JP2019089840A - Method of treating a condition using an antibody that binds colony stimulating factor 1 receptor (CSF1R) - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for treating rheumatoid arthritis and related conditions, a method for treating systemic lupus erythematosus and related conditions, and a method for treating multiple sclerosis. A method for treating a condition related to rheumatoid arthritis, which comprises a step of administering an antibody that binds a colony stimulating factor 1 receptor (CSF1R) to a subject having rheumatoid arthritis. However, blocking the binding of colony stimulating factor 1 (CSF1) to CSF1R, blocking the binding of IL-34 to CSF1R, and treating the condition associated with rheumatoid arthritis reduces 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 bone A method comprising at least one effect selected from reducing loss. [Selection diagram] None

Description

TECHNICAL FIELD Methods are provided for treating conditions using antibodies that bind colony stimulating factor 1 receptor (CSF1R). Such methods include, but are not limited to, methods of treating rheumatoid arthritis, multiple sclerosis, and generalized lupus erythematosus.

BACKGROUND Colony stimulating factor 1 receptor (also referred to herein as CSF1R, also referred to in the art as FMS, FIM2, C-FMS, M-CSF receptor, and CD115) is an N-terminal cell. It is a single transmembrane receptor with tyrosine kinase activity, which has an exodomain (ECD) and a C-terminal intracellular domain. The ligand binding of CSF1 or interleukin 34 ligand (herein referred to as IL-34, Lin et al., Science 320: 807-11 (2008)) to CSF1 R is acceptable It leads to body dimerization, upregulation of CSF1R protein tyrosine kinase activity, phosphorylation of CSF1R tyrosine residues, and downstream of signaling events. Both CSF1 and IL-34 stimulate monocyte survival, proliferation, and differentiation to macrophages, as well as differentiation to other monocyte cell lineages such as osteoclasts, dendritic cells, and microglia.

  Many tumor cells have been found to secrete CSF1 which activates monocytes / macrophage cells through CSF1R. Levels of CSF1 in tumors have been shown to correlate with levels of tumor associated macrophages (TAM) in tumors. Higher levels of TAM have been found to correlate with a poorer prognosis for the patient. In addition, CSF1 has been found to promote tumor growth and progression to metastasis, for example, in human breast cancer xenografts in mice. For example, Paulus et al. , Cancer Res. 66: 4349-56 (2006) (non-patent document 2). Furthermore, CSF1R is involved in osteolytic bone destruction in bone metastases. For example, Ohno et al. , Mol. Cancer Ther. 5: 2634-43 (2006) (non-patent document 3).

  CSF1 and its receptor have also been found to be involved in various inflammatory and autoimmune diseases. For example, Hamilton, Nat. Rev. 8: 533-44 (2008) (Non-patent Document 4). For example, synovial endothelial cells from joints affected with rheumatoid arthritis have been found to produce CSF1, suggesting a role for CSF1 and its receptor in disease. Blockade of CSF1R activity by antibodies results in positive clinical effects in mouse models of arthritis, including reduced bone and cartilage destruction and reduced numbers of macrophages. For example, Kitaura et al. , J. Clin. Invest. 115: 3418-3427 (2005) (non-patent document 5).

  Mature differentiated myeloid lineage cells such as macrophages, microglia and osteoclasts contribute to the pathology of various diseases such as rheumatoid arthritis, multiple sclerosis and bone loss diseases. Differentiated myeloid lineage cells are derived from peripheral blood monocyte intermediates. CSF1R stimulation contributes to monocyte development from bone marrow precursors, monocyte proliferation and survival, and differentiation of peripheral blood monocytes to differentiated myeloid lineage cells, such as macrophages, microglia cells, and osteoclasts . As such, CSF1R stimulation contributes to proliferation, survival, activation, and maturation of differentiated myeloid lineage cells, and in pathological situations CSF1R stimulation contributes to the ability of differentiated myeloid lineage cells to mediate disease pathologies. doing.

