Patent Application
20210147558
The invention pertains to the field of immunotherapy. The present invention provides new anti-chemerin receptor antibodies which have an agonist activity on chemokine like receptor-1 (CMKLR1). The present invention also provides uses of such an antibody in therapy, in particular for treating autoimmune diseases and chronic inflammatory diseases, infectious diseases, cancers, and any condition wherein the resolution phase of inflammation is disrupted or delayed.
The critical role of inflammatory processes in health and diseases has long been recognized. The detailed molecular mechanisms and biological events that regulate the progression and the resolution of inflammation remain of critical interest. Recent investigations have provided strong evidence that the resolution of inflammation is not a passive process, as believed earlier. Resolution of the inflammation is instead a biosynthetically active process, regulated by biochemical mediators and receptors-signaling pathways. Resolution is therefore driven by specialized pro-resolving mediators. Inflammation is a spontaneous mechanism that occurs during an infection, an injury or a traumatism. Inflammation is inevitable and usually salutary, and its response is orchestrated by a delicate balance between positive and negative feedback loops. The inflammation is usually divided in 3 steps: initiation, amplification and resolution.
The initiation step is characterized by a vasodilatation of blood vessel. Resident cells (Dendritic Cells (DCs) and Macrophages) recognize the pathogen infecting the body or danger signals. This step induces the secretion of cytokines, chemokines and the production of pro-inflammatory lipid mediators named Prostaglandins (PG) and leukotrienes. Chemokines return in blood circulation and induce recruitment of cellular actors of innate immunity. The amplification step begins with the recruitment of cells of inflammation of the immune system. The first actors recruited to the site of infection are the Polymorphonuclears (PMN). During the cellular phase the PMN recognize the pathogen and are in charge of its elimination. Other cells travel to the inflamed site for helping the PMN such as DCs and the pro-inflammatory macrophages M1. DCs go in the blood circulation and reach the lymph node to activate the adaptive immunity. Lymphocytes T and B reach the site of inflammation and eradicate the infected cells. When the pathogen is completely eliminated, it's become primordial that inflammation is stopped to prevent chronic inflammation that would become pathogenic for organism. To do so, an active mechanism named Resolution of the inflammation takes place. Resolution is accordingly described as the last step of inflammation.
The resolution process which allows the ending of the inflammatory response is a complex process involving the sequential and chronological engagement of cellular (e.g. granulocytes or macrophages) and chemical (e.g. cytokines or specialized pro-solving mediators or factors) effectors.
A resolution defect can result in an increased penetration of granulocytes at the inflammatory site (measured for example by histology, cytometry or indirect biochemical techniques such as elastase quantification by enzyme immunoassay or molecular quantification by PCR of granulocyte receptor 1), a delay in the apoptosis of such cells (measured for example by cytology using specific antibodies against annexin 5). A defect in the resolution of the inflammation may also result in sustained or increased synthesis of pro-inflammatory cytokines such as TNF-alpha, IL8 or IL12 and a decrease of anti-inflammatory cytokines such as IL-10 (measured by enzyme immunoassay or by PCR), sustained or increased activation of transcription factors involved in the synthesis of inflammatory cytokines such as NF-kappaB (measured for example by nuclear translocation or by Western blot and quantification of the level of degradation of IkappaB). It can also be measured by quantification of specialized pro-resolving mediators (such as lipoxins, resolvins, protectins or maresins) or their precursors (like 17-HDOHE or 14-HDOHE) by mass spectrometry or enzyme immunoassay. A defect of resolution then results in a defect of the synthesis of one or more of these mediators. A resolution defect can also result from a decrease of the expression of the receptors of the resolution molecules (ALX/CMK1R1, GPR32, GPR18) or internalization and processing of those receptors into the cytoplasm or to an overexpression of some receptors of inflammatory cytokines or lipids. These conditions may be measured by histology, cytology or PCR. The resolution defect can also result in a decreased or inhibited switch of M1 to M2 macrophages, a damage in phagocytosis or efferocytosis of the same cells.
It is now clear that a family of chemicals actively promotes resolution and tissue repair of inflammation without compromising host defense in addition to checkpoints activator and/or inhibitors involved in such mechanism. Events at the onset of acute inflammation establish biosynthetic pathways for a series of chemical mediators that could serve as antagonists as well as agonists meaning that they do not merely inhibit inflammation pathways but they strike inflammation leading to the restoration of tissue homeostasis and function. Anti-inflammation and pro-resolution factors are therefore not equivalent (Buckley et al., 2014) (Serhan, 2014a). Failure in the resolution of acute inflammation participates in chronic inflammation development. Anti-inflammatory compounds accordingly refer to inhibitors or blockers of the resolution of inflammation, as do molecules stopping immune extravasation; while pro-resolving factors stimulate and/or activate specific processes such as apoptosis or efferocytosis which initiate or enhance the resolution of the inflammation.
Resolution is initiated shortly after the beginning of the inflammatory response by the PMN, it eliminates the pathogen and in parallel starts the synthesis of specialized pro-resolving mediators (SPM). These SPM are the main actors of the resolution phase. Neutrophils govern the initiation of the resolution phase of inflammation by enabling activation of pro-resolving circuits to ensure safe conclusion of the inflammatory response. In the early stage of resolution, neutrophils undergo a phenotype switch to produce different profiles of lipid mediators depending on the cells and substrates present in the immediate environment. PMN-LT switch into PMN-LO (lipoxygenase) pathway under the action of lipoxin and Resolvins. They are exposed to autacoid gradient initiating phenotypic changes. Lipoxins are generated via biosynthetic route engaged during cell-cell interaction (PMN-5-LO/tissue resident cell—5-LO). It has been observed that wound healing is delayed in neutrophil deletion model, and that neutrophils release proteases that deactivate inflammatory cytokines.
Various molecules are involved in the initiation or the inhibition of the resolution of inflammation. The following molecules are illustrative of such active molecules. COX-2 (Cyclooxygenase-2), a prostaglandin-endoperoxide synthase (PTGS), is an enzyme responsible for formation of prostanoids, including thromboxane and prostaglandins such as prostacyclin, and has a dual role as a contributor to the onset of inflammation, and later as a helper to resolve the process. COX-2 inhibitors may have a benefic effect in the early phase of inflammation. COX-2 inhibitors have also deleterious consequences, such as decreasing early PMN trafficking, disrupting the production of LXA, decreasing macrophage phagocytosis, and reducing PGE2 and LXA. An anti-inflammatory prostaglandin called PGD2/15dPGJ is also involved in the negative feedback, but other prostaglandins, like PGE2, are involved in the positive feedback during the resolution of the inflammation.
G protein-coupled receptors (GPCRs) constitute a vast protein family that encompasses a wide range of functions, including various autocrine, paracrine and endocrine processes. They show considerable diversity at the sequence level, on the basis of which they can be separated into distinct groups. Typically, GPCRs activation engages broad networks of signaling pathways which are mediated by either G proteins or B-arrestin or both.
Chemokine-like receptor 1 (CMKLR1), also known as ChemR23, and chemokine receptor-like 2 (CCRL2) are 7-transmembrane receptors identified by their homology to known G-protein-coupled receptors (AJ Kennedy and AP Davenport, 2018). The Chemokine-like Receptor 1 (CMKLR1; also named Dez in Murine animals), is an orphan G protein—coupled receptor related to GPR-1 (38% overall amino acid identity), C3a receptor (38%), C5a anaphylatoxin receptor (36%) and formyl Met-Leu-Phe receptors (35%). ChemR23 is more distantly related to the chemokine receptors subfamily (Samson et al., 1998). CMKLR1 is expressed on monocytes, on macrophages, on dendritic cells, and NK on cells, as well as on adipocytes and endothelial cells. CMKLR1 expression was also described in many cell populations other than leukocytes, including preadipocytes and adipocytes (Goralski et al., 2007; Roh et al., 2007), skeletal muscle cells (Sell et al., 2009), and endothelial cells (Kaur et al., 2010), and additional roles for the chemerin/CMKLR1 system were proposed in the control of lipid and glucose metabolism (Bozaoglu et al., 2007; Ernst and Sinal, 2010), blood pressure (Watts et al., 2013), and angiogenesis (Kaur et al., 2010).
Recent studies identified ligands for these receptors and their functions have begun to be unveiled. Accordingly, the plasma protein-derived chemoattractant chemerin is a ligand for CMKLR1 and activation of CMKLR1 with chemerin has been shown to induce the migration of macrophages and dendritic cells (DCs) in vitro, suggesting a pro-inflammatory role of Chemerin. In vivo studies using CMKLR-deficient mice suggest on the contrary that these receptors may have an anti-inflammatory role, possibly due to the recruitment of plasmacytoid DCs. The chemerin/CMKLR1 interaction also promotes adipogenesis and angiogenesis.
Chemerin acts as a chemokine during inflammation, to recruit cells at the site of infection. This ligand promotes resolution of inflammation in animal models of acute inflammation by enhancement of PMN apoptosis and M2-dependent efferocytosis (phagocytosis non-phlogistic; without releasing proinflammatory mediators), and by decreasing the DC's migration and the secretion of IL12 (proinflammatory cytokine) (Serhan, 2014b).
Chemerin is present in high amounts in inflammatory fluids, has antimicrobial activities, was shown to attract CMKLR1 expressing leukocytes, and promotes adhesion of macrophages to extracellular matrix proteins (Wittamer et al., 2003). In addition, chemerin was recently discovered to be an adipokine. Secreted by mature adipocytes, it stimulates preadipocytes to differentiation. Increased serum levels of chemerin have been associated with chronic inflammatory diseases, coronary artery disease, the metabolic syndrome, and obesity. There is indication that high chemerin production in obese adipose tissue might contribute to the increased infiltration of macrophages observed in obese adipose tissue leading to low-level inflammation. When Chemerin binds CMKLR1, two signaling pathways are activated: the G-protein signaling pathway and the β-arrestin signaling pathway.
The second ligand of CMKLR1 is the lipid mediator Resolvin E1 (RvE1) that belongs to the Resolvin family. The anti-inflammatory lipid mediator Resolvin E1 inhibits leukocyte infiltration and pro-inflammatory gene expression. These divergent results suggest that CMKLR1 is a multifunctional receptor. It is involved in increasing PMN apoptosis and M2 efferocytosis and is decreasing DC migration and secretion of pro-inflammatory cytokines like IL12. RvE1 is produced by neutrophils and endothelial cells. It is also induced in vitro with aspirin treatment because aspirin activate the COX-2 pathway responsible for the production of RvE1. Furthermore, neutrophil enable the conversion of 18R-HEPE to RvE1. In localized aggressive periodontitis (LAP) patients, it has been observed that macrophages reduce phagocytosis. RvE1 rescues impaired phagocytic activity of LAP macrophages. RvE1 additionally promotes resolution via reducing IL-23 and IL-6 in allergic airways of mice as well as increasing IFN-γ. RvE1 regulates natural killer (NK) cell migration and cytotoxicity. When RvE1 binds CMKLR1, only the G-protein signaling pathway is activated and the B-arrestin pathway is inhibited; and in particular conditions, the B-arresting pathway is inhibited.
Initially, the interest in the chemerin system was focused on its role in inflammation and chemotaxis of immune cells following its discovery in psoriasis disease. Most recently, in connection with its role in inflammation, in obesity, metabolic syndrome, it's potential role in association with cardiovascular functions has been considered, as well as role in reproductive biology. Therefore, the chemerin system is of major interest for its role in the inflammation process, in particular for its role in the resolution of the inflammation. A number of diseases are related to delay or disruption of the resolution process. Most of the specialized pro-resolving factor mediators currently known are derived from polyunsaturated fatty acids, including lipoxins, the resolving family, including E-series resolvins and D-series resolvins, protectins, and maresins. Nonetheless, pro-resolving molecules are difficult to synthesize because of their lipidic nature. Production of pro-resolving molecules in sufficient quantities, for a clinical trial for example, is a burden, and very few SPM have gone through efficient production. Besides that, antibodies specifically targeting G-protein-coupled receptors are difficult to produce. There is therefore a need for molecules having the capability to initiate or enhance the resolution stage of the inflammatory response like pro-resolving factors.
