WO2020232051A1

WO2020232051A1 - Antibody binding csf-1r and use thereof

Info

Publication number: WO2020232051A1
Application number: PCT/US2020/032570
Authority: WO
Inventors: Mingjiu Chen; Wei Tan; Shukai Xia
Original assignee: Biosion Inc.
Priority date: 2019-05-15
Filing date: 2020-05-13
Publication date: 2020-11-19
Also published as: CN113939539A

Abstract

The present invention relates to an isolated monoclonal antibody or an antigen-binding portion thereof that specifically binds CSF-1R. A nucleic acid molecule encoding the antibody or the antigen-binding portion thereof, an expression vector, a host cell and a method for expressing the antibody or the antigen-binding portion thereof are also provided. The present disclosure further provides a bispecific molecule, an immunoconjugate, a chimeric antigen receptor, an oncolytic virus and a pharmaceutical composition comprising the antibody or the antigen-binding portion thereof, as well as a treatment method using the antibody or the antigen-binding portion thereof of the disclosure.

Description

ANTIBODY BINDING CSF-1 R AND USE THEREOF

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

[0001] This application claims priority to US provisional patent application Serial No. 62/847,950 filed

May 15, 2019.

[0002] The foregoing applications, and all documents cited therein or during their prosecution (“appln cited documents”) and all documents cited or referenced herein (including without limitation all literature documents, patents, published patent applications cited herein) (“herein cited documents”), and all documents cited or referenced in herein cited documents, together with any manufacturer’s instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. Any Genbank sequences mentioned in this disclosure are incorporated by reference with the Genbank sequence to be that of the earliest effective filing date of this disclosure.

TECHNICAL FIELD

[0003] The present disclosure relates generally to an isolated monoclonal antibody, particularly a monoclonal antibody, or an antigen-binding portion thereof, that specifically binds to human CSF-1R with high affinity and functionality. A nucleic acid molecule encoding the antibody or the antigen binding portion thereof, an expression vector, a host cell and a method for expressing the antibody or the antigen-binding portion thereof are also provided. The present disclosure further provides an immunoconjugate, a bispecific molecule, a chimeric antigen receptor, an oncolytic virus, and a pharmaceutical composition which may comprise the antibody or the antigen-binding portion thereof, as well as a treatment method using an anti-CSF-lR antibody or the antigen-binding portion thereof of the disclosure.

BACKGROUND

[0004] Colony stimulating factor 1 receptor (CSF-1R), a type III receptor tyrosine kinase, contains an intracellular kinase domain and a ligand-binding extracellular region organized in five immunoglobulin-like subdomains. It is expressed on hematopoietic stem cells at low levels, on e.g., monocytes and tissue-resident macrophages, osteoclasts, and myeloid dendritic cells at higher levels, and controls the development of these cell types (Stanley ER and Chitu V, (2014) Cold Spring Harb Perspect Biol 6(6):a021857).

[0005] CSF1 and IL34 are two known ligands of CSF-1R. IL34 expression is restricted to the central nervous system and the skin, while CSF1 is systematically expressed (Baghdadi M et al., (2018) Scientific Reports 8(1): 418). The differential expression of these two ligands results in differential spatiotemporal regulation through CSF-1R. [0006] The engagement of CSF-1R with CSF1 or IL34 activates CSF-1R signaling and plays roles in embryonic development, bone physiology, innate immunity, inflammation, tissue repair and in tumor microenvironment (Stanley ER and Chitu V, (2014) supra). For example, studies have shown the CSF- 1R signaling regulates the production, differentiation and homeostasis of most circulating and tissue- resident macrophages. The elevated CSF1 and/or IL34 expression may lead to the functional dysregulation of macrophages and accordingly development of chronic inflammatory diseases such as rheumatoid arthritis, osteoarthritis and inflammatory bowel disease. Dual blockade of CSF1 and IL34 was proved to eliminate arthritis or inflammatory bowel disease in mouse models {Lin W et al., (2019) Frontiers in Immunology 10: 2019).

[0007] The CSF1-CSF-1R interaction is also implicated in cancer development and progression. It has been recently discovered that CSF1, but not IL34, is required for accumulation of tumor associated macrophages, which are known to produce immunosuppressive factors and promote immune suppressive cells such as Trigs, in mice inoculated with MC38 tumor cells {Lin W et al., (2019) supra). CSF1 also supports angiogenesis in tumor microenvironments. In addition to CSF1, CSF-1R is found to be over-expressed in many tumors such as ovarian and endometrial cancers, and CSF-1R expression is associated with larger tumor sizes and decreased survival (KLUGER, et al. (2004) Clinical cancer research. 10(1): 173-177; SCHOLL, et al. (1994) Journal of the National Cancer Institute. 86(2): 120- 126; BAIOCCHI, et al. (1991) Cancer, 67(4): 990-996).

[0008] CSF-1R signaling has further been proved to play a physiological role in bone remodeling. The knockout animals for either CSF-1 or CSF-1R have shown osteopetrotic phenotypes. And CSF1 blockade is reported to significantly reduce bone loss in metastatic bone disease and rheumatoid arthritis models (Patel S and Player MR, (2009) Current Topics in Medicinal Chemistry 9(7): 599-610).

[0009] CSF-1R inhibitors have been studied for years as promising candidates for treatment of cancers, inflammatory diseases and bone loss. One CSF-1R inhibitor, Pexidartinib, was approved by FDA in 2019 for tenosynovial giant cell tumor treatment. Another CSF-1R inhibitor, Cabiralizumab, a monoclonal antibody targeting tumor-associated macrophages, however, missed primary end point in phase II trial of advanced pancreatic cancer. Therefore, there is always a need for additional CSF-1R inhibitors especially antibodies with enhanced binding affinity and other desirable pharmaceutical characteristics, for treatment of the mentioned diseases.

[0010] Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.

SUMMARY OF THE INVENTION

[0011] The present disclosure provides an isolated monoclonal antibody, for example, a mouse, chimeric or humanized monoclonal antibody, or an antigen-binding portion thereof, that binds to CSF- 1R (e.g., the human CSF-1R) and has comparable, if not higher, binding affinity/capacity to human and/or monkey CSF-1R, and comparable, if not higher, blocking activity on CSF-1R-CSF1/IL34 interaction, as compared to prior art anti-CSF-lR antibodies such as Cabiralizumab.

[0012] Accordingly, in one aspect, the disclosure pertains to an isolated monoclonal antibody (e.g., a mouse, chimeric or humanized antibody), or an antigen-binding portion thereof, that binds CSF-1R, having a heavy chain variable region that may comprise a CDR1 region, a CDR2 region and a CDR3 region, wherein the CDR1 region, the CDR2 region and the CDR3 region may comprise amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to (1) SEQ ID NOs: 1, 8 and 14, respectively; (2) SEQ ID NOs: 2, 9 and 15, respectively; (3) SEQ ID NOs: 3, 10 and 16, respectively; (4) SEQ ID NOs: 4, 11 and 17, respectively; (5) SEQ ID NOs: 5, 12 and 18, respectively; (6) SEQ ID NOs: 5, 12 and 19, respectively; (7) SEQ ID NOs: 6, 12 and 18, respectively; or (8) SEQ ID NOs: 7, 13 and 20, respectively.

[0013] The isolated monoclonal antibody, or the antigen-binding portion thereof, of the present disclosure may comprise a heavy chain variable region which may comprise an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NOs:37, 38 (X1=V, X2=R, X3=A; X1=R, X2=T, X3=A; or X1=R, X2=T, X3=V), 39, 40, 41, 42, 43, 44 or 45. The amino acid sequence of SEQ ID NO: 37 may be encoded by the nucleotide sequences of SEQ ID NOs: 63 or 64. The amino acid sequence of SEQ ID NO: 38 (X1=R, X2=T, X3=A) may be encoded by the nucleotide sequence of SEQ ID NO: 65.

[0014] The isolated monoclonal antibody, or the antigen-binding portion thereof, of the present disclosure may comprise a light chain variable region that may comprise a CDR1 region, a CDR2 region and a CDR3 region, wherein the CDR1 region, the CDR2 region, and the CDR3 region may comprise amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to (1) SEQ ID NOs: 21, 27 and 32, respectively; (2) SEQ ID NOs: 22, 28 and 33, respectively; (3) SEQ ID NOs: 23, 29 and 34, respectively; (4) SEQ ID NOs: 24, 30 and 35, respectively; (5) SEQ ID NOs: 25, 29 and 36, respectively; or (6) SEQ ID NOs: 26, 31 and 34, respectively.

[0015] The isolated monoclonal antibody, or the antigen-binding portion thereof, of the present disclosure may comprise a light chain variable region which may comprise an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NOs: 46, 47 (X1=I, X2=S, X3=K, K4=P; X1=I, X2=Y, X3=T, X4=L; X1=L, X2=S, X3=T, X4=L; X1=I, X2=S, X3=T, X4=L; or X1=L, X2=Y, X3=T, X4=L), 48, 49, 50, 51, 52, 53 or 54. The amino acid sequence of SEQ ID NO: 46 may be encoded by the nucleotide sequences of SEQ ID NOs: 66 or 67. The amino acid sequence of SEQ ID NO: 47 (X1=L, X2=Y, X3=T, X4=L) may be encoded by the nucleotide sequence of SEQ ID NO: 68.

[0016] In some embodiments, the isolated monoclonal antibody, or the antigen-binding portion thereof, of the present disclosure may comprise a heavy chain variable region and a light chain variable region each which may comprise a CDR1 region, a CDR2 region and a CDR3 region, wherein the heavy chain variable region CDR1, CDR2 and CDR3, and the light chain variable region CDR1, CDR2 and CDR3 may comprise amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to (1) SEQ ID NOs: 1, 8, 14, 21, 27 and 32, respectively; (2) SEQ ID NOs: 2, 9, 15, 22, 28 and 33, respectively; (3) SEQ ID NOs: 3, 10, 16, 23, 29 and 34, respectively; (4) SEQ ID NOs: 4, 11, 17, 24, 30 and 35, respectively; (5) SEQ ID NOs: 5, 12, 18, 25, 29 and 36, respectively; (6) SEQ ID NOs: 5, 12, 19, 25, 29 and 36, respectively; (7) SEQ ID NOs: 6, 12, 18, 25, 29 and 36, respectively; or (8) SEQ ID NOs: 7, 13, 20, 26, 31 and 34, respectively.

[0017] In some embodiments, the isolated monoclonal antibody, or the antigen-binding portion thereof, of the present disclosure may comprise a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region which may comprise amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to (1) SEQ ID NOs: 37 and 46, respectively; (2) SEQ ID NOs: 38 (X1=V, X2=R, X3=A; X1=R, X2=T, X3=A; or X1=R, X2=T, X3=V) and 47 (X1=I, X2=S, X3=K, K4=P), respectively; (3) SEQ ID NOs: 38 (X1=V, X2=R, X3=A; X1=R, X2=T, X3=A; or X1=R, X2=T, X3=V) and 47 (X1=I, X2=Y, X3=T, X4=L), respectively; (4) SEQ ID NOs: 38 (X1=V, X2=R, X3=A; X1=R, X2=T, X3=A; or X1=R, X2=T, X3=V) and 47 (X1=L, X2=S, X3=T, X4=L), respectively; (5) SEQ ID NOs: 38 (X1=V, X2=R, X3=A; X1=R, X2=T, X3=A; or X1=R, X2=T, X3=V) and 47 (X1=I, X2=S, X3=T, X4=L), respectively; (6) SEQ ID NOs: 38 (X1=V, X2=R, X3=A; X1=R, X2=T, X3=A; or X1=R, X2=T, X3=V) and 47 (X1=L, X2=Y, X3=T, X4=L), respectively; (7) SEQ ID NOs: 39 and 48, respectively; (8) SEQ ID NOs: 40 and 49, respectively; (9) SEQ ID NOs: 41 and 50, respectively; (10) SEQ ID NOs: 42 and 51, respectively; (11) SEQ ID NOs: 43 and 52, respectively; (12) SEQ ID NOs: 44 and 53, respectively; or (13) SEQ ID NOs: 45 and 54, respectively.

[0018] In some embodiments, the isolated monoclonal antibody, or the antigen-binding portion thereof, of the present disclosure may comprise a heavy chain and a light chain linked by disulfide bonds, the heavy chain which may comprise a heavy chain variable region and a heavy chain constant region, the light chain which may comprise a light chain variable region and a light chain constant region, wherein the C terminus of the heavy chain variable region is linked to the N terminus of the heavy chain constant region, and the C terminus of the light chain variable region is linked to the N terminus of the light chain constant region, wherein the heavy chain variable region and the light chain variable region may comprise amino acid sequences described above. The heavy chain constant region may be human IgG4 constant region having an amino acid sequence set forth in e.g., SEQ ID NO.: 55, and the light chain constant region may be human kappa constant region having an amino acid sequences set forth in e.g., SEQ ID NO. : 56. The heavy chain constant region may also be human IgGl or IgG2 constant region. The amino acid sequences of SEQ ID NOs: 55 and 56 may be encoded by the nucleotide sequences of SEQ ID NOs: 69 and 70, respectively. [0019] The antibody of the present disclosure in some embodiments may comprise or consists of two heavy chains and two light chains, wherein each heavy chain may comprise the heavy chain constant region, heavy chain variable region or CDR sequences mentioned above, and each light chain may comprise the light chain constant region, light chain variable region or CDR sequences mentioned above. The antibody of the disclosure can be a full-length antibody, for example, of an IgGl, IgG2 or IgG4 isotype. The antibody or the antigen-binding portion of the present disclosure in other embodiments may be a single chain variable fragment (scFv), or antibody fragments such as Fab or Fab'2 fragments.

[0020] The disclosure also provides a bispecific molecule which may comprise the antibody, or the antigen-binding portion thereof, of the disclosure, linked to a second functional moiety (e.g., a second antibody) having a different binding specificity than the antibody, or antigen-binding portion thereof. The disclosure also provides an immunoconjugate, such as an antibody -drug conjugate, which may comprise the antibody of the disclosure, or the antigen-binding portion thereof, linked to a therapeutic agent, such as a cytotoxin. In another aspect, the antibody or an antigen binding portions thereof of the present disclosure can be made into part of a chimeric antigen receptor (CAR). Also provided is an immune cell which may comprise the antigen chimeric receptor, such as a T cell. The antibody or the antigen binding portion thereof of the present disclosure can also be encoded by or used in conjunction with an oncolytic virus.

