Antibodies targeting CCR2

Abstract

The present disclosure relates to antibodies and antibody fragments specific for CCR2. The antibodies deplete CCR2 positive cells and block MCP-1 (CCL2) induced downregulation of CCR2 on human leukocytes. Furthermore, they have advantageous biophysical properties. The antibodies are useful for the treatment of 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. The antibodies deplete CCR2 positive cells and block MCP-1 (CCL2) induced downregulation of CCR2 on human leukocytes. Furthermore, they have advantageous biophysical properties. The antibodies are useful for the treatment of inflammatory diseases, autoimmune diseases, hematological malignancies and potentially other diseases. [Background technology]

[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, causing monocytes to exit the bone marrow. MCP-1 is secreted by cells during inflammatory diseases, autoimmune diseases, hematological malignancies, and other diseases.

[0003] Several molecules targeting the MCP-1 / CCR2 pathway have been or are still being tested, and although some have shown promising results, none have been approved so far. Most of the drugs developed are small organic molecules. For example, cenicrivirc, a small molecule developed by Takeda and Tobira Therapeutics, is currently in phase 3 clinical trials. However, cenicrivirc is not specific for CCR2, but also inhibits CCR5.

[0004] STI-B0201 is an antibody made by Sorrento, but has not been reported in clinical development. Sorrento's anti-CCR2 antibody is disclosed in WO 2013 / 192596. Unlike the antibodies of the present disclosure, the antibodies of WO 2013 / 192596 cross-react with mouse CCR2. Anti-CCR2 antibody antibodies are also disclosed in WO 2007 / 115713 and U.S. Pat. No. 9,068,002. However, these are mouse antibodies that were also never developed. MLNM1202 (prosalizumab) is another anti-CCR2 antibody, but development was discontinued by Millennium. Takeda / Millennium's antibody is disclosed in WO 2016 / 079276. Amgen's antibody is disclosed in U.S. Pat. App. Pub. No. 2011 / 0274696. The antibody of US Patent Application No. 2011 / 0274696 shows reactivity to cynomolgus monkey CCR2, but the antibody of the present disclosure does not cross-react with cynomolgus monkey CCR2. However, the antibody of the present disclosure cross-reacts with marmoset CCR2. The antibody of University of Regensburg / Yeda / Tel Aviv Medical Center against CCR2 is disclosed in US Patent No. 9,068,002. The antibody of Pfizer against CCR2 is disclosed in WO2010021697. Again, no development has been reported.

[0005] Therefore, there is a need and great interest in developing specific anti-CCR2 targeting moieties, but currently no anti-CCR2 antibodies are in development. The present invention discloses novel antibodies that are highly specific for CCR2 and have high affinity and biophysical properties beneficial for successful clinical development. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2013 / 192596 [Patent Document 2] International Publication No. 2007 / 115713 [Patent Document 3] U.S. Patent No. 9,068,002 [Patent Document 4] International Publication No. 2016 / 079276 [Patent Document 5] US Patent Application Publication No. 2011 / 0274696 [Patent Document 6] U.S. Patent No. 9,068,002 [Patent Document 7] International Publication No. 2010021697 Summary of the Invention

[0007] The present disclosure relates to novel antibodies and antibody fragments specific for human CCR2. The antibodies are cross-reactive to marmoset CCR2, which is a relevant animal model for clinical development. Compared to antibodies in the prior art, the antibodies and antibody fragments of the present disclosure have several advantages. One advantage is high affinity to the target antigen. The affinity of the antibodies and antibody fragments of the present invention is at least one order of magnitude higher than that of any prior art antibodies. In addition, the antibodies and antibody fragments of the present disclosure have excellent biophysical properties. This includes high temperature stability, high stability upon repeated freezing and thawing, stability over a wide pH range, and high stability in human plasma. Thus, the exploitability of the antibody is greatly increased. The stability of the binder also results in extended serum levels of the antibody, thereby allowing the antibody to be administered at a lower dose, thereby reducing potential side effects.

[0008] The present disclosure relates to antibodies and antibody fragments specific for human CCR2 that block MCP-1-induced downregulation of CCR2 on human leukocytes.

[0009] The present disclosure also relates to antibodies and antibody fragments specific for human CCR2 that bind to the ECL-2 domain of human CCR2 (SEQ ID NO:2).

[0010] The present disclosure also relates to antibodies and antibody fragments specific for human CCR2 that do not bind to human CCR5 (SEQ ID NO:6).

[0011] The present disclosure also relates to antibodies and antibody fragments specific for human CCR2 that are cross-reactive with marmoset CCR2.

[0012] The present disclosure also relates to antibodies and antibody fragments specific for human CCR2 having at least 10-fold higher affinity for human CCR2 than antibody DOC2. The present disclosure also relates to antibodies and antibody fragments, wherein the antibodies or antibody fragments have higher affinity for human CCR2 expressed on human monocytes than an antibody comprising a variable heavy chain of SEQ ID NO: 28 and a variable heavy chain of SEQ ID NO: 29. Preferably, the antibodies or antibody fragments have at least 2-fold, at least 3-fold, at least 5-fold, or at least 10-fold higher affinity at an antibody concentration of 0.1-1.0 μg / ml.

[0013] Preferred antibodies and antibody fragments of the present disclosure are of the human IgG class. Even more preferred antibodies or antibody fragments of the present disclosure are of the IgG1 class.

[0014] Preferred antibodies and antibody fragments of the present disclosure are monoclonal antibodies or antibody fragments.

[0015] Preferred antibodies and antibody fragments of the present disclosure are humanized antibodies or antibody fragments.

[0016] The present disclosure also relates to antibodies and antibody fragments specific for human CCR2 that have excellent biophysical properties, including high temperature stability, high stability upon repeated freezing and thawing, stability over a wide pH range, and high stability in human plasma.

[0017] Preferred antibodies and antibody fragments of the disclosure comprise an HCDR1 region of SEQ ID NO:10, an HCDR2 region of SEQ ID NO:11, an HCDR3 region of SEQ ID NO:12, an LCDR1 region of SEQ ID NO:13, an LCDR2 region of SEQ ID NO:14 and an LCDR3 region of SEQ ID NO:15.

[0018] Preferred antibodies and antibody fragments of the disclosure comprise a variable heavy chain of SEQ ID NO:22 and a variable light chain of SEQ ID NO:23.

[0019] The present disclosure also relates to a nucleic acid composition comprising a nucleic acid sequence or multiple nucleic acid sequences encoding an antibody or antibody fragment comprising an HCDR1 region of SEQ ID NO:10, an HCDR2 region of SEQ ID NO:11, an HCDR3 region of SEQ ID NO:12, an LCDR1 region of SEQ ID NO:13, an LCDR2 region of SEQ ID NO:14, and an LCDR3 region of SEQ ID NO:15.

[0020] The present disclosure relates to a nucleic acid composition comprising a nucleic acid sequence or sequences encoding an antibody or antibody fragment comprising a variable heavy chain of SEQ ID NO:22 and a variable light chain of SEQ ID NO:23.

[0021] The present disclosure also relates to vectors comprising such nucleic acids or nucleic acid compositions.

[0022] The present disclosure also relates to host cells comprising such nucleic acids, nucleic acid compositions or vectors.

[0023] The antibodies and antibody fragments of the present disclosure are for use in medicine. Preferred are the antibodies and antibody fragments of the present disclosure for use in the treatment of inflammatory diseases, autoimmune diseases, hematological malignancies and potentially other diseases.

[0024] The present disclosure also relates to pharmaceutical compositions comprising the antibodies and antibody fragments and pharma- ceutically acceptable antibodies and antibody fragments of the present disclosure. [Brief description of the drawings]

[0025] [Figure 1]Specific binding of the antibodies of the present invention to human CCR2. No binding to human CCR5 was observed. The anti-CCR5 antibody MC-1 was specific for human CCR5 but did not show binding to human CCR2. [Diagram 2] It is shown that antibodies of the invention bind to CD16 negative monocytes (upper panel) but not to CD16 positive monocytes (lower panel). [Diagram 3] Binding of antibody Y4T3 to CD16-negative marmoset monocytes (top) but not to CD16-positive marmoset monocytes (bottom) Antibodies Y1T2 and Y2T63 do not bind to CD16-negative and CD16-positive marmoset monocytes. [Figure 4] We show that the ECL2 domain of human CCR2 is important for Y4T3 binding. Y4T3 did not bind to a mutant of human CCR2b in which the ECL2 domain was replaced with the ECL2 domain of rhesus CCR2b. [Diagram 5] We demonstrate that downmodulation of human CCR2 by MCP-1 for 30 min at 37°C reduces binding of Y4T3 to human monocytes. [Figure 6] Figure 1 shows the binding of humanized variants of Y3T3 to CCR2 expressed on human monocytes. All antibodies were tested at 0.5 μg / ml. H0L0 represents the human IgG1 chimeric version of Y4T3. H1L1-H5L5 are humanized variants of Y4T3. [Figure 7] Binding of antibody hY4T3 to CCR2+ human monocytes is demonstrated. The concentration of antibody used is indicated on the x-axis. [Figure 8] Binding of antibody hY4T3 to CCR2+ marmoset monocytes is demonstrated. The concentration of antibody used is indicated on the x-axis. [Figure 9] Binding of human IgG1 chimeric variants of Y4T3 (Y4T3-chim) and hY4T3 to CHO cells transfected with human CCR2 is shown. Y4T3-chim and hY4T3 do not bind to CHO cells transfected with human CCR5. The concentration of antibodies used is shown on the x-axis. [Figure 10]We demonstrate the blocking of MCP-1-induced downregulation of CCR2 by hY4T3 in various leukocyte subsets. Cells were first incubated with hY4T3, MCP-1 or medium (RPMI) at various concentrations (μg / ml) as indicated for 30 min at 37° C. Then, MCP-1, hY4T3 or RPMI was added, followed by further incubation for 30 min at 37° C. Cells were not washed between the first and second incubation steps. Surface-bound hY4T3 was detected with a PE-labeled goat anti-human IgG antibody. [Figure 11] Figure 1 shows the effect of treatment with hY4T3 on PBMC from marmoset monkeys. Marmosets were treated with 5 mg / kg hY4T3 at time 0. At various time points, leukocyte subpopulations were quantified in whole blood by flow cytometry before and after treatment. Relative cell numbers are shown as % of PBMC. [Figure 12] SEC chromatograms of hY4T3 are shown after 3 hours of incubation at the indicated temperatures (top left = reference; top right = 55°C; bottom left = 65°C; bottom right = 75°C). [Figure 13] Binding of hY4T3 to CCR2 on CHO cells is shown after incubation of the antibody for 2 hours at the indicated temperatures. [Figure 14] 1 shows binding of hY4T3 to CCR2 on CHO cells following repeated freeze-thaw cycles of the antibody preparation. [Figure 15] Binding of hY4T3 to CCR2 on CHO cells is shown after antibody incubation at various antibody concentrations for 2 hours at the indicated pH. [Figure 16] Figure 1 shows the binding of hY4T3 to CCR2 on CHO cells after incubation of the antibody for various time intervals in PBS and human plasma. The antibody was incubated at a concentration of 1 mg / ml for the indicated time intervals. The antibody binding was then tested at various dilutions. [Figure 17] 1 shows a comparison of binding of antibody Y4T4 to prior art antibodies DOC2, 1D9 and clone 48607 on human monocytes. [Figure 18]We show that hY4T3 binds to human CCR2, but not to mouse CCR2. MC-21 binds to mouse CCR2, but not to human CCR2. [Figure 19] We show that hY4T3 efficiently blocks the chemokine receptor CCR2.

[0026] The present disclosure relates to antibodies that specifically bind to CCR2.

[0027] The term "CCR2" refers to a protein known as CC motif chemokine receptor 2, also known as 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 the cytoplasmic C-terminal portion. The extracellular portion accessible to therapeutic antibodies and antibody fragments is identical in both isoforms.