In a first aspect, the invention relates to an anti-CMKLR1 antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic or modified antibody. In the following disclosure, an anti-CMKLR1 compound is considered as being either an anti-CMKLR1 antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic or modified antibody. In a particular embodiment of the invention, said compound is defined by the sequences of its CDRS. In a more particular embodiment of the invention, the anti-CMKLR1 compound is an antibody defined by the sequences of its CDRs and its framework regions (FR). An anti-CMKLR1 compound is a compound which binds specifically to the Chemokin-like receptor 1 (CMKLR1). In the following disclosure, the terms Chemokin-like receptor 1, CMKLR1 and ChemR23 are used interchangeably, and all designate the receptor encoded by gene CMKLR1 in human or cmkIr1 in non-human animals. In a particular embodiment of the invention, the anti-CMKLR1 compound binds specifically to human CMKLR1 or in other words the invention relates to an anti-human CMKLR1 compound. As used herein, the term “CMKLR1” refers to a Chemokin-Like Receptor 1 protein (also designated as chemR23), a member of the G-protein coupled receptor family from a mammal species, preferably a human CMKLR1. A reference sequence of the human CMKLR1 protein, used in the examples of the present application, corresponds to the sequence associated to the Uniprot Accession number Q99788 (SEQ ID No: 1).
In a second aspect, the invention relates to an anti-CMKLR1 antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic or modified antibody defined by at least one functional feature. In a preferred embodiment, said anti-CMKLR1 compound is defined by its capability to inhibit secretion of pro-inflammatory cytokines, in particular IL12, and/or its capability to enhance secretion of anti-inflammatory cytokines, in particular IL10 and/or CCL17. In a more particular embodiment, the anti-CMKLR1 compound inhibits or enhances cytokine secretion by macrophages, in particular M1 and/or M2 macrophages. In a particular embodiment, the anti-CMKLR1 compound of the invention enhances the polarization of macrophages into anti-inflammatory macrophages, in particular M2 macrophages.
In a third aspect, the invention relates to an anti-CMKLR1 antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic that has agonist properties towards Resolvin E1 (RvE1) thereby mimicking the binding of RvE1 to CMKLR1 on CMKLR1 positive cells. “Agonist properties towards RvE1-CMKLR1 interaction” means that the antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic or modified antibody of the invention, which targets the CMKLR1, has the effect of mimicking the binding of RvE1 to CMKLR1, thereby activating the receptor signaling pathway normally activated by RvE1, especially the binding of human RvE1 to human CMKLR1, in particular on dendritic cells, monocytes and macrophages. As a result of binding and activation of the receptor to produce a biological response, the compounds of the invention may lead to the activation of the G protein signaling pathway, in particular Ga, signaling pathway and/or Gao, without activating the 3-arrestin pathway, in particular the compound of the invention may lead to the inhibition of the 3-arrestin pathway. In particular, the binding of a compound according to the invention induces the activation of Akt and/or Erk protein(s) in vitro and/or in vivo. In other words, a Resolvin-E1 like agonist antibody may be defined as an antibody able to bind CMKLR1, and thereby able to induce the phosphorylation of the Akt and/or Erk protein(s), as compared to a control antibody. A control antibody may be an antibody which does not specifically bind CMKLR1. Phosphorylation of a protein may be determined according to methods well known by the skilled artisan, for example by the method disclosed in the examples of the present description.
In a particular embodiment, a compound of the invention is a RvE1-like agonist, i.e. a compound of the invention is an agonist of CMKLR1 signaling pathway induced by RvE1. In other words, the anti-CMKLR1 compound of the invention is an agonist of the interaction between RvE1 and CMKLR1, in particular between human RvE1 and human CMKLR1. In a particular embodiment, the compound of the invention enhances activation of the G protein pathway induced by CMKLR1. In another embodiment, the compound of the invention does not induce the activation of the β-arrestin pathway induced by CMKLR1. In another embodiment, the compound of the invention inhibits the β-arrestin pathway induced by CMKLR1. In another embodiment, since a compound of the invention induces at least one agonist effect of the binding of RvE1 to CMKLR1, and since RvE1 is a pro-resolution factor or pro-resolution mediator, a compound of the invention is a pro-resolution factor or a pro-resolution mediator.
In a particular embodiment, a compound of the invention does not interfere with the binding of Chemerin to CMKLR1. Chemerin is one of the natural ligand of CMKLR1. In other words, a compound according to the invention is not an agonist of the interaction between Chemerin and CMKLR1. The absence of such an agonist capability may be assessed according to the examples of the invention, wherein a competition assay to measure Chemerin-dependent B-arrestin recruitment by CMKLR1 receptor in presence of anti-CMKLR1 antibody of the invention is disclosed. In a preferred embodiment, the anti-CMKLR1 compound of the invention does not compete with Chemerin for the binding to CMKLR1. The absence of competition between an anti-CMKLR1 compound of the invention and chemerin may be determined when, in presence of the CMKLR1 compound of the invention, the binding of Chemerin 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 Chemerin to CMKLR1, under the same experimental conditions but without the presence of the anti-CMKLR1 of the invention. Alternatively, the absence of competition between an anti-CMKLR1 compound of the invention and Chemerin may be determined according to the method illustrated in example 11.
In a particular embodiment, the anti-CMKLR1 compound has the capability in vitro and/or in vivo to activate at least one of the Akt signaling pathway proteins (also known as PI3K-Akt Pathway) and/or Erk signaling pathway proteins, preferably Akt protein and/or Erk protein, preferably both the Akt and the Erk proteins. Activation of a pathway may be assessed according to methods known in the art, and in particular with methods disclosed in the examples of the present invention.
In a particular embodiment, the antibody, antigen-binding fragment thereof, antigen-binding antibody mimetic or modified antibody of the invention enhances RvE1-induced secretion of IL10 cytokine in vitro and/or in vivo, in particular secretion of IL10 cytokine by macrophages. In a particular embodiment, the antibody, antigen-binding fragment thereof, antigen-binding antibody mimetic or modified antibody of the invention enhances RvE1-induced secretion of IL10 and CCL17 cytokines in vitro and/or in vivo, in particular secretion of IL10 and CCL17 by macrophages. In a particular embodiment, the antibody, antigen-binding fragment thereof, antigen-binding antibody mimetic or modified antibody of the invention inhibits the secretion of IL12 cytokine in vitro and/or in vivo, in particular secretion of IL12 by macrophages. The inhibition may be partial, i.e. the level of secretion of IL12 in the presence of the anti-CMKLR1 compound is decreased over a baseline level (i.e. the level without RvE1 or the anti-CMKLR1 compound), or the inhibition may be complete (no secretion of IL12).
As used herein, the term “antibody” comprises polyclonal antibodies, monoclonal antibodies or recombinant antibodies. As used herein, a “monoclonal antibody” is intended to refer to a preparation of antibody molecules to obtain antibodies which share a common heavy chain and common light chain amino acid sequence, in contrast with “polyclonal” antibody preparations which contain a mixture of antibodies of different amino acid sequence. Monoclonal antibodies can be generated by several known technologies like phage, bacteria, yeast or ribosomal display, as well as by classical methods exemplified by hybridoma-derived antibodies. They may also be synthetized using the disclosed amino acid sequences as reference. Thus, the term “monoclonal” is used to refer to all antibodies derived from one nucleic acid clone.
The antibodies of the present invention include recombinant antibodies. As used herein, the term “recombinant antibody” refers to antibodies which are produced, expressed, generated or isolated by recombinant means, such as antibodies which are expressed using a recombinant expression vector transfected into a host cell; antibodies isolated from a recombinant combinatorial antibody library; antibodies isolated from an animal (e.g. a mouse) which is transgenic due to human immunoglobulin genes; or antibodies which are produced, expressed, generated or isolated in any other way in which particular immunoglobulin gene sequences (such as human immunoglobulin gene sequences) are assembled with other DNA sequences. Recombinant antibodies include, for example, chimeric and humanized antibodies.
As used herein, a “chimeric antibody” refers to an antibody in which the sequence of the variable domain derived from the germline of a mammalian species, such as a mouse, have been grafted onto the sequence of the constant domain derived from the germline of another mammalian species, such as a human.
As used herein, a “humanized antibody” refers in a first embodiment to an antibody in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human or humanized framework sequences. In a further embodiment, a “humanized antibody” refers to an antibody wherein at least one CDR and all or part of framework sequences have been humanized.
As used herein, an “antigen-binding fragment of an antibody” means a part of an antibody, i.e. a molecule corresponding to a portion of the structure of the antibody of the invention, that exhibits antigen-binding capability for CMKLR1, possibly in its native form; such fragment especially exhibits the same or substantially the same antigen-binding specificity for said antigen compared to the antigen-binding specificity of the corresponding four-chain antibody. Advantageously, the antigen-binding fragments have a similar binding affinity as the corresponding 4-chain antibodies. However, antigen-binding fragment that have a reduced antigen-binding affinity with respect to corresponding 4-chain antibodies are also encompassed within the invention. The antigen-binding capability can be determined by measuring the affinity between the antibody and the target fragment. These antigen-binding fragments may also be designated as “functional fragments” of antibodies.
Antigen-binding fragments of antibodies are fragments which comprise their hypervariable domains designated CDRs (Complementary Determining Regions) or part(s) thereof encompassing the recognition site for the antigen, i.e. the extracellular domain of CMKLR1, in particular the third loop of the extracellular domain of CMKLR1 (designated EL3), thereby defining antigen recognition specificity. EL3 is localized between amino acid residue 283 and amino acid residue 300 of SEQ ID No: 1. EL3 corresponds to the amino acid residues of SEQ ID No: 2. EL3 is also comprised within the polypeptide of SEQ ID No: 18.
Each Light and Heavy chain variable domains (respectively VL and VH) of a four-chain immunoglobulin has three CDRs designated VLCDR1, VLCDR2 and VLCDR3 for the light chain variable domain; and VHCDR1, VHCDR2, VHCDR3 for the heavy chain variable domain. Each Light chain and Heavy chain variable domains of a four-chain immunoglobulin has four framework regions (FR), designated LFR1, LFR2, LFR3 and LFR4 for the light chain variable domain; and HFR1, HFR2, HFR3 and HFR4 for the heavy chain variable domain.
The skilled person is able to determine the location of the various regions/domains of antibodies by reference to the standard definitions in this respect set forth, including a reference numbering system, a reference to the numbering system of KABAT or by application of the IMGT “collier de perle” algorithm. In this respect, for the definition of the sequences of the invention, it is noted that the delimitation of the regions/domains may vary from one reference system to another. Accordingly, the regions/domains as defined in the present invention encompass sequences showing variations in length or localization of the concerned sequences within the full-length sequence of the variable domains of the antibodies, of approximately +/−10%.
Based on the structure of four-chain immunoglobulins, antigen-binding fragments can thus be defined by comparison with sequences of antibodies in the available databases and prior art, and especially by comparison of the location of the functional domains in these sequences, noting that the positions of the framework and constant domains are well defined for various classes of antibodies, especially for IgGs, in particular for mammalian IgGs. Such comparison also involves data relating to 3-dimensional structures of antibodies.
For illustration purpose of specific embodiments of the invention, antigen binding fragments of an antibody that contain the variable domains comprising the CDRs of said antibody encompass Fv, dsFv, scFv, Fab, Fab′, F(ab′)2. Fv fragments consist of the VL and VH domains of an antibody associated together by hydrophobic interactions; in dsFv fragments, the VH:VL heterodimer is stabilised by a disulphide bond; in scFv fragments, the VL and VH domains are connected to one another via a flexible peptide linker thus forming a single-chain protein. Fab fragments are monomeric fragments obtainable by papain digestion of an antibody; they comprise the entire L chain, and a VH-CH1 fragment of the H chain, bound together through a disulfide bond. The F(ab′)2 fragment can be produced by pepsin digestion of an antibody below the hinge disulfide; it comprises two Fab′ fragments, and additionally a portion of the hinge region of the immunoglobulin molecule. The Fab′ fragments are obtainable from F(ab′)2 fragments by cutting a disulfide bond in the hinge region. F(ab′)2 fragments are divalent, i.e. they comprise two antigen binding sites, like the native immunoglobulin molecule; on the other hand, Fv (a VHVL dimmer constituting the variable part of Fab), dsFv, scFv, Fab, and Fab′ fragments are monovalent, i.e. they comprise a single antigen-binding site. These basic antigen-binding fragments of the invention can be combined together to obtain multivalent antigen-binding fragments, such as diabodies, tribodies or tetrabodies. These multivalent antigen-binding fragments are also part of the present invention.
