In summary, these results from two different colon cancer models suggest that anti-CMKLR1 agonist antibodies can be used alone or in combination with another therapeutic agent to prevent tumor development.

[0229] Example 8. Meta-analysis of CMKLR1 expression in biopsies of human patients with UC or CD treated with anti-TNFα or anti-α4β7 antibody therapy.

[0230] In the two main forms of human inflammatory bowel disease (IBD), Crohn's disease (CD) and ulcerative colitis (UC), the signaling network that enables the persistence of chronic gastrointestinal inflammation remains unclear. Based on an analysis of nearly 500 IBD patients and 100 controls, the inventors report in this paper that CMKLR1 transcripts accumulate in the inflamed colonic tissue of patients with severe IBD who do not respond to immunosuppressive / corticosteroid and immunotherapies such as anti-TNFα (infliximab) or anti-α4β7 integrin (vedolizumab) therapy.

[0231] The inventors first analyzed mucosal CMKLR1 transcript expression through a meta-analysis of publicly available transcriptome datasets from three UC patient cohorts: GSE16879 (Arijs et al., 2009a) and GSE12251 (Arijs et al., 2009b) and GSE73661, in which colonic mucosal biopsies were performed within one week prior to antiTNF therapy in patients who did not respond to corticosteroids and / or immunosuppressive therapy. In these three cohorts, antiTNF response was defined as histological healing analyzed 4–6 weeks after their first antiTNF infusion (total: n=18, non-IBD controls; n=41, UC refractory; and n=28 UC, responders).

[0232] Results: Analysis showed that, compared with non-IBD controls or UC patients who had not yet received anti-TNF therapy and were expected to respond to anti-TNF therapy, patients with primary UC should have significantly increased CMKLR1 transcript expression in colon biopsies before and after receiving anti-TNF therapy. Figure 10 A). In patients who will not respond to anti-TNF therapy, mucosal CMKLR1 expression in colonic or ileal biopsies was significantly increased in Crohn's disease patients (n=24, non-IBD controls; n=17, CD refractory patients; and n=20, CD responsive patients; GSE16879 (Arijs et al., 2009a)) before and after anti-TNF therapy, compared with non-IBD controls or future-responding patients ( Figure 10 Compared to B), finally, colonic mucosal gene expression analysis in the UC patient cohort (GSE7366146) treated with anti-α4β7 (vedolizumab) therapy also confirmed that CMKLR1 expression was significantly increased in the non-responsive group before and after vedolizumab treatment. Figure 11 ).

[0233] In summary, the meta-analysis showed that CMKLR1 is overexpressed in inflamed tissues of IBD patients, particularly in patients who do not respond to current immunosuppression or immunotherapy, even before treatment initiation. Our meta-analysis provides evidence that CMKLR1 expression in the colon of patients with refractory UC or CD, or in the ileum of CD patients, may conversely enable these patients to respond to agonist anti-CMKLR1 antibody therapy (such as the antibody of this invention).

[0234] Example 9. Study on CMKLR1 expression and antibody binding - ELISA combined CMKLR1 ( Figure 12 ) CMKLR1 peptide (273NH2-PYHTLNLLELHHTAMPGSVFSLGLPLATALAIA-COOH305) (SEQ ID No: 60) (5 μg / ml) was coated overnight in borate buffer. It was then saturated at 37°C with 0.1% PBS-Tween and 0.25% gelatin for 2 hours. Next, different concentrations of 2G1 or hIgG1 antibody were added at 37°C for 2 hours. Then, a peroxidase-conjugated secondary antibody (0.8 µg / ml) was added at 37°C for 1 hour, and the reaction was visualized using TMB substrate. The colorimetric reaction was read using TECAN.

[0235] - CMKLR1 expression via FACS ( Figure 13 A) Cells were resuspended in PBS-FBS-EDTA and incubated on ice for 30 minutes with Fc blocking agent (1 / 50). T cell lines (Thp-1 and U937) were stained with A488-labeled 2G1 (5 µg) or A488-labeled hIgG1 (5 µg).

[0236] - Western blot analysis of CMKLR1 ( Figure 13 B) As previously described, after protein migration and transfer, the 2G1 antibody (10 μg / membrane) was incubated overnight at 4°C and visualized with a peroxidase-conjugated second antibody (1:2000). CMKLR1 expression was then detected using chemiluminescence and an image reader. The protein blot images were quantified using Multi Gauge software.

[0237] result: Figure 12 The results showed that the anti-CMKLR1 antibody clone 2G1 can bind to the polypeptide that forms the cyclic EL3 of CMKLR1. Assessment of CMKLR1 expression in different cell lines using the 2G1 antibody by FACS and Western blotting showed that human tumor T cell lines Trp1 and U937 express CMKLR1, as do transduced CMKLR1 CHO cells, human lung fibroblast line MRC5, and human NK cell line NKL. Figure 13 ).

[0238] Example 10. Study on CMKLR1 expression in bone marrow lineages Example 10.1. Differentiation and polarization of human monocytes Monocytes were collected from peripheral blood mononuclear cells (PBMCs) in the erythrocyte sedimentation rate (ESR) layer of healthy volunteers and isolated by magnetic separation or elutriation. The PBMCs were then cultured with different cytokine mixtures to generate differentiated non-polarized or polarized macrophages. This protocol allows for the generation of polarized macrophages to produce pro-inflammatory (M1) or pro-remission (M2) inflammatory macrophages in different wells. Plates were seeded in complete RPMI (RPMI containing 10% FBS, 1% glutamine, and 1% antibiotics) at 0, 5, and 10 μL depths. 6 Cells / mL of monocytes were seeded, with 500 μL of cell suspension in each well of a 24-well plate. 100 ng / mL M-CSF was added to the culture medium for cell differentiation. Cells were incubated for 5 days, and on day 3, the culture medium was replaced with fresh medium supplemented with 100 ng / mL M-CSF. For the polarization phase, during day 3, pro-inflammatory macrophages were generated by supplementing the LPS-IFNg solution (100 ng / mL LPS and 20 ng / mL IFNg) with isotype controls (mIgG1 or hIgG4) (2 μg / mL) or anti-CMKLR1 antibody (2 μg / mL) (2G1 or 2G4, H6, BZ332 or 84939) or with C15 peptide (10 nM) or RvE1 (10 ng / mL). Pro-inflammatory-IFNg macrophages can also be generated by adding only IFNg (20 ng / mL) to the culture medium. To promote the reduction of macrophage polarization, cells were incubated with 20 ng / mL IL-4. After differentiation and / or polarization, the release of phenotypical and functional cytokines / chemokines was investigated by FACS analysis, ELISA, and Western blotting.