Lin et al. , Science 320: 807-11 (2008). Paulus et al. , Cancer Res. 66: 4349-56 (2006). Ohno et al. , Mol. Cancer Ther. 5: 2634-43 (2006) Hamilton, Nat. Rev. 8: 533-44 (2008) Kitaura et al. , J. Clin. Invest. 115: 3418-3427 (2005).

SUMMARY In some embodiments, methods of treating conditions associated with rheumatoid arthritis are provided. In some embodiments, the method comprises administering to a subject having rheumatoid arthritis, an antibody that binds colony stimulating factor 1 receptor (CSF1R), wherein the antibody stimulates colony stimulation to CSF1R. It blocks the binding of factor 1 (CSF1) and blocks the binding of IL-34 to CSF1R. In some embodiments, treating a condition associated with rheumatoid arthritis reduces inflammation, reduces pannus formation, reduces cartilage damage, reduces bone resorption, suffers from rheumatoid arthritis. And at least one effect selected from reducing the number of macrophages in at least one joint, reducing autoantibody levels, and reducing bone loss. In some embodiments, treating a condition associated with rheumatoid arthritis comprises reducing inflammation. In some embodiments, the method of reducing inflammation associated with rheumatoid arthritis comprises determining an erythrocyte sedimentation rate, wherein a reduction in sedimentation rate is indicative of a reduction in inflammation.

  In some embodiments, 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 for bone resorption is reduced. In some embodiments, the bone resorption marker is tartrate-resistant acid phosphatase 5b (TRAP 5b), total pyridinoline in urine, total deoxypyridinoline in urine, free pyridinoline in urine, type I collagen cross-linked N-telopeptide in serum It is selected from urine type I collagen cross-linked N-telopeptide and carboxy terminal telopeptide of serum type I collagen.

  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, at least one effect is measured using an imaging technique. In some such embodiments, imaging techniques include x-ray imaging, magnetic resonance imaging, computed tomography (CT) scanning, arthroscopy, scintigraphy, sonography, bone densitometry, singleton One-photon absorption measurement (SPA), double-photon absorption measurement (DPA), single-energy X-ray absorption measurement (SXA), dual-energy X-ray absorption measurement (DXA), scintigraphy, ultrasonography , Duplex ultrasound, and power Doppler imaging.

  In some embodiments of the method of treating a condition associated with rheumatoid arthritis, the number of CD16 + monocytes is reduced by at least 30%. In some embodiments of the method, the number of CD16-monocytes is not reduced or reduced by less than 20%. In some embodiments, the CD16 + monocytes are CD16 + peripheral blood monocytes.

  In some embodiments, a method of treating rheumatoid arthritis is provided, the method comprising administering to a subject having rheumatoid arthritis an antibody that binds CSF1R, the antibody to CSF1R Block binding of CSF1 and block binding of IL-34 to CSF1R, and the antibody reduces the number of CD16 + monocytes by at least 30% in the subject and does not reduce CD16-monocytes Or decrease by less than 20%. In some embodiments, the CD16 + monocytes are CD16 + peripheral blood monocytes.

  In some embodiments of the method of treating rheumatoid arthritis, the method comprises methotrexate, an anti-TNF agent, glucocorticoid, cyclosporin, leflunomide, azathioprine, a JAK inhibitor, a SYK inhibitor, an anti-IL-6 antibody, an anti-IL- At least one additional treatment selected from 6R antibody, anti-CD-20 antibody, anti-CD19 antibody, anti-GM-CSF antibody, IL-1 receptor antagonist, CTLA-4 antagonist, and anti-GM-CSF-R antibody It further comprises the step of administering the substance.

  In some embodiments, methods of treating skin lesions associated with acne are provided. In some embodiments, the method comprises administering to a subject having lupus, an antibody that binds CSF1R, which blocks binding of CSF1 to CSF1R, and to CSF1R. Block binding of IL-34. In some embodiments, treating the skin lesions associated with wrinkles is selected from reducing the number of skin lesions, reducing the rate of formation of skin lesions, and reducing the severity of the skin lesions. Containing at least one effect.

