Antibodies targeting CCR2

Abstract

This invention provides antibodies and antibody fragments specific to CCR2. [Solution] This disclosure relates to antibodies and antibody fragments specific to CCR2. Almost all attempts to humanize the original antibodies have failed due to loss of reactivity with marmoset CCR2, a species essential for further preclinical and clinical development. The antibodies are useful for treating inflammatory diseases, autoimmune diseases, hematological malignancies, and potentially other diseases.

Description

Technical Field

[0001] The present disclosure relates to antibodies and antibody fragments specific for CCR2. Almost all attempts to humanize the original antibody have failed, for reasons such as loss of cross-reactivity with marmoset CCR2, which is an essential species for further preclinical and clinical development. The antibodies produced in the present invention are useful for the treatment of inflammatory diseases, autoimmune diseases, hematological malignancies, and potentially other diseases.

Background Art

[0002] Monocytes migrate to sites of inflammation under the influence of chemokines and cytokines. One such chemokine is MCP-1, which binds to the chemokine receptor CCR2 and releases monocytes from the bone marrow. MCP-1 is secreted by cells among inflammatory diseases, autoimmune diseases, hematological malignancies, and other diseases. A plurality of molecules targeting the MCP-1 / CCR2 pathway have been tested or are currently under test, and some have shown promising results, but none have been approved to date. Most of the drugs developed are small organic molecules. For example, cenicriviroc, a low molecular weight substance developed by Takeda and Tobira Therapeutics, is currently in a Phase 3 clinical trial. However, cenicriviroc is not specific for CCR2 and also inhibits CCR5.

[0003] STI-B0201 is an antibody produced by Sorrento, but clinical development has not been reported. Sorrento's anti-CCR2 antibody is disclosed in WO2013 / 192596. Unlike the antibody in this disclosure, the antibody in WO2013 / 192596 cross-reacts with mouse CCR2. Anti-CCR2 antibodies are also disclosed in WO2007 / 115713 and U.S. Patent No. 9068002. However, these are also mouse antibodies that were never developed. MLNM1202 (prozarizumab) is another anti-CCR2 antibody, but development was discontinued by Millennium. Takeda / Millennium's antibody is disclosed in WO2016 / 079276. Amgen's antibody is disclosed in U.S. Patent Application Publication No. 2011 / 0274696. The antibody in U.S. Patent Application Publication 2011 / 0274696 shows reactivity to cynomolgus monkey CCR2, but the antibody in this disclosure does not cross-react with cynomolgus monkey CCR2. However, the antibody in this disclosure does cross-react with marmoset CCR2. The University of Regensburg / Yeda / Tel Aviv Medical Center antibody against CCR2 is disclosed in U.S. Patent No. 9,068,002. Pfizer's antibody against CCR2 is disclosed in WO2010021697. Again, no development has been reported. Therefore, there is a need and great interest in developing a specific anti-CCR2 targeting moiety, but currently, no anti-CCR2 antibodies are in development. This invention discloses a novel antibody that is highly specific to CCR2. Since essentially all humanized derivatives have lost their reactivity to marmoset CCR2, a species essential for further preclinical and clinical development, humanizing the original antibody has been extremely difficult. [Overview of the Initiative]

[0004] This disclosure relates to a humanized antibody or antibody fragment specific to human CCR2, wherein the antibody includes the HCDR1 region of SEQ ID NO: 2, the HCDR2 region of SEQ ID NO: 3, the HCDR3 region of SEQ ID NO: 4, the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9. This disclosure also relates to a humanized antibody or antibody fragment, which includes the VH of SEQ ID NO: 21 and the VL of SEQ ID NO: 43. In certain embodiments, the humanized antibody is of the human IgG1 class. In certain embodiments, the humanized antibody or antibody fragment binds to the first extracellular loop (SEQ ID NO: 53) and the second extracellular loop (SEQ ID NO: 54) of human CCR2. In certain embodiments, the humanized antibody or antibody fragment according to the present invention is cross-reactive with marmoset CCR2. In certain embodiments, the humanized antibody or antibody fragment according to the present invention binds to the first extracellular loop (SEQ ID NO: 53) and the second extracellular loop (SEQ ID NO: 55) of marmoset CCR2.

[0005] In certain embodiments, the humanized antibody or antibody fragment does not bind to the rhesus monkey CCR2. This disclosure also relates to humanized antibodies or antibody fragments disclosed herein for use in medical settings, preferably for the treatment of inflammatory diseases, autoimmune diseases, or hematological malignancies. This disclosure also relates to nucleic acid compositions comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment disclosed herein. This disclosure also relates to a vector comprising the aforementioned nucleic acid composition. This disclosure also relates to host cells comprising the aforementioned vector or nucleic acid composition. This disclosure also relates to pharmaceutical compositions comprising a humanized antibody or antibody fragment and a pharmaceutically acceptable carrier or excipient as disclosed herein. [Brief explanation of the drawing]

[0006] [Figure 1]This figure shows the specific binding of the antibody DOC2 chimera and DOC3 chimera, as well as the control antibody AMBA, to human monocytes. [Figure 2] This figure compares the specific binding of the antibody DOC2 chimera and DOC2 reformation, as well as the control antibody AMBA, to human monocytes. [Figure 3] This figure compares the specific binding of DOC3-chimeric, DOC3-RH03XRKA, and DOC3-RHLXRKA, as well as the control antibody AMBA, to human monocytes. [Figure 4] This figure compares the specific binding of the antibody DOC2 chimera and DOC2 reformation, as well as the control antibody AMBA, to primary marmoset monocytes. [Figure 5] This figure compares the specific binding of DOC3-chimera, DOC3-RH03XRKA, and DOC3-RHLXRKA, as well as the control antibody AMBA, to primary marmoset monocytes. [Figure 6] This figure compares the specific binding of additionally humanized DOC2 antibodies to primary human monocytes. [Figure 7] This figure compares the specific binding of additionally humanized DOC2 antibodies to primary marmoset monocytes. [Figure 8] This figure compares the specific binding of additionally humanized DOC3 antibodies to primary human monocytes. [Figure 9] This figure compares the specific binding of additionally humanized DOC3 antibodies to primary marmoset monocytes. [Figure 10] This figure compares the specific binding of antibodies DOC2 RHAXRKA FR1, DOC2 RHAXRKA FR2, DOC2 RHAXRKA FR3a, DOC2 RHAXRKA FR3b1, and a DOC2 chimera to primary human monocytes. [Figure 11] This figure compares the specific binding of antibodies DOC2 RHAXRKA FR1, DOC2 RHAXRKA FR2, DOC2 RHAXRKA FR3a, DOC2 RHAXRKA FR3b1, and a DOC2 chimera to primary marmoset monocytes. [Figure 12]This figure shows that DOC2-RHAXRKA FR2 significantly depletes classical CD14-positive monocytes, while rituximab or human IgG1 isotype control antibodies do not. hDOC2 = DOC2-RHAXRKA FR2; hIgG1 Iso = human IgG1 isotype control antibody. [Figure 13] This figure shows that rituximab depletes B cells, but neither DOC2-RHAXRKA FR2 nor human IgG1 isotype control antibodies do. hDOC2 = DOC2-RHAXRKA FR2; hIgG1 Iso = human IgG1 isotype control antibody. [Figure 14] This figure shows that none of the tested antibodies depleted T cells. hDOC2 = DOC2-RHAXRKA FR2; hIgG1 Iso = Human IgG1 isotype control antibody. [Figure 15] This figure shows that DOC2-RHAXRKA FR2 upregulates the median CD11b fluorescence intensity on NK cells. hDOC2 = DOC2-RHAXRKA FR2; hIgG1 Iso = human IgG1 isotype control antibody. [Figure 16] This figure shows that DOC2-RHAXRKA FR2 downregulates the median CD16 fluorescence intensity on NK cells. hDOC2 = DOC2-RHAXRKA FR2; hIgG1 Iso = human IgG1 isotype control antibody. [Modes for carrying out the invention]