[0239] Example 10.2. Isolation and differentiation of mouse macrophages and dendritic cells - Isolate mouse bone marrow-derived macrophages Bone marrow cells were harvested and cultured for 5 days in RPMI medium supplemented with 10% FBS, glutamine, and antibiotics to induce macrophage differentiation. The RPMI medium contained 100 ng / mL macrophage colony-stimulating factor (M-CSF). Macrophages were harvested and cultured for 2 days with either IFNg (20 ng / ml) and LPS (100 ng / ml) to induce pro-inflammatory polarization, or with IL-4 (20 ng / ml) to induce depolarization. A therapeutic agent of 2 μg / ml was added during macrophage polarization.

[0240] - Generation of dendritic cells derived from bone marrow Bone marrow cells were harvested and cultured in RPMI medium supplemented with 10% FBS, glutamine, and antibiotics. Dendritic cell differentiation was induced with 20 ng / ml GM-CSF for 7 days. Immature dendritic cells (iDCs) were then collected and cultured with LPS (100 ng / ml) for 24 hours to induce iDC maturation into mDCs. A therapeutic agent of 2 μg / ml was added during differentiation and maturation.

[0241] Following differentiation into pro-inflammatory or pro-regressive macrophages in mice, as described above, cells were incubated in culture medium, with isotype controls, and in the presence of anti-CMKLR1 antibody: clones H6 and BZ194, C15 peptide, and target anti-CMKLR1 antibody 2G1 or RvE1 were used. The secretion of IL10, CCL17, and IL12p40 was then assessed by ELISA. Cytokine secretion in the supernatant was measured using an ELISA kit from BD. For IL10 cytokine, the supernatant was diluted 1 / 10; for CCL17 cytokine, 1 / 50; and for IL12p40 cytokine, 1 / 100.

[0242] - Research on ELISA cytokine secretion Cytokine secretion was detected by ELISA according to the manufacturer's instructions. In short, the supernatant was diluted in an appropriate buffer and coated overnight with capture antibody followed by saturation and incubation for 2 hours. Cytokines were then visualized using a biotin-conjugated antibody, and the signal was amplified using a biotin-streptomycin-conjugated peroxidase system. TMB provided by BD Biosciences was used as the substrate, and the colorimetric reaction was read using TECAN.

[0243] - Activated cell markers analyzed by FACS Dendritic cells were resuspended in PBS-FBS-EDTA and incubated on ice for 30 minutes with live and dead cells (LIVE / DEAD™ fixable yellow dead cell staining kit, Life Technologies). Staining was performed on CD11c-BV711, CD11b-APCCy7, I / Ab-APC, CD103-PerCPCy5.5, CCR7-V450, CD40-PeCy7, CD80-PE, and CD86-FITC (all provided by BD Pharmingen).

[0244] - Protein blot analysis ERK / Akt Mouse pro-inflammatory macrophages (M1) were generated from bone marrow using M-CSF and polarized with IFN-gamma (IFNg) and LPS. Briefly, bone marrow cells were collected by flushing the femur and cultured in 100 ng / mL mM-CSF for 5 days, followed by polarization with 20 ng / mL IFNg and 100 ng / mL LPS for 24 hours. Their FBS was then removed with RPMI FBS 2% medium for 24 hours. Finally, the mouse pro-inflammatory macrophages were treated with 2 µg / mL 2G1 antibody at different time points: 5 min, 10 min, and 30 min. Cells were collected in RIPA buffer. Protein concentration was measured using a BCA protein assay kit. Proteins were denatured by heating at 95°C for 5 min and diluted in DTT and Laemmli solutions. After migration and transfer, the cells were blocked in nitrocellulose membranes containing 5% BSA in TBS-T for 2 hours. Antiphosphorylated ERK antibody and antiphosphorylated Akt antibody (1:1000) were incubated with the membrane overnight at 4°C and visualized using a peroxidase-conjugated second antibody (1:2000). The Western blot images were quantified using Multi Gauge software.

[0245] Example 10.3: CMLKR1 expression on human blood mononuclear cells and mouse bone marrow cells and neutrophils after inflammatory stimulation.

[0246] Human monocytes were collected from peripheral blood mononuclear cells in the erythrocyte sedimentation rate (ESR) layer of healthy volunteers and separated by magnetic separation or panning. The monocytes (CD14-positive cells) were then cultured in a culture medium and treated with different pro-inflammatory stimuli for 16 or 48 hours: LPS (100 ng / ml), TNFα (100 U / ml), or IL6 (20 ng / ml).

[0247] Mouse monocytes (CD11b+ Ly6G- SSClow) and neutrophils (CD11b+ Ly6G- SSClow) were obtained from bone marrow cells harvested and cultured in RPMI medium supplemented with 10% FBS, glutamine, and antibiotics. The cells were then cultured in the medium and treated with different pro-inflammatory stimuli for 16 or 48 hours: LPS (100 ng / ml), TNFα (100 U / ml), or IL6 (20 ng / ml).

[0248] CMLKR1 expression was measured by FACS using commercial anti-CMKLR1 antibodies (human anti-ChemR23: clone 84939 and mouse anti-ChemR23: clone 477806).

[0249] Result: For Figure 14The analysis of CMKLR1 expression in mouse bone marrow lineages shown above revealed good expression of the protein in monocytes, macrophages, and dendritic cells. Figure 15 The image above shows the expression of CMKLR1 in human monocytes and mouse bone marrow myelocytes and neutrophils. Inflammatory stimuli (such as LPS, TNFα, or IL6) significantly increased this expression (at least twice that of controls at 48 hours later), confirming that CMKLR1 expression and overexpression in bone marrow cell lineages during inflammation can represent a therapeutic approach of downregulation and / or induction of inflammatory resolution. DC activation markers were analyzed by FACS, and... Figure 16 The results shown above indicate that, compared with the excipient or isotype control, the expression of CD80, CD86, CD103, CD40, and IAb was significantly reduced when cells were treated with RvE1 lipid or 2G1 antibody. These results suggest that the 2G1 antibody has the same activity on the CMKLR1 pathway in DCs as RVE1. The inventors then analyzed the CMKLR1 activation pathway in mouse macrophages using Western blotting. Figure 17 The results showed that the anti-CMKLR1 antibody 2G1 could induce the activation of Akt and Erk proteins after incubation for 10 to 30 minutes. These results indicate that the 2G1 antibody can exhibit agonist properties against the CMKLR1 receptor, just like the RvE1 lipid.