  In some embodiments, methods of treating lupus nephritis are provided. In some embodiments, the method comprises administering to a subject having lupus nephritis an antibody that binds CSF1R, wherein the antibody blocks binding of CSF1 to CSF1R and to CSF1R. Block the binding of IL-34. In some embodiments, renal function is improved in the subject. In some embodiments, proteinuria is decreased in the subject. In some embodiments, the glomerular filtration rate is improved in the subject.

  In some embodiments, methods of treating acne are provided. In some embodiments, one method comprises administering to a subject having lupus, an antibody that binds CSF1R, which blocks binding of CSF1 to CSF1R, and to CSF1R. Block binding of IL-34. In some embodiments, the antibody reduces the number of CD16 + monocytes by at least 30% and the CD16-monocytes are not reduced or reduced by less than 20% in the subject. In some embodiments, CD16 + monocytes are reduced by at least 50%. In some embodiments, the CD16 + monocytes are CD16 + peripheral blood monocytes.

  In some embodiments, the method of treating lupus, lupus nephritis, or lupus-related skin lesions is hydroxychloroquine (Plaquenil), corticosteroids, cyclophosphamide (Cytoxan), azathioprine (Imuran, Azasan), It further comprises the step of administering at least one additional therapeutic substance selected from mycophenolate (Cellcept), leflunomide (Arava) and methotrexate (Trexall), and belimumab (Benlysta).

  In some embodiments, a method of treating an inflammatory condition is provided. In some such embodiments, one method comprises administering to a subject having an inflammatory condition an antibody that binds CSF1R, wherein the antibody blocks binding of CSF1 to CSF1R, and CSF1R Block binding of IL-34 to. In some embodiments, the antibody reduces the number of CD16 + monocytes by at least 30% and the CD16-monocytes are not reduced or reduced by less than 20%. In some embodiments, CD16 + monocytes are reduced by at least 50%. In some embodiments, the CD16 + monocytes are CD16 + peripheral blood monocytes.

  In some embodiments, methods of treating a CD16 + disorder are provided. In some embodiments, one method comprises administering to a subject with a CD16 + disorder an antibody that binds CSF1R, which blocks CSF1 binding to CSF1R and to CSF1R. Block the binding of IL-34. In some embodiments, the antibody reduces the number of CD16 + monocytes by at least 30% and the CD16-monocytes are not reduced or reduced by less than 20%. In some embodiments, CD16 + monocytes are reduced by at least 50%. In some embodiments, the CD16 + monocytes are CD16 + peripheral blood monocytes.

  In some embodiments, the methods of treatment discussed herein comprise treating a condition that does not respond to methotrexate.

  In some embodiments, methods of reducing the number of CD16 + monocytes are provided. In some embodiments, one method comprises administering to the subject an antibody that binds CSF1R, which blocks binding of CSF1 to CSF1R and binding of IL-34 to CSF1R. Cut off. In some embodiments, the antibody reduces the number of CD16 + monocytes by at least 30% and the CD16-monocytes are not reduced or reduced by less than 20%. In some embodiments, CD16 + monocytes are reduced by at least 50%. In some embodiments, the CD16 + monocytes are CD16 + peripheral blood monocytes.

  In some embodiments, a method is provided for delaying the progression of a renal condition associated with hemorrhoids. In some embodiments, the method comprises administering to the subject an antibody that binds CSF1R, which blocks binding of CSF1 to CSF1R and binding of IL-34 to CSF1R. Cut off. In some embodiments, proteinuria does not increase in the subject or does not increase in the subject at the same rate as a subject to which the antibody is not administered. In some embodiments, the glomerular filtration rate does not decrease in the subject or does not decrease in the subject at the same rate as the subject to which the antibody is not administered.