[0021] Compositions which may comprise the antibody or the antigen-binding portion thereof, or the immunoconjugate, the bispecific molecule, the oncolytic virus, the CAR or the immune cell with the CAR of the disclosure, and a pharmaceutically acceptable carrier, are also provided. In some embodiments, the pharmaceutical composition may further contain a therapeutic agent for treating a specific disease, such as an anti-inflammatory agent, or an anti-cancer agent.

[0022] Nucleic acid molecules encoding the antibody, or the antigen-binding portion thereof, of the disclosure are also encompassed by the disclosure, as well as expression vectors which may comprise such nucleic acids and host cells which may comprise such expression vectors. A method for preparing the anti-CSF-lR antibody or the antigen-binding portion thereof using the host cell which may comprise the expression vector is also provided, which may comprise steps of (i) expressing the antibody or the antigen-binding portion thereof in the host cell and (ii) isolating the antibody or the antigen-binding portion thereof from the host cell or its cell culture.

[0023] In yet another aspect, the disclosure provides a method for treating tumor or cancer in a subject, which may comprise administering to the subject a therapeutically effective amount of an antibody, or an antigen-binding portion thereof, of the disclosure. In some embodiments, the method may comprise administering a pharmaceutical composition, a bispecific molecule, an immunoconjugate, an immune cell with the CAR, or an antibody -encoding or antibody -bearing oncolytic virus of the disclosure, or alternatively a nucleic acid molecule or a vector capable of expressing the same of the disclosure. In some embodiments, at least one additional anti-cancer antibody can be administered, such as an anti- PD-1 antibody, an anti-LAG-3 antibody and/or an anti-CTLA-4 antibody. In yet another embodiment, the subject is further administered with a cytokine (e.g., IL-2 and/or IL-21), or a costimulatory antibody (e.g., an anti-CD 137 and/or anti-GITR antibody), if appropriate. The antibody or the antigen-binding portion thereof of the present disclosure can be, for example, a mouse, chimeric or humanized antibody. The cancer or tumor includes, but not limited to, ovarian cancer, endometrial cancer, tenosynovial giant cell tumor, pancreatic cancer, breast cancer, cervical cancer, lung cancer and prostate cancer.

[0024] In a further aspect, the disclosure provides a method for treating an inflammatory disease in a subject, comprising administering to the subject a therapeutically effective amount of an antibody, or an antigen-binding portion thereof, of the disclosure. In some embodiments, the method may comprise administering a pharmaceutical composition, a bispecific molecule, an immunoconjugate, an immune cell with the CAR, or an antibody -encoding or antibody -bearing oncolytic virus of the disclosure, or alternatively a nucleic acid molecule or a vector capable of expressing the same of the disclosure. In some embodiments, at least one additional anti-inflammatory agent can be further administered, such as an STAT3 inhibitor especially an anti-STAT3 antibody. The antibody or the antigen-binding portion thereof of the present disclosure can be, for example, a mouse, chimeric or humanized antibody. The inflammatory disease includes, but not limited to, rheumatoid arthritis, atherosclerosis, osteoarthritis and inflammatory bowel disease.

[0025] In yet another aspect, the disclosure provides a method for treatment of bone loss, including, but not limited to, periodontitis, histiocytosis X, osteoporosis, Paget's disease of bone (PDB), bone loss due to cancer therapy, periprosthetic osteolysis, and glucocorticoid-induced osteoporosis, which may comprise administering to the subject a therapeutically effective amount of an antibody, or an antigen binding portion thereof, of the disclosure. In some embodiments, the method may comprise administering a pharmaceutical composition, a bispecific molecule, an immunoconjugate, an immune cell with the CAR, or an antibody -encoding or antibody -bearing oncolytic virus of the disclosure, or alternatively a nucleic acid molecule or a vector capable of expressing the same of the disclosure. In yet another embodiment, the subject can be further administered with a costimulatory antibody (e.g., an anti-CD 137 and/or anti-GITR antibody). The antibody or the antigen-binding portion thereof of the present disclosure can be, for example, a mouse, chimeric or humanized antibody.

[0026] Other features and advantages of the instant disclosure will be apparent from the following detailed description and examples which should not be construed as limiting. The contents of all references, GenBank entries, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.

[0027] Accordingly, it is an object of the invention not to encompass within the invention any previously known product, process of making the product, or method of using the product such that Applicants reserve the right and hereby disclose a disclaimer of any previously known product, process, or method. It is further noted that the invention does not intend to encompass within the scope of the invention any product, process, or making of the product or method of using the product, which does not meet the written description and enablement requirements of the USPTO (35 U.S.C. §112, first paragraph) or the EPO (Article 83 of the EPC), such that Applicants reserve the right and hereby disclose a disclaimer of any previously described product, process of making the product, or method of using the product. It may be advantageous in the practice of the invention to be in compliance with Art. 53(c) EPC and Rule 28(b) and (c) EPC. All rights to explicitly disclaim any embodiments that are the subject of any granted patent(s) of applicant in the lineage of this application or in any other lineage or in any prior filed application of any third party is explicitly reserved. Nothing herein is to be construed as a promise.

[0028] It is noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as "comprises", "comprised", "comprising" and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean "includes", "included", "including", and the like; and that terms such as "consisting essentially of and "consists essentially of have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The following detailed description, given by way of example, but not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings.

[0030] FIGs. 1A-1B show the binding capacity of mouse antibodies 2B6, 3B1, 1H8 and 1F7-2 (A), 1G8, 2B7, lDlO-1 and 2B12 (B) to cell surface human CSF-1R.

[0031] FIGs. 2A-2C show the blocking ability of mouse antibodies 3B1 and 2B7 (A), lDlO-1, 2B12, 1F7-2 and 1H8 (B), and 1G8 (C) on human CSF-1R - CSF-1 binding.

[0032] FIGs. 3A-3C show the ability of mouse antibodies 3B1, 2B6 and 2B7 (A), lDlO-1, 2B12, 1F7- 2 and 1H8 (B), and 1G8 (C) to block Cabiralizumab-human CSF-1R binding.

[0033] FIGs. 4A-4C show the inhibitory effect of mouse antibodies 2B6, 1G8 and 2B7 (A), ID 10-1 and 2B12 (B), 3B1, 1H8 and 1F7-2 (C) on CSF1R tyrosine phosphorylation induced by CSFi binding.

[0034] FIGs. 5A-5C show the binding capacity of chimeric antibodies 2B6 (A), 1H8 (B) and 1G8 (C) to human CSF-1R.

[0035] FIGs. 6A-6B show the blocking ability of chimeric antibodies 2B6 and 1H8 (A), and 1G8 (B) on human CSF-1R - CSF-1 binding.

[0036] FIGs. 7 shows the inhibitory effect of chimeric antibodies 2B6 and 1G8 on CSF1R tyrosine phosphorylation induced by CSFI binding. [0037] FIGs. 8A-8B show the binding capacity of humanized antibodies hulG8-Vl - hulG8-V9 (A), and hulG8-V10 - hulG8-V15 (B) to human CSF-1R.

[0038] FIG. 9 shows the binding capacity of humanized antibody hulG8-V13 to human CSF-1R.

[0039] FIG. 10 shows the binding capacity of humanized antibody hulG8-V13 to cynomolgus CSF- 1R.

[0040] FIG. 11 shows the blocking ability of humanized antibody hulG8-V13 on human CSF-1R - CSF-1 binding.

[0041] FIG. 12 shows the blocking ability of humanized antibody hulG8-V13 on human CSF-1R - IL34 binding.

[0042] FIG. 13 shows the ability of humanized antibody hulG8-V13 to block Cabiralizumab-human CSF-1R binding.

[0043] FIG. 14 shows inhibition of CSF1 binding induced CSF1R tyrosine phosphorylation by humanized antibody hulG8-V13.

[0044] FIG. 15 shows the binding capacity of the humanized antibody hulG8-V13 to cell surface human CSF-1R.

[0045] FIG. 16 shows the protein thermal shift assay result of the humanized antibody hulG8-V13.

DETAILED DESCRIPTION OF THE INVENTION

[0046] To ensure that the present disclosure may be more readily understood, certain terms are set forth throughout the detailed description.

[0047] The term "CSF-1R" refers to colony stimulating factor 1 receptor, also known as macrophage colony stimulating factor receptor (M-CSFR) and CD115. The term "CSF-1R" may comprise variants, isoforms, homologs, orthologs and paralogs. For example, an antibody specific for a human CSF-1R protein may, in certain cases, cross-reacts with a CSF-1R protein from a species other than human, such as cynomolgus monkey. In other embodiments, an antibody specific for a human CSF-1R protein may be completely specific for the human CSF-1R protein and exhibit no cross-reactivity to other species or of other types, or may cross-react with CSF-1R from certain other species but not all other species.

[0048] The term“human CSF-1R” refers to a CSF-1R protein having an amino acid sequence from human, such as the amino acid sequence of human CSF-1R having UniProtKB/Swiss-Prot Accession Number of P07333.2. The term“monkey CSF-1R” or“cyno CSF1R” refer to a CSF-1R protein having an amino acid sequence from macaca fascicularis, such as the amino acid sequence having Genbank Accession No. EHH54662.

[0049] The term“antibody” as referred to herein includes whole antibodies and any antigen binding fragment (i.e.,“antigen-binding portion”) or single chains thereof. Whole antibodies are glycoproteins which may comprise two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain may be comprised of a heavy chain variable region (abbreviated herein as V_H) and a heavy chain constant region. The heavy chain constant region may be comprised of three domains, CHI, C_H2 and C_H3. Each light chain may be comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region may be comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each V_H and V _L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies can 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 (Clq) of the classical complement system.

[0050] The term“antigen-binding portion” of an antibody (or simply“antibody portion”), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a CSF1R protein). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and C HI domains; (ii) a F(ab')₂ fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V_H and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al, (1989) Nature 341 :544-546), which consists of a VH domain; (vi) an isolated complementarity determining region (CDR); and (viii) a nanobody, a heavy chain variable region containing a single variable domain and two constant domains. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al, (1988) Science 242:423-426; and Huston et al, (1988) Proc. Natl Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term“antigen-binding portion” of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0051] An "isolated antibody", as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds a CSF-1R protein is substantially free of antibodies that specifically bind antigens other than CSF-1R proteins). An isolated antibody that specifically binds a human CSF-1R protein may, however, have cross-reactivity to other antigens, such as CSF-1R proteins from other species. Moreover, an isolated antibody can be substantially free of other cellular material and/or chemicals. [0052] The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0053] The term“mouse antibody”, as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from mouse germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from mouse germline immunoglobulin sequences. The mouse antibodies of the disclosure can include amino acid residues not encoded by mouse germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term“mouse antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species have been grafted onto mouse framework sequences.

[0054] The term“chimeric antibody” refers to an antibody made by combining genetic material from a nonhuman source with genetic material from a human being. Or more generally, a chimeric antibody is an antibody having genetic material from a certain species with genetic material from another species.

[0055] The term“humanized antibody”, as used herein, refers to an antibody from non-human species whose protein sequences have been modified to increase similarity to antibody variants produced naturally in humans.

[0056] The term "isotype" refers to the antibody class (e.g., IgM or IgGl) that is encoded by the heavy chain constant region genes.

[0057] The phrases "an antibody recognizing an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody which binds specifically to an antigen."

[0058] The term "antibody derivatives" refers to any modified form of the antibody, e.g., a conjugate of the antibody and another agent or antibody.

[0059] As used herein, an antibody that "specifically binds to human CSF-1R" is intended to refer to an antibody that binds to human CSF-1R protein (and possibly a CSF-1R protein from one or more non human species) but does not substantially bind to non-CSF-lR proteins. Preferably, the antibody binds to a human CSF-1R protein with "high affinity", namely with a K_D of 1 xlO ⁸ M or less, and more preferably 5 x 10⁹ M or less.

[0060] The term "does not substantially bind" to a protein or cells, as used herein, means does not bind or does not bind with a high affinity to the protein or cells, i.e. binds to the protein or cells with a K_D of 1 x 10⁶ M or more, more preferably 1 x 10 ⁵ M or more, more preferably 1 x 10 ⁴ M or more, more preferably 1 x 10³ M or more, even more preferably 1 x 10 ² M or more.

[0061] The term "K_asSoc" or "K_a", as used herein, is intended to refer to the association rate of a particular antibody -antigen interaction, whereas the term "K_diS" or "K_d," as used herein, is intended to refer to the dissociation rate of a particular antibody -antigen interaction. The term "K_D", as used herein, is intended to refer to the dissociation constant, which is obtained from the ratio of K_d to K_a (i.e., K_d/K_a) and is expressed as a molar concentration (M). K_D values for antibodies can be determined using methods well established in the art. A preferred method for determining the K_D of an antibody is by using surface plasmon resonance, preferably using a biosensor system such as a Biacore™ system.

[0062] The term "high affinity" for an IgG antibody refers to an antibody having a K_D of 1 x 10 ⁶ M or less, more preferably 5 x 10 ⁸ M or less, even more preferably 1 x 10 ⁸ M or less, even more preferably 5 x 10 ⁹ M or less and even more preferably 1 x 10 ⁹ M or less for a target antigen. However, "high affinity" binding can vary for other antibody isotypes. For example, "high affinity" binding for an IgM isotype refers to an antibody having a K_D of 10 ⁶ M or less, more preferably 10 ⁷ M or less, even more preferably 10 ⁸ M or less.

[0063] The term “IC50”, also known as half maximal inhibitory concentration, refers to the concentration of an antibody which inhibits a specific biological or biochemical function by 50% relative to the absence of the antibody.

[0064] The term "EC50", also known as half maximal effective concentration, refers to the concentration of an antibody which induces a response halfway between the baseline and maximum after a specified exposure time.

[0065] The term "subject" includes any human or nonhuman animal. The term "nonhuman animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, although mammals are preferred, such as nonhuman primates, sheep, dogs, cats, cows and horses.