[0250] Example 11. Study on the competition between chemokine-induced CMKLR1 activation and anti-CMKLR1 antibody. method. The competition of the CMKLR1 receptor for chemokine-dependent β-repressor protein recruitment was measured in the presence of anti-CMKLR1 antibody. Determination: The day before the assay, CHO-K1 CMKLR1 cells (Discover'X, reference number 93-0313E2) were seeded on preheated cell reagent plates, followed by seeding 100 µl / well of cells in 96-well plates (Discover'X, reference number 15-103) and incubating at 37°C in a humidified 5% CO2 incubator for 48 hours. Anti-CMLKR1 antibody was diluted (22X, 1 µM to 1 nM, 7 spots, a series of 3-fold dilutions), and cells were incubated with the antibody at 37°C for 30 minutes. Cells were then stimulated with chemokines (2 nM or 6 nM) at 37°C for 90 minutes according to the supplier's protocol (Discover'X, reference number 92-1036). After adding the working assay solution to the cells, luminescence was measured using a plate reader with an integration time set to 0.5 seconds.

[0251] Measuring the competition between anti-CMKLR1 antibody and chemokine in the production of AMPc from the CMKLR1 receptor: The day before the experiment, CHO-K1 CMKLR1 Gi cells (Discover'X, reference number 95-0080C2) were seeded on plates in preheated cell reagent, and then 100 µl / well of cells (Discover'X, reference number 15-103) were seeded on plates in 96-well plates and incubated at 37°C in a 5% CO2 humidified incubator for 24 hours.

[0252] Chemokinin agonists (6x, 10) -7 µM to 10 -10 A mixture of M (7 spots, a series of 3-fold dilutions) (Discover'x from R&D Systems, USA, reference number 92-1036 or 2324-CM-025) and forskolin (40 µM) (a cAMP activator) (Discover'x, reference number 92-0005) was added to cells at 37°C over 30 minutes; or cells were pre-incubated at 37°C for 30 minutes with anti-CMKLR1 antibody (serial dilutions: 6X, 1 µM to 1 nM, 7 spots, a series of 3-fold dilutions). Then, a mixture of chemokine (2 nM) + forskolin (60 nM) was added to cells at 37°C over 30 minutes. For cAMP detection, the antibody reagent and cAMP working assay solution were added to the plate at room temperature and incubated for 1 hour, followed by the addition of cAMP solution A, and the cells were incubated at room temperature in the dark for 3 hours. Bioluminescence was read using a plate reader with the integration time set to 0.5 seconds.

[0253] Results: To test whether the antibody of the present invention is an antagonist of chemokine-induced CMKLR1 activation, two assays were performed, and the results are shown in... Figure 18 superior. Figure 18 A shows the inhibition of trichokinin-dependent cAMP production induced by chemokines (black circles or white squares); the anti-CMLKR1 antibody of the present invention cannot reverse this inhibition (black circles or white squares) compared to the control (gray diamonds). Figure 18 Figure B shows chemokine-induced beta-inhibitor protein activation, indicating that the anti-CMKLR1 antibody of the present invention does not significantly alter the chemokine-dependent activation of the beta-inhibitor protein (white circle compared to black diamond). The antibody of the present invention does not possess antagonistic activity against CMLKR1-chemokine interactions. Furthermore, the antibody of the present invention cannot induce the chemokine-induced CMLKLR1 signaling pathway, thus confirming that these antibodies are not chemokine agonists of the CMLKR1 pathway.

[0254] Example 12. CD45Rb 高 T-cell metastasis chronic colitis mouse model Method: CD45Rb 高 CD4 T cells were isolated from the spleens of mice undergoing the first experiment and sorted on ARIA FACS after negative selection by magnetic sorting. Then, 0, 5, and 10... 6 100 µL of cells (in 100 µL PBS) were intraperitoneally injected into 6-week-old female Rag1 knockout mice. From CD45Rb 高 Anti-CMKLR1 antibody (2G1) or isotype control was administered on day 32 post-CD4 T cell transfer for 3 weeks, three times a week, at a dose of 1 mg / kg. Weight was assessed three times a week, and weight change was determined from initial weight. * p < 0.05, ** p < 0.01.

[0255] result: Figure 19 The percentage change in body weight over time is shown for animals treated with either anti-CMLKR1 antibody or isotype control. Both groups showing the same initial body weight change were treated with either anti-CMLKR1 antibody or isotype control within the first 30 days. Mice treated with anti-CMKLR1 continued to gain weight, while control mice began to lose weight, indicating that the control group developed chronic colitis as expected. Figure 19 A). A decrease in tissue thickness corresponding to colon repair was observed in mice treated with anti-CMKLR1. A reduction in fibrotic tissue was also observed. Figure 19 B). Different scores represent anatomical and pathological scores used to calculate symptom severity. Mice treated with anti-CMKLR1 had lower scores (including inflammation scores). Figure 19 C). The inventors demonstrated in a third colitis model (a chronic inflammation model in this paper) that the anti-CMKLR1 antibody of the present invention is meaningful for the treatment of chronic inflammation and autoimmune diseases (such as colitis).

[0256] Example 13. Antitumor effect on overall survival of mouse liver cancer tumor model Methods: Mice were anesthetized with a toluidine / ketamine mixture. After laparotomy, hepatocellular carcinoma tumor cells (2, 5, 10) placed in PBS were injected via the portal vein. 6 Treatment began 4 days after tumor injection. Anti-CMKLR1 antibody (2G1 clone) and hIgG1 isotype control were administered three times weekly for 2 weeks. Anti-PD1 monoclonal antibody (8 mg / kg) in PBS was administered intraperitoneally twice weekly for 2 weeks. Combinations of anti-CMKLR1 and anti-PD1 antibodies (0.8 mg / kg and 8 mg / kg, respectively) were also tested. Overall survival was tracked over 60 days, and... Figure 22The report shows the survival percentage for each scenario.

[0257] Result: As Figure 20 As shown, for animals treated with either anti-CMKLR1 or anti-PD1 antibodies, a prolonged survival was observed in only 1 out of 7 treated animals (15% of treated animals), indicating a partial response (PR). However, animals treated with a combination of anti-PD1 and anti-CMKLR1 antibodies showed a significantly improved survival rate after 60 days of treatment (from 15% to 45%), indicating a complete response (CR). These results demonstrate the unexpected efficiency of the treatment combination (anti-PD1 / anti-CMKLR1 antibody) in HCC tumor models.

[0258] Example 14. Antibody production in different cell lines.

