TECHNICAL FIELD

[001] Methods of treating conditions with antibodies that bind colony stimulating factor 1 receptor (CSFIR) are provided. Such methods include, but are not limited to, methods of treating rheumatoid arthritis, multiple sclerosis, and systemic lupus erythematosus.

BACKGROUND

[002] Colony stimulating factor 1 receptor (referred to herein as CSFIR; also referred to in the art as FMS, FIM2, C-FMS, M-CSF receptor, and CD115) is a single-pass transmembrane receptor with an N-terminal extracellular domain (ECD) and a C-terminal intracellular domain with tyrosine kinase activity. Ligand binding of CSF 1 or the interleukin 34 ligand (referred to herein as IL-34; Lin et al., Science 320: 807-11 (2008)) to CSFIR leads to receptor dimerization, upregulation of CSFIR protein tyrosine kinase activity, phosphorylation of CSFIR tyrosine residues, and downstream signaling events. Both CSF l and IL-34 stimulate monocyte survival, proliferation, and differentiation into macrophages, as well as other monocytic cell lineages such as osteoclasts, dendritic cells, and microglia.

[003] Many tumor cells have been found to secrete CSF 1, which activates monocyte/macrophage cells through CSFIR. The level of CSFl in tumors has been shown to correlate with the level of tumor-associated macrophages (TAMs) in the tumor. Higher levels of TAMs have been found to correlate with poorer patient prognoses. In addition, CSF l has been found to promote tumor growth and progression to metastasis in, for example, human breast cancer xenografts in mice. See, e.g., Paulus et al, Cancer Res. 66: 4349-56 (2006). Further, CSF IR plays a role in osteolytic bone destruction in bone metastasis. See, e.g., Ohno et al, Mol. Cancer Ther. 5: 2634-43 (2006).

[004] CSFl and its receptor have also been found to be involved in various inflammatory and autoimmune diseases. See, e.g., Hamilton, Nat. Rev. 8: 533-44 (2008). For example, synovial endothelial cells from joints afflicted with rheumatoid arthritis have been found to produce CSFl, suggesting a role for CSFl and its receptor in the disease. Blocking CSF IR activity with an antibody results in positive clinical effects in mouse models of arthritis, including a reduction in the destruction of bone and cartilage and a reduction in macrophage numbers. See, e.g., Kitaura et al, J. Clin. Invest. 1 15: 3418-3427 (2005). [005] Mature differentiated myeloid lineage cells such as macrophages, microglial cells, and osteoclasts contribute to pathology of various diseases such as rheumatoid arthritis, multiple sclerosis and diseases of bone loss. Differentiated myeloid lineage cells are derived from peripheral blood monocyte intermediates. CSF1R stimulation contributes to development of monocytes from bone marrow precursors, to monocyte proliferation and survival, and to differentiation of peripheral blood monocytes into differentiated myeloid lineage cells such as macrophages, microglial cells, and osteoclasts. CSF1R stimulation thus contributes to proliferation, survival, activation, and maturation of differentiated myeloid lineage cells, and in the pathologic setting, CSF1R stimulation contributes to the ability of differentiated myeloid lineage cells to mediate disease pathology.

SUMMARY

[006] In some embodiments, a method of treating a condition associated with rheumatoid arthritis is provided. In some embodiments, the method comprises

administering an antibody that binds colony stimulating factor 1 receptor (CSF 1R) to a subject with rheumatoid arthritis, wherein the antibody blocks binding of colony stimulating factor 1 (CSF 1) to CSF1R and blocks binding of IL-34 to CSF 1R. In some embodiments, treating a condition associated with rheumatoid arthritis comprises at least one effect selected from reducing inflammation, reducing pannus formation, reducing cartilage damage, reducing bone resorption, reducing the number of macrophages in at least one joint affected by rheumatoid arthritis, reducing autoantibody levels, and reducing bone loss. In some embodiments, treating a condition associated with rheumatoid arthritis comprises reducing inflammation. In some embodiments, a method of reducing inflammation associated with rheumatoid arthritis comprises determining an erythrocyte sedimentation rate, wherein a reduced sedimentation rate indicates reduced inflammation.

[007] In some embodiments, a method of treating a condition associated with rheumatoid arthritis comprises at least one effect selected from reducing pannus formation, reducing bone resorption, and reducing bone loss. In some embodiments, treating a condition associated with rheumatoid arthritis comprises reducing bone resorption. In some such embodiments, the level of at least one marker of bone resorption is reduced. In some embodiments, the bone resorption marker is selected from tartrate resistant acid phosphatase 5b (TRAP5b), urinary total pyridinoline, Urinary total deoxypyridinoline, urinary free pyridinoline, serum collagen type I cross-linked N-telopeptide, urinary collagen type I cross-linked N-telopeptide, and serum carboxyterminal telopeptide of type I collagen. [008] In some embodiments, treating a condition associated with rheumatoid arthritis comprises at least one effect selected from reducing pannus formation, and reducing bone loss. In some embodiments, the at least one effect is measured using an imaging technique. In some such embodiments, the imaging technique comprises a method selected from x-ray imaging, magnetic resonance imaging, computed tomography (CT) scan, arthroscopy, scintigraphy, ultrasonography, bone densitometry, single photon

absorptiometry (SPA), dual photon absorptiometry (DPA), single energy x-ray

absorptiometry (SXA), dual energy x-ray absorptiometry (DXA), scintigraphy,

ultrasonography, duplex ultrasonography, and power doppler imaging.

[009] In some embodiments of a method of treating a condition associated with rheumatoid arthritis, the number of CD 16+ monocytes is reduced by at least 30%. In some embodiments of the method, the number of CD 16- monocytes is not reduced or is reduced by less than 20%. In some embodiments, the CD 16+ monocytes are CD 16+ peripheral blood monocytes.

[010] In some embodiments, a method of treating rheumatoid arthritis is provided, comprising administering an antibody that binds CSF1R to a subject with rheumatoid arthritis, wherein the antibody blocks binding of CSF 1 to CSF1R and blocks binding of IL- 34 to CSF 1R, and wherein the antibody reduces the number of CD16+ monocytes in the subject by at least 30%, and wherein CD 16- monocytes are not reduced or are reduced by less than 20%. In some embodiments, the CD 16+ monocytes are CD 16+ peripheral blood monocytes.

[011] In some embodiments of a method of treating rheumatoid arthritis, the method further comprises administering at least one additional therapeutic agent selected from methotrexate, an anti-TNF agent, a glucocorticoid, cyclosporine, leflunomide, azathioprine, a JAK inhibitor, a SYK inhibitor, an anti-IL-6 antibody, an anti-IL-6R antibody, an anti-CD-20 antibody, an anti-CD 19 antibody, an anti-GM-CSF antibody, an IL-1 receptor antagonist, a CTLA-4 antagonist, and an anti-GM-CSF-R antibody.

[012] In some embodiments, methods of treating skin lesions associated with lupus are provided. In some embodiments, the method comprises administering an antibody that binds CSF1R to a subject with lupus, wherein the antibody blocks binding of CSF1 to CSF1R and blocks binding of IL-34 to CSF1R. In some embodiments, treating skin lesions associated with lupus comprises at least one effect selected from reducing the number of skin lesions, reducing the rate of formation of skin lesions, and reducing the severity of skin lesions. [013] In some embodiments, methods of treating lupus nephritis are provided. In some embodiments, the method comprises administering an antibody that binds CSFIR to a subject with lupus nephritis, wherein the antibody blocks binding of CSF l to CSFIR and blocks binding of IL-34 to CSFIR. In some embodiments, kidney function is improved in the subject. In some embodiments, proteinuria is reduced in the subject. In some embodiments, glomerular filtration rate is improved in the subject.

[014] In some embodiments, methods of treating lupus are provided. In some embodiments, a method comprises administering an antibody that binds CSFIR to a subject with lupus, wherein the antibody blocks binding of CSFl to CSFIR and blocks binding of IL-34 to CSF IR. In some embodiments, the antibody reduces the number of CD16+ monocytes in the subject by at least 30%, and CD 16- monocytes are not reduced or are reduced by less than 20%. In some embodiments, CD 16+ monocytes are reduced by at least 50%. In some embodiments, the CD 16+ monocytes are CD 16+ peripheral blood monocytes.

[015] In some embodiments, the method of treating lupus, lupus nephritis, or skin lesions associated with lupus further comprises administering at least one additional therapeutic agent selected from hydroxychloroquine (Plaquenil), a corticosteroids, cyclophosphamide (Cytoxan), azathioprine (Imuran, Azasan), mycophenolate (Cellcept), leflunomide (Arava) and methotrexate (Trexall), and belimumab (Benlysta).

[016] In some embodiments, methods of treating inflammatory conditions are provided. In some such embodiments, a method comprises administering an antibody that binds CSFIR to a subject with an inflammatory condition, wherein the antibody blocks binding of CSFl to CSFIR and blocks binding of IL-34 to CSFIR. In some embodiments, the antibody reduces the number of CD 16+ monocytes by at least 30%, and CD 16- monocytes are not reduced or are reduced by less than 20%. In some embodiments, CD 16+ monocytes are reduced by at least 50%. In some embodiments, the CD 16+ monocytes are CD 16+ peripheral blood monocytes.

[017] In some embodiments, methods of treating CD 16+ disorders are provided. In some embodiments, a method comprises administering an antibody that binds CSFIR to a subject with a CD 16+ disorder, wherein the antibody blocks binding of CSFl to CSFIR and blocks binding of IL-34 to CSFIR. In some embodiments, the antibody reduces the number of CD 16+ monocytes by at least 30%, and CD 16- monocytes are not reduced or are reduced by less than 20%. In some embodiments, CD 16+ monocytes are reduced by at least 50%. In some embodiments, the CD 16+ monocytes are CD 16+ peripheral blood monocytes. [018] In some embodiments, a method of treating discussed herein comprises treating a condition that does not respond to methotrexate.

[019] In some embodiments, methods of reducing the number of CD 16+ monocytes are provided. In some embodiments, a method comprises administering an antibody that binds CSF 1R to a subject, wherein the antibody blocks binding of CSF 1 to CSFIR and blocks binding of IL-34 to CSFIR. In some embodiments, the antibody reduces the number of CD 16+ monocytes by at least 30%, and CD 16- monocytes are not reduced or are reduced by less than 20%. In some embodiments, CD 16+ monocytes are reduced by at least 50%. In some embodiments, the CD 16+ monocytes are CD 16+ peripheral blood monocytes.

[020] In some embodiments, methods of slowing the progression of a kidney condition associated with lupus are provided. In some embodiments, the method comprises administering an antibody that binds CSFIR to a subject, wherein the antibody blocks binding of CSF 1 to CSFIR and blocks binding of IL-34 to CSFIR. In some embodiments, proteinuria does not increase in the subject or does not increase in the subject at the same rate as in subjects not administered the antibody. In some embodiments, glomerular filtration rate does not decrease in the subject or does not decrease in the subject at the same rate as in a subjects not administered the antibody.

[021 ] In some embodiments, methods of slowing the progression of pannus formation in a subject with rheumatoid arthritis are provided. In some such embodiments, a method comprises administering an antibody that binds CSF IR to a subject with rheumatoid arthritis, wherein the antibody blocks binding of CSF 1 to CSFIR and blocks binding of IL- 34 to CSF IR.

[022] In some embodiments, methods of slowing the progression of bone loss in a subject with rheumatoid arthritis are provided. In some embodiments, a method comprises administering an antibody that binds colony stimulating factor 1 receptor (CSF IR) to a subject with rheumatoid arthritis, wherein the antibody blocks binding of colony stimulating factor 1 (CSF 1) to CSF IR and blocks binding of IL-34 to CSFIR.

[023] In some embodiments of the methods described herein, the antibody heavy chain and/or the antibody light chain have the following structure.

[024] In some embodiments, the heavy chain comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from SEQ ID NOs: 9, 11, 13, and 39 to 45. In some embodiments, the light chain comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from SEQ ID NOs: 10, 12, 14, and 46 to 52. In some embodiments, the heavy chain comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from SEQ ID NOs: 9, 1 1, 13, and 39 to 45, and the light chain comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence selected from SEQ ID NOs: 10, 12, 14, and 46 to 52.

[025] In some embodiments, the HC CDR1, HC CDR2, and HC CDR3 comprise a set of sequences selected from: (a) SEQ ID NOs: 15, 16, and 17; (b) SEQ ID NOs: 21, 22, and 23; and (c) SEQ ID NOs: 27, 28, and 29. In some embodiments, the LC CDR1, LC CDR2, and LC CDR3 comprise a set of sequences selected from: (a) SEQ ID NOs: 18, 19, and 20; (b) SEQ ID NOs: 24, 25, and 26; and (c) SEQ ID NOs: 30, 31, and 32.

[026] In some embodiments, the heavy chain comprises an HC CDR1, HC CDR2, and HC CDR3, wherein the HC CDR1, HC CDR2, and HC CDR3 comprise a set of sequences selected from: (a) SEQ ID NOs: 15, 16, and 17; (b) SEQ ID NOs: 21, 22, and 23; and (c) SEQ ID NOs: 27, 28, and 29; and the light chain comprises an LC CDR1, LC CDR2, and LC CDR3, wherein the LC CDR1, LC CDR2, and LC CDR3 comprise a set of sequences selected from: (a) SEQ ID NOs: 18, 19, and 20; (b) SEQ ID NOs: 24, 25, and 26; and (c) SEQ ID NOs: 30, 31, and 32.

[027] In some embodiments, an isolated antibody is provided, wherein the antibody comprises a heavy chain and a light chain, wherein the antibody comprises: (a) a heavy chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 9 and a light chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 10; (b) a heavy chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 11 and a light chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 12; (c) a heavy chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 13 and a light chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 14; (d) a heavy chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 39 and a light chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 46; (e) a heavy chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 40 and a light chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 46; (f) a heavy chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 41 and a light chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 46; (g) a heavy chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 39 and a light chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 47; (h) a heavy chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 40 and a light chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 47; (i) a heavy chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 41 and a light chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 47; and (j) a heavy chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 42 and a light chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 48; (k) a heavy chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 42 and a light chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 49; (1) a heavy chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 42 and a light chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 50; (m) a heavy chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 43 and a light chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 48; (n) a heavy chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 43 and a light chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 49; (o) a heavy chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 43 and a light chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 50; (p) a heavy chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 44 and a light chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 51; (q) a heavy chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 44 and a light chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 52; (r) a heavy chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 45 and a light chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 51 ; or (s) a heavy chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 45 and a light chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 52.

[028] In some embodiments, an antibody is provided, wherein the antibody comprises a heavy chain and a light chain, wherein the antibody comprises: (a) a heavy chain comprising a heavy chain (HC) CDRl having the sequence of SEQ ID NO: 15, an HC CDR2 having the sequence of SEQ ID NO: 16, and an HC CDR3 having the sequence of SEQ ID NO: 17, and a light chain comprising a light chain (LC) CDRl having the sequence of SEQ ID NO: 18, a LC CDR2 having the sequence of SEQ ID NO: 19, and a LC CDR3 having the sequence of SEQ ID NO: 20; (b) a heavy chain comprising a heavy chain (HC) CDRl having the sequence of SEQ ID NO: 21, an HC CDR2 having the sequence of SEQ ID NO: 22, and an HC CDR3 having the sequence of SEQ ID NO: 23, and a light chain comprising a light chain (LC) CDRl having the sequence of SEQ ID NO: 24, a LC CDR2 having the sequence of SEQ ID NO: 25, and a LC CDR3 having the sequence of SEQ ID NO: 26; or (c) a heavy chain comprising a heavy chain (HC) CDRl having the sequence of SEQ ID NO: 27, an HC CDR2 having the sequence of SEQ ID NO: 28, and an HC CDR3 having the sequence of SEQ ID NO: 29, and a light chain comprising a light chain (LC) CDRl having the sequence of SEQ ID NO: 30, a LC CDR2 having the sequence of SEQ ID NO: 31, and a LC CDR3 having the sequence of SEQ ID NO: 32.

[029] In some embodiments, an antibody comprises a heavy chain and a light chain, wherein the antibody comprises: (a) a heavy chain comprising a sequence of SEQ ID NO: 53 and a light chain comprising a sequence of SEQ ID NO: 60; (b) a heavy chain comprising a sequence of SEQ ID NO: 53 and a light chain comprising a sequence of SEQ ID NO: 61; or (c) a heavy chain comprising a sequence of SEQ ID NO: 58 and a light chain comprising a sequence of SEQ ID NO: 65. In some embodiments, an antibody comprises a heavy chain and a light chain, wherein the antibody comprises: (a) a heavy chain consisting of the sequence of SEQ ID NO: 53 and a light chain consisting of the sequence of SEQ ID NO: 60; (b) a heavy chain consisting of the sequence of SEQ ID NO: 53 and a light chain consisting of the sequence of SEQ ID NO: 61 ; or (c) a heavy chain consisting of the sequence of SEQ ID NO: 58 and a light chain consisting of the sequence of SEQ ID NO: 65.

[030] In some embodiments, an antibody is a humanized antibody. In some embodiments, an antibody is selected from a Fab, an Fv, an scFv, a Fab', and a (Fab')2. In some embodiments, an antibody is a chimeric antibody. In some embodiments, an antibody is selected from an IgA, an IgG, and an IgD. In some embodiments, an antibody is an IgG. In some embodiments, an antibody is an IgG4. In some embodiments, an antibody is an IgG4 comprising an S241P mutation in at least one IgG4 heavy chain constant region.

[031] In some embodiments, an antibody binds to human CSFIR and/or binds to cynomolgus CSFIR. In some embodiments, an antibody blocks ligand binding to CSFIR. In some embodiments, an antibody blocks binding of CSFl and/or IL-34 to CSFIR. In some embodiments, an antibody blocks binding of both CSF l and IL-34 to CSFIR. In some embodiments, an antibody inhibits ligand-induced CSF IR phosphorylation. In some embodiments, an antibody inhibits CSF l- and/or IL-34-induced CSFIR phosphorylation. In some embodiments, an antibody binds to human CSFIR with an affinity (¾) of less than 1 nM. In some embodiments, antibody inhibits monocyte proliferation and/or survival responses in the presence of CSFl or IL-34.

[032] In some embodiments, a pharmaceutical composition comprising an antibody that binds CSFIR is provided.

[033] In some embodiments, compositions comprising antibodies that bind CSFIR are provided for use in methods of treatment of human or animals. In some embodiments, antibodies that bind CSFIR and compositions comprising antibodies that bind CSFIR are provided for use in a method of treating conditions associated with rheumatoid arthritis in a human or animal. In some embodiments, antibodies that bind CSF IR and compositions comprising antibodies that bind CSF IR are provided for use in a method of treating multiple sclerosis in a human or animal. In some embodiments, antibodies that bind CSFIR and compositions comprising antibodies that bind CSFIR are provided for use in a method of treating conditions associated with systemic lupus erythematosus are provided.

BRIEF DESCRIPTION OF THE FIGURES

[034] FIG. 1A-C shows an alignment of the humanized heavy chain variable regions for each of humanized antibodies huAbl to huAbl6, as discussed in Example 1. Boxed residues are amino acids in the human acceptor sequence that were changed back to the corresponding mouse residue.

[035] FIG. 2A-C shows an alignment of the humanized light chain variable regions for each of humanized antibodies huAbl to huAbl6, as discussed in Example 1 Boxed amino acids are residues in the human acceptor sequence that were changed back to the corresponding mouse residue. [036] FIG. 3 shows reduction in CD 16+ monocytes in cynomolgus monkeys administered huAbl, as described in Example 2.

[037] FIG. 4 shows dose-dependent binding of surrogate antibody cAbl to mouse CSFIR, as described in Example 3.

[038] FIG. 5 shows dose-dependent inhibition of CSF 1- and IL-34-induced proliferation in mNFS60 cells, as described in Example 4.

[039] FIG. 6 shows suppression of clinical disease scores in a mouse model of rheumatoid arthritis following administration of cAbl, as described in Example 5.

[040] FIG. 7 shows suppression of bone loss, indicated by plasma TRAP5b levels, in a mouse model of rheumatoid arthritis following administration of cAbl, as described in Example 6.

[041] FIG. 8 shows suppression of inflammation, pannus formation, cartilage damage, and bone damage in a mouse model of rheumatoid arthritis following

administration of cAbl, as described in Example 7.

[042] FIG. 9 shows suppression of macrophage numbers in paw joints and knee joints in a mouse model of rheumatoid arthritis following administration of cAbl, as described in Example 8.

[043] FIG. 10 shows suppression of autoantibody formation in a mouse model of rheumatoid arthritis following administration of cAbl, as described in Example 9.

[044] FIG. 11 shows suppression of bone loss in a mouse model of established rheumatoid arthritis following administration of cAbl, as described in Example 10.

[045] FIG. 12 shows suppression of pannus formation and bone destruction in a mouse model of established rheumatoid arthritis following administration of cAbl, as described in Example 11.

[046] FIG. 13 shows suppression of glomerulonephritis, interstitial nephritis, and perivascular infiltrates in a mouse model of systemic lupus erythematosus following administration of cAbl, as described in Example 12.

[047] FIG. 14 shows suppression of skin lesions in a mouse model of systemic lupus erythematosus following administration of cAbl, as described in Example 13.

[048] FIG. 15 shows suppression of clinical disease scores in a mouse model of multiple sclerosis following administration of cAbl, as described in Example 14.