  In some embodiments, provided is a method of delaying the progression of pannus formation in a subject having rheumatoid arthritis. In some such embodiments, one method comprises administering to a subject having rheumatoid arthritis, an antibody that binds CSF1R, wherein the antibody blocks binding of CSF1 to CSF1R, and CSF1R. Block binding of IL-34 to.

  In some embodiments, provided is a method of delaying the progression of bone loss in a subject having rheumatoid arthritis. In some embodiments, one method comprises administering to a subject having rheumatoid arthritis, an antibody that binds colony stimulating factor 1 receptor (CSF1R), wherein the antibody stimulates colony stimulation to CSF1R. It blocks the binding of factor 1 (CSF1) and blocks the binding of IL-34 to CSF1R.

  In some embodiments of the methods described herein, the antibody heavy chain and / or antibody light chain has the following structure:

  In some embodiments, the heavy chain is at least 90%, at least 95%, at least 97%, at least 99%, or 100% with a sequence selected from SEQ ID NOs: 9, 11, 13, and 39-45 Contains sequences that are identical. In some embodiments, the light chain is at least 90%, at least 95%, at least 97%, at least 99%, or 100% with a sequence selected from SEQ ID NO: 10, 12, 14, and 46-52. Contains sequences that are identical. In some embodiments, the heavy chain is at least 90%, at least 95%, at least 97%, at least 99%, or 100% with a sequence selected from SEQ ID NOs: 9, 11, 13, and 39-45 Comprising a sequence that is identical, the light chain being at least 90%, at least 95%, at least 97%, at least 99%, or 100% with a sequence selected from SEQ ID NO: 10, 12, 14, and 46-52 Contains sequences that are identical.

  In some embodiments, HC CDR1, HC CDR2, and HC CDR3 are (a) SEQ ID NOs: 15, 16, and 17, (b) SEQ ID NOs: 21, 22, and 23, and (c) It comprises a set of sequences selected from SEQ ID NO: 27, 28 and 29. In some embodiments, LC CDRl, LC CDR2 and LC CDR3 are (a) SEQ ID NO: 18, 19 and 20, (b) SEQ ID NO: 24, 25 and 26 and (c) It comprises a set of sequences selected from SEQ ID NO: 30, 31 and 32.

  In some embodiments, the heavy chain comprises HC CDR1, HC CDR2, and HC CDR3, and HC CDR1, HC CDR2, and HC CDR3 are (a) SEQ ID NOs: 15, 16, and 17, (b) ) The light chain comprises a set of sequences selected from SEQ ID NOs: 21, 22 and 23 and (c) SEQ ID NOs: 27, 28 and 29, wherein the light chain comprises LC CDR1, LC CDR2 and LC CDR3. And LC CDR1, LC CDR2 and LC CDR3 (a) SEQ ID NO: 18, 19 and 20, (b) SEQ ID NO: 24, 25 and 26 and (c) SEQ ID NO: It comprises a set of sequences selected from 30, 31, and 32.