[0007] definition This disclosure relates to an antibody that specifically binds to CCR2. The term "CCR2" refers to a protein known as CC motif chemokine receptor 2, also known as the CD192 or MCP-1 receptor. Human CCR2 exists in two major isoforms, CCR2a and CCR2b, produced by alternative splicing. CCR2a and CCR2b differ only in their cytosolic C-terminal regions. The extracellular portions accessible to therapeutic antibodies and antibody fragments are identical in both isoforms. Human CCR2b has the amino acid sequence (UniProtKB / Swiss-Prot:P41597). MLSTSRSRFIRNTNESGEEVTTFFDYDYGAPCHKFDVKQIGAQLLPPLYSLVFIFGFVGNMLVVLILINCKKLKCLTDIYLLNLAISDLLFLITLPLWAHSAANEWVFGNAMCKLFTGLYHIGYFGGIFFIILLTIDRYLAIVHAVFALKARTVTFGVVTSVITWLVAVFASVPGIIFTKCQKEDSVYVCGPYFPRGWNNFHTIMRNILGLVLPLLIMVICYSGILKTLLRCRNEKKRHRAVRVIFTIMIVYFLFWTPYNIVILLNTFQEFFGLSNCESTSQLDQATQVTETLGMTHCCINPIIYAFVGEKFRRYLSVFFRKHITKRFCKQCPVFYRETVDGVTSTNTPSTGEQEVSAGL(Sequence ID 53)

[0008] The marmoset monkey (common marmoset (Callithrix jacchus)) CCR2b has the following amino acid sequence. MLSTSHSRFIRNTESGEEVTTIFDYDYGAPCHKFDVKQIGAQLLPPLYSLVFIFGFVGNMLVVLILINCKKLKSLTDIYLLNLAVSDLLFLITLPLWAHSAANEWVFGNAVCKLFTGLYHIGYFGGIFFIILLTIDRYLAIVHAVFALKARTVTFGVVTSVITWFVAVFASVPGIIFTKSQKEDSVYVCGPYFPRGWNHFHTIMRNLLGLVLPLLVMIICYSGILKTLLRCRNEKKRHRAVRLIFTIMIVYFLFWTPYNIVVLLNTFQEFFGLSNCESTSQLDQATQVTETLGMTHCCINPIIYAFVGEKFRRYLSVFFRKHIAKRFCKQCPVFYRETVDGVTSTNTPSTGEQEVSAGL (Sequence ID 54)

[0009] The rhesus macaque (Macaca mulatta) CCR2b has the following amino acid sequence. MLSTSRSRFIRNTNGSGEEVTTFFDYDYGAPCHKFDVKQIGAQLLPPLYSLVFIFGFVGNMLVVLILINCKKLKSLTDIYLLNLAISDLLFLITLPLWAHSAANEWVFGNAMCKLFTGLYHIGYLGGIFFIILLTIDRYLAIVHAVFALKARTVTFGVVTSVITWLVAVFASVPGIIFTKCQEEDSVYICGPYFPRGWNNFHTIMRNILGLVLPLLIMVICYSGILKTLLRCRNEKKRHRAVRLIFTIMIVYFLFWTPYNIVILLNTFQEFFGLSNCESTRQLDQATQVTETLGMTHCCINPIIYAFVGEKFRRYLSMFFRKYITKRFCKQCPVFYRETVDGVTSTNTPSTAEQEVSVGL (SEQ ID NO: 55) CCR2 is the major functional receptor for MCP-1. Its binding to MCP-1 mediates chemotaxis and migration of monocytes and macrophages. The term "MCP-1" refers to the protein known as monocyte chemoattractant protein 1. It is also known as CCL2 (C-C motif chemokine ligand 2). Human MCP-1 has the amino acid sequence of (UniProtKB / Swiss-Prot: P13500). MKVSAALLCLLLIAATFIPQGLAQPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVAKEICADPKQKWVQDSMDHLDKQTQTPKT (SEQ ID NO: 56)

[0010] As used herein, the term “antibody” refers to a protein that interacts with an antigen, comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (hereinafter abbreviated as “VH”) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (hereinafter abbreviated as “VL”) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q). The term "antibody" includes, for example, monoclonal antibodies, human antibodies, humanized antibodies, camelized antibodies, and chimeric antibodies. Antibodies may be any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., Igd, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. Both the light and heavy chains are divided into regions of structural and functional homology.

[0011] As used herein, the term “antibody fragment” refers to one or more portions of an antibody that possess the ability to specifically interact with an antigen (e.g., by binding, steric hindrance, or stabilization of spatial distribution). Examples of binding fragments include, but are not limited to, Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; F(ab)2 fragments, bivalent fragments containing two Fab fragments linked by disulfide crosslinking at a hinge region; Fd fragments consisting of VH and CH1 domains; Fv fragments consisting of VL and VH domains of a single arm of the antibody; dAb fragments consisting of a VH domain (Ward et al., (1989) Nature 341:544-546); and isolated complementarity-determining regions (CDRs). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked by a synthetic linker using recombination, thereby allowing the VL and VH regions to pair up and form a single protein chain that forms a monovalent molecule (known as single-chain Fv (scFv); see, for example, Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. 85:5879-5883). Such single-chain antibodies are also intended to be included in the term “antibody fragment.” These antibody fragments are obtained using conventional techniques known to those skilled in the art, and these fragments are screened for utility in the same manner as intact antibodies. Antibody fragments can also be incorporated into single-domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, for example, Hollinger and Hudson, (2005) Nature Biotechnology 23:1 126-1 136). Antibody fragments can be grafted onto polypeptide-based scaffolds, such as fibronectin type III (Fn3) (see U.S. Patent No. 6,703,199 describing fibronectin polypeptide monobodies).An antibody fragment can be incorporated into a single-chain molecule containing a pair of tandem Fv segments (VH-CH1-VH-CH1) that together with a complementary light chain polypeptide form a pair of antigen-binding sites (Zapata et al., (1995) Protein Eng. 8:1057-1062; and U.S. Patent No. 5,641,870).

[0012] The structure and location of immunoglobulin variable domains, e.g., CDRs, can be defined using known numbering schemes, e.g., the Kabat numbering scheme, the Chothia numbering scheme, or a combination of Kabat and Chothia (see, e.g., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services (1991), eds. Kabat et al.; Lazikani et al., (1997) J. Mol. Bio. 273:927-948; Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th edit., NIH Publication no. 91-3242 U.S. Department of Health and Human Services; Chothia et al., (1987) J. Mol. Biol. 196:901-917; Chothia et al., (1989) Nature 342:877-883; and Al-Lazikani et al., (1997) J. Mol. Biol. 273:927-948; Annals of the New York Academy of Sciences, 764, 47-49 (1995); Nucleic Acids Research, 25, 206-211 (1997)). As used herein, “human antibody” or “human antibody fragment” refers to antibodies and antibody fragments having variable regions in which both the framework region and the CDR region are derived from human sequences. Human antibodies can also be isolated from synthetic libraries or transgenic mice (e.g., xenomouse), provided that each system produces antibodies having variable regions in which both the framework region and the CDR region are derived from human sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from such a sequence. Human origins include, for example, human germline sequences, or variant versions of human germline sequences, or antibodies containing consensus framework sequences derived from human framework sequence analysis, such as that described in Knappik et al., (2000) J Mol Biol 296:57-86.

[0013] In this specification, a "humanized antibody" or "humanized antibody fragment" is defined as an antibody molecule having a constant antibody region derived from a human-derived sequence, with only the variable antibody region, part thereof, or CDR derived from another species. For example, a humanized antibody may be CDR-grafted, where the CDR of the variable domain is of non-human origin, while one or more frameworks of the variable domain are of human origin, and the constant domain (if present) is of human origin. The terms "chimeric antibody" or "chimeric antibody fragment" are defined herein as an antibody molecule having a constant antibody region derived from or corresponding to a sequence found in one species and a variable antibody region derived from another species. Preferably, the constant antibody region is derived from or corresponds to a sequence found in humans, and the variable antibody region (e.g., VH, VL, CDR, or FR region) is derived from a sequence found in a non-human animal, such as a mouse, rat, rabbit, or hamster.

[0014] The term "isolated antibody" refers to an antibody or antibody fragment that substantially contains no other antibodies or antibody fragments having different antigen specificities. Furthermore, an isolated antibody or antibody fragment may substantially contain no other cellular material and / or chemical substances. Therefore, in some embodiments, the antibody provided is an isolated antibody separated from an antibody having different specificities. The isolated antibody may be a monoclonal antibody. The isolated antibody may be a recombinant monoclonal antibody. However, an isolated antibody that specifically binds to a target epitope, isoform, or variant may cross-reactive to other relevant antigens from other species (e.g., species homologs).

[0015] As used herein, the term “recombinant antibody” includes all antibodies prepared, expressed, produced, or isolated by means not found in nature. For example, antibodies isolated from host cells transformed to express the antibody, antibodies selected and isolated from a recombinant combinatorial human antibody library, and antibodies prepared, expressed, produced, or isolated by any other means including sequence splicing all or part of a human immunoglobulin gene to another DNA sequence, or antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomes to human immunoglobulin genes, or hybridomas prepared therefrom. Preferably, such recombinant antibodies have a variable region in which the framework and CDR region are derived from a human germline immunoglobulin sequence. However, in certain embodiments, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, if transgenic animals for human Ig sequences are used, in vitro somatic mutagenesis), and therefore the amino acid sequences of the VH and VL regions of the recombinant antibody are derived from and related to human germline VH and VL sequences, but may not be naturally present in the human antibody germline repertoire in vivo. The recombinant antibody may also be a monoclonal antibody. In one embodiment, the antibodies and antibody fragments disclosed herein are isolated from a HuCAL library (Rothe et al, J. Mol. Biol. (2008) 376, 1 182-1200).