As used herein, the term modified antibody includes “bispecific” antibodies and refers to antibodies that recognize two different antigens by virtue of possessing at least one region (e.g. derived from a variable region of a first antibody) that is specific for a first antigen, and at least a second region (e.g. derived from a variable region of a second antibody) that is specific for a second antigen. A bispecific antibody specifically binds to two target antigens and is thus one type of multispecific antibody. Multispecific antibodies, which recognize two or more different antigens, can be produced by recombinant DNA methods or include, but are not limited to, antibodies produced chemically by any convenient method. Bispecific antibodies include all antibodies or conjugates of antibodies, or polymeric forms of antibodies which are capable of recognizing two different antigens. Bispecific antibodies include antibodies that have been reduced and reformed so as to retain their bivalent characteristics and to antibodies that have been chemically coupled so that they can have several antigen recognition sites for each antigen such as 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), kih (knobs into holes).
Antigen-binding antibody mimetics are organic compounds that specifically bind antigens, but that are not structurally related to antibodies. They are usually artificial peptides or small proteins with a molar mass of about 3 to 20 kDa. Nucleic acids and small molecules are sometimes considered antibody mimetics as well, but not artificial antibodies, antibody fragments and fusion proteins composed from these. Common advantages over antibodies are better solubility, tissue penetration, stability towards heat and enzymes, and comparatively low production costs. Antibody mimetics are being developed as therapeutic and diagnostic agents. Antigen-binding antibody mimetics may also be selected among the group comprising affibodies, affilins, affimers, affitins, DARPins, and Monobodies.
An antigen-binding antibody mimetic is more preferentially selected from the groups comprising affitins and anticalins. Affitins are artificial proteins with the ability to selectively bind antigens. They are structurally derived from the DNA binding protein Sac7d, found in Sulfolobus acidocaldarius, a microorganism belonging to the archaeal domain. By randomizing the amino acids on the binding surface of Sac7d, e.g. by generating variants corresponding to random substitutions of 11 residues of the binding interface of Sac7d, an affitin library may be generated and subjecting the resulting protein library to rounds of ribosome display, the affinity can be directed towards various targets, such as peptides, proteins, viruses and bacteria. Affitins are antibody mimetics and are being developed as tools in biotechnology. They have also been used as specific inhibitors for various enzymes (Krehenbrink et al., J. mol. Biol., 383:5, 2008). The skilled person may readily develop affitins with the required binding properties using methods know in the art, in particular as disclosed in patent application WO2008068637 and the above-cited publication, in particular the generation of phage display and/or ribosome display libraries and their screening using an antigen as disclosed herein. Anticalins are artificial proteins that are able to bind to antigens, either to proteins or to small molecules. They are antibody mimetics derived from human lipocalins which are a family of naturally binding proteins. Anticalins are about eight times smaller with a size of about 180 amino acids and a mass of about 20 kDa (Skerra, Febs J., 275:11, 2008). Anticalin phage display libraries have been generated which allow for the screening and selection, in particular of anticalins with specific binding properties. The skilled person may readily develop anticalins with the required binding properties using methods know in the art, in particular as disclosed in EP patent EP1270725 B1, US patent U.S. Pat. No. 8,536,307 B2, Schlehuber and Skerra, Biophys. Chem., 96:2-3, 2002 and the above-cited publication, in particular the generation of phage display and/or ribosome display libraries and their screening using an antigen as disclosed herein. Anticalins and affitins may both be produced in a number of expression system comprising bacterial expression systems. Thus, the invention includes the use of affitins, anticalins and other similar antibody mimetics with the features of the antibodies described herein, in particular with regard to their binding capability to CMKLR1, to their agonist capability towards the binding between RvE1 and CMKLR1, their capability to induce or inhibit secretion of particular cytokines as described herein, to their use in the treatment or the prevention of a disease as described herein, all of which are contemplated as mimetics according to the invention.
As used herein, a “modified antibody” refers to antibodies the amino sequence of which has been modified by mutation of at least one amino acid residue. Accordingly, “modified antibody” encompasses chimeric antibodies or humanized antibodies as defined herein, “modified antibody” may also correspond to a molecule comprising an antibody or an antigen-binding fragment thereof, wherein said monoclonal antibody or functional fragment thereof is associated with a functionally different molecule. A modified antibody of the invention may be either a fusion chimeric protein or a conjugate resulting from any suitable form of attachment including covalent attachment, grafting, chemical bonding with a chemical or biological group or with a molecule, such as a PEG polymer or another protective group or molecule suitable for protection against proteases cleavage in vivo, for improvement of stability and/or half-life of the antibody or functional fragment. With similar techniques, especially by chemical coupling or grafting, a modified antibody can be prepared with a biologically active molecule, said active molecule being for example chosen among toxins, in particular Pseudomonas exotoxin A, the A-chain of plant toxin ricin or saporin toxin, especially a therapeutic active ingredient, a vector (including especially a protein vector) suitable for targeting the antibody or functional fragment to specific cells or tissues of the human body, or it may be associated with a label or with a linker, especially when fragments of the antibody are used. PEGylation of the antibody or functional fragments thereof is a particular interesting embodiment as it improves the delivery conditions of the active substance to the host, especially for a therapeutic application. PEGylation can be site specific to prevent interference with the recognition sites of the antibodies or functional fragments, and can be performed with high molecular weight PEG. PEGylation can be achieved through free cysteine residues present in the sequence of the antibody or functional fragment or through added free Cysteine residues in the amino sequence of the antibody or functional fragment. According to the present invention, when the term “antibody” is used, it means either an antibody, an antigen-binding fragment thereof, an antigen-binding antibody mimetic or a modified antibody.
“Humanized” forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)2 or other target-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all the CDR regions correspond to those of a non-human immunoglobulin and/or humanized version of those; and all or substantially all of the FR regions are those of a human immunoglobulin template sequence. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin template chosen. In a particular embodiment, the 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, in particular a Fc region.
The terms “Specifically binding” and “specifically bind to” refer to the ability of an antibody, antigen-binding fragment thereof, antigen-binding antibody mimetic or a modified antibody according to the invention to bind to CMKLR1 with an affinity of at least 1×10−6 M, 1×10−7 M, 1×10−8 M, 1×10−9 M, 1×10−10 M, 1×10−11 M, 1×10−12 M, or more, and/or to bind to CMKLR1 with an affinity which is at least two-fold greater to its affinity for a non-specific target (e.g. another protein than CMKLR1). The affinity may be assessed according to various methods well known from those skilled in the art. These methods include but are not limited to biosensors such as Biacore analysis, Blitz analysis and Scatchard plot.
The term “therapeutically effective amount” is used to refer to an amount of any given compound as defined herein sufficient for at least the improvement of the clinical or physiological condition of a treated patient. The therapeutically effective amount of the antibody, antigen-binding fragment thereof, antigen-binding antibody mimetic or modified antibody according to the invention to be administered is governed by considerations such as the disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.
All the embodiments disclosed herein for antibodies or antigen-binding fragments thereof are transposed mutatis mutandis to the macromolecules of the invention, in particular to antigen-binding antibody mimetics and to modified antibodies.
In a first aspect, the invention relates to an antibody, an antigen-binding fragment thereof, an antigen-binding antibody mimetic or a modified antibody which specifically binds to CMKLR1, in particular to the EL3 loop of CMKLR1, in particular to a polypeptide comprising the amino acid residues of SEQ ID No: 2, and more particularly to an epitope localized within SEQ ID No: 2 or within the EL3 loop of CMKLR1 and consisting of the sequence of amino acid residues “AMPGS” (SEQ ID No: 152) and comprising:
In another aspect, the invention relates to an anti-CMKLR1 compound selected from the group of an antibody or an antigen-binding fragment thereof or an antigen-binding antibody mimetic or a chimeric or humanized antibody, which specifically binds to CMKLR1, said compound comprising:
In another aspect, the invention relates to an anti-CMKLR1 compound selected from the group of an antibody or an antigen-binding fragment thereof or an antigen-binding antibody mimetic or a chimeric or humanized antibody, which specifically binds to CMKLR1, said compound comprising an antibody heavy chain variable domain comprising a VHCDR3 comprising or consisting of the amino acid sequences set forth in SEQ ID No: 8 or a mutated sequence thereof wherein amino acid residue(s) is(are) substituted providing amino acid residues at position 1 and 2 of SEQ ID No: 8 are respectively L and I or L; and
wherein the anti-CMKLR1 compound specifically binds to an epitope localized within the third extra-cellular loop (EL3) of CMKLR1, in particular wherein the compound binds specifically to a polypeptide comprising or consisting of amino acid residues of sequence SEQ ID No: 2 or SEQ ID No: 152; and
wherein the anti-CMKLR1 compound is a Resolvin E1-like agonist of CMKLR1, in particular wherein the anti-CMKLR1 compound is a pro-resolution factor, in particular on myeloid cell lineages; and
wherein said compound competes with an antibody comprising the heavy variable domain corresponding to SEQ ID No: 9 and the light chain variable domain corresponding to SEQ ID No: 16, more particularly with antibody 2G1, for the binding to a polypeptide comprising or consisting of amino acid residues of sequence SEQ ID No: 2 or SEQ ID No: 152 or to a polypeptide comprising or consisting of the third loop (EL3) of the extracellular domain of CMKLR1.
In another aspect, the invention relates to an anti-CMKLR1 compound selected from the group of an antibody or an antigen-binding fragment thereof or an antigen-binding antibody mimetic or a chimeric or humanized antibody, which specifically binds to CMKLR1, said compound comprising:
In another aspect, the invention relates to an antibody, antigen-binding fragment thereof, antigen-binding antibody mimetic or chimeric or humanized antibody which specifically binds to CMKLR1, in particular to human CMKLR1, comprising:
In a particular embodiment, the invention relates to an antibody, antigen-binding fragment thereof, antigen-binding antibody mimetic or modified antibody which specifically binds to CMKLR1, in particular to human CMKLR1, comprising:
In a particular embodiment of the invention, the antibody heavy chain variable domain comprises VHCDR1 of the amino acid sequence set forth in SEQ ID No: 4; VHCDR2 of the amino acid sequence set forth in SEQ ID No: 67 and VHCDR3 of the amino acid sequence set forth in SEQ ID No: 144 or SEQ ID No: 148, more particularly of the amino acid sequence set forth in SEQ ID No: 144.
In a particular embodiment of the invention, the antibody heavy chain variable domain comprises VHCDR1 of the amino acid sequence set forth in SEQ ID No: 4; VHCDR2 of the amino acid sequence set forth in SEQ ID No: 70 and VHCDR3 of the amino acid sequence set forth in SEQ ID No: 144 or SEQ ID No: 148, more particularly of the amino acid sequence set forth in SEQ ID No: 144.
In a particular embodiment of the invention, the antibody heavy chain variable domain comprises VHCDR1 of the amino acid sequence set forth in SEQ ID No: 4; VHCDR2 of the amino acid sequence set forth in SEQ ID No: 72 and VHCDR3 of the amino acid sequence set forth in SEQ ID No: 144 or SEQ ID No: 148, more particularly of the amino acid sequence set forth in SEQ ID No: 144.
In a particular embodiment of the invention, the antibody heavy chain variable domain comprises VHCDR1 of the amino acid sequence set forth in SEQ ID No: 62; VHCDR2 of the amino acid sequence set forth in SEQ ID No: 67 and VHCDR3 of the amino acid sequence set forth in SEQ ID No: 144 or SEQ ID No: 148, more particularly of the amino acid sequence set forth in SEQ ID No: 144.
In a particular embodiment of the invention, the antibody heavy chain variable domain comprises VHCDR1 of the amino acid sequence set forth in SEQ ID No: 62; VHCDR2 of the amino acid sequence set forth in SEQ ID No: 70 and VHCDR3 of the amino acid sequence set forth in SEQ ID No: 144 or SEQ ID No: 148, more particularly of the amino acid sequence set forth in SEQ ID No: 144.