[0066] The term "therapeutically effective amount" means an amount of the antibody of the present disclosure sufficient to prevent or reduce the symptoms associated with a disease or condition (such as a cancer) and/or lessen the severity of the disease or condition. A therapeutically effective amount is understood to be in context to the condition being treated, where the actual effective amount is readily discerned by those of skill in the art.

[0067] Various aspects of the disclosure are described in further detail in the following subsections.

[0068] Antibodies of the disclosure specifically bind to human CSF-1R and monkey CSF-1R and have comparable or improved affinity compared to prior art anti-CSF-lR antibodies such as Cabiralizumab.

[0069] The antibodies of the disclosure may block the binding of CSF-1R to both CSF1 and IL34, thus inhibiting CSF-1R tyrosine phosphorylation induced by ligand binding.

[0070] The antibodies of the disclosure are monoclonal antibodies. Additionally or alternatively, the antibodies can be, for example, mouse, chimeric or humanized monoclonal antibodies.

[0071] The exemplary antibody of the disclosure is the monoclonal antibody structurally and chemically characterized as described below and in the following Examples. The amino acid sequence ID numbers of the heavy /light chain CDRs and variable regions of the antibodies are summarized in Table 1 below, some antibodies sharing the same V_H or V_L. The heavy chain constant region for the antibodies may be human IgG4 heavy chain constant region having an amino acid sequence set forth in, e.g., SEQ ID NO: 55, and the light chain constant region for the antibodies may be human kappa constant region having an amino acid sequence set forth in, e.g., SEQ ID NO: 56. The antibodies of the disclosure may also contain human IgGl heavy chain constant region and human kappa light chain constant region.

[0072] The heavy chain variable region CDRs and the light chain variable region CDRs in Table 1 have been defined by the Rabat numbering system. However, as is well known in the art, CDR regions can also be determined by other systems such as Chothia, and IMGT, AbM, or Contact numbering system/method, based on heavy chain/light chain variable region sequences.

[0073] The V_H and V_L sequences (or CDR sequences) of other anti-CSF-lR antibodies which bind to human CSF-1R can be "mixed and matched" with the V_H and V_L sequences (or CDR sequences) of the anti-CSF-lR antibody of the present disclosure. Preferably, when V_H and V_L chains (or the CDRs within such chains) are mixed and matched, a V_H sequence from a particular V_H/V_L pairing is replaced with a structurally similar V_H sequence. Likewise, preferably a V_L sequence from a particular VH/VL pairing is replaced with a structurally similar V_L sequence.

[0074] Accordingly, in one embodiment, an antibody of the disclosure, or an antigen binding portion thereof, may comprise:

(a) a heavy chain variable region which may comprise an amino acid sequence listed above in Table 1; and

(b) a light chain variable region which may comprise an amino acid sequence listed above in Table 1, or the V_L of another anti-CSF-lR antibody, wherein the antibody specifically binds human CSF-1R.

PATENT

Docket No. 55532-00016

Table 1. SEQ ID numbers of CDRs and heavy /light chain variable regions of anti-CSF-lR antibodies of the disclosure

[0075] In another embodiment, an antibody of the disclosure, or an antigen binding portion thereof, may comprise:

(a) the CDR1, CDR2, and CDR3 regions of the heavy chain variable region listed above in Table 1; and

(b) the CDR1, CDR2, and CDR3 regions of the light chain variable region listed above in Table 1 or the CDRs of another anti-CSF-lR antibody, wherein the antibody specifically binds human CSF-1R.

[0076] In yet another embodiment, the antibody, or antigen binding portion thereof, includes the heavy chain variable CDR2 region of anti-CSF-lR antibody combined with CDRs of other antibodies which bind human CSF-1R, e.g., CDR1 and/or CDR3 from the heavy chain variable region, and/or CDR1, CDR2, and/or CDR3 from the light chain variable region of a different anti-CSF-lR antibody.

[0077] Accordingly, in another embodiment, antibodies of the disclosure may comprise the CDR2 of the heavy chain variable region of the anti-CSF-lR antibody and at least the CDR3 of the heavy and/or light chain variable region of the anti-CSF-lR antibody, or the CDR3 of the heavy and/or light chain variable region of another CSF-1R antibody, wherein the antibody is capable of specifically binding to human CSF-1R. These antibodies preferably (a) compete for binding with CSF-1R; (b) retain the functional characteristics; (c) bind to the same epitope; and/or (d) have a similar binding affinity as the anti-CSF-lR antibody of the present disclosure. In yet another embodiment, the antibodies further may comprise the CDR2 of the light chain variable region of the anti-CSF-lR antibody, or the CDR2 of the light chain variable region of another CSF-1R antibody, wherein the antibody is capable of specifically binding to human CSF-1R. In another embodiment, the antibodies of the disclosure further may include the CDR1 of the heavy and/or light chain variable region of the anti-CSF-lR antibody, or the CDR1 of the heavy and/or light chain variable region of another CSF-1R antibody, wherein the antibody is capable of specifically binding to human CSF-1R.

[0078] In another embodiment, an antibody of the disclosure may comprise a heavy and/or light chain variable region sequences of CDR1, CDR2 and CDR3 sequences which differ from those of the anti- CSF-1R antibodies of the present disclosure by one or more conservative modifications. It is understood in the art that certain conservative sequence modification can be made which do not remove antigen binding. See, e.g., Brummell et al. (1993) Biochem 32: 1180-8; de Wildt et al. (1997) Prot. Eng. 10:835-41; Komissarov et al. (1997) J. Biol. Chem. 272:26864-26870; Hall et al. (1992) J. Immunol. 149: 1605-12; Kelley and O'Connell (1993) Biochem .32:6862-35; Adib-Conquy et al. (1998) Ini. Immunol.10:341-6; and Beers et al. (2000) Clin. Can. Res. 6:2835-43.

[0079] Accordingly, in one embodiment, the antibody may comprise a heavy chain variable region which may comprise CDR1, CDR2, and CDR3 sequences and/or a light chain variable region which may comprise CDR1, CDR2, and CDR3 sequences, wherein:

(a) the heavy chain variable region CDR1 sequence may comprise a sequence listed above in Table 1, and/or conservative modifications thereof; and/or (b) the heavy chain variable region CDR3 sequence may comprise a sequence listed above in Table 1, and conservative modifications thereof; and/or

(c) the light chain variable region CDR1, and/or CDR2, and/or CDR3 sequences may comprise the sequences listed above in Table 1; and/or conservative modifications thereof; and

(d) the antibody specifically binds human CSF-1R.

[0080] The antibody of the present disclosure possesses one or more of the following functional properties described above, such as high affinity binding to human CSF-1R, the ability to inhibit binding of CSF-1R to CSF1 or IL34 and/or the ability to inhibit CSF-1R tyrosine phosphorylation induced by ligand binding.

[0081] In various embodiments, the antibody can be, for example, a mouse, human, humanized or chimeric antibody.

[0082] As used herein, the term "conservative sequence modifications" is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody of the disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CDR regions of an antibody of the disclosure can be replaced with other amino acid residues from the same side chain family and the altered antibody can be tested for retained function (i.e., the functions set forth above) using the functional assays described herein.

[0083] Antibodies of the disclosure can be prepared using an antibody having one or more of the V_H/V_L sequences of the anti-CSF-lR antibody of the present disclosure as starting material to engineer a modified antibody. An antibody can be engineered by modifying one or more residues within one or both variable regions (i.e., V_H and/or V_L), for example within one or more CDR regions and/or within one or more framework regions. Additionally or alternatively, an antibody can be engineered by modifying residues within the constant region(s), for example to alter the effector function(s) of the antibody.

[0084] In certain embodiments, CDR grafting can be used to engineer variable regions of antibodies. Antibodies interact with target antigens predominantly through amino acid residues that are located in the six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences within CDRs are more diverse between individual antibodies than sequences outside of CDRs. Because CDR sequences are responsible for most antibody -antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors that include CDR sequences from the specific naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann et al. (1998) Nature 332:323-327; Jones et al. (1986) Nature 321 :522-525; Queen et al. (1989) Proc. Natl. Acad. See also U.S.A. 86: 10029-10033; U.S. Pat. Nos. 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

[0085] Accordingly, another embodiment of the disclosure pertains to an isolated monoclonal antibody, or antigen binding portion thereof, which may comprise a heavy chain variable region which may comprise CDR1, CDR2, and CDR3 sequences which may comprise the sequences of the present disclosure, as described above, and/or a light chain variable region which may comprise CDR1, CDR2, and CDR3 sequences which may comprise the sequences of the present disclosure, as described above. While these antibodies contain the V_H and V_L CDR sequences of the monoclonal antibody of the present disclosure, they can contain different framework sequences.

[0086] Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, germline DNA sequences for human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrc-cpe.cam.ac.uk/vbase), as well as in Rabat et al. (1991), cited supra; Tomlinson et al. (1992) J. Mol. Biol. 227:776-798; and Cox et al. (1994) Eur. J. Immunol. 24:827-836; the contents of each of which are expressly incorporated herein by reference. As another example, the germline DNA sequences for human heavy and light chain variable region genes can be found in the Genbank database. For example, the following heavy chain germline sequences found in the HCo7 HuMAb mouse are available in the accompanying Genbank Accession Nos.: 1-69 (NG— 0010109, NT-024637 & BC070333), 3-33 (NG-0010109 & NT-024637) and 3-7 (NG- 0010109 & NT— 024637). As another example, the following heavy chain germline sequences found in the HCol2 HuMAb mouse are available in the accompanying Genbank Accession Nos.: 1-69 (NG- 0010109, NT-024637 & BC070333), 5-51 (NG-0010109 & NT-024637), 4-34 (NG-0010109 & NT- -024637), 3-30.3 (CAJ556644) & 3-23 (AJ406678).

[0087] Antibody protein sequences are compared against a compiled protein sequence database using one of the sequence similarity searching methods called the Gapped BLAST (Altschul et al. (1997), supra), which is well known to those skilled in the art.

[0088] Preferred framework sequences for use in the antibodies of the disclosure are those that are structurally similar to the framework sequences used by antibodies of the disclosure. The VH CDRl, CDR2, and CDR3 sequences can be grafted onto framework regions that have the identical sequence as that found in the germline immuno globulin gene from which the framework sequence derives, or the CDR sequences can be grafted onto framework regions that contain one or more mutations as compared to the germline sequences. For example, it has been found that in certain instances it is beneficial to mutate residues within the framework regions to maintain or enhance the antigen binding ability of the antibody (see e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

[0089] Another type of variable region modification is to mutate amino acid residues within the VH and/or V_L CDR1, CDR2 and/or CDR3 regions to thereby improve one or more binding properties (e.g., affinity) of the antibody of interest. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutation(s) and the effect on antibody binding, or other functional property of interest, can be evaluated in in vitro or in vivo assays as known in the art. Preferably conservative modifications (as known in the art) are introduced. The mutations can be amino acid substitutions, additions or deletions, but are preferably substitutions. Moreover, typically no more than one, two, three, four or five residues within a CDR region are altered.

[0090] Accordingly, in another embodiment, the disclosure provides isolated anti-CSF-lR monoclonal antibodies, or antigen binding portions thereof, which may comprise a heavy chain variable region which may comprise: (a) a VH CDRl region which may comprise the sequence of the present disclosure, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions; (b) a VH CDR2 region which may comprise the sequence of the present disclosure, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions; (c) a VH CDR3 region which may comprise the sequence of the present disclosure, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions; (d) a VL CDRl region which may comprise the sequence of the present disclosure, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions; (e) a VL CDR2 region which may comprise the sequence of the present disclosure, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions; and (f) a VL CDR3 region which may comprise the sequence of the present disclosure, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions.

[0091] Engineered antibodies of the disclosure include those in which modifications have been made to framework residues within VH and/or VL, e.g. to improve the properties of the antibody. Typically, such framework modifications are made to decrease the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation can contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived. [0092] Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T cell epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in further detail in U.S. Patent Publication No. 20030153043.

[0093] In addition, or as an alternative to modifications made within the framework or CDR regions, antibodies of the disclosure can be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity. Furthermore, an antibody of the disclosure can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody.

[0094] In one embodiment, the hinge region of CHI is modified in such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. This approach is described further in U.S. Pat. No. 5,677,425. The number of cysteine residues in the hinge region of CHI is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

[0095] In another embodiment, the Fc hinge region of an antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the Cm-Cm domain interface region of the Fc-hinge fragment such that the antibody has impaired Staphylococcyl protein A (SpA) binding relative to native Fc-hinge domain SpA binding. This approach is described in further detail in U.S. Pat. No. 6,165,745.

[0096] In still another embodiment, the glycosylation of an antibody is modified. For example, a glycosylated antibody can be made (i.e., the antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for antigen. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for antigen. See, e.g., U.S. Pat. Nos. 5,714,350 and 6,350,861.

[0097] Additionally or alternatively, an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the disclosure to thereby produce an antibody with altered glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (a (1,6)- fucosyltransferase), such that antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8-/- cell lines were created by the targeted disruption of the FUT8 gene in CHO/DG44 cells using two replacement vectors (see U.S. Patent Publication No. 20040110704 and Yamane-Ohnuki et al. (2004) Biotechnol Bioeng 87:614-22). As another example, EP 1,176,195 describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation by reducing or eliminating the a-1, 6 bond-related enzyme. EP 1,176,195 also describes cell lines which have a low enzyme activity for adding fucose to the N-acetylglucosamine that binds to the Fc region of the antibody or does not have the enzyme activity, for example the rat myeloma cell line YB2/0 (ATCC CRL 1662). PCT Publication WO 03/035835 describes a variant CHO cell line, Lecl3 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields et al. (2002) J. Biol. Chem. 277:26733-26740). Antibodies with a modified glycosylation profile can also be produced in chicken eggs, as described in PCT Publication WO 06/089231. Alternatively, antibodies with a modified glycosylation profile can be produced in plant cells, such as Lemna. Methods for production of antibodies in a plant system are disclosed in the U.S. patent application corresponding to Alston & Bird LLP attorney docket No. 040989/314911, filed on Aug. 11, 2006. PCT Publication WO 99/54342 describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (e.g., (l,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC activity of the antibodies (see also Umana et al. (1999) Nat. Biotech. 17: 176-180). Alternatively, the fucose residues of the antibody can be cleaved off using a fucosidase enzyme; e.g., the fucosidase a-L-fucosidase removes fucosyl residues from antibodies (Tarentino et al. (1975) Biochem. 14:5516-23).