  In some embodiments, an isolated antibody is provided, said antibody comprising a heavy chain and a light chain, said antibody comprising (a) at least 95%, at least 97% of SEQ ID NO: 9, A heavy chain comprising a sequence which is at least 99%, or 100% identical, and a light chain comprising a sequence which 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 at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 11, and at least 95%, at least 97%, at least 99%, or SEQ ID NO: 12 A light chain comprising a sequence that is 100% identical, (c) at least 95%, at least 97%, at least 99% to SEQ ID NO: 13 Or a light chain comprising a sequence that is 100% identical to a heavy chain and a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 14; (d) SEQ ID NO: A heavy chain comprising a sequence at least 95%, at least 97%, at least 99%, or 100% identical to 39 and at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 46 A light chain comprising a sequence, (e) a heavy chain comprising a sequence at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 40 and at least 95% with SEQ ID NO: 46 A light chain comprising a sequence which is at least 97%, at least 99% or 100% identical, (f) SEQ A heavy chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to D NO: 41, and at least 95%, at least 97%, at least 99%, or 100 to SEQ ID NO: 46 A light chain comprising a sequence that is% identical, (g) a heavy chain comprising a sequence at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 39 A light chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical; (h) at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 40 A heavy chain comprising a sequence, and at least 95%, at least 97%, less than SEQ ID NO: 47 A light chain comprising a sequence which is at least 99% or 100% identical, and (i) a heavy chain which comprises a sequence at least 95%, at least 97%, at least 99% or 100% identical to SEQ ID NO: 41 A light chain comprising a sequence at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 47, and (j) at least 95%, at least 97%, at least 95% to SEQ ID NO: 42 A heavy chain comprising a sequence which is 99% or 100% identical and a light chain comprising a sequence which is at least 95%, at least 97%, at least 99% or 100% identical to SEQ ID NO: 48, (k) Sequences at least 95%, at least 97%, at least 99%, or 100% identical to ID NO: 42 A heavy chain and a light chain comprising a sequence at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 49, (l) at least 95% with SEQ ID NO: 42, at least 97 A heavy chain comprising a sequence that is%, at least 99%, or 100% identical to a light chain comprising a sequence 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 at least 95%, at least 97%, at least 99% to SEQ ID NO: 48 Or a light chain comprising a sequence that is 100% identical, (n) at least 95 with SEQ ID NO: 43 , A heavy chain comprising a sequence that is at least 97%, at least 99%, or 100% identical to light comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 49 Chain, (o) a heavy chain comprising a sequence at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 43, and at least 95%, at least 97%, to SEQ ID NO: 50 A light chain comprising a sequence which is at least 99%, or 100% identical, (p) a heavy chain comprising a sequence which is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 44; SEQ ID NO: 51 at least 95%, at least 97%, at least 99%, or 100% identical A light chain comprising a sequence that is (q) a heavy chain comprising a sequence at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 44; %, At least 97%, at least 99%, or 100% identical to a light chain, (r) at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 45 Or a light chain comprising a sequence which is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 51, or (s) at least 95% with SEQ ID NO: 45 A heavy chain comprising a sequence that is at least 97%, at least 99%, or 100% identical; SEQ ID N : 52 and at least 95%, and a light chain comprising at least 97%, at least 99%, or a sequence that is 100% identical.

  In some embodiments, an antibody is provided, said antibody comprising a heavy chain and a light chain, said antibody comprising (a) a heavy chain (HC) CDR1 having the sequence of SEQ ID NO: 15, SEQ ID NO: 15 A heavy chain comprising HC CDR2 having the sequence of NO: 16 and HC CDR3 having the sequence of SEQ ID NO: 17 and a light chain (LC) CDR1 having the sequence of SEQ ID NO: 18 of SEQ ID NO: 19 A light chain comprising LC CDR2 having the sequence and LC CDR3 having the sequence of SEQ ID NO: 20, (b) a heavy chain (HC) CDR1 having the sequence of SEQ ID NO: 21 and the sequence of SEQ ID NO: 22 A heavy chain comprising HC CDR2 having and HC CDR3 having the sequence of SEQ ID NO: 23, a sequence of SEQ ID NO: 24 A light chain (LC) having CDR1; an LC CDR2 having the sequence of SEQ ID NO: 25; and a light chain comprising an LC CDR3 having the sequence of SEQ ID NO: 26; or (c) a sequence of SEQ ID NO: 27 Has a heavy chain (HC) having a CDR1, an HC CDR2 having a sequence of SEQ ID NO: 28, and a heavy chain comprising an HC CDR3 having a sequence of SEQ ID NO: 29 and a light chain having a sequence of SEQ ID NO: 30 ( LC) comprising a CDR1, an LC CDR2 having the sequence of SEQ ID NO: 31, and a light chain comprising an LC CDR3 having the sequence of SEQ ID NO: 32.