DETAILED DESCRIPTION

[049] Methods of treating conditions comprising administering antibodies that bind CSFIR and block CSFl and IL-34 ligand binding are provided. As discussed herein, antibodies that bind CSF1R and block CSF 1 and IL-34 ligand binding are effective for treating rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis. The present inventors found that administering such antibodies to cynomolgus monkeys reduced the number of CD 16+ peripheral blood monocytes in cynomolgus monkeys, but does not affect CD 16- peripheral blood monocyte numbers CD 16+ peripheral blood monocytes are highly inflammatory monocytes. See, e.g., Ziegler-Heitbrock, J. Leukocyte Biol., 2007, 81 : 584-592. Further, administering such antibodies in a mouse model of rheumatoid arthritis suppressed clinical disease scores, including suppression of erythema and swelling;

suppressed bone loss as measured by a reduction in tartrate resistant acid phosphatase 5b (TRAP5b) levels; and suppressed inflammation, cartilage destruction, pannus formation, and bone destruction. Notably, when the antibodies were administered after the appearance of clinical manifestations of rheumatoid arthritis, bone loss (as measured by a reduction in TRAP5b levels), pannus formation, and bone destruction were reduced. Administering such antibodies in a mouse model of rheumatoid arthritis also reduced joint macrophage numbers and autoantibody formation.

[050] Administering antibodies that bind CSF1R and block CSF1 and IL-34 ligand binding in a systemic lupus erythematosus mouse model suppressed glomerulonephritis, interstitial nephritis, and perivascular infiltrates. Further, administering such antibodies in a systemic lupus erythematosus mouse model suppressed skin lesions. Finally, administering the antibodies in a mouse model of multiple sclerosis suppressed clinical disease scores of MS.

[051] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.

Definitions

[052] Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[053] Exemplary techniques used in connection with recombinant DNA, oligonucleotide synthesis, tissue culture and transformation (e.g., electroporation, lipofection), enzymatic reactions, and purification techniques are known in the art. Many such techniques and procedures are described, e.g., in Sambrook et al. Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), among other places. In addition, exemplary techniques for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients are also known in the art.

[054] In this application, the use of "or" means "and/or" unless stated otherwise. In the context of a multiple dependent claim, the use of "or" refers back to more than one preceding independent or dependent claim in the alternative only. Also, terms such as "element" or "component" encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise.

[055] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[056] The terms "nucleic acid molecule" and "polynucleotide" may be used interchangeably, and refer to a polymer of nucleotides. Such polymers of nucleotides may contain natural and/or non-natural nucleotides, and include, but are not limited to, DNA, RNA, and PNA. "Nucleic acid sequence" refers to the linear sequence of nucleotides that comprise the nucleic acid molecule or polynucleotide.

[057] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by the definition. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of the present invention, a "polypeptide" refers to a protein which includes modifications, such as deletions, additions, and substitutions (generally conservative in nature), to the native sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.

[058] The term "CSF1R" refers herein to the full-length CSF 1R, which includes the N-terminal ECD, the transmembrane domain, and the intracellular tyrosine kinase domain, with or without an N-terminal leader sequence. In some embodiments, the CSF1R is a human CSF1R having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. [059] The term "CSFIR extracellular domain" ("CSFIR ECD") as used herein refers to a CSFIR polypeptide that lacks the intracellular and transmembrane domains. CSFIR ECDs include the full-length CSFIR ECD and CSFIR ECD fragments that are capable of binding CSFIR and/or IL-34. The human full-length CSF IR ECD is defined herein as comprising either amino acids 1 to 512 (i.e., including the leader sequence) or amino acids 20 to 512 (i.e., lacking the leader sequence) of SEQ ID NO: 2. In some embodiments, a human CSFIR ECD fragment comprises amino acids 20 to 506 of SEQ ID NO: 2 (see SEQ ID NO:5). In some embodiments, a human CSF IR fragment ends at amino acid 507, 508, 509, 510, or 511. In some embodiments, a cyno CSF 1R ECD comprises the sequence of SEQ ID NO: 7 (with leader sequence) or amino acids 20 to 506 of SEQ ID NO: 7 (without leader sequence).

[060] With reference to anti-CSF IR antibodies the terms "active" or "activity" or "function", and grammatical variants thereof, are used to refer to the ability to inhibit (blocking or antagonist antibodies) or mimic (agonist antibodies) at least one of the foregoing activities. Antibodies and antibody fragments referred to as "functional" are characterized by having such properties.

[061] An "immunological" activity refers only to the ability to induce the production of an antibody against an antigenic epitope possessed by a native or naturally- occurring CSFIR polypeptide.

[062] The term "antibody" as used herein refers to a molecule comprising at least complementarity-determining region (CDR) 1, CDR2, and CDR3 of a heavy chain and at least CDR1, CDR2, and CDR3 of a light chain, wherein the molecule is capable of binding to antigen. The term antibody includes, but is not limited to, fragments that are capable of binding antigen, such as Fv, single-chain Fv (scFv), Fab, Fab', and (Fab')2. The term antibody also includes, but is not limited to, chimeric antibodies, humanized antibodies, and antibodies of various species such as mouse, human, cynomolgus monkey, etc.

[063] In some embodiments, an antibody comprises a heavy chain variable region and a light chain variable region. In some embodiments, an antibody comprises at least one heavy chain comprising a heavy chain variable region and at least a portion of a heavy chain constant region, and at least one light chain comprising a light chain variable region and at least a portion of a light chain constant region. In some embodiments, an antibody comprises two heavy chains, wherein each heavy chain comprises a heavy chain variable region and at least a portion of a heavy chain constant region, and two light chains, wherein each light chain comprises a light chain variable region and at least a portion of a light chain constant region. As used herein, a single-chain Fv (scFv), or any other antibody that comprises, for example, a single polypeptide chain comprising all six CDRs (three heavy chain CDRs and three light chain CDRs) is considered to have a heavy chain and a light chain. In some such embodiments, the heavy chain is the region of the antibody that comprises the three heavy chain CDRs and the light chain in the region of the antibody that comprises the three light chain CDRs.

[064] The term "heavy chain variable region" as used herein refers to a region comprising heavy chain CDR1, framework (FR) 2, CDR2, FR3, and CDR3. In some embodiments, a heavy chain variable region also comprises at least a portion of an FR1 and/or at least a portion of an FR4. In some embodiments, a heavy chain CDR1 corresponds to Kabat residues 26 to 35; a heavy chain CDR2 corresponds to Kabat residues 50 to 65; and a heavy chain CDR3 corresponds to Kabat residues 95 to 102. See, e.g., Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, Md.); and Figure 1. In some embodiments, a heavy chain CDR1 corresponds to Kabat residues 31 to 35; a heavy chain CDR2 corresponds to Kabat residues 50 to 65; and a heavy chain CDR3 corresponds to Kabat residues 95 to 102. See id.

[065] The term "heavy chain constant region" as used herein refers to a region comprising at least three heavy chain constant domains, CHI, CH2, and CH3. Nonlimiting exemplary heavy chain constant regions include γ, δ, and a. Nonlimiting exemplary heavy chain constant regions also include ε and μ. Each heavy constant region corresponds to an antibody isotype. For example, an antibody comprising a γ constant region is an IgG antibody, an antibody comprising a δ constant region is an IgD antibody, and an antibody comprising an a constant region is an IgA antibody. Further, an antibody comprising a μ constant region is an IgM antibody, and an antibody comprising an ε constant region is an IgE antibody. Certain isotypes can be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgGl (comprising a γι constant region), IgG2 (comprising a γ ₂ constant region), IgG3 (comprising a γ ₃ constant region), and IgG4 (comprising a γ ₄ constant region) antibodies; IgA antibodies include, but are not limited to, IgAl (comprising an ai constant region) and IgA2 (comprising an ο¾ constant region) antibodies; and IgM antibodies include, but are not limited to, IgMl and IgM2.

[066] In some embodiments, a heavy chain constant region comprises one or more mutations (or substitutions), additions, or deletions that confer a desired characteristic on the antibody. A nonlimiting exemplary mutation is the S241P mutation in the IgG4 hinge region (between constant domains CHI and CH2), which alters the IgG4 motif CPSCP to CPPCP, which is similar to the corresponding motif in IgGl. That mutation, in some embodiments, results in a more stable IgG4 antibody. See, e.g., Angal et al, Mol. Immunol. 30: 105-108 (1993); Bloom et al, Prot. Sci. 6: 407-415 (1997); Schuurman et al, Mol. Immunol. 38: 1-8 (2001).

[067] The term "heavy chain" as used herein refers to a polypeptide comprising at least a heavy chain variable region, with or without a leader sequence. In some

embodiments, a heavy chain comprises at least a portion of a heavy chain constant region. The term "full-length heavy chain" as used herein refers to a polypeptide comprising a heavy chain variable region and a heavy chain constant region, with or without a leader sequence.

[068] The term "light chain variable region" as used herein refers to a region comprising light chain CDR1, framework (FR) 2, CDR2, FR3, and CDR3. In some embodiments, a light chain variable region also comprises an FR1 and/or an FR4. In some embodiments, a light chain CDR1 corresponds to Kabat residues 24 to 34; a light chain CDR2 corresponds to Kabat residues 50 to 56; and a light chain CDR3 corresponds to Kabat residues 89 to 97. See, e.g., Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, Md.); and Figure 1.

[069] The term "light chain constant region" as used herein refers to a region comprising a light chain constant domain, CL. Nonlimiting exemplary light chain constant regions include λ and κ.

[070] The term "light chain" as used herein refers to a polypeptide comprising at least a light chain variable region, with or without a leader sequence. In some embodiments, a light chain comprises at least a portion of a light chain constant region. The term "full- length light chain" as used herein refers to a polypeptide comprising a light chain variable region and a light chain constant region, with or without a leader sequence.

[071] A "chimeric antibody" as used herein refers to an antibody comprising at least one variable region from a first species (such as mouse, rat, cynomolgus monkey, etc.) and at least one constant region from a second species (such as human, cynomolgus monkey, etc.). In some embodiments, a chimeric antibody comprises at least one mouse variable region and at least one human constant region. In some embodiments, a chimeric antibody comprises at least one cynomolgus variable region and at least one human constant region. In some embodiments, a chimeric antibody comprises at least one rat variable region and at least one mouse constant region. In some embodiments, all of the variable regions of a chimeric antibody are from a first species and all of the constant regions of the chimeric antibody are from a second species.

[072] A "humanized antibody" as used herein refers to an antibody in which at least one amino acid in a framework region of a non-human variable region has been replaced with the corresponding amino acid from a human variable region. In some embodiments, a humanized antibody comprises at least one human constant region or fragment thereof. In some embodiments, a humanized antibody is an Fab, an scFv, a (Fab') ₂ , etc.

[073] A "CDR-grafted antibody" as used herein refers to a humanized antibody in which the complementarity determining regions (CDRs) of a first (non-human) species have been grafted onto the framework regions (FRs) of a second (human) species.

[074] A "human antibody" as used herein refers to antibodies produced in humans, antibodies produced in non-human animals that comprise human immunoglobulin genes, such as XenoMouse®, and antibodies selected using in vitro methods, such as phage display, wherein the antibody repertoire is based on a human immunoglobulin sequences.

[075] The term "leader sequence" refers to a sequence of amino acid residues located at the N terminus of a polypeptide that facilitates secretion of a polypeptide from a mammalian cell. A leader sequence may be cleaved upon export of the polypeptide from the mammalian cell, forming a mature protein. Leader sequences may be natural or synthetic, and they may be heterologous or homologous to the protein to which they are attached. Exemplary leader sequences include, but are not limited to, antibody leader sequences, such as, for example, the amino acid sequences of SEQ ID NOs: 3 and 4, which correspond to human light and heavy chain leader sequences, respectively. Nonlimiting exemplary leader sequences also include leader sequences from heterologous proteins. In some embodiments, an antibody lacks a leader sequence. In some embodiments, an antibody comprises at least one leader sequence, which may be selected from native antibody leader sequences and heterologous leader sequences.

[076] The term "vector" is used to describe a polynucleotide that may be engineered to contain a cloned polynucleotide or polynucleotides that may be propagated in a host cell. A vector may include one or more of the following elements: an origin of replication, one or more regulatory sequences (such as, for example, promoters and/or enhancers) that regulate the expression of the polypeptide of interest, and/or one or more selectable marker genes (such as, for example, antibiotic resistance genes and genes that may be used in colorimetric assays, e.g., β-galactosidase). The term "expression vector" refers to a vector that is used to express a polypeptide of interest in a host cell.

[077] A "host cell" refers to a cell that may be or has been a recipient of a vector or isolated polynucleotide. Host cells may be prokaryotic cells or eukaryotic cells. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate animal cells; fungal cells, such as yeast; plant cells; and insect cells. Nonlimiting exemplary mammalian cells include, but are not limited to, NSO cells, PER.C6® cells (Crucell), and 293 and CHO cells, and their derivatives, such as 293-6E and DG44 cells, respectively.

[078] The term "isolated" as used herein refers to a molecule that has been separated from at least some of the components with which it is typically found in nature. For example, a polypeptide is referred to as "isolated" when it is separated from at least some of the components of the cell in which it was produced. Where a polypeptide is secreted by a cell after expression, physically separating the supernatant containing the polypeptide from the cell that produced it is considered to be "isolating" the polypeptide. Similarly, a polynucleotide is referred to as "isolated" when it is not part of the larger polynucleotide (such as, for example, genomic DNA or mitochondrial DNA, in the case of a DNA polynucleotide) in which it is typically found in nature, or is separated from at least some of the components of the cell in which it was produced, e.g., in the case of an RNA polynucleotide. Thus, a DNA polynucleotide that is contained in a vector inside a host cell may be referred to as "isolated" so long as that polynucleotide is not found in that vector in nature.

[079] The terms "subject" and "patient" are used interchangeably herein to refer to a human. In some embodiments, methods of treating other mammals, including, but not limited to, rodents, simians, felines, canines, equines, bovines, porcines, ovines, caprines, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and mammalian pets, are also provided.

[080] As used herein, "rheumatoid arthritis" or "RA" refers to a recognized disease state that may be diagnosed according to the 2000 revised American Rheumatoid Association criteria for the classification of RA, or any similar criteria. In some

embodiments, the term "rheumatoid arthritis" refers to a chronic autoimmune disease characterized primarily by inflammation of the lining (synovium) of the joints, which can lead to joint damage, resulting in chronic pain, loss of function, and disability. Because RA can affect multiple organs of the body, including skin, lungs, and eyes, it is referred to as a systemic illness. [081] The term "rheumatoid arthritis" includes not only active and early RA, but also incipient RA, as defined below. Physiological indicators of RA include, symmetric joint swelling which is characteristic though not invariable in RA. Fusiform swelling of the proximal interphalangeal (PIP) joints of the hands as well as metacarpophalangeal (MCP), wrists, elbows, knees, ankles, and metatarsophalangeal (MTP) joints are commonly affected and swelling is easily detected. Pain on passive motion is the most sensitive test for joint inflammation, and inflammation and structural deformity often limits the range of motion for the affected joint. Typical visible changes include ulnar deviation of the fingers at the MCP joints, hyperextension, or hyperflexion of the MCP and PIP joints, flexion

contractures of the elbows, and subluxation of the carpal bones and toes. The subject with RA may be resistant to a disease-modifying anti-rheumatic drug (DMARD), and/or a nonsteroidal anti-inflammatory drug (NSAID). Nonlimiting exemplary "DMARDs" include hydroxycloroquine, sulfasalazine, methotrexate (MTX), leflunomide, etanercept, infliximab (plus oral and subcutaneous MTX), azathioprine, D-penicillamine, gold salts (oral), gold salts (intramuscular), minocycline, cyclosporine including cyclosporine A and topical cyclosporine, staphylococcal protein A (Goodyear and Silverman, J. Exp. Med.,

197(9): 1 125-1139 (2003)), including salts and derivatives thereof, etc. Further candidates for therapy according to this invention include those who have experienced an inadequate response to previous or current treatment with TNF inhibitors such as etanercept, infliximab and/or adalimumab because of toxicity or inadequate efficacy.

[082] A patient with "active rheumatoid arthritis" means a patient with active and not latent symptoms of RA. Subjects with "early active rheumatoid arthritis" are those subjects with active RA diagnosed for at least 8 weeks but no longer than four years, according to the revised 1987 ACR criteria for the classification of RA.

[083] Subjects with "early rheumatoid arthritis" are those subjects with RA diagnosed for at least eight weeks but no longer than four years, according to the revised 1987 ACR criteria for classification of RA. RA includes, for example, juvenile-onset RA, juvenile idiopathic arthritis (JIA), or juvenile RA (JRA).

[084] Patients with "incipient RA" have early polyarthritis that does not fully meet ACR criteria for a diagnosis of RA, in association with the presence of RA-specific prognostic biomarkers such as anti-CCP and shared epitope. They include patients with positive anti-CCP antibodies who present with polyarthritis, but do not yet have a diagnosis of RA, and are at high risk for going on to develop bona fide ACR criteria RA (95% probability). [085] "Joint damage" is used in the broadest sense and refers to damage or partial or complete destruction to any part of one or more joints, including the connective tissue and cartilage, where damage includes structural and/or functional damage of any cause, and may or may not cause joint pain/arthalgia. It includes, without limitation, joint damage associated with or resulting from inflammatory joint disease as well as non-inflammatory joint disease. This damage may be caused by any condition, such as an autoimmune disease, especially arthritis, and most especially RA. Exemplary such conditions include acute and chronic arthritis, rheumatoid arthritis (including juvenile-onset RA, juvenile idiopathic arthritis (JIA), and juvenile rheumatoid arthritis (JRA)), and stages such as rheumatoid synovitis, gout or gouty arthritis, acute immunological arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, septic arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, vertebral arthritis, osteoarthritis, arthritis chronica progrediente, arthritis deformans, polyarthritis chronica primaria, reactive arthritis, menopausal arthritis, estrogen-depletion arthritis, and ankylosing spondylitis/rheumatoid spondylitis), rheumatic autoimmune disease other than RA, and significant systemic involvement secondary to RA (including but not limited to vasculitis, pulmonary fibrosis or Felty's syndrome). For purposes herein, joints are points of contact between elements of a skeleton (of a vertebrate such as an animal) with the parts that surround and support it and include, but are not limited to, for example, hips, joints between the vertebrae of the spine, joints between the spine and pelvis (sacroiliac joints), joints where the tendons and ligaments attach to bones, joints between the ribs and spine, shoulders, knees, feet, elbows, hands, fingers, ankles and toes, but especially joints in the hands and feet.

[086] The term "lupus" as used herein is an autoimmune disease or disorder that in general involves antibodies that attack connective tissue. The principal form of lupus is a systemic one, systemic lupus erythematosus (SLE), including cutaneous SLE and subacutecutaneous SLE, as well as other types of lupus (including nephritis, extrarenal, cerebritis, pediatric, non-renal, discoid, and alopecia). In certain embodiments, the term "systemic lupus erythematosus" refers to a chronic autoimmune disease that can result in skin lesions, joint pain and swelling, kidney disease (lupus nephritis), fluid around the heart and/or lungs, inflammation of the heart, and various other systemic conditions. In certain embodiments, the term "lupus nephritis" refers to inflammation of the kidneys that occurs in patients with SLE. Lupus nephritis may include, for example, glomerulonephritis and/or interstitial nephritis, and can lead to hypertension, proteinuria, and kidney failure. Lupus nephritis may be classified based on severity and extent of disease, for example, as defined by the International Society of Nephrology/Renal/Pathology Society. Lupus nephritis classes include class I (minimal mesangial lupus nephritis), class II (mesangial proliferative lupus nephritis), class III (focal lupus nephritis), class IV (diffuse segmental (IV-S) or diffuse global (IV-G) lupus nephritis), class V (membranous lupus nephritis), and class VI (advanced sclerosing lupus nephritis). The term "lupus nephritis" encompasses all of the classes.

[087] The term "multiple sclerosis" ("MS") refers to the chronic and often disabling disease of the central nervous system characterized by the progressive destruction of the myelin. "Dem elination" occurs when the myelin sheath becomes inflamed, injured, and detaches from the nerve fiber. There are four internationally recognized forms of MS, namely, primary progressive multiple sclerosis (PPMS), relapsing-remitting multiple sclerosis (RRMS), secondary progressive multiple sclerosis (SPMS), and progressive relapsing multiple sclerosis (PRMS).

[088] "Primary progressive multiple sclerosis" or "PPMS" is characterized by a gradual progression of the disease from its onset with no superimposed relapses and remissions at all. There may be periods of a leveling off of disease activity and there may be good and bad days or weeks. PPMS differs from RRMS and SPMS in that onset is typically in the late thirties or early forties, men are as likely as women to develop it, and initial disease activity is often in the spinal cord and not in the brain. PPMS often migrates into the brain, but is less likely to damage brain areas than RRMS or SPMS; for example, people with PPMS are less likely to develop cognitive problems. PPMS is the sub-type of MS that is least likely to show inflammatory (gadolinium enhancing) lesions on MRI scans. The primary progressive form of the disease affects between 10 and 15% of all people with multiple sclerosis. PPMS may be defined according to the criteria in McDonald et al. Ann Neurol 50: 121-7 (2001). The subject with PPMS treated herein is usually one with a probable or definitive diagnosis of PPMS.

[089] "Relapsing-remitting multiple sclerosis" or "RRMS" is characterized by relapses (also known as exacerbations) during which time new symptoms can appear and old ones resurface or worsen. The relapses are followed by periods of remission, during which time the person fully or partially recovers from the deficits acquired during the relapse. Relapses can last for days, weeks, or months, and recovery can be slow and gradual or almost instantaneous. The vast majority of people presenting with MS are first diagnosed with RRMS. This is typically when they are in their twenties or thirties, though diagnoses much earlier or later are known. Twice as many women as men present with this sub-type of MS. During relapses, myelin, a protective insulating sheath around the nerve fibers (neurons) in the white matter regions of the central nervous system (CNS), may be damaged in an inflammatory response by the body's own immune system. This causes a wide variety of neurological symptoms that vary considerably depending on which areas of the CNS are damaged. Immediately after a relapse, the inflammatory response dies down and a special type of glial cell in the CNS (called an oligodendrocyte) sponsors remyelination - a process whereby the myelin sheath around the axon may be repaired. It is this remyelination that may be responsible for the remission. Approximately 50% of patients with RRMS convert to SPMS within 10 years of disease onset. After 30 years, this figure rises to 90%. At any one time, the relapsing-remitting form of the disease accounts around 55% of all people with MS.