In a particular embodiment of the invention, the antibody heavy chain variable domain comprises VHCDR1 of the amino acid sequence set forth in SEQ ID No: 62; VHCDR2 of the amino acid sequence set forth in SEQ ID No: 72 and VHCDR3 of the amino acid sequence set forth in SEQ ID No: 144 or SEQ ID No: 148, more particularly of the amino acid sequence set forth in SEQ ID No: 144.
In a particular embodiment of the invention, the antibody heavy chain variable domain comprises VHCDR1 of the amino acid sequence set forth in SEQ ID No: 63; VHCDR2 of the amino acid sequence set forth in SEQ ID No: 67 and VHCDR3 of the amino acid sequence set forth in SEQ ID No: 144 or SEQ ID No: 148, more particularly of the amino acid sequence set forth in SEQ ID No: 144.
In a particular embodiment of the invention, the antibody heavy chain variable domain comprises VHCDR1 of the amino acid sequence set forth in SEQ ID No: 63; VHCDR2 of the amino acid sequence set forth in SEQ ID No: 70 and VHCDR3 of the amino acid sequence set forth in SEQ ID No: 144 or SEQ ID No: 148, more particularly of the amino acid sequence set forth in SEQ ID No: 144.
In a particular embodiment of the invention, the antibody heavy chain variable domain comprises VHCDR1 of the amino acid sequence set forth in SEQ ID No: 63; VHCDR2 of the amino acid sequence set forth in SEQ ID No: 72 and VHCDR3 of the amino acid sequence set forth in SEQ ID No: 144 or SEQ ID No: 148, more particularly of the amino acid sequence set forth in SEQ ID No: 144.
In a particular embodiment of the invention, the antibody light chain variable domain comprises VLCDR1 of the amino acid sequence set forth in SEQ ID No: 77; VLCDR2 of the amino acid sequence set forth in SEQ ID No: 14 and VLCDR3 of the amino acid sequence set forth in SEQ ID No: 15.
In a particular embodiment of the invention, the antibody light chain variable domain comprises VLCDR1 of the amino acid sequence set forth in SEQ ID No: 77; VLCDR2 of the amino acid sequence set forth in SEQ ID No: 14 and VLCDR3 of the amino acid sequence set forth in SEQ ID No: 89.
In a particular embodiment of the invention, the antibody light chain variable domain comprises VLCDR1 of the amino acid sequence set forth in SEQ ID No: 77; VLCDR2 of the amino acid sequence set forth in SEQ ID No: 81 and VLCDR3 of the amino acid sequence set forth in SEQ ID No: 15.
In a particular embodiment of the invention, the antibody light chain variable domain comprises VLCDR1 of the amino acid sequence set forth in SEQ ID No: 77; VLCDR2 of the amino acid sequence set forth in SEQ ID No: 81 and VLCDR3 of the amino acid sequence set forth in SEQ ID No: 89.
In a particular embodiment of the invention, the antibody light chain variable domain comprises VLCDR1 of the amino acid sequence set forth in SEQ ID No: 77; VLCDR2 of the amino acid sequence set forth in SEQ ID No: 84 and VLCDR3 of the amino acid sequence set forth in SEQ ID No: 15.
In a particular embodiment of the invention, the antibody light chain variable domain comprises VLCDR1 of the amino acid sequence set forth in SEQ ID No: 77; VLCDR2 of the amino acid sequence set forth in SEQ ID No: 84 and VLCDR3 of the amino acid sequence set forth in SEQ ID No: 89.
In a particular embodiment of the invention, the antibody, or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody is a humanized antibody of a murine antibody, in particular wherein at least one framework region (FR1 and/or FR2 and/or FR3 and/or FR4) of the antibody light chain and/or heavy chain variable domain is derived from human chain framework regions, said light chain and/or heavy chain further comprising any combination of CDRs described herein, or wild type CDRs.
The wild type antibodies, referenced in the present disclosure as 2G1 and 2G4 (having the same variable region but with a different heavy chain constant region) have been defined and synthetized starting from multiple options in the amino acid sequence of the third CDR of the heavy chain. Several putative antibody sequences have accordingly been generated, synthetized and tested for their binding and for their biological properties. Within the plurality of newly synthetized antibodies, the presence of the sequence of amino acid residues “5′ RLIY 3′” or “5′ RLLY 3′ ” within the third CDR (according to IMGT) of the heavy chain variable domain has been determined as being highly implicated in the agonist property of the antibody towards the RvE1-CMKLR1 interaction. Antibodies having a different amino acid residue sequence (like “5′ RIIY 3′”; “5′ RILY 3′”) showed less appreciable capability to be agonists of the RvE1 binding to CMKLR1.
The wild type anti-CMKLR1 antibodies synthetized (referenced 2G1 and 4G1 in the present disclosure) are therefore a chimeric antibody with murine variable regions and human constant regions (human IgG1 for 2G1 or human IgG4 for 2G4). 2G1 has the following amino acid sequences in its variable sequences:
SSYG
MSWVRQTPDRRLELVATINRYGGSTYYPDSVKGRFTISRDNAKNTL
wherein the signal peptide is represented in lower case, the CDRs are in bold characters (according to Kabat) or underlined (according to IMGT). Full sequence of the heavy chain corresponds to SEQ ID No: 10, with the signal peptide, while SEQ ID No: 9 corresponds to the heavy chain without the signal peptide. CDR1, CDR2 and CDR3 according to Kabat correspond to SEQ ID No: 4, SEQ ID No: 6 and SEQ ID No: 144 respectively; while CDR1, CDR2 and CDR3 according to IMGT correspond to SEQ ID No: 3, SEQ ID No: 5 and SEQ ID No: 7 respectively. CDR3 may be modified by substituting the amino acid residue ILE in second position by the amino acid residue LEU (second position according to Kabat numbering) leading to a heavy chain of sequence:
SSYG
MSWVRQTPDRRLELVATINRYGGSTYYPDSVKGRFTISRDNAKNTL
SF
MHWYQQKSGTSPKRWIYDTTKLTSGVPARFSGSGSGTFYSLTISSMEA
wherein the signal peptide is represented in lower case, the CDRs are in bold characters (according to Kabat) or underlined (according to IMGT). Full sequence of the light chain corresponds to SEQ ID No: 17, with the signal peptide, while SEQ ID No: 16 corresponds to the heavy chain without the signal peptide. CDR1, CDR2 and CDR3 according to Kabat correspond to SEQ ID No: 12, SEQ ID No: 14 and SEQ ID No: 15 respectively; while CDR1, CDR2 and CDR3 according to IMGT correspond to SEQ ID No: 11, SEQ ID No: 13 and SEQ ID No: 15 respectively.
As known in the art, and as exemplified with the present chimeric antibody, a heavy chain variable domain and a light chain variable domain both comprise 3 CDRs (CDR1, CDR2 and CDR3 from 5′ end to 3′ end respectively) and 4 framework regions (FR1, FR2, FR3 and FR4 from 5′ end to 3′ end respectively). Humanization of the murine antibody may consist of humanizing at least one framework region within the light chain variable region or within the heavy chain variable region or both. In a particular embodiment, several framework regions may be humanized, in particular within the heavy chain variable region and within the light chain variable region. The wild type CDRs may be conserved, but the CDRs may also be replaced by the CDRs already described herein. Hence, the anti-CKLMR1 compound according to the invention may comprise at least 1, or at least 2, or at least 3 or at least 4, or at least 5, or 6 wild type CDRs when the framework regions are humanized. In other words, the anti-CMKLR1 compound is a humanized version of the parental chimeric antibody 2G1, wherein at least 1 framework region is humanized, in particular wherein at least 1 framework region and at least 1 CDR are humanized. In a particular embodiment of the invention, the variable regions of the antibody may be associated with antibody constant regions, for example the constant regions set forth in SEQ ID No: 134 (encoded by the nucleotide sequence of SEQ ID No: 133); SEQ ID No: 136 (encoded by the nucleotide sequence of SEQ ID No: 135); SEQ ID No: 138 (encoded by the nucleotide sequence of SEQ ID No: 137); SEQ ID No: 139; SEQ ID No: 140 and SEQ ID No: 141. Such a combination is illustrated with the light chain of SEQ ID No: 142 and of the heavy chain of SEQ ID No: 143.
In particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises at least one humanized framework region within its antibody heavy chain variable domain, wherein:
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises at least one humanized framework region within its antibody light chain variable domain, wherein:
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises at least one humanized framework region within its antibody light chain variable region and within its antibody heavy chain variable region, wherein:
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises:
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises:
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises:
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises:
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises:
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises:
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises:
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises:
In a particular embodiment, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises a humanized antibody light chain variable domain and a humanized antibody heavy chain variable domain; wherein:
It should be noted that these combinations of CDRs (VHCDRs and VLCDRs) may be combined with the humanized framework regions described herein, in particular with the following framework regions:
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises a heavy chain variable domain comprising or consisting of the amino acid sequences set forth in SEQ ID No: 20; SEQ ID No: 21; SEQ ID No: 22; SEQ ID No: 23; 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; SEQ ID No: 33; SEQ ID No: 34; SEQ ID No: 35; SEQ ID No: 36; SEQ ID No: 37; SEQ ID No: 38; SEQ ID No: 39; SEQ ID No: 40; SEQ ID No: 41 or SEQ ID No: 42; in particular the heavy chain variable domain comprises or consists of the amino acid sequences set forth in SEQ ID No: 20; SEQ ID No: 21; SEQ ID No: 22; SEQ ID No: 23; SEQ ID No: 28: SEQ ID No; 29; SEQ ID No: 30; SEQ ID No: 36; SEQ ID No: 37; SEQ ID No: 38; or SEQ ID No: 39.
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises a light chain variable domain comprising or consisting of the amino acid sequences set forth in SEQ ID No: 43; SEQ ID No: 44; SEQ ID No: 45; SEQ ID No: 46; SEQ ID No: 47; SEQ ID No: 48; 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: 59; SEQ ID No: 60 or SEQ ID No: 61; in particular the heavy chain variable domain comprises or consists of the amino acid sequences set forth in SEQ ID No: 43; SEQ ID No: 44; SEQ ID No: 45; SEQ ID No: 46; SEQ ID No: 47; SEQ ID No: 48; SEQ ID No: 50; SEQ ID No: 51: SEQ ID No: 52; SEQ ID No: 53; SEQ ID No: 54; SEQ ID No: 56; SEQ ID No: 57; SEQ ID No: 58; SEQ ID No: 59 or SEQ ID No: 60.
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises:
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises a heavy chain variable domain of SEQ ID No: 10 and a light chain variable domain of SEQ ID No: 17.
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises:
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises a heavy chain variable domain wherein HFR1 comprises or consists of amino acid sequence set forth in SEQ ID No: 19 (E/Q VQLV A/E/Q SG GINS G L/E V/L Q/K P/K PG G/A S L/V K/R/V L/V SC A/K AS).
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises a heavy chain variable domain wherein HFR2 comprises or consists of amino acid sequence set forth in SEQ ID No: 125 (WVR Q/A TP D/G R/K R/G/Q LELVA).
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises a heavy chain variable domain wherein HFR3 comprises or consists of amino acid sequence set forth in SEQ ID No: 126 (R FN TN I S/T RDN NS K/T/V N/S TLY L/M Q/E M/L/I SSL K/R S/A EDTA MN YYCPR).
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises a heavy chain variable domain wherein HFR4 comprises or consists of amino acid sequence set forth in SEQ ID No: 127 (WGQGT T/L LN TVSS).
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises a heavy chain variable domain wherein LFR1 comprises or consists of amino acid sequence set forth in SEQ ID No: 128 (Q/A/E I V/Q LTQSP A/S/D I/S/F/T M/L/Q S A/S/L SN P/V/T G/P E/D/K K/R V/A T M/I/L TC).
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises a heavy chain variable domain wherein LFR2 comprises or consists of amino acid sequence set forth in SEQ ID No: 129 (WYQQK S/P G/D T/K S/A P K/R RWIY).
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises a heavy chain variable domain wherein LFR3 comprises or consists of amino acid sequence set forth in SEQ ID No: 130 (G V/I P NS RFSGSGSGT F/D Y S/T LTI S/N S M/L E/Q A/P ED NF A TN YYC).