[0098] Another modification of the antibodies herein that is contemplated by this disclosure is pegylation. An antibody can be pegylated to, for example, increase the biological (e.g., serum) half- life of the antibody. To pegylate an antibody, the antibody, or fragment thereof, typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment. Preferably, the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (Cl -CIO) alkoxy- or ary loxy -poly ethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies of the disclosure. See, e.g., EPO 154 316 and EP 0 401 384. [0099] Antibodies of the disclosure can be characterized by their various physical properties, to detect and/or differentiate different classes thereof.

[00100] For example, antibodies can contain one or more glycosylation sites in either the light or heavy chain variable region. Such glycosylation sites may result in increased immunogenicity of the antibody or an alteration of the pK of the antibody due to altered antigen binding (Marshall et al (1972) Annu Rev Biochem 41:673-702; Gala and Morrison (2004) J Immunol 172:5489-94; Wallick et al (1988) J Exp Med 168: 1099-109; Spiro (2002) Glycobiology 12:43R-56R; Parekh et al (1985) Nature 316:452- 7; Mimura et al. (2000) Mol Immunol 37:697-706). Glycosylation has been known to occur at motifs containing an N-X-S/T sequence. In some instances, it is preferred to have an anti-CSF-lR antibody that does not contain variable region glycosylation. This can be achieved either by selecting antibodies that do not contain the glycosylation motif in the variable region or by mutating residues within the glycosylation region.

[00101] In a preferred embodiment, the antibodies do not contain asparagine isomerism sites. The deamidation of asparagine may occur on N-G or D-G sequences and result in the creation of an isoaspartic acid residue that introduces a kink into the polypeptide chain and decreases its stability (isoaspartic acid effect).

[00102] Each antibody will have a unique isoelectric point (pi), which generally falls in the pH range between 6 and 9.5. The pi for an IgGl antibody typically falls within the pH range of 7-9.5 and the pi for an IgG4 antibody typically falls within the pH range of 6-8. There is speculation that antibodies with a pi outside the normal range may have some unfolding and instability under in vivo conditions. Thus, it is preferred to have an anti-CSF-lR antibody that contains a pi value that falls in the normal range. This can be achieved either by selecting antibodies with a pi in the normal range or by mutating charged surface residues.

[00103] In another aspect, the disclosure provides nucleic acid molecules that encode heavy and/or light chain variable regions, or CDRs, of the antibodies of the disclosure. The nucleic acids can be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "rendered substantially pure" when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques. A nucleic acid of the disclosure can be, e.g., DNA or RNA and may or may not contain intronic sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

[00104] Nucleic acids of the disclosure can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes as described further below), cDNAs encoding the light and heavy chains of the antibody made by the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (e.g., using phage display techniques), a nucleic acid encoding such antibodies can be recovered from the gene library. [00105] Preferred nucleic acids molecules of the disclosure include those encoding the VH and VL sequences of the CSF-1R monoclonal antibody or the CDRs. Once DNA fragments encoding V_H and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes or to a scFv gene. In these manipulations, a VL- or V_H-encoding DNA fragment is operatively linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operatively linked", as used in this context, is intended to mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in-frame.

[00106] The isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operatively linking the V_H-encoding DNA to another DNA molecule encoding heavy chain constant regions (CHI, CH2 and Cm). The sequences of mouse/human heavy chain constant region genes are known in the art and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgGl, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably is an IgGl or IgG4 constant region. For a Fab fragment heavy chain gene, the V_H-encoding DNA can be operatively linked to another DNA molecule encoding only the heavy chain CHI constant region.

[00107] The isolated DNA encoding the VL region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operatively linking the V_L-encoding DNA to another DNA molecule encoding the light chain constant region, CL. The sequences of mouse/human light chain constant region genes are known in the art and DNA fragments encompassing these regions can be obtained by standard PCR amplification. In preferred embodiments, the light chain constant region can be a kappa or lambda constant region.

[00108] To create a scFv gene, the VH- and V_L-encoding DNA fragments are operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser)3, such that the V_H and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions joined by the flexible linker (see e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).

[00109] Monoclonal antibodies (mAbs) of the present disclosure can be produced using the well- known somatic cell hybridization (hybridoma) technique of Kohler and Milstein (1975) Nature 256: 495. Other embodiments for producing monoclonal antibodies include viral or oncogenic transformation of B lymphocytes and phage display techniques. Chimeric or humanized antibodies are also well known in the art. See e.g., U.S. Pat. Nos. 4,816,567; 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6, 180,370, the contents of which are specifically incorporated herein by reference in their entirety. [00110] Antibodies of the disclosure also can be produced in a host cell transfectoma using, for example, a combination of recombinant DNA techniques and gene transfection methods as is well known in the art (e.g., Morrison, S. (1985) Science 229: 1202). In one embodiment, DNA encoding partial or full-length light and heavy chains obtained by standard molecular biology techniques is inserted into one or more expression vectors such that the genes are operatively linked to transcriptional and translational regulatory sequences. In this context, the term "operatively linked" is intended to mean that an antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene.

[00111] The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, e.g., in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, e.g., the adenovirus major late promoter (AdMLP) and polyoma. Alternatively, non-viral regulatory sequences can be used, such as the ubiquitin promoter or b-globin promoter. Still further, regulatory elements composed of sequences from different sources, such as the SRa promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T cell leukemia virus type 1 (Takebe et al. (1988) Mol. Cell. Biol. 8:466-472). The expression vector and expression control sequences are chosen to be compatible with the expression host cell used.

[00112] The antibody light chain gene and the antibody heavy chain gene can be inserted into the same or separate expression vectors. In preferred embodiments, the variable regions are used to create full- length antibody genes of any antibody isotype by inserting them into expression vectors already encoding heavy chain constant and light chain constant regions of the desired isotype such that the VH segment is operatively linked to the CH segment(s) within the vector and the VL segment is operatively linked to the CL segment within the vector. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[00113] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the disclosure can carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216; 4,634,665 and 5,179,017). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).

[00114] For expression of the light and heavy chains, the expression vector(s) encoding the heavy and light chains is transfected into a host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium -phosphate precipitation, DEAE-dextran transfection and the like. Although it is theoretically possible to express the antibodies of the disclosure in either prokaryotic or eukaryotic host cells, expression of antibodies in eukaryotic cells, and most preferably mammalian host cells, is the most preferred because such eukaryotic cells, and in particular mammalian cells, are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active antibody.

[00115] Preferred mammalian host cells for expressing the recombinant antibodies of the disclosure include Chinese Hamster Ovary (CHO cells) (including dhfr- CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in R. J. Kaufman and P. A. Sharp (1982) J. Mol. Biol. 159:601-621), NSO myeloma cells, COS cells and SP2 cells. In particular for use with NSO myeloma cells, another preferred expression system is the GS gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338,841. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods.

[00116] Antibodies of the disclosure can be conjugated to a therapeutic agent to form an immunoconjugate such as an antibody-drug conjugate (ADC). Suitable therapeutic agents include antimetabolites, alkylating agents, DNA minor groove binders, DNA intercalators, DNA crosslinkers, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerase I or II inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics, and anti-mitotic agents. In the ADC, the antibody and therapeutic agent preferably are conjugated via a linker cleavable such as a peptidyl, disulfide, or hydrazone linker. More preferably, the linker is a peptidyl linker such as Val- Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val, Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser, or Glu. The ADCs can be prepared as described in U.S. Pat. Nos. 7,087,600; 6,989,452; and 7,129,261; PCT Publications WO 02/096910; WO 07/038,658; WO 07/051,081; WO 07/059,404; WO 08/083,312; and WO 08/103,693; U.S. Patent Publications 20060024317; 20060004081; and 20060247295; the disclosures of which are incorporated herein by reference.

[00117] In another aspect, the present disclosure features bispecific molecules which may comprise one or more antibodies of the disclosure linked to at least one other functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand for a receptor) to generate a bispecific molecule that binds to at least two different binding sites or target molecules. Thus, as used herein, "bispecific molecule" includes molecules that have three or more specificities.

[00118] In an embodiment, a bispecific molecule has, in addition to an anti-Fc binding specificity and an anti-CSF-lR binding specificity, a third specificity. The third specificity can be for an anti enhancement factor (EF), e.g., a molecule that binds to a surface protein involved in cytotoxic activity and thereby increases the immune response against the target cell. For example, the anti-enhancement factor can bind a cytotoxic T-cell (e.g. via CD2, CD3, CD8, CD28, CD4, CD40, or ICAM-1) or other immune cell, resulting in an increased immune response against the target cell.

[00119] Bispecific molecules can come in many different formats and sizes. At one end of the size spectrum, a bispecific molecule retains the traditional antibody format, except that, instead of having two binding arms of identical specificity, it has two binding arms each having a different specificity. At the other extreme are bispecific molecules consisting of two single-chain antibody fragments (scFv's) linked by a peptide chain, a so-called Bs(scFv) 2 construct. Intermediate-sized bispecific molecules include two different F(ab) fragments linked by a peptidyl linker. Bispecific molecules of these and other formats can be prepared by genetic engineering, somatic hybridization, or chemical methods. See, e.g., Kufer et al, cited supra; Cao and Suresh, Bioconjugate Chemistry, 9 (6), 635-644 (1998); and van Spriel et al., Immunology Today , 21 (8), 391-397 (2000), and the references cited therein.

[00120] An oncolytic virus preferabtially infects and kills cancer cells. Antibodies of the present disclosure can be used in conjunction with oncolytic viruses. Alternatively, oncolytic viruses encoding antibodies of the present disclosure can be introduced into human body.

[00121] Also provided herein are a chimeric antigen receptor (CAR) containing an anti-CSFIR scFv, the anti-CSFIR scFv which may comprise CDRs and heavy/light chain variable regions described herein.

[00122] The anti-CSFIR CAR may comprise (a) an extracellular antigen binding domain which may comprise an anti-CSFIR scFv; (b) a transmembrane domain; and (c) an intracellular signaling domain.

[00123] The CAR may contain a signal peptide at the N-terminus of the extracellular antigen binding domain that directs the nascent receptor into the endoplasmic reticulum, and a hinge peptide at the N- terminus of the extracellular antigen binding domain that makes the receptor more available for binding. The CAR preferably comprises, at the intracellular signaling domain, a primary intracellular signaling domain and one or more co-stimulatory signaling domains. The mainly used and most effective primary intracellular signaling domain is CD3-zeta cytoplasmic domain which contains IT AMs, the phosphorylation of which results in T cell activation. The co-stimulatory signaling domain may be derived from the co-stimulatory proteins such as CD28, CD137 and 0X40.

[00124] The CARs may further add factors that enhance T cell expansion, persistence, and anti-tumor activity, such as cytokines, and co-stimulatory ligands.

[00125] Also provided are engineered immune effector cells, which may comprise the CAR provided herein. In some embodiments, the immune effector cell is a T cell, an NK cell, a peripheral blood mononuclear cell (PBMC), a hematopoietic stem cell, a pluripotent stem cell, or an embryonic stem cell. In some embodiments, the immune effector cell is a T cell.

[00126] In another aspect, the present disclosure provides a pharmaceutical composition which may comprise one or more antibodies of the present disclosure formulated together with a pharmaceutically acceptable carrier. The composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or a drug. The pharmaceutical compositions of the disclosure also can be administered in a combination therapy with, for example, another anti-cancer agent, another anti-inflammatory agent, or a vaccine.

[00127] The pharmaceutical composition may comprise any number of excipients. Excipients that can be used include carriers, surface active agents, thickening or emulsifying agents, solid binders, dispersion or suspension aids, solubilizers, colorants, flavoring agents, coatings, disintegrating agents, lubricants, sweeteners, preservatives, isotonic agents, and combinations thereof. The selection and use of suitable excipients is taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy , 20th Ed. (Lippincott Williams & Wilkins 2003), the disclosure of which is incorporated herein by reference.

[00128] Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active ingredient can be coated in a material to protect it from the action of acids and other natural conditions that may inactivate it. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion. Alternatively, an antibody of the disclosure can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, e.g., intranasally, orally, vaginally, rectally, sublingually or topically.

[00129] Pharmaceutical compositions can be in the form of sterile aqueous solutions or dispersions. They can also be formulated in a microemulsion, liposome, or other ordered structure suitable to high drug concentration. [00130] The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated and the particular mode of administration and will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.01% to about ninety -nine percent of active ingredient, preferably from about 0.1% to about 70%, most preferably from about 1% to about 30% of active ingredient in combination with a pharmaceutically acceptable carrier.

[00131] Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus can be administered, several divided doses can be administered over time or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Alternatively, antibody can be administered as a sustained release formulation, in which case less frequent administration is required.

[00132] For administration of the antibody, the dosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight. For example dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg. An exemplary treatment regime entails administration once per week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months or once every three to 6 months. Preferred dosage regimens for an anti-CSF-lR antibody of the disclosure include 1 mg/kg body weight or 3 mg/kg body weight via intravenous administration, with the antibody being given using one of the following dosing schedules: (i) every four weeks for six dosages, then every three months; (ii) every three weeks; (iii) 3 mg/kg body weight once followed by 1 mg/kg body weight every three weeks. In some methods, dosage is adjusted to achieve a plasma antibody concentration of about 1-1000 pg/ml and in some methods about 25-300 pg/ml.

[00133] A "therapeutically effective dosage" of an anti-CSF-lR antibody of the disclosure preferably results in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. For example, for the treatment of tumor-bearing subjects, a "therapeutically effective dosage" preferably inhibits tumor growth by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80% relative to untreated subjects. A therapeutically effective amount of a therapeutic antibody can decrease tumor size, or otherwise ameliorate symptoms in a subject, which is typically a human or can be another mammal.

[00134] The pharmaceutical composition can be a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, poly glycolic acid, collagen, poly orthoesters, and poly lactic acid. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[00135] Therapeutic compositions can be administered via medical devices such as (1) needleless hypodermic injection devices (e.g., U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; and 4,596,556); (2) micro-infusion pumps (U.S. Pat. No. 4,487,603); (3) transdermal devices (U.S. Pat. No. 4,486,194); (4) infusion apparatuses (U.S. Pat. Nos. 4,447,233 and 4,447,224); and (5) osmotic devices (U.S. Pat. Nos. 4,439,196 and 4,475, 196); the disclosures of which are incorporated herein by reference.