[090] "Secondary progressive multiple sclerosis" or "SPMS" is characterized by a steady progression of clinical neurological damage with or without superimposed relapses and minor remissions and plateau. People who develop SPMS will have previously experienced a period of RRMS which may have lasted anywhere from two to forty years or more. Any superimposed relapses and remissions tend to tail off over time. From the onset of the secondary progressive phase of the disease, disability starts advancing much quicker than it did during RRMS though the progress can still be quite slow in some individuals. SPMS tends to be associated with lower levels of inflammatory lesion formation than in RRMS but the total burden of disease continues to progress. At any one time, SPMS accounts around 30% of all people with multiple sclerosis.

[091 ] "Progressive relapsing multiple sclerosis" or "PRMS" is characterized by a steady progression of clinical neurological damage with superimposed relapses and remissions. There is significant recovery immediately following a relapse but between relapses there is a gradual worsening of symptoms. PRMS affects around 5% of all people with multiple sclerosis. Some neurologists believe PRMS is a variant of PPMS.

[092] The term "CD16+ disorder" means a disease in which CD 16+ monocytes of a mammal cause, mediate or otherwise contribute to a morbidity in the mammal. Also included are diseases in which reduction of CD 16+ monocytes has an ameliorative effect on progression of the disease. Included within this term are CD 16+ inflammatory diseases, infectious diseases, immunodeficiency diseases, neoplasia, etc. In certain embodiments, CD 16+ inflammatory diseases include inflammatory diseases that are not responsive to methotrexate therapy. In certain embodiments, CD 16+ inflammatory diseases include methotrexate-resistant rheumatoid arthritis, methotrexate-resistant multiple sclerosis, methotrexate-resistant lupus, methotrexate-resistant inflammatory bowel disease, methotrexate-resistant Crohn's disease, methotrexate-resistant asthma, and methotrexate- resistant psoriasis. In certain embodiments, patients having methotrexate-resistant diseases, such as methotrexate-resistant rheumatoid arthritis, are referred to as methotrexate incomplete responders or methotrexate inadequate responders.

[093] Examples of CD 16+ disorders that can be treated according to the invention include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, juvenile chronic arthritis, spondyloarthropathies, systemic sclerosis (scleroderma), idiopathic inflammatory myopathies (dermatomyositis, polymyositis), Sjogren's syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia (immune pancytopenia, paroxysmal nocturnal hemoglobinuria), autoimmune thrombocytopenia (idiopathic thrombocytopenic purpura, immune-mediated thrombocytopenia), thyroiditis (Grave's disease, Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis), diabetes mellitus, immune-mediated renal disease (glomerulonephritis, tubulointerstitial nephritis), demyelinating diseases of the central and peripheral nervous systems such as multiple sclerosis, idiopathic demyelinating polyneuropathy or Guillain-Barre syndrome, and chronic inflammatory demyelinating polyneuropathy, hepatobiliary diseases such as infectious hepatitis (hepatitis A, B, C, D, E and other non-hepatotropic viruses), autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis, inflammatory bowel disease (IBD), including ulcerative colitis: Crohn's disease, gluten-sensitive enteropathy, and Whipple's disease, autoimmune or immune-mediated skin diseases including bullous skin diseases, erythema multiforme and contact dermatitis, psoriasis, allergic diseases such as asthma, allergic rhinitis, atopic dermatitis, food hypersensitivity and urticaria, immunologic diseases of the lung such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and hypersensitivity pneumonitis, transplantation associated diseases including graft rejection and graft-versus-host-disease; fibrosis, including kidney fibrosis and hepatic fibrosis, cardiovascular disease, including

atherosclerosis and coronary artery disease, cardiovascular events associated with chronic kidney disease, myocardial infarction, and congestive heart failure, diabetes, including type II diabetes, Bronchiolitis obliterans with organizing pneumonia (BOOP), hemophagocytic syndrome, macrophage activation syndrome, sarcoidosis, and periodontitis. Infectious diseases including viral diseases such as AIDS (HIV infection), hepatitis A, B, C, D, and E, herpes, etc., bacterial infections, fungal infections, protozoal infections and parasitic infections.

[094] "Treatment," as used herein, refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. In certain embodiments, the term "treatment" covers any administration or application of a therapeutic for disease in a mammal, including a human, and includes inhibiting or slowing the disease or progression of the disease; partially or fully relieving the disease, for example, by causing regression, or restoring or repairing a lost, missing, or defective function; stimulating an inefficient process; or causing the disease plateau to have reduced severity. The term "treatment" also includes reducing the severity of any phenotypic characteristic and/or reducing the incidence, degree, or likelihood of that characteristic. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented.

[095] "Chronic" administration refers to administration of an agent in a continuous mode as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time. "Intermittent" administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature.

[096] The term "effective amount" or "therapeutically effective amount" refers to an amount of a drug effective to treat a disease or disorder in a subject. In certain embodiments, an effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A

therapeutically effective amount of an anti-CSFIR antibody of the invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the anti-CSF IR antibody to elicit a desired response in the individual. A therapeutically effective amount encompasses an amount in which any toxic or detrimental effects of the anti-CSF IR antibody are outweighed by the therapeutically beneficial effects.

[097] A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result.

Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount would be less than the therapeutically effective amount.

[098] Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order. [099] A "pharmaceutically acceptable carrier" refers to a non-toxic solid, semisolid, or liquid filler, diluent, encapsulating material, formulation auxiliary, or carrier conventional in the art for use with a therapeutic agent that together comprise a

"pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. The pharmaceutically acceptable carrier is appropriate for the formulation employed. For example, if the therapeutic agent is to be administered orally, the carrier may be a gel capsule. If the therapeutic agent is to be administered subcutaneously, the carrier ideally is not irritable to the skin and does not cause injection site reaction.

Anti-CSFIR Antibodies

[0100] Anti-CSFIR antibodies include, but are not limited to, humanized antibodies, chimeric antibodies, mouse antibodies, human antibodies, and antibodies comprising the heavy chain and/or light chain CDRs discussed herein.

Exemplary Humanized Antibodies

[0101] In some embodiments, humanized antibodies that bind CSF 1R are provided. Humanized antibodies are useful as therapeutic molecules because humanized antibodies reduce or eliminate the human immune response to non-human antibodies (such as the human anti-mouse antibody (HAMA) response), which can result in an immune response to an antibody therapeutic, and decreased effectiveness of the therapeutic.

[0102] Nonlimiting exemplary humanized antibodies include huAbl through huAbl6, described herein. Nonlimiting exemplary humanized antibodies also include antibodies comprising a heavy chain variable region of an antibody selected from huAbl to huAbl6 and/or a light chain variable region of an antibody selected from huAbl to huAbl6. Nonlimiting exemplary humanized antibodies include antibodies comprising a heavy chain variable region selected from SEQ ID NOs: 39 to 45 and/or a light chain variable region selected from SEQ ID NOs: 46 to 52. Exemplary humanized antibodies also include, but are not limited to, humanized antibodies comprising heavy chain CDR1, CDR2, and CDR3, and/or light chain CDR1, CDR2, and CDR3 of an antibody selected from 0301, 0302, and 0311.

[0103] In some embodiments, a humanized anti-CSFIR antibody comprises heavy chain CDR1, CDR2, and CDR3 and/or a light chain CDR1, CDR2, and CDR3 of an antibody selected from 0301, 0302, and 0311. Nonlimiting exemplary humanized anti- CSFIR antibodies include antibodies comprising sets of heavy chain CDR1, CDR2, and CDR3 selected from: SEQ ID NOs: 15, 16, and 17; SEQ ID NOs: 21, 22, and 23; and SEQ ID NOs: 27, 28, and 29. Nonlimiting exemplary humanized anti-CSFIR antibodies also include antibodies comprising sets of light chain CDRl, CDR2, and CDR3 selected from: SEQ ID NOs: 18, 19, and 20; SEQ ID NOs: 24, 25, and 26; and SEQ ID NOs: 30, 31, and 32.

[0104] Nonlimiting exemplary humanized anti-CSFIR antibodies include antibodies comprising the sets of heavy chain CDRl, CDR2, and CDR3, and light chain CDRl, CDR2, and CDR3 in Table 1 (SEQ ID NOs shown; see Table 8 for sequences). Each row of Table 1 shows the heavy chain CDRl, CDR2, and CDR3, and light chain CDRl, CDR2, and CDR3 of an exemplary antibody.

Table 1 : Heavy chain and light chain CDRs

Further exemplary humanized antibodies

[0105] In some embodiments, a humanized anti-CSFIR antibody comprises a heavy chain comprising a variable region sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 9, 1 1, 13, and 39 to 45, and wherein the antibody binds CSF1R. In some embodiments, a humanized anti-CSFIR antibody comprises a light chain comprising a variable region sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 10, 12, 14, and 46 to 52, wherein the antibody binds CSF 1R. In some embodiments, a humanized anti-CSF IR antibody comprises a heavy chain comprising a variable region sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 9, 1 1, 13, and 39 to 45; and a light chain comprising a variable region sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 10, 12, 14, and 46 to 52; wherein the antibody binds CSF 1R.

[0106] As used herein, whether a particular polypeptide is, for example, at least 95% identical to an amino acid sequence can be determined using, e.g., a computer program. When determining whether a particular sequence is, for example, 95% identical to a reference sequence, the percentage of identity is calculated over the full length of the reference amino acid sequence.

[0107] In some embodiments, a humanized anti-CSFIR antibody comprises at least one of the CDRs discussed herein. That is, in some embodiments, a humanized anti-CSFIR antibody comprises at least one CDR selected from a heavy chain CDRl discussed herein, a heavy chain CDR2 discussed herein, a heavy chain CDR3 discussed herein, a light chain CDRl discussed herein, a light chain CDR2 discussed herein, and a light chain CDR3 discussed herein. Further, in some embodiments, a humanized anti-CSFIR antibody comprises at least one mutated CDR based on a CDR discussed herein, wherein the mutated CDR comprises 1, 2, 3, or 4 amino acid substitutions relative to the CDR discussed herein. In some embodiments, one or more of the amino acid substitutions are conservative amino acid substitutions. One skilled in the art can select one or more suitable conservative amino acid substitutions for a particular CDR sequence, wherein the suitable conservative amino acid substitutions are not predicted to significantly alter the binding properties of the antibody comprising the mutated CDR.

[0108] Exemplary humanized anti-CSF IR antibodies also include antibodies that compete for binding to CSF1R with an antibody described herein. Thus, in some embodiments, a humanized anti-CSF IR antibody is provided that competes for binding to CSF1R with an antibody selected from Fabs 0301, 0302, and 0311 ; and bivalent (i.e., having two heavy chains and two light chains) antibody versions of those Fabs.

Exemplary humanized antibody constant regions

[0109] In some embodiments, a humanized antibody described herein comprises one or more human constant regions. In some embodiments, the human heavy chain constant region is of an isotype selected from IgA, IgG, and IgD. In some embodiments, the human light chain constant region is of an isotype selected from κ and λ. In some embodiments, a humanized antibody described herein comprises a human IgG constant region. In some embodiments, a humanized antibody described herein comprises a human IgG4 heavy chain constant region. In some such embodiments, a humanized antibody described herein comprises an S241P mutation in the human IgG4 constant region. In some embodiments, a humanized antibody described herein comprises a human IgG4 constant region and a human K light chain.

[0110] The choice of heavy chain constant region can determine whether or not an antibody will have effector function in vivo. Such effector function, in some embodiments, includes antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement- dependent cytotoxicity (CDC), and can result in killing of the cell to which the antibody is bound. In some methods of treatment, including methods of treating some cancers, cell killing may be desirable, for example, when the antibody binds to a cell that supports the maintenance or growth of the tumor. Exemplary cells that may support the maintenance or growth of a tumor include, but are not limited to, tumor cells themselves, cells that aid in the recruitment of vasculature to the tumor, and cells that provide ligands, growth factors, or counter-receptors that support or promote tumor growth or tumor survival. In some embodiments, when effector function is desirable, an anti-CSFIR antibody comprising a human IgGl heavy chain or a human IgG3 heavy chain is selected.

[011 1] In some methods of treatment, effector function may not be desirable. For example, in some embodiments, it may be desirable that antibodies used in the treatment of lupus and/or MS and/or RA and/or osteolysis do not have effector function. Thus, in some embodiments, anti-CSFIR antibodies developed for the treatment of cancer may not be suitable for use in treatment of lupus and/or MS and/or RA and/or osteolysis. Accordingly, in some embodiments, an anti-CSFIR antibody that lacks significant effector function is used in treatment of lupus and/or MS and/or RA and/or osteolysis. In some embodiments, an anti-CSFIR antibody for treatment of lupus and/or MS and/or RA and/or osteolysis comprises a human IgG4 or IgG2 heavy chain constant region. In some embodiments, an IgG4 constant region comprises an S241P mutation.

[0112] An antibody may be humanized by any method. Nonlimiting exemplary methods of humanization include methods described, e.g., in U.S. Patent Nos. 5,530, 101; 5,585,089; 5,693,761 ; 5,693,762; 6,180,370; Jones et al, Nature 321 : 522-525 (1986); Riechmann et al, Nature 332: 323-27 (1988); Verhoeyen et al, Science 239: 1534-36 (1988); and U.S. Publication No. US 2009/0136500.

[0113] As noted above, a humanized antibody is an antibody in which at least one amino acid in a framework region of a non-human variable region has been replaced with the amino acid from the corresponding location in a human framework region. In some embodiments, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10, at least 11, at least 12, at least 15, or at least 20 amino acids in the framework regions of a non-human variable region are replaced with an amino acid from one or more corresponding locations in one or more human framework regions.

[0114] In some embodiments, some of the corresponding human amino acids used for substitution are from the framework regions of different human immunoglobulin genes. That is, in some such embodiments, one or more of the non-human amino acids may be replaced with corresponding amino acids from a human framework region of a first human antibody or encoded by a first human immunoglobulin gene, one or more of the non-human amino acids may be replaced with corresponding amino acids from a human framework region of a second human antibody or encoded by a second human immunoglobulin gene, one or more of the non-human amino acids may be replaced with corresponding amino acids from a human framework region of a third human antibody or encoded by a third human immunoglobulin gene, etc. Further, in some embodiments, all of the corresponding human amino acids being used for substitution in a single framework region, for example, FR2, need not be from the same human framework. In some embodiments, however, all of the corresponding human amino acids being used for substitution are from the same human antibody or encoded by the same human immunoglobulin gene.

[0115] In some embodiments, an antibody is humanized by replacing one or more entire framework regions with corresponding human framework regions. In some embodiments, a human framework region is selected that has the highest level of homology to the non-human framework region being replaced. In some embodiments, such a humanized antibody is a CDR-grafted antibody.

[0116] In some embodiments, following CDR-grafting, one or more framework amino acids are changed back to the corresponding amino acid in a mouse framework region. Such "back mutations" are made, in some embodiments, to retain one or more mouse framework amino acids that appear to contribute to the structure of one or more of the CDRs and/or that may be involved in antigen contacts and/or appear to be involved in the overall structural integrity of the antibody. In some embodiments, ten or fewer, nine or fewer, eight or fewer, seven or fewer, six or fewer, five or fewer, four or fewer, three or fewer, two or fewer, one, or zero back mutations are made to the framework regions of an antibody following CDR grafting.

[0117] In some embodiments, a humanized antibody also comprises a human heavy chain constant region and/or a human light chain constant region. Exemplary Chimeric Antibodies

[0118] In some embodiments, an anti-CSF IR antibody is a chimeric antibody. In some embodiments, an anti-CSFIR antibody comprises at least one non-human variable region and at least one human constant region. In some such embodiments, all of the variable regions of an anti-CSFIR antibody are non-human variable regions, and all of the constant regions of an anti-CSFIR antibody are human constant regions. In some embodiments, one or more variable regions of a chimeric antibody are mouse variable regions. The human constant region of a chimeric antibody need not be of the same isotype as the non-human constant region, if any, it replaces. Chimeric antibodies are discussed, e.g., in U.S. Patent No. 4,816,567; and Morrison et al. Proc. Natl Acad. Sci. USA 81 : 6851- 55 (1984).

[0119] Nonlimiting exemplary chimeric antibodies include chimeric antibodies comprising the heavy and/or light chain variable regions of an antibody selected from 0301, 0302, and 031 1. Additional nonlimiting exemplary chimeric antibodies include chimeric antibodies comprising heavy chain CDR1, CDR2, and CDR3, and/or light chain CDR1, CDR2, and CDR3 of an antibody selected from 0301, 0302, and 031 1.

[0120] Nonlimiting exemplary chimeric anti-CSFIR antibodies include antibodies comprising the following pairs of heavy and light chain variable regions: SEQ ID NOs: 9 and 10; SEQ ID NOs: 1 1 and 12; and SEQ ID NOs: 13 and 14.

[0121] Nonlimiting exemplary anti-CSFIR antibodies include antibodies comprising a set of heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3 shown above in Table 1.

Further exemplary chimeric antibodies

[0122] In some embodiments, a chimeric anti-CSFIR antibody comprises a heavy chain comprising a variable region sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 9, 1 1, 13, and 39 to 45, wherein the antibody binds CSFIR. In some embodiments, a chimeric anti-CSFIR antibody comprises a light chain comprising a variable region sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 10, 12, 14, and 46 to 52, wherein the antibody binds CSFIR. In some embodiments, a chimeric anti-CSFIR antibody comprises a heavy chain comprising a variable region sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 9, 1 1, 13, and 39 to 45; and a light chain comprising a variable region sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 10, 12, 14, and 46 to 52; wherein the antibody binds CSF 1R.

[0123] In some embodiments, a chimeric anti-CSFIR antibody comprises at least one of the CDRs discussed herein. That is, in some embodiments, a chimeric anti-CSFIR antibody comprises at least one CDR selected from a heavy chain CDR1 discussed herein, a heavy chain CDR2 discussed herein, a heavy chain CDR3 discussed herein, a light chain CDR1 discussed herein, a light chain CDR2 discussed herein, and a light chain CDR3 discussed herein. Further, in some embodiments, a chimeric anti-CSF IR antibody comprises at least one mutated CDR based on a CDR discussed herein, wherein the mutated CDR comprises 1, 2, 3, or 4 amino acid substitutions relative to the CDR discussed herein. In some embodiments, one or more of the amino acid substitutions are conservative amino acid substitutions. One skilled in the art can select one or more suitable conservative amino acid substitutions for a particular CDR sequence, wherein the suitable conservative amino acid substitutions are not predicted to significantly alter the binding properties of the antibody comprising the mutated CDR.

[0124] Exemplary chimeric anti-CSFIR antibodies also include chimeric antibodies that compete for binding to CSF1R with an antibody described herein. Thus, in some embodiments, a chimeric anti-CSFIR antibody is provided that competes for binding to CSF1R with an antibody selected from Fabs 0301, 0302, and 031 1; and bivalent (i.e., having two heavy chains and two light chains) antibody versions of those Fabs.

Exemplary chimeric antibody constant regions

[0125] In some embodiments, a chimeric antibody described herein comprises one or more human constant regions. In some embodiments, the human heavy chain constant region is of an isotype selected from IgA, IgG, and IgD. In some embodiments, the human light chain constant region is of an isotype selected from κ and λ. In some embodiments, a chimeric antibody described herein comprises a human IgG constant region. In some embodiments, a chimeric antibody described herein comprises a human IgG4 heavy chain constant region. In some such embodiments, a chimeric antibody described herein comprises an S241P mutation in the human IgG4 constant region. In some embodiments, a chimeric antibody described herein comprises a human IgG4 constant region and a human κ light chain. [0126] As noted above, whether or not effector function is desirable may depend on the particular method of treatment intended for an antibody. Thus, in some embodiments, when effector function is desirable, a chimeric anti-CSFIR antibody comprising a human IgGl heavy chain constant region or a human IgG3 heavy chain constant region is selected. In some embodiments, when effector function is not desirable, a chimeric anti-CSFIR antibody comprising a human IgG4 or IgG2 heavy chain constant region is selected.

Exemplary Human Antibodies

[0127] Human antibodies can be made by any suitable method. Nonlimiting exemplary methods include making human antibodies in transgenic mice that comprise human immunoglobulin loci. See, e.g., Jakobovits et al, Proc. Natl. Acad. Sci. USA 90: 2551-55 (1993); Jakobovits et al, Nature 362: 255-8 (1993); Lonberg et al., Nature 368: 856-9 (1994); and U.S. Patent Nos. 5,545,807; 6,713,610; 6,673,986; 6, 162,963; 5,545,807; 6,300, 129; 6,255,458; 5,877,397; 5,874,299; and 5,545,806.

[0128] Nonlimiting exemplary methods also include making human antibodies using phage display libraries. See, e.g., Hoogenboom et al, J. Mol. Biol. 227: 381-8 (1992); Marks et al, J. Mol. Biol. 222: 581-97 (1991); and PCT Publication No. WO 99/10494.

[0129] In some embodiments, a human anti-CSF IR antibody binds to a polypeptide having the sequence of SEQ ID NO: 1. Exemplary human anti-CSF IR antibodies also include antibodies that compete for binding to CSFIR with an antibody described herein. Thus, in some embodiments, a human anti-CSF IR antibody is provided that competes for binding to CSFIR with an antibody selected from Fabs 0301, 0302, and 0311, and bivalent (i.e., having two heavy chains and two light chains) antibody versions of those Fabs.