[0259] The CDR of the 2G1 heavy chain was grafted into three human germline backbones and named IGHV3-23*04 (corresponding to SEQ ID No. 41), IGHV1-46*01, and IGHV7-4-1. The CDR of the 2G1 light chain was grafted into three human germline backbones and named IGKV1-13*02 (corresponding to SEQ ID No. 50), IGKV6-21*01, and IGKV3-11*01 (IMGT nomenclature). Each sequence was fused with a constant fragment of human immunoglobulin and co-transfected in mammalian cells to generate humanized antibodies. More specifically, to construct the anti-ChemR23 A heavy chain, the antibody variable domain VH sequence was synthesized via EcoRV and cloned into the pFUSE-CHIg-hG1 expression plasmid containing the Fc of human IgG1 (pFUSE-CHIg-hG1 mediator, from Invitrogen, Toulouse). To construct the light chain of the anti-ChemR23 antibody, the variable domain VL was synthesized via BsiWI and cloned into the pFuse2CLIg-hk expression plasmid containing human CLkappa (pFuse2CLIg-hk, from Invitrogen, Toulouse). In mammalian HEK or CHO cells, we co-transfected plasmids containing VH-hFcG1 and VL-CLkappa using liposomes. After incubation for 3–7 days, the supernatant was recovered and quantified by sandwich ELISA. The supernatant was then purified by affinity chromatography using protein A (HiTrap, GeHealthcare) with 0.1 M citrate pH 3 elution buffer. The purified antibodies were dialyzed and concentrated in PBS. They were quantified by UV (A280 nm) and tested in an activity assay targeting the C7 antigen-specific peptide.

[0260] Result. Figure 21As shown, the human line IGHV3-23*04, representing the heavy chain, produces antibodies more efficiently in mammalian cells, as demonstrated by VHvAv3-23 (SEQ ID NO: 37). Mutations related to other backbones induce the production of fewer chains. For the light chain, the human line IGKV1-13*02 produces the best humanized antibodies, as demonstrated by VlvAv1-13 (SEQ ID NO: 49), while other lines show a 1-log reduction in yield. The combination of humanized IGHV3-23*04 and IGKV1-13*02 is suitable for high-yield production of humanized anti-CMKLR1 antibodies. Figure 22 As shown, this combination (as demonstrated by VHvAv3-23 (SEQ ID NO: 37) + VlvAv1-13 (SEQ ID NO: 49)) can indeed produce a sufficient number of antibodies with satisfactory yield.

[0261] Therefore, germline IGHV3-23*04 and germline IGKV1-13*02 were selected for further humanization of the antibody.

[0262] A few mutations were added to either the heavy or light chain. In the heavy chain, mutations G33A (in CDR1), P60A (in CDR2), and R94K (in FR3) were substituted to increase humanization, and amino acid D61 (in CDR2) could be substituted with amino acid E or A to reduce the risk of deamination in the antibody (sequence variants vB-vD). In the light chain, mutations S24R, S27Q, M33L (in CDR1), T51A (in CDR2), and Y71F (in FR3) were substituted to increase humanization, and amino acid N92 could be substituted with amino acid Q to reduce the risk of glycosylation in the antibody (sequence variants vB-vD). Each sequence was fused with a constant fragment of human immunoglobulin and co-transfected in mammalian cells to generate humanized antibodies. The results showed that all combinations of the heavy and light chains produced antibodies. Productivity in mammalian cells appears to be affected differently depending on the combination of heavy and light chains, but for therapeutic applications of antibodies, the amount is always sufficient to produce them effectively.

[0263] Example 15. Recognition ability of anti-CMKLR1 antibodies generated in vitro and from specific strains of heavy chain variable domains and light chain variable domains.

[0264] For quantitative ELISA assays, Fc-specific donkey anti-human IgG (Jackson Immunoresearch; USA; Reference No. 709-005-098) was immobilized at 1.3 µg / ml on a plastic substrate in borate buffer (pH 9), and a supernatant containing the antibody was added to measure binding (compared to standard antibodies). After incubation and washing, mouse anti-human kappa antibody (OSE Immunotherapeutics, Reference No. NaM76-5F3) was added, and detection was performed using peroxidase-labeled donkey anti-mouse IgG antibody (Jackson Immunoresearch; USA; Reference No. 715-036-151). ELISA visualization was performed using standard methods.

[0265] For the active ELISA assay, Fc-specific donkey anti-human IgG (Jackson Immunoresearch; USA; Reference No. 709-005-098) was immobilized at 1.3 µg / ml on a plastic substrate in borate buffer (pH 9), and purified antibody was added to measure binding in 1% BSA buffer (compared to wild-type 2G1). After incubation and washing, a biotinylated antigen-specific peptide (Biot-C7 peptide: biotinylated NH2-PYHTLNLLELHHTAMPGSVFSLGLPLATALAIA-COOH, synthesized from Synpeptide, SEQ ID No. 60) was added, followed by the addition of peroxidase-streptomycin (Jackson Immunoresearch; USA; Reference No. 016-030-084) and visualization was performed using standard methods.

[0266] Combinations of heavy and light chains derived from VHvAv3-23*04 (SEQ ID No. 41 - 89.8% humanized) and VLvAv1-13*01 (SEQ ID No. 50 - 82.1% humanized) yielded humanized antibodies with good binding activity to antigen-specific peptides (C7 peptides), such as wild-type antibody 2G1. Figure 23 As shown, the combination of humanized antibody variable domain chains is derived from 2G1 (for the heavy variable domain: HA corresponds to VHvAv3-23*04 and SEQ ID No. 4; HC corresponds to SEQ ID No. 42; HD corresponds to SEQ ID No. 43; for the light variable chain: LA corresponds to SEQ ID No. 50; LC corresponds to SEQ ID No. 52; LD corresponds to SEQ ID No. 53).

[0267] All combinations exhibit at least the same binding activity to the antigen-specific peptide (C7 peptide) as wild-type antibody 2G1. In some cases (combinations of HCLC, HCLD, HDLC, and HDLD), binding is even superior to germline antibody HALA and wild-type antibody 2G1. Figure 23 ).like Figure 24 As shown, the ED50 (ng / ml) of humanized antibodies is at least comparable to that of 2G1 antibodies, and in most cases the ED50 (ng / ml) of humanized antibodies is better.

[0268] Example 16. Biological effects of CCR7 internalization Materials and methods Macrophages were generated from monocytes of healthy volunteers using 100 ng / mL M-CSF for 5 days. The macrophages were then collected and incubated with 10 mg / mL coated monoclonal antibody (mAb) in the presence of 20 ng / mL IFNγ to obtain M1 inflammatory macrophages. Phenotypic analysis of M1 macrophages for CXCR4 and CCR7 was performed by flow cytometry, and cytokines released from the supernatant were quantified by ELISA. Dendritic cells (DCs) were generated from monocytes of healthy volunteers using 50 ng / mL GM-CSF and 20 ng / mL IL-4 for 6 days. Phenotypic analysis of DCs for CCR7 was then performed by flow cytometry.