[0028] Human CCR2b has the amino acid sequence of (UniProtKB / Swiss-Prot:P41597). MLSTSRSRFIRNTNESGEEVTTFFDYDYGAPCHKFDVKQIGAQLLPPLYSLVFIFGFVGNMLVVLILINCKKLKCLTDIYLLNLAISDLLFLITLPLWAHSAANEWVFGNAMCKLFTGLYHIGYFGGIFFIILLTIDRYLAIVHAVFALKARTVTFGVVTSVITWLVAVFASVPGIIFTKCQKEDSVYVCGPYFPRGWNNFHTIMRNILGLVLPLLIMVICYSGILKTLLRCRNEKKRHRAVRVIFTIMIVYFLFWTPYNIVILLNTFQEFFGLSNCESTSQLDQATQVTETLGMTHCCINPIIYAFVGEKFRRYLSVFFRKHITKRFCKQCPVFYRETVDGVTSTNTPSTGEQEVSAGL (SEQ ID NO: 1)

[0029] The extracellular domain 2 (ECL2) of human CCR2 has the following amino acid sequence: TKCQKEDSVYVCGPYFPRGWNNFHTIMR (SEQ ID NO: 2)

[0030] Marmoset monkey (Callithrix jacchus) CCR2b has the following amino acid sequence: MLSTSHSRFIRNTESGEEVTTIFDYDYGAPCHKFDVKQIGAQLLPPLYSLVFIFGFVGNMLVVLILINCKKLKSLTDIYLLNLAVSDLLFLITLPLWAHSAANEWVFGNAVCKLFTGLYHIGYFGGIFFIILLTIDRYLAIVHAVFALKARTVTFGVVTSVITWFVAVFASVPGIIFTKSQKEDSVYVCGPYFPRGWNHFHTIMRNLLGLVLPLLVMIICYSGILKTLLRCRNEKKRHRAVRLIFTIMIVYFLFWTPYNIVVLLNTFQEFFGLSNCESTSQLDQATQVTETLGMTHCCINPIIYAFVGEKFRRYLSVFFRKHIAKRFCKQCPVFYRETVDGVTSTNTPSTGEQEVSAGL (SEQ ID NO: 3)

[0031] Macaca mulatta CCR2b has the following amino acid sequence: MLSTSRSRFIRNTNGSGEEVTTFFDYDYGAPCHKFDVKQIGAQLLPPLYSLVFIFGFVGNMLVVLILINCKKLKSLTDIYLLNLAISDLLFLITLPLWAHSAANEWVFGNAMCKLFTGLYHIGYLGGIFFIILLTIDRYLAIVHAVFALKARTVTFGVVTSVITWLVAVFASVPGIIFTKCQEEDSVYICGPYFPRGWNNFHTIMRNILGLVLPLLIMVICYSGILKTLLRCRNEKKRHRAVRLIFTIMIVYFLFWTPYNIVILLNTFQEFFGLSNCESTRQLDQATQVTETLGMTHCCINPIIYAFVGEKFRRYLSMFFRKYITKRFCKQCPVFYRETVDGVTSTNTPSTAEQEVSVGL (SEQ ID NO: 4)

[0032] CCR2 is the key functional receptor for MCP-1, whose binding to MCP-1 mediates chemotaxis and migration of monocytes and macrophages.

[0033] The term "MCP-1" refers to a protein known as monocyte chemotactic protein 1. It is also known as CCL2 (CC motif chemokine ligand 2). Human MCP-1 has the amino acid sequence (UniProtKB / Swiss-Prot:P13500): MKVSAALLCLLLIAATFIPQGLAQPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVAKEICADPKQKWVQDSMDHLDKQTQTPKT (SEQ ID NO: 5)

[0034] The term "CCR5" refers to a protein known as CC motif chemokine receptor 5. Also known as CD195. Human CCR5 has the amino acid sequence of (UniProtKB / Swiss-Prot:P51681). MDYQVSSPIYDINYYTSEPCQKINVKQIAARLLPPLYSLVFIFGFVGNMLVILILINCKRLKSMTDIYLLNLAISDLFFLLTVPFWAHYAAAQWDFGNTMCQLLTGLYFIGFFSGIFFIILLTIDRYLAVVHAVFALKARTVTFGVVTSVITWVVAVFASLPGIIFTRSQKEGLHYTCSSHFPYSQYQFWKNFQTLKIVILGLVLPLLVMVICYSGILKTLLRCRNEKKRHRAVRLIFTIMIVYFLFWAPYNIVLLLNTFQEFFGLNNCSSSNRLDQAMQVTETLGMTHCCINPIIYAFVGEKFRNYLLVFFQKHIAKRFCKCCSIFQQEAPERASSVYTRSTGEQEISVGL (SEQ ID NO: 6)

[0035] In certain experiments of this disclosure, the following additional constructs were used:

[0036] Human CCR2b with rhesus monkey ECL2 domain: MLSTSRSRFIRNTNESGEEVTTFFDYDYGAPCHKFDVKQIGAQLLPPLYSLVFIFGFVGNMLVVLILINCKKLKCLTDIYLLNLAISDLLFLITLPLWAHSAANEWVFGNAMCKLFTGLYHIGYFGGIFFIILLTIDRYLAIVHAVFALKARTVTFGVVTSVITWLVAVFASVPGIIFTKCQEEDSVYICGPYFPRGWNNFHTIMRNILGLVLPLLIMVICYSGILKTLLRCRNEKKRHRAVRVIFTIMIVYFLFWTPYNIVILLNTFQEFFGLSNCESTSQLDQATQVTETLGMTHCCINPIIYAFVGEKFRRYLSVFFRKHITKRFCKQCPVFYRETVDGVTSTNTPSTGEQEVSAGL (SEQ ID NO: 7)

[0037] The rhesus monkey ECL2 domain differs from the human ECL2 domain by two amino acid residues.

[0038] Human CCR2b with rhesus monkey ECL3 domain: MLSTSRSRFIRNTNESGEEVTTFFDYDYGAPCHKFDVKQIGAQLLPPLYSLVFIFGFVGNMLVVLILINCKKLKCLTDIYLLNLAISDLLFLITLPLWAHSAANEWVFGNAMCKLFTGLYHIGYFGGIFFIILLTIDRYLAIVHAVFALKARTVTFGVVTSVITWLVAVFASVPGIIFTKCQKEDSVYVCGPYFPRGWNNFHTIMRNILGLVLPLLIMVICYSGILKTLLRCRNEKKRHRAVRVIFTIMIVYFLFWTPYNIVILLNTFQEFFGLSNCESTRQLDQATQVTETLGMTHCCINPIIYAFVGEKFRRYLSVFFRKHITKRFCKQCPVFYRETVDGVTSTNTPSTGEQEVSAGL (SEQ ID NO: 8)

[0039] The rhesus monkey ECL3 domain differs from the human ECL2 domain by one amino acid residue.

[0040] Human CCR2b with rhesus N-terminus: MLSTSRSRFIRNTNGSGEEVTTFFDYDYGAPCHKFDVKQIGAQLLPPLYSLVFIFGFVGNMLVVLILINCKKLKCLTDIYLLNLAISDLLFLITLPLWAHSAANEWVFGNAMCKLFTGLYHIGYFGGIFFIILLTIDRYLAIVHAVFALKARTVTFGVVTSVITWLVAVFASVPGIIFTKCQKEDSVYVCGPYFPRGWNNFHTIMRNILGLVLPLLIMVICYSGILKTLLRCRNEKKRHRAVRVIFTIMIVYFLFWTPYNIVILLNTFQEFFGLSNCESTSQLDQATQVTETLGMTHCCINPIIYAFVGEKFRRYLSVFFRKHITKRFCKQCPVFYRETVDGVTSTNTPSTGEQEVSAGL (SEQ ID NO: 9)

[0041] The N-terminus of rhesus CCR2 differs from human CCR2 by one amino acid residue.

[0042] The term "antibody" as used herein refers to a protein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds that interact with an antigen. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains: CH1, CH2 and CH3. Each light chain consists of a light chain variable region (abbreviated herein 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) interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable regions of the heavy and light chains comprise the binding domains that interact with an antigen. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The term "antibody" includes, for example, monoclonal antibodies, human antibodies, humanized antibodies, camelized antibodies, and chimeric antibodies. Antibodies can be of 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.

[0043] The term "antibody fragment" as used herein refers to one or more portions of an antibody that retain the ability to specifically interact with an antigen (e.g., by binding, steric hindrance, stabilization of spatial distribution). Examples of binding fragments include, but are not limited to, Fab fragments, VL, VH, CL and CH1 domains; F(ab)2 fragments, bivalent fragments containing two Fab fragments linked by a disulfide bridge at the hinge region; Fd fragments consisting of the VH and CH1 domains; Fv fragments consisting of the VL and VH domains of a single arm of an antibody; dAb fragments consisting of the VH domain (Ward et al., (1989), Nature 341:544-546); and monovalent fragments consisting of 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 that allows them to be made using recombinant methods as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as a single-chain Fv (scFv); see, e.g., 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 encompassed by the term "antibody fragment". These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the 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, e.g., Hollinger and Hudson, (2005) Nature Biotechnology 23:1 126-1 136). Antibody fragments can be grafted onto scaffolds based on polypeptides such as fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide monobodies).Antibody fragments can be assembled into single-chain molecules comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen-binding sites (Zapata et al., (1995) Protein Eng. 8:1057-1062; and U.S. Pat. No. 5,641,870).

[0044] The structures and locations of immunoglobulin variable domains, e.g., CDRs, can be numbered according to well-known numbering schemes, such as the Kabat numbering scheme, the Chothia numbering scheme, or a combination of Kabat and Chothia (e.g., Sequences of Proteins of Immunological Interest, USDepartment 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, 5 th edit., NIH Publication no. 91-3242 USDepartment 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).

[0045] As used herein, a "human antibody" or "human antibody fragment" refers to antibodies and antibody fragments having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Human antibodies can also be isolated from synthetic libraries or transgenic mice (e.g., Xenomouse), providing a respective yield in antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody comprises a constant region, the constant region is also derived from such sequences. Human origin includes, for example, mutated versions of human germline sequences or antibodies that contain human germline sequences or consensus framework sequences derived from human framework sequence analysis, e.g., as described in Knappik et al. (2000) J Mol Biol 296:57-86).

[0046] A "humanized antibody" or "humanized antibody fragment" is defined herein as an antibody molecule having constant antibody regions derived from sequences of human origin, and only the variable antibody regions or parts thereof, or the CDRs, are derived from another species. For example, a humanized antibody can be CDR-grafted, in which the CDRs of the variable domain are of non-human origin, while one or more frameworks of the variable domain are of human origin, and the constant domains (if any) are of human origin.

[0047] The term "chimeric antibody" or "chimeric antibody fragment" is defined herein as an antibody molecule having constant antibody regions derived from or corresponding to sequences found in one species and variable antibody regions derived from another species. Preferably, the constant antibody regions are derived from or correspond to sequences found in humans and the variable antibody regions (e.g. VH, VL, CDR or FR regions) are derived from sequences found in a non-human animal, such as mouse, rat, rabbit or hamster.

[0048] The term "isolated antibody" refers to an antibody or antibody fragment that is substantially free of other antibodies or antibody fragments with different antigen specificities. Furthermore, an isolated antibody or antibody fragment may be substantially free of other cellular material and / or chemicals. Thus, in some aspects, the antibody provided is an isolated antibody that is separated from antibodies with different specificities. An isolated antibody may be a monoclonal antibody. An isolated antibody may be a recombinant monoclonal antibody. However, an isolated antibody that specifically binds to a target epitope, isoform, or variant may have cross-reactivity with other related antigens, such as antigens from other species (e.g., species homologs).

[0049] The term "recombinant antibody" as used herein includes all antibodies prepared, expressed, created or isolated by non-naturally occurring means, such as antibodies isolated from host cells transformed to express the antibody, antibodies prepared, expressed, created or isolated by any other means including splicing of all or part of a human immunoglobulin gene, sequences relative to other DNA sequences, or antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal for human immunoglobulin genes or hybridomas prepared therefrom. Preferably, such recombinant antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies can be subjected to in vitro mutagenesis (or in vivo somatic mutagenesis when using animals transgenic for human Ig sequences) such that the amino acid sequences of the VH and VL regions of the recombinant antibodies are derived from and related to human germline VH and VL sequences, but are sequences that may not naturally occur within the human antibody germline repertoire in vivo. The recombinant antibodies can be monoclonal antibodies. 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).

[0050] As used herein, binding specificity is a relative, not absolute, property, so an antibody "specifically binds to," "specifically binds to," "is specific in / for," or "specifically recognizes" an antigen, such as human CCR2, if such an antibody can discriminate between such antigen and one or more reference antigens. For example, a standard ELISA assay or a standard flow cytometry assay can be performed. Scoring can be performed by standard color development (e.g. secondary antibody with tetramethylbenzidine with horseradish peroxide and 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 mean fluorescence intensity (MFI) in flow cytometry. A typical background (=negative reaction) can be 0.1 OD. A typical positive reaction can be 1 OD. Background and positive reaction MFI are highly dependent on the instrument settings. The positive / negative difference can be more than 10-fold. Typically, the determination of binding specificity is performed using a set of about 3-5 unrelated antigens, such as milk powder, BSA, transferrin, etc., rather than a single reference antigen. A variety of antigen-negative cells can be used for flow cytometry. However, antibodies that specifically bind to an antigen may have cross-reactivity to the respective orthologous antigens from other species (e.g., species homologs). In certain embodiments, such cross-reactivity to orthologous antigens is even preferred.

[0051] As used herein, an antibody has "cross-reactivity" or is "cross-reactive" if it binds to orthologous antigens from other species. For example, an antibody is cross-reactive if it binds to human CCR2 and marmoset CCR2.

[0052] As used herein, the term "affinity" refers to the strength of 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 through weak non-covalent forces. The more interactions, the stronger the affinity.

[0053] The term "epitope" includes any proteinaceous region that is specifically recognized by an antibody or antibody fragment thereof or that otherwise interacts with a molecule. Generally, epitopes are chemically active surface groupings of molecules such as amino acids or carbohydrate or sugar side chains and generally may have specific three-dimensional structural characteristics, as well as specific charge characteristics. As will be appreciated by those skilled in the art, virtually anything to which an antibody can specifically bind can be an epitope.

[0054] The term "domain" or "protein domain" refers to a region of a polypeptide chain of a protein that forms a functional unit and / or forms an independent three-dimensional structure. For example, ECL2 of CCR2 is a protein domain.