[00136] In certain embodiments, the human monoclonal antibodies of the disclosure can be formulated to ensure proper distribution in vivo. For example, to ensure that the therapeutic antibody of the disclosure cross the blood-brain barrier, they can be formulated in liposomes, which may additionally comprise targeting moieties to enhance selective transport to specific cells or organs. See, e.g. U.S. Pat. Nos. 4,522,811; 5,374,548; 5,416,016; and 5,399,331; V. V. Ranade (1989) J. Clin.Pharmacol.29:685; Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153: 1038; Bloeman et al. (1995) FEBS l.ell.357: 140 M. Owais et al. (1995) Antimicrob. Agents Chemother. 39: 180; Briscoe et al. ( 1995) Am. J. Physiol. 1233: 134; Schreier et al. (1994) J. Biol. Chem. 269:9090; Keinanen and Laukkanen (1994) FEBS Lett. 346: 123; and Killion and Fidler (1994) Immunomethods 4:273.

[00137] Antibodies (compositions, bispecifics, immunoconjugates, immune cells with CARs and oncolytic viruses) of the present disclosure have numerous in vitro and in vivo utilities involving, for example, treatment of cancers, inflammatory diseases or bone loss. The antibodies (compositions, bispecifics, immunoconjugates, immune cells with CARs and oncolytic viruses) can be administered to human subjects, e.g., in vivo, to inhibit tumor growth or bone loss, or to treat inflammatory diseases.

[00138] There are three distinct mechanisms by which CSF-1R signaling is likely involved in tumor growth and metastasis. The first is that expressions of CSF-1 and CSF-1R have been found in tumor cells originating in the female reproductive system (breast, ovarian, endometrium, cervical) (Scholl, S. M., et al., (1994) J. Natl. Cancer Inst. 86, 120-126; Kacinski, B. M., (1997) Mol. Reprod. Dev. 46, 71- 74; Ngan, H. Y., et al., (1999) Eur. J. Cancer. 35, 1546-1550; Kirma, N., et al., (2007) Cancer Res. 67, 1918-1926) and the expression has been associated with breast cancer xenograft growth as well as poor prognosis in breast cancer patients. Two-point mutations were seen in CSF-1R in about 10-20% of acute myelocytic leukemia, chronic myelocytic leukemia and myelodysplasia patients tested in one study, and one of the mutations was found to disrupt receptor turnover (Ridge, S. A., et al., (1990) Proc. Natl. Acad. Sci USA. 87, 1377-1380). However the incidence of the mutations could not be confirmed in later studies (Abu-Duhier, F. M., et al., (2003) Br. J. Haematol. 120, 464-470). Mutations were also found in some cases of hepatocellular cancer (Yang, D. H., et al., (2004) Hepatobiliary Pancreat. Dis. Int. 3, 86-89) and idiopathic myelofibrosis (Abu-Duhier, F. M., et al., (2003) Br. J. Haematol. 120, 464- 470).

[00139] Pigmented villonodular synovitis (PVNS) and Tenosynovial Giant cell tumors (TGCT) can occur as a result of a translocation that fuses the CSF-1 gene to a collagen gene COL6A3 and results in overexpression of CSF-1 (West, R. B., et al., (2006) Proc. Natl. Acad. Sci. USA. 103, 690-695). A landscape effect is proposed to be responsible for the resulting tumor mass that consists of monocytic cells attracted by cells that express CSF-1.

[00140] The second mechanism is based on blocking signaling through CSF-1-CSF-1R at metastatic sites in bone which induces osteoclastogenesis, bone resorption and osteolytic bone lesions. Breast, multiple myeloma and lung cancers are examples of cancers that have been found to metastasize to the bone and cause osteolytic bone disease resulting in skeletal complications. CSF-1 released by tumor cells and stroma induces the differentiation of hematopoietic myeloid monocyte progenitors to mature osteoclasts in collaboration with the receptor activator of nuclear factor kappa-B ligand-RANKL. During this process, M-CSF acts as a permissive factor by giving the survival signal to osteoclasts (Tanaka, S., et al., (1993) J. Clin. Invest. 91, 257-263). Inhibition of CSF-1R activity during osteoclast differentiation and maturation with an anti-CSF-lR antibody is likely to prevent unbalanced activity of osteoclasts that cause osteolytic disease and the associated skeletal related events in metastatic disease. Whereas breast, lung cancer and multiple myeloma typically result in osteolytic lesions, metastasis to the bone in prostate cancer initially has an osteoblastic appearance in which increased bone forming activity results in“woven bon” which is different from typical lamellar structure of normal bone. During disease progression, bone lesions display a significant osteolytic component as well as high serum levels of bone resorption and suggests that anti-re sorptive therapy may be useful. Bisphosphonates have been shown to inhibit the formation of osteolytic lesions and reduced the number of skeletal-related events only in men with hormone -refractory metastatic prostate cancer but at this point their effect on osteoblastic lesions is controversial and bisphosphonates have not been beneficial in preventing bone metastasis or hormone responsive prostate cancer to date. The effect of anti- resorptive agents in mixed osteolytic/osteoblastic prostate cancer is studied in the clinic (Choueiri, M. B., et al., (2006) Cancer Metastasis Rev. 25, 601-609; Vessella, R. L. and Corey, E., Clin. (2006) Cancer Res. 12, 6285s-6290s).

[00141] The third mechanism is based on the recent observation that tumor associated macrophages (TAM) found in solid tumors of the breast, prostate, ovarian and cervical cancers correlated with poor prognosis (Bingle, L., et al., (2002) J. Pathol. 196, 254-265; Pollard, J. W., (2004) Nat. Rev. Cancer. 471-78). Macrophages are recruited to the tumor by CSF-1 and other chemokines. The macrophages can then contribute to tumor progression through the secretion of angiogenic factors, proteases and other growth factors and cytokines and may be blocked by inhibition of CSF-1R signaling. It was shown by Zins et al (Zins, K., et al. (2007), Cancer Res. 67, 1038-1045) that expression of siRNA of Tumor necrosis factor alpha (TNF alpha), CSF-1 or both would reduce tumor growth in a mouse xenograft model by 34%-50% after intratumoral injection of the respective siRNA. SiRNA targeting the TNF alpha secreted by the human SW620 cells reduced mouse CSF-1 levels and led to reduction of macrophages in the tumor. In addition, treatment of MCF7 tumor xenografts with an antigen binding fragment directed against CSF-1 did result in 40% tumor growth inhibition, reversed the resistance to chemotherapeutics and improved survival of the mice when given in combination with chemotherapeutics (Paulus, P., et al., (2006) Cancer Res. 66, 4349-4356).

[00142] TAMs are only one example of an emerging link between chronic inflammation and cancer. There is additional evidence for the correlation between inflammation and cancer. For example, many chronic diseases are associated with an increased risk of cancer, cancers arise at sites of chronic inflammation, and chemical mediators of inflammation are found in many cancers. Also, deletion of the cellular or chemical mediators of inflammation inhibits development of cancers, and long-term use of anti-inflammatory agents reduce the risk of some cancers. A link to cancer exists for a number of inflammatory conditions among those H. pylori induced gastritis for gastric cancer, Schistosomiasis for bladder cancer, HHVX for Kaposi's sarcoma, endometriosis for ovarian cancer and prostatitis for prostate cancer (Balkwill, F., et al., (2005) Cancer Cell. 7, 211-217). Macrophages are key cells in chronic inflammation and respond differentially to their microenvironment. There are two types of macrophages that are considered as extremes in a continuum of functional states: Ml macrophages are involved in Type 1 reactions. These reactions involve the activation by microbial products and consequent killing of pathogenic microorganisms that result in reactive oxygen intermediates. On the other end of the extreme are M2 macrophages involved in Type 2 reactions that promote cell proliferation, tune inflammation and adaptive immunity and promote tissue remodeling, angiogenesis and repair (Mantovani, A., et al., (2004) Trends Immunol. 25, 677-686). Chronic inflammation resulting in established neoplasia is usually associated with M2 macrophages. A pivotal cytokine that mediates inflammatory reactions is TNF alpha that true to its name can stimulate anti-tumor immunity and hemorrhagic necrosis at high doses but has also been found to be expressed by tumor cells and acting as a tumor promoter (Zins, K., et al., (2007) Cancer Res. Cl, 1038-1045; Balkwill, F., (2006) Cancer Metastasis Rev. 25, 409-416). The specific role of macrophages with respect to the tumor still needs to be better understood including the potential spatial and temporal dependence on their function and the relevance to specific tumor types.

[00143] Thus, one embodiment of the disclosure is the CSF-1R antibodies of the present disclosure for use in the treatment of cancer. The term“cancer” as used herein may be, for example, lung cancer, non-small cell lung (NSCL) cancer, bronchioloalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, mesothelioma, hepatocellular cancer, biliary cancer, neoplasms of the central nervous system (CNS), spinal axis tumors, brain stem glioma, glioblastoma multiforme, astrocytomas, schwanomas, ependymonas, medulloblastomas, meningiomas, squamous cell carcinomas, pituitary adenoma, lymphoma, lymphocytic leukemia, including refractory versions of any of the above cancers, or a combination of one or more of the above cancers. Preferably such cancer is breast cancer, ovarian cancer, cervical cancer, lung cancer or prostate cancer. Preferably such cancers are further characterized by CSF-1 or CSF-1R expression or overexpression. One further embodiment the disclosure is the CSF-1R antibodies of the present disclosure for use in the simultaneous treatment of primary tumors and new metastases. Alternatively, an anti-CSF-lR antibody can be used in conjunction with other immunogenic agents used in cancer treatments such as oncolytic viruses, or other antibodies.

[00144] Another embodiment of the disclosure is the CSF-1R antibodies of the present disclosure for use in the treatment of bone loss, such as periodontitis, histiocytosis X, osteoporosis, Paget's disease of bone (PDB), bone loss due to cancer therapy, periprosthetic osteolysis, and glucocorticoid-induced osteoporosis.

[00145] According to another embodiment, the present disclosure relates to the use of the antibody, the nucleic acid sequence, the vector or the pharmaceutical composition according to the disclosure for the treatment of diseases associated to inflammation and/or autoimmunity . Such diseases may comprise but are not limited to seronegative spondyloarthropathy (psoriatic arthritis, ankylosing spondylitis, reiters syndrome, spondyloarthropathy associated with inflammatory bowel disease), prosthetic joint loosening, connective tissue diseases (juvenile rheumatoid arthritis, rheumatoid arthritis, systemic lupus erythematosus (SLE) and lupus nephritis, scleroderma, Sjogren's syndrome, mixed connective tissue disease, polymyositis, dermatomyositis), inflammatory bowel disease (e.g. Crohn's disease; ulcerative colitis), whipples disease, arthritis associated with granulomatous ileocolitis, inflammatory skin conditions (autoimmune bullous pemphigoid, autoimmune pemphigus vulgaris, eczema, dermatitis), inflammatory lung disease (alveolitis, pulmonary fibrosis, sarcoidoisis, asthma, bronchitis, bronchiolitis obliterans), inflammatory renal disease (glomerulonethritis, renal allograft rejection, renal tubular inflammation), atherosclerosis, systemic vasculitis (temporal arteritis/giant cell arteritis, takayasu arteritis, polyarteritis nodosa, Kawasaki disease, Wegener's granulomatosis, churg strauss syndrome, microscopic polyangiitis, necrotising glomerulonephritis, henoch schonlein purpura, essential cryoglobulinaemic vasculitis and other small vessel vasculitis, Behcets disease), macrophage activation diseases (macrophage activation syndrome (MAS), adult onset stills disease, haemophagocytic syndrome), polymyalgia rheumatica, primary biliary sclerosis, sclerosing cholangitis, autoimmune hepatitis, Type 1 Diabetes Mellitus, Hashimoto's thyroiditis, Graves' disease, multiple sclerosis (MS), Guillain-Barre syndrome, Addison's disease, and/or Raynaud's phenomenon, Goodpasture's syndrome.

[00146] It has demonstrated that SNPs in the CSF-1 gene exhibited a positive association with aggressive periodontitis, an inflammatory disease of the periodontal tissues that causes tooth loss due to resorption of the alveolar bone (Rabello, D., et al., (2006) Biochem. Biophys. Res. Commun. 347, 791-796).

[00147] Histiocytosis X (also called Langerhans cell histiocytosis, LCH) is a proliferative disease of Langerhans dendritic cells that appear to differentiate into osteoclasts in bone and extra osseous LCH lesions. Langerhans cells are derived from circulating monocytes. Increased levels of M-CSF that have been measured in sera and lesions where found to correlate with disease severity (da Costa, C. E., et al., (2005) J. Exp. Med. 201, 687-693). The disease occurs primarily in a pediatric patient population and has to be treated with chemotherapy when the disease becomes systemic or is recurrent.

[00148] The pathophysiology of osteoporosis is mediated by loss of bone forming osteoblasts and increased osteoclast dependent bone resorption. Supporting data has been described by Cenci et al., showing that an anti-CSF-1 antibody injection preserves bone density and inhibits bone resorption in ovariectomized mice (Cenci, S., et al., (2000) J. Clin. Invest. 105, 1279-1287). Recently a potential link between postmenopausal bone loss due to estrogen deficiency was identified and found that the presence of TNF alpha producing T-cell affected bone metabolism (Roggia, C., et al., (2004) Minerva Med. 95, 125-132). A possible mechanism could be the induction of CSF-1 by TNF alpha in vivo. An important role for CSF-1 in TNF-alpha-induced osteoclastogenesis was confirmed by the effect of an antibody directed against CSF-1 that blocked the TNF alpha induced osteolysis in mice and thereby making inhibitors of CSF-1R signaling potential targets for inflammatory arthritis (Kitaura, H., et al., (2005) J. Clin. Invest. 115, 3418-3427).