[0130] In some embodiments, a human anti-CSFIR antibody comprises one or more human constant regions. In some embodiments, the human heavy chain constant region is of an isotype selected from IgA, IgG, and IgD. In some embodiments, the human light chain constant region is of an isotype selected from κ and λ. In some embodiments, a human antibody described herein comprises a human IgG constant region. In some embodiments, a human antibody described herein comprises a human IgG4 heavy chain constant region. In some such embodiments, a human antibody described herein comprises an S241P mutation in the human IgG4 constant region. In some embodiments, a human antibody described herein comprises a human IgG4 constant region and a human κ light chain.

[0131] In some embodiments, when effector function is desirable, a human anti- CSFIR antibody comprising a human IgGl heavy chain constant region or a human IgG3 heavy chain constant region is selected. In some embodiments, when effector function is not desirable, a human anti-CSFlR antibody comprising a human IgG4 or IgG2 heavy chain constant region is selected.

Additional Exemplary Anti-CSFlR Antibodies

[0132] Exemplary anti-CSF lR antibodies also include, but are not limited to, mouse, humanized, human, chimeric, and engineered antibodies that comprise, for example, one or more of the CDR sequences described herein. In some embodiments, an anti-CSFlR antibody comprises a heavy chain variable region described herein. In some embodiments, an anti-CSFlR antibody comprises a light chain variable region described herein. In some embodiments, an anti-CSF lR antibody comprises a heavy chain variable region described herein and a light chain variable region described herein. In some embodiments, an anti- CSF1R antibody comprises heavy chain CDR1, CDR2, and CDR3 described herein. In some embodiments, an anti-CSF lR antibody comprises light chain CDR1, CDR2, and CDR3 described herein. In some embodiments, an anti-CSFlR antibody comprises heavy chain CDR1, CDR2, and CDR3 described herein and light chain CDR1, CDR2, and CDR3 described herein.

[0133] In some embodiments, an anti-CSFlR antibody comprises a heavy chain variable region of an antibody selected from Fabs 0301, 0302, and 0311. Nonlimiting exemplary anti-CSF lR antibodies also include antibodies comprising a heavy chain variable region of an antibody selected from humanized antibodies huAbl to huAbl6. Nonlimiting exemplary anti-CSF lR antibodies include antibodies comprising a heavy chain variable region comprising a sequence selected from SEQ ID NOs: 9, 11, 13, and 39 to 45.

[0134] In some embodiments, an anti-CSF lR antibody comprises a light chain variable region of an antibody selected from Fabs 0301, 0302, and 31 1. Nonlimiting exemplary anti-CSFlR antibodies also include antibodies comprising a light chain variable region of an antibody selected from humanized antibodies huAbl to huAbl6. Nonlimiting exemplary anti-CSF lR antibodies include antibodies comprising a light chain variable region comprising a sequence selected from SEQ ID NOs: 10, 12, 14, and 46 to 52.

[0135] In some embodiments, an anti-CSFlR antibody comprises a heavy chain variable region and a light chain variable region of an antibody selected from Fabs 0301, 0302, and 031 1. Nonlimiting exemplary anti-CSFlR antibodies also include antibodies comprising a heavy chain variable region and a light chain variable region of an antibody selected from humanized antibodies huAbl to huAbl6. Nonlimiting exemplary anti-CSFlR antibodies include antibodies comprising the following pairs of heavy and light chain variable regions: SEQ ID NOs: 9 and 10; SEQ ID NOs: 11 and 12; and SEQ ID NOs: 13 and 14; SEQ ID NOs: 39 and 40; SEQ ID NOs: 41 and 42; SEQ ID NOs: 43 and 44; SEQ ID NOs: 45 and 46; SEQ ID NOs: 47 and 48; SEQ ID NOs: 49 and 50; and SEQ ID NOs: 51 and 52. Nonlimiting exemplary anti-CSF IR antibodies also include antibodies comprising the following pairs of heavy and light chains: SEQ ID NOs: 33 and 34; SEQ ID NOs: 35 and 36; and SEQ ID NOs: 37 and 38.

[0136] In some embodiments, an anti-CSF IR antibody comprises heavy chain CDRl, CDR2, and CDR3 of an antibody selected from Fabs 0301, 0302, and 031 1.

Nonlimiting exemplary anti-CSFIR antibodies include antibodies comprising sets of heavy chain CDRl, CDR2, and CDR3 selected from: SEQ ID NOs: 15, 16, and 17; SEQ ID NOs: 21, 22, and 23; and SEQ ID NOs: 27, 28,and 29.

[0137] In some embodiments, an anti-CSFIR antibody comprises light chain CDRl, CDR2, and CDR3 of an antibody selected from Fabs 0301, 0302, and 031 1. Nonlimiting exemplary anti-CSFIR antibodies include antibodies comprising sets of light chain CDRl, CDR2, and CDR3 selected from: SEQ ID NOs: 18, 19, and 20; SEQ ID NOs: 24, 25, and 26; and SEQ ID NOs: 30, 31, and 32.

[0138] In some embodiments, an anti-CSF IR antibody comprises heavy chain CDRl, CDR2, and CDR3, and light chain CDRl, CDR2, and CDR3 of an antibody selected from Fabs 0301, 0302, and 031 1.

[0139] Nonlimiting exemplary anti-CSFIR antibodies include antibodies comprising the sets of heavy chain CDRl, CDR2, and CDR3, and light chain CDRl, CDR2, and CDR3 shown above in Table 1.

Further exemplary antibodies

[0140] In some embodiments, an anti-CSFIR antibody comprises a heavy chain comprising a variable region sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 9, 1 1, 13, and 39 to 45, wherein the antibody binds CSF1R. In some embodiments, an anti-CSFIR antibody comprises a light chain comprising a variable region sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 10, 12, 14, and 46 to 52, wherein the antibody binds CSF1R. In some embodiments, an anti-CSFIR antibody comprises a heavy chain comprising a variable region sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 9, 11, 13, and 39 to 45; and a light chain comprising a variable region sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 10, 12, 14, and 46 to 52; wherein the antibody binds CSF1R.

[0141] In some embodiments, an anti-CSFIR antibody comprises at least one of the CDRs discussed herein. That is, in some embodiments, an anti-CSF IR antibody comprises at least one CDR selected from a heavy chain CDRl discussed herein, a heavy chain CDR2 discussed herein, a heavy chain CDR3 discussed herein, a light chain CDRl discussed herein, a light chain CDR2 discussed herein, and a light chain CDR3 discussed herein. Further, in some embodiments, an anti-CSFIR antibody comprises at least one mutated CDR based on a CDR discussed herein, wherein the mutated CDR comprises 1, 2, 3, or 4 amino acid substitutions relative to the CDR discussed herein. In some embodiments, one or more of the amino acid substitutions are conservative amino acid substitutions. One skilled in the art can select one or more suitable conservative amino acid substitutions for a particular CDR sequence, wherein the suitable conservative amino acid substitutions are not predicted to significantly alter the binding properties of the antibody comprising the mutated CDR.

[0142] Exemplary anti-CSF IR antibodies also include antibodies that compete for binding to CSF1R with an antibody described herein. Thus, in some embodiments, an anti- CSFIR antibody is provided that competes for binding to CSF1R with an antibody selected from Fabs 0301, 0302, and 031 1, and bivalent (i.e., having two heavy chains and two light chains) antibody versions of those Fabs.

Exemplary antibody constant regions

[0143] In some embodiments, an antibody described herein comprises one or more human constant regions. In some embodiments, the human heavy chain constant region is of an isotype selected from IgA, IgG, and IgD. In some embodiments, the human light chain constant region is of an isotype selected from κ and λ. In some embodiments, an antibody described herein comprises a human IgG constant region. In some embodiments, an antibody described herein comprises a human IgG4 heavy chain constant region. In some such embodiments, an antibody described herein comprises an S241P mutation in the human IgG4 constant region. In some embodiments, an antibody described herein comprises a human IgG4 constant region and a human κ light chain. [0144] As noted above, whether or not effector function is desirable may depend on the particular method of treatment intended for an antibody. Thus, in some embodiments, when effector function is desirable, an anti-CSFIR antibody comprising a human IgGl heavy chain constant region or a human IgG3 heavy chain constant region is selected. In some embodiments, when effector function is not desirable, an anti-CSFIR antibody comprising a human IgG4 or IgG2 heavy chain constant region is selected.

Exemplary Anti-CSF IR Heavy Chain Variable Regions

[0145] In some embodiments, anti-CSFIR antibody heavy chain variable regions are provided. In some embodiments, an anti-CSFIR antibody heavy chain variable region is a mouse variable region, a human variable region, or a humanized variable region.

[0146] An anti-CSFIR antibody heavy chain variable region comprises a heavy chain CDRl, FR2, CDR2, FR3, and CDR3. In some embodiments, an anti-CSFIR antibody heavy chain variable region further comprises a heavy chain FR1 and/or FR4. Nonlimiting exemplary heavy chain variable regions include, but are not limited to, heavy chain variable regions having an amino acid sequence selected from SEQ ID NOs: 9, 1 1, 13, and 39 to 45.

[0147] In some embodiments, an anti-CSF IR antibody heavy chain variable region comprises a CDRl comprising a sequence selected from SEQ ID NOs: 15, 21, and 27.

[0148] In some embodiments, an anti-CSF IR antibody heavy chain variable region comprises a CDR2 comprising a sequence selected from SEQ ID NOs: 16, 22, and 28.

[0149] In some embodiments, an anti-CSF IR antibody heavy chain variable region comprises a CDR3 comprising a sequence selected from SEQ ID NOs: 17, 23, and 29.

[0150] Nonlimiting exemplary heavy chain variable regions include, but are not limited to, heavy chain variable regions comprising sets of CDRl, CDR2, and CDR3 selected from: SEQ ID NOs: 15, 16, and 17; SEQ ID NOs: 21, 22, and 23; and SEQ ID NOs: 27, 28, and 29.

[0151] In some embodiments, an anti-CSFIR antibody heavy chain comprises a variable region sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 9, 11, 13, and 39 to 45, wherein the heavy chain, together with a light chain, is capable of forming an antibody that binds CSF1R.

[0152] In some embodiments, an anti-CSFIR antibody heavy chain comprises at least one of the CDRs discussed herein. That is, in some embodiments, an anti-CSF IR antibody heavy chain comprises at least one CDR selected from a heavy chain CDRl discussed herein, a heavy chain CDR2 discussed herein, and a heavy chain CDR3 discussed herein. Further, in some embodiments, an anti-CSF IR antibody heavy chain comprises at least one mutated CDR based on a CDR discussed herein, wherein the mutated CDR comprises 1, 2, 3, or 4 amino acid substitutions relative to the CDR discussed herein. In some embodiments, one or more of the amino acid substitutions are conservative amino acid substitutions. One skilled in the art can select one or more suitable conservative amino acid substitutions for a particular CDR sequence, wherein the suitable conservative amino acid substitutions are not predicted to significantly alter the binding properties of the heavy chain comprising the mutated CDR.

[0153] In some embodiments, a heavy chain comprises a heavy chain constant region. In some embodiments, a heavy chain comprises a human heavy chain constant region. In some embodiments, the human heavy chain constant region is of an isotype selected from IgA, IgG, and IgD. In some embodiments, the human heavy chain constant region is an IgG constant region. In some embodiments, a heavy chain comprises a human igG4 heavy chain constant region. In some such embodiments, the human IgG4 heavy chain constant region comprises an S241P mutation.

[0154] In some embodiments, when effector function is desirable, a heavy chain comprises a human IgGl or IgG3 heavy chain constant region. In some embodiments, when effector function is less desirable, a heavy chain comprises a human IgG4 or IgG2 heavy chain constant region.

Exemplary Anti-CSF IR Light Chain Variable Regions

[0155] In some embodiments, anti-CSFIR antibody light chain variable regions are provided. In some embodiments, an anti-CSFIR antibody light chain variable region is a mouse variable region, a human variable region, or a humanized variable region.

[0156] An anti-CSFIR antibody light chain variable region comprises a light chain CDR1, FR2, CDR2, FR3, and CDR3. In some embodiments, an anti-CSFIR antibody light chain variable region further comprises a light chain FR1 and/or FR4. Nonlimiting exemplary light chain variable regions include light chain variable regions having an amino acid sequence selected from SEQ ID NOs: 10, 12, 14, and 46 to 52.

[0157] In some embodiments, an anti-CSFIR antibody light chain variable region comprises a CDR1 comprising a sequence selected from SEQ ID NOs: 18, 24 and 30.

[0158] In some embodiments, an anti-CSFIR antibody light chain variable region comprises a CDR2 comprising a sequence selected from SEQ ID NOs: 19, 25, and 31.

[0159] In some embodiments, an anti-CSFIR antibody light chain variable region comprises a CDR3 comprising a sequence selected from SEQ ID NOs: 20, 26, and 32. [0160] Nonlimiting exemplary light chain variable regions include, but are not limited to, light chain variable regions comprising sets of CDRl, CDR2, and CDR3 selected from: SEQ ID NOs: 18, 19, and 20; SEQ ID NOs: 24, 25, and 26; and SEQ ID NOs: 30, 31, and 32.

[0161] In some embodiments, an anti-CSF IR antibody light chain comprises a variable region sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 10, 12, 14, and 46 to 52, wherein the light chain, together with a heavy chain, is capable of forming an antibody that binds CSF1R.

[0162] In some embodiments, an anti-CSF IR antibody light chain comprises at least one of the CDRs discussed herein. That is, in some embodiments, an anti-CSFIR antibody light chain comprises at least one CDR selected from a light chain CDRl discussed herein, a light chain CDR2 discussed herein, and a light chain CDR3 discussed herein. Further, in some embodiments, an anti-CSF IR antibody light chain comprises at least one mutated CDR based on a CDR discussed herein, wherein the mutated CDR comprises 1, 2, 3, or 4 amino acid substitutions relative to the CDR discussed herein. In some embodiments, one or more of the amino acid substitutions are conservative amino acid substitutions. One skilled in the art can select one or more suitable conservative amino acid substitutions for a particular CDR sequence, wherein the suitable conservative amino acid substitutions are not predicted to significantly alter the binding properties of the light chain comprising the mutated CDR.

[0163] In some embodiments, a light chain comprises a human light chain constant region. In some embodiments, a human light chain constant region is selected from a human κ and a human λ light chain constant region.

Exemplary Additional CSF 1R Binding Molecules

[0164] In some embodiments, additional molecules that bind CSF 1R are provided. Such molecules include, but are not limited to, non-canonical scaffolds, such as anti-calins, adnectins, ankyrin repeats, etc. See, e.g., Hosse et al, Prot. Set 15: 14 (2006); Fiedler, M. and Skerra, A., "Non-Antibody Scaffolds," pp.467-499 in Handbook of Therapeutic Antibodies, Dubel, S., ed., Wiley-VCH, Weinheim, Germany, 2007.

Exemplary Properties of anti-CSFIR antibodies

[0165] In some embodiments, an antibody having a structure described above binds to the CSF1R with a binding affinity (KD) of less than 1 nM, blocks binding of CSF l and/or IL-34 to CSF 1R, and inhibits CSF 1R phosphorylation induced by CSF1 and/or IL-34. [0166] In some embodiments, an anti-CSF lR antibody binds to the extracellular domain of CSFIR (CSF1R-ECD). In some embodiments, an anti-CSF lR antibody has a binding affinity (K _D ) for CSFIR of less than 1 nM, less than 0.5 nM, less than 0.1 nM, or less than 0.05 nM. In some embodiments, an anti-CSFlR antibody has a ¾ of between 0.01 and 1 nM, between 0.01 and 0.5 nM, between 0.01 and 0.1 nM, between 0.01 and 0.05 nM, or between 0.02 and 0.05 nM.

[0167] In some embodiments, an anti-CSFlR antibody blocks ligand binding to CSFIR. In some embodiments, an anti-CSF lR antibody blocks binding of CSFl to CSF IR. In some embodiments, an anti-CSFlR antibody blocks binding of IL-34 to CSFIR. In some embodiments, an anti-CSF lR antibody blocks binding of both CSFl and IL-34 to CSFIR. In some embodiments, an antibody that blocks ligand binding binds to the extracellular domain of CSFIR. An antibody is considered to "block ligand binding to CSFIR" when it reduces the amount of detectable binding of a ligand to CSF 1R by at least 50%, using the assay described in Example 7. In some embodiments, an antibody reduces the amount of detectable binding of a ligand to CSFIR by at least 60%, at least 70%, at least 80%, or at least 90%, using the assay described in Example 7. In some such embodiments, the antibody is said to block ligand binding by at least 50%, at least 60%, at least 70%, etc.

[0168] In some embodiments, an anti-CSF lR antibody inhibits ligand-induced CSF IR phosphorylation. In some embodiments, an anti-CSFlR antibody inhibits CSF 1- induced CSFIR phosphorylation. In some embodiments, an anti-CSF lR antibody inhibits IL-34-induced CSFIR phosphorylation. In some embodiments, an anti-CSFlR antibody inhibits both CSFl -induced and IL-34-induced CSFIR phosphorylation. An antibody is considered to "inhibit ligand-induced CSF IR phosphorylation" when it reduces the amount of detectable ligand-induced CSF IR phosphorylation by at least 50%, using the assay described in Example 6. In some embodiments, an antibody reduces the amount of detectable ligand-induced CSFIR phosphorylation by at least 60%, at least 70%, at least 80%, or at least 90%, using the assay described in Example 6. In some such embodiments, the antibody is said to inhibit ligand-induced CSF IR phosphorylation by at least at least 50%, at least 60%, at least 70%, etc.

[0169] In some embodiments, an antibody inhibits monocyte proliferation and/or survival responses in the presence of CSFl and/or IL-34. An antibody is considered to "inhibit monocyte proliferation and/or survival responses" when it reduces the amount of monocyte proliferation and/or survival responses in the presence of CSFl and/or IL-34 by at least 50%, using the assay described in Example 10. In some embodiments, an antibody reduces the amount of monocyte proliferation and/or survival responses in the presence of CSF1 and/or IL-34 by at least 60%, at least 70%, at least 80%, or at least 90%, using the assay described in Example 10. In some such embodiments, the antibody is said to inhibit monocyte proliferation and/or survival responses by at least at least 50%, at least 60%, at least 70%, etc.

Exemplary Antibody Conjugates

[0170] In some embodiments, an anti-CSFIR antibody is conjugated to a label and/or a cytotoxic agent. As used herein, a label is a moiety that facilitates detection of the antibody and/or facilitates detection of a molecule to which the antibody binds. Nonlimiting exemplary labels include, but are not limited to, radioisotopes, fluorescent groups, enzymatic groups, chemiluminescent groups, biotin, epitope tags, metal-binding tags, etc. One skilled in the art can select a suitable label according to the intended application.

[0171] As used herein, a cytotoxic agent is a moiety that reduces the proliferative capacity of one or more cells. A cell has reduced proliferative capacity when the cell becomes less able to proliferate, for example, because the cell undergoes apoptosis or otherwise dies, the cell fails to proceed through the cell cycle and/or fails to divide, the cell differentiates, etc. Nonlimiting exemplary cytotoxic agents include, but are not limited to, radioisotopes, toxins, and chemotherapeutic agents. One skilled in the art can select a suitable cytotoxic according to the intended application.

[0172] In some embodiments, a label and/or a cytotoxic agent is conjugated to an antibody using chemical methods in vitro. Nonlimiting exemplary chemical methods of conjugation are known in the art, and include services, methods and/or reagents

commercially available from, e.g., Thermo Scientific Life Science Research Produces (formerly Pierce; Rockford, IL), Prozyme (Hayward, CA), SACRI Antibody Services (Calgary, Canada), AbD Serotec (Raleigh, NC), etc. In some embodiments, when a label and/or cytotoxic agent is a polypeptide, the label and/or cytotoxic agent can be expressed from the same expression vector with at least one antibody chain to produce a polypeptide comprising the label and/or cytotoxic agent fused to an antibody chain. One skilled in the art can select a suitable method for conjugating a label and/or cytotoxic agent to an antibody according to the intended application.

Exemplary Leader Sequences

[0173] In order for some secreted proteins to express and secrete in large quantities, a leader sequence from a heterologous protein may be desirable. In some embodiments, a leader sequence is selected from SEQ ID NOs: 3 and 4, which are light chain and heavy chain leader sequences, respectively. In some embodiments, employing heterologous leader sequences may be advantageous in that a resulting mature polypeptide may remain unaltered as the leader sequence is removed in the ER during the secretion process. The addition of a heterologous leader sequence may be required to express and secrete some proteins.

[0174] Certain exemplary leader sequence sequences are described, e.g., in the online Leader sequence Database maintained by the Department of Biochemistry, National University of Singapore. See Choo et al, BMC Bioinformatics, 6: 249 (2005); and PCT Publication No. WO 2006/081430.

Nucleic Acid Molecules Encoding Anti-CSFIR Antibodies

[0175] Nucleic acid molecules comprising polynucleotides that encode one or more chains of anti-CSF IR antibodies are provided. In some embodiments, a nucleic acid molecule comprises a polynucleotide that encodes a heavy chain or a light chain of an anti- CSF1R antibody. In some embodiments, a nucleic acid molecule comprises both a polynucleotide that encodes a heavy chain and a polynucleotide that encodes a light chain, of an anti-CSFIR antibody. In some embodiments, a first nucleic acid molecule comprises a first polynucleotide that encodes a heavy chain and a second nucleic acid molecule comprises a second polynucleotide that encodes a light chain.