[0269] result 2G1 cells and all humanized 2G1 variants (HALA, HCLC, HCLD, HDLC, and HDLD) were immobilized on plates. Isotype controls were added as references. Two isotypes that inhibit FcRγ binding were also added: 2G1-N297A (2G1wt mutated in N297A to reduce FcγR binding) and 2G4 (wild-type, where isotype IgG4 is mutated in S228P to stabilize the hinge region). Pro-inflammatory macrophages M1 were added to the coated plates for 48 hours, and CCR7 expression on the macrophage surface was measured by flow cytometry.

[0270] like Figure 25 As shown, 2G1 and all humanized 2G1 variants can reduce CCR7 expression on the surface of pro-inflammatory macrophages (M1). However, no internalization of CCR7 was observed in homotype IgG1-N297A or IgG4, which block FcRγ binding, suggesting that homotype IgG1 is preferred to obtain this biological activity.

[0271] Example 17. Neutrophil Apoptosis and Mortality Neutrophils are typically located at sites of inflammation, and their presence sustains the inflammatory process, preventing the initiation of or actively maintaining inflammatory resolution, thus potentially leading to chronic inflammation. Neutrophil apoptosis prevents the release of toxic neutrophil contents and exerts an anti-inflammatory effect.

[0272] like Figure 26 As shown in Figure A, the survival / death ratio of neutrophils is higher in cells treated with the antibody of the present invention, thus illustrating the effect of the agonist of the present invention on these cells.

[0273] CASPASE-3 expression Materials and methods: PMNs from healthy volunteers were incubated for different times in medium containing 10 µg / mL coated antibody, and the PMNs were collected for caspase-3 staining analysis by Western blot. The intensity of Capase-3 expression was calculated on Western blotting.

[0274] Result: As Figure 26 As shown in B, administration of the anti-CMKLR1 agonist enhanced caspase-3 activity compared to cells treated with the control antibody. The HALA antibody had a greater effect on caspase-3 activity than the 2G1 antibody. Both 2G1 WT and HALA increased caspase-3 cleavage, implying that ChemR23 triggers caspase-3-dependent apoptosis.

[0275] Percentage of PMN deaths and ROS test ( Figure 26 C): PMNs from healthy volunteers were incubated in medium containing 10 µg / mL coated antibodies for 24 hours or 5 hours, and stained with specific markers of reactive oxygen species (ROS) using a death / survival assay kit (LIVE / DEAD (Invitrogen)). The percentage of positive cells was obtained by analyzing the images using ImageJ software.

[0276] Results: 2G1 and all humanized 2G1 variants increased PMN mortality at 24 hours. At 48 hours, the percentage of dead cells incubated was similar to that of the IgG1 control and HALA variants, indicating that the antibody only accelerated programmed cell death in the PMN by triggering CMKLR1 signaling. 2G1 and all humanized 2G1 variants accelerated PMN death, thus the humanized variants retained the pro-regression properties. 2G1 and HALA variants increased ROS production in the PMN at 5 hours.

[0277] like Figure 27As shown in Figure B, the percentage of ChemR23-positive cells (macrophages and neutrophils) increases upon induction of inflammation. However, in animals treated with anti-CMKLR1 antibody, the percentage of neutrophils in the exudate did not decrease significantly. Figure 27 C; black square), while the overall percentage of macrophages in the exudate was slightly increased ( Figure 27 D). This indicates that administration of CMKLR1 agonists does not reduce the total number of bone marrow cells in the exudate and does affect apoptosis of neutrophils, primarily at sites of inflammation. Figure 27 E and Figure 27 As shown in F, the percentage of dead neutrophils and the increase in their mortality were observed when CMKLR1 agonists were administered. These results suggest a positive effect of CMKLR1 agonists in treating delayed inflammation resolution, since the neutrophil population is located at the site of inflammation, not in the exudate.

[0278] In summary, apart from inducing neutrophil apoptosis at the site of inflammation, treatment with the antibodies of this invention does not lead to apoptosis in all neutrophil populations. This feature may help reduce side effects. Example 18. Neutrophil Migration Neutrophils migrate after being recruited to sites of inflammation, thereby initiating, enhancing, and / or maintaining the inflammatory process.

[0279] Materials and methods Human endothelial cells (HDMECs) were incubated for 24 hours in gelatin-coated transwell chambers and activated overnight with 100 U / mL TNF-alpha or in the absence of inflammation. PMNs from healthy volunteers or ANCA patients were then incubated for 4 hours in transwell chambers containing an HDMEC monolayer. During the 4-hour migration assay, 10 μg / mL of antibody (isotype control and 2G1) plus / - 100 U / mL TNF-alpha were added. The lower migrating fractions of the transwell chambers were collected, and the migrating PMNs were counted using counting beads by flow cytometry.

[0280] result Neutrophils treated with 2G1 had lower migration ability compared to cells treated with the control compound. Figure 28 A). When PMNs and endothelial cells are activated with TNFα in healthy volunteers and HIV patients, 2G1 prevents PMNs from migrating across the endothelial monolayer, especially in inflammatory conditions. Figure 28 B).

[0281] The antibody of this invention has the ability to reduce the migration ability of neutrophils.

[0282] Example 19 - Expression of CD62L Materials and Methods: PMNs from healthy volunteers were incubated for different times in a medium containing 10 µg / mL coated antibody and collected for CD62L staining by flow cytometry analysis. Figure 29 (Left figure). Compared to cells incubated without antibodies, cell surface expression of CD62L was reduced in cells incubated with the antibody of the present invention. Soluble CD62L released through detachment was detected by ELISA in the supernatant of PMN incubated with the coated antibody. Treatment of PMN with anti-ChemR23 antibody increased the concentration of soluble CD62L compared to isotype control conditions. Figure 29 (See right image).

[0283] Example 20 - Survival rate of mesothelioma model Mice treated with the CMKLR1 agonist according to the method explained in the accompanying drawings had a higher survival rate than mice treated with the control antibody, thus demonstrating the positive effect of the compounds according to the invention in treating mesothelioma. Figure 30 ).

[0284] Example 21 - CRC Model like Figure 31 As shown in Figure A, the tumor volume of animals treated with the anti-CMKLR1 antibody of the present invention was reduced compared to the control antibody. Complete remission was also observed in some cases, demonstrating the positive effect of the compounds according to the present invention in treating CRC.