[0055] The "compositions" of the present disclosure can be used for therapeutic or prophylactic applications. Thus, the present disclosure includes pharmaceutical compositions containing the antibodies or antibody fragments disclosed herein and a pharma- ceutically acceptable carrier or excipient therefor. In a related aspect, the present disclosure provides methods of treating inflammatory diseases, autoimmune diseases, hematological malignancies, and potentially other diseases. Such methods include administering to a subject in need thereof an effective amount of a pharmaceutical composition containing an antibody or antibody fragment as described herein.

[0056] The present disclosure provides a method of treatment comprising administering a therapeutically effective amount of an antibody or antibody fragment disclosed herein to a subject in need of such treatment. As used herein, a "therapeutically effective amount" or "effective amount" refers to the amount of CCR2 antibody required to induce a desired biological response. According to the subject disclosure, a therapeutically effective amount is the amount of CCR2 antibody required to treat and / or prevent a disease.

[0057] "Administered" or "administration" includes, but is not limited to, delivery of a drug in an injectable form, e.g., intravenous, intramuscular, intradermal or subcutaneous routes, or by mucosal routes, e.g., as a nasal spray or aerosol for inhalation, or as an ingestible solution, capsule or tablet. Preferably, administration is by injectable form.

[0058] As used herein, "treatment", "treat" or "treating" and the like refer to a clinical intervention that seeks to alter the natural course of a disease in the subject being treated, and can be performed prophylactically or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of direct or indirect pathological consequences of the disease, prevention of metastasis, slowing the rate of disease progression, amelioration or alleviation of the disease state, and remission or improved prognosis. In some embodiments, the antibodies or antibody fragments of the present disclosure are used to delay the onset of the disease or to delay the progression of the disease.

[0059] "Preventing" or "prevention" refers to a reduction in the risk of acquiring or developing a disease (i.e., not developing at least one clinical symptom of a disease in a subject who may be exposed to a disease-causing agent or who is susceptible to the disease prior to the onset of the disease). "Prevention" also refers to methods aimed at preventing the onset of a disease or its symptoms or delaying the onset of a disease or its symptoms.

[0060] "Subject" or "species" as used in this context refers to any mammal, including rodents such as mice or rats, and primates such as cynomolgus monkeys (Macaca fascicularis), marmoset monkeys (Callithrix jacchus), rhesus monkeys (Macaca mulatta) or humans (Homo sapiens). Preferably, the subject is a primate, most preferably a human.

[0061] The term "effector function" refers to biological activities attributable to the Fc region of an antibody, which vary depending on the antibody isotype. Non-limiting examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding and antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cellular phagocytosis (ADCP); downregulation of cell surface receptors (e.g., B cell receptors); direct cell activation or direct cell inhibition.

[0062] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which antibodies bound to Fc receptors (FcR) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages) enable these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins. The primary cells for mediating ADCC, NK cells, express only FcyRIII, whereas monocytes / macrophages express FcyRI, FcyRII, and FcyRIII.

[0063] "Complement dependent cytotoxicity" or "CDC" refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody of the present disclosure (of the appropriate subclass) that is bound to its cognate antigen.

[0064] "Antibody-dependent cellular phagocytosis" or "ADCP" refers to a mechanism for elimination of antibody-coated target cells by internalization by phagocytes such as macrophages or dendritic cells.

[0065] Throughout this specification, unless the context requires otherwise, the words "comprise", "have" and "include", as well as their respective variations such as "comprises", "comprising", "has", "having", "includes" and "including", are understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

[0066] The terms "engineered" or "modified" as used herein include the manipulation of nucleic acids or polypeptides by synthetic means (e.g., by recombinant techniques, in vitro peptide synthesis, enzymatic or chemical coupling of peptides, or some combination of these techniques). Preferably, an antibody or antibody fragment according to the present disclosure is engineered or modified to improve one or more properties, such as antigen binding, stability, half-life, effector functions, immunogenicity, safety, etc.

[0067] As used herein, "variant" refers to a polypeptide that differs from a reference polypeptide by one or more modifications, such as amino acid substitutions, insertions, or deletions. A variant polypeptide typically retains most of the properties of the reference polypeptide, such as binding to a target antigen, but introduces a new additional feature or property, e.g., the variant polypeptide has a higher affinity for a target antigen compared to the reference polypeptide, or the variant polypeptide is a humanized version of the reference polypeptide.

[0068] The term "amino acid mutation" as used herein is meant to encompass amino acid substitution, deletion, insertion, and modification. Any combination of substitution, deletion, insertion, and modification can be made, so long as the final construct has the desired characteristics, e.g., reduced binding to Fc receptors. Deletions and insertions of amino acid sequences include N-terminal and / or C-terminal deletions and insertions of amino acid residues. A particular amino acid mutation is an amino acid substitution. Amino acid substitutions include substitutions with unnatural amino acids or with natural amino acid derivatives of the 20 standard amino acids. Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, and the like. It is contemplated that methods of modifying the side chain groups of amino acid residues by methods other than genetic engineering, such as chemical modification, may also be useful. Various names may be used herein to refer to the same amino acid mutation. For example, a glycine to alanine substitution at position 327 of the Fc region may be represented as 237A, G337, G337A, or Gly329Ala.

[0069] As used herein, the term "EC50" refers to the concentration of an antibody or antibody fragment that induces a response in an assay halfway between the baseline and maximum, and thus represents the antibody or ligand concentration at which 50% of the maximal effect is observed.

[0070] The term "inhibition" or "inhibiting" or "reduction" or "reducing" or "neutralization" or "neutralizing" refers to the reduction or cessation of any phenotypic characteristic (such as binding or biological activity or function), or the reduction or cessation of the incidence, extent, or likelihood of that characteristic. "Inhibition", "reduction" or "neutralization" need not be complete, so long as it is detectable using an appropriate assay. In some embodiments, "reduce" or "inhibit" or "neutralizing" refers to the ability to cause a reduction of 20% or more. In another embodiment, "reduce" or "inhibit" or "neutralizing" refers to the ability to cause a reduction of 50% or more. In yet another embodiment, "reduce" or "inhibit" or "neutralizing" refers to the ability to cause an overall reduction of 75%, 85%, 90%, 95% or more.

[0071] The term "antagonist" antibody, as used herein, refers to an antibody or antibody fragment that interacts with an antigen and partially or completely inhibits or neutralizes the biological activity or function or any other phenotypic property of the target antigen.

[0072] A "wild type" protein is a version or variant of a protein as found in nature. The amino acid sequence of a wild type protein, such as the Fc region of a human IgG1 antibody, is the amino acid sequence of a naturally occurring protein. Due to allotypic differences, there may be more than one amino acid sequence of 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).

[0073] "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. The Fc region of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain. Although the boundaries of the Fc region of an IgG heavy chain may vary slightly, the human IgG heavy chain Fc region is usually defined to extend 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 is as follows: th The best way to identify HIV-1 positive controls is to follow the EU numbering system, also known as the EU index, as described in Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. EMBODIMENTS OF THE PRESENT DISCLOSURE Polypeptides

[0074] In one embodiment, the present disclosure relates to an isolated antibody or antibody fragment specific to human CCR2, wherein the antibody or antibody fragment comprises an HCDR1 region of SEQ ID NO: 10, an HCDR2 region of SEQ ID NO: 11, an HCDR3 region of SEQ ID NO: 12, an LCDR1 region of SEQ ID NO: 13, an LCDR2 region of SEQ ID NO: 14, and an LCDR3 region of SEQ ID NO: 15.

[0075] In one embodiment, the present disclosure relates to an isolated antibody or antibody fragment specific for human CCR2 as defined by Kabat, wherein the antibody or antibody fragment comprises an HCDR1 region of SEQ ID NO: 10, an HCDR2 region of SEQ ID NO: 11, an HCDR3 region of SEQ ID NO: 12, an LCDR1 region of SEQ ID NO: 13, an LCDR2 region of SEQ ID NO: 14, and an LCDR3 region of SEQ ID NO: 15.

[0076] In one embodiment, the present disclosure relates to an isolated antibody or antibody fragment specific for human CCR2, as defined by IMGT, said antibody or antibody fragment comprising an HCDR1 region of SEQ ID NO: 16, an HCDR2 region of SEQ ID NO: 17, an HCDR3 region of SEQ ID NO: 18, an LCDR1 region of SEQ ID NO: 19, an LCDR2 region of SEQ ID NO: 20, and an LCDR3 region of SEQ ID NO: 21, as defined by IMGT.

[0077] In one embodiment, the disclosure relates to an isolated antibody or antibody fragment specific for human CCR2 that comprises the six CDRs defined by Kabat of any one of the antibodies disclosed in Table 3.

[0078] In one embodiment, the disclosure relates to an isolated antibody or antibody fragment specific for human CCR2 that comprises the six CDRs defined by IMGT of any one of the antibodies disclosed in Table 3.

[0079] In one embodiment, the disclosure relates to an isolated antibody or antibody fragment specific for human CCR2 that comprises the six Chothia-defined CDRs of any one of the antibodies disclosed in Table 3.

[0080] In one embodiment of the disclosure, the isolated antibody or antibody fragment is a monoclonal antibody or antibody fragment.

[0081] In one embodiment of the disclosure, the isolated antibody or antibody fragment is a human antibody, a humanized antibody, or a chimeric antibody or antibody fragment. In another preferred embodiment of the disclosure, the isolated antibody or antibody fragment is a humanized antibody or antibody fragment.

[0082] In one embodiment of the disclosure, the isolated antibody or antibody fragment is a recombinant antibody or antibody fragment.

[0083] In one embodiment of the disclosure, the isolated antibody or antibody fragment is of the IgG isotype.

[0084] In one embodiment of the disclosure, the isolated antibody or antibody fragment is of the IgG1 class.

[0085] In another embodiment of the disclosure, the isolated antibody or antibody fragment binds to the ECL-2 domain of human CCR2 (SEQ ID NO:2).

[0086] In another embodiment of the disclosure, the isolated antibody or antibody fragment binds to a polypeptide comprising the ECL-2 domain of human CCR2 (SEQ ID NO:2).

[0087] In another embodiment of the disclosure, the isolated antibody or antibody fragment specific for human CCR2 is cross-reactive with marmoset CCR2.

[0088] In another embodiment of the disclosure, the isolated antibody or antibody fragment specific for human CCR2 is cross-reactive with marmoset CCR2, but not with rhesus monkey CCR2.

[0089] In another embodiment of the disclosure, the isolated antibody or antibody fragment specific for human CCR2 does not bind to rhesus CCR2.

[0090] In another embodiment of the disclosure, the isolated antibody or antibody fragment specific for human CCR2 does not bind to mouse CCR2.

[0091] In another embodiment of the disclosure, the isolated antibody or antibody fragment specific for human CCR2 does not bind to human CCR5 (SEQ ID NO:6).

[0092] In another embodiment of the disclosure, an isolated antibody or antibody fragment specific for human CCR2 blocks MCP-1 induced downregulation of CCR2 on human leukocytes.

[0093] In another embodiment of the disclosure, the isolated antibody or antibody fragment specific for human CCR2 binds to human CCR2 at least 2-fold, at least 3-fold, at least 5-fold, or at least 10-fold better than DOC2. Preferably, the isolated antibody or antibody fragment specific for human CCR2 binds to human CCR2 at least 10-fold better than antibody DOC2.

[0094] In another embodiment of the disclosure, the isolated antibody or antibody fragment specific for human CCR2 binds to human CCR2 at least 2-fold, at least 3-fold, at least 5-fold, or at least 10-fold better than an antibody comprising a variable heavy chain of SEQ ID NO: 28 and a variable heavy chain of SEQ ID NO: 29. Preferably, the isolated antibody or antibody fragment specific for human CCR2 binds to human CCR2 at least 10-fold better than an antibody comprising a variable heavy chain of SEQ ID NO: 28 and a variable heavy chain of SEQ ID NO: 29.

[0095] The present disclosure also relates to antibodies and antibody fragments specific for human CCR2 that have at least 10-fold greater affinity for human CCR2 than an antibody comprising a variable heavy chain of SEQ ID NO:28 and a variable heavy chain of SEQ ID NO:29.

[0096] The present disclosure also relates to antibodies and antibody fragments, wherein said antibody or antibody fragment has a higher affinity for human CCR2 expressed on human monocytes than an antibody comprising a variable heavy chain of SEQ ID NO: 28 and a variable heavy chain of SEQ ID NO: 29. Preferably, said antibody or antibody fragment has at least 2-fold, at least 3-fold, at least 5-fold or at least 10-fold higher affinity for human CCR2 expressed on human monocytes than an antibody comprising a variable heavy chain of SEQ ID NO: 28 and a variable heavy chain of SEQ ID NO: 29.

[0097] The present disclosure also relates to antibodies and antibody fragments, wherein the antibodies or antibody fragments have a higher affinity for human CCR2 expressed on human monocytes than an antibody comprising a variable heavy chain of SEQ ID NO: 28 and a variable heavy chain of SEQ ID NO: 29 at an antibody concentration of 0.1 to 1.0 μg / ml. Preferably, the antibodies or antibody fragments have at least 2-fold, at least 3-fold, at least 5-fold, or at least 10-fold higher affinity for human CCR2 expressed on human monocytes than an antibody comprising a variable heavy chain of SEQ ID NO: 28 and a variable heavy chain of SEQ ID NO: 29.