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises a heavy chain variable domain wherein LFR4 comprises or consists of amino acid sequence set forth in SEQ ID No: 131 (FG P/G GTK LN E L/I KR).
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises:
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises:
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises:
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises:
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises:
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises:
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises:
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises:
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises:
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises:
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises:
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises:
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises:
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises:
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises:
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises:
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises:
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises:
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises:
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises:
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises:
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises:
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises:
The invention relates to any of the above defined compound of the invention, for use in the prevention and/or the treatment of a disease wherein the resolution of inflammation is delayed or disrupted, and/or a disease selected from the group of inflammatory diseases, in particular acute inflammatory diseases, chronic inflammatory diseases such as asthma, keratoconjunctivitis, periodontal disease, eczema, inflammatory bowel disease, in particular Crohn's disease or colitis, in particular ulcerative colitis or spontaneous colitis; autoimmune diseases such as diabetes, in particular type I diabetes, psoriasis, lupus, rheumatoid arthritis, multiple sclerosis, Sjögren's syndrome, celiac disease, vasculitis, myasthenia gravis; infection diseases such as sepsis, peritonitis; degenerative diseases; wound healing disorders, dry eye syndrome; cancer diseases, in particular solid and liquid cancers, metastatic cancers, in particular carcinoma, in particular mammary carcinoma or colon carcinoma, or lung cancer or myeloid cancer, in particular leukemia, in particular a cancer wherein cancer cells express CMKLR1 or where the microenvironment of the tumor is invaded by cells expressing or overexpressing CMKLR1. In a particular embodiment, the invention relates to any of the above defined compound of the invention, for use in the prevention and/or the treatment of a disease wherein the resolution of inflammation is delayed or disrupted, and/or a disease selected from the group of inflammatory diseases, in particular acute inflammatory diseases, chronic inflammatory diseases such as asthma, keratoconjunctivitis, periodontal disease, eczema, inflammatory bowel disease, in particular Crohn's disease or colitis, in particular ulcerative colitis or spontaneous colitis; autoimmune diseases such as diabetes, in particular type I diabetes, psoriasis, lupus, rheumatoid arthritis, multiple sclerosis, Sjögren's syndrome, celiac disease, vasculitis, myasthenia gravis; infection diseases such as sepsis, peritonitis; degenerative diseases; wound healing disorders and dry eye syndrome. In another particular embodiment of the invention, the invention relates to any of the above defined compound of the invention, for use in the prevention and/or the treatment of a cancer, in particular solid and liquid cancers, metastatic cancers, in particular carcinoma, in particular mammary carcinoma or colon carcinoma, or lung cancer or myeloid cancer, in particular leukemia, in particular a cancer wherein cancer cells express CMKLR1 or where the microenvironment of the tumor is invaded by cells expressing or overexpressing CMKLR1.
In a particular embodiment, the invention relates to an anti-CMKLR1 compound selected from the group of an antibody, or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody, which specifically binds to CMKLR1, said compound comprising:
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises:
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises a heavy chain variable domain of SEQ ID No: 10 and a light chain variable domain of SEQ ID No: 17;
for use in the prevention and/or the treatment of a disease wherein the resolution of inflammation is delayed or disrupted, and/or a disease selected from the group of inflammatory diseases, in particular acute inflammatory diseases, chronic inflammatory diseases such as asthma, keratoconjunctivitis, periodontal disease, eczema, inflammatory bowel disease, in particular Crohn's disease or colitis, in particular ulcerative colitis or spontaneous colitis; autoimmune diseases such as diabetes, in particular type I diabetes, psoriasis, lupus, rheumatoid arthritis, multiple sclerosis, Sjögren's syndrome, celiac disease, vasculitis, myasthenia gravis; infection diseases such as sepsis, peritonitis; degenerative diseases; wound healing disorders, dry eye syndrome; cancer diseases, in particular solid and liquid cancers, metastatic cancers, in particular carcinoma, in particular mammary carcinoma or colon carcinoma, or lung cancer or myeloid cancer, in particular leukemia, in particular a cancer wherein cancer cells express CMKLR1 or where the microenvironment of the tumor is invaded by cells expressing or overexpressing CMKLR1. In a particular embodiment, the invention relates to any of the above defined compound of the invention, for use in the prevention and/or the treatment of a disease wherein the resolution of inflammation is delayed or disrupted, and/or a disease selected from the group of inflammatory diseases, in particular acute inflammatory diseases, chronic inflammatory diseases such as asthma, keratoconjunctivitis, periodontal disease, eczema, inflammatory bowel disease, in particular Crohn's disease or colitis, in particular ulcerative colitis or spontaneous colitis; autoimmune diseases such as diabetes, in particular type I diabetes, psoriasis, lupus, rheumatoid arthritis, multiple sclerosis, Sjögren's syndrome, celiac disease, vasculitis, myasthenia gravis; infection diseases such as sepsis, peritonitis; degenerative diseases; wound healing disorders and dry eye syndrome. In another particular embodiment of the invention, the invention relates to any of the above defined compound of the invention, for use in the prevention and/or the treatment of a cancer, in particular solid and liquid cancers, in particular carcinoma, in particular mammary carcinoma or colon carcinoma, or myeloid cancer, in particular leukemia, in particular a cancer wherein cancer cells express CMKLR1 or where the microenvironment of the tumor is invaded by cells expressing or overexpressing CMKLR1.
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises:
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody comprises:
In a particular embodiment of the invention, the antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic or modified antibody has:
In another aspect, the invention relates to an anti-CMKLR1 compound as defined herein, which enhances the secretion of anti-inflammatory cytokines, in particular the secretion of 110, in particular by macrophages. In another aspect, the invention relates to an anti-CMKLR1 compound as defined herein, which enhances the secretion of anti-inflammatory cytokines, in particular the secretion of CCL17, in particular by macrophages. In another aspect, the invention relates to an anti-CMKLR1 compound as defined herein, which enhances the secretion of anti-inflammatory cytokines, in particular the secretion of I10 and/or CCL17, in particular by macrophages. The invention also relates to an anti-CMKLR1 compound as defined herein, which inhibits the secretion of pro-inflammatory cytokines, in particular IL12, in particular by macrophages. The cytokine secretion may be assessed by method known in the art, or by methods disclosed in the examples of the present invention.
In another aspect, the invention relates to an anti-CMKLR1 compound as defined herein, which inhibits the proliferation and/or the activation of dendritic cells, in particular human dendritic cells. Proliferation and/or activation of dendritic cells may be assessed by the method disclosed in the examples of the present invention.
In another aspect, the invention relates to a composition comprising an anti-CMKLR1 compound as described herein, in particular a pharmaceutical composition comprising an anti-CMKLR1 compound according to the invention and a further therapeutic agent, or pharmaceutical acceptable carrier. In a particular embodiment, the invention relates to a composition comprising an anti-CMKLR1 compound according to the invention and a therapeutic agent selected from the group consisting of immunomodulatory agent, immune checkpoint blocker, immune checkpoint activator, antibody, in particular anti-CD137 antibody (which targets 4-1BB, a TNF receptor superfamily member also known as CD137) or anti-SIRPa antibody (P84—anti-mouse SIRPa from Merck Millipore).
In another aspect, the invention relates to a combination of compounds comprising an anti-CMKLR1 compound as described herein, in particular a pharmaceutical composition comprising an anti-CMKLR1 compound according to the invention and an anti-PD1 or an anti-PDL1 compound, in particular an anti-PD1 compound; such a compound is in particular selected from the group consisting of an antibody, an antigen-binding antibody fragment, an antigen-binding antibody mimetic, a small molecule like an aptamer or a peptide, a modified antibody, like but not limited to a humanized or a chimeric antibody, able to bind to PD1 or PDL1.
In another aspect, the invention relates to a combination of compounds comprising an anti-CMKLR1 compound as described herein, in particular a pharmaceutical composition comprising an anti-CMKLR1 compound according to the invention and an anti-SIRPa compound; such a compound is in particular selected from the group consisting of an antibody, an antigen-binding antibody fragment, an antigen-binding antibody mimetic, a small molecule like an aptamer or a peptide, a modified antibody, like but not limited to a humanized or a chimeric antibody, able to bind to SIRPa, in particular human SIRPa.
In another aspect, the invention concerns the therapeutic use of the anti-CMKLR1 compound of the invention, especially for inducing and/or enhancing the resolution of inflammation, in particular for inducing and/or enhancing the resolution of inflammation when said resolution is delayed or disrupted, in view of treating diseases wherein the extension of the inflammation is pathologic, or wherein the duration of the resolution of inflammation is pathologic.
In a particular embodiment of the invention, the anti-CKLMR1 compound binds CMKLR1 with an affinity (KD value) of at least 10E-8 M, more preferably with an affinity of at least 10E-9 M. The specific binding between the antibody, or antigen-binding fragment thereof, or antigen-binding antibody mimetic or modified antibody of the invention and CMKLR1 (or a region of CMKLR1 comprising the third extra-cellular loop, including amino acid sequences set forth in SEQ ID No: 2 and 18) implies that the antibody exhibits appreciable affinity for CMKLR1. “Appreciable affinity” includes binding with an affinity of about 10−8 M (KD) or stronger. Preferably, binding is considered specific when the binding affinity is between 10−8 M and 10−12 M, optionally between 10−9 M and 10−1° M, in particular at least 10−9 M. Whether a binding domain specifically reacts with or binds to a target can be tested readily by, inter alia, comparing the reaction of said binding domain with a target protein or antigen with the reaction of said binding domain with proteins or antigens other than the target protein. Such an antibody of the invention specifically binds CMKLR1 and has an agonist effect towards the interaction between RvE1 and CMKLR1. Methods for determining antibody specificity and affinity by competitive inhibition are known in the art (see, e.g., Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1998); Colligan et al., Current Protocols in Immunology, Green Publishing Assoc., NY (1992; 1993); Muller, Meth. Enzym., 92:589-601 (1983)). These methods include, but are not limited to, Biacore Analysis, Blitz analysis, flow cytometry and ELISA assay.
In a particular embodiment of the invention, the anti-CMKLR1 compound binds specifically to an epitope localized within the third extra-loop of CMKLR1, in particular to an epitope localized within amino acid residue sequences set forth in SEQ ID No: 2 or SEQ ID No: 18, in particular SEQ ID No: 2. An anti-CMKLR1 compound binding within this particular region of CMKLR1 may have an agonist property on CMKLR1, thereby mimicking the binding of RvE1 to CMKLR1.
In another aspect, the invention relates to an anti-CMKLR1 antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic or modified antibody as defined here above, which has an agonist capability towards the interaction between RvE1 and CMKLR1, for use as a medicament.
In another aspect, the invention relates to an anti-CMKLR1 antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic or modified antibody as defined here above, which has the capability to induce the activation of Akt and/or Erk protein(s) in vitro and/or in vivo. The activation of these proteins may be assessed by the methods described in the examples of the present invention. In particular, the anti-CMKLR1 antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic or modified antibody has the capability to activate either Akt and/or Erk protein, or both, in macrophages, in particular in human macrophages.
The present invention also relates to a method of treatment in a subject in need thereof comprising administering to said subject an effective amount of an anti-CMKLR1 antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic as defined above, which has an agonist capability towards the interaction between RvE1 and CMKLR1, or in other words which is an RvE1-agonist like factor or modulator.
Modifying the polarization of macrophages to favor anti-inflammatory cells can be useful in a number of pathologies or situations. As described above, this modification is particularly useful in the context of a disease selected from the group of inflammatory diseases, including but not limited to acute inflammatory diseases and chronic inflammatory diseases, inflammatory bowel disease, Crohn's disease, asthma, keratoconjunctivitis, periodontal disease, eczema, colitis, in particular ulcerative colitis or spontaneous colitis, diabetes, in particular type I diabetes, peritonitis, psoriasis, carcinoma, in particular mammary carcinoma or colon carcinoma, cancers, metastatic cancers, lung cancer, degenerative disease, infection disease, in particular sepsis, autoimmune diseases.