[00149] Paget's disease of bone (PDB) is the second most common bone metabolism disorder after osteoporosis in which focal abnormalities of increased bone turnover lead to complications such as bone pain, deformity, pathological fractures and deafness. Mutations in four genes have been identified that regulate normal osteoclast function and predispose individuals to PDB and related disorders: insertion mutations in TNFRSF11A, which encodes receptor activator of nuclear factor (NF) kappaB (RANK)— a critical regulator of osteoclast function, inactivating mutations of TNFRSF11B which encodes osteoprotegerin (a decoy receptor for RANK ligand), mutations of the sequestosome 1 gene (SQSTM1), which encodes an important scaffold protein in the NFkappaB pathway and mutations in the valosin- containing protein (VCP) gene. This gene encodes VCP, which has a role in targeting the inhibitor of NFkappaB for degradation by the proteasome (Daroszewska, A. and Ralston, S. H., (2006) Nat. Clin. Pract. Rheumatol. 2, 270-277). Targeted CSF-1R inhibitors provide an opportunity to block the deregulation of the RANKL signaling indirectly and add an additional treatment option to the currently used bisphosphonates.

[00150] Cancer therapy induced bone loss especially in breast and prostate cancer patients is an additional indication where a targeted CSF-1R inhibitor could prevent bone loss (Lester, J. E., et al., (2006) Br. J. Cancer. 94, 30-35). With the improved prognosis for early breast cancer the long-term consequences of the adjuvant therapies become more important as some of the therapies including chemotherapy, irradiation, aromatase inhibitors and ovary ablation affect bone metabolism by decreasing the bone mineral density, resulting in increased risk for osteoporosis and associated fractures (Lester, J. E., et al., (2006) Br. J. Cancer. 94, 30-35). The equivalent to adjuvant aromatase inhibitor therapy in breast cancer is androgen ablation therapy in prostate cancer which leads to loss of bone mineral density and significantly increases the risk of osteoporosis-related fractures (Stoch, S. A., et al., (2001) J. Clin. Endocrinol. Metab. 86, 2787-2791).

[00151] Targeted inhibition of CSF-1R signaling is likely to be beneficial in other indications as well when targeted cell types include osteoclasts and macrophages, e.g., treatment of specific complications in response to joint replacement as a consequence of rheumatoid arthritis. Implant failure due to periprosthetic bone loss and consequent loosing of prostheses is a major complication of joint replacement and requires repeated surgery with high socioeconomic burdens for the individual patient and the health-care system.

[00152] Glucocorticoid-induced osteoporosis (GIOP) is another indication in which a CSF-1R inhibitor could prevent bone loss after long-term glucocorticocosteroid use that is given as a result of various conditions among those chronic obstructive pulmonary disease, asthma and rheumatoid arthritis (Guzman-Clark, J. R., et al., (2007) Arthritis Rheum. 57, 140-146; Feldstein, A. C., et al., (2005) Osteoporos. Int. 16, 2168-2174).

[00153] Rheumatoid arthritis, psoriatic arthritis and inflammatory arthridities are potential indications for CSF-1R signaling inhibitors in that they consist of a macrophage component and to a varying degree bone destruction (Ritchlin, C. T., et al., (2003) J. Clin. Invest. I l l, 821-831). Osteoarthritis and rheumatoid arthritis are inflammatory autoimmune disease caused by the accumulation of macrophages in the connective tissue and infiltration of macrophages into the synovial fluid, which is at least partially mediated by CSF-1. It was demonstrated that CSF-1 is produced by human-joint tissue cells (chondrocytes, synovial fibroblasts) in vitro and is found in synovial fluid of patients with rheumatoid arthritis, suggesting that it contributes to the synovial tissue proliferation and macrophage infiltration which is associated with the pathogenesis of the disease (Campbell, L, K., et al., (2000) J. Leukoc. Biol. 68, 144-150). Inhibition of CSF-1R signaling is likely to control the number of macrophages in the joint and alleviate the pain from the associated bone destruction. In order to minimize adverse effects and to further understand the impact of the CSF-1R signaling in these indications, one method is to specifically inhibit CSF-1R without targeting myriad kinases, such as Raf kinase. [00154] Literature also reported correlate increased circulating M-CSF with poor prognosis and atherosclerotic progression in chronic coronary artery disease (Saitoh, T., et al., J (2000). Am. Coll. Cardiol. 35, 655-665; Ikonomidis, I., et al., (2005) Eur. Heart. J. 26, 1618-1624). CSF-1 influences the atherosclerotic process by aiding the formation of foam cells (macrophages with ingested oxidized LDL) that express CSF-1R and represent the initial plaque (Murayama, T., et al., (1999) Circulation 99, 1740-1746).

[00155] Expression and signaling of CSF-1 and CSF-1R is also found in activated microglia. Microglia, which are resident macrophages of the central nervous system, can be activated by various factors, including infection and traumatic injury. CSF-1 is considered as a key regulator of inflammatory responses in the brain and the CSF-1 level increases in encephalitis, Alzheimer's disease (AD) and brain tumors. Microgliosis as a consequence of autocrine signaling by CSF-1/CSF-1R results in induction of inflammatory cytokines and nitric oxides being released as demonstrated by e.g. using an experimental neuronal damage model (Hao, A. J., et al., (2002) Neuroscience. 112, 889-900; Murphy, G. M., Jr., et al., (1998) J. Biol. Chem. 273, 20967-20971). Microglia that have increased expression of CSF-1R are found to surround plaques in AD and in the amyloid precursor protein V717F transgenic mouse model of AD (Murphy, G. M., Jr., et al., (2000) Am. J. Pathol. 157, 895-904). On the other hand, op/op mice with fewer microglia in the brain resulted in fibrillar deposition of A-beta and neuronal loss compared to normal control, suggesting that microglia do have a neuroprotective function in the development of AD lacking in the op/op mice (Kaku, M., et al., (2003) Brain Res. Brain Res. Protoc. 12, 104-108).

[00156] Further, expression and signaling of CSF-1 and CSF-1R is also associated with inflammatory bowel disease (IBD) (see WO 2005/046657).

[00157] Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined in the appended claims.

[00158] The present invention will be further illustrated in the following Examples which are given for illustration purposes only and are not intended to limit the invention in any way. Examples

Example 1 Generation of Mouse Anti-CSF-IR Monoclonal Antibodies Using Hvbridoma Technology

[00159] Immunization

[00160] Mice were immunized according to the method as described in E Harlow, D. Lane, Antibody: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998. Recombinant human CSF-1R protein (AA lie 20 - Glu 512) with human IgGl Fc tag at the C-terminus (Aero biosystems, Cat#CSR-H5258) was used as the immunogen. Human CSF-lR-his protein (Aero biosystems, Cat#CSR-H5228) was used for determining anti-sera titer and for screening hybridomas secreting antigen-specific antibodies. [00161] Immunizing dosages contained 50 pg human CSF-IR-Fc protein/mouse/injection for primary immunizations, and 25 pg human CSF-IR-Fc protein/mouse/injection for boost immunizations. To increase immune response, the complete Freud's adjuvant and incomplete Freud's adjuvant (Sigma, St. Louis, Mo., USA) were used respectively for primary and boost immunizations. Briefly, adjuvant- antigen mixture was prepared by first gently mixing the adjuvant in a vial using a vortex. The desired amount of adjuvant was transferred to an autoclaved 1.5 mL micro-centrifuge tube. The antigen was prepared in PBS or saline with a concentration ranging from 0.25-0.34 mg/ml, and the calculated amount of antigen was then added to the micro-centrifuge tube with the adjuvant. The obtained solution was mixed by gently vortexing for 2 minutes to generate water-in-oil emulsion. The adjuvant-antigen solution was then drawn into the proper syringe for animal injection. A total of 50 or 25 pg of antigen was injected in a volume of 150-200 pi. Each animal was immunized, and then boosted for 2 to 3 times depending on the anti-sera titer. Animals with good titers were given a final boost by intraperitoneal injection before fusion.

[00162] Hybridoma fusion and screening

[00163] Cells of murine myeloma cell line (SP2/0-Agl4, ATCC#CRL-1581) were cultured to reach the log phase stage right before fusion. Spleen cells from immunized mice were prepared sterilely and fused with myeloma cells according to the method as described in Kohler G, and Milstein C, "Continuous cultures of fused cells secreting antibody of predefined specificity," Nature, 256: 495- 497(1975).

[00164] Fused "hybrid cells" were subsequently dispensed into 96-well cell plates in DMEM/20% FCS/HAT media. Surviving hybridoma colonies were observed under the microscope seven to ten days post-fusion. After two weeks, the supernatant from each well was subjected to indirect ELISA and Capture ELISA using recombinant human CSFIR-his protein. Positive hybridomas secreting antibodies that bound to human CSFIR-his were then selected and transferred to 24-well plates. These hybridomas were further tested for the activity of blocking human CSFl-his protein binding to CSF1R- Fc by ligand blocking ELISA. Hybridoma clones producing antibodies that showed high specific CSF1R binding and CSF1-CSF1R blocking activity were subcloned by limiting dilution to ensure the clonality of the cell line, and then monoclonal antibodies were purified. Briefly, Protein A sepharose column (from bestchrom (Shanghai) Biosciences, Cat#AA0273) was washed using PBS buffer in 5 to 10 column volumes. Cell supernatants were passed through the columns, and then the columns were washed using PBS buffer until the absorbance for protein reached the baseline. The columns were eluted with elution buffer (0.1 M Glycine-HCl, pH 2.7), and immediately collected into 1.5 ml tubes with neutralizing buffer (1 M Tris-HCl, pH 9.0). Fractions containing immunoglobulins were pooled and dialyzed in PBS overnight at 4°C. Subsequently, the in vitro functional activities of purified monoclonal antibodies were characterized as follows. Example 2 Affinity Determination of Mouse Anti-CSF-IR Monoclonal Antibodies Using BIACORE Surface Plasmon Resonance Technology

[00165] The purified anti-CSF-lR mouse monoclonal antibodies (mAbs) generated in Example 1 were characterized for affinities and binding kinetics by Biacore T200 system (GE healthcare, Pittsburgh, PA, USA).

[00166] Briefly, goat anti-mouse IgG (GE healthcare, Cat#BR100838, Mouse Antibody Capture Kit) was covalently linked to a CM5 chip (carboxy methyl dextran coated chip from GE healthcare #BR100530) via primary amines using a standard amine coupling kit (GE healthcare, Pittsburgh, PA, USA) provided by Biacore, a Protein G chip (GE healthcare, Cat#29-1793-15) was used for the benchmark’s Affinity determination. Un-reacted moieties on the biosensor surface were blocked with ethanolamine. Then purified mouse anti-CSF-lR antibodies generated in Example 1 and Cabiralizumab (in house made with heavy chain’s amino acid sequence set forth in SEQ ID NO: 57 and light chain’s amino acid sequence set forth in SEQ ID NO: 58, also referred to as benchmark) at the concentration of 10 pg/ml were flowed onto the chip at a flow rate of 10 pL/min. Then, serially diluted recombinant human CSF-lR-his (Aero biosystems, Cat#CSR-H5228, starting at 120 nM) or cynomolgus monkey CSF-lR-his protein (Aero biosystems, Cat#CSR-C52El, starting at 200 nM), 2-fold dilution in HBS- EP⁺ buffer (provided by Biacore), was flowed onto the chip at a flow rate of 30 pL/min. The antigen- antibody association kinetics was followed for 2 minutes and the dissociation kinetics was followed for 10 minutes. The association and dissociation curves were fit to a 1 : 1 Langmuir binding model using BIAcore evaluation software.

[00167] The K_a, ¾ and K_D values were determined and summarized in Table 2 below.

[00168] All the mouse antibodies of the disclosure specifically bound to human CSF-1R with comparable or higher binding affinity compared to the benchmark, with the antibodies 2B6, 1G8, 2B7, lDlO-1 and 2B12 having the highest binding affinity. All the mouse antibodies of the disclosure also specifically bound to monkey CSF-1R with high binding affinity.

Table 2. Binding affinity of mouse Anti-CSF-lR monoclonal Antibodies

Kinetics on Biacore

Human CSF-lR-his Cynomolgus CSF-lR-his

Mouse mAb

Ka K_d K_D K_a ¾ K_D

(M-ls-1) (s-1) (M) (M-ls-1) (s-1) (

2B6 3.37E+05 6.93E-05 2.05E-10 3.13E+05 2.49E-04 7.95E-10

3B 1 9.60E+04 6.66E-04 6.94E-09 4.17E+04 5.45E-04 1.31E-08

1H8 9.41E+04 7.01E-04 7.45E-09 8.00E+05 3.52E-03 4.40E-09

1F7-2 3.51E+04 0.002416 6.88E-08 5.34E+04 0.002621 4.91E-08

1G8 1.49E+05 4.69E-04 3.16E-09 5.87E+04 3.58E-04 6.10E-09

2B7 1.99E+05 1.45E-04 7.30E-10 2.25E+05 1.66E-04 7.35E-10 lDlO-1 2.65E+05 1.45E-04 5.48E-10 1.89E+05 7.40E-04 3.91E-09

2B12 2.76E+05 7.18E-05 2.60E-10 6.46E+04 9.59E-05 1.48E-09 Cabiralizumab 4.42E+05 6.36E-04 1.44E-09 Not tested Not tested Not tested

Example 3 Binding of Anti-CSF-IR Monoclonal Antibodies to Surface of human CSF-1R Over expressing Cell Lines by Flow Cytometry (FACS)

[00169] The binding activity of the mouse anti-CSF-lR antibodies of the disclosure to CSF-1R expressed on 293F-CSF-1R cell surface was tested by flow cytometry (FACS), using Biosion in-house prepared 293F-CSF-1R cells stably expressing full length human CSF-1R (uniprot# P07333, SEQ ID NO.: 59) on cell membrane. The 293F-CSF-1R cells were prepared by transfecting 293F cells (Thermofisher Inc., Cat# 11625019) with a pCDNA 3.1 plasmid inserted with CSF-1R coding sequence between Notl and Xbal sites, following the instruction of lipofectamine 3000 transfection reagent (Thermo Fisher). A stable cell pool named 293F-CSF-1R was chosen for subsequent cell based binding FACS.