[0176] In some such embodiments, the heavy chain and the light chain are expressed from one nucleic acid molecule, or from two separate nucleic acid molecules, as two separate polypeptides. In some embodiments, such as when an antibody is an scFv, a single polynucleotide encodes a single polypeptide comprising both a heavy chain and a light chain linked together.

[0177] In some embodiments, a polynucleotide encoding a heavy chain or light chain of an anti-CSFIR antibody comprises a nucleotide sequence that encodes a leader sequence, which, when translated, is located at the N terminus of the heavy chain or light chain. As discussed above, the leader sequence may be the native heavy or light chain leader sequence, or may be another heterologous leader sequence.

[0178] Nucleic acid molecules may be constructed using recombinant DNA techniques conventional in the art. In some embodiments, a nucleic acid molecule is an expression vector that is suitable for expression in a selected host cell.

Anti-CSFIR Antibody Expression and Production

Vectors

[0179] Vectors comprising polynucleotides that encode anti-CSF IR heavy chains and/or anti-CSFIR light chains are provided. Vectors comprising polynucleotides that encode anti-CSFIR heavy chains and/or anti-CSF IR light chains are also provided. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, etc. In some embodiments, a vector comprises a first polynucleotide sequence encoding a heavy chain and a second polynucleotide sequence encoding a light chain. In some embodiments, the heavy chain and light chain are expressed from the vector as two separate polypeptides. In some embodiments, the heavy chain and light chain are expressed as part of a single polypeptide, such as, for example, when the antibody is an scFv.

[0180] In some embodiments, a first vector comprises a polynucleotide that encodes a heavy chain and a second vector comprises a polynucleotide that encodes a light chain. In some embodiments, the first vector and second vector are transfected into host cells in similar amounts (such as similar molar amounts or similar mass amounts). In some embodiments, a mole- or mass-ratio of between 5: 1 and 1 :5 of the first vector and the second vector is transfected into host cells. In some embodiments, a mass ratio of between 1 : 1 and 1 :5 for the vector encoding the heavy chain and the vector encoding the light chain is used. In some embodiments, a mass ratio of 1 :2 for the vector encoding the heavy chain and the vector encoding the light chain is used.

[0181] In some embodiments, a vector is selected that is optimized for expression of polypeptides in CHO or CHO-derived cells, or in NSO cells. Exemplary such vectors are described, e.g., in Running Deer et al, Biotechnol. Prog. 20:880-889 (2004).

[0182] In some embodiments, a vector is chosen for in vivo expression of anti- CSFIR heavy chains and/or anti-CSFIR light chains in animals, including humans. In some such embodiments, expression of the polypeptide is under the control of a promoter that functions in a tissue-specific manner. For example, liver-specific promoters are described, e.g., in PCT Publication No. WO 2006/076288.

Host Cells

[0183] In various embodiments, anti-CSFIR heavy chains and/or anti-CSFIR light chains may be expressed in prokaryotic cells, such as bacterial cells; or in eukaryotic cells, such as fungal cells (such as yeast), plant cells, insect cells, and mammalian cells. Such expression may be carried out, for example, according to procedures known in the art. Exemplary eukaryotic cells that may be used to express polypeptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S and DG44 cells; PER.C6® cells (Crucell); and NSO cells. In some embodiments, anti-CSF IR heavy chains and/or anti-CSF IR light chains may be expressed in yeast. See, e.g., U.S. Publication No. US 2006/0270045 Al . In some embodiments, a particular eukaryotic host cell is selected based on its ability to make desired post- translational modifications to the anti-CSFlR heavy chains and/or anti-CSFlR light chains. For example, in some embodiments, CHO cells produce polypeptides that have a higher level of sialylation than the same polypeptide produced in 293 cells.

[0184] Introduction of one or more nucleic acids into a desired host cell may be accomplished by any method, including but not limited to, calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, etc. Nonlimiting exemplary methods are described, e.g., in

Sambrook et al, Molecular Cloning, A Laboratory Manual, 3 ^rd ed. Cold Spring Harbor Laboratory Press (2001). Nucleic acids may be transiently or stably transfected in the desired host cells, according to any suitable method.

[0185] In some embodiments, one or more polypeptides may be produced in vivo in an animal that has been engineered or transfected with one or more nucleic acid molecules encoding the polypeptides, according to any suitable method.

Purification of Anti-CSFlR Antibodies

[0186] Anti-CSFlR antibodies may be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrices or hydrophobic interaction chromatography. Suitable affinity ligands include the CSFIR ECD and ligands that bind antibody constant regions. For example, a Protein A, Protein G, Protein A/G, or an antibody affinity column may be used to bind the constant region and to purify an anti- CSF 1R antibody. Hydrophobic interactive chromatography, for example, a butyl or phenyl column, may also suitable for purifying some polypeptides. Many methods of purifying polypeptides are known in the art.

Cell-free Production of Anti-CSFlR Antibodies

[0187] In some embodiments, an anti-CSF lR antibody is produced in a cell-free system. Nonlimiting exemplary cell-free systems are described, e.g., in Sitaraman et al, Methods Mol. Biol. 498: 229-44 (2009); Spirin, Trends Biotechnol. 22: 538-45 (2004); Endo et al, Biotechnol. Adv. 21 : 695-713 (2003).

Therapeutic Compositions and Methods

Methods of Treating Diseases using Anti-CSFlR Antibodies

[0188] Provided herein are methods of treating systemic lupus erythematosus (SLE), rheumatoid arthritis, and multiple sclerosis with an antibody that binds CSFIR and blocks CSF1 and IL-34 ligand binding. [0189] In some embodiments, a method of treating SLE is provided. In some such embodiments, the method comprises administering an antibody that binds CSF1R and blocks CSF1 and IL-34 ligand binding, such as an antibody selected from huAbl to huAbl6, to a subject with SLE. Treating SLE, in some embodiments, means reducing the incidence and/or severity of skin lesions associated with SLE, and/or reducing a kidney condition selected from glomerulonephritis, interstitial nephritis, and perivascular infiltrates. In some embodiments, a method of treating lupus nephritis is provided, wherein the method comprises administering an antibody that binds CSF1R and blocks CSF 1 and IL-34 ligand binding, such as an antibody selected from huAbl to huAbl6, to a subject with lupus nephritis. In some embodiments, a method of treating a class of lupus nephritis selected from class I, class II, class III, class IV, class V, and class VI lupus nephritis is provided, wherein the method comprises administering an antibody that binds CSF 1R and blocks CSF1 and IL-34 ligand binding, such as an antibody selected from huAbl to huAbl6, to a subject with lupus nephritis. Class III and class IV lupus nephritis have significantly greater CD 16+ monocyte counts than other classes of lupus nephritis. See Yoshimoto et al, Am. J. Kidney Dis. 50: 47-58 (2007). Accordingly, in some embodiments, a method of treating class III or class IV lupus nephritis is provided, wherein the method comprises administering an antibody that binds CSF1R and blocks CSF1 and IL-34 ligand binding, such as an antibody selected from huAbl to huAbl6, to a subject with lupus nephritis. In some such embodiments, the method comprises administering huAbl to the subject.

[0190] In some embodiments, a method of treating skin lesions associated with SLE is provided, wherein the method comprises administering an antibody that binds CSF 1R, such as an antibody selected from huAbl to huAbl6, to a subject with SLE. In some embodiments, a method of treating skin lesions associated with SLE comprises

administering huAbl to a subject with SLE. In some embodiments, a method of reducing the number, severity, and/or rate of formation of skin lesions associated with SLE is provided, wherein the method comprises administering an antibody that binds CSF1R, such as an antibody selected from huAbl to huAbl6, to a subject with SLE. In some

embodiments, a method of reducing the number, severity, and/or rate of formation of skin lesions associated with SLE comprises administering huAbl to a subject with SLE. The number, severity, and/or rate of formation of skin lesions may be determined, in some embodiments, by visual inspection. Such visual inspection may be carried out, for example, by a clinician or by the subject (e.g., self-reported visual inspection). [0191] In some embodiments, a method of treating a kidney condition associated with SLE is provided. In some such embodiments, the method comprises administering an antibody that binds CSF 1R and blocks CSF1 and IL-34 ligand binding, such as an antibody selected from huAbl to huAbl6, to a subject with a kidney condition associated with SLE. In some embodiments, antibody huAbl is administered to the subject. In some

embodiments, the kidney condition is selected from glomerulonephritis, interstitial nephritis, and perivascular infiltrates. In some embodiments, a method of treating lupus nephritis is provided, wherein the method comprises administering an antibody that binds CSF1R and blocks CSF1 and IL-34 ligand binding, such as an antibody selected from huAbl to huAbl6, to a subject with lupus nephritis. In some embodiments, a method of treating lupus nephritis comprises administering antibody huAbl to a subject with lupus nephritis. The extent of kidney damage in lupus nephritis may be determined, in some embodiments, using ultrasonography, scintigraphy, pyelography, or needle biopsy. The extent of kidney damage in lupus nephritis may also be determined, in some embodiments, by measuring kidney function, such as, for example, by measuring proteinuria and/or glomerular filtration rate.

[0192] In some embodiments, a method of reducing proteinuria associated with lupus nephritis is provided, comprising administering an antibody that binds CSF1R and blocks CSF1 and IL-34 ligand binding, such as an antibody selected from huAbl to huAbl6, to a subject exhibiting proteinuria. Proteinuria may be detected by any method in the art, including, but not limited to, dipstick testing, protein/creatinine ratio, serum albumin/creatinine ratio, and protein electrophoresis. Levels of protein in urine may be indicated, in some embodiments, using the following scale: trace (5-20 mg/dl), 1+ (30 mg/dl), 2+ (100 mg/dl), 3+ (300 mg/dl), and 4+ (>2000 mg/dl). A protein/creatinine ratio of < 200 mg/g is considered normal. A serum albumin/creatinine ratio of < 17 mg/g and < 25 mg/g is considered normal for men and women, respectively. Proteinuria is defined by the National Kidney Foundation Kidney Disease Outcome Quality Initiative as the presence of >300 mg/d total protein or albumin for 24-hour urine excretions; as >30 mg/dL total protein for spot urine dipstick measurements (i.e., 1+ or greater on the scale described above); as >200 mg/g total protein-to-creatinine ratio; as >300 mg/g albumin-to-creatinine ratio for spot urine measurements (or >250 mg/g albumin-to-creatinine ratio for spot urine measurements from men; and >355 mg/g albumin-to-creatinine ratio for spot urine measurements from women).

[0193] In some embodiments, administration of an antibody that binds CSF 1R and blocks CSF1 and IL-34 ligand binding to a subject reduces proteinuria by at least one number or at least two numbers on the "trace, 1+, 2+, 3+, 4+" scale. In other words, in some embodiments, administration of an antibody that binds CSF1R and blocks CSF1 and IL-34 ligand binding to a subject with 3+ proteinuria reduces the proteinuria to a 2+ level or lower, or a 1+ level or lower. In some embodiments, administration of an antibody that binds CSF1R and blocks CSF 1 and IL-34 ligand binding to a subject reduces the protein/creatinine ratio and/or albumin/creatinine ratio by at least 50 mg/g, at least 100 mg/g, at least 150 mg/g, at least 200 mg/g, at least 250 mg/g, or at least 300 mg/g. In some embodiments, administration of an antibody that binds CSF1R and blocks CSF1 and IL-34 ligand binding to a subject reduces the protein/creatinine ratio and/or albumin/creatinine ratio to below 250 mg/g, below 200 mg/g, below 150 mg/g, or below 100 mg/g. In some embodiments, administration of an antibody that binds CSF 1R and blocks CSF 1 and IL-34 ligand binding to a subject reduces the protein/creatinine ratio and/or albumin/creatinine ratio to within 2-fold, within 3-fold, within 4-fold, within 5-fold of a normal ratio. In some embodiments, the reduction in proteinuria is determined in the first week, in the first two weeks, in the first month, in the first two months, in the first three months, in the first 4 months, or in the first six months after beginning treatment. In some embodiments, a subject is monitored for continued suppression of proteinuria while being treated with an antibody that binds CSF 1R and blocks CSF 1 and IL-34 ligand binding.

[0194] In some embodiments, a method of improving glomerular filtration rate in a subject with lupus nephritis is provided, comprising administering an antibody that binds CSF 1R and blocks CSF1 and IL-34 ligand binding, such as an antibody selected from huAbl to huAbl6 to a subject with lupus nephritis. Glomerular filtration rate may be determined by any method in the art, including, but not limited to, serum or urine levels of insulin or sinistrin (in some embodiments, insulin or sinistrin is injected prior to analysis), creatinine clearance estimated from creatinine concentrations in 24 urine collection and serum, and serum cystatin C levels. Normal glomerular filtration rates in adults are in the range of 90-130 ml/min/1.73 m ² . Glomerular filtration rates below 90 ml/min/1.73 m ² , below 80 ml/min/1.73 m ² , below 70 ml/min/1.73 m ² , or below 60 ml/min/1.73 m ² are, in some embodiments, indicative of chronic kidney disease, while rates below 15 ml/min/1.73 m ² are indicative of kidney failure. In some embodiments, a method of improving glomerular filtration rate in a subject with lupus nephritis is provided, comprising administering an antibody that binds CSF1R and blocks CSF1 and IL-34 ligand binding, such as an antibody selected from huAbl to huAbl6, to a subject with lupus nephritis. In some embodiments, following administration of an antibody that binds CSF 1R and blocks CSFl and IL-34 ligand binding, a subject's glomerular filtration rate improves by at least 10 ml/min/1.73 m ² , at least 15 ml/min/1.73 m ² , at least 20 ml/min/1.73 m ² , at least 25 ml/min/1.73 m ² , at least 30 ml/min/1.73 m ² , at least 40 ml/min/1.73 m ² , or at least 50 ml/min/1.73 m ² . In some embodiments, following administration of an antibody that binds CSFIR and blocks CSFl and IL-34 ligand binding, a subject's glomerular filtration rate is at least 60 ml/min/1.73 m ² , at least 70 ml/min/1.73 m ² , at least 80 ml/min/1.73 m ² , or at least 90 ml/min/1.73 m ² . In some embodiments, the reduction in glomerular filtration rate is determined in the first week, in the first two weeks, in the first month, in the first two months, in the first three months, in the first 4 months, or in the first six months after beginning treatment. In some embodiments, a subject is monitored for continued improvement of glomerular filtration rate while being treated with an antibody that binds CSFIR and blocks CSFl and IL-34 ligand binding.

[0195] In some embodiments, a method of treating lupus nephritis is provided, comprising administering an antibody that binds CSFIR and blocks CSFl and IL-34 ligand binding, such as an antibody selected from huAbl to huAbl6 to a subject with lupus nephritis, wherein administration of the antibody slows or prevents the progression of a kidney condition associated with lupus nephritis. In some such embodiments, proteinuria and/or the glomerular filtration rate in the subject does not worsen (i.e., proteinuria does not increase and/or the glomerular filtration rate does not decrease) following administration of the antibody and/or during a particular time interval during which the subject is undergoing treatment with the antibody. The treatment may be a single dose or multiple doses.

[0196] In some embodiments, methods of treating rheumatoid arthritis are provided, wherein the method comprises administering an antibody that binds CSF IR and blocks CSFl and IL-34 ligand binding, such as an antibody selected from huAbl to huAbl6, to a subject with rheumatoid arthritis. In some embodiments, methods of treating rheumatoid arthritis are provided, wherein the method comprises administering antibody huAb 1 to a subject with rheumatoid arthritis. Treating rheumatoid arthritis, in some embodiments, means reducing clinical disease scores, which may be measured, for example, by measuring erythema and swelling in joints affected by rheumatoid arthritis. Treating rheumatoid arthritis, in some embodiments, means reducing inflammation, reducing pannus formation, reducing cartilage damage, reducing bone resorption, reducing macrophage numbers in the joints, reducing autoantibody formation, and/or reducing bone loss.

[0197] In some embodiments, a method of reducing inflammation associated with rheumatoid arthritis is provided, wherein the method comprises administering an antibody that binds CSFIR and blocks CSFl and IL-34 ligand binding, such as an antibody selected from huAbl to huAbl6, to a subject with rheumatoid arthritis. In some embodiments, a method of reducing inflammation associated with rheumatoid arthritis is provided, wherein the method comprises administering antibody huAbl to a subject with rheumatoid arthritis. Reducing inflammation, in some embodiments, comprises reducing erythrocyte sedimentation rate and/or reducing the levels of C-reactive proteins in blood. When inflammation is present in a subject, the erythrocyte sedimentation rate increases, possibly due to increased levels of fibrinogen in the blood. The erythrocyte sedimentation rate may be determined by any method in the art, including, but not limited to, calculating the rate by measuring the change in height of anticoagulated erythrocytes in one hour in a Westergren tube. See also Procedures for the Erythrocyte Sedimentation Rate Test; Approved

Standard-- Fifth Edition, CLSI document H02-A5. Wayne, PA: Clinical and Laboratory Standards Institute; 2011 . Levels of C-reactive protein in blood may be determined by any methods in the art, including but not limited to using the RAPITEX® CRP test kit

(Siemens).

[0198] Reducing inflammation, in some embodiments, comprises reducing peripheral edema, which is tissue swelling due to the buildup of fluids. Peripheral edema may occur, in some instances, in the ankles, feet, legs, and/or calves of a subject with rheumatoid arthritis. Reducing inflammation, in some embodiments, comprises reducing infiltration of inflammatory cells in the synovium of one or more affected joints. Synovial fluid may be collected, in some embodiments, by athrocentesis.

[0199] In some embodiments, a method of reducing pannus formation associated with rheumatoid arthritis is provided, wherein the method comprises administering an antibody that binds CSF IR and blocks CSFl and IL-34 ligand binding, such as an antibody selected from huAbl to huAbl6, to a subject with rheumatoid arthritis. In some embodiments, a method of reducing pannus formation associated with rheumatoid arthritis is provided, wherein the method comprises administering antibody huAbl to a subject with rheumatoid arthritis. Reducing pannus formation, in some embodiments, comprises reducing infiltration of pannus into cartilage and/or subchondrial bone, and/or reducing hard tissue destruction resulting from pannus infiltration. Pannus formation can be measured by any method in the art, including, but not limited to, imaging one or more affected joints. Nonlimiting exemplary imaging techniques for detecting pannus formation include magnetic resonance imaging (MRI), computed tomography (CT) scan, arthroscopy, ultrasonography, duplex ultrasonography, and power doppler imaging. [0200] In some embodiments, a method of slowing the progression of pannus formation associated with rheumatoid arthritis is provided, wherein the method comprises administering an antibody that binds CSFIR and blocks CSFl and IL-34 ligand binding, such as an antibody selected from huAbl to huAbl6, to a subject with rheumatoid arthritis. In some embodiments, the antibody is huAbl. In some embodiments, the formation of pannus is slower following administration of the antibody and/or during a particular time interval during which the subject is undergoing treatment with the antibody. The treatment may be a single dose or multiple doses.

[0201] In some embodiments, a method of reducing cartilage damage associated with rheumatoid arthritis is provided, wherein the method comprises administering an antibody that binds CSF IR and blocks CSFl and IL-34 ligand binding, such as an antibody selected from huAbl to huAbl6, to a subject with rheumatoid arthritis. In some embodiments, a method of reducing cartilage damage associated with rheumatoid arthritis is provided, wherein the method comprises administering antibody huAbl to a subject with rheumatoid arthritis. Reducing cartilage damage, in some embodiments, comprises reducing chondrocyte loss, reducing collagen disruption, and/or reducing cartilage loss. Cartilage damage can be measured by any method in the art, including, but not limited to, imaging one or more affected joints. Nonlimiting exemplary imaging techniques for detecting cartilage damage include MRI, CT scan, arthroscopy, and x-ray imaging.

[0202] In some embodiments, a method of slowing the progression of cartilage damage associated with rheumatoid arthritis is provided, wherein the method comprises administering an antibody that binds CSFIR and blocks CSFl and IL-34 ligand binding, such as an antibody selected from huAbl to huAbl6, to a subject with rheumatoid arthritis. In some embodiments, the antibody is huAbl. In some embodiments, the progression of cartilage damage is slower following administration of the antibody and/or during a particular time interval during which the subject is undergoing treatment with the antibody. The treatment may be a single dose or multiple doses.

[0203] In some embodiments, a method of reducing bone resorption associated with rheumatoid arthritis is provided, wherein the method comprises administering an antibody that binds CSFIR and blocks CSFl and IL-34 ligand binding, such as an antibody selected from huAbl to huAbl6, to a subject with rheumatoid arthritis. In some embodiments, a method of reducing bone resorption associated with rheumatoid arthritis is provided, wherein the method comprises administering antibody huAbl to a subject with rheumatoid arthritis. Reducing bone resorption, in some embodiments, comprises reducing the number of osteoclasts in joints affected by rheumatoid arthritis.

[0204] In some embodiments, bone resorption may be measured by determining the level of TRAP5b in plasma from the subject, wherein an elevated level of TRAP5b indicates elevated bone resorption in the subject. Thus, in some embodiments, a reduced level of TRAP5b indicates a reduction in bone resorption. TRAP5b levels may be determined, in certain instances, before and after treatment with an antibody that binds CSFIR, and/or may be determined periodically throughout the course of treatment to monitor the effectiveness of the treatment in reducing bone loss. TRAP5b levels may be determined using any method in the art, including, but not limited to, ELISA (including FAICEA, or fragments absorbed immunocapture enzymatic assay; see, e.g., Quidel® TRAP5b assay,

TECOmedical Group, Sissach, Switzerland).