The present invention also relates to the use of an anti-CMKLR1 antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic as defined above, which has an agonist capability towards the interaction between RvE1 and CMKLR1 in the manufacture of a medicament.
In another aspect, the invention relates to an anti-CMKLR1 antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic or modified antibody, like but not limited to humanized or chimeric antibody, as defined here above, for its use in the treatment of a chronic inflammatory disease, in particular for treating chronic colitis.
In another aspect, the invention relates to an anti-CMKLR1 antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic as defined above, which has an agonist activity towards the interaction between RvE1 and CMKLR1 for use in the treatment of a delayed or disrupted resolution of an inflammatory condition, in particular an inflammatory disease wherein its resolution is delayed or disrupted, and/or in the treatment or the prevention of a disease selected from the group of inflammatory diseases, including but not limited to acute inflammatory diseases and chronic inflammatory diseases, inflammatory bowel disease, Crohn's disease, asthma, keratoconjunctivitis, periodontal disease, eczema, colitis, in particular ulcerative colitis or spontaneous colitis, diabetes, in particular type I diabetes, peritonitis, psoriasis, carcinoma, in particular mammary carcinoma or colon carcinoma, cancers, degenerative disease, infection disease, in particular sepsis, autoimmune diseases.
As defined herein, “a delay or a disruption in the resolution of an inflammatory condition” occurs when the resolution of inflammation is delayed or disrupted as compared to a normal resolution (i.e. the resolution occurring in a patient who experiences normal resolution after an inflammatory event). A resolution delay or defect can result in an increased penetration of granulocytes at the inflammatory site. Hence, a resolution delay or defect may be assessed by quantification of granulocytes at the inflammatory site. Granulocyte population may be measured for example by histology, cytometry or indirect biochemical techniques such as elastase quantification by enzyme immunoassay or molecular quantification by PCR of granulocyte receptor 1). A resolution delay or defect may also be assessed by the determination of a delay in the apoptosis of granulocytes, measured for example by cytology using specific antibodies against annexin 5. A defect or a delay in the resolution of the inflammation may also be determined by assessing by quantifying the synthesis of pro-inflammatory cytokines such as TNF-alpha, IL8 or IL12 and anti-inflammatory cytokines such as IL-10. Cytokine secretion may be assessed by enzyme immunoassay or by PCR. A defect or a delay in the resolution of the inflammation may also be determined by assessing the activation of transcription factors involved in the synthesis of inflammatory cytokines, such as NF-kappaB which can be measured for example by nuclear translocation or by Western blot and/or by quantification of the level of degradation of IkappaB). A defect or a delay in the resolution of the inflammation may also be determined by quantifying specialized pro-resolving mediators (such as lipoxins, resolvins, protectins or maresins) or their precursors (like 17-HDOHE or 14-HDOHE) by mass spectrometry or enzyme immunoassay. A defect or a delay of the resolution then results in a defect of the synthesis of one or more of these mediators. A resolution defect or delay can also be determined when expression of the receptors of the resolution molecules is decreased. These receptors may be selected from the group comprising ALX, CMK1R1, GPR32 or GPR18. Alternatively or complementarily, the internalization and processing of those receptors into the cytoplasm may also be assessed. Alternatively or complementarily, expression of some receptors of inflammatory cytokines or lipids may also be assessed, an overexpression compared to a normal condition being significant of a delay or a defect in the resolution of the inflammation. These conditions may be measured by histology, cytology or PCR. The resolution defect can also result in a decreased or inhibited switch of M1 to M2 macrophages, a damage in phagocytosis or efferocytosis of the same cells. Hence, a delay or a defect of the resolution may be assessed by analyzing the switch of M1 to M2 macrophages in a particular condition as compared to a normal condition, as exemplified in the examples of the present invention.
According to a particular embodiment, anti-CMKLR1 compound can be used to treat an individual who has a cancer selected from the group consisting of mammary cancer, in particular mammary carcinoma cancer, melanoma, colon cancer, in particular colon carcinoma cancer, leukemia, in particular acuter myeloid leukemia, in particular when cancer cells over-express CMKLR1.
In an embodiment, the invention relates to an anti-human CMKLR1 antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic or modified antibody as defined above, for its uses as defined above, wherein said anti-human CMKLR1 antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic or modified antibody of the invention is administered to a patient presenting a CMKLR1-positive tumor.
The antibody or antigen-binding fragment thereof of the invention can be administered in a variety of suitable routes, e.g., intravenously (IV), subcutaneously (SC), or, intramuscularly (IM) to the subject. The anti-CMKLR1 compound can be administered alone or in combination with another therapeutic agent, e.g., a second human monoclonal antibody or antigen binding fragment thereof. In another example, the antibody is administered together with another agent, for example, an immunosuppressive agent, an erythropoiesis-stimulating agent (ESA), in combination with therapeutic cell compositions, and the like. In an embodiment, the invention relates to an anti-CMKLR1 compound or antigen-binding fragment thereof or antigen-binding antibody mimetic for its use as defined above, wherein the anti-CMKLR1 antibody or antigen-binding fragment is combined with a second therapeutic agent.
The administration of the second therapeutic agent can be simultaneous or not with the administration of the anti-CMKLR1 compound. Depending on the nature of the second agent, a co-administration can be prepared in the form of a combination drug (product), also known as a “combo”. A combo is a fixed-dose combination that includes two or more active pharmaceutical ingredients combined in a single dosage form, which is manufactured and distributed in fixed doses. But the dose regimen and/or the administration route can also differ.
In a preferred embodiment, this second therapeutic agent is selected from the group consisting of chemotherapeutic agents, radiotherapy agents, immunotherapeutic agents, cell therapy agents (such as CAR-T cells), antibiotics and probiotics.
In particular, immunotherapeutic agents useful in the context of the invention are selected from the group consisting of therapeutic vaccines (DNA, RNA or peptide vaccines), immune checkpoint blockers or activators, in particular of adaptive immune cells (T or B lymphocytes) or immunoconjugates such as antibody-drug conjugates.
As used herein, the term “immunotherapeutic agents” refers in particular to agents that could take cancer vaccines from interesting biological phenomena to effective therapeutic agents including: T-cell growth factors to increase number and repertoire of naive T cells, growth factors to increase the number of dendritic cells (DCs), agonists to activate DCs and other antigen-presenting cells (APCs), adjuvants to allow and augment cancer vaccines, agonists to activate and stimulate T cells, inhibitors of T-cell checkpoint blockade, T-cell growth factors to increase the growth and survival of immune T cells, agents to inhibit, block, or neutralize cancer cell and immune cell-derived immunosuppressive cytokine.
Numerous immune checkpoint blocker or activator are known in the art. In the context of the invention, examples of immune checkpoint blockers or activators of adaptive immune cells (B or T lymphocytes) that could be useful are 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 agonist, CD40-L, TLR agonists, anti-ICOS, ICOS-L and B-cell receptor agonists, in particular anti-CD137 and anti-SIRPa. In a particular embodiment of the invention, the second therapeutic agent is an anti-PDL1 or an anti-PD1 compound, in particular 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, in particular an anti-SIRPa antibody
Said immunotherapeutic agent can also be an antibody targeting tumoral antigen, particularly selected from the group consisting of anti-Her2, anti-EGFR, anti-CD20, anti-CD19, anti-CD52.
The antibody may be provided at an effective dose from about 1 ng/kg body weight to about 30 mg/kg body weight, or more. In specific embodiments, the dosage may range from 1 μg/kg to about 20 mg/kg, optionally from 10 μg/kg up to 10 mg/kg or from 100 μg/kg up to 5 mg/kg.
The term “effective dose” or “effective dosage” or “effective amount” is defined as an amount sufficient to achieve or at least partially achieve the desired effect. The term “effective dose” is meant to encompass an amount sufficient to cure or at least partially arrest the disease and its complications or alleviate the symptoms of the disease in a patient already suffering from the disease. Amounts or doses effective for this use will depend on the condition to be treated, the delivered antibody construct, the therapeutic context and objectives, the severity of the disease, prior therapy, the patient's clinical history and response to the therapeutic agent, the route of administration, the size (body weight, body surface or organ size) and/or condition (the age and general health) of the patient, and the general state of the patient's own immune system. The proper dose can be adjusted such that it can be administered to the patient once or over a series of administrations, and in order to obtain the optimal therapeutic effect.
Dosing for such purposes may be repeated as required, e.g. daily, semi-weekly, weekly, semi-monthly, monthly, or as required during relapses.
In another aspect, the invention relates to a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as defined above and a pharmaceutically acceptable carrier.
As used herein, a “pharmaceutical composition” is meant to encompass a composition suitable for administration to a subject or patient, such as a mammal, especially a human. In general, a “pharmaceutical composition” is sterile and is usually free of contaminants that are capable of eliciting an undesirable response within the subject (e.g., the compound(s) in the pharmaceutical composition is pharmaceutical grade). Pharmaceutical compositions can be designed for administration to subjects or patients in need thereof via a number of different routes of administration including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, intracheal and the like.
As used herein, a “pharmaceutically acceptable carrier” is meant to encompass an excipient, diluent, carrier, and adjuvant that are useful in preparing a pharmaceutical composition that are generally safe, non-toxic and neither biologically nor otherwise undesirable, and include an excipient, diluent, carrier, and adjuvant that are acceptable for veterinary use as well as human pharmaceutical use. “A pharmaceutically acceptable carrier” as used herein includes both one and more than one such excipient, diluent, carrier, and adjuvant.
In particular, the invention relates to a pharmaceutical composition which comprises as an active ingredient an antibody or antigen-binding fragment thereof as defined above and a pharmaceutically acceptable carrier.
In another aspect, the invention relates to a therapeutic means, in particular a combination product means, which comprises as active ingredients: an anti-SIRPa antibody or antigen-binding fragment thereof or antigen-binding antibody mimetic as defined above and a second therapeutic agent, wherein said active ingredients are formulated for separate, sequential or combined therapy, in particular for combined or sequential use.
In particular, the invention relates to a combination product comprising an anti-CMKLR1 compound as defined above and a second therapeutic agent for simultaneous, separate or sequential use a medicament.
In an embodiment, the 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, immunotherapeutic agents, antibiotics and probiotics.
In an embodiment, the invention relates to a combination product as defined above, wherein said immunotherapeutic agent is selected from the group consisting of therapeutic vaccines, immune checkpoint blockers or activators, in particular of adaptive immune cells (T and B lymphocytes) and antibody-drug conjugates.
In an embodiment, the invention relates to a combination product as defined above, wherein said immune checkpoint blocker or activator of adaptive immune cells (T and B lymphocytes) is selected from the group consisting of anti-PDL1, anti-PD1, anti-SIRPA, anti-CTLA4, anti-CD137, anti-CD2, anti-CD28, anti-CD40, anti-HVEM, anti-BTLA, anti-CD160, anti-TIGIT, anti-TIM-1/3, anti-LAG-3, anti-2B4, and anti-OX40, anti-CD40 agonist, CD40-L, TLR agonists, anti-ICOS, ICOS-L and B-cell receptor agonists, in particular selected from the group consisting of anti-PDL1, anti-PD1 and anti-CD137. In a particular embodiment of the invention, the second therapeutic agent is an anti-PDL1 or an anti-PD1 compound, in particular 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, in particular an anti-SIRPa antibody.
In one embodiment, said immunotherapeutic agent is an antibody targeting tumoral antigen, particularly selected from the group consisting of anti-Her2, anti-EGFR, anti-CD20, anti-CD19, anti-CD52.
In an aspect, the invention relates to a combination product as defined above, for simultaneous, separate or sequential use in the treatment of any condition susceptible of being improved or prevented by modifying macrophage polarization to anti-inflammatory macrophages.
In an embodiment, the invention relates to a method of treatment of any condition susceptible of being improved or prevented by modifying macrophage polarization to anti-inflammatory macrophages in a subject in need thereof comprising administering simultaneously, separately or sequentially to said subject an effective amount of a combination product as defined above.
In an embodiment, the invention relates to the use of a combination product as defined above in the manufacture of a medicament for the treatment any condition susceptible of being improved or prevented by modifying macrophage polarization to anti-inflammatory macrophages.