[0098] In another embodiment of the disclosure, the isolated antibody or antibody fragment specific for human CCR2 has at least 2-fold, at least 3-fold, at least 5-fold, or at least 10-fold higher affinity for human CCR2 than for DOC2. Preferably, the isolated antibody or antibody fragment specific for human CCR2 has at least 10-fold higher affinity for human CCR2 than for antibody DOC2.

[0099] In another embodiment of the disclosure, the isolated antibody or antibody fragment specific for human CCR2 has at least 2-fold, at least 3-fold, at least 5-fold, or at least 10-fold higher affinity for human CCR2 than an antibody comprising a variable heavy chain of SEQ ID NO: 28 and a variable heavy chain of SEQ ID NO: 29. Preferably, the isolated antibody or antibody fragment specific for human CCR2 has at least 10-fold higher affinity for human CCR2 than an antibody comprising a variable heavy chain of SEQ ID NO: 28 and a variable heavy chain of SEQ ID NO: 29.

[0100] In another embodiment of the disclosure, the isolated antibody or antibody fragment specific for human CCR2 binds to human CCR2 at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, or at least 30-fold better than antibody 1D9. Preferably, the isolated antibody or antibody fragment specific for human CCR2 binds to human CCR2 at least 30-fold better than antibody 1D9.

[0101] In another embodiment of the disclosure, the isolated antibody or antibody fragment specific for human CCR2 binds to human CCR2 at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 50-fold, or at least 100-fold better than antibody clone 48607. Preferably, the isolated antibody or antibody fragment specific for human CCR2 binds to human CCR2 at least 30-fold better than antibody clone 48607. Preferably, the isolated antibody or antibody fragment specific for human CCR2 binds to human CCR2 at least 100-fold better than antibody clone 48607.

[0102] The antibody referred to herein as "clone 48607" or "antibody clone 40867" refers to CD designated antibody clone 48607. This antibody clone is commercially available (R&D Systems; catalog number: MAB150).

[0103] In one embodiment, the disclosure relates to an isolated antibody or antibody fragment specific for human CCR2 comprising a variable heavy chain (VH) of SEQ ID NO:22 and a variable light chain (VL) of SEQ ID NO:23.

[0104] In one embodiment, the disclosure relates to an isolated antibody or antibody fragment specific for human CCR2 having a variable heavy chain (VH) of SEQ ID NO:22 and a variable light chain (VL) of SEQ ID NO:23.

[0105] In one embodiment, the disclosure relates to an isolated antibody or antibody fragment specific for human CCR2 comprising the variable heavy chain (VH) and variable light chain (VL) of any one of the antibodies disclosed in Table 3.

[0106] In one embodiment, the disclosure relates to an isolated antibody or antibody fragment specific for human CCR2, comprising a variable heavy chain (VH) having at least 80% identity to the VH of SEQ ID NO: 22 and a variable light chain (VL) having at least 80% identity to the VL of SEQ ID NO: 23.

[0107] In one embodiment, the disclosure relates to an isolated antibody or antibody fragment specific for human CCR2, comprising a variable heavy chain (VH) having at least 85% identity to the VH of SEQ ID NO: 22 and a variable light chain (VL) having at least 85% identity to the VL of SEQ ID NO: 23.

[0108] In one embodiment, the disclosure relates to an isolated antibody or antibody fragment specific for human CCR2, comprising a variable heavy chain (VH) having at least 90% identity to the VH of SEQ ID NO: 22 and a variable light chain (VL) having at least 90% identity to the VL of SEQ ID NO: 23.

[0109] In one embodiment, the disclosure relates to an isolated antibody or antibody fragment specific for human CCR2, comprising a variable heavy chain (VH) having at least 95% identity to the VH of SEQ ID NO: 22 and a variable light chain (VL) having at least 95% identity to the VL of SEQ ID NO: 23.

[0110] In one embodiment, the disclosure relates to an isolated antibody or antibody fragment specific for human CCR2, the antibody or antibody fragment comprising a variable heavy chain (VH) having at least 80% identity to an HCDR1 region of SEQ ID NO: 10, an HCDR2 region of SEQ ID NO: 11, an HCDR3 region of SEQ ID NO: 12, an LCDR1 region of SEQ ID NO: 13, an LCDR2 region of SEQ ID NO: 14, an LCDR3 region of SEQ ID NO: 15, and a variable light chain (VL) having at least 80% identity to a VH of SEQ ID NO: 22, and a variable light chain (VL) having at least 80% identity to a VL of SEQ ID NO: 23.

[0111] In one embodiment, the disclosure relates to an isolated antibody or antibody fragment specific for human CCR2, the antibody or antibody fragment comprising a variable heavy chain (VH) having at least 85% identity to an HCDR1 region of SEQ ID NO: 10, an HCDR2 region of SEQ ID NO: 11, an HCDR3 region of SEQ ID NO: 12, an LCDR1 region of SEQ ID NO: 13, an LCDR2 region of SEQ ID NO: 14, an LCDR3 region of SEQ ID NO: 15, and a variable light chain (VL) having at least 85% identity to a VH of SEQ ID NO: 22, and a variable light chain (VL) having at least 85% identity to a VL of SEQ ID NO: 23.

[0112] In one embodiment, the disclosure relates to an isolated antibody or antibody fragment specific for human CCR2, the antibody or antibody fragment comprising an HCDR1 region of SEQ ID NO: 10, an HCDR2 region of SEQ ID NO: 11, an HCDR3 region of SEQ ID NO: 12, an LCDR1 region of SEQ ID NO: 13, an LCDR2 region of SEQ ID NO: 14, an LCDR3 region of SEQ ID NO: 15, and a variable heavy chain (VH) having at least 90% identity to a VH of SEQ ID NO: 22, and a variable light chain (VL) having at least 90% identity to a VL of SEQ ID NO: 23.

[0113] In one embodiment, the disclosure relates to an isolated antibody or antibody fragment specific for human CCR2, the antibody or antibody fragment comprising an HCDR1 region of SEQ ID NO: 10, an HCDR2 region of SEQ ID NO: 11, an HCDR3 region of SEQ ID NO: 12, an LCDR1 region of SEQ ID NO: 13, an LCDR2 region of SEQ ID NO: 14, an LCDR3 region of SEQ ID NO: 15, and a variable heavy chain (VH) having at least 95% identity to a VH of SEQ ID NO: 22, and a variable light chain (VL) having at least 95% identity to a VL of SEQ ID NO: 23. nucleic acid

[0114] In one embodiment, the present disclosure relates to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific to human CCR2, wherein the antibody or antibody fragment comprises an HCDR1 region of SEQ ID NO: 10, an HCDR2 region of SEQ ID NO: 11, an HCDR3 region of SEQ ID NO: 12, an LCDR1 region of SEQ ID NO: 13, an LCDR2 region of SEQ ID NO: 14, and an LCDR3 region of SEQ ID NO: 15.

[0115] In one embodiment, the present disclosure relates to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human CCR2, said antibody or antibody fragment comprising an HCDR1 region of SEQ ID NO: 10, an HCDR2 region of SEQ ID NO: 11, an HCDR3 region of SEQ ID NO: 12, an LCDR1 region of SEQ ID NO: 13, an LCDR2 region of SEQ ID NO: 14, and an LCDR3 region of SEQ ID NO: 15, as defined by Kabat.

[0116] In one embodiment, the present disclosure relates to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific to human CCR2, said antibody or antibody fragment comprising an HCDR1 region of SEQ ID NO: 16, an HCDR2 region of SEQ ID NO: 17, an HCDR3 region of SEQ ID NO: 18, an LCDR1 region of SEQ ID NO: 19, an LCDR2 region of SEQ ID NO: 20, and an LCDR3 region of SEQ ID NO: 21, as defined by IMGT.

[0117] In one embodiment, the present disclosure relates to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human CCR2, said antibody or antibody fragment comprising a variable heavy chain (VH) of SEQ ID NO: 22 and a variable light chain (VL) of SEQ ID NO: 23.

[0118] In one embodiment, the present disclosure relates to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human CCR2, said antibody or antibody fragment comprising a variable heavy chain (VH) of SEQ ID NO: 22 and a variable light chain (VL) of SEQ ID NO: 23.

[0119] In one embodiment, the disclosure relates to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human CCR2 that comprises the six Kabat-defined CDRs of any one of the antibodies disclosed in Table 3.

[0120] In one embodiment, the present disclosure relates to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human CCR2 that comprises the six CDRs defined by IMGT of any one of the antibodies disclosed in Table 3.

[0121] In one embodiment, the disclosure relates to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human CCR2 that comprises the six Chothia-defined CDRs of any one of the antibodies disclosed in Table 3.

[0122] In one embodiment, said nucleic acid composition and / or said nucleic acid sequence and / or nucleic acid sequences are isolated. vector

[0123] In one embodiment, the present disclosure provides a vector composition comprising a vector or a plurality of vectors comprising a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific to human CCR2 according to the present disclosure.

[0124] In one embodiment, the present disclosure provides a vector composition comprising a vector or a plurality of vectors comprising a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding any one of the isolated antibodies or antibody fragments specific for human CCR2 disclosed in Table 3.

[0125] In one embodiment, the vector composition and / or vector and / or vectors are isolated. host cell

[0126] In one embodiment, the present disclosure provides a host cell comprising a vector or a vector composition comprising a plurality of vectors comprising a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific to human CCR2 according to the present disclosure.

[0127] In one embodiment, the present disclosure refers to a host cell comprising a vector or a vector composition comprising a plurality of vectors comprising a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for CCR2 as disclosed in Table 3.

[0128] In one embodiment, a host cell according to the present disclosure is capable of expressing an isolated antibody or antibody fragment specific for human CCR2 encoded by a vector composition or a nucleic acid composition.

[0129] In a further embodiment, the host cell is an isolated host cell. In a further embodiment, 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 a HKB11 cell.

[0130] One skilled in the art will understand that the nucleic acid sequence or sequences encoding the heavy and / or light chains of an antibody or antibody fragment of the present disclosure can be cloned into different vectors or into the same vector.

[0131] The vector can be introduced into a suitable host cell, such as a prokaryotic (e.g., bacterial) or eukaryotic (e.g., yeast or mammalian) cell, by methods well known in the art (e.g., "Current Protocols in Molecular Biology", Ausubel et al. (eds.) Greene Publishing Assoc and John Wiley Interscience, New York, 1989 and 1992). A large number of cloning vectors are known to those skilled in the art, and the selection of an appropriate cloning vector is a matter of choice. The gene may be placed under the control of a promoter, a ribosome binding site (for bacterial expression) and optionally an operator (collectively referred to herein as "control" elements) such that the nucleic acid sequence encoding the desired protein is transcribed into RNA in a host cell transformed by a vector containing this expression construct. The coding sequence may or may not include a signal peptide or leader sequence. Upon expression in a host cell, the antibody or antibody fragment of the present disclosure is obtained. These steps can be achieved in different ways, as known to those skilled in the art. In general, such steps typically include transforming or transfecting a suitable host cell with a nucleic acid composition or vector composition or infectious particle encoding the antibody or antibody fragment. Furthermore, such steps typically include culturing said host cell under conditions suitable for the growth (growth, growth) of said host cell, and culturing under conditions suitable for the production (expression, synthesis) of the encoded antibody or antibody fragment. Cultivation of host cells under conditions suitable for growth or expression is typically accomplished in the presence of a medium containing components suitable for inducing cell growth or expression. In particular, in embodiments, the method of producing an antibody or antibody fragment of the present disclosure further includes isolating and purifying the produced antibody or antibody fragment from the host cell 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 a cell lysate or recovered from a cell membrane fraction. The selection of appropriate growth conditions and recovery methods is within the skill of the art.The antibodies or antibody fragments of the present disclosure can then be purified by a number of techniques known to those of skill in the art.

[0132] In one embodiment, the present disclosure relates to a method of producing an isolated antibody or antibody fragment specific to human CCR2 of any of the antibodies disclosed in Table 3. In one embodiment, a method of producing an isolated antibody or antibody fragment according to the present disclosure is provided, comprising culturing a host cell comprising a vector or vector composition comprising a plurality of vectors comprising a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an antibody or antibody fragment according to the present disclosure under conditions suitable for expression of the antibody or antibody fragment according to the present disclosure, and isolating the antibody or antibody fragment from the host cell or host cell culture medium. Antibodies or antibody fragments isolated as described herein may be purified by techniques known in the art, such as high performance liquid chromatography (HPLC), ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The conditions used to purify a particular antibody or antibody fragment will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, etc., and will be apparent to one of skill 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 with protein A or protein G can be used for affinity chromatography purification of an antibody or antibody fragment according to the present disclosure. The purity of the antibody or antibody fragment can be determined by any of a variety of well-known analytical methods, including gel electrophoresis, high pressure liquid chromatography, and the like. specificity

[0133] In one embodiment, the present disclosure relates to isolated antibodies or antibody fragments specific for human CCR2 as disclosed in Table 3.