[00170] In specific, the 293F-CSF-1R cells were harvested from cell culture flasks, washed twice and resuspended in phosphate buffered saline (PBS) containing 2% v/v Fetal Bovine Serum (FACS buffer). 2 x 10⁵ cells per well in 96 well-plates were incubated in 100 pL of the anti-CSF-lR antibodies or controls of various concentrations (starting at 120 nM with a 4-fold serial dilution) in FACS buffer for 40 minutes on ice. Cells were washed twice with FACS buffer, and added with 100 pL/well R- Phycoerythrin AffiniPure F(ab Fragment Goat Anti-Mouse IgG (H+L) (1 : 1000 dilution in FACS buffer, Jackson Immunoresearch, Cat#l 15-116-146). Following an incubation of 40 minutes at 4°C in dark, cells were washed three times and resuspended in FACS buffer. Fluorescence was measured using a Becton Dickinson FACS Canto II-HTS equipment. Data was analyzed using Graphpad Prism software and EC₅o values were reported.

[00171] The results were shown in Figs. 1A-1B.

[00172] It can be seen that the mouse anti-CSF-lR antibodies of the disclosure specifically bound human CSF-1R with comparable or lower binding activity compared to Cabiralizumab. As shown in Fig. IB, the antibody ID 10-1 showed higher Bmax (maximum binding) than the benchmark.

Example 4 Blocking Activity of Mouse Anti-CSF-lR Antibodies on CSF-IR-Benchmark or CSF-1R - CSF-1 Binding

[00173] 4.1 Ligand Blocking ELISA

[00174] The ability of the anti-CSF-lR antibodies of the disclosure to block the CSF-lR-CSF-1 binding was measured in a competitive ELISA assay. Briefly, 100 mΐ human CSF-l-his proteins (prepared in house, amino acid sequence set forth in SEQ ID NO: 60) were coated on 96-well micro plates at 100 ng/well in carbonate/bicarbonate buffer (pH 9.6) overnight at 4°C. The next day, plates were washed with wash buffer (PBS+0.05% Tween-20, PBST), and blocked with 5% w/v non-fatty milk in PBST for 2 hours at 37°C.

[00175] During plate blocking, the anti-CSF-lR antibodies of the disclosure or Cabiralizumab were diluted with biotin labeled human CSF-IR-Fc proteins (prepared in house with SEQ ID NO: 61, 0.48 nM in PBST with 2.5% non-fatty milk), starting at 80 nM with a 4-fold serial dilution, and incubated at room temperature for 40 minutes. After plate washing, the antibody /CSF-1R-Fc mixtures were added to human CSF-l-his coated plates, 100 mΐ per well. After incubation at 37°C for 40 minutes, plates were washed using wash buffer. Then streptavidin conjugated HRP was added and incubated for 40 minutes at 37°C. Plates were washed again using wash buffer. Finally, TMB was added and the reaction was stopped using 1M H2SO4, and the absorbance of the each well was read on a microplate reader using dual wavelengths mode with at 450 nm for TMB and 630 nm as the reference wavelength. The OD (450-630) values were plotted against antibody concentration. Data was analyzed using Graphpad Prism software and IC50 values were reported.

[00176] 4.2 Benchmark Blocking ELISA

[00177] The ability of the anti-CSF-lR antibodies of the present disclosure to block Benchmark (Cabiralizumab)-human CSF-1R binding was measured in a competitive ELISA assay. Briefly, Cabiralizumab was coated on 96-well micro plates at 1 pg/mL in PBS, 100 mΐ/well, for 2 hours at 37°C. Plates were washed with wash buffer, and blocked with 200 mΐ/well PBST with 5% w/v non-fatty milk overnight at 4°C.

[00178] The next day, anti-CSF-lR antibodies or controls were diluted in biotin labeled human CSF- lR-Fc solution (amino acid sequence set forth in SEQ ID NO.:61, 10 nM in 2.5% non-fatty milk in PBST), starting at 50 nM with a 4-fold serial dilution. The antibody /CSF-1R-Fc mixture was incubated at room temperature for 40 minutes. After plate washing, the CSF-lR-Fc/antibody mixture was added to the Cabiralizumab coated plates, 100 mΐ/well. After incubation at 37°C for 40 minutes, plates were washed 4 times using wash buffer. Then streptavidin conjugated HRP (1: 10000 dilution in PBST, Jackson Immuno Research, Cat# 016-030-084, 100 mΐ/well) was added, and the plates were incubated for 40 minutes at 37°C. Plates were finally washed using wash buffer. TMB was added and the reaction was stopped using 1M H2SO4, and the absorbance at each well was read on a microplate reader using dual wavelengths mode with at 450 nm for TMB and 630 nm as the reference wavelength. The OD (450-630) values were plotted against antibody concentration. Data was analyzed using Graphpad Prism software and IC50 values were reported.

[00179] The results of the two assays were shown in Figs. 2A-2C and 3A-3C.

[00180] It can be seen from Figs. 2A-2C that all the antibodies of the disclosure were capable of blocking human CSF-1-CSF-1R binding, and 1G8 and 2B7’s blocking activities were similar to the benchmark.

[00181] The data also showed that all anti-CSF-lR antibodies of the disclosure were unable to block human CSF-1R- Cabiralizumab binding, suggesting that none bound to the same or similar epitope as the benchmark did.

Example 5 Cell Based Functional Assay of Mouse Anti-CSF-lR Antibodies [00182] The anti-CSF-lR antibodies of the disclosure were tested for their ability to inhibit CSF-1R tyrosine phosphorylation induced by ligand binding, using the 293F-CSF-1R cells mentioned in Example 3.

[00183] Briefly, 293F-CSF1R cells at the log phase were harvested from cell culture, and resuspended in assay buffer (OPM-293 CD05 medium (Shanghai OPM Biosciences Co., Ltd. Cat#81075-001). 1.2 x 10⁶ cells per well in 96 well-plates were incubated with serially diluted anti-CSF-lR antibodies or controls in assay buffer (50 mΐ/well) for 1 hour at 4°C. Then, 50 mΐ/well human CSF-l-his protein (prepared in house with SEQ ID NO: 60) at 200 ng/ml were added and incubated with the cells for 10 min to stimulate CSF-1R tyrosine phosphorylation. After incubation, cells were centrifuged. After the supernatants were removed, the cells were washed twice with 200 mΐ of ice-cold PBS. 120 mΐ of freshly prepared ice-cold NP-40 lysis buffer (Beyotime Biotechnology, Cat#P0013F) supplemented with Protease/Phosphatase Inhibitor Cocktail (Cell signaling Technology, Cat#5872) was added to each well of the plates. After 15 min on ice, the plates were centrifuged for 5 min at 2000 g, and the supernatants were transferred to clean test tubes. The supernatants were assayed immediately or stored at -80^°C . Before the assay, the supernatants were further diluted in NP-40 lysis buffer with a dilution radio of 2.5. The amount of phosphorylated CSF-1R tyrosine in the cell lysates was quantified using a Human Phospho-M-CSF R DuoSet IC ELISA kit (R&D systems, Cat#DYC3268-5) according to manufacturer’s instruction. Data was analyzed using Graphpad Prism software and IC50 values were reported.

[00184] The results were shown in Figs.4A-4C.

[00185] It can be seen that all the antibodies of the disclosure were capable of inhibiting CSF1 binding induced CSF-1R tyrosine phosphorylation in 293F-CSF-1R cells, with comparable or higher inhibitory activity as compared to Cabiralizumab. In specific, the antibody 2B6 had the highest inhibitory activity.

Example 6 Generation and Characterization of Chimeric Antibodies

[00186] The variable domains of the heavy and light chain of the anti-CSF-lR mouse mAbs were sequenced, and their sequence ID numbers were summarized in Table 1.

[00187] The variable domains of the heavy and light chain of the anti-CSF-lR mouse mAbs 2B6, 1G8 and 1H8 were cloned in frame to human IgG4 heavy -chain (SEQ ID NO.: 55) and human kappa light- chain constant regions (SEQ ID NO.: 56), respectively, wherein the C terminus of variable region was linked to the N terminus of the respective constant region.

[00188] The vectors each containing a nucleotide encoding a heavy chain variable region linked to human IgG4 heavy -chain constant region, and the vectors each containing a nucleotide encoding a light chain variable region linked to human kappa light-chain constant region were transiently transfected into 50 ml of 293F suspension cell cultures in a ratio of 1.1: 1 light to heavy chain construct, with 1 mg/mL PEI. [00189] Cell supernatants containing the chimeric antibodies were harvested after six days in shaking flasks, and then chimeric antibodies were purified from cell supernatants. The antibodies were tested in capture ELISA, Octet affinity test, Ligand blocking ELISA and Cell-based functional assay following the protocols in the foregoing Examples and described below.

[00190] Lor the Capture ELISA, 96-well plates were coated with 100 mΐ 2 pg/ml AffiniPure goat antihuman IgG F(ab’)₂ fragment specific (Jackson Immiino Research, Cat#109-005-097) in PBS for 2 hours at 37^°C . Plates were washed once with wash buffer (PBS+0.05% Tween-20, PBST) and then blocked with 200 mΐ/well blocking buffer (PBST with 5% w/v non-fatty milk) for 2 hours at 37°C. Plates were washed again and incubated with 100 mΐ/well serially diluted chimeric anti-CSF-lR antibodies of the disclosure, the benchmark, or negative control hlgG (human immunoglobulin (pH4) for intravenous injection, Hualan Biological Engineering Inc.) (5-fold dilution in PBST with 2.5% non-fatty milk, starting at 10000 ng/ml) for 40 minutes at 37°C, and then washed 4 times again. Plates containing captured anti-CSF-lR antibodies were added with 100 mΐ/well biotin labeled human CSF-IR-Fc protein (in house made, amino acid sequence set forth in SEQ ID NO:61, 0.24 nM in PBST with 2.5% nonfatty milk) and incubated for 40 minutes at 37°C, washed 4 times, and incubated with streptavidin conjugated HRP (1: 10000 dilution in PBST, Jackson Immuno Research, Cat#016-030-084, 100 mΐ/well) for 40 minutes at 37°C. After a final wash, plates were incubated with 100 mΐ/well ELISA substrate TMB (Innoreagents, Cat#TMB-S-002). The reaction was stopped in 3-10 minutes at room temperature with 50 mΐ/well 1M H2SO4, and the absorbance of the each well was read on a microplate reader using dual wavelengths mode with at 450 nm for TMB and 630 nm as the reference wavelength. The OD (450-630) values were plotted against antibody concentration. Data was analyzed using Graphpad Prism software and EC50 values were reported. The results were shown in Figs.5A-5C.

[00191] The Octet affinity test was performed using Octet system (Fortebio, Octet RED 96). Briefly, AHC biosensors (anti-human IgG Fc capture, from ForteBio) were presoaked with 10 mM glycine (pH 1.5) for 3 seconds, and then dipped in a well with running buffer (0.5% w/v BSA in PBST) for 3 seconds. The soaking and dipping steps were repeated for three times. Then, the sensors were dipped in a well with the chimeric anti-CSF-lR antibodies or the benchmark in HBS-EP⁺ at 5 pg/ml for 100 seconds, and then immersed in a well with running buffer for 5 min. A new baseline was rim for 180 seconds in another well with running buffer. Then the sensors were dipped in a well with serially diluted human CSF-lR-his proteins (Aero biosystems, Cat#CSR-H5228, starting at 40 nM with a two-fold serial dilution) in running buffer for 100 seconds, and then immersed in a baseline well for 10 min. Finally, sensors were presoaked with 10 mM glycine (pH 1.5) for 3 seconds, and then were dipped in a well with running buffer for 3 seconds. The soaking and dipping steps repeated for three times. The association and dissociation curves were fit to a 1: 1 Langmuir binding model using Octet evaluation software. The K_D values were determined and summarized in Table 3 below. [00192] The results of the CSF1R-CSF1 ligand blocking ELISA and the CSF-1R tyrosine phosphorylation inhibition assay were respectively shown in Figs.6A-6B and Fig.7.

[00193] The data showed that the chimeric anti-CSF-lR antibodies had similar binding affinity /capacity and ligand blocking activity to their parental mouse mAbs. Particularly, the chimeric antibodies 2B6, 1H8 and 1G8 showed higher binding capacity than Cabiralizumab in the Capture ELISA assay.

Table 3. Binding affinity of Chimeric Antibodies mAb Octet Affinity to human CSF-1R (KD, M)

Chimeric 2B6 6.46E-09

Chimeric 1H8 2.60E-09

Chimeric 1G8 3.48E-09

Cabiralizumab 1.77E-09

Example 7 Humanization of Anti-CSF-IR Mouse Monoclonal Antibody 1G8

[00194] Mouse anti-CSF-lR antibody 1G8 was humanized and further characterized. Humanization of the mouse antibodies were conducted using the well-established CDR-grafting method as described in detail below.

[00195] To select acceptor frameworks for humanization of mouse antibody 1G8, the light and heavy chain variable region sequences of 1G8 were blasted against the human immunoglobulin gene database. The human germlines with the highest homology were selected as the acceptor frameworks for humanization. The mouse antibody heavy /light chain variable region CDRs were inserted into the selected frameworks, and the residue(s) in the frameworks was/were further backmutated to obtain more candidate heavy chain/light chain variable regions. A total of 15 humanized 1G8 antibodies, namely hulG8-Vl to hulG8-V15 were obtained, whose heavy /light chain variable region sequence ID numbers were also summarized in Table 1.

[00196] The vectors each containing a nucleotide encoding a humanized heavy chain variable region linked to human IgG4 heavy-chain constant region (SEQ ID NO: 55), and the vectors each containing a nucleotide encoding a humanized light chain variable region linked to human kappa light-chain constant region (SEQ ID NO: 56) were transiently transfected into 200 ml of 293F suspension cell cultures in a ratio of 1.1 : 1 light to heavy chain construct, with 1 mg/mL PEI.

Example 8 Characterization of humanized antibodies

[00197] Cell supernatants containing humanized antibodies were harvested after six days in shaking flasks and tested for binding capacity /affinity to human CSF-1R by Capture ELISA and Octet affinity test following the protocols described above with some modifications.