[0016] As used herein, binding specificity is a relative rather than an absolute characteristic; therefore, if an antibody can distinguish an antigen from one or more reference antigens, the antibody is said to "binds specifically to," "specifically binds to," "be specific to," or "specifically recognize" an antigen such as human CCR2. For example, a standard ELISA assay or a standard flow cytometry assay can be performed. Scoring can be done by standard color development (e.g., a secondary antibody with horseradish peroxidase and tetramethylbenzidine with hydrogen peroxide) or by binding of a secondary antibody labeled with PE or another dye or marker. The reaction in a particular well is scored, for example, by optical density (OD) at 450 nm or by mean fluorescence intensity (MFI) in flow cytometry. A typical background (= negative reaction) may be 0.1 OD, and a typical positive reaction may be 1 OD. Background and positive reaction MFIs are highly dependent on instrument settings. The positive / negative difference can exceed 10-fold. Typically, the determination of binding specificity is performed by using a set of about 3-5 unrelated antigens, such as powdered milk, BSA, and transferrin, rather than a single reference antigen. Various antigen-negative cells can be used for flow cytometry. However, antibodies that specifically bind to an antigen may exhibit cross-reactivity to their respective orthologous antigens from other species (e.g., species homologs). In certain embodiments, such cross-reactivity to orthologous antigens is rather preferred.

[0017] As used herein, antibodies are “cross-reactive” when binding to orthologous antigens of other species. For example, an antibody is cross-reactive when binding to human CCR2 and marmoset CCR2. As used herein, the term "affinity" refers to the strength of the interaction between a polypeptide and its target at a single site. Within each site, the binding region of the polypeptide interacts with its target at multiple sites via weak non-covalent forces, and the more interactions there are, the stronger the affinity. The term "epitope" includes any proteinaceous region that is specifically recognized by an antibody or an antibody fragment, or otherwise interacts with a molecule. Generally, an epitope is a chemically active surface group of a molecule, such as an amino acid or a carbohydrate or sugar side chain, and may generally have specific three-dimensional structural properties as well as specific charge properties. As will be understood by those skilled in the art, anything to which an antibody can specifically bind may, in effect, be an epitope. The term "domain" or "protein domain" refers to a region of a protein polypeptide chain that forms a functional unit and / or independently forms a three-dimensional structure. For example, ECL2 of CCR2 is a protein domain.

[0018] The “compositions” of this disclosure can be used for therapeutic or preventive purposes. Accordingly, this disclosure includes pharmaceutical compositions comprising antibodies or antibody fragments disclosed herein and pharmaceutically acceptable carriers or excipients therefor. In relevant embodiments, this disclosure provides methods for treating inflammatory diseases, autoimmune diseases, hematological malignancies, and potentially other diseases. Such methods include the step of administering an effective amount of a pharmaceutical composition containing the antibodies or antibody fragments described herein to a subject requiring administration. This disclosure provides a therapeutic method comprising administering a therapeutically effective dose of an antibody or antibody fragment disclosed herein to a subject requiring such treatment. As used herein, “therapeutic dose” or “effective dose” refers to the amount of CCR2 antibody required to induce a desired biological response. According to this disclosure, a therapeutically effective dose is the amount of CCR2 antibody required to treat and / or prevent a disease. "Administered" or "administered" includes, but is not limited to, delivery of a drug in an injectable form, such as intravenous, intramuscular, intradermal or subcutaneous route or mucosal route, for example, as a nasal spray or aerosol for inhalation, or as an ingestible solution, capsule or tablet. Preferably, administration is in an injectable form.

[0019] As used herein, “treatment,” “to treat,” or “to treat” refers to a clinical intervention aimed at altering the natural course of a disease in the subject being treated, and may be performed for preventive purposes or during the course of a clinicopathological condition. Desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, relief of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, slowing of the rate of disease progression, improvement or temporary relief of the disease state, and remission or improved prognosis. In some embodiments, antibodies or antibody fragments according to this disclosure are used to delay the onset of a disease or to slow the progression of a disease. "Prevention" or "prevention" refers to reducing the risk of acquiring or developing a disease (i.e., preventing the development of at least one of the clinical symptoms of a disease in a person who may be exposed to a disease-causing agent or who is susceptible to the disease before the disease develops). "Prevention" also refers to methods aimed at preventing the development of a disease or its symptoms, or methods aimed at delaying the development of a disease or its symptoms. When used in this context, "subject" or "species" refers to any mammal, including rodents such as mice or rats, and primates such as crab-eating macaques (Macaca fascicularis), marmosets (common marmosets), rhesus macaques (Macaca mulatta), or humans (Homo sapiens). Preferably, the subject is a primate, and most preferably a human.

[0020] The term "effector function" refers to the biological activity attributable to the Fc region of an antibody, which varies depending on the antibody isotype. Non-limiting examples of antibody effector functions include C1q binding and complement-dependent cell-mediated cytotoxicity (CDC); Fc receptor binding and antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent phagocytosis (ADCP); downregulation of cell surface receptors (e.g., B cell receptors); and direct cell activation or direct cell inhibition. Antibody-dependent cell-mediated cytotoxicity, or ADCC, refers to a form of cytotoxicity in which antibodies bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages) allow these cytotoxic effector cells to specifically bind to target cells containing antigens, and subsequently kill the target cells with cytotoxins. NK cells, the primary cells for mediating ADCC, express only FcγRIII, while monocytes / macrophages express FcγRI, FcγRII, and FcγRIII.

[0021] "Complement-dependent cell injury" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated when the first component of the complement system (C1q) binds to an antibody (of the appropriate subclass) of this disclosure that is bound to its congener antigen. "Antibody-dependent phagocytosis" or "ADCP" refers to the mechanism by which antibody-coated target cells are eliminated through internal migration by phagocytic cells such as macrophages or dendritic cells. Throughout this specification, unless otherwise required by context, the words “comprise,” “have,” and “include,” as well as their respective variations such as “comprises,” “comprising,” “has,” “having,” “includes,” and “including,” mean the inclusion of the element or integer or group of elements or integers described, but not the exclusion of any other element or integer or group of elements or integers. As used herein, the terms “engineered” or “modified” include the manipulation of nucleic acids or polypeptides by synthetic means (e.g., by recombinant techniques, by in vitro peptide synthesis, by enzymatic or chemical coupling of peptides, or by some combination of these techniques). Preferably, the antibodies or antibody fragments according to this disclosure are engineered or modified to improve one or more properties such as antigen binding, stability, half-life, effector function, immunogenicity, and safety.

[0022] As used herein, “variant” refers to a polypeptide that differs from a reference polypeptide by one or more modifications, such as amino acid substitution, insertion, or deletion. A variant polypeptide typically retains most of the properties of the reference polypeptide, such as binding to a target antigen, but introduces novel additional features or properties, such as having a higher affinity for a target antigen compared to the reference polypeptide, or being a humanized version of the reference polypeptide. As used herein, the term “amino acid mutation” encompasses amino acid substitutions, deletions, insertions, and modifications. Any combination of substitutions, deletions, insertions, and modifications can be performed, provided that the final construct has a desired characteristic, such as reduced binding to the Fc receptor. Deletions and insertions of amino acid sequences include deletions and insertions of amino acid residues at the N-terminus and / or C-terminus. A specific amino acid mutation is an amino acid substitution. Amino acid substitutions include substitutions with amino acids that do not exist naturally, or with naturally occurring amino acid derivatives of 20 standard amino acids. Amino acid mutations can be produced using genetic or chemical methods known in the art. Genetic methods include site-directed mutagenesis, PCR, and gene synthesis. Methods other than genetic engineering, such as chemical modification, that alter the side chain groups of amino acid residues may also be useful. In this specification, various names can be used to indicate the same amino acid mutation. For example, a substitution of glycine to alanine at position 327 of the Fc region can be represented as 237A, G337, G337A, or Gly329Ala.