In an aspect, the invention relates to a combination product as defined above, for simultaneous, separate or sequential use in the treatment of a pathology selected from the group consisting of inflammatory diseases, including but not limited to acute inflammatory diseases and chronic inflammatory diseases, inflammatory bowel disease, Crohn's disease, asthma, keratoconjunctivitis, periodontal disease, eczema, colitis, in particular ulcerative colitis or spontaneous colitis, diabetes, in particular type I diabetes, peritonitis, psoriasis, carcinoma, in particular mammary carcinoma or colon carcinoma, cancers, metastatic cancers, lung cancer, degenerative disease, infection disease, in particular sepsis, autoimmune diseases or for use in vaccination.
In an embodiment, the invention relates to a method of treatment of a pathology selected from the group of inflammatory diseases, including but not limited to acute inflammatory diseases and chronic inflammatory diseases, inflammatory bowel disease, Crohn's disease, asthma, keratoconjunctivitis, periodontal disease, eczema, colitis, in particular ulcerative colitis or spontaneous colitis, diabetes, in particular type I diabetes, peritonitis, psoriasis, carcinoma, in particular mammary carcinoma or colon carcinoma, cancers, metastatic cancers, lung cancer, degenerative disease, infection disease, in particular sepsis, autoimmune diseases of a subject in need thereof comprising administering simultaneously, separately or sequentially to said subject an effective amount of a combination product as defined above.
In an embodiment, the invention relates to the use of a combination product as defined above, in the manufacture of a medicament for the treatment of a pathology selected from the group of inflammatory diseases, including but not limited to acute inflammatory diseases and chronic inflammatory diseases, inflammatory bowel disease, Crohn's disease, asthma, keratoconjunctivitis, periodontal disease, eczema, colitis, in particular ulcerative colitis or spontaneous colitis, diabetes, in particular type I diabetes, peritonitis, psoriasis, carcinoma, in particular mammary carcinoma or colon carcinoma, cancers, metastatic cancers, lung cancer, degenerative disease, infection disease, in particular sepsis, autoimmune diseases.
The invention is also related to a method for selecting an anti-CMKLR1 compound, said method comprising the following steps:
The capabilities of the compound may be tested according to the examples disclosed in the examples of the present disclosure.
The invention also relates to a polynucleotide encoding an anti-CMKLR1 compound as defined herein. To this end, the invention also relates to a nucleic acid molecule, or a group of nucleic acid molecules, more particularly an isolated nucleic acid molecule(s) and/or a recombinant a nucleic acid molecule(s), which encode(s) any anti-CMKLR1 compound according to the present disclosure, more particularly which encode(s) a heavy chain variable domain comprising or consisting of the amino acid residues of sequences set forth in SEQ ID No: 20; SEQ ID No: 21; SEQ ID No: 22; SEQ ID No: 23; 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; SEQ ID No: 33; SEQ ID No: 34; SEQ ID No: 35; SEQ ID No: 36; SEQ ID No: 37; SEQ ID No: 38; SEQ ID No: 39; SEQ ID No: 40; SEQ ID No: 41 or SEQ ID No: 42 and/or a light chain variable domain comprising or consisting of the amino acid residues of sequences set forth in SEQ ID No: SEQ ID No: 43; SEQ ID No: 44; SEQ ID No: 45; SEQ ID No: 46; SEQ ID No: 47; SEQ ID No: 48; 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: 59; SEQ ID No: 60 or SEQ ID No: 61. The nucleic acid molecule(s) may further comprise regulation sequences, like but not limited to enhancers, silencers, promoters, in particular expression promoters, signal peptide, for transcription and expression of the encoded heavy chain variable domain and/or the light chain variable domain.
The invention also relates to a vector comprising the polynucleotide as disclosed herein, or comprising the nucleic acid molecule(s) as disclosed herein. As used herein, a vector is a nucleic acid molecule used as a vehicle to transfer a genetic material into a cell, and in a preferred embodiment allows the expression of a polynucleotide inserted within the vector. The term vector encompasses plasmids, viruses, cosmids and artificial chromosomes. A vector generally comprises an origin of replication, a multicloning site, and a selectable marker. The vector itself is generally a nucleotide sequence, commonly a DNA sequence, that comprises an insert {transgene) and a larger sequence that serves as the “backbone” of the vector. Modern vectors may encompass additional features besides the transgene insert and a backbone: promoter, genetic marker, antibiotic resistance, reporter gene, targeting sequence, protein purification tag. Vectors called expression vectors (expression constructs) specifically are for the expression of the transgene in the target cell, and generally have control sequences.
In another aspect, the invention relates to a cell, an isolated cell, a host cell, an isolated host cell, or a cell line comprising a vector as defined above. As used herein, these terms related to cells are intended to include any individual cell or cell culture that can be or has been recipient of vectors, exogenous nucleic acid molecules, and polynucleotides encoding the antibody construct of the present invention; and/or recipients of the antibody construct itself. The introduction of the respective material into the cell can be carried out by way of transformation, transfection and the like. These terms are also intended to include progeny or potential progeny of a single cell. Suitable host cells include prokaryotic or eukaryotic cells, and also include but are not limited to bacteria, yeast cells, fungi cells, plant cells, and animal cells such as insect cells and mammalian cells, e.g., murine, rat, rabbit, macaque or human.
The following Figures and Examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.
After 16 or 48 hours of stimulation of the cells with LPS, TNFa or IL6, expression of the CMKLR1 (ChemR23) was measured by FACS. A. Results on human blood monocytes, B. and C. Results on myeloid and neutrophil cells from mice bone marrow.
Weight variation of treated animals was followed up to sixty days. Animals were treated with isotype control hIgG1 (x) or anti-CMKLR1 antibody (▪).
Production and Selection of Anti-CMKLR1 Antibody
Several antibodies having different CDR sequences within their heavy and light chain variable domains have been synthetized. The distinct antibodies were tested for their ability to induce maturation and differentiation of dendritic cells towards a pro-inflammatory pathway or an anti-inflammatory pathway. Considering the divergent results, the inventors compared obtained amino acid sequences of the heavy and light chain variable domains and determined that the amino acid difference in HCDR3 might be material to said experimental results. They selected antibody 2G1 (SEQ ID No: 10 and SEQ ID No: 17) to assess its properties in resolving inflammatory status of at least influencing said status in resolution phase.
As shown in
The 2G1 was humanized using in silico CDR grafting method, a method of humanization well known in the art. The resulting humanized sequences from the CDR and FR regions, as well as from the variable heavy chain and light chain are described in the following tables.
Examples of Therapeutic Efficacy of Anti-CMKLR1 Antibody Treatment on Preclinical Models of Autoimmune and Inflammatory Diseases
Colitis was induced in 8-10 weeks-old-C57Bl/6 male mice by adding 2% (wt/vol) of DSS to the sterile drinking ad libitum water for 6 days. Treatments were injected intra-peritoneally: Isotype control hIgG1 (10 μg per mouse), RvE1 (1 μg per mouse) daily, or 2G1 antibody (10 μg per mouse) three times for 5 days. Colitis follow-up consisting of body weight and stool score (0: normal stool; 4: blood in stool) parameters were performed daily. When mice were euthanized, colon length representing the pathology severity was measured. Resolution index was determined in the different conditions as described in Bannenberg et al., 2005.
RESULTS: The DSS animal model presented on
Colitis was induced in 8-10 weeks-old-C57Bl/6 male mice by intrarectal injection of 200 μL of the haptenating agent TNBS at 5% in 50% ethanol on day 0. Treatments were injected intra-peritoneally; RvE1 (1 μg per mouse) daily for three days, or 2G1 antibody (10 μg per mouse) twice for 3 days. Colitis follow-up consisting of body weight and stool score (0: normal stool; 4: blood in stool) parameters were performed daily (data not represented). When mice are euthanized, colon length representing the pathology severity was measured.
RESULTS: Colitis induced by TNBS is another model of acute inflammation.
IL-10KO mice develop a spontaneous colitis from 20 weeks of age mostly due to the absence of regulatory T cells function through IL-10 secretion in the intestine. IL-10KO mice were followed-up three times a week from 18-week old for their weight loss and stool consistency which are clinical hallmarks of this pathology. Anti-CMKLR1 antibody (2G1) or isotype control (hIgG1) were injected intra-peritoneally when the weight loss was superior to 5% and the stool score was superior or equal to 1 for 2 weeks (25 μg/injection, 3 times a week).
RESULTS: A chronic inflammatory model was used to study the efficacy of anti-CMKLR1 antibody treatment.
8 weeks-old NOD female mice were obtained from Charles River laboratory. These mice develop a spontaneous type 1 diabetes at age between 12 to 20 weeks. The diabetes initiation can be measured by the high glycaemia. When the glycaemia was between 180 and 234 mg/dL, anti-CMKLR1 and isotype control were given intra-peritoneally at 20 μg/injection three times a week for 2 weeks. Mice were euthanized when the glycaemia was superior to 600 mg/dL corresponding to an irreversible diabetes.
RESULTS: this type 1 diabetes model is considered as a mice autoimmune disease model as well. Results presented
Aldara® cream which is known to induce psoriasis in mice was used on male C57Bl/6 mice (8-10-week old). Mice received a daily topical dose of Aldara on the shaved back and the left ear for 6 consecutive days. Treatments were injected intra-peritoneally: RvE1 (1 μg per mouse) and PBS daily. Ear (
RESULTS: Animals treated with the resolvin RvE1 had a lower thickness of the ears and skin than animals treated with a control molecule. These results suggest a potential application for agonist anti-CMKLR1 antibody therapy on psoriasis mice model, illustrative of an autoimmune disease.
Common peritonitis is induced by intraperitoneal injection of Zymosan A® (1 mg per mouse in 1 mL). Preventive injection of anti-CMKLR1 was performed 5 minutes before Zymosan A injection: RvE1 (1 μg per mouse), 2G1 antibody (10 μg per mouse). Murine peritoneal polymorphonuclear neutrophil (PMN) and macrophages were collected at 2-4-8-16-24 and 48h after Zymosan A injection and numbered by flow cytometry analysis to determinate the resolution index (Bannenberg et al., 2005).
RESULTS: Results presented on
Mice were anesthetized with 3% of isoflurane. Mice were shaved on the abdomen and 4T1 cells (0.25 millions) were injected in the mammary gland with an insulinic syringe (30 Gauges) in 50 μL of PBS. The anti-CMKLR1 antibody (2G1) or an anti-41BB antibody (3H3) or both antibodies were injected twice at day 4 and day 7 (10 μg/injection); a control antibody was injected three times a week for three weeks intraperitoneally in PBS (100 μg/injection). In a second study to measure the lung metastasis following mammary carcinoma development, animals were treated with anti-CMKLR1 antibody at 0.8 mg/kg or control antibody (100 μg/injection) three times a week for three weeks.
RESULTS: As shown in
8-week old C57bl/6J male mice were anesthetized with 3% of isoflurane. Mice were shaved on the flank and MC38 cells (0.5.10{circumflex over ( )}6 cells/mouse) cell lines were injected subcutaneously with an insulinic syringe (30 Gauges) in 50 μL of PBS. Another model was used, in which 8-week old Balb/c male mice were anesthetized with 3% of isoflurane. Mice were shaved on the flank and CT26 cells (1.10″6 cells/mouse) were injected subcutaneously with an insulinic syringe (30 Gauges) in 50 μL of PBS.
The agonistic anti-CMKLR1 antibody (2G1) or an anti-SIRPa antibody (p84-anti-mouse SIRPa from Merck Millipore) (SIRPa is a new checkpoint inhibitor) were injected once a week (20 μg/injection) intraperitoneally for 3 weeks starting at d4 after tumor inoculation alone or combined.
RESULTS: As shown in
Signaling networks perpetuating chronic gastrointestinal inflammation in Crohn's disease (CD) and ulcerative colitis (UC), the two main forms of inflammatory bowel diseases (IBD), remain unclear in human. According to an analysis of nearly 500 patients with IBD and 100 controls, inventors report here that CMKLR1 transcript are accumulated in inflamed colon tissues of severe IBD patients who were not responding to immunosuppressive/corticosteroids and immunotherapies such as anti-TNFα (infliximab) or anti-α4β7 integrin (vedolizumab) therapies.