[00198] In the capture ELISA assay, the 96-well micro plates were coated with 0.2 pg/ml goat antihuman IgG (AffmiPure Goat Anti-Human IgG, F(ab fragment specific, Jackson Immunoresearch, Cat#109-005-097) instead of the goat anti-mouse IgG F(ab fragment, 100 mΐ/well. The results were shown in Figs. 8A-8B. [00199] In the Octet affinity test, cell supernatants containing humanized antibodies were used, while the chimeric antibody and Cabiralizumab were prepared in HBS-EP⁺ at 5 pg/ml concentration. The K_a, K_d and K_D values of the Octet affinity test were determined and summarized in Table 4 below. The data indicated that all cell supernatants containing humanized 1G8 antibodies showed comparable binding affinity to human CSF-1R compared to Cabiralizumab.

Table 4. Binding Affinity of Humanized 1G8 mAbs

Kinetics on Octet

Human CSF-1R

(M-ls-1) (s-1) (M)

huCSF-lR 1G8-V4 1.47E+05 1.13E-03 7.66E-09 huCSF-lR 1G8-V7 1.41E+05 1.04E-03 7.43E-09 huCSF-lR 1G8-V10 1.74E+05 1.27E-03 7.30E-09 huCSF- l R 1 G8-V 13 1.48E+05 9.95E-04 6.75E-09

Chimeric 1G8 1.97E+05 5.35E-04 2.71E-09

Cabiralizumab 3.30E+05 4.47E-04 1.35E-09

[00200] The humanized antibody hulG8-V13 was purified as described above and tested in Biacore, binding capture ELISA, cyno-cross indirect ELISA, benchmark blocking ELISA, cell-based binding FACS and cell-based functional assay following the protocols in foregoing Examples with minor modifications and protocols described below.

[00201] In specific, the indirect ELISA for testing the antibody’s cross-reaction to monkey CSF-1R proteins was performed as follows. Briefly, 96-well micro plates were coated with 1 pg/ml cynomolgus CSF-lR-his protein (in house made, amino acid sequence set forth in SEQ ID NO:62, in carbonate/bicarbonate buffer), 100 mΐ/well, for 2 hours at 37°C. ELISA plates were washed once with wash buffer (PBS+0.05% Tween-20, PBST) and then blocked with 200 mΐ/well blocking buffer (5% w/v non-fatty milk in PBST) overnight at 4°C. The next day, the plates were washed again and incubated with serially diluted anti-CSF-lR antibodies of the disclosure or controls (starting at 66.7 nM with 5-fold serial dilution in PBST with 2.5% non-fatty milk in) for 40 minutes at 37°C. ELISA plates were washed 4 times and incubated with Peroxidase AffiniPure F(ab')2 Fragment Goat Anti-Human IgG, Fey Fragment Specific (1:5000 dilution in PBST buffer, Jackson Immunoresearch, Cat#109-036- 098, 100 mΐ/well) for 40 minutes at 37^°C . After the final wash, plates were incubated with 100 mΐ/well TMB (Innoreagents). The reaction was stopped 3 minutes later at room temperature with 50 mΐ/well 1M H2SO4, and the absorbance of the each well was read on a microplate reader using dual wavelengths mode with at 450 nm for TMB and 630 nm as the reference wavelength. The OD (450-630) values were plotted against antibody concentration. Data was analyzed using Graphpad Prism software and EC50 values were reported. The results were shown in Fig. 10. [00202] For the Biacore test, CM5 chips were used for the test antibodies, including the benchmark, and goat anti-human IgG (GE healthcare, Cat#BRl 00839, Mouse Antibody Capture Kit) was covalently linked to the CM5 chips. The purified hulG8-V13 and Cabiralizumab, at the concentration of 2 pg/ml, were flowed onto the chip at a flow rate of 10 pL/min, and serially diluted recombinant human CSF-lR-his and cynomolgus monkey CSF-lR-his protein both had an initial concentration of 320 nM. The data was summarized in Table 6 below.

[00203] For the cell-based binding FACS, R-Phycoerythrin AffiniPure Goat Anti-human IgG Fey fragment specific (Jackson ImmunoResearch Laboratories, Inc., Cat#109-115-098) was used instead of R-Phycoerythrin AffiniPure F(ab Fragment Goat Anti-Mouse IgG (H+L), 1 : 1000 dilution in FACS buffer, 100 mΐ/well. The results were shown in Fig. 15.

[00204] In the ligand blocking ELISA, the inhibitory effect of hulG8-V13 on CSF-1R-IL34 binding was also tested, with 100 mΐ human IL34-his (Sino biological inc., Cat#10948-H08S) coated on 96-well micro plates at 100 ng/well in carbonate/bicarbonate buffer (pH 9.6) at the beginning of the assay. The results on CSF-1R-CSF1 and CSF-1R-IL34 blocking were shown in Figs. 11 and 12, respectively.

[00205] To determine the thermal stability of humanized antibody hulG8-V13, a protein thermal shift assay was used to determine melting temperature (Tm) using a GloMelt™ Thermal Shift Protein Stability Kit (Biotium, Cat# 33022 -T). Briefly, the GloMelt™ dye was allowed to thaw and reach room temperature. The vial containing the dye was vortexed and centrifuged. Then, lOx dye was prepared by adding 5 pL 200x dye to 95 pL PBS. 2 pL lOx dye was added with 10 pg humanized antibodies, and PBS was added to a total reaction volume of 20 pL. The tubes containing the dye and antibodies were briefly spun and placed in real-time PCR thermocycler (Roche, LightCycler 480 II) set up with a melt curve program having the parameters in Table 5. The results were shown in Fig. 16.

Table 5. Parameters for Melt Curve Program

[00206] The results of the capture ELISA, benchmark-CSF-lR blocking and CSF-1R phosphorylation inhibition were respectively shown in Figs. 9, 13 and 14 as well as Table 6 below.

Table 6. Binding activity of Humanized mAh hulG8-V13

Binding assay

Human CSF-1R Cynomolgus CSF-1R mAbs -

Capture ELISA Biacore (K_D, Indirect ELISA

Biacore (K_D, M)

(EC₅₀, nM) M) (EC₅₀, nM)

Mouse 1G8 0.17 Not tested 0.23 Not tested

Chimeric 1G8 0.22 1.82E-09 0.48 9.92E-09 hulG8-V13 0.28 2.52E-09 0.42 8.72E-09

Cabiralizumab 0.06 3.27E-10 0.19 4.32E-10 [00207] The data showed that the antibody hulG8-V13 had comparable or a bit lower binding affmity/capacity to human CSF-1R or monkey CSF-1R compared to benchmark in the Biacore test, cell-based binding FACS assay and the Capture ELISA assay, while this antibody’s binding capacity to monkey CSF-1R was proved to be higher than the benchmark (e.g., higher Bmax) in the indirect ELISA. Further, this antibody effectively blocked CSF-1R binding to CSF-1 or IL34 with comparable blocking capacity as compared to the benchmark, and accordingly inhibited CSF-1R tyrosine phosphorylation.

[00208] The data also showed that the antibody hulG8-V13 was unable to block human CSF-1R- Cabiralizumab interaction, suggesting that it bound to a different epitope. For the melting temperature, T1 and T2 were respectively 66°C and 77°C.

[00209] While the disclosure has been described above in connection with one or more embodiments, it should be understood that the disclosure is not limited to those embodiments, and the description is intended to cover all alternatives, modifications, and equivalents, as may be included within the spirit and scope of the appended claims. All referenced cited herein are further incorporated by reference in their entirety.

[00210] Sequences in the present application are summarized below.

[00211] Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the above paragraphs is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention.

Claims

What is claimed is:

1. An isolated monoclonal antibody, or an antigen-binding portion thereof, binding to CSF-1R, comprising a heavy chain variable region comprising a CDR1 region, a CDR2 region and a CDR3 region, wherein the CDR1 region, the CDR2 region and the CDR3 region comprise amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to (1) SEQ ID NOs: 1, 8 and 14, respectively; (2) SEQ ID NOs: 2, 9 and 15, respectively; (3) SEQ ID NOs: 3, 10 and 16, respectively; (4) SEQ ID NOs: 4, 11 and 17, respectively; (5) SEQ ID NOs: 5, 12 and 18, respectively; (6) SEQ ID NOs: 5, 12 and 19, respectively; (7) SEQ ID NOs: 6, 12 and 18, respectively; or (8) SEQ ID NOs: 7, 13 and 20, respectively.

2. The isolated monoclonal antibody, or the antigen-binding portion thereof, of claim 1, wherein the heavy chain variable region comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NOs: 37, 38 (X1=V, X2=R, X3=A; X1=R, X2=T, X3=A; or X1=R, X2=T, X3=V), 39, 40, 41, 42, 43, 44 or 45.

3. The isolated monoclonal antibody, or the antigen-binding portion thereof, of claim 1, comprising a light chain variable region comprising a CDR1 region, a CDR2 region and a CDR3 region, wherein the CDR1 region, the CDR2 region and the CDR3 region comprise amino acid sequences amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to (1) SEQ ID NOs: 21, 27 and 32, respectively; (2) SEQ ID NOs: 22, 28 and 33, respectively; (3) SEQ ID NOs: 23, 29 and 34, respectively; (4) SEQ ID NOs: 24, 30 and 35, respectively; (5) SEQ ID NOs: 25, 29 and 36, respectively; or (6) SEQ ID NOs: 26, 31 and 34, respectively.

4. The isolated monoclonal antibody, or the antigen-binding portion thereof, of claim 3, wherein the light chain variable region comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NOs: 46, 47 (X1=I, X2=S, X3=K, K4=P; X1=I, X2=Y, X3=T, X4=L; X1=L, X2=S, X3=T, X4=L; X1=I, X2=S, X3=T, X4=L; or X1=L, X2=Y, X3=T, X4=L), 48, 49, 50, 51, 52, 53 or 54.

5. The isolated monoclonal antibody, or an antigen-binding portion thereof, of claim 3, wherein the heavy chain variable region and the light chain variable region comprise amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to (1) SEQ ID NOs: 1, 8, 14, 21, 27 and 32, respectively; (2) SEQ ID NOs: 2, 9, 15, 22, 28 and 33, respectively; (3) SEQ ID NOs: 3, 10, 16, 23, 29 and 34, respectively; (4) SEQ ID NOs: 4, 11, 17, 24, 30 and 35, respectively; (5) SEQ ID NOs: 5, 12, 18, 25, 29 and 36, respectively; (6) SEQ ID NOs:

5, 12, 19, 25, 29 and 36, respectively; (7) SEQ ID NOs: 6, 12, 18, 25, 29 and 36, respectively; or (8) SEQ ID NOs: 7, 13, 20, 26, 31 and 34, respectively.

6. The isolated monoclonal antibody, or an antigen-binding portion thereof, of claim 5, wherein the heavy chain variable region and the light chain variable region comprise amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to (1) SEQ ID NOs: 37 and 46, respectively; (2) SEQ ID NOs: 38 (X1=V, X2=R, X3=A; X1=R, X2=T, X3=A; or X1=R, X2=T, X3=V) and 47 (X1=I, X2=S, X3=K, K4=P), respectively; (3) SEQ ID NOs: 38 (X1=V, X2=R, X3=A; X1=R, X2=T, X3=A; or X1=R, X2=T, X3=V) and 47 (X1=I, X2=Y, X3=T, X4=L), respectively; (4) SEQ ID NOs: 38 (X1=V, X2=R, X3=A; X1=R, X2=T, X3=A; or X1=R, X2=T, X3=V) and 47 (X1=L, X2=S, X3=T, X4=L), respectively; (5) SEQ ID NOs: 38 (X1=V, X2=R, X3=A; X1=R, X2=T, X3=A; or X1=R, X2=T, X3=V) and 47 (X1=I, X2=S, X3=T, X4=L), respectively; (6) SEQ ID NOs: 38 (X1=V, X2=R, X3=A; X1=R, X2=T, X3=A; or X1=R, X2=T, X3=V) and 47 (X1=L, X2=Y, X3=T, X4=L), respectively; (7) SEQ ID NOs: 39 and 48, respectively; (8) SEQ ID NOs: 40 and 49, respectively; (9) SEQ ID NOs: 41 and 50, respectively; (10) SEQ ID NOs: 42 and 51, respectively; (11) SEQ ID NOs: 43 and 52, respectively; (12) SEQ ID NOs: 44 and 53, respectively; or (13) SEQ ID NOs: 45 and 54, respectively.

7. The isolated monoclonal antibody, or the antigen-binding portion thereof, of claim 1, which is an IgGl, IgG2 or IgG4 isotype.

8. The isolated monoclonal antibody, or an antigen-binding portion thereof, of claim 7, comprising a heavy chain constant region having an amino acid sequence of SEQ ID NO: 55, linked to the heavy chain variable region, and a light chain constant region having an amino acid sequence of SEQ ID NO: 56, linked to the light chain variable region.

9. The isolated monoclonal antibody, or the antigen-binding portion thereof, of claim 1, which (a) binds human CSF1R; (b) binds monkey CSF1R; (c) blocks CSF1R-CSF1 binding; (d) blocks CSF1R-IL34 binding; and/or (e) inhibits CSF1R tyrosine phosphorylation induced by ligand binding.

10. The isolated monoclonal antibody, or the antigen-binding portion thereof, of claim 1, which is a mouse, chimeric or humanized antibody.

11. A nucleotide encoding the isolated monoclonal antibody or the antigen-binding portion thereof of claim 1.

12. An expression vector comprising the nucleotide of claim 11.

13. A host cell comprising the expression vector of claim 12.

14. A pharmaceutical composition comprising the isolated monoclonal antibody, or antigen-binding portion thereof, of claim 1, and a pharmaceutically acceptable carrier.

15. The pharmaceutical composition of claim 14, further comprising an anti-tumor agent, an anti- inflammation agent, or an anti-bone loss agent.

16. A method for treating a disease associated with excessive CSF-1R signaling, comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 14.

17. The method of claim 16, wherein the disease is an inflammatory disease, cancer or bone loss.

18. The method of claim 17, wherein the inflammatory disease is rheumatoid arthritis, atherosclerosis, osteoarthritis, or inflammatory bowel disease.

19. The method of claim 17, wherein the cancer is ovarian cancer, endometrial cancer, tenosynovial giant cell tumor, pancreatic cancer, breast cancer, cervical cancer, lung cancer or prostate cancer

20. The method of claim 17, wherein the bone loss is periodontitis, histiocytosis X, osteoporosis, Paget's disease of bone (PDB), bone loss due to cancer therapy, periprosthetic osteolysis, or glucocorticoid-induced osteoporosis.