[0023] As used herein, the term "EC50" refers to the concentration of antibody or antibody fragment that induces an intermediate response between baseline and maximum in the assay. Therefore, it represents the antibody or ligand concentration at which 50% of the maximum effect is observed. The terms “inhibit” or “to inhibit,” “reduce” or “to reduce,” or “neutralize” or “to neutralize” refer to a reduction or cessation of any phenotypic characteristic (e.g., binding or biological activity or function), or a reduction or cessation of the incidence, degree, or possibility of such characteristic. “Inhibition,” “reduction,” or “neutralization” does not need to be complete, as long as it is detectable using a suitable assay. In some embodiments, “reduce,” “inhibit,” or “neutralize” means the ability to cause a reduction of 20% or more. In another embodiment, “reduce,” “inhibit,” or “neutralize” means the ability to cause a reduction of 50% or more. In yet another embodiment, “reduce,” “inhibit,” or “neutralize” means the ability to cause an overall reduction of 75%, 85%, 90%, 95%, or more.

[0024] As used herein, the term “antagonist” antibody refers to an antibody or antibody fragment that interacts with an antigen to partially or completely inhibit or neutralize the biological activity or function or any other phenotypic characteristics of the target antigen. A "wild-type" protein is a naturally occurring version or variant of a protein. The amino acid sequence of a wild-type protein, for example, the Fc region of a human IgG1 antibody, is the amino acid sequence of a naturally occurring protein. Due to allotyping, multiple amino acid sequences may exist for a wild-type protein. For example, there are several allotypes of the naturally occurring human IgG1 heavy chain constant region (see, e.g., Jeffries et al. (2009) mAbs 1:1). The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. The Fc region of an immunoglobulin generally contains two constant domains: the CH2 domain and the CH3 domain. While the boundaries of the Fc region of an IgG heavy chain may vary slightly, the human IgG heavy chain Fc region is typically defined as extending from Cys226 or Pro230 to the C-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region follows the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0025] Embodiments of the present invention polypeptide In certain embodiments, the disclosure relates to a humanized antibody or antibody fragment specific to human CCR2, the antibody or antibody fragment comprising the HCDR1 region of SEQ ID NO: 2, the HCDR2 region of SEQ ID NO: 3, the HCDR3 region of SEQ ID NO: 4, the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9. In certain embodiments, the disclosure relates to a humanized antibody or antibody fragment specific to human CCR2, the antibody or antibody fragment comprising the HCDR1 region of SEQ ID NO: 12, the HCDR2 region of SEQ ID NO: 13, the HCDR3 region of SEQ ID NO: 14, the LCDR1 region of SEQ ID NO: 17, the LCDR2 region of SEQ ID NO: 18, and the LCDR3 region of SEQ ID NO: 19. In certain embodiments, the disclosure relates to a humanized antibody or antibody fragment specific to human CCR2, the antibody or antibody fragment comprising the VH of SEQ ID NO: 21 and the VL of SEQ ID NO: 23.

[0026] In certain embodiments, the disclosure relates to a humanized antibody or antibody fragment specific to human CCR2, the antibody or antibody fragment comprising SEQ ID NO: 25 (VH) and SEQ ID NO: 29 (VL). In certain embodiments, the disclosure relates to a humanized antibody or antibody fragment specific to human CCR2, wherein the antibody or antibody fragment includes VH of SEQ ID NO: 27 and VL of SEQ ID NO: 29. In certain embodiments, the disclosure relates to a humanized antibody or antibody fragment specific to human CCR2, wherein the antibody or antibody fragment includes VH of SEQ ID NO: 21 and VL of SEQ ID NO: 31. In certain embodiments, the disclosure relates to a humanized antibody or antibody fragment specific to human CCR2, the antibody or antibody fragment comprising the VH of SEQ ID NO: 33 and the VL of SEQ ID NO: 23. In certain embodiments, the disclosure relates to a humanized antibody or antibody fragment specific to human CCR2, the antibody or antibody fragment comprising SEQ ID NO: 33 (VH) and SEQ ID NO: 31 (VL). In certain embodiments, the disclosure relates to a humanized antibody or antibody fragment specific to human CCR2, wherein the antibody or antibody fragment includes VH of SEQ ID NO: 33 and VL of SEQ ID NO: 35. In certain embodiments, the disclosure relates to a humanized antibody or antibody fragment specific to human CCR2, the antibody or antibody fragment comprising the VH of SEQ ID NO: 37 and the VL of SEQ ID NO: 29.

[0027] In certain embodiments, the disclosure relates to a humanized antibody or antibody fragment specific to human CCR2, the antibody or antibody fragment comprising SEQ ID NO: 37 (VH) and SEQ ID NO: 39 (VL). In certain embodiments, the disclosure relates to a humanized antibody or antibody fragment specific to human CCR2, the antibody or antibody fragment comprising the VH of SEQ ID NO: 21 and the VL of SEQ ID NO: 41. In certain embodiments, the disclosure relates to a humanized antibody or antibody fragment specific to human CCR2, wherein the antibody or antibody fragment includes the VH of SEQ ID NO: 21 and the VL of SEQ ID NO: 43. In certain embodiments, the disclosure relates to a humanized antibody or antibody fragment specific to human CCR2, the antibody or antibody fragment comprising the VH of SEQ ID NO: 21 and the VL of SEQ ID NO: 45. In certain embodiments, the disclosure relates to a humanized antibody or antibody fragment specific to human CCR2, the antibody or antibody fragment comprising the VH of SEQ ID NO: 21 and the VL of SEQ ID NO: 47. In certain embodiments, the CCR2-specific humanized antibody is of the IgG1 class. In certain embodiments, the constant heavy chain domain is the G1m3 allotype. In certain embodiments, the humanized antibody contains the amino acid sequence of SEQ ID NO: 49. In certain embodiments, the constant light chain domain is the Km3 allotype. In certain embodiments, the humanized antibody contains the amino acid sequence of SEQ ID NO: 51.

[0028] In certain embodiments, the disclosure relates to humanized antibodies specific to human CCR2, the antibodies comprising SEQ ID NO: 21 (VH) and SEQ ID NO: 43 (VL), and the humanized antibodies are of the IgG1 isotype. In certain embodiments, the disclosure relates to humanized antibodies specific to human CCR2, the antibodies comprising SEQ ID NO: 21 (VH) and SEQ ID NO: 43 (VL), and the humanized antibodies are of the G1m3 allotype. In certain embodiments, the disclosure relates to humanized antibodies specific to human CCR2, the antibodies comprising SEQ ID NO: 21 (VH) and SEQ ID NO: 43 (VL), and the humanized antibodies are Km3 allotypes. In certain embodiments, the disclosure relates to humanized antibodies specific to human CCR2, the antibodies comprising SEQ ID NO: 21 (VH) and SEQ ID NO: 43 (VL), and the humanized antibodies being G1m3 allotype and Km3 allotype. In certain embodiments, the disclosure relates to a humanized antibody or antibody fragment that binds to a first extracellular loop (SEQ ID NO: 53) and a second extracellular loop (SEQ ID NO: 54) of human CCR2.

[0029] In certain embodiments, the disclosure relates to a humanized antibody or antibody fragment that binds to a first extracellular loop (SEQ ID NO: 53) and a second extracellular loop (SEQ ID NO: 55) of marmoset CCR2. In certain embodiments, the disclosure relates to a humanized antibody or antibody fragment that is cross-reactive with marmoset CCR2. In certain embodiments, the disclosure relates to a humanized antibody or antibody fragment that does not bind to rhesus monkey CCR2. In certain embodiments, the disclosure relates to a humanized antibody or antibody fragment, which specifically binds to a polypeptide comprising the amino acid sequence of SEQ ID NO: 53 and a polypeptide comprising the amino acid sequence of SEQ ID NO: 54. In certain embodiments, the disclosure relates to a humanized antibody or antibody fragment that specifically binds to a polypeptide containing the amino acid sequence of SEQ ID NO: 53 and a polypeptide containing the amino acid sequence of SEQ ID NO: 54, but does not specifically bind to a polypeptide containing the amino acid sequence of SEQ ID NO: 55.

[0030] In certain embodiments, the disclosure relates to a humanized antibody or antibody fragment specific to human CCR2, the antibody or antibody fragment comprising SEQ ID NO: 21 (VH) and SEQ ID NO: 43 (VL), the antibody or antibody fragment binding to a first extracellular loop of human CCR2 (SEQ ID NO: 53) and a second extracellular loop of human CCR2 (SEQ ID NO: 54). In certain embodiments, the disclosure relates to a humanized antibody or antibody fragment specific to human CCR2, wherein the antibody or antibody fragment comprises VH of SEQ ID NO: 21 and VL of SEQ ID NO: 43, and the antibody or antibody fragment binds to a first extracellular loop of marmoset CCR2 (SEQ ID NO: 53) and a second extracellular loop of marmoset CCR2 (SEQ ID NO: 55). In certain embodiments, the disclosure relates to a humanized antibody or antibody fragment specific to human CCR2, wherein the antibody or antibody fragment comprises the VH of SEQ ID NO: 21 and the VL of SEQ ID NO: 43, and the antibody or antibody fragment is cross-reactive with marmoset CCR2. In certain embodiments, the disclosure relates to a humanized antibody or antibody fragment specific to human CCR2, wherein the antibody or antibody fragment comprises the VH of SEQ ID NO: 21 and the VL of SEQ ID NO: 43, and the antibody or antibody fragment does not bind to rhesus monkey CCR2.