[0134] In one embodiment, the present disclosure relates to an isolated antibody or antibody fragment specific for human CCR2.

[0135] In one embodiment, the disclosure relates to an isolated antibody or antibody fragment specific for a polypeptide encoded by the amino acid sequence of SEQ ID NO:1.

[0136] In one embodiment, the disclosure relates to an isolated antibody or antibody fragment specific for a polypeptide comprising the amino acid sequence of SEQ ID NO:1.

[0137] In one embodiment, the isolated antibody or antibody fragment binds to the extracellular domain of human CCR2. In one embodiment, the isolated antibody or antibody fragment binds to the extracellular domain 2 (ECL2) of human CCR2.

[0138] In one embodiment, the present disclosure relates to an isolated antibody or antibody fragment specific for the ECL-2 domain of human CCR2 (SEQ ID NO:2).

[0139] In one embodiment, the present disclosure relates to an isolated antibody or antibody fragment, wherein said antibody or antibody fragment binds to the ECL-2 domain of human CCR2 (SEQ ID NO:2).

[0140] In one embodiment, the disclosure relates to an isolated antibody or antibody fragment specific for marmoset CCR2.

[0141] In one embodiment, the disclosure relates to an isolated antibody or antibody fragment specific for a polypeptide encoded by the amino acid sequence of SEQ ID NO:2.

[0142] In one embodiment, the disclosure relates to an isolated antibody or antibody fragment specific for a polypeptide comprising the amino acid sequence of SEQ ID NO:2.

[0143] In one embodiment, the disclosure relates to an isolated antibody or antibody fragment that is cross-reactive to marmoset CCR2.

[0144] In one embodiment, the present disclosure relates to isolated antibodies or antibody fragments specific for human CCR2 and marmoset CCR2.

[0145] In one embodiment, the present disclosure relates to an isolated antibody or antibody fragment specific to human CCR2 and marmoset CCR2, wherein the antibody or antibody fragment comprises an HCDR1 region of SEQ ID NO: 10, an HCDR2 region of SEQ ID NO: 11, an HCDR3 region of SEQ ID NO: 12, an LCDR1 region of SEQ ID NO: 13, an LCDR2 region of SEQ ID NO: 14, and an LCDR3 region of SEQ ID NO: 15.

[0146] In one embodiment, the present disclosure relates to an isolated antibody or antibody fragment specific to human CCR2 and marmoset CCR2, wherein the antibody or antibody fragment comprises an HCDR1 region of SEQ ID NO: 10, an HCDR2 region of SEQ ID NO: 11, an HCDR3 region of SEQ ID NO: 12, an LCDR1 region of SEQ ID NO: 13, an LCDR2 region of SEQ ID NO: 14, and an LCDR3 region of SEQ ID NO: 15.

[0147] In one embodiment, the disclosure relates to an isolated antibody or antibody fragment that is specific for human CCR2 and does not bind to rhesus CCR2.

[0148] In one embodiment, the present disclosure relates to an isolated antibody or antibody fragment that is specific to human CCR2 and does not bind to rhesus monkey CCR2, wherein the antibody or antibody fragment comprises an HCDR1 region of SEQ ID NO: 10, an HCDR2 region of SEQ ID NO: 11, an HCDR3 region of SEQ ID NO: 12, an LCDR1 region of SEQ ID NO: 13, an LCDR2 region of SEQ ID NO: 14, and an LCDR3 region of SEQ ID NO: 15.

[0149] In one embodiment, the present disclosure relates to an isolated antibody or antibody fragment that is specific to human CCR2 and does not bind to rhesus monkey CCR2, wherein the antibody or antibody fragment comprises an HCDR1 region of SEQ ID NO: 10, an HCDR2 region of SEQ ID NO: 11, an HCDR3 region of SEQ ID NO: 12, an LCDR1 region of SEQ ID NO: 13, an LCDR2 region of SEQ ID NO: 14, and an LCDR3 region of SEQ ID NO: 15.

[0150] In one embodiment, the present disclosure relates to an isolated antibody or antibody fragment specific for human CCR2 that does not bind to human CCR5.

[0151] In one embodiment, the disclosure relates to an isolated antibody or antibody fragment specific for a polypeptide encoded by the amino acid sequence of SEQ ID NO:1 that does not bind to a polypeptide encoded by the amino acid sequence of SEQ ID NO:6.

[0152] In one embodiment, the disclosure relates to an isolated antibody or antibody fragment that binds to a polypeptide encoded by the amino acid sequence of SEQ ID NO:1 and does not bind to a polypeptide encoded by the amino acid sequence of SEQ ID NO:6.

[0153] In one embodiment, the disclosure relates to an isolated antibody or antibody fragment that binds to the extracellular domain 2 (ECL2) of human CCR2 and does not bind to the polypeptide encoded by the amino acid sequence of SEQ ID NO:6.

[0154] In one embodiment, the present disclosure relates to an isolated antibody or antibody fragment, wherein the antibody or antibody fragment binds to the ECL-2 domain of human CCR2 (SEQ ID NO:2) and does not bind to the polypeptide encoded by the amino acid sequence of SEQ ID NO:6.

[0155] In one embodiment, the present disclosure relates to an isolated antibody or antibody fragment, which binds to the ECL-2 domain of human CCR2 (SEQ ID NO:2), binds to a polypeptide encoded by the amino acid sequence of SEQ ID NO:3, and does not bind to a polypeptide encoded by the amino acid sequence of SEQ ID NO:6.

[0156] In one embodiment, the disclosure relates to an isolated antibody or antibody fragment, wherein the antibody or antibody fragment binds to a polypeptide encoded by the amino acid sequence of SEQ ID NO:1, binds to a polypeptide encoded by the amino acid sequence of SEQ ID NO:3, and does not bind to a polypeptide encoded by the amino acid sequence of SEQ ID NO:6.

[0157] In one embodiment, the disclosure relates to an isolated antibody or antibody fragment, wherein the antibody or antibody fragment binds to a polypeptide encoded by the amino acid sequence of SEQ ID NO:1, binds to a polypeptide encoded by the amino acid sequence of SEQ ID NO:3, and does not bind to a polypeptide encoded by the amino acid sequence of SEQ ID NO:6, and wherein the antibody or antibody fragment comprises an HCDR1 region of SEQ ID NO:10, an HCDR2 region of SEQ ID NO:11, an HCDR3 region of SEQ ID NO:12, an LCDR1 region of SEQ ID NO:13, an LCDR2 region of SEQ ID NO:14, and an LCDR3 region of SEQ ID NO:15.

[0158] In one embodiment, the disclosure relates to an isolated antibody or antibody fragment, wherein the antibody or antibody fragment binds to a polypeptide encoded by the amino acid sequence of SEQ ID NO:1, binds to a polypeptide encoded by the amino acid sequence of SEQ ID NO:3, and does not bind to a polypeptide encoded by the amino acid sequence of SEQ ID NO:6, wherein the antibody or antibody fragment has an HCDR1 region of SEQ ID NO:10, an HCDR2 region of SEQ ID NO:11, an HCDR3 region of SEQ ID NO:12, an LCDR1 region of SEQ ID NO:13, an LCDR2 region of SEQ ID NO:14, and an LCDR3 region of SEQ ID NO:15.

[0159] In one embodiment, the disclosure relates to an isolated antibody or antibody fragment, wherein the antibody or antibody fragment binds to a polypeptide encoded by the amino acid sequence of SEQ ID NO:1, binds to a polypeptide encoded by the amino acid sequence of SEQ ID NO:3, and does not bind to a polypeptide encoded by the amino acid sequence of SEQ ID NO:6, wherein the antibody or antibody fragment comprises a variable heavy chain (VH) of SEQ ID NO:22 and a variable light chain (VL) of SEQ ID NO:23.

[0160] In one embodiment, the disclosure relates to an isolated antibody or antibody fragment, wherein the antibody or antibody fragment binds to a polypeptide encoded by the amino acid sequence of SEQ ID NO:1, binds to a polypeptide encoded by the amino acid sequence of SEQ ID NO:3, and does not bind to a polypeptide encoded by the amino acid sequence of SEQ ID NO:6, wherein the antibody or antibody fragment has a variable heavy chain (VH) of SEQ ID NO:22 and a variable light chain (VL) of SEQ ID NO:23. Effects on white blood cells

[0161] CCR2 and MCP-1 play important roles in the migration of monocytes to sites of inflammation. The antibodies and antibody fragments of the present disclosure play an important role in this process and form the basis of the therapeutic utility of the antibodies and antibody fragments of the present disclosure.

[0162] In one embodiment, the present disclosure relates to an isolated antibody or antibody fragment that blocks MCP-1-induced downregulation of CCR2 on leukocytes. In one embodiment, the present disclosure relates to an isolated antibody or antibody fragment that blocks MCP-1-induced downregulation of CCR2 on human leukocytes.

[0163] In one embodiment, the present disclosure relates to an isolated antibody or antibody fragment that blocks MCP-1-induced downregulation of CCR2 on monocytes. In one embodiment, the present disclosure relates to an isolated antibody or antibody fragment that blocks MCP-1-induced downregulation of CCR2 on human monocytes.

[0164] In one embodiment, the present disclosure relates to an isolated antibody or antibody fragment that blocks MCP-1 induced downregulation of CCR2 on basophils and plasmacytoid dendritic cells. In one embodiment, the present disclosure relates to an isolated antibody or antibody fragment that blocks MCP-1 induced downregulation of CCR2 on human basophils and plasmacytoid dendritic cells.

[0165] In one embodiment, the disclosure relates to an isolated antibody or antibody fragment that inhibits CCR2-induced chemotaxis in leukocytes. In one embodiment, the disclosure relates to an isolated antibody or antibody fragment that inhibits CCR2-induced chemotaxis in monocytes. In one embodiment, the disclosure relates to an isolated antibody or antibody fragment that inhibits CCR2-induced chemotaxis in basophils and plasmacytoid dendritic cells. Effector Function

[0166] The Fc region of an immunoglobulin generally confers favorable pharmacokinetic properties to an antibody, such as an extended half-life in serum, and the ability to induce effector functions through binding to Fc receptors expressed on cells. However, binding to Fc receptors can also result in undesired activation of certain cell surface receptors that lead to unwanted cytokine release and severe side effects upon systemic administration.

[0167] Thus, in certain therapeutic situations, it may be desirable to reduce or eliminate the normal binding of a wild-type Fc region of an antibody, such as a wild-type IgG Fc region, to one or more or all of the Fc receptors and / or binding to complement components, such as C1q, to ​​reduce or abolish the ability of the antibody to induce effector functions. For example, it may be desirable to reduce or eliminate binding of an Fc region of an antibody to one or more or all of the Fcy receptors, such as FcyRI, FcyRIla, FcyRIIb, FcyRIIIa, etc. Effector functions may include, but are not limited to, one or more of complement dependent cytotoxicity (CDC), antibody dependent cell-mediated cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen presenting cells, binding to NK cells, binding to macrophages, binding to monocytes, binding to polymorphonuclear cells, direct signaling to induce apoptosis, cross-linking of target-bound antibodies, dendritic cell maturation, or T cell priming.

[0168] The reduction or elimination of binding of an Fc region to an Fc receptor and / or C1q is typically achieved by mutating a wild-type Fc region, such as an IgG1 Fc region, more particularly a human IgG1 Fc region, to result in a mutant or engineered Fc region of said wild-type Fc region, such as a mutant human IgG1 Fc region. Substitutions that result in reduced binding may be useful. To reduce or eliminate the binding properties of an Fc region to an Fc receptor, non-conservative amino acid substitutions, i.e. replacing one amino acid with another amino acid with different structural and / or chemical properties, are preferred.

[0169] Thus, in one embodiment, an isolated antibody or antibody fragment specific for human CCR2 according to the present disclosure comprises a variant Fc region that has reduced or eliminated binding to an Fc receptor and / or C1q when compared to a wild-type Fc region. In one such embodiment, an isolated antibody or antibody fragment according to the present disclosure comprises a variant Fc region that reduces or eliminates the ability of the antibody to induce effector function. In a further embodiment, an isolated antibody or antibody fragment according to the present disclosure does not substantially induce effector function.

[0170] In certain embodiments, the effector function is one or more selected from the group consisting of CDC, ADCC, and ADCP. In one embodiment, the effector function is ADCC. In one embodiment, the effector function is CDC. In one embodiment, the effector function is ADCP. In one embodiment, an isolated antibody or antibody fragment according to the present disclosure does not substantially induce ADCC and / or CDC and / or ADCP. In one embodiment, an isolated antibody or antibody fragment according to the present disclosure does not induce ADCC or ADCP in vitro.

[0171] In one embodiment, the variant Fc region of an isolated antibody or antibody fragment according to the present disclosure comprises one or more amino acid substitutions that reduce or eliminate binding of the variant Fc region to one or more Fc receptors and / or C1q when compared to the wild-type Fc region. In one embodiment, the variant Fc region of an isolated antibody or antibody fragment according to the present disclosure comprises one or more amino acid substitutions that reduce or abolish the ability of the antibody to induce effector function when compared to the wild-type Fc region. In certain embodiments, the one or more amino acid substitutions can reduce the binding affinity of the variant Fc region to one or more Fc receptors and / or C1q by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, or even at least 50-fold when compared to the wild-type Fc region. In alternative embodiments, the one or more amino acid substitutions can reduce the ability of the isolated antibody or antibody fragment according to the present disclosure to induce effector function by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, or even at least 50-fold when compared to the wild-type Fc region.