[0205] In some embodiments, a method of reducing bone loss associated with rheumatoid arthritis is provided, wherein the method comprises administering an antibody that binds CSFIR and blocks CSF1 and IL-34 ligand binding, such as an antibody selected from huAbl to huAbl6, to a subject with rheumatoid arthritis. In some embodiments, a method of reducing bone loss associated with rheumatoid arthritis is provided, wherein the method comprises administering antibody huAbl to a subject with rheumatoid arthritis. Bone loss may be determined using any method in the art, including, but not limited to, x- ray imaging, MRI, CT, bone densitometry, single and dual photon absorptiometry (SPA, DPA), single and dual energy x-ray absorptiometry (SXA, DXA), ultrasonography, scintigraphy, and by measuring levels of serum markers of bone formation and resorption. Nonlimiting exemplary serum markers of bone formation and bone resorption are shown in Table 2.

Table 2: Serum markers of bone formation and resorption

1 jC- terminal telopeptide (CTx, also referred to as I isCrossLaps®)

Serum carboxyterminal telopeptide of type I collagen (ITCP)

Tartrate -resistant acid phosphatase (TRAP or TRACP)

[0206] In some embodiments, a method of slowing the progression of bone loss associated with rheumatoid arthritis is provided, wherein the method comprises

administering an antibody that binds CSFIR and blocks CSFl and IL-34 ligand binding, such as an antibody selected from huAbl to huAbl6, to a subject with rheumatoid arthritis. In some embodiments, the antibody is huAbl. In some embodiments, bone loss is slower following administration of the antibody and/or during a particular time interval during which the subject is undergoing treatment with the antibody. The treatment may be a single dose or multiple doses.

[0207] In some embodiments, a method of reducing the number of monocyte lineage cells, such as macrophages and/or CD 16+ monocytes, in joints affected by rheumatoid arthritis is provided, wherein the method comprises administering an antibody that binds CSFIR and blocks CSFl and IL-34 ligand binding, such as an antibody selected from huAbl to huAbl6, to a subject with rheumatoid arthritis. In some embodiments, a method of reducing the number of monocyte lineage cells, such as macrophages and/or CD 16+ monocytes, in joints affected by rheumatoid arthritis is provided, wherein the method comprises administering antibody huAbl to a subject with rheumatoid arthritis. In some embodiments, a method of reducing the number of monocyte lineage cells, such as macrophages and/or CD 16+ monocytes, in synovial fluid of joints affected by rheumatoid arthritis is provided, wherein the method comprises administering an antibody that binds CSFIR and blocks CSFl and IL-34 ligand binding, such as an antibody selected from huAbl to huAbl6, to a subject with rheumatoid arthritis. In some embodiments, a method of reducing the number of monocyte lineage cells, such as macrophages and/or CD 16+ monocytes, in synovial fluid of joints affected by rheumatoid arthritis is provided, wherein the method comprises administering antibody huAbl to a subject with rheumatoid arthritis.

[0208] In some embodiments, a method of reducing autoantibody levels in a subject with rheumatoid arthritis is provided, wherein the method comprises administering an antibody that binds CSF IR and blocks CSFl and IL-34 ligand binding, such as an antibody selected from huAbl to huAbl6, to a subject with rheumatoid arthritis. In some embodiments, a method of reducing autoantibody levels in a subject with rheumatoid arthritis is provided, wherein the method comprises administering antibody huAbl to a subject with rheumatoid arthritis.

[0209] In some embodiments, a method of reducing autoantibody levels in a subject with rheumatoid arthritis are provided, comprising administering an antibody that binds CSF1R and blocks CSF1 and IL-34 ligand binding, such as an antibody selected from huAbl to huAbl6, to a subject with rheumatoid arthritis. In some such embodiments, the method comprises administering huAbl to a subject with rheumatoid arthritis. The levels of autoantibodies may be determined by any method in the art. In some embodiments, autoantibody levels are determined by the level of rheumatoid factor (RF) and/or anti- citrullinated protein antibodies (ACPA) and/or anti-nuclear antibodies (ANA).

[0210] In some embodiments, methods of treating multiple sclerosis are provided, wherein the method comprises administering an antibody that binds CSF 1R and blocks CSF1 and IL-34 ligand binding, such as an antibody selected from huAbl to huAbl6, to a subject with multiple sclerosis. Treating multiple sclerosis, in some embodiments, means reducing clinical disease scores.

[021 1] In some embodiments, methods of decreasing CD 16+ monocytes are provided, wherein the methods comprise administering an antibody that binds CSF1R and blocks CSF1 and IL-34 ligand binding, such as an antibody selected from huAbl to huAbl6, to a subject with increased CD16+ monocytes. In some embodiments, the subject has an autoimmune condition selected from rheumatoid arthritis and SLE. In some embodiments, administering an antibody that binds CSF1R and blocks CSF1 and IL-34 ligand binding does not decrease the number of CD 16- monocytes. In some embodiments, CD 16+ monocytes are reduced to a greater extent than CD 16- monocytes are reduced when an antibody that binds CSF1R and blocks CSF 1 and IL-34 ligand binding is administered to the subject. In some embodiments, CD 16+ monocytes are reduced by at least 20%, at least 30%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. In some embodiments, CD 16- monocytes are reduced by less than 30%, less than 20%, or less than 10%. In some embodiments, the CD 16+ monocytes are CD 16+ peripheral blood monocytes. In some embodiments, the CD16- monocytes are CD16- peripheral blood monocytes.

Routes of Administration and Carriers

[0212] In various embodiments, anti-CSFIR antibodies may be administered in vivo by various routes, including, but not limited to, oral, intra-arterial, parenteral, intranasal, intramuscular, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, intradermal, topical, transdermal, and intrathecal, or otherwise by implantation or inhalation. The subject compositions may be formulated into preparations in solid, semi-solid, liquid, or gaseous forms; including, but not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants, and aerosols. A nucleic acid molecule encoding an anti-CSFIR antibody may be coated onto gold microparticles and delivered intradermally by a particle bombardment device, or "gene gun," as described in the literature (see, e.g., Tang et al, Nature 356: 152-154 (1992)). The appropriate formulation and route of administration may be selected according to the intended application.

[0213] In various embodiments, compositions comprising anti-CSFIR antibodies are provided in formulations with a wide variety of pharmaceutically acceptable carriers (see, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and

Comparisons: Drugfacts Pius, 20th ed. (2003); Ansel et al, Pharmaceutical Dosage Forms and Drug Delivery Systems, 7 ^th ed., Lippencott Williams and Wilkins (2004); Kibbe et al, Handbook of Pharmaceutical Excipients, 3 ^rd ed., Pharmaceutical Press (2000)). Various pharmaceutically acceptable carriers, which include vehicles, adjuvants, and diluents, are available. Moreover, various pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are also available. Non-limiting exemplary carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.

[0214] In various embodiments, compositions comprising anti-CSFIR antibodies may be formulated for injection, including subcutaneous administration, by dissolving, suspending, or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids, or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives. In various

embodiments, the compositions may be formulated for inhalation, for example, using pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen, and the like. The compositions may also be formulated, in various embodiments, into sustained release microcapsules, such as with biodegradable or non-biodegradable polymers. A non- limiting exemplary biodegradable formulation includes poly lactic acid-glycolic acid polymer. A non-limiting exemplary non-biodegradable formulation includes a polyglycerin fatty acid ester. Certain methods of making such formulations are described, for example, in EP 1 125 584 Al. [0215] Pharmaceutical packs and kits comprising one or more containers, each containing one or more doses of an anti-CSFIR antibody are also provided. In some embodiments, a unit dosage is provided wherein the unit dosage contains a predetermined amount of a composition comprising an anti-CSFIR antibody, with or without one or more additional agents. In some embodiments, such a unit dosage is supplied in single-use prefilled syringe for injection. In various embodiments, the composition contained in the unit dosage may comprise saline, sucrose, or the like; a buffer, such as phosphate, or the like; and/or be formulated within a stable and effective pH range. Alternatively, in some embodiments, the composition may be provided as a lyophilized powder that may be reconstituted upon addition of an appropriate liquid, for example, sterile water. In some embodiments, the composition comprises one or more substances that inhibit protein aggregation, including, but not limited to, sucrose and arginine. In some embodiments, a composition of the invention comprises heparin and/or a proteoglycan.

[0216] Pharmaceutical compositions are administered in an amount effective for treatment or prophylaxis of the specific indication. The therapeutically effective amount is typically dependent on the weight of the subject being treated, his or her physical or health condition, the extensiveness of the condition to be treated, or the age of the subject being treated. In general, anti-CSF IR antibodies may be administered in an amount in the range of about 10 μg/kg body weight to about 100 mg/kg body weight per dose. In some embodiments, anti-CSF IR antibodies may be administered in an amount in the range of about 50 μg/kg body weight to about 5 mg/kg body weight per dose. In some embodiments, anti-CSF IR antibodies may be administered in an amount in the range of about 100 μg/kg body weight to about 10 mg/kg body weight per dose. In some embodiments, anti-CSFIR antibodies may be administered in an amount in the range of about 100 μg/kg body weight to about 20 mg/kg body weight per dose. In some embodiments, anti-CSFIR antibodies may be administered in an amount in the range of about 0.5 mg/kg body weight to about 20 mg/kg body weight per dose.

[0217] The anti-CSFIR antibody compositions may be administered as needed to subjects. Determination of the frequency of administration may be made by persons skilled in the art, such as an attending physician based on considerations of the condition being treated, age of the subject being treated, severity of the condition being treated, general state of health of the subject being treated and the like. In some embodiments, an effective dose of an anti-CSF IR antibody is administered to a subject one or more times. In various embodiments, an effective dose of an anti-CSFIR antibody is administered to the subject once a month, less than once a month, such as, for example, every two months or every three months. In other embodiments, an effective dose of an anti-CSFIR antibody is administered more than once a month, such as, for example, every three weeks, every two weeks or every week. An effective dose of an anti-CSFIR antibody is administered to the subject at least once. In some embodiments, the effective dose of an anti-CSFIR antibody may be administered multiple times, including for periods of at least a month, at least six months, or at least a year.

Combination Therapy

[0218] Anti-CSFIR antibodies may be administered alone or with other modes of treatment. They may be provided before, substantially contemporaneous with, or after other modes of treatment, for example, surgery, chemotherapy, radiation therapy, or the administration of a biologic, such as another therapeutic antibody. For treatment of rheumatoid arthritis, anti-CSFIR antibodies may be administered with other therapeutic agents, for example, methotrexate, anti-TNF agents such as Remicade (infliximab), Humira (adalimumab), Simponi (golimumab), and Enbrel (etanercept); glucocorticoids such as prednisone; leflunomide; azothioprine; JAK inhibitors such as CP 590690; SYK inhibitors such as R788; anti-IL-6 antibodies; anti-IL-6R antibodies such as tocilizumab; anti-CD-20 antibodies such as rituximab; anti-CD 19 antibodies; anti-GM-CSF antibodies; anti-GM- CSF-R antibodies; IL-1 receptor antagonists such as anakinra; CTLA-4 antagonists, such as abatacept; immunosuppressants such as cyclosporine.

[0219] For treatment of systemic lupus erythematosus, anti-CSF IR antibodies may be administered with other therapeutic agents, for example, hydroxychloroquine (Plaquenil); corticosteroids, such as prednisone, methylprednisone, and prednisolone;

immunosuppressants, such as cyclophosphamide (Cytoxan), azathioprine (Imuran, Azasan), mycophenolate (Cellcept), leflunomide (Arava) and methotrexate (Trexall), and belimumab (Benlysta).

[0220] For treatment of multiple sclerosis, anti-CSFIR antibodies may be administered with other therapeutic agents, for example, interferon alpha; interferon beta; prednisone; anti-alpha4 integrin antibodies such as Tysabri; anti-CD20 antibodies such as Rituxan; FTY720 (Fingolimod); and Cladribine (Leustatin).

EXAMPLES

[0221] The examples discussed below are intended to be purely exemplary of the invention and should not be considered to limit the invention in any way. The examples are not intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (for example, amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

Example 1: Humanized anti-CSFIR antibodies

[0222] Various humanized anti-CSFIR antibodies were developed previously. See, e.g., PCT Publication No. WO 2011/140249.

[0223] The sequences for each of the humanized heavy chain variable regions and humanized light chain variable regions, aligned with the sequences of the parental chimeric antibody variable regions and the sequences of the human acceptor variable framework regions are shown in Figures 1 (heavy chains) and 2 (light chains). The changes in humanized variable region sequences relative to the human acceptor variable framework region sequences are boxed. Each of the CDRs for each of the variable regions is shown in a boxed region, and labeled as "CDR" above the boxed sequences.

[0224] Table 5, below, shows the full sequences for the humanized heavy chains and humanized light chains of antibodies huAbl to huAbl6. The name and SEQ ID NOs of the humanized heavy chain and humanized light chain of each of those antibodies is shown in

Table 3.

Table 3: Humanized heavy chains and light chains of huAbl to huAb!6

huAM2 h0302-H2 57 h0302-L2 64

huAM3 h0311-Hl 58 h0311-L0 65

huAM4 h0311-Hl 58 h0311-Ll 66

huAbl5 h0311-H2 59 h0311-L0 65

huAbl6 h0311-H2 59 h0311-Ll 66

[0225] The 16 humanized antibodies were tested for binding to human, cynomolgus monkey, and mouse CSFIR ECD, as described previously. See, e.g., PCT Publication No. WO 201 1/140249. The antibodies were found to bind to both human and cynomolgus monkey CSFIR ECD, but not to mouse CSFIR ECD. The humanized antibodies were also found to block binding of CSFl and IL-34 to both human and mouse CSFIR and to inhibit CSFl -induced and IL-34-induced phosphorylation of human CSFIR expressed in CHO cells. See, e.g., PCT Publication No. WO 201 1/140249.

[0226] The k _a , kj, and K _D for binding to human CSFIR ECD were previously determined and are shown in Table 4. See, e.g., PCT Publication No. WO 201 1/140249. Table 4: Humanized antibody binding affinity for human CSFIR

Example 2: HuAbl decreases CD16+ but not CD16- peripheral blood monocytes in cynomolgus monkeys

[0227] 0 mg/kg, 3 mg/kg, 10 mg/kg, or 30 mg/kg huAbl was administered intravenously to cynomolgus monkeys (2 monkeys per group). Peripheral blood was drawn 14 days later, and CD 16+ or CD 16- monocyte numbers were analyzed by flow cytometry. Briefly, 0.2 ml peripheral blood was labeled with 20 μΐ anti-CD 16-PE (Becton-Dickenson) for 30 minutes at room temperature in the dark. Erythrocytes were lysed by incubation with 2 ml of 1 X BD PharmLyse (Becton-Dickenson) for 20 min, and cells were washed with lml FACS buffer (PBS/0.1% BSA) prior to analysis. Data was collected on an LSRII flow cytometer using the Diva software program (Becton-Dickenson), and results were analyzed using FloJo data analysis program (Tree Star).

[0228] Figure 3 shows that treatment of monkeys with huAbl resulted in a dose- dependent decrease in CD 16+ but not CD 16- peripheral blood monocytes compared to untreated animals. These data show that huAbl can reduce monocytes that are mainly response for autoimmune disorders while leaving other subsets of monocytes that are responsible for pathogen clearance intact. This suggests that an antibody that binds CSFIR and blocks ligand binding, such as huAbl, may be effective for treating autoimmune disorders while maintaining a good safety profile with respect to infections.

Example 3: Characterization of a surrogate anti-mouse CSFIR antibody

[0229] Because humanized antibodies huAbl to huAbl6 do not bind to mouse CSFIR, a chimeric anti-mouse CSFIR antibody ("cAbl") was selected as a surrogate antibody for use in mouse models. The antibody comprises rat variable regions and mouse constant regions. To generate cAbl, rats were immunized with a mouse CSFIR-ECD-Fc fusion protein, and hybridomas secreting antibodies that blocked binding of both mouse CSF1 and mouse IL-34 to mouse CSFIR were identified by ELISA assay. The variable regions of one such blocking antibody were grafted onto mouse IgG constant regions using standard molecular biologic techniques (Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Third Edition, CSH Press, Cold Spring Harbor, New York, 2001). Recombinant chimeric antibody cAbl was then expressed in CHO cells using standard techniques (Sambrook and Russell, Ibid.).

[0230] Binding of cAbl to mouse CSF IR was determined as follows. Ninety-six well clear-bottom ELISA plates were coated overnight with 1 μg/ml recombinant mouse CSFIR ECD-Fc Chimera (R&D Systems) in PBS. The next morning, wells were washed four times with 0.05% Tween20 in PBS (PBST) and blocked with Blocker-Blotto (Pierce). Fifty μΐ of 0.5x serial dilutions of cAbl, starting with a concentration of 2.6 μg/ml, diluted 1 : 1 in Blocker-Blotto were added to the CSFlR-coated wells. After incubation at room temperature (RT) for 90 min, wells were washed four times with PBST, and a 1 :5000 dilution of a peroxidase-conjugated goat anti-mouse IgG (Sigma) in Blocker-Blotto was added to each well. After incubation at RT for 60 min, wells were washed four times with PBST, and 50 μΐ o-phenylenediamine dihydrochloride peroxidase substrate (Sigma) was added to each well. After incubation at RT for 30 min, A450 values of each well were read directly on a SpectraMaxPlus spectrophotometer with SoftMaxPro software (Molecular Devices).

[0231] The results of that experiment are shown in Figure 4. cAbl bound to mouse

CSF1R ECD in a dose-dependent manner.

Example 4: cAbl blocks ligand-induced proliferation of a factor dependent cell line

[0232] cAbl was tested for the ability to block ligand-induced proliferation of the factor dependent mouse cell line mNFS60 as follows. mNFS60 cells were stimulated with 10 ng/ml recombinant mouse CSF1 or 100 ng/ml recombinant mouse IL-34 (both from R&D Systems) in the presence or absence of serial dilutions of cAbl. After incubation at 37°C for 48 hours, relative cellular ATP content of each individual culture was assessed using Cell Titer Glo reagent (Promega) according to the manufacturer's instructions. In this assay, relative cellular ATP content is directly proportional to the number of viable cells in culture, and thus reflects the mNFS60 cell proliferation response.

[0233] The results of that experiment are shown in Figure 5. cAbl blocked CSF1 or

IL-34 induced proliferation of mNFS60 cells in a dose-dependent manner.

Example 5: cAbl suppresses Clinical Disease Scores in a mouse model of rheumatoid arthritis

[0234] Arthritis was induced in male DBA/1 mice by intradermal injection of 150 μΐ of 2 mg/ml bovine Type II collagen (Elastin Products, Owensville, MO) in Freund's complete adjuvant (Sigma) on study day 0 and again on study day 21. Mice were randomized into treatment groups on study day 0 and dosing was initiated. Mice were dosed intraperitoneally 3 times per week (3x/wk) with vehicle or with cAbl (3, 6, 10, or 30 mg/kg) through day 32 of the study, and Clinical Disease Scores were measured for each paw on study days 18 to 35. Clinical Disease Scores were assigned as follows:

0 = normal

1 = 1 hind or fore paw joint affected or minimal diffuse erythema and swelling

2 = 2 hind or fore paw joints affected or mild diffuse erythema and swelling 3 = 3 hind or fore paw joints affected or moderate diffuse erythema and swelling

4 = 4 hind or fore paw joints affected or marked diffuse erythema and swelling

5 = Entire paw affected, severe diffuse erythema and severe swelling, unable to flex digits

[0235] Figure 6 shows the results of that experiment. cAbl suppressed Clinical Disease Scores in the Collagen Induced Arthritis model of rheumatoid arthritis. These data suggest that in human patients, an antibody that binds CSF1R and blocks ligand binding, such as huAbl, may be effective for reducing the signs and symptoms of rheumatoid arthritis.

Example 6: cAbl suppresses bone loss in a mouse model of rheumatoid arthritis

[0236] High levels of Tartrate Resistant Acid Phosphatase 5b (TRAP5b) are associated with bone remodeling, and have been observed in various conditions

characterized by increased bone resorption. TRAP5b levels can therefore be used as a marker for bone resorption (i.e., bone loss).

[0237] Arthritis was induced in mice, and mice were treated with cAbl as described in Example 5. Blood was collected at day 35, and plasma levels of TRAP5b (Tartrate Resistant Acid Phosphatase 5b) were measured using a commercial solid phase

immunofixed enzyme activity assay for the determination of osteoclast-derived TRAP5b in mouse serum (Immunodiagnostics Systems) according to the manufacturer's instructions. TRAP5b is a marker for bone turnover.

[0238] Figure 7 shows the results of that experiment. cAbl suppressed bone loss in the collagen induced arthritis mode of rheumatoid arthritis, as measured by the suppression of the TRAP5b bone turnover marker. These data suggest that in human patients, an antibody that binds CSF 1R and blocks ligand binding, such as huAbl, may suppress bone erosions and joint destruction in rheumatoid arthritis.

Example 7: cAbl suppresses inflammation, cartilage damage, pannus formation, and bone destruction in a mouse model of rheumatoid arthritis

[0239] Arthritis was induced in mice, and mice were treated with cAbl as described in Example 5. At the end of the study, paws and knees were collected and placed into neutral buffered formalin (NBF; available from Sigma-Aldrich, cat # HT-501128) overnight and then moved to 70% ethanol for histopathology and immunohistochemistry.