[0031] In certain embodiments, the disclosure relates to a humanized antibody or antibody fragment specific to human CCR2, wherein the antibody or antibody fragment comprises the VH of SEQ ID NO: 21 and the VL of SEQ ID NO: 43, and the antibody or antibody fragment specifically binds to a polypeptide comprising the amino acid sequence of SEQ ID NO: 53 and a polypeptide comprising the amino acid sequence of SEQ ID NO: 54. In certain embodiments, the Disclosure relates to a humanized antibody or antibody fragment specific to human CCR2, wherein the antibody or antibody fragment comprises the VH of SEQ ID NO: 21 and the VL of SEQ ID NO: 43, and the antibody or antibody fragment specifically binds to a polypeptide containing the amino acid sequence of SEQ ID NO: 53 and a polypeptide containing the amino acid sequence of SEQ ID NO: 54, but does not specifically bind to a polypeptide containing the amino acid sequence of SEQ ID NO: 55. Nucleic acids, vectors, and host cells In certain embodiments, this disclosure relates to a plurality of nucleic acids encoding humanized antibodies or antibody fragments specific to human CCR2 as disclosed herein. In certain embodiments, this disclosure relates to a plurality of nucleic acid sequences encoding humanized antibodies or antibody fragments specific to human CCR2 as disclosed herein. In certain embodiments, the Disclosure relates to a nucleic acid encoding a humanized antibody or antibody fragment specific to the disclosed human CCR2, wherein the humanized antibody or antibody fragment comprises a variable heavy chain containing the amino acid sequence of SEQ ID NO: 21 and a variable light chain containing the amino acid sequence of SEQ ID NO: 43.

[0032] In certain embodiments, the Disclosure relates to a nucleic acid encoding a humanized antibody or antibody fragment specific to the disclosed human CCR2, wherein the humanized antibody or antibody fragment comprises a variable heavy chain consisting of the amino acid sequence of SEQ ID NO: 21 and a variable light chain consisting of the amino acid sequence of SEQ ID NO: 43. In certain embodiments, the Disclosure relates to a plurality of nucleic acids encoding a humanized antibody or antibody fragment specific to the disclosed human CCR2, wherein the humanized antibody or antibody fragment comprises a variable heavy chain containing the amino acid sequence of SEQ ID NO: 21 and a variable light chain containing the amino acid sequence of SEQ ID NO: 43. In certain embodiments, the Disclosure relates to a plurality of nucleic acids encoding a humanized antibody or antibody fragment specific to the disclosed human CCR2, wherein the humanized antibody or antibody fragment comprises a variable heavy chain consisting of the amino acid sequence of SEQ ID NO: 21 and a variable light chain consisting of the amino acid sequence of SEQ ID NO: 43. In certain embodiments, the disclosure relates to a nucleic acid encoding a humanized antibody or antibody fragment specific to the disclosed human CCR2, wherein the nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 22 and the nucleic acid sequence of SEQ ID NO: 44.

[0033] In certain embodiments, the disclosure relates to a plurality of nucleic acids encoding humanized antibodies or antibody fragments specific to the disclosed human CCR2, wherein the nucleic acids include the nucleic acid sequences of SEQ ID NO: 22 and SEQ ID NO: 44. In certain embodiments, the Disclosure relates to a vector comprising a nucleic acid composition containing a nucleic acid sequence or a plurality of nucleic acid sequences encoding a humanized antibody or antibody fragment specific to human CCR2 according to the Disclosure. In certain embodiments, the disclosure relates to a host cell comprising a vector or a vector composition comprising a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding a humanized antibody or antibody fragment specific to human CCR2 according to the disclosure. In one embodiment, the disclosure relates to a host cell comprising a vector composition. In one embodiment, the host cells according to this disclosure can express a humanized antibody or antibody fragment specific to human CCR2 encoded by a vector composition or nucleic acid composition.

[0034] In further embodiments, the host cell is an isolated host cell. In further embodiments, the host cell is a mammalian cell. In one embodiment, the mammalian cell is a human cell. In another embodiment, the mammalian cell is a CHO cell. In one embodiment, the cell is a HEK cell. In another embodiment, the cell is a PERC.6 cell. In one embodiment, the cell is an HKB11 cell. Those skilled in the art will understand that nucleic acid sequences or multiple nucleic acid sequences encoding the heavy and / or light chains of the antibody or antibody fragment of this disclosure can be cloned into different vectors or the same vector.

[0035] The vector can be introduced into suitable host cells, such as prokaryotic (e.g., bacteria) or eukaryotic (e.g., yeast or mammalian) cells, by methods known in the art (see, for example, “Current Protocol in Molecular Biology”, Ausubel et al. (eds.), Greene Publishing Assoc and John Wiley Interscience, New York, 1989 and 1992). Numerous cloning vectors are known to those skilled in the art, and the selection of a suitable cloning vector is a matter of preference. The gene can be placed under the control of a promoter, a ribosome binding site (for bacterial expression), and optionally an operator (collectively referred to herein as “regulatory” elements), and as a result, the nucleic acid sequence encoding the desired protein is transcribed into RNA in host cells transformed with the vector containing this expression construct. The encoding sequence may or may not include a signal peptide or a leader sequence. Upon expression in host cells, the antibody or antibody fragment of this disclosure is obtained. These steps can be achieved in a variety of ways, as known to those skilled in the art. Generally, such a process typically involves transforming or transfecting suitable host cells with a nucleic acid composition or vector composition or infectious particles encoding an antibody or antibody fragment. Furthermore, such a process typically includes culturing the host cells under conditions suitable for proliferation (growth, development) and culturing under conditions suitable for the production (expression, synthesis) of the encoded antibody or antibody fragment. Culturing host cells under conditions suitable for proliferation or expression is typically achieved in the presence of a medium containing components suitable for inducing cell proliferation or expression. In particular, in some embodiments, the method for producing the antibody or antibody fragment of this disclosure further includes the step of isolating and purifying the produced antibody or antibody fragment from the host cells or medium. If the expression system secretes the protein into the growth medium, the protein can be purified directly from the medium. If the protein is not secreted, it is isolated from the cell lysate or recovered from the cell membrane fraction. The selection of appropriate growth conditions and recovery methods is within the scope of the art.Subsequently, the antibodies or antibody fragments of this disclosure can be purified by some techniques known to those skilled in the art.

[0036] In certain embodiments, the Disclosure refers to a method for producing a humanized antibody or antibody fragment specific to human CCR2 according to the Disclosure. In certain embodiments, a method for producing a humanized antibody or antibody fragment according to the Disclosure is provided, the method comprising culturing a host cell containing a vector or vector composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding the humanized antibody or antibody fragment according to the Disclosure under conditions suitable for the expression of the humanized antibody or antibody fragment, and isolating the humanized antibody or antibody fragment from the host cell or host cell culture medium. The humanized antibodies or antibody fragments disclosed herein can be purified by techniques known in the art, such as high-performance liquid chromatography (HPLC), ion exchange chromatography, gel electrophoresis, affinity chromatography, and size exclusion chromatography. The conditions used to purify a particular antibody or antibody fragment depend in part on factors such as net charge, hydrophobicity, and hydrophilicity, and will be apparent to those skilled in the art. For affinity chromatography purification, an antibody, ligand, receptor, or antigen to which the antibody or antibody fragment binds can be used. For example, a matrix containing protein A or protein G can be used for affinity chromatography purification of an antibody or antibody fragment according to this disclosure. The purity of the antibody or antibody fragment can be determined by any of the various known analytical methods, including gel electrophoresis and high-pressure liquid chromatography.