[0285] In addition, such as Figure 31 As illustrated in B, treatment with the anti-CMKLR1 monoclonal antibody (OSE-230) resulted in a decrease in stool scores and a reduction in the number of tumors.

[0286] Example 22 - Experimental Model of Autoimmune Encephalomyelitis In this model, therapeutic administration of the antibody of the present invention did not result in weight loss in the EAE model compared to animals treated with the control antibody. Figure 32 A). However, when treated with anti-CMKLR1 antibodies, the disease score was significantly reduced ( Figure 32 B). Compared to the control group, the anti-CMKLR1 antibody significantly improved disease scores 10 days after treatment, as the scores of animals treated with the agonist compound were approximately 30% lower. Therefore, this demonstrates that treatment with the anti-CMKLR1 compound is effective in treating autoimmune encephalomyelitis.

[0287] Example 23: Optimization of CDR amino acid residues A small number of mutations were added to the heavy or light chain of the HDLD variant to replace the mutations found in the computer using IEDB software and HLA-II prediction software (NetMHCpanII method). in silico The predicted immunogenicity-related amino acids.

[0288] It is further emphasized that, among a very large number of possible amino acid mutations, the inventors have studied and identified a number of highly advantageous mutations that do not significantly and unduly affect the bioactivity of the product. The selections described below are based on expertise.

[0289] In the heavy chain, the following amino acid substitutions were implemented to reduce immunogenicity: D61E (HD-61E, SEQ ID NO: 89) in CDR2, R52G (HD-R52G, SEQ ID NO: 90) in CDR2, or R52aG, A49S, N52S, and Y53S (HEF, SEQ ID NO: 91) in CDR2. In the light chain, amino acid N92 in CDR3 was substituted with amino acid S (LD-N92S, SEQ ID NO: 92) or Q (LD-T52S, SEQ ID NO: 93) to prevent post-translational modification. T52S in CDR2 was also substituted to reduce the immunogenicity of the LD-N92S variant. N92Q in CDR3 and T52S, T55E in CDR2, and W47L in backbone 2 were substituted to reduce the immunogenicity of the LEF variant (SEQ ID NO: 55). The sequences are shown in the table below: Table 3: Optimized heavy and light chains of HDLD variant sequences.

[0290] As will be detailed in the following examples, antibodies containing the light chain LDT52S, and especially the HEF-LDT52S variant, are particularly advantageous among all possible combinations. HEF-LDT52S, optimized for reduced immunogenicity, exhibits high binding activity and stability while maintaining biological function compared to other antibodies tested.

[0291] Each sequence was fused with a constant fragment of human immunoglobulin and co-transfected in mammalian cells to produce humanized antibodies. Results showed that all combinations of heavy and light chains produced antibodies. The production rate in mammalian cells appeared to be affected differently depending on the combination of heavy and light chains, but the amount was always suitable for effective production in terms of therapeutic applications of the antibodies.

[0292] Example 24: Recognition ability of in vitro generated anti-CMKLR1 antibody For the active ELISA assay, Fc-specific donkey anti-human IgG (Jackson Immunoresearch; USA; Reference No. 709-005-098) was immobilized at 1.3 µg / ml on a plastic substrate in borate buffer (pH 9), and purified antibody was added to measure binding in 1% BSA buffer (compared to wild-type 2G1). After incubation and washing, a biotinylated antigen-specific peptide (Biot-C7 peptide: biotinylated NH2-PYHTLNLLELHHTAMPGSVFSLGLPLATALAIA-COOH, synthesized from Synpeptide, SEQ ID No. 60) was added, followed by the addition of peroxidase-streptomycin (Jackson Immunoresearch; USA; Reference No. 016-030-084) and visualization was performed using standard methods.

[0293] like Figure 33 As shown, the combination of humanized antibody variable domain chains derived from 2G1 (for the heavy variable domain: HD corresponds to SEQ ID No. 43 and HEF corresponds to SEQ ID NO: 91; for the light variable chain: LD corresponds to SEQ ID No. 53; LD-T52S corresponds to SEQ ID No. 93 and LEF corresponds to SEQ ID NO: 55) produced humanized antibodies with better binding activity to the antigen-specific peptide (C7 peptide) compared to germline antibody HALA and wild-type antibody 2G1.

[0294] Example 25: Stability Test Each purified humanized anti-ChemR23 antibody (HALA, HDLD, HD-LDT52S, HEF-LDT52S, HEF-LEF) was incubated at 4°C or 37°C for 7 days. After 7 days, the binding of the purified antibodies was analyzed by ELISA, and the formation of aggregates was analyzed by gel filtration (Superdex 200 10 / 300GL, GeHealthcare).

[0295] like Figure 34 As shown in Figure A, all purified antibodies exhibited similar binding activity at 37°C, 4°C, or -80°C. Figure 34 As shown in B, for HDLD and HEF-LDT52S, the percentage of aggregates did not change after 7 days at 37°C.

[0296] Example 26: Biological effects of CCR7 internalization The humanized 2G1 variant HEF-LD-T52S was immobilized on a plate. An isotype control was added as a reference. Pro-inflammatory macrophages M1 were added to the coated plate and incubated for 48 h, and CCR7 expression on the macrophage surface was measured by flow cytometry.

[0297] like Figure 35 As shown, compared with the isotype control, the humanized 2G1 variant HEF-LD-T52S reduced CCR7 expression on the surface of pro-inflammatory macrophages (M1). The humanized 2G1 variant HEF-LD-T52S, optimized to reduce immunogenicity, retained the functional properties of the 2G1 antibody.

[0298] Example 27: Biological effects on PMN activity PMNs from healthy volunteers were incubated for 24 hours in medium containing 10 µg / mL of HEF-LDT52S, HEF-LEF, and HDLD antibody variants and stained with a death / survival assay kit (LIVE / DEAD (Invitrogen)). The percentage of positive cells was obtained by analyzing the images using Fiji software. An isotype control was added as a control. A mutant HEF-LDT52S antibody (HEF-LDT52S N297A) that does not bind to the Fc receptor (FcR) was also added. Figure 36 As shown, 2G1 and all humanized 2G1 variants HEF-LDT52S, HEF-LEF and HDLD accelerate the death of PMN, thus the humanized variants retain the extinction-promoting properties.

[0299] The humanized HEF-LDT52S variant has been optimized to reduce immunogenicity. This optimized variant maintains high binding activity and stability while preserving biological function.