The inventors first analyzed the mucosal CMKLR1 transcript expression by performing a meta-analysis of publicly available transcriptional datasets of three cohorts of UC patients (GSE16879(Arijs et al., 2009a) and GSE12251(Arijs et al., 2009b), and GSE73661 with colon mucosa biopsies performed before anti-TNF treatment (within a week) in patients refractory to corticosteroids and/or immunosuppression. In these three cohorts, anti-TNF response was defined as histological healing analyzed 4-6 weeks after their first anti-TN F infusion (altogether: n=18 non-IBD controls, n=41 UC non-responders and n=28 UC responders).
RESULTS: The analysis showed that CMKLR1 transcript expression is significantly increased in colon biopsies of primary UC non-responder patients before and after treatment with anti-TNF therapy as compared to non-IBD controls or patients with UC before anti-TNF and who will respond to anti-TNF therapy (
Altogether, the meta-analysis shows that CMKLR1 is over-expressed in inflamed tissues of IBD patients, in particular in patients non-responding to current immunosuppressive or immunotherapies even before initiation of the treatment. Our meta-analysis provide evidence that the CMKLR1 expression in the colon, or rather ileon for CD, from UC or CD patients who are treatment refractory may in contrast qualify these patients as being responsive to an agonist anti-CMKLR1 antibody treatment such as an antibody of the invention.
CMKLR1 peptide (273NH2-PYHTLNLLELHHTAMPGSVFSLGLPLATALAIA-COOH305) (SEQ ID No: 18) (5 μg/ml) was coated in borate buffer overnight. Saturation was performed with PBS-Tween 0.1%-Gelatin 0.25% for 2 hours at 37° C. Then, 2G1 or hIgG1 antibodies were added at different concentrations for 2 hours at 37° C. Then peroxidase-conjugated secondary antibody (0.8 μg/ml) was added for 1 hour at 37° C. and revealed by a TMB substrate. The colorimetric reaction was read with TECAN.
Cells were resuspended in PBS-FBS-EDTA and incubated with Fc block (1/50) for 30 minutes on ice. Staining on monocytes, macrophages and dendritic cells were performed using A488-labeled 2G1 (5 μg) or A488-labeled hIgG1 (5 μg).
After protein migration and transfer as described previously, 2G1 antibody (10 μg/membrane) was incubated overnight at 4° C. and revealed with a peroxidase-conjugated secondary antibody (1:2000). The CMKLR1 expression was then detected by using Chemiluminescence and Image Reader. Western blot Images were quantified by Multi Gauge software.
RESULTS: the results illustrated on
Monocytes were collected from PBMC of buffy coat of healthy volunteers and isolated by magnetic separation or by elutriation. Then, monocytes were cultured with different cocktails of cytokines to generate differentiated unpolarized macrophages or polarized macrophages. This protocol allowed to generate polarized differentiated macrophages in order to have pro-(M1) or anti-(M2) inflammatory macrophages in different wells. Monocytes were plated at 0.5.106 cells/mL in complete RPMI (RPMI with 10% FBS, 1% glutamine, 1% antibiotics) and 500 μL of the cell suspension was plated per well in a 24-well plate. 100 ng/mL of M-CSF was added with medium for differentiation of the cells. Cells were incubated 5 days and medium was replaced with fresh medium complemented with 100 ng/mL of M-CSF at day 3. For the polarization phases, solution of LPS-IFNg at 100 ng/mL of LPS and 20 ng/mL of IFNg complemented with isotype controls (m IgG1 or hIgG4) (2 μg/ml) or anti-CMKLR1 antibodies (2 μg/ml) (2G1 or 2G4, H6, BZ332 or 84939) or with C15 peptide (10 nM) or RvE1 (10 ng/ml) during 3 days to generate M1 macrophages. M1-IFNg macrophages could also be generated by adding only IFNg (20 ng/mL) in culture medium. For M2 polarization cells were incubated with IL-4 at 20 ng/mL. Following differentiation and/or polarization, phenotype and functional cytokines/chemokines release was studied by FACS analysis, ELISA and Western blot.
—Isolation of Murine Bone Marrow Derived Macrophages
Bone marrow cells were harvested and cultured in the RPMI medium supplemented with 10% FBS, glutamine and antibiotics containing macrophages colony-stimulating factor (M-CSF) at 100 ng/mL for 5 days inducing the macrophages differentiation. The macrophages were harvested and cultured for 2 days with either IFNg (20 ng/ml) and LPS (100 ng/ml) inducing the M1 polarization or with IL-4 (20 ng/ml) inducing the M2 polarization. Treatments were added during macrophage polarization at 2 μg/ml.
—Bone Marrow Derived Dendritic Cells Generation
Bone marrow cells were harvested and cultured in RPMI medium supplemented with 10% FBS, glutamine and antibiotics and the dendritic cell differentiation was induced by GM-CSF at 20 ng/ml for 7 days. Then, immature dendritic cells (iDC) were collected and cultured for 24 hours with LPS (100 ng/ml) to induce the maturation from iDC to mDC. Treatments were added during differentiation and maturation at 2 μg/ml.
After differentiation of mice M1 or M2 as explain above, cells were incubated in presence of medium, isotype control, anti-CMKLR1 antibodies: clones H6 and BZ194, C15 peptides, 2G1 the anti-CMKLR1 antibody of interest or RvE1 were used. Then the secretion of IL10, CCL17 and IL12p40 was assessed by ELISA. Cytokines secretion was measured in the supernatant using an ELISA kit from BD. Supernatants were diluted at 1/10 for IL10 cytokines, 1/50 for CCL17 cytokines and 1/100 for IL12p40 cytokines.
—ELISA Cytokines Secretion Study
Cytokines secretion was detected by ELISA according to the BD manufacturer instructions. Briefly, supernatants were diluted in the appropriate buffer and incubated for 2 hours after overnight coating with capture antibody and saturation. Then, cytokines were revealed with a detection biotin-coupled antibody and the signal was amplified with the biotin-streptavidin-coupled peroxidase system. TMB supplied by BD Bioscience was used as substrate and colorimetric reaction was read with TECAN.
—Activation Cell Markers Analyzed by FACS
Dendritic cells were resuspended in PBS-FBS-EDTA and incubated with live and dead (LIVE/DEAD® Fixable Dead Cell Stains Yellow-Life Technologies) for 30 minutes on ice. Staining of CD11c-BV711, CD11b-APCCy7, I/Ab-APC, CD103-PerCPCy5.5, CCR7-V450, CD40-PeCy7, CD80-PE, CD86-FITC (all provided by BD Pharmingen) were performed.
—Western Blot Analysis ERK/Akt
Mice pro-inflammatory macrophages (M1) were generated from bone marrow with M-CSF and polarized with IFN-gamma (IFNg) and LPS. Briefly, bone marrow cells were collected by flushing the femoral bone and cultured with 100 ng/mL of mM-CSF for 5 days and then polarized with 20 ng/mL of IFNg and 100 ng/mL of LPS for 24h. Then, they were deprived of FBS for 24 hours with RPMI FBS 2% medium. Finally, mice M1 were treated with 2 μg/mL of 2G1 antibody at different times: 5, 10 and 30 minutes. Cells were collected in a RIPA buffer. The protein concentration was measured by a BCA protein kit assay. Proteins were denatured by heating at 95° C. for 5 minutes and diluted in DTT and Laemmli solution. After migration and transfer, the nitrocellulose membrane was blocked with 5% BSA in TBS-T for 2 hours. Anti-phospho-ERK antibody and anti-phospho-Akt antibody (1:1000) were incubated with the membrane overnight at 4° C. and revealed with a peroxidase-conjugated secondary antibody (1:2000). Western blot Images were quantified by Multi Gauge software
Human monocytes were collected from PBMC of buffy coat of healthy volunteers and isolated by magnetic separation or by elutriation. Then the monocytes (CD14 positive cells) were cultured in medium and treated with different pro-inflammatory stimuli 16 hours or 48 hours: LPS (100 ng/ml) or TNFa (10.000 U/ml) or IL6 (20 ng/ml).
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. Cells were then cultured in medium and treated with different pro-inflammatory stimuli 16 hours or 48 hours: LPS (100 ng/ml) or TNFa (10.000 U/mI) or IL6 (20 ng/ml).
The expression of CMLKR1 was measured by FACS using commercial anti-CMKLR1 antibodies (Human anti-ChemR23: clone 84939 and Mouse anti-ChemR23: clone 477806).
RESULTS: Analysis of the expression of CMKLR1 in mice myeloid lineage illustrated on
Methods.
Competition Assay to Measure Chemerin-Dependent B-Arrestin Recruitment by CMKLR1 Receptor in Presence of Anti-CMKLR1 Antibody:
The day before the assay, CHO-K1 CMKLR1 cells (Discover′X ref 93-0313E2) were plated in pre-warmed cell reagent then plated in a 96-well plate at 100 μl/well of cells as (Discover′X ref 15-103) and incubated 48 hours at 37° C., in a 5% CO2 humidified incubator. The anti-CMLKR1 antibody was diluted (22× in 7-point series of 3-fold dilutions from 1 μM to 1 nM) and cells were incubated 30 min at 37° C. with the antibody. Then cells were stimulated with Chemerin (2 or 6 nM) according to provider protocol (Discover′x ref 92-1036) 90 min at 37° C. The luminescence was measured after Working Detection Solution addition to the cells with a plate reader with 0.5 s integration.
Measurement of the Competition of the Anti-CMKLR1 Antibody with Chemerin in the Production of AMPc by CMKLR1 Receptor:
The day before the experiment CHO-K1 CMKLR1 Gi cells (Discover′X ref 95-0080C2) were plated in pre-warmed cell reagent then plated in a 96-well plate at 100 μl/well of cells as (Discover′X ref 15-103) and incubated 24 hours at 37° C., in a 5% CO2 humidified incubator for 24 hours.
A mix of Chemerin agonist (6× in 7-point series of 3-fold dilutions from 10−7 μM to 10−10M) (Discover′x ref 92-1036 or 2324-CM-025 from R&D Systems) and forskolin (40 μM) (a cAMP activator) (Discover′x ref 92-0005) was added to the cells during 30 min at 37° C.; or cells were pre-incubated 30 min at 37° C. with anti-CMKLR1 antibody (serial dilution: 6λ in 7-point series of 3-fold dilutions from 1 μM to 1 nM). Then a mix of chemerin (2 nM)+forskolin (60 nM) was added to the cells 30 min at 37° C. For the detection cAMP, antibody reagent and cAMP working detection solution was added to the plate for 1 hour at room temperature, then cAMP solution A was added, and cells incubated 3 hours at room temperature in the dark. Bioluminescence was read with a plate reader with 0.5s integration.
Results: In order to test if the antibody of the invention is an antagonist of the Chemerin-induced CMKLR1 activation, two assays were performed, and the results are presented on
Method: CD45Rbhigh CD4 T cells were isolated from the spleen of naive mice and sorted on an ARIA FACS after a negative selection of the CD4 T cells by magnetic sorting, then injected intraperitoneally at 0.5.106 cells in 100 μL of PBS into 6-weeks old female Rag1 knock-out mice. Anti-CMKLR1 antibody (2G1) or an isotype control were administered from day 32 after the CD45Rbhigh CD4 T cell transfer for 3 weeks three times a week at 1 mg/kg. The follow-up of weight was evaluated three times a week and the weight variation was determined over the initial weight. * p<0.05, ** p<0.01.
Results:
Method: Mice were anesthetized with a cocktail of xylazine/ketamine. After a laparotomy, tumoral Hepa 1.6 cells were injected in PBS through the portal vein (2.5.106 cells/100 μL) in PBS. The treatment was started 4 days after tumor injection. The anti-CMKLR1 antibody (2G1clone) and the hIgG1 istotype control were injected at 0.8 mg/kg three times per week during 2 weeks. The anti-PD1 monoclonal antibody was injected twice a week during 2 weeks intraperitoneally in PBS (8 mg/kg). Combination anti-CMKLR1 and anti-PD1 antibodies was tested as well (0.8 mg/kg and 8 mg/kg respectively). The Overall survival was followed during sixty days and the percentage of survival in each condition was reported
Results: As shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 18305395.8 | Apr 2018 | FR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2019/058358 | 4/3/2019 | WO | 00 |