[0037] therapeutic use In one embodiment, the disease is associated with the undesirable presence of CCR2, particularly human CCR2. In another embodiment, the disease is associated with the undesirable presence of CCR2-positive cells, particularly human CCR2-positive cells. Examples of human CCR2-positive cells include CD16-negative monocytes, basophils, and plasmacytoid dendritic cells. Additionally, approximately 20% of CD4-positive and CD8-positive T cells express CCR2. In one embodiment, the disease being treated is an autoimmune or inflammatory disease. Non-limiting examples of autoimmune or inflammatory diseases include autoinflammatory diseases such as rheumatoid arthritis (RA), psoriasis, psoriatic arthritis, systemic lupus erythematosus (SLE), lupus nephritis, type 1 diabetes mellitus, Graves' disease, inflammatory bowel disease (IBD), Crohn's disease (CD), ulcerative colitis (UC), irritable bowel syndrome, multiple sclerosis (MS), Guillain-Barré syndrome, familial Mediterranean fever (FMF), cryopyrin-associated periodic syndromes (CAPS), IL-1 receptor antagonist deficiency (DIRA), hyperimmune IgD syndrome (HIDS), autoimmune myocarditis, Kawasaki disease, coronary artery disease, chronic obstructive pulmonary disease (COPD), interstitial lung disease, and autoimmune myocarditis. Examples include autoimmune thyroiditis, scleroderma, systemic sclerosis, osteoarthritis, atopic dermatitis, vitiligo, graft-versus-host disease, Sjögren's syndrome, autoimmune nephritis, Goodpasture syndrome, chronic inflammatory demyelinating polyneuropathy, ANCA-associated vasculitis, uveitis, scleroderma, bullous pemphigoid, Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, cystic fibrosis, gout, age-related macular degeneration, allergies, asthma, antiphospholipid syndrome (APS), atherosclerosis, C3 glomerulopathy and IgA nephropathy, ischemia / reperfusion injury, peritonitis, sepsis, and other autoimmune diseases resulting from either acute or chronic inflammation.

[0038] In one embodiment, the disease being treated is a proliferative disorder. In a particular embodiment, the disease is cancer. Non-limiting examples of cancer include hematological malignancies such as chronic myelomonocytic leukemia (CMML), acute myeloid leukemia (AML), myelodysplastic syndrome, and mastocytosis, as well as non-hematological malignancies such as bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, hematological malignancies, sarcomas, skin cancers, squamous cell carcinomas, bone cancers, melanomas, renal cell carcinomas, and kidney cancers. In one embodiment, the present disclosure provides a method for treating a disease. In one embodiment, the present disclosure provides a method for treating a disease, comprising administering a humanized antibody or antibody fragment of the present disclosure to a patient. In one embodiment, the Disclosure provides a method for treating a disease, comprising administering a humanized antibody or antibody fragment of the Disclosure to a subject in need of treatment for the disease. In one embodiment, the present disclosure provides a method for preventing a disease.

[0039] In one embodiment, the Disclosure provides a method for preventing a disease, which includes administering a humanized antibody or antibody fragment of the Disclosure to a target. In one embodiment, the Disclosure provides a humanized antibody or antibody fragment according to the Disclosure for treating a disease. In one embodiment, the Disclosure provides a humanized antibody or antibody fragment according to the Disclosure for use in treating a disease. In one embodiment, the Disclosure provides a humanized antibody or antibody fragment according to the Disclosure for use in treating a disease in a subject requiring treatment of the disease. In one embodiment, the Disclosure provides the use of a humanized antibody or antibody fragment according to the Disclosure for the manufacture of a pharmaceutical. In one embodiment, the Disclosure provides a humanized antibody or antibody fragment according to the Disclosure for use as a pharmaceutical. In one embodiment, the Disclosure provides a humanized antibody or antibody fragment according to the Disclosure for use in medical treatment. In one embodiment, the Disclosure provides a humanized antibody or antibody fragment according to the Disclosure for use as a pharmaceutical for the treatment of an object requiring treatment. In one embodiment, the Disclosure provides a human CCR2-specific humanized and isolated antibody or antibody fragment according to the Disclosure for use in a method of treating a subject having a disease, which involves administering a therapeutically effective amount of the humanized antibody or antibody fragment according to the Disclosure to the subject.

[0040] In one embodiment, the method further comprises administering a therapeutically effective amount of at least one additional therapeutic agent to a target. The target requiring treatment is typically a mammal, more specifically a human. For use in the therapeutic method, the isolated antibody or antibody fragment according to the disclosure is formulated, administered, and given in a manner consistent with good medical practice. The humanized antibodies and antibody fragments provided herein can be used in therapeutic methods. The antibodies or antibody fragments provided herein can be used to treat inflammatory diseases, autoimmune diseases, hematological malignancies and potentially other diseases. In certain embodiments, this disclosure relates to a humanized antibody or antibody fragment specific to human CCR2 for use in medical applications, wherein the antibody or antibody fragment includes the HCDR1 region of SEQ ID NO: 2, the HCDR2 region of SEQ ID NO: 3, the HCDR3 region of SEQ ID NO: 4, the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9. In certain embodiments, the medical use is the treatment of inflammatory diseases, autoimmune diseases, or hematological malignancies. Accordingly, in certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific to human CCR2 for use in the treatment of inflammatory diseases, autoimmune diseases, or hematological malignancies, wherein the antibody or antibody fragment includes the HCDR1 region of SEQ ID NO: 2, the HCDR2 region of SEQ ID NO: 3, the HCDR3 region of SEQ ID NO: 4, the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9.

[0041] In certain embodiments, the disclosure relates to a humanized antibody or antibody fragment specific to human CCR2 for use in medical applications, wherein the antibody or antibody fragment includes the VH of SEQ ID NO: 21 and the VL of SEQ ID NO: 43. In certain embodiments, the Disclosure relates to a humanized antibody or antibody fragment specific to human CCR2 for use in the treatment of inflammatory diseases, autoimmune diseases, or hematological malignancies, wherein the antibody or antibody fragment includes the VH of SEQ ID NO: 21 and the VL of SEQ ID NO: 43. In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific to human CCR2 for use in the treatment of an inflammatory disease, wherein the antibody or antibody fragment includes the VH of SEQ ID NO: 21 and the VL of SEQ ID NO: 43. In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific to human CCR2 for use in the treatment of an autoimmune disease, wherein the antibody or antibody fragment includes the VH of SEQ ID NO: 21 and the VL of SEQ ID NO: 43. In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific to human CCR2 for use in the treatment of hematological malignancies, wherein the antibody or antibody fragment includes the VH of SEQ ID NO: 21 and the VL of SEQ ID NO: 43.

[0042] In certain embodiments, the present disclosure relates to a method for treating a disease or disorder, comprising administering a humanized antibody or antibody fragment containing SEQ ID NO: 21 VH and SEQ ID NO: 43 VL to a patient in need of treatment for the disease or disorder. In certain embodiments, the Disclosure relates to a method for treating inflammatory diseases, autoimmune diseases, or hematological malignancies, comprising administering a humanized antibody or antibody fragment comprising SEQ ID NO: 21 VH and SEQ ID NO: 43 VL to a patient requiring treatment for an inflammatory disease, autoimmune disease, or hematological malignancy. Pharmaceutical composition In certain embodiments, the Disclosure provides a pharmaceutical composition comprising a humanized antibody or antibody fragment and a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition may further comprise at least one other pharmaceutically active compound. The pharmaceutical composition according to this disclosure can be used in the diagnosis, prevention and / or treatment of diseases associated with the undesirable presence of CCR2, particularly human CCR2. The pharmaceutical composition according to this disclosure can be used in the diagnosis, prevention and / or treatment of diseases associated with the undesirable presence of CCR2-positive cells, particularly CCR2-positive human cells. In particular, this disclosure provides a pharmaceutical composition comprising a humanized antibody or antibody fragment according to this disclosure that is suitable for prophylactic, therapeutic and / or diagnostic use in mammals, more specifically in humans.

[0043] Generally, antibodies or antibody fragments according to the present disclosure can be formulated as a pharmaceutical composition comprising at least one antibody or antibody fragment according to the present disclosure, at least one pharmaceutically acceptable carrier or excipient, and optionally one or more further pharmaceutically active compounds. Such formulations may be suitable for oral, parenteral, topical, or inhalation administration. Therefore, a pharmaceutical composition comprising at least one antibody or antibody fragment according to the present disclosure can be administered parenterally, such as intravenously, intramuscularly, or subcutaneously. Alternatively, the antibodies of the present invention can be administered via non-parenteral routes, such as oral or topical administration. In preferred embodiments, a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure is administered intravenously or subcutaneously. In particular, the antibodies or antibody fragments provided herein may be used in combination with one or more pharmaceutically active compounds used or usable for the prevention and / or treatment of diseases involving the target antigen of interest, resulting in or without synergistic effects. Examples of such compounds, as well as routes, methods, and pharmaceutical formulations or compositions for their administration, are readily apparent to clinicians.