[0300] References Arijs, I., De Hertogh, G., Lemaire, K., Quintens, R., Van Lommel, L., Van Steen, K., Leemans, P., Cleynen, I., Van Assche, G., Vermeire, S. et al. (2009a). Mucosal gene expression of antimicrobial peptides in inflammatory bowel disease before and after first infliximab treatment. PLOS One 4, e7984. Arijs, I., Li, K., Toedter, G., Quintens, R., Van Lommel, L., VanSteen, K., Leemans, P., De Hertogh, G., Lemaire, K., Ferrante, M. et al. (2009b). Mucosal gene signatures to predict response to infliximab in patients with ulcerative colitis. Gut 58, 1612–1619. Arijs, I., Hertogh, GD, Lemmens, B., Lommel, LV, Bruyn, M. de, Vanhove, W., Cleynen, I., Machiels, K., Ferrante, M., Schuit, F. et al. (2018). Effect of vedolizumab (anti-α4β7-integrin) therapy on histological healing and mucosal gene expression in patients with UC. Gut 67, 43–52. Bannenberg, GL, Chiang, N., Ariel, A., Arita, M., Tjonahen, E., Gotlinger, KH, Hong, S., and Serhan, CN (2005). Molecular circuits of resolution: formation and actions of resolvins and protectins. Journal of Immunology-Baltimore-Medicine, 1950, 174, 4345–4355. Bozaoglu, K., Bolton, K., McMillan, J., Zimmet, P., Jowett, J., Collier, G., Walder, K., and Segal, D. (2007). Chemerin Is a Novel Adipokine Associated with Obesity and Metabolic Syndrome. Endocrinology 148, 4687–4694. Buckley, CD, Gilroy, DW, and Serhan, CN (2014). Proresolving Lipid Mediators and Mechanisms in the Resolution of Acute Inflammation. Immunity 40, 315–327. Cash, JL et al., 2008. Synthetic chemerin-derived peptides suppress inflammation through ChemR23. Journal of Experimental Medicine, 205, 767–775. Ernst, MC and Sinal, CJ (2010). Chemins: at the crossroads of inflammation and obesity. Trends Endocrinol. Metab. TEM 21, 660–667. Goralski, KB, McCarthy, TC, Hanniman, EA, Zabel, BA, Butcher, EC, Parlee, SD, Muruganandan, S., and Sinal, CJ (2007). Chemerin, a novel adipokine that regulates adipogenesis and adipocyte metabolism. Journal of Biochemistry, 282, 28175–28188. Ichim, G. and Tait, SWG (2016). A fate worse than death: apoptosis as an oncogenic process. Nature Review Cancer 16, 539–548. Kaur, J., Adya, R., Tan, BK, Chen, J., and Randeva, HS (2010). Identification of chemerin receptor (ChemR23) in human endothelial cells: Chemerin-induced endothelial angiogenesis. Biochem. Biophys. Res. Commun. 391, 1762–1768. Peyrassol, X., Laeremans, T., Gouwy, M., Lahura, V., Debulpaep, M., Damme, JV, Steyaert, J., Parmentier, M., and Langer, I. (2016). Development by Genetic Immunization of Monovalent Antibodies (Nanobodies) Behaving as Antagonists of the Human ChemR23 Receptor. Journal of Immunology, 196, 2893–2901. Roh, S., Song, S.-H., Choi, K.-C., Katoh, K., Wittamer, V., Parmentier, M., and Sasaki, S. (2007). Chemerin—a new adipokine that modulates adipogenesis via its own receptor. Biochem. Biophys. Res. Commun. 362, 1013–1018. Samson, M., Edinger, AL, Stordeur, P., Rucker, J., Verhasselt, V., Sharron, M., Govaerts, C., Mollereau, C., Vassart, G., Doms, RW et al. (1998). ChemR23 is a putative chemoattractant receptor expressed in monocyte-derived dendritic cells and macrophages and is a coreceptor for SIV and some primary HIV-1 strains. (Eur. J. Immunol.) 28, 1689–1700. Sell, H., Laurencikiene, J., Taube, A., Eckardt, K., Cramer, A., Horrighs, A., Arner, P., and Eckel, J. (2009). Chemerin is a novel adipocyte-derived factor that induces insulin resistance in primary human skeletal muscle cells. Diabetes 58, 2731–2740. Serhan, CN (2014a). Pro-resolving lipid mediators are leads for resolution physiology. Nature 510, 92–101. Serhan, CN (2014b). Pro-resolving lipid mediators are leads for resolution physiology. Nature 510, 92–101. Sulciner, ML, Serhan, CN, Gilligan, MM, Mudge, DK, Chang, J., Gartung, A., Lehner, KA, Bielenberg, DR, Schmidt, B., Dalli, J. et al. (2017). Resolvins suppress tumor growth and enhance cancer therapy. Journal of Experimental Medicine (J. Exp. Med. jem.) 20170681. Watts, SW, Dorrance, AM, Penfold, ME, Rourke, JL, Sinal, CJ, Seitz, B., Sullivan, TJ, Charvat, TT, Thompson, JM, Burnett, R. et al. (2013). Chemins connect fat to arterial contraction. Arterioscler. Thromb. Vasc. Biol. 33, 1320–1328. Wittamer, V., Franssen, J.-D., Vulcano, M., Mirjolet, J.-F., Poul, EL., Migeotte, I., Brézillon, S., Tyldesley, R., Blanpain, C., Detheux, M. et al. (2003). Specific Recruitment of Antigen-presenting Cells by Chemerin, a Novel Processed Ligand from Human Inflammatory Fluids. Journal of Experimental Medicine (J. Exp. Med.) 198, 977–985. Zabel, BA, Rott, A. and Butcher, EC (2015). Leukocyte chemoattractant receptors in human disease pathogenesis. Annual Review of Pathology – Disease Mechanisms (Annu. Rev. Pathol.), 10, 51–81.

Claims

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

35.

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

3. The anti-CMKLR1 antibody or its antigen-binding fragment according to claim 1, wherein the antibody or its antigen-binding fragment specifically binds to an epitope located within a polypeptide comprising the amino acid sequence SEQ ID No: 2 or SEQ ID No: 59 or a polypeptide composed of the amino acid sequences SEQ ID No: 2 or SEQ ID No: 59, or specifically binds to an epitope located within the amino acid sequence SEQ ID No:

60.

4. The anti-CMKLR1 antibody or its antigen-binding fragment according to claim 1, wherein the anti-CMKLR1 antibody or its antigen-binding fragment is a CMKLR1-like agonist of E1, which, compared with the control antibody, can induce phosphorylation of Akt and / or Erk proteins.

5. The anti-CMKLR1 antibody or its antigen-binding fragment according to claim 1, wherein the anti-CMKLR1 antibody or its antigen-binding fragment has the effect of a pro-inflammatory remission factor, which enhances inflammation remission in the subject.