[0240] Six joints from each animal were processed for histopathologic evaluation. The joints were then scored as follows: Inflammation

0=Normal

l=Minimal infiltration of inflammatory cells in synovium and periarticular tissue of affected joints

2=Mild infiltration of inflammatory cells. If referring to paws, generally restricted to affected joints (1-3 affected)

3=Moderate infiltration with moderate edema. If referring to paws, restricted to affected joints, generally 3-4 joints, including at least one wrist or ankle joint

4=Marked infiltration affecting most areas with marked edema, 1 or 2 unaffected joints may be present

5=Severe diffuse infiltration with severe edema affecting all joints and periarticular tissues

Pannus

0=Normal

l=Minimal infiltration of pannus in cartilage and subchondral bone, marginal zones

2=Mild infiltration with marginal zone destruction of hard tissue in affected joints

3=Moderate infiltration with moderate hard tissue destruction in affected joints

4=Marked infiltration with marked destruction of joint architecture, affecting most joints

5=Severe infiltration associated with total or near total destruction of joint architecture, affects all joints

Cartilage Damage

0=Normal

l=Minimal=generally minimal to mild loss of toluidine blue staining with no obvious chondrocyte loss or collagen disruption in affected joints

2=Mild=generally mild loss of toluidine blue staining with focal areas of chondrocyte loss and/or collagen disruption in some affected joints

3=Moderate=generally moderate loss of toluidine blue staining with multifocal chondrocyte loss and/or collagen disruption in affected joints, some matrix remains on any affected surface with areas of severe matrix loss

4=Marked=marked loss of toluidine blue staining with multifocal marked (depth to deep zone) chondrocyte loss and/or collagen disruption in most joints, if knee-one surface with total to near total cartilage loss

5=Severe=severe diffuse loss of toluidine blue staining with multifocal severe (depth to tide mark) chondrocyte loss and/or collagen disruption in all joints, if knee-2 or more surfaces with total to near total cartilage loss

Bone Resorption 0=Normal

l=Minimal=small areas of resorption, not readily apparent on low magnification, rare osteoclasts in affected joints, restricted to marginal zones

2=Mild=more numerous areas of resorption, not readily apparent on low magnification, osteoclasts more numerous in affected joints, restricted to marginal zones

3=Moderate=obvious resorption of medullary trabecular and cortical bone without full thickness defects in cortex, loss of some medullary trabeculae, lesion apparent on low magnification, osteoclasts more numerous in affected joints

4=Marked=Full thickness defects in cortical bone, often with distortion of profile of remaining cortical surface, marked loss of medullary bone, numerous osteoclasts, affects most joints

5=Severe=Full thickness defects in cortical bone and destruction of joint architecture of all joints

A sum total histologic score for each parameter for all six joints was calculated.

[0241] Figure 8 shows the results of that experiment. cAbl suppressed

inflammation, cartilage damage, pannus formation, and bone destruction in the Collagen

Induced Arthritis model of rheumatoid arthritis.

Example 8: cAbl suppresses paw and knee joint macrophage numbers in a mouse model of rheumatoid arthritis

[0242] Arthritis was induced in mice, and mice were treated with cAb 1 as described in Example 5, and paw and knee joint sections were prepared as described in Example 7. Paw and knee joint sections were stained with a peroxidase conjugated anti-F4/80 antibody, and macrophage numbers were enumerated by counting positive staining cells under a microscope. Tissue sectioning and staining were done by Histotox Labs, Boulder, CO. F4/80 is a mouse macrophage marker (Leenen et al, J. Immunol. Methods, 1994, 174: 5- 19).

[0243] The results of that experiment are shown in Figure 9. cAbl reduced both paw and knee joint macrophage numbers in the Collagen Induced Arthritis model of rheumatoid arthritis.

Example 9: cAbl suppresses autoantibody formation in a mouse model of rheumatoid arthritis

[0244] Arthritis was induced in mice, and mice were treated with cAb 1 as described in Example 5. Blood was collected at day 35, and plasma levels of anti-mouse collagen II antibodies were measured using a mouse anti-type 1 and type2 collagen IgG assay kit (Chondrex) according to the manufacturer's instructions. [0245] The results of that experiment are shown in Figure 10. cAbl suppressed the formation of anti-mouse collagen II autoantibodies in the Collagen Induced Arthritis mode of rheumatoid arthritis. These data suggest that in human patients, an antibody that binds

CSF1R and blocks ligand binding, such as huAbl, may reduce an important trigger of joint inflammation, namely autoantibodies. In addition, autoantibodies such as RF and ACPA may, in some embodiments, be used to monitor drug effect.

Example 10: cAbl suppresses bone loss in a mouse model of established rheumatoid arthritis

[0246] Arthritis was induced in mice as described in Example 5. Mice were treated intraperitoneally 3x/week with 30 mg/kg cAbl starting when clinical disease scores reached 0.05 to 1.0. Blood was collected 23 days after initial treatment with cAbl, and plasma levels of TRAP5b (Tartrate Resistant Acid Phosphatase 5b) were measured using a commercial solid phase immunofixed enzyme activity assay for the determination of osteoclast-derived TRAP5b in mouse serum (Immunodiagnostics Systems) according to the manufacturer's instructions. TRAP5b is a marker for bone turnover.

[0247] The results of that experiment are shown in Figure 11. cAbl administered after development of clinical disease scores suppressed bone loss in the Collagen Induced

Arthritis mode of rheumatoid arthritis as measured by the suppression of the TRAP5b bone turnover marker. These data suggest that in human patients, an antibody that binds CSF1R and blocks ligand binding, such as huAbl, may suppress bone erosion and joint destruction in rheumatoid arthritis.

Example 11: cAbl suppresses pannus formation and bone destruction in a mouse model of established rheumatoid arthritis

[0248] Arthritis was induced in mice, and mice were treated with cAbl as described in Example 10. At the end of the study paws and knees were collected and placed into neutral buffered formalin ( BF) overnight and then moved to 70% ethanol for

histopathology and immunohistochemistry.

[0249] Six joints from each animal were processed for histopathologic evaluation, and a sum total histologic score for all six joints was calculated. The joints were scored as described in Example 7.

[0250] The results of that experiment are shown in Figure 12. cAbl administered after development of clinical disease scores suppressed pannus formation, and bone destruction in the Collagen Induced Arthritis model of rheumatoid arthritis. Histologic scores of inflammation and cartilage damage were unaffected. These data also suggest that in human patients, an antibody that binds CSFIR and blocks ligand binding, such as huAbl, may suppress bone erosion and joint destruction in rheumatoid arthritis.

Example 12: cAbl suppresses glomerulonephritis, interstitial nephritis, and perivascular infiltrates in a mouse model of systemic lupus erythematosus

[0251] A subset of lupus patients develop lupus nephritis, which involves inflammation in one or more structures in the kidney and can lead to kidney failure. Lupus nephritis may detected in patients and severity of disease assessed , for example, by measuring proteinuria, or protein in the urine, and/or determining the glomerular filtration rate, which is a measurement of how much fluid is filtered through the kidneys per unit time. Lupus nephritis may also be detected histologically through examination of kidney biopsies, or through imaging techniques such as ultrasonography (Hahn et al, Arthr. Care Res., 2012, 64: 797-808).

[0252] The MRL/MpJ- as^/J strain of mice spontaneously develops lupus-like symptoms, including glomerular, tubulointerstitial, and perivascular kidney disease;

lymphadenopathy; splenomegaly; circulating autoantibodies; and skin lesions. Female MRL/MpJ-i ^/J mice were dosed intraperitoneally every day for 10 weeks from the age of 10 weeks to the age of 20 weeks with vehicle or with 50 mg/kg cAbl. At the end of the study (mouse age 20 weeks), kidneys were collected, weighed, and placed in 10% neutral buffered formalin (NBF) for Histopathologic evaluation. Kidneys were scored according to the following system:

Glomerulonephritis

0 = Normal.

1 = Focal or multifocal, minimal to mild, early proliferative.

2 = Multifocal mild to moderate or mild diffuse proliferative.

3 = Diffuse moderate proliferative with or without multifocal severe areas.

4 = Marked diffuse proliferative, with crescents/sclerosis, multifocal protein casts present.

5 = Severe diffuse proliferative, with crescents/sclerosis, diffuse protein casts present.

Protein Cast Severity

0 = Normal tubules.

1 = Minimal = One to 5 profiles of tubules with protein casts.

2 = Mild = Multifocal tubules with protein casts (6-20).

3 = Moderate = Multifocal tubules with protein casts (21-40). 4 = Marked = Multifocal tubules with protein casts (>40 but affects less than 50% of all tubules).

5 = Severe = Diffuse tubules with protein casts, affects greater than 50% of all Tubules.

Interstitial Nephritis (inflammation not obviously associated with vessels)

0 = Normal.

0.5 = Very Minimal = Small focal area of MNC in pelvis only.

1 = Minimal = Occasional small focal, accumulations of MNC: affects less than 10% of total interstitium and generally localized around pelvis.

2 = Mild = Multifocal small to larger infiltrates distributed around pelvis and cortex: affects 10-25% of cortex area.

3 = Moderate = Multifocal small to extensive infiltrates in pelvis and cortex: affects 26-50% of cortex area.

4 = Marked = Multifocal to diffuse infiltration: affects 51-75% of pelvis and cortex area.

5 = Severe = Diffuse infiltration: affects 76-100% of pelvis and cortex area.

Vessels

0 = Normal.

0.5 = Very Minimal = One vessel with minimal perivascular infiltrate.

1 = Minimal = Small but definite perivascular infiltrates (1-2).

2 = Mild = Several (3-4) foci of perivascular infiltrate, no necrosis.

3 = Moderate = Multifocal (5-6) foci of perivascular infiltrate, more extensive, may have some necrosis of vessel wall.

4 = Marked = Multifocal (7-8) foci of perivascular infiltrate, extensive with necrosis.

5 = Severe = Multifocal (>8) foci of perivascular infiltrate, extensive with necrosis.

[0253] The results of that experiment are shown in Figure 13. cAbl suppressed glomerulonephritis, intersitital nephritis, and perivascular infiltrates in the MRL/lpr model of lupus. Histologic scores of protein cast severity were unaffected. These data suggest that in human patients, an antibody that binds CSFIR and blocks ligand binding, such as huAbl, may reduce renal inflammation associated with systemic lupus erythematosus and restore renal function.

Example 13: cAbl suppresses skin lesions in a mouse model of systemic lupus erythematosus

[0254] Female MRL/MpJ-i ^/J mice were dosed with cAb 1 described in Example 12. Mice were scored for the presence and severity of skin lesions from the age of 10 weeks through the age of 20 weeks. Skin lesions were scored as follows: 0=none

l=mild (snout and ears)

2=moderate (<2 cm snout, ears, and intrascapular)

3=severe (>2 cm snout, ears, and intrascapular)

[0255] The results of that experiment are shown in Figure 14. cAbl suppressed the incidence and severity of skin lesions in the MRL/lpr model of lupus. These data suggest that in human patients, an antibody that binds CSF1R and blocks ligand binding, such as huAbl, may reduce the systemic manifestations of SLE, including skin inflammation, rash, and discoid lesions.

Example 14: cAbl suppresses Clinical Disease Scores in a mouse model of multiple sclerosis

[0256] Experimental autoimmune encephalitis (EAE) was induced in female C57B1/6 mice by subcutaneous injection of 300 ng myelin oligodendrocyte glycoprotein peptide 35-55 (MOG 35-55, Sigma-Aldrich, Cat # M4939) in complete Freund's adjuvant on study day 0 and again on study day 7. To enhance the induction and consistency of EAE, 500 ng pertussis toxin (List Biological Laboratories, Cat # 180) was administered intraperitoneally on days 0 and 2. EAE is a model of multiple sclerosis. Mice were randomized into treatment groups on study day 0 and dosing was initiated. Mice were dosed intraperitoneally 3 times per week (3x/wk) with vehicle or with 30 mg/kg cAbl through day 15 of the study. Clinical Disease Scores were measured on study days 6-15. Clinical Disease Scores were assigned as follows:

0.0 = No clinical signs

0.5 = Partially limp tail

1.0 = Paralyzed tail

2.0 = Loss in coordinated movement; hind limb paresis

2.5 = One hind limb paralyzed

3.0 = Both hind limbs paralyzed

3.5 = Hind limbs paralyzed; weakness in forelimbs

4.0 = Forelimbs and hindlimbs paralyzed

5.0 = Moribund

[0257] The results of that experiment are shown in Figure 15. cAbl suppressed Clinical Disease Scores in the Experimental Autoimmune Encephalitis model of multiple sclerosis. These data suggest that in human patients, an antibody that binds CSF1R and blocks ligand binding, such as huAbl, may reduce inflammation in the CNS associated with multiple sclerosis and may reverse paralysis caused by MS-associated demyelination and neuronal damage.

TABLE OF SEQUENCES

[0258] Table 5 provides certain sequences discussed herein. All polypeptide and antibody sequences are shown without leader sequences, unless otherwise indicated.

Table 5: Sequences and Descriptions

SEQ ID Description Sequence

NO

IPVIEPSVPE LVVKPGATVT LRCVGNGSVE WDGPPSPHWT LYSDGSSSIL STNNATFQNT GTYRCTEPGD PLGGSAAIHL YVKDPARPWN VLAQEWVFE DQDALLPCLL TDPVLEAGVS LVRVRGRPLM RHTNYSFSPW HGFTIHRAKF IQSQDYQCSA LMGGRKVMS I SIRLKVQKVI PGPPALTLVP AELVRIRGEA AQIVCSASSV DVNFDVFLQH NNTKLAIPQQ SDFHNNRYQK VLTLNLDQVD FQHAGNYSCV ASNVQGKHST SMFFRVVESA YLNLSSEQNL IQEVTVGEGL NLKVMVEAYP GLQGFNWTYL GPFSDHQPEP KLANATTKDT YRHTFTLSLP RLKPSEAGRY SFLARNPGGW RALTFELTLR YPPEVSVIWT FINGSGTLLC

hCSFIR AASGYPQPNV TWLQCSGHTD RCDEAQVLQV WDDPYPEVLS QEPFHKVTVQ (full-length, SLLTVETLEH NQTYECRAHN SVGSGSWAFI PISAGAHTHP PDEFLFTPVV no leader VACMS IMALL LLLLLLLLYK YKQKPKYQVR WKI IESYEGN SYTFIDPTQL sequence) PYNEKWEFPR NNLQFGKTLG AGAFGKWEA TAFGLGKEDA VLKVAVKMLK

STAHADEKEA LMSELKIMSH LGQHENIVNL LGACTHGGPV LVITEYCCYG DLLNFLRRKA EAMLGPSLSP GQDPEGGVDY KNIHLEKKYV RRDSGFSSQG VDTYVEMRPV STSSNDSFSE QDLDKEDGRP LELRDLLHFS SQVAQGMAFL ASKNCIHRDV AARNVLLTNG HVAKIGDFGL ARDIMNDSNY IVKGNARLPV KWMAPES IFD CVYTVQSDVW SYGILLWEIF SLGLNPYPGI LVNSKFYKLV KDGYQMAQPA FAPKNIYSIM QACWALEPTH RPTFQQICSF LQEQAQEDRR ERDYTNLPSS SRSGGSGSSS SELEEESSSE HLTCCEQGDI AQPLLQPNNY QFC

MGPGVLLLLL VATAWHGQGI PVIEPSVPEL VVKPGATVTL RCVGNGSVEW DGPPSPHWTL YSDGSSSILS TNNATFQNTG TYRCTEPGDP LGGSAAIHLY VKDPARPWNV LAQEWVFED QDALLPCLLT DPVLEAGVSL VRVRGRPLMR HTNYSFSPWH GFTIHRAKFI QSQDYQCSAL MGGRKVMSIS IRLKVQKVIP GPPALTLVPA ELVRIRGEAA QIVCSASSVD VNFDVFLQHN NTKLAIPQQS DFHNNRYQKV LTLNLDQVDF QHAGNYSCVA SNVQGKHSTS MFFRVVESAY LNLSSEQNLI QEVTVGEGLN LKVMVEAYPG LQGFNWTYLG PFSDHQPEPK LANATTKDTY RHTFTLSLPR LKPSEAGRYS FLARNPGGWR ALTFELTLRY

hCSFIR PPEVSVIWTF INGSGTLLCA ASGYPQPNVT WLQCSGHTDR CDEAQVLQVW (full-length, DDPYPEVLSQ EPFHKVTVQS LLTVETLEHN QTYECRAHNS VGSGSWAFIP + leader ISAGAHTHPP DEFLFTPVVV ACMS IMALLL LLLLLLLYKY KQKPKYQVRW sequence) KIIESYEGNS YTFIDPTQLP YNEKWEFPRN NLQFGKTLGA GAFGKWEAT

AFGLGKEDAV LKVAVKMLKS TAHADEKEAL MSELKIMSHL GQHENIVNLL GACTHGGPVL VITEYCCYGD LLNFLRRKAE AMLGPSLSPG QDPEGGVDYK NIHLEKKYVR RDSGFSSQGV DTYVEMRPVS TSSNDSFSEQ DLDKEDGRPL ELRDLLHFSS QVAQGMAFLA SKNCIHRDVA ARNVLLTNGH VAKIGDFGLA RDIMNDSNYI VKGNARLPVK WMAPESIFDC VYTVQSDVWS YGILLWEIFS LGLNPYPGIL VNSKFYKLVK DGYQMAQPAF APKNIYSIMQ ACWALEPTHR PTFQQICSFL QEQAQEDRRE RDYTNLPSSS RSGGSGSSSS ELEEESSSEH LTCCEQGDIA QPLLQPNNYQ FC

hCSFIR IPVIEPSVPE LVVKPGATVT LRCVGNGSVE WDGPPSPHWT LYSDGSSSIL ECD.506 STNNATFQNT GTYRCTEPGD PLGGSAAIHL YVKDPARPWN VLAQEWVFE

DQDALLPCLL TDPVLEAGVS LVRVRGRPLM RHTNYSFSPW HGFTIHRAKF IQSQDYQCSA LMGGRKVMS I SIRLKVQKVI PGPPALTLVP AELVRIRGEA AQIVCSASSV DVNFDVFLQH NNTKLAIPQQ SDFHNNRYQK VLTLNLDQVD FQHAGNYSCV ASNVQGKHST SMFFRVVESA YLNLSSEQNL IQEVTVGEGL NLKVMVEAYP GLQGFNWTYL GPFSDHQPEP KLANATTKDT YRHTFTLSLP RLKPSEAGRY SFLARNPGGW RALTFELTLR YPPEVSVIWT FINGSGTLLC AASGYPQPNV TWLQCSGHTD RCDEAQVLQV WDDPYPEVLS QEPFHKVTVQ SLLTVETLEH NQTYECRAHN SVGSGSWAFI PISAGAH

IPVIEPSVPE LVVKPGATVT LRCVGNGSVE WDGPPSPHWT LYSDGSSSIL STNNATFQNT GTYRCTEPGD PLGGSAAIHL YVKDPARPWN VLAQEWVFE DQDALLPCLL TDPVLEAGVS LVRVRGRPLM RHTNYSFSPW HGFTIHRAKF IQSQDYQCSA LMGGRKVMS I SIRLKVQKVI PGPPALTLVP AELVRIRGEA AQIVCSASSV DVNFDVFLQH NNTKLAIPQQ SDFHNNRYQK VLTLNLDQVD FQHAGNYSCV ASNVQGKHST SMFFRVVESA YLNLSSEQNL IQEVTVGEGL NLKVMVEAYP GLQGFNWTYL GPFSDHQPEP KLANATTKDT YRHTFTLSLP

hCSFIR RLKPSEAGRY SFLARNPGGW RALTFELTLR YPPEVSVIWT FINGSGTLLC ECD.506-Fc AASGYPQPNV TWLQCSGHTD RCDEAQVLQV WDDPYPEVLS QEPFHKVTVQ

SLLTVETLEH NQTYECRAHN SVGSGSWAFI PISAGAHEPK SSDKTHTCPP CPAPELLGGP SVFLFPPKPK DTLMISRTPE VTCWVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSRDEL TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK

MGPGVLLLLL WTAWHGQGI PVIEPSGPEL VVKPGETVTL RCVGNGSVEW DGPISPHWTL YSDGPSSVLT TTNATFQNTR TYRCTEPGDP LGGSAAIHLY VKDPARPWNV LAKEWVFED QDALLPCLLT DPVLEAGVSL VRLRGRPLLR HTNYSFSPWH GFTIHRAKFI QGQDYQCSAL MGSRKVMSIS IRLKVQKVIP

cynoCSFIR

GPPALTLVPA ELVRIRGEAA QIVCSASNID VDFDVFLQHN TTKLAIPQRS ECD (with

DFHDNRYQKV LTLSLGQVDF QHAGNYSCVA SNVQGKHSTS MFFRVVESAY

leader

LDLSSEQNLI QEVTVGEGLN LKVMVEAYPG LQGFNWTYLG PFSDHQPEPK

sequence) LANATTKDTY RHTFTLSLPR LKPSEAGRYS FLARNPGGWR ALTFELTLRY

PPEVSVIWTS INGSGTLLCA ASGYPQPNVT WLQCAGHTDR CDEAQVLQVW VDPHPEVLSQ EPFQKVTVQS LLTAETLEHN QTYECRAHNS VGSGSWAFIP ISAGAR

MGPGVLLLLL WTAWHGQGI PVIEPSGPEL VVKPGETVTL RCVGNGSVEW DGPISPHWTL YSDGPSSVLT TTNATFQNTR TYRCTEPGDP LGGSAAIHLY VKDPARPWNV LAKEWVFED QDALLPCLLT DPVLEAGVSL VRLRGRPLLR HTNYSFSPWH GFTIHRAKFI QGQDYQCSAL MGSRKVMSIS IRLKVQKVIP GPPALTLVPA ELVRIRGEAA QIVCSASNID VDFDVFLQHN TTKLAIPQRS DFHDNRYQKV LTLSLGQVDF QHAGNYSCVA SNVQGKHSTS MFFRVVESAY

cynoCSFIR

LDLSSEQNLI QEVTVGEGLN LKVMVEAYPG LQGFNWTYLG PFSDHQPEPK ECD-Fc LANATTKDTY RHTFTLSLPR LKPSEAGRYS FLARNPGGWR ALTFELTLRY (with leader