[0044] In certain embodiments, the Disclosure provides a pharmaceutical composition comprising a humanized antibody or antibody fragment according to the Disclosure for use in the prevention and / or treatment of diseases associated with the undesirable presence of CCR2, particularly human CCR2. In one embodiment, the Disclosure provides a pharmaceutical composition comprising a humanized antibody or antibody fragment according to the Disclosure for use in the prevention and / or treatment of diseases associated with the undesirable presence of CCR2-positive cells, particularly CCR2-positive human cells. In one embodiment, the Disclosure provides a pharmaceutical composition comprising a humanized antibody or antibody fragment according to the Disclosure for use as a pharmaceutical. In one embodiment, the Disclosure provides a pharmaceutical composition comprising a humanized antibody or antibody fragment according to the Disclosure for use in the prevention and / or treatment of autoimmune diseases and / or inflammatory diseases and / or cancer. In certain embodiments, the Disclosure provides a method for treating autoimmune diseases and / or inflammatory diseases and / or cancer in a subject requiring treatment, using a pharmaceutical composition comprising a humanized antibody or antibody fragment according to the Disclosure.

[0045] Furthermore, the present disclosure provides a method for producing a humanized antibody or antibody fragment in a form suitable for in vivo administration, the method comprising (a) obtaining a humanized antibody or antibody fragment by the method of the present disclosure, and (b) formulating the humanized antibody or antibody fragment with at least one pharmaceutically acceptable carrier or excipient, thereby formulating a preparation of the humanized antibody or antibody fragment for in vivo administration. The pharmaceutical composition of the present disclosure comprises a therapeutically effective amount of one or more humanized antibodies or antibody fragments of the present disclosure dissolved in a pharmaceutically acceptable carrier or excipient. Diagnostic use In certain embodiments, the Disclosure provides the use of a humanized antibody or antibody fragment specific to human CCR2 according to the Disclosure for diagnosing disease. In one embodiment, the Disclosure provides the use of a humanized antibody or antibody fragment according to the Disclosure for detecting CCR2, particularly human CCR2 and / or marmoset CCR2. In one embodiment, the Disclosure provides a method for detecting CCR2 in a subject or sample, comprising the step of contacting the subject or sample with a humanized antibody or antibody fragment specific to human CCR2 according to the Disclosure. In one embodiment, the Disclosure provides a method for detecting disease in a subject, comprising the step of contacting the subject or sample with a humanized antibody or antibody fragment according to the Disclosure. The antibody can also be used to determine the level of CCR2 expression in patient-derived cells. The level of CCR2 expression may be useful as a therapeutic biomarker, for example, for patient stratification. [Examples]

[0046] (Example 1) Production of anti-CCR2 antibody using hybridoma technology Anti-CCR2 antibodies were produced using classical hybridoma techniques. Briefly, BALB / c mice were immunized with CHO cells stably transfected with human CCR2. Splenocytes were fused with X63Ag8 myeloma cells, and hybridomas were screened for specific binding to CCR2-transfected CHO cells and non-binding to CCR5-transfected CHO cells. Hybridomas were cloned and re-cloned using limiting dilution techniques. Of these antibodies, only DOC2 and DOC3 showed cross-reactivity with marmoset CCR2 when tested on primary marmoset monocytes. Furthermore, antibodies DOC2 and DOC3 showed downregulation of binding to CCR2 on marmoset monocytes by MCP-1. The amino acid and nucleic acid sequences of antibodies DOC2 and DOC3 are shown in the table below (CDR by Kabat).

[0047] [Table 1-1] [Table 1-2]

[0048] (Example 2) Preparation of chimeric versions of antibodies DOC2 and DOC3 Chimeric antibodies containing the human IgG1κ constant domain were prepared from antibodies DOC2 and DOC3. Both the DOC-2 and DOC3 chimeric antibodies showed excellent binding to human and marmoset CCR2 using primary human and marmoset PBMCs. The results are shown in Figure 1. The DOC3 antibody chimeric antibody was found to show slightly better binding to human and marmoset PBMCs than the DOC2 chimeric antibody. The antibody AMBA (provided by LifeArc / MRCT) was used as a negative control. The constant region amino acid sequences and nucleic acid sequences of the chimeric variants of DOC2 and DOC3, as well as the humanized variants of the following examples, are as follows.

[0049] [Table 2]

[0050] (Example 3) Classical humanization of DOC2 and DOC3 Next, various humanized derivatives of DOC2 and DOC3 were prepared by applying commonly known strategies and procedures for antibody humanization. The following humanized variable heavy chains were prepared.

[0051] [Table 3-1] [Table 3-2]

[0052] Using the aforementioned humanized variable heavy chain and variable light chain, a total of three antibodies were produced. The antibody DOC2-reformation comprises a fully human constant domain, a variable heavy chain of DOC2RHA (SEQ ID NO: 21), and a variable light chain of DOC2RKA (SEQ ID NO: 23). The antibody DOC3-RH03XRKA contains a fully human constant domain, a variable heavy chain of DOC3RH03 (SEQ ID NO: 25), and a variable light chain of DOC3RKA (SEQ ID NO: 29). The antibody DOC3-RHLXRKA contains a fully human constant domain, a variable heavy chain of DOC3RHL (SEQ ID NO: 27), and a variable light chain of DOC3RKA (SEQ ID NO: 29). The binding of DOC2-reformation to human PBMCs was compared with that of DOC2-chimeras. The antibody AMBA was used as a negative control. The results are shown in Figure 2. Binding of DOC2-reformation was comparable to, and slightly inferior to, that of DOC2-chimeras. The same experiment was performed using the antibodies DOC3-RH03XRKA and DOC3-RHLXRKA, compared to the DOC3 chimeric antibody. The results are shown in Figure 3. DOC3 RH03XRKA and DOC3 RHLXRKA showed reduced binding compared to the DOC3 chimeric antibody, indicating that some binding activity was lost during humanization.

[0053] In the following experiment, the humanized binder was tested for cross-reactivity with marmoset CCR2 using marmoset blood. The results for the humanized DOC2 antibody are shown in Figure 4. The binding activity of the antibody DOC2-reformation against primary marmoset monocytes was significantly reduced compared to that of the DOC2 chimera. This result was surprising because DOC2-reformation showed the same primary human monocyte binding activity as the DOC2 chimera. The results for humanized DOC3 antibodies are shown in Figure 5. The results were even more pronounced. DOC3-RH03XRKA and DOC3-RHLXRKA showed complete loss of binding to primary marmoset monocytes. Taken together, these data suggest that general strategies for humanization are not suitable for obtaining humanized antibodies that bind to human CCR2 and still cross-react with marmoset CCR2. Cross-reactivity with marmoset CCR2 is considered essential for preclinical antibody development, such as collecting safety and other preclinical data in non-human primates.

[0054] (Example 4) Further attempts to humanize DOC2 and DOC3 Next, further humanized derivatives of DOC2 and DOC3 were prepared. The following humanized variable chains were also prepared.

[0055] [Table 4-1] [Table 4-2]

[0056] The following antibodies: • The antibody DOC2-RHAXRKA, also known as DOC2-reformation, contains the fully human constant domain, the variable heavy chain of DOC2RHA (SEQ ID NO: 21), and the variable light chain of DOC2RKA (SEQ ID NO: 23). • The antibody DOC2-RHAXRKB contains a fully human constant domain, the variable heavy chain of DOC2RHA (SEQ ID NO: 21), and the variable light chain of DOC2RKB (SEQ ID NO: 31). • The antibody DOC2-RHBXRKA contains a fully human constant domain, the variable heavy chain of DOC2RHB (SEQ ID NO: 33), and the variable light chain of DOC2RKA (SEQ ID NO: 23). • The antibody DOC2-RHBXRKB contains a fully human constant domain, the variable heavy chain of DOC2RHB (SEQ ID NO: 33), and the variable light chain of DOC2RKA (SEQ ID NO: 31). • The antibody DOC2-RHBXRKC contains a fully human constant domain, the variable heavy chain of DOC2RHB (SEQ ID NO: 33), and the variable light chain of DOC2RKC (SEQ ID NO: 35). • The antibody DOC2-ChXRKA contains a fully human constant domain, a chimeric variable heavy chain, and a variable light chain of DOC2RKA (SEQ ID NO: 23). • Antibody DOC-2 chimera, • The antibody DOC3-RH09XRKA contains a fully human constant domain, the variable heavy chain of DOC3RH09 (SEQ ID NO: 37), and the variable light chain of DOC3RKA (SEQ ID NO: 29). • The antibody DOC3-RH09XRKC, which contains a fully human constant domain, the variable heavy chain of DOC3RH09 (SEQ ID NO: 37), and the variable light chain of DOC3RKC (SEQ ID NO: 39), and The antibody DOC3-CHXRKA, containing a fully human constant domain, a chimeric variable heavy chain, and a variable light chain of DOC3RKA (SEQ ID NO: 29), was subsequently tested in experiments.