[0172] In one embodiment, the variant Fc region of an isolated antibody or antibody fragment according to the present disclosure does not substantially bind to one or more Fc receptors and / or C1q. In one embodiment, the variant Fc region of an antibody according to the present disclosure substantially abolishes the ability of said antibody to induce effector function. In one embodiment, an antibody or antibody fragment according to the present disclosure does not substantially induce effector function. In one embodiment, said effector function is ADCC and / or ADCP and / or CDC. In one embodiment, an antibody or antibody fragment according to the present disclosure does not substantially induce effector function, meaning that the level of induced effector function is not significantly above background measured in the absence of said antibody.

[0173] In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is a Fey receptor. In one embodiment, the Fc receptor is human FcyRIIIa, FcyRI, FcyRIla and / or FcyRIlb.

[0174] In one embodiment, an isolated antibody or antibody fragment according to the disclosure comprises a variant human IgG1 Fc region comprising one or more amino acid substitutions compared to a wild-type human IgG1 Fc region, which in one embodiment reduce or eliminate binding of the variant Fc region to an Fc receptor and / or C1q and / or reduce the ability of the antibody to induce effector function when compared to the wild-type Fc region.

[0175] An isolated antibody or antibody fragment according to the present disclosure may or may not be fused to one or more other amino acid residues, polypeptides or moieties. Such fusion proteins may be prepared by any suitable method, including genetic or chemical approaches. The linked moieties may include secretory or leader sequences, sequences that aid in detection, expression, separation or purification, or sequences that provide increased protein stability, for example during recombinant production. Non-limiting examples of possible moieties include β-galactosidase, glutathione-S-transferase, luciferase, T7 polymerase fragments, secretory signal peptides, antibodies or antibody fragments, toxins, reporter enzymes, moieties capable of binding metal ions such as polyhistidine tags, tags suitable for detection and / or purification, homo- or hetero-association domains, moieties that increase the solubility of the protein, or moieties that include an enzymatic cleavage site.

[0176] Thus, an isolated antibody or antibody fragment according to the present disclosure may optionally include one or more moieties for binding to other targets or target proteins of interest. It will be apparent that such additional moieties may or may not provide additional functionality to the antibody and may or may not alter the properties of the isolated antibody or antibody fragment according to the present disclosure. Treatment method

[0177] The isolated antibodies or antibody fragments according to the present disclosure may be used in therapeutic methods. The antibodies or antibody fragments according to the present disclosure may be used to treat inflammatory diseases, autoimmune diseases, hematological malignancies, and potentially other diseases.

[0178] In one embodiment, the disease is associated with the undesired presence of CCR2 in human CCR2. In another embodiment, the disease is associated with the undesired presence of CCR2 positive cells, particularly human CCR2 positive cells. Human CCR2 positive cells include CD16 negative monocytes, basophils and plasmacytoid dendritic cells. Also, about 20% of CD4+ and CD8+ T cells express CCR2.

[0179] In one embodiment, the disease to be treated is an autoimmune disease or an inflammatory disease. Non-limiting examples of autoimmune diseases or inflammatory diseases include autoinflammatory diseases such as rheumatoid arthritis (RA), psoriasis, psoriatic arthritis, systemic lupus erythematosus (SLE), lupus nephritis, type I diabetes, Graves' disease, inflammatory bowel disease (IBD), Crohn's disease (CD), ulcerative colitis (UC), irritable bowel syndrome, multiple sclerosis (MS), Guillain-Barre syndrome, and familial Mediterranean fever (FMF), cryopyrin-associated periodic syndrome (CAPS), IL-1 receptor antagonist (DIRA) deficiency, hyper IgD syndrome (HIDS), autoimmune myocarditis, Kawasaki disease, coronary artery disease, chronic obstructive pulmonary disease (COPD), and interstitial lung disease. These include autoimmune thyroiditis, scleroderma, systemic sclerosis, osteoarthritis, atopic dermatitis, vitiligo, graft versus host disease, Sjogren's syndrome, autoimmune nephritis, Goodpasture's syndrome, chronic inflammatory demyelinating polyneuropathy, ANCA-associated vasculitis, uveitis, scleroderma, bullous pemphigoid, Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's chorea, 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 acute or chronic inflammation.

[0180] In one embodiment, the disease to be treated is a proliferative disease. In a particular embodiment, the disease is cancer. Non-limiting examples of cancer include hematological malignancies such as chronic myelomonocytic leukemia (CMML), acute myelogenous leukemia (AML), myelodysplastic syndrome, mastocytosis, and 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, blood cancer, sarcoma, skin cancer, squamous cell carcinoma, bone cancer, melanoma, renal cell carcinoma, and kidney cancer.

[0181] In one embodiment, the disclosure provides a method for the treatment of a disease.

[0182] In one embodiment, the disclosure provides a method for the treatment of a disease comprising administering to a patient an antibody or antibody fragment of the disclosure.

[0183] In one embodiment, the disclosure provides a method for the treatment of a disease comprising administering to a subject in need thereof an antibody or antibody fragment of the disclosure.

[0184] In one embodiment, the present disclosure provides a method for the prevention of a disease.

[0185] In one embodiment, the present disclosure provides a method for the prevention of a disease comprising administering to a subject an antibody or antibody fragment of the present disclosure.

[0186] In one embodiment, the disclosure provides an isolated antibody or antibody fragment according to the disclosure for the treatment of a disease. In one embodiment, the disclosure provides an isolated antibody or antibody fragment according to the disclosure for use in the treatment of a disease. In one embodiment, the disclosure provides an isolated antibody or antibody fragment according to the disclosure for use in the treatment of a disease in a subject in need thereof.

[0187] In one embodiment, the disclosure provides for the use of an isolated antibody or antibody fragment according to the disclosure for the manufacture of a medicament. In one embodiment, the disclosure provides for an isolated antibody or antibody fragment according to the disclosure for use as a medicament. In one embodiment, the disclosure provides for an isolated antibody or antibody fragment according to the disclosure for use as a medicament. In one embodiment, the disclosure provides for an isolated antibody or antibody fragment according to the disclosure for use as a medicament for the treatment of a subject in need thereof.

[0188] In one embodiment, the present disclosure provides an isolated antibody or antibody fragment specific to human CCR2 according to the present disclosure for use in a method of treating a subject having a disease comprising administering to the subject a therapeutically effective amount of an antibody or antibody fragment according to the present disclosure.

[0189] In one embodiment, the method further comprises administering to the subject a therapeutically effective amount of at least one additional therapeutic agent. The subject in need of treatment is typically a mammal, more particularly a human. For use in a therapeutic method, the isolated antibody or antibody fragment according to the present disclosure will be formulated, dosed, and administered in a manner consistent with good medical practice. Pharmaceutical Compositions

[0190] In one embodiment, the present disclosure provides a pharmaceutical composition comprising an isolated antibody or antibody fragment according to the present disclosure and a pharma- ceutically acceptable carrier or excipient.

[0191] The pharmaceutical composition may further comprise at least one other pharma- ceutical active compound. The pharmaceutical composition according to the present disclosure can be used for the diagnosis, prevention and / or treatment of diseases associated with the undesired presence of CCR2, particularly human CCR2. The pharmaceutical composition according to the present disclosure can be used for the diagnosis, prevention and / or treatment of diseases associated with the undesired presence of CCR2-positive cells, particularly CCR2-positive human cells. In particular, the present disclosure provides a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure suitable for prophylactic, therapeutic and / or diagnostic use in a mammal, more particularly in a human.

[0192] In general, an antibody or antibody fragment according to the present disclosure may be formulated as a pharmaceutical composition comprising at least one antibody or antibody fragment according to the present disclosure, at least one pharma- ceutically acceptable carrier or excipient, and optionally one or more additional pharma- ceutical active compounds. Such a formulation may be suitable for oral, parenteral, topical or inhalation administration. Thus, a pharmaceutical composition comprising at least one antibody or antibody fragment according to the present disclosure may be administered parenterally, for example, intravenously, intramuscularly, or subcutaneously. Alternatively, an antibody of the present invention may be administered by a non-parenteral route, for example, orally or topically. In a preferred embodiment, a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure is administered intravenously or subcutaneously.

[0193] In particular, the antibodies or antibody fragments according to the present disclosure may be used or may be used for the prevention and / or treatment of diseases involving the target antigen of interest, and may be used in combination with one or more pharma- ceutical active compounds, which may or may not result in synergistic effects. Examples of such compounds, as well as routes, methods and pharmaceutical formulations or compositions for administering them, will be apparent to the clinician.

[0194] In one embodiment, the present disclosure provides a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure for use in the prevention and / or treatment of a disease associated with the undesired presence of CCR2, particularly human CCR2. In one embodiment, the present disclosure provides a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure for use in the prevention and / or treatment of a disease associated with the undesired presence of CCR2 positive cells, particularly CCR2 positive human cells. In one embodiment, the present disclosure provides a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure for use as a medicament. In one embodiment, the present disclosure provides a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure for use in the prevention and / or treatment of an autoimmune disease and / or an inflammatory disease and / or cancer.

[0195] In one embodiment, the present disclosure provides a method for treating an autoimmune disease and / or inflammatory disease and / or cancer in a subject in need thereof using a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure.

[0196] Further provided is a method of producing an antibody or antibody fragment according to the present disclosure in a form suitable for in vivo administration, comprising (a) obtaining an antibody or antibody fragment by a method according to the present disclosure, and (b) formulating said antibody or antibody fragment with at least one pharma- ceutically acceptable carrier or excipient, whereby an antibody or antibody fragment preparation is formulated for in vivo administration. Pharmaceutical compositions according to the present disclosure comprise a therapeutically effective amount of one or more antibodies or antibody fragments according to the present disclosure dissolved in a pharma- ceutical carrier or excipient. Diagnostic Uses

[0197] In one embodiment, the present disclosure provides the use of an isolated antibody or antibody fragment specific to human CCR2 according to the present disclosure for the diagnosis of disease. In one embodiment, the present disclosure provides the use of an antibody or antibody fragment according to the present disclosure for the detection of CCR2, particularly human CCR2 and / or marmoset CCR2. In one embodiment, the present disclosure provides a method for detecting CCR2 in a subject or sample, comprising contacting the subject or sample with an isolated antibody or antibody fragment specific to human CCR2 according to the present disclosure. In one embodiment, the present disclosure provides a method for diagnosing disease in a subject, comprising contacting the subject or sample with an isolated antibody or antibody fragment according to the present disclosure. The antibody can also be used to determine CCR2 expression levels in cells from a patient. CCR2 expression levels can serve as a therapeutic biomarker, for example, for patient stratification. Working Example

[0198] Example 1: Generation of anti-CCR2 antibodies using hybridoma technology Anti-CCR2 antibodies were generated using classical hybridoma technology. Briefly, BALB / c mice were immunized six times at 4-week intervals with 10 million CHO cells stably transfected with human CCR2b. Splenocytes were fused to X63Ag8 myeloma cells, and hybridomas were screened for specific binding to CCR2-transfected CHO cells and absence of binding to CCR5-transfected CHO cells. Hybridomas were cloned and recloned by limiting dilution technique.

[0199] Several anti-CCR2 antibodies have been identified and characterized, including antibodies Y4T3, Y2T63 and Y1T2.

[0200] Example 2: Specificity of human CCR2 and absence of cross-reactivity with CCR5 Hybridoma antibodies were tested for binding to human CCR2 expressed in transfected CHO cells. As a control, CHO cells were also transfected with human CCR5. As another control, anti-CCR5 antibody MC-1 (Mol Biol Cell. 2002 Feb;13(2):723-737, in-house) was also tested for binding to transfected CHO cells. Antibodies were tested at concentrations of 10 μg / ml and 1 μg / ml and incubated with the cells for 45 min on ice. After three washing steps with cold PBS, bound antibodies were detected with a PE-labeled polyclonal rabbit anti-mouse immunoglobulin antibody (R0439 from Agilent Dako, diluted 1:200 in PBS) for 30 min on ice. After two washing steps with PBS, cells were analyzed by flow cytometry. The mean fluorescence intensity of PE was determined and shown.

[0201] The results for the exemplary antibody Y4T3 are shown in Figure 1. The anti-CCR2 antibody Y4T3 was specific for human CCR2 but showed no binding to human CCR5. The anti-CCR5 antibody MC-1 was specific for human CCR5 but showed no binding to human CCR2.