PPEVSVIWTS INGSGTLLCA ASGYPQPNVT WLQCAGHTDR CDEAQVLQVW

sequence) VDPHPEVLSQ EPFQKVTVQS LLTAETLEHN QTYECRAHNS VGSGSWAFIP

ISAGARGSEP KSSDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVWDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK

Light chain

leader METDTLLLWV LLLWVPGSTG

sequence

Heavy chain

leader MAVLGLLLCL VTFPSCVLS

sequence

Fab 0301 EVQLQQSGPE LVRPGASVKM SCKASGYTFT DNYMIWVKQS HGKSLEWIGD heavy chain INPYNGGTTF NQKFKGKATL TVEKSSSTAY MQLNSLTSED SAVYYCARES variable PYFSNLYVMD YWGQGTSVTV SS

region

Fab 0301

NIVLTQSPAS LAVSLGQRAT ISCKASQSVD YDGDNYMNWY QQKPGQPPKL

light chain

LIYAASNLES GIPARFSGSG SGTDFTLNIH PVEEEDAATY YCHLSNEDLS

variable TFGGGTKLEI K

region

Fab 0302

EIQLQQSGPE LVKPGASVKM SCKASGYTFS DFNIHWVKQK PGQGLEWIGY

heavy chain INPYTDVTVY NEKFKGKATL TSDRSSSTAY MDLSSLTSED SAVYYCASYF variable DGTFDYALDY WGQGTSITVS S

region

Fab 0302

DWVTQTPAS LAVSLGQRAT ISCRASESVD NYGLSFMNWF QQKPGQPPKL

light chain

LIYTASNLES GIPARFSGGG SRTDFTLTID PVEADDAATY FCQQSKELPW

variable TFGGGTRLEI K

region

Fab 0311

EIQLQQSGPD LMKPGASVKM SCKASGYIFT DYNMHWVKQN QGKSLEWMGE

heavy chain INPNNGVWY NQKFKGTTTL TVDKSSSTAY MDLHSLTSED SAVYYCTRAL variable YHSNFGWYFD SWGKGTTLTV SS

region

Fab 0311

DIVLTQSPAS LAVSLGQRAT ISCKASQSVD YDGDSHMNWY QQKPGQPPKL

light chain

LIYTASNLES GIPARFSGSG SGADFTLTIH PVEEEDAATY YCQQGNEDPW

variable TFGGGTRLEI K

region

0301 heavy GYTFTDNYMI

chain CDR1

0301 heavy DINPYNGGTT FNQKFKG

chain CDR2

0301 heavy ESPYFSNLYV MDY

chain CDR3

0301 light

KASQSVDYDG DNYMN

chain CDR1

0301 light

AASNLES

chain CDR2

0301 light

HLSNEDLST

chain CDR3

0302 heavy GYTFSDFNIH

chain CDR1

0302 heavy YINPYTDVTV YNEKFKG

chain CDR2

0302 heavy YFDGTFDYAL DY

chain CDR3

0302 light

RASESVDNYG LSFMN

chain CDR1

0302 light

TASNLES

chain CDR2

0302 light

QQSKELPWT

chain CDR3

0311 heavy GYIFTDYNMH

chain CDR1 0311 heavy

EINPNNGWV YNQKFKG

chain CDR2

0311 heavy ALYHSNFGWY FDS

chain CDR3

0311 light

KASQSVDYDG DSHMN

chain CDR1

0311 light

TASNLES

chain CDR2

0311 light

QQGNEDPWT

chain CDR3

EVQLQQSGPE LVRPGASVKM SCKASGYTFT DNYMIWVKQS HGKSLEWIGD INPYNGGTTF NQKFKGKATL TVEKSSSTAY MQLNSLTSED SAVYYCARES PYFSNLYVMD YWGQGTSVTV SSASTKGPSV FPLAPCSRST SESTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT

cAb 0301

KTYTCNVDHK PSNTKVDKRV ESKYGPPCPP CPAPEFLGGP SVFLFPPKPK

heavy chain DTLMISRTPE VTCVWDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNS

TYRWSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK

NIVLTQSPAS LAVSLGQRAT ISCKASQSVD YDGDNYMNWY QQKPGQPPKL LIYAASNLES GIPARFSGSG SGTDFTLNIH PVEEEDAATY YCHLSNEDLS

cAb 0301

TFGGGTKLEI KRTVAAPSVF IFPPSDEQLK SGTASWCLL NNFYPREAKV

light chain

QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC

EIQLQQSGPE LVKPGASVKM SCKASGYTFS DFNIHWVKQK PGQGLEWIGY INPYTDVTVY NEKFKGKATL TSDRSSSTAY MDLSSLTSED SAVYYCASYF DGTFDYALDY WGQGTSITVS SASTKGPSVF PLAPCSRSTS ESTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTK

cAb 0302

TYTCNVDHKP SNTKVDKRVE SKYGPPCPPC PAPEFLGGPS VFLFPPKPKD

heavy chain TLMISRTPEV TCWVDVSQE DPEVQFNWYV DGVEVHNAKT KPREEQFNST

YRVVSVLTVL HQDWLNGKEY KCKVSNKGLP SSIEKTISKA KGQPREPQVY TLPPSQEEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSR LTVDKSRWQE GNVFSCSVMH EALHNHYTQK SLSLSLGK

DWVTQTPAS LAVSLGQRAT ISCRASESVD NYGLSFMNWF QQKPGQPPKL LIYTASNLES GIPARFSGGG SRTDFTLTID PVEADDAATY FCQQSKELPW

cAb 0302

TFGGGTRLEI KRTVAAPSVF IFPPSDEQLK SGTASWCLL NNFYPREAKV

light chain

QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC

EIQLQQSGPD LMKPGASVKM SCKASGYIFT DYNMHWVKQN QGKSLEWMGE INPNNGVWY NQKFKGTTTL TVDKSSSTAY MDLHSLTSED SAVYYCTRAL YHSNFGWYFD SWGKGTTLTV SSASTKGPSV FPLAPCSRST SESTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT

cAb 0311

KTYTCNVDHK PSNTKVDKRV ESKYGPPCPP CPAPEFLGGP SVFLFPPKPK

heavy chain DTLMISRTPE VTCVWDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNS

TYRWSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK

DIVLTQSPAS LAVSLGQRAT ISCKASQSVD YDGDSHMNWY QQKPGQPPKL

LIYTASNLES GIPARFSGSG SGADFTLTIH PVEEEDAATY YCQQGNEDPW

cAb 0311

TFGGGTRLEI KRTVAAPSVF IFPPSDEQLK SGTASWCLL NNFYPREAKV

light chain

QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV

THQGLSSPVT KSFNRGEC

h0301-H0 QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DNYMIWVRQA PGQGLEWMGD heavy chain INPYNGGTTF NQKFKGRVTI TADKSTSTAY MELSSLRSED TAVYYCARES variable PYFSNLYVMD YWGQGTLVTV SS

region

h0301-Hl

QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DNYMIWVRQA PGQGLEWMGD

heavy chain INPYNGGTTF NQKFKGRVTI TVDKSTSTAY MELSSLRSED TAVYYCARES variable PYFSNLYVMD YWGQGTLVTV SS

region

h0301-H2

QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DNYMIWVRQA PGQGLEWIGD

heavy chain INPYNGGTTF NQKFKGRATL TVDKSTSTAY MELSSLRSED TAVYYCARES variable PYFSNLYVMD YWGQGTLVTV SS

region

H0302-H1

QVQLVQSGAE VKKPGSSVKV SCKASGYTFS DFNIHWVRQA PGQGLEWMGY

heavy chain

INPYTDVTVY NEKFKGRVTI TSDKSTSTAY MELSSLRSED TAVYYCASYF

variable

DGTFDYALDY WGQGTLVTVS S

region

H0302-H2

QVQLVQSGAE VKKPGSSVKV SCKASGYTFS DFNIHWVRQA PGQGLEWIGY

heavy chain

INPYTDVTVY NEKFKGRATL TSDKSTSTAY MELSSLRSED TAVYYCASYF

variable

DGTFDYALDY WGQGTLVTVS S

region

H0311-H1

QVQLVQSGAE VKKPGSSVKV SCKASGYIFT DYNMHWVRQA PGQGLEWMGE

heavy chain INPNNGVWY NQKFKGRVTI TVDKSTSTAY MELSSLRSED TAVYYCTRAL variable YHSNFGWYFD SWGQGTLVTV SS

region

H0311-H2

QVQLVQSGAE VKKPGSSVKV SCKASGYIFT DYNMHWVRQA PGQGLEWMGE

heavy chain INPNNGVWY NQKFKGTTTL TVDKSTSTAY MELSSLRSED TAVYYCTRAL variable YHSNFGWYFD SWGQGTLVTV SS

region

h0301-L0

EIVLTQSPAT LSLSPGERAT LSCKASQSVD YDGDNYMNWY QQKPGQAPRL

light chain

LIYAASNLES GIPARFSGSG SGTDFTLTIS SLEPEDFAVY YCHLSNEDLS

variable TFGGGTKVEI K

region

h0301-Ll

NIVLTQSPAT LSLSPGERAT LSCKASQSVD YDGDNYMNWY QQKPGQAPRL

light chain

LIYAASNLES GIPARFSGSG SGTDFTLTIS SLEPEDFAVY YCHLSNEDLS

variable TFGGGTKVEI K

region

H0302-L0

EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGLSFMNWY QQKPGQAPRL

light chain

LIYTASNLES GIPARFSGSG SGTDFTLTIS SLEPEDFAVY YCQQSKELPW

variable TFGQGTKVEI K

region

H0302-L1

EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGLSFMNWY QQKPGQAPRL

light chain

LIYTASNLES GIPARFSGSG SRTDFTLTIS SLEPEDFAVY YCQQSKELPW

variable TFGQGTKVEI K

region

H0302-L2

EIVVTQSPAT LSLSPGERAT LSCRASESVD NYGLSFMNWF QQKPGQAPRL

light chain

LIYTASNLES GIPARFSGSG SRTDFTLTIS SLEPEDFAVY YCQQSKELPW

variable TFGQGTKVEI K

region

H0311-L0

EIVLTQSPAT LSLSPGERAT LSCKASQSVD YDGDSHMNWY QQKPGQAPRL

light chain

LIYTASNLES GIPARFSGSG SGTDFTLTIS SLEPEDFAVY YCQQGNEDPW

variable TFGQGTKVEI K

region

H0311-L1

DIVLTQSPAT LSLSPGERAT LSCKASQSVD YDGDSHMNWY QQKPGQAPRL

light chain

LIYTASNLES GIPARFSGSG SGADFTLTIS SLEPEDFAVY YCQQGNEDPW

variable region TFGQGTKVEI K

QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DNYMIWVRQA PGQGLEWMGD INPYNGGTTF NQKFKGRVTI TADKSTSTAY MELSSLRSED TAVYYCARES PYFSNLYVMD YWGQGTLVTV SSASTKGPSV FPLAPCSRST SESTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT

h0301-H0 KTYTCNVDHK PSNTKVDKRV ESKYGPPCPP CPAPEFLGGP SVFLFPPKPK heavy chain DTLMISRTPE VTCVWDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNS

TYRWSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK

QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DNYMIWVRQA PGQGLEWMGD INPYNGGTTF NQKFKGRVTI TVDKSTSTAY MELSSLRSED TAVYYCARES PYFSNLYVMD YWGQGTLVTV SSASTKGPSV FPLAPCSRST SESTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT

h0301-Hl

KTYTCNVDHK PSNTKVDKRV ESKYGPPCPP CPAPEFLGGP SVFLFPPKPK

heavy chain DTLMISRTPE VTCVWDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNS

TYRWSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK

QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DNYMIWVRQA PGQGLEWIGD INPYNGGTTF NQKFKGRATL TVDKSTSTAY MELSSLRSED TAVYYCARES PYFSNLYVMD YWGQGTLVTV SSASTKGPSV FPLAPCSRST SESTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT

h0301-H2

KTYTCNVDHK PSNTKVDKRV ESKYGPPCPP CPAPEFLGGP SVFLFPPKPK

heavy chain DTLMISRTPE VTCVWDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNS

TYRWSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK

QVQLVQSGAE VKKPGSSVKV SCKASGYTFS DFNIHWVRQA PGQGLEWMGY INPYTDVTVY NEKFKGRVTI TSDKSTSTAY MELSSLRSED TAVYYCASYF DGTFDYALDY WGQGTLVTVS SASTKGPSVF PLAPCSRSTS ESTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTK

H0302-H1

TYTCNVDHKP SNTKVDKRVE SKYGPPCPPC PAPEFLGGPS VFLFPPKPKD

heavy chain TLMISRTPEV TCWVDVSQE DPEVQFNWYV DGVEVHNAKT KPREEQFNST

YRVVSVLTVL HQDWLNGKEY KCKVSNKGLP SSIEKTISKA KGQPREPQVY TLPPSQEEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSR LTVDKSRWQE GNVFSCSVMH EALHNHYTQK SLSLSLGK

QVQLVQSGAE VKKPGSSVKV SCKASGYTFS DFNIHWVRQA PGQGLEWIGY INPYTDVTVY NEKFKGRATL TSDKSTSTAY MELSSLRSED TAVYYCASYF DGTFDYALDY WGQGTLVTVS SASTKGPSVF PLAPCSRSTS ESTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTK

H0302-H2

TYTCNVDHKP SNTKVDKRVE SKYGPPCPPC PAPEFLGGPS VFLFPPKPKD

heavy chain TLMISRTPEV TCWVDVSQE DPEVQFNWYV DGVEVHNAKT KPREEQFNST

YRVVSVLTVL HQDWLNGKEY KCKVSNKGLP SSIEKTISKA KGQPREPQVY TLPPSQEEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSR LTVDKSRWQE GNVFSCSVMH EALHNHYTQK SLSLSLGK

QVQLVQSGAE VKKPGSSVKV SCKASGYIFT DYNMHWVRQA PGQGLEWMGE INPNNGVWY NQKFKGRVTI TVDKSTSTAY MELSSLRSED TAVYYCTRAL YHSNFGWYFD SWGQGTLVTV SSASTKGPSV FPLAPCSRST SESTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT

H0311-H1 KTYTCNVDHK PSNTKVDKRV ESKYGPPCPP CPAPEFLGGP SVFLFPPKPK heavy chain DTLMISRTPE VTCVWDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNS

TYRWSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK

QVQLVQSGAE VKKPGSSVKV SCKASGYIFT DYNMHWVRQA PGQGLEWMGE

H0311-H2

INPNNGVWY NQKFKGTTTL TVDKSTSTAY MELSSLRSED TAVYYCTRAL heavy chain YHSNFGWYFD SWGQGTLVTV SSASTKGPSV FPLAPCSRST SESTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT KTYTCNVDHK PSNTKVDKRV ESKYGPPCPP CPAPEFLGGP SVFLFPPKPK DTLMISRTPE VTCVWDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNS TYRWSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK

EIVLTQSPAT LSLSPGERAT LSCKASQSVD YDGDNYMNWY QQKPGQAPRL LIYAASNLES GIPARFSGSG SGTDFTLTIS SLEPEDFAVY YCHLSNEDLS

h0301-L0

TFGGGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASWCLL NNFYPREAKV

light chain

QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC

NIVLTQSPAT LSLSPGERAT LSCKASQSVD YDGDNYMNWY QQKPGQAPRL LIYAASNLES GIPARFSGSG SGTDFTLTIS SLEPEDFAVY YCHLSNEDLS

h0301-Ll

TFGGGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASWCLL NNFYPREAKV

light chain

QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC

EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGLSFMNWY QQKPGQAPRL LIYTASNLES GIPARFSGSG SGTDFTLTIS SLEPEDFAVY YCQQSKELPW

H0302-L0

TFGQGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASWCLL NNFYPREAKV

light chain QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV

THQGLSSPVT KSFNRGEC

EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGLSFMNWY QQKPGQAPRL LIYTASNLES GIPARFSGSG SRTDFTLTIS SLEPEDFAVY YCQQSKELPW

H0302-L1

TFGQGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASWCLL NNFYPREAKV

light chain

QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC

EIVVTQSPAT LSLSPGERAT LSCRASESVD NYGLSFMNWF QQKPGQAPRL LIYTASNLES GIPARFSGSG SRTDFTLTIS SLEPEDFAVY YCQQSKELPW

H0302-L2

TFGQGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASWCLL NNFYPREAKV

light chain QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV

THQGLSSPVT KSFNRGEC

EIVLTQSPAT LSLSPGERAT LSCKASQSVD YDGDSHMNWY QQKPGQAPRL LIYTASNLES GIPARFSGSG SGTDFTLTIS SLEPEDFAVY YCQQGNEDPW

H0311-L0

TFGQGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASWCLL NNFYPREAKV

light chain

QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC

DIVLTQSPAT LSLSPGERAT LSCKASQSVD YDGDSHMNWY QQKPGQAPRL LIYTASNLES GIPARFSGSG SGADFTLTIS SLEPEDFAVY YCQQGNEDPW

H0311-L1 TFGQGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASWCLL NNFYPREAKV light chain QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV

THQGLSSPVT KSFNRGEC

EEVSEYCSHM IGSGHLQSLQ RLIDSQMETS CQITFEFVDQ EQLKDPVCYL

Human KKAFLLVQDI MEDTMRFRDN TPNAIAIVQL QELSLRLKSC FTKDYEEHDK CSF1 ACVRTFYETP LQLLEKVKNV FNETKNLLDK DWNIFSKNCN NSFAECSSQG

HERQSEGS

NEPLEMWPLT QNEECTVTGF LRDKLQYRSR LQYMKHYFPI NYKISVPYEG

Human IL- VFRIANVTRL QRAQVSEREL RYLWVLVSLSATESVQDVLL EGHPSWKYLQ 34 EVQTLLLNVQ QGLTDVEVSP KVESVLSLLN APGPNLKLVR PKALLDNCFR VMELLYCSCC KQSSVLNWQD CEVPSPQSCS PEPSLQYAAT QLYPPPPWSP SSPPHSTGSV RPVRAQGEGL LP

Human

acceptor A QVQLVQSGAE VKKPGSSVKV SCKAS

FR1

Human WVRQAPGQGL EWMG acceptor A

FR2

Human

acceptor A RVTITADKST STAYMELSSL RSEDTAVYYC AR

71 FR3

Human

acceptor A WGQGTLVTVS S

72

FR4

Human

acceptor B QVQLVQSGAE VKKPGSSVKV SCKAS

73

FR1

Human

acceptor B WVRQAPGQGL EWMG

74

FR2

Human

acceptor B RVTITADKST STAYMELSSL RSEDTAVYYC AR

75

FR3

Human

acceptor B WGQGTLVTVSS

76

FR4

Human

acceptor C QVQLVQSGAE VKKPGSSVKV SCKAS

77 FR1

Human

acceptor C WVRQAPGQGL EWMG

78

FR2

Human

acceptor C RVTITADKST STAYMELSSL RSEDTAVYYC AR

79

FR3

Human

acceptor C WGQGTLVTVS S

80

FR4

Human

acceptor D EIVLTQSPAT LSLSPGERAT LSC

81

FR1

Human

acceptor D WYQQKPGQAP RLLIY

82

FR2

Human

acceptor D GIPARFSGSG SGTDFTLTIS SLEPEDFAVY YC

83

FR3

Human

acceptor D FGGGTKVEIK

84

FR4

Human

acceptor E EIVLTQSPAT LSLSPGERAT LSC

85

FR1

Human

acceptor E WYQQKPGQAP RLLIY

86

FR2 Human

acceptor E GIPARFSGSG SGTDFTLTIS SLEPEDFAVY YC

87

FR3

Human

acceptor E FGQGTKVEIK

FR4

Human

acceptor F EIVLTQSPAT LSLSPGERAT LSC

89

FR1

Human

acceptor F WYQQKPGQAP RLLIY

90

FR2

Human

acceptor F GIPARFSGSG SGTDFTLTIS SLEPEDFAVY YC

91 FR3

Human

acceptor F FGQGTKVEIK

92

FR4

APVIEPSGPE LVVEPGETVT LRCVSNGSVE WDGPISPYWT LDPESPGSTL TTRNATFKNT GTYRCTELED PMAGSTTIHL YVKDPAHSWN LLAQEVTWE GQEAVLPCLI TDPALKDSVS LMREGGRQVL RKTVYFFSPW RGFI IRKAKV LDSNTYVCKT MVNGRESTST GIWLKVNRVH PEPPQIKLEP SKLVRIRGEA AQIVCSATNA EVGFNVILKR GDTKLEIPLN SDFQDNYYKK VRALSLNAVD FQDAGIYSCV ASNDVGTRTA TMNFQVVESA YLNLTSEQSL LQEVSVGDSL ILTVHADAYP SIQHYNWTYL GPFFEDQRKL EFITQRAIYR YTFKLFLNRV

mCSFIR

KASEAGQYFL MAQNKAGWNN LTFELTLRYP PEVSVTWMPV NGSDVLFCDV

93 ECD-Fc SGYPQPSVTW MECRGHTDRC DEAQALQVWN DTHPEVLSQK PFDKVIIQSQ

LPIGTLKHNM TYFCKTHNSV GNSSQYFRAV SLGQSKQEPK SSDKTHTCPP CPAPELLGGP SVFLFPPKPK DTLMISRTPE VTCWVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSRDEL TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK

ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFLGGPSV FLFPPKPKDT LMISRTPEVT CWVDVSQED

Human

PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RWSVLTVLH QDWLNGKEYK

94 IgG4 S241P

CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGK

RTVAAPSVFI FPPSDEQLKS GTASWCLLN NFYPREAKVQ WKVDNALQSG

Human IgK NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK

95 SFNRGEC