6. The anti-CMKLR1 antibody or its antigen-binding fragment according to claim 1, wherein the antibody or its antigen-binding fragment comprises: a. A heavy chain variable domain comprising SEQ ID NO: 43 or consisting of SEQ ID NO: 43, and a light chain variable domain comprising SEQ ID NO: 53 or consisting of SEQ ID NO: 53; b. A heavy chain variable domain comprising SEQ ID NO: 43 or consisting of SEQ ID NO: 43, and a light chain variable domain comprising SEQ ID NO: 52 or consisting of SEQ ID NO: 52; c. A heavy chain variable domain comprising SEQ ID NO: 43 or consisting of SEQ ID NO: 43, and a light chain variable domain comprising SEQ ID NO: 93 or consisting of SEQ ID NO: 93; d. A heavy chain variable domain comprising SEQ ID NO: 42 or consisting of SEQ ID NO: 42, and a light chain variable domain comprising SEQ ID NO: 52 or consisting of SEQ ID NO: 52; e. A heavy chain variable domain comprising SEQ ID NO: 42 or consisting of SEQ ID NO: 42, and a light chain variable domain comprising SEQ ID NO: 53 or consisting of SEQ ID NO: 53; f. A heavy chain variable domain comprising SEQ ID NO: 41 or consisting of SEQ ID NO: 41, and a light chain variable domain comprising SEQ ID NO: 50 or consisting of SEQ ID NO: 50; or g. A heavy chain variable domain comprising SEQ ID NO: 91 or consisting of SEQ ID NO: 91, and a light chain variable domain comprising SEQ ID NO: 55 or consisting of SEQ ID NO:

55.

7. The anti-CMKLR1 antibody or its antigen-binding fragment according to claim 1, wherein the anti-CMKLR1 antibody or its antigen-binding fragment 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.

8. The anti-CMKLR1 antibody or its antigen-binding fragment according to claim 1, wherein the anti-CMKLR1 antibody or its antigen-binding fragment comprises the following backbone regions: 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.

9. The anti-CMKLR1 antibody or its antigen-binding fragment according to claim 1, wherein the anti-CMKLR1 antibody or its antigen-binding fragment does not activate the β-repressor protein signaling pathway in CMKLR1 positive cells in vitro and / or in vivo.

10. The anti-CMKLR1 antibody or its antigen-binding fragment according to claim 1, wherein the anti-CMKLR1 antibody or its antigen-binding fragment does not exhibit significant depletion of CMKLR1-positive cells in vitro and / or in vivo.

11. The anti-CMKLR1 antibody or its antigen-binding fragment according to claim 1, wherein the anti-CMKLR1 antibody or its antigen-binding fragment does not compete with chemokines for binding to CMKLR1, and / or does not interfere with the binding of chemokines to CMKLR1.

12. The anti-CMKLR1 antibody or its antigen-binding fragment according to claim 1, wherein the anti-CMKLR1 antibody or its antigen-binding fragment is a humanized monoclonal antibody or its antigen-binding fragment, and wherein the antibody heavy chain constant domain is derived from the human IgG1, IgG2, IgG3 or IgG4 heavy chain constant domain.

13. The anti-CMKLR1 antibody or its antigen-binding fragment according to claim 12, wherein the antibody light chain constant domain is derived from the human κ light chain constant region domain.

14. The anti-CMKLR1 antibody or its antigen-binding fragment according to claim 1, wherein the antibody light chain constant domain comprises or is composed of the sequence of SEQ ID No: 79, and wherein the antibody heavy chain constant domain comprises or is composed 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.

15. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment specifically binding to CMKLR1, said antibody or antigen-binding fragment comprising an antibody heavy chain variable domain, said antibody heavy chain variable domain comprising: (i) VHCDR2, said VHCDR2 comprising or consisting of an amino acid sequence listed in SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 11, SEQ ID No: 12 or SEQ ID No: 61, and (ii) VHCDR3, said VHCDR3 comprising or consisting of a mutant sequence listed in SEQ ID No: 13, SEQ ID No: 14, SEQ ID No: 15 or SEQ ID No: 16, or a sequence listed in SEQ ID No: 16, or a sequence listed in SEQ ID No: 16, or a sequence listed in SEQ ID No: 16, or a sequence listed in SEQ ID No:

16. 16 consists of the amino acid sequence listed or its mutant sequence, wherein amino acid residues are substituted, provided that the amino acid residues at positions 1 and 2 of the mutant sequence are L and I, respectively; and The antibody or its antigen-binding fragment specifically binds to an epitope located within a polypeptide comprising the amino acid sequence SEQ ID No: 2 or SEQ ID No: 59 or composed of the amino acid sequences SEQ ID No: 2 or SEQ ID No: 59, or specifically binds to an epitope located within the amino acid sequence SEQ ID No: 60; and The antibody or its antigen-binding fragment thereof competes with antibodies containing a heavy chain variable domain corresponding to SEQ ID No: 37 and a light chain variable domain corresponding to SEQ ID No: 49 for binding to the following polypeptides: polypeptides containing an amino acid sequence of SEQ ID No: 2, SEQ ID No: 59, or SEQ ID No: 60, or polypeptides consisting of an amino acid sequence of SEQ ID No: 2, SEQ ID No: 59, or SEQ ID No:

60.

16. The antibody or antigen-binding fragment thereof according to claim 15, wherein the antibody or antigen-binding fragment thereof comprises the following CDR: - 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.

17. The antibody or antigen-binding fragment thereof according to claim 15, wherein the antibody or antigen-binding fragment thereof comprises the following backbone regions: 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.

18. Use of the antibody or antigen-binding fragment thereof according to claim 1 or 15 in the preparation of a medicament for the prevention or therapeutic treatment of inflammatory diseases, autoimmune diseases, infectious diseases or cancer.

19. The use according to claim 18, wherein - The inflammatory diseases mentioned are selected from acute inflammatory diseases, chronic inflammatory diseases, keratoconjunctivitis, periodontitis, eczema, inflammatory bowel disease, cystic fibrosis, non-alcoholic steatohepatitis, scleroderma and diseases related to antineutrophil cytoplasmic antibodies; - The infectious disease mentioned is sepsis or peritonitis; - The autoimmune diseases mentioned are selected from diabetes, psoriasis, lupus, rheumatoid arthritis, multiple sclerosis, Sjögren's syndrome, celiac disease, vasculitis, and myasthenia gravis; or - The cancers mentioned are selected from liver cancer, breast cancer, colon cancer, colorectal cancer, lung cancer, mesothelioma, and bone marrow cancer.

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