  In some embodiments, the antibody comprises a heavy chain and a light chain, said antibody comprising (a) a heavy chain comprising the sequence of SEQ ID NO: 53 and a light chain comprising the sequence of SEQ ID NO: 60, (B) a heavy chain comprising the sequence of SEQ ID NO: 53 and a light chain comprising the sequence of SEQ ID NO: 61, or (c) a heavy chain comprising the sequence of SEQ ID NO: 58; And a light chain comprising the sequence. In some embodiments, the 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 Or (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.

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

In some embodiments, the antibody binds human CSF1R and / or binds cynomolgus monkey CSF1R. In some embodiments, the antibody blocks ligand binding to CSF1R. In some embodiments, the antibody blocks CSF1 and / or IL-34 binding to CSF1R. In some embodiments, the antibody blocks both CSF1 and IL-34 binding to CSF1R. In some embodiments, the antibody inhibits ligand induced phosphorylation of CSF1R. In some embodiments, the antibody inhibits CSF1 and / or IL-34 induced phosphorylation of CSF1R. In some embodiments, the antibody binds human CSF1R with an affinity (K _D ) of less than 1 nM. In some embodiments, the antibody suppresses monocyte proliferation and / or survival response in the presence of CSF1 or IL-34.

  In some embodiments, pharmaceutical compositions comprising an antibody that binds CSF1R are provided.