[0202] Example 3: Binding to human monocytes To exert their functional effect, the antibodies of the invention must bind to CCR2 expressed on human monocytes. Binding to CD16-positive and CD16-negative monocytes was measured. 60 μl of lithium heparin-anticoagulated human whole blood was incubated with the indicated antibodies at the indicated concentrations in PBS for 45 min on ice. After three washing steps with cold PBS, bound antibodies were detected with a PE-labeled polyclonal rabbit anti-mouse immunoglobulin antibody (R0439 from Agilent Dako, diluted 1:200 in PBS) for 30 min on ice. After three washing steps with PBS, 10% mouse serum was added and incubated for 10 min on ice. Without further washing, directly labeled antibodies against CD3, CD8, CD4, CD123, CD304, CD116 and CD16 were added for 30 min on ice. After two washing steps with 0.9% NaCl, red blood cells were lysed with lysing solution (BD Bioscience) for 10 min in the dark. Cells were washed once with PBS and analyzed by flow cytometry to identify CD16 positive and CD16 negative monocytes. The mean fluorescence intensity of PE was determined for these cell populations and is shown. CCR2 is expressed only on CD16 negative, but not on CD-16 positive monocytes. Two additional anti-CCR2 antibodies were also tested: 1D9, the parent antibody of MLN1202 previously developed by Millennium (BD Bioscience), and Clone48607, a commercially available anti-CCR2 antibody (R&D Systems; Catalog Number: MAB150).

[0203] The results are shown in Figure 2. Exemplary antibody Y4T3 was reactive with CD16-negative monocytes but did not bind to CD16-positive monocytes. Control antibodies 1D9 and clone 48607 also showed binding to CD16-negative monocytes but did not show binding to CD16-positive monocytes. However, the binding of Y4T3 was clearly stronger than that of 1D9 and clone 48607.

[0204] Example 4: Binding to marmoset monocytes Marmoset monkeys are an established animal model, therefore binding to marmoset CCR2 is highly desirable.

[0205] Binding to CD16-positive and CD16-negative marmoset monocytes was measured. DOC-3, an anti-CCR2 antibody with known reactivity against marmoset CCR2 (WO 2007 / 115713), was used as a control. Marmoset splenocytes were incubated with the indicated antibodies at the indicated concentrations in PBS for 30 min on ice. After three washing steps with cold PBS, bound antibodies were detected with a PE-labeled polyclonal rabbit anti-mouse immunoglobulin antibody (R0439 from Agilent Dako, diluted 1:200 in PBS) for 30 min on ice. After three washing steps with PBS, 10% mouse serum was added and incubated for 10 min on ice. Without further washing, directly labeled antibodies against CD14, CD11b, CD16 and CD20 were added for 20 min on ice. After two washing steps with 0.9% NaCl, red blood cells were lysed with lysing solution (BD Bioscience) for 10 min in the dark. Cells were washed once with PBS and analyzed by flow cytometry to identify CD16 positive and CD16 negative monocytes. The mean fluorescence intensity of PE was determined for these cell populations and is shown.

[0206] The results are shown in Figure 3. Exemplary antibodies DOC-3 and Y4T3 demonstrated binding to CD16-negative marmoset monocytes, but not to CD16-positive marmoset monocytes. Antibodies Y1T2 and Y2T63 did not bind to CD16-negative, CD16-positive marmoset monocytes.

[0207] Antibody Y4T3 did not bind to CCR2 of cynomolgus monkeys (data not shown). CCR2 of cynomolgus monkeys, rhesus monkeys and Janaver have identical amino acid sequences in the extracellular domains.

[0208] Example 5: Binding of Y4T3 to the ECL2 domain of human CCR2 To further investigate the specificity of the antibodies of the invention, the binding of Y4T3 to various CCR2 constructs was tested. Antibody 1D9 and clone 48607 were also tested (see above). CHO cells were stably transfected with the CCR2 constructs shown in table 1. Non-transfected CHO cells (blank) were also analyzed. Antibodies were tested at a concentration of 10 μg / ml and incubated with the cells for 30 min on ice. After three washing steps with cold PBS, bound antibodies were detected with a PE-labeled polyclonal rabbit anti-mouse immunoglobulin antibody (R0439 from Agilent Dako, diluted 1:200 in PBS) for 30 min on ice. After two washing steps with PBS, the cells were analyzed by flow cytometry. The mean fluorescence intensity of PE was determined on the cells and is shown graphically.

[0209] The following constructs were tested: [Table 1]

[0210] The results are shown in FIG. 4. Antibody Y4T3 bound to human CCR2b and marmoset CCR2b, but not to rhesus CCR2b. Antibody Y4T3 also bound to a human CCR2b mutant in which the ECL3 domain was replaced with the ECL3 domain of rhesus CCR2b, and to a human CCR2b mutant in which the N-terminus of CCR2b was replaced with the N-terminus of rhesus CCR2b. Antibody Y4T3 no longer bound to a human CCR2b mutant in which the ECL2 domain was replaced with the ECL2 domain of rhesus CCR2b. Thus, the ECL2 domain contains amino acid residues important for Y4T3 binding. In contrast, both antibody 1D9 and clone 48607 showed reactivity with rhesus CCR2b.

[0211] Example 6: MCP-1-induced downregulation of CCR2 from the surface of human monocytes results in decreased binding of Y4T3 to human monocytes To investigate whether binding of Y4T3 to human monocytes is reduced when CCR2 is downregulated from the cell surface by MCP-1, the following experiment was performed: Human PBMCs were preincubated with various concentrations of human MCP-1 (Peprotec) for 30 min at 37°C. Afterwards, cells were cooled to 4°C and Y4T3 was added at 10 μg / ml for 30 min on ice. After three washing steps with cold PBS, bound antibodies were detected with a PE-labeled polyclonal rabbit anti-mouse immunoglobulin antibody (R0439 from Agilent Dako, diluted 1:200 in PBS) for 30 min on ice. After two washing steps with PBS, cells were analyzed by flow cytometry and monocytes were identified by their light scattering properties. The mean fluorescence intensity of PE was determined in monocytes and is shown graphically.

[0212] The results are shown in Figure 5. At a concentration of 2 μg / ml MCP-1, more than 70% of the surface CCR2 signal obtained with Y4T3 was lost. When lower concentrations of MCP-1 were used, the surface CCR2 signal returned.

[0213] This experiment demonstrates that Y4T3 binds to a surface molecule that can be downregulated by MCP-1.

[0214] Example 7: Selection of candidate antibodies for humanization Candidate antibodies were selected for humanization based on experimental data obtained to date, and the characteristics of the lead antibodies are summarized in the table below. [Table 2]

[0215] Based on experimental data, the available antibody Y4T3 was selected for humanization. Criteria included the high affinity of the binder for CCR2 on human PBMCs as well as the excellent cross-reactivity of the binder for marmoset CCR2.

[0216] Example 8: Humanization of Y4T3 Y4T3 was humanized by CDR grafting to obtain hY4T3. Databases of human IgG sequences and human germline sequences were searched for comparison with the mouse VH and VL domains using the BLAST search algorithm and candidate human variable domains selected from the top 200 BLAST results. These were reduced to five candidates for VH (HC1-HC5 or H1-H5) and five candidates for VL (LC1-LC5 or L1-L5) based on combinations of framework homology, preserving key framework residues and canonical loop structures. A total of 25 humanized variants of Y4T3 were generated. Humanized Y4T3 variants were tested for binding to human CCR2+ monocytes in human whole blood by flow cytometry. 60 μl of lithium heparin anticoagulated human whole blood was incubated with the indicated antibodies at 0.5 μg / ml in PBS for 45 min on ice. After three washing steps with cold PBS, bound antibodies were detected with PE-F(ab)2 goat anti-human IgG (Jackson ImmunoResearch, 109-116-098) along with directly labeled antibodies against CD8, CD4, CD123 and CD116. After two washing steps with 0.9% NaCl, red blood cells were lysed with lysing solution (BD Bioscience) for 10 min in the dark. Cells were washed once with PBS and analyzed by flow cytometry to identify CCR2 expression on various cell types, including monocytes. The mean fluorescence intensity (MFI) of PE was determined for these cell populations and is shown.

[0217] The results are shown in Figure 6. The majority of the binders were able to bind to CCR2 expressed on human monocytes.

[0218] The selected binder, H3L1, showed excellent binding not only to CCR2 positive monocytes but also to other CCR2 positive cells such as basophils, pDCs and T cells. The selected binder is designated hY4T3.

[0219] Example 9: Sequencing of Y4T3 and hY4T3 The sequences of Y4T3 and hY4T3 were determined by standard sequencing techniques.

[0220] The variable heavy and variable light chains and CDR regions according to Kabat and IMGT nomenclature are shown in the table below. [Table 3]

[0221] The closest germline for the variable heavy chain of hY4T3 is IGHV 1-3*01. The closest germline gene for the variable light chain of hY4T3 is IGKV2-30*01. See Table 4. [Table 4]

[0222] Compared to the closest germline sequence, the variable heavy chain of hY4T3 differs by one amino acid: in framework region 2, a methionine residue is replaced by an isoleucine residue to reduce oxidation. Similarly, in framework region 3 of the variable light chain, a serine residue is replaced by a threonine residue.

[0223] Example 10: hY4T3 binds to CCR2+ human monocytes Binding of hY4T3 to CCR2+ human monocytes was tested with human PBMCs by flow cytometry. Human PBMCs were incubated with various concentrations of hY4T3 in PBS for 30 min on ice. After three washing steps with cold PBS, bound antibodies were detected with PE-F(ab)2 goat anti-human IgG (Jackson ImmunoResearch, 109-116-098). After three washing steps with PBS, 5% mouse and 5% rat serum was added for 10 min on ice. Directly labeled antibodies against CD20, CD14, CD11b, CD16 were added without washing. After two washing steps with 0.9% NaCl, red blood cells were lysed with lysing solution (BD Bioscience) for 10 min in the dark. Cells were washed once with PBS and analyzed by flow cytometry to identify CCR2 expression on monocytes. The mean fluorescence intensity (MFI) of PE was determined and is shown. hY4T3 retained the binding properties to human monocytes. The results are shown in Figure 7.

[0224] Example 11: hY4T3 binds to CCR2+ marmoset monocytes Binding of hY4T3 to CCR2+ marmoset monocytes was tested using marmoset splenocytes as described in Example 10. hY4T3 retained binding properties to marmoset monocytes. The results are shown in FIG.

[0225] Example 12: hY4T3 does not bind to CCR5 expressed in transfected CHO cells Similar to Y4T3, the human IgG1 chimeric variant of Y4T3 (Y4T3-chim) and hY4T3 also do not bind to CHO cells transfected with human CCR5. Y4T3-chim and hY4T3 bind to CHO cells transfected with human CCR2. Antibodies were tested at concentrations of 10 μg / ml and 1 μg / ml and incubated with the cells for 45 min on ice. After three washing steps with cold PBS, bound antibodies were detected with PE-F(ab)2 goat anti-human IgG (Jackson ImmunoResearch, 109-116-098) for 30 min on ice. After two washing steps with PBS, the cells were analyzed by flow cytometry. The mean fluorescence intensity (MFI) of PE was determined and shown. The results are shown in FIG. 9.

[0226] Example 13: hY4T3 blocks MCP-1-induced downregulation of CCR2 in leukocytes To examine the functional activity of hY4T3, MCP-1-induced downregulation of CCR2 was measured in various leukocyte subsets. Basophils, plasmacytoid dendritic cells and CD16 low monocytes were tested.

[0227] Cells were first incubated with various concentrations of hY4T3, MCP-1 or medium (RPMI) for 30 min at 37°C. Then, MCP-1, hY4T3 or RPMI was added, followed by further incubation at 37°C for 30 min. Cells were not washed between the first and second incubation steps. After the second incubation, cells were washed three times with PBS. Surface-bound antibodies were detected with PE-F(ab)2 goat anti-human IgG (Jackson ImmunoResearch, 109-116-098) together with directly labeled antibodies against CD3, CD8, CD4, CD123, CD304, CD116 and CD16. After two washing steps with 0.9% NaCl, red blood cells were lysed with lysing solution (BD Bioscience) for 10 min in the dark. Cells were washed once with PBS and analyzed by flow cytometry to identify CCR2 expression on various cell types, including basophils, plasmacytoid dendritic cells, and CD16 low monocytes. The mean fluorescence intensity (MFI) of PE was determined for these cell populations and is shown.

[0228] The results are shown in Figure 10. Preincubation with MCP-1 resulted in a dose-dependent decrease in the binding of hY4T3 to CCR2. When cells were first incubated with hY4T3, the binding strength was largely unaffected by subsequent incubation with MCP-1.

[0229] This indicates that hY4T3 blocks MCP-1-induced downregulation of CCR2 in leukocytes, indicating that hY4T3 blocks activation of CCR2 by MCP-1.

[0230] Example 14: Cell depletion in marmosets treated with hY4T3 Marmoset monkeys were treated at time 0 with 5 mg / kg hY4T3 by slow intravenous injection. At various time points, pre- and post-treatment whole blood was analyzed by flow cytometry. EDTA-decoagulated whole blood was incubated with 3 μg / ml hY4T3 in PBS for 30 min on ice. After three washing steps with cold PBS, bound antibodies were detected with PE-F(ab)2 goat anti-human IgG (Jackson ImmunoResearch, 109-116-098) together with directly labeled antibodies against CD3, CD20, CD14, CD11b and CD16. After two washing steps with 0.9% NaCl, red blood cells were lysed with lysing solution (BD Bioscience) for 10 min in the dark. Cells were washed once with PBS and analyzed by flow cytometry to identify the different cell populations. Experiments were performed on three monkeys.