[0057] In the following experiment, additional humanized DOC2 and DOC3 binders were tested for binding to primary human monocytes and primary marmoset monocytes. Figure 6 shows the binding of the humanized DOC2 binder to human monocytes. Figure 7 shows the binding of the humanized DOC2 binder to marmoset monocytes. Figure 8 shows the binding of the humanized DOC3 binder to human monocytes. Figure 9 shows the binding of the humanized DOC3 binder to marmoset monocytes. Regarding DOC2 derivatives, all humanized variants, including the variant containing only the humanized light chain (DOC2 CHXRKA), showed good binding to primary human monocytes, but not to primary marmoset monocytes. These results suggest that the DOC2 light chain is important for cross-reactivity with primary marmoset monocytes.

[0058] Regarding DOC3 derivatives, all humanized variants (Doc3RH09XRKA, Doc3RH09XRKC) showed good binding to primary human monocytes but not to primary marmoset monocytes. The variant containing only the humanized light chain (DOC3 CHXRKA) showed complete cross-reactivity with primary marmoset monocytes. These data suggest that the heavy chain of DOC3 is important for cross-reactivity with primary marmoset monocytes. In summary, the results indicate that it is extremely difficult to maintain the cross-reactivity of DOC2 and DOC3 to primary marmoset monocytes during the humanization procedure.

[0059] (Example 5) Further humanization of DOC2 using a more variable light chain Since the DOC2 light chain appears to be important for cross-reactivity with primary marmoset monocytes, several additional humanized variants of the DOC2 light chain were constructed and tested for binding to marmoset monocytes together with the DOC2RHA heavy chain. The following further humanized variable light chains were constructed in a reasonable manner to return to the original mouse framework. [Table 5-1] [Table 5-2]

[0060] The following antibodies: • The antibody DOC2-RHAXRKA FR1 contains a fully human constant domain, the variable heavy chain of DOC2RHA (SEQ ID NO: 21), and the variable light chain of DOC2RKA FR1 (SEQ ID NO: 41). • The antibody DOC2-RHAXRKA FR2 contains a fully human constant domain, the variable heavy chain of DOC2RHA (SEQ ID NO: 21), and the variable light chain of DOC2RKA FR2 (SEQ ID NO: 43). • The antibody DOC2-RHAXRKA FR3a, which contains a fully human constant domain, the variable heavy chain of DOC2RHA (SEQ ID NO: 21), and the variable light chain of DOC2RKA FR3a (SEQ ID NO: 45), and The antibody DOC2-RHAXRKA FR3b, containing the fully human constant domain, the variable heavy chain of DOC2RHA (SEQ ID NO: 21), and the variable light chain of DOC2RKA FR3b (SEQ ID NO: 47), was subsequently tested in experiments.

[0061] The antibody was again tested for binding to primary human monocytes and primary marmoset monocytes. Binding to human monocytes is shown in Figure 10. Binding to marmoset monocytes is shown in Figure 11. The data indicate that DOC2 RHAXRKA FR2 exhibits acceptable cross-reactivity with primary marmoset monocytes. Therefore, this variant was selected for monocyte depletion in marmosets, as well as for pharmacokinetic and toxicological studies in marmosets. (Example 6) Overview of the humanization procedure Standard approaches to humanizing the anti-CCR2 antibodies DOC2 and DOC3 were unsuccessful. While the humanized variants showed good binding to human CCR2-transfected CHO cells and primary human monocytes, they almost completely lost cross-reactivity to primary marmoset monocytes. Antibody-mediated monocyte depletion has not been performed in humans or non-human primates, and preclinical data on the safety of monocyte depletion in non-human primates are essential for clinical development; therefore, cross-reactivity with marmoset monocytes is a crucial characteristic. Furthermore, the feasibility of monocyte depletion with monoclonal antibodies against CCR2 had not been previously demonstrated in non-human primates.

[0062] Classical strategies for humanizing DOC-2 and DOC-3 only yielded antibodies that did not cross-react with marmoset CCR2, requiring the creation of multiple variants through trial and error. Finally, a humanized DOC2 antibody retaining cross-reactivity with marmoset CCR2 was successfully produced. This humanized DOC2 antibody, DOC2 RHAXRKA FR2, retains the light chain framework region necessary for marmoset cross-reactivity. No humanized DOC3 antibody retaining cross-reactivity with marmoset CCR2 could be identified. The antibody DOC-2 recognizes a three-dimensional epitope consisting of the first extracellular loop and the first half of the second extracellular loop of CCR2. DOC-2 cross-reacts with marmoset CCR2 but not with rhesus macaque CCR2. Sequence comparisons of human, marmoset, and rhesus macaque CCR2 have revealed that the first extracellular loop is identical in all three species. In the second extracellular loop, rhesus macaque CCR2 has two amino acid exchanges (Lys→Glu and Val→Ile), while marmoset has only one conservative exchange (Cys→Ser).

[0063] [Table 6] The exchange of Cys to Ser in marmoset CCR2 may explain the loss of cross-reactivity to marmoset CCR2, which is considered an essential characteristic for preclinical safety studies of cell-depleting antibodies, due to a slight change in the binding mode of the humanized DOC-2 antibody.

[0064] (Example 7) ADCC activity of humanized DOC2 antibody In this experiment, the activity of the humanized antibody DOC2-RHAXRKA FR2 was compared with that of rituximab and a human IgG1 isotype control antibody. PBMCs were prepared by Ficoll-Paque density gradient centrifugation. L-glutamine in RPMI1640 + 10% FCS + 1% PenStrep + 1% was used as the culture medium. 500,000 freshly prepared PBMCs were grown in 200 μl of medium in a 96-well plate (U-bottom). Antibodies were added to the wells in serial dilutions. Cells were incubated at 37°C for 24 hours, then stained on ice for 30 minutes with the following directly labeled antibodies: anti-CD16-APC, anti-CD11b-PECy7, anti-CD3-APCCy7, anti-CD14-AmCyan, and anti-CD19-PacificBlue. Cells were washed with PBS, and the absolute cell count / well was quantified by adding counting beads (10,000 / well). Cells were analyzed using a BD CANTO-II FACS instrument and FACS-DIVA analysis software.

[0065] The results are shown in Figures 12-16. DOC2-RHAXRKA FR2 (hDOC2) significantly depleted classical CD14-positive monocytes (expressing high levels of CCR2; Figure 12), but did not deplete B cells (Figure 13) or T cells (Figure 14). DOC2-RHAXRKA FR2 also strongly activates NK cells, as shown by the upregulation of CD11b on NK cells (Figure 15) and the downregulation of CD16 (Figure 16). Rituximab depletes B cells, but not other cell types.

Claims

1. A humanized antibody or antibody fragment specific to human CCR2, comprising the HCDR1 region of SEQ ID NO: 2, the HCDR2 region of SEQ ID NO: 3, the HCDR3 region of SEQ ID NO: 4, the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO:

9.

2. A humanized antibody or antibody fragment according to claim 1, comprising the variable heavy chain (VH) of SEQ ID NO: 21 and the variable light chain (VL) of SEQ ID NO:

43.

3. A humanized antibody according to claim 1 or 2, which is of the human IgG1 class.

4. A humanized antibody or antibody fragment according to any one of claims 1 to 3, which binds to the first extracellular loop of human CCR2 (SEQ ID NO: 53) and the second extracellular loop of human CCR2 (SEQ ID NO: 54).

5. A humanized antibody or antibody fragment according to any one of claims 1 to 4, which is cross-reactive with marmoset CCR2.

6. A humanized antibody or antibody fragment according to any one of claims 1 to 5, which binds to the first extracellular loop (SEQ ID NO: 53) and the second extracellular loop (SEQ ID NO: 55) of marmoset CCR2.

7. A humanized antibody or antibody fragment according to any one of claims 1 to 6, which does not bind to rhesus macaque CCR2.

8. A humanized antibody or antibody fragment according to any one of claims 1 to 7, for use in medical treatment, preferably the treatment being for an inflammatory disease, an autoimmune disease, or a hematological malignancy.

9. A nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment according to any one of claims 1 to 7.

10. A vector comprising the nucleic acid composition described in claim 9.

11. A host cell comprising the vector according to claim 10 or the nucleic acid composition according to claim 9.

12. A pharmaceutical composition comprising a humanized antibody or antibody fragment according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier or excipient.

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