In some embodiments, a composition comprising an antibody that binds CSF1R is provided for use in methods of treatment of humans or animals. In some embodiments, a composition comprising an antibody that binds CSF1R and an antibody that binds CSF1R is provided for use in a method of treating a condition associated with rheumatoid arthritis in humans or animals. In some embodiments, compositions comprising CSF1R binding antibodies and CSF1R binding antibodies are provided for use in a method of treating multiple sclerosis in humans or animals. In some embodiments, compositions comprising CSF1R binding antibodies and CSF1R binding antibodies are provided and provided for use in a method of treating a condition associated with systemic lupus erythematosus.
[Invention 1001]
A method of treating a condition associated with rheumatoid arthritis comprising administering to a subject having rheumatoid arthritis an antibody that binds colony stimulating factor 1 receptor (CSF1R), said antibody to CSF1R. Blocks binding of colony-stimulating factor 1 (CSF1) and blocks binding of IL-34 to CSF1 R and treating conditions associated with rheumatoid arthritis reduces inflammation, pannus formation Reducing, reducing cartilage damage, reducing bone resorption, reducing the number of macrophages in at least one joint affected with rheumatoid arthritis, reducing autoantibody levels, and reducing bone loss A method comprising at least one effect selected from:
[Invention 1002]
The method of the invention 1001, wherein treating a condition associated with rheumatoid arthritis comprises reducing inflammation.
[Invention 1003]
The method of the invention 1002, wherein the number of CD16 + monocytes is reduced by at least 30%.
[Invention 1004]
The method of the invention 1003 wherein the number of CD16-monocytes is not reduced or reduced by less than 20%.
[Invention 1005]
The method of invention 1002, further comprising determining an erythrocyte sedimentation rate, wherein the reduction in sedimentation rate is indicative of a reduction in inflammation.
[Invention 1006]
The method of the invention 1001, wherein treating a condition associated with rheumatoid arthritis comprises at least one effect selected from reducing pannus formation, reducing bone resorption, and reducing bone loss.
[Invention 1007]
The method of the invention 1006, wherein treating a condition associated with rheumatoid arthritis comprises reducing bone resorption.
[Invention 1008]
The method of the invention 1007, wherein the level of at least one marker for bone resorption is reduced.
[Invention 1009]
The bone resorption marker includes tartaric acid-resistant acid phosphatase 5b (TRAP 5b), total pyridinoline in urine, total deoxypyridinoline in urine, free pyridinoline in urine, type I collagen cross-linked N-telopeptide in serum, type I collagen in urine The method of the invention 1008 selected from a cross-linked N-telopeptide and a carboxy-terminal telopeptide of serum type I collagen.
[Invention 1010]
The method of the invention 1006, wherein treating a condition associated with rheumatoid arthritis comprises at least one effect selected from reducing pannus formation and reducing bone loss.
[Invention 1011]
The method of invention 1010 wherein the at least one effect is measured using an imaging technique.
[Invention 1012]
Said imaging techniques include X-ray imaging, magnetic resonance imaging, computed tomography (CT) scanning, arthroscopy, scintigraphy, sonography, bone densitometry, single photon absorptiometry (SPA) ), Double photon absorption measurement (DPA), single energy X-ray absorption measurement (SXA), dual energy X-ray absorption measurement (DXA), scintigraphy, ultrasonography, duplex ultrasonography, And the method of the invention 1011 comprising a method selected from power Doppler imaging.
[Invention 1013]
A method of treating rheumatoid arthritis comprising administering to a subject having rheumatoid arthritis an antibody that binds CSF1R, wherein the antibody blocks binding of CSF1 to CSF1R and to CSF1R. A method which blocks binding of IL-34 and wherein said antibody reduces the number of CD16 + monocytes by at least 30% and said CD16-monocytes are not reduced or reduced by less than 20% in said subject.
[Invention 1014]
Methotrexate, anti-TNF substance, glucocorticoid, cyclosporin, leflunomide, azathioprine, JAK inhibitor, SYK inhibitor, anti-IL-6 antibody, anti-IL-6R antibody, anti-CD-20 antibody, anti-CD19 antibody, anti-GM-CSF antibody Any of Inventions 1001 to 1013, further comprising the step of administering at least one additional therapeutic agent selected from an IL-1 receptor antagonist, a CTLA-4 antagonist, and an anti-GM-CSF-R antibody Method.
[Invention 1015]
CLAIMS: 1. A method of treating skin lesions associated with lupus comprising administering to a subject having lupus, an antibody that binds CSF1 R, wherein the antibody blocks binding of CSF1 to CSF1 R, and Blocking the binding of IL-34 to CSF1R and treating acne-related skin lesions reduces the number of skin lesions, reduces the rate of formation of skin lesions, and the severity of skin lesions A method comprising at least one effect selected from reducing the degree.
[Invention 1016]
A method of treating lupus nephritis comprising administering to a subject having lupus nephritis an antibody that binds CSF1R, wherein the antibody blocks binding of CSF1 to CSF1R, and to CSF1R. A method of blocking IL-34 binding.
[Invention 1017]
The method of the invention 1016 wherein renal function is improved in said subject.
[Invention 1018]