[0231] Representative results from one monkey are shown in Figure 11. The percentages of CD3 positive cells, CD3 positive / CCR2 positive cells, CD16 positive NK cells, and plasmacytoid dendritic cells (pDCs) did not change significantly. The percentage of B cells increased with treatment with hY4T3, but dropped to the original level (i.e., pretreatment level) on day 23. The number of basophils was strongly decreased with treatment with hY4T3, but returned to the original level about 1-2 weeks after treatment. The same pattern was observed for CD16 negative monocytes, which were also strongly decreased with treatment with hY4T3. The number returned to the original level about 2 weeks after treatment.

[0232] This indicates that the antibodies and antibody fragments of the present disclosure are useful for treating inflammatory diseases, autoimmune diseases, hematological malignancies, and potentially other diseases associated with expression of CCR2.

[0233] Example 15: Developability - hY4T3 has good temperature stability To test whether hY4T3 has a melting point indicative of successful clinical development, the melting point was measured by differential scanning calorimetry using a Microcal VP-DSC (Malvern). The temperature of an 8.44 mg / ml solution of hY4T3 in PBS + 100 mM L-arginine was increased from 25°C to 130°C at a rate of 1°C / min.

[0234] The melting temperature of the CH2 domain was determined to be 70.9° C. (Tm1). The Fab and CH3 domains have melting temperatures of 75.1° C. (Tm2). Complete melting of the molecule occurred at 82.5° C. (Tm3).

[0235] The stability of hY4T3 was further investigated by analytical size exclusion chromatography (SEC). An 8.44 mg / ml solution of hY4T3 in PBS + 100 mM L-arginine was incubated at 55, 65 or 75°C for 3 hours, respectively, followed by analytical SEC using a Superdex 200 Increase and photometric detection of protein at 280 nm. The results are shown in Figure 12. The SEC pattern of hY4T3 incubated at 55°C for 3 hours was indistinguishable from the reference, i.e., antibody kept at 4°C. After 3 hours of incubation at 65°C, more than 50% of the signal was lost due to the formation of insoluble denatured protein. Incubation of hY4T3 at 75°C for 3 hours resulted in the loss of most of the antibody due to the formation of insoluble denatured protein. There were almost no cleavage products or soluble aggregates.

[0236] Binding of hY4T3 to CCR2 on CHO cells was investigated similarly. 8.44 mg / ml solutions of hY4T3 in PBS+100 mM L-arginine were incubated at 55, 65 and 75° C. for 2 hours, respectively. The antibody preparations were then tested for binding to CCR2 on CHO cells as described above. The results are shown in FIG. 13. hY4T3 incubated at 55° C. for 2 hours showed similar binding to CHO cells as the reference, i.e., antibody kept at 4° C. After 2 hours of incubation at 65° C., a slight loss of binding was observed. Incubation of hY4T3 at 75° C. for 2 hours led to a loss of binding.

[0237] hY4T3 was also stable at 37°C for at least 14 days (longest time point tested) at various antibody concentrations (0.015625, 0.0625, 0, 25 and 1 μg / ml) in PBS as well as in serum (data not shown). These data indicate that hY4T3 has a very favorable melting point, supporting successful clinical development. The antibody is stable at 55°C for at least 2 hours.

[0238] Example 16: Developability - hY4T3 has good freeze-thaw stability To test the behavior of hY4T3 against repeated freezing and thawing, hY4T3 (8.44 mg / ml in PBS containing 100 mM L-arginine) was subjected to repeated freeze-thaw cycles. After 10 cycles of freezing and thawing, samples were subjected to SEC, but no differences were observed compared to the reference (data not shown).

[0239] A sample of hY4T3 (8.44 mg / ml in PBS containing 100 mM L-arginine) was also tested for binding to CCR2 on CHO cells after various freeze-thaw cycles. The results are shown in Figure 14. Up to 10 cycles of freezing and thawing, the preparation showed no loss of binding to CCR2 expressed on CHO cells.

[0240] These data further demonstrate that hY4T3 is stable and support successful clinical development.

[0241] Example 17: Developability - hY4T3 is stable over a wide pH range To test the behavior of hY4T3 over different pH ranges, hY4T3 was incubated with different antibody concentrations in buffers of different pH for 2 hours.

[0242] To do so, a 8.44 mg / ml solution of hY4T3 in PBS + 100 mM L-arginine was dissolved 1:5 with water and the pH was adjusted to the desired value with HCl / NaOH. The sample was then incubated at room temperature for 2 hours before readjusting the pH to 7.2 with HCl / NaOH.

[0243] Binding of the samples to CCR2 expressed on CHO cells was measured using various concentrations of pH-exposed antibody, and the results are shown in FIG.

[0244] Compared to the reference sample (pH 6,82), incubation at a low pH of 2 did not affect binding to CCR2 expressed on CHO cells, whereas incubation at pH 9 showed a slight loss of binding.

[0245] Again, these data show that hY4T3 is highly stable even at very low pH, again strongly supporting successful clinical development. The antibody is stable at pH 3.0 and 22° C. for at least 2 hours.

[0246] Example 18: Developability - hY4T3 is stable in plasma To test the behavior of hY4T3 in plasma, various experiments were performed: hY4T3 was incubated at various concentrations in PBS and human plasma for 48 hours, 7 days, and 14 days.

[0247] To do so, 23.7 μl of hY4T3 (8.44 mg / ml in PBS containing 100 mM L-arginine) was mixed with 176.3 μl of PBS or human lithium heparin plasma (1 mg / ml) and incubated at 37° C. for 0 h, 48 h or 14 days. Binding of the samples to CCR2 expressed on CHO cells was then measured using various concentrations of challenge antibody. The results are shown in FIG.

[0248] Binding to CCR2 expressed on CHO cells was independent of the incubation time interval, and there was no difference between incubation in PBS and in human plasma.

[0249] This experiment further confirms the advantageous biochemical properties of hY4T3.

[0250] Example 19: Comparison with prior art antibodies Antibody Y4T3 was compared to prior art antibodies DOC2, 1D9 (BD Bioscience) and clone 48607 (R&D Systems; Catalog Number: MAB150). The DOC-2 antibody has the following amino acid sequence: VH: EVQLVESGGGLVKPGGSLKLSCVASGFTLSNYAMSWVRQSPEKRLEWVAEVSSSGIYIYYSDTVTGRFSISRDNAKNTLYLEMSSLRSEDTAIYYCARDRYAYAMDYWGHGTSVIVSS (SEQ ID NO:28) VL: DIVMTQSPSSLAISVGQRVTLSCKSSQSLLNSYNQKNSLAWYQQKPGQSPKLLVYFASTRESGVPDRFIGSGSGTYFTLTITSVQAGDLADYFCQQHYSNPRTFGGGTRLEIK (SEQ ID NO:29)

[0251] Human heparinized whole blood was incubated with the indicated antibodies for 45 min on ice, washed three times with cold PBS, then stained with secondary PE-labeled rabbit anti-mouse Ig (R0439, DakoCytomation 1:200) for 30 min on ice and washed three times with cold PBS. 10% mouse serum was added for 10 min, followed by directly labeled antibodies against CD3-APCCy7, CD8-PE-Cy7, CD4-V500, CD123-PerCP, CD304-APC, CD116-FITC, CD16-PB. After washing and lysis of red blood cells with BD lysis solution, cells were acquired on a FACSCanto-II and analyzed by FACS-DIVA software. Classical monocytes were identified by light scattering properties, expression of CD116, and absence of CD16 and CD123. Median fluorescence intensity of PE is shown on classical monocytes.

[0252] The results are shown in Figure 17. The affinity of Y3T3 was more than 5-fold higher than that of the prior art antibodies tested.

[0253] Example 20: hY4T3 does not cross-react with mouse CCR2 MouseCCR2 CHO cells and human CCR2 CHO cells were incubated with PBS or various concentrations of MC-21 (rat monoclonal antibody against mouse CCR2; University of Regensburg) on ​​ice for 45 min. After three washing steps with cold PBS, cells were stained with Biotin-anti-rat IgG2b antibody (5 μg / ml; BD clone RG7 / 11.1, catalog number 553898) on ice for 30 min. After three washing steps with cold PBS, cells were stained with streptavidin-PE (1:100) on ice for 30 min. After three washing steps with cold PBS, cells were analyzed by FACS to measure the mean fluorescence intensity (MFI).

[0254] Mouse CCR2 CHO cells and human CCR2 CHO cells were incubated with PBS or various concentrations of hY4T3 on ice for 45 min. After three washing steps with cold PBS, cells were stained with PE-F(ab)2 goat anti-human IgG (1:300; Jackson, Cat. No. 109-116-098) on ice for 30 min. After three washing steps with cold PBS, cells were analyzed by FACS to measure the mean fluorescence intensity (MFI).

[0255] The results are shown in Figure 18. hY4T3 does not bind to mouse CCR2. MC-21 does not bind to human CCR2.

[0256] Example 21: Antagonistic effect of hY4T3 to efficiently block chemokine receptor CCR2 The PathHunter® eXpress CCR2 CHO-K1 β-arrestin GPCR assay (DiscoverX; Cat. No. 93-0192E2CP0M) was used to assess the antagonist activity of hY4T3 according to the manufacturer's protocol. CCR2 CHO K1 β-arrestin GPCR cells from the kit were thawed, plated and incubated for 48 hours at 37°C and 5% CO2. After incubation, different concentrations of CCL2 (MCP-1), hY4T3, hY4T3 pre-incubated with fixed concentrations of CCL2 (EC20, EC50 and EC80) for 30 minutes or hY4T3 pre-incubated with anti-IgG antibody (Biorad; #MCA 5748 G) for 30 minutes were prepared and added to the cells. After 90 min of incubation at 37° C. and 5% CO2, detection reagent was added to the plate and luminescence was measured after 60 min on an Envision plate reader.

[0257] The results are shown in Figure 19. This experiment demonstrates that hY4T3 is a potent CCR2 antagonist with no agonist activity under any of the conditions tested.

Claims

1. 1. An isolated antibody or antibody fragment specific for human CCR2, comprising: the antibody or antibody fragment comprises an HCDR1 region of SEQ ID NO: 10, an HCDR2 region of SEQ ID NO: 11, an HCDR3 region of SEQ ID NO: 12, an LCDR1 region of SEQ ID NO: 13, an LCDR2 region of SEQ ID NO: 14, and an LCDR3 region of SEQ ID NO: 15; the antibody or antibody fragment is cross-reactive with marmoset CCR2 but not with rhesus CCR2; the antibody or antibody fragment does not bind to mouse CCR2, An antibody or antibody fragment, wherein the antibody or antibody fragment has a higher affinity for human CCR2 expressed on human monocytes than an antibody comprising the variable heavy chain of SEQ ID NO: 28 and the variable light chain of SEQ ID NO:

29.

2. 2. The isolated antibody or antibody fragment of claim 1, wherein the antibody or antibody fragment comprises a VH of SEQ ID NO: 22 and a VL of SEQ ID NO:

23.

3. 2. The isolated antibody or antibody fragment of claim 1, wherein the antibody is of the human IgG1 class.

4. 2. The isolated antibody or antibody fragment of claim 1, wherein the antibody binds to the ECL-2 domain of human CCR2 (SEQ ID NO: 2).

5. 2. The isolated antibody or antibody fragment of claim 1, wherein the antibody does not bind to human CCR5 (SEQ ID NO: 6).

6. 2. The isolated antibody or antibody fragment of claim 1, wherein the antibody or antibody fragment has at least 5 times higher affinity for human CCR2 expressed on human monocytes than an antibody comprising a variable heavy chain of SEQ ID NO: 28 and a variable light chain of SEQ ID NO:

29.

7. 2. The isolated antibody or antibody fragment of claim 1, wherein the antibody blocks MCP-1-induced downregulation of CCR2 on human leukocytes.

8. 2. The isolated antibody or antibody fragment of claim 1, wherein the isolated antibody or antibody fragment is a monoclonal antibody or antibody fragment and / or a humanized antibody or antibody fragment.

9. 9. The isolated antibody or antibody fragment of any one of claims 1 to 8 for use in a medicine used in the treatment of an inflammatory disease, an autoimmune disease or a hematological malignancy.

10. A nucleic acid composition comprising a nucleic acid sequence or sequences encoding the isolated antibody or antibody fragment of any one of claims 1 to 8.

11. A vector comprising the nucleic acid composition of claim 10.

12. A host cell comprising the vector of claim 11.

13. A host cell comprising the nucleic acid composition described in claim 10.

14. A pharmaceutical composition comprising the isolated antibody or antibody fragment of any one of claims 1 to 8 and a pharmaceutically acceptable carrier or excipient.

15. 1. An isolated antibody or antibody fragment specific for the ECL-2 domain of human CCR2 (SEQ ID NO: 2), comprising: the antibody or antibody fragment comprises a VH of SEQ ID NO: 22 and a VL of SEQ ID NO: 23; the antibody or antibody fragment is cross-reactive with marmoset CCR2 but not with rhesus CCR2; the antibody or antibody fragment does not bind to mouse CCR2, The antibody or antibody fragment has a higher affinity for human CCR2 expressed on human monocytes than an antibody comprising the variable heavy chain of SEQ ID NO: 28 and the variable light chain of SEQ ID NO: 29 at an antibody concentration of 0.1 to 1.0 μg / ml.

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