The method of the invention 1016 or 1017, wherein proteinuria is reduced in said subject.
[Invention 1019]
The method of any of inventions 1016-1018, wherein the glomerular filtration rate is enhanced in said subject.
[Invention 1020]
A method of treating lupus comprising administering an antibody that binds CSF1R to a subject having lupus, said antibody blocking binding of CSF1 to CSF1R and IL-to CSF1R. 34. A method which blocks 34 binding and wherein said antibody reduces the number of CD16 + monocytes by at least 30% and said CD16-monocytes are not reduced or reduced by less than 20% in said subject.
[Invention 1021]
The method of the invention 1020, wherein the CD16 + monocytes are reduced by at least 50%.
[Invention 1022]
Selection from Hydroxychloroquine (Plaquenil), Corticosteroids, Cyclophosphamide (Cytoxan), Azathioprine (Imuran, Azasan), Mycophenolate (Cellcept), Leflunomide (Arava), and Methotrexate (Trexall), and Belimumab (Benlysta) The method of any of the inventions 1015-1021, further comprising administering at least one additional therapeutic agent.
[Invention 1023]
A method of treating an inflammatory condition comprising administering to a subject having an inflammatory condition an antibody that binds CSF1R, wherein the antibody blocks binding of CSF1 to CSF1R and to CSF1R. A method which blocks the binding of IL-34 and wherein said antibody reduces the number of CD16 + monocytes by at least 30% and does not reduce or less than 20% of CD16-monocytes.
[Invention 1024]
The method of the invention 1023, wherein the CD16 + monocytes are reduced by at least 50%.
[Invention 1025]
A method of treating a CD16 + disorder comprising administering to the subject having a CD16 + disorder an antibody that binds CSF1R, wherein the antibody blocks binding of CSF1 to CSF1R, and to CSF1R. A method which blocks the binding of IL-34 and wherein said antibody reduces the number of CD16 + monocytes by at least 30% and does not reduce or less than 20% of CD16-monocytes.
[Invention 1026]
The method of the invention 1025, wherein the CD16 + monocytes are reduced by at least 50%.
[Invention 1027]
The method of any of inventions 1020-1026, wherein said lupus, said inflammatory condition, or said CD16 + disorder do not respond to methotrexate.
[Invention 1028]
A method of reducing the number of CD16 + monocytes, comprising administering to a subject an antibody that binds CSF1R, wherein the antibody blocks binding of CSF1 to CSF1R and IL-34 to CSF1R. A method that blocks binding and wherein the antibody reduces the number of CD16 + monocytes by at least 30% and does not reduce or reduce CD16-monocytes by less than 20%.
[Invention 1029]
The method of the invention 1028, wherein the CD16 + monocytes are reduced by at least 50%.
[Invention 1030]
The method of any of the present invention 1021-1029, wherein said CD16 + monocytes are CD16 + peripheral blood monocytes.
[Invention 1031]
A method of delaying the progression of a lupus related renal condition comprising administering to a subject an antibody that binds CSF1R, wherein the antibody blocks binding of CSF1 to CSF1R and IL to CSF1R. A method of blocking -34 binding.
[Invention 1032]
The method of the invention 1031, wherein proteinuria does not increase in said subject, or does not increase in said subject at the same rate as a subject to which said antibody is not administered.
[Invention 1033]
The method of invention 1031 or invention 1032 wherein the glomerular filtration rate does not decrease in said subject, or does not decrease in said subject at the same rate as a subject not receiving said antibody.
[Invention 1034]
A method of delaying the progression of pannus formation in a subject having rheumatoid arthritis comprising administering to a subject having rheumatoid arthritis an antibody that binds colony stimulating factor 1 receptor (CSF1R). , Wherein the antibody blocks binding of colony stimulating factor 1 (CSF1) to CSF1R and blocks binding of IL-34 to CSF1R.
[Invention 1035]
A method of delaying the progression of bone loss in a subject having rheumatoid arthritis comprising administering to a subject having rheumatoid arthritis an antibody that binds colony stimulating factor 1 receptor (CSF1R) , Wherein the antibody blocks binding of colony stimulating factor 1 (CSF1) to CSF1R and blocks binding of IL-34 to CSF1R.
[Invention 1036]
Said antibody is
(a) an antibody comprising a heavy chain comprising the sequence of SEQ ID NO: 39 and a light chain comprising the sequence of SEQ ID NO: 46,
(b) Heavy chain (HC) CDR1 having the sequence of SEQ ID NO: 15, HC CDR2 having the sequence of SEQ ID NO: 16, and a heavy chain comprising HC CDR3 having the sequence of SEQ ID NO: 17 An antibody comprising a light chain (LC) CDR1 having the sequence of ID NO: 18, an LC CDR2 having the sequence of SEQ ID NO: 19, and a light chain comprising an LC CDR3 having the sequence of SEQ ID NO: 20,
(c) A method according to any of the inventions 1001 to 1035, selected from antibodies comprising a heavy chain comprising the sequence of SEQ ID NO: 53 and a light chain comprising the sequence of SEQ ID NO: 60.
[Invention 1037]
The method of the invention 1036 wherein the antibody is a humanized antibody.
[Invention 1038]
The method of the invention 1036 or the invention 1037, wherein the antibody is selected from Fab, Fv, scFv, Fab ′ and (Fab ′) ₂ .

FIG. 7 shows the alignment of the humanized heavy chain variable region against each of the humanized antibodies huAb1 to huAb16 described in Example 1. The residues in the box are the amino acids in the human receptor sequence that are reverted to the corresponding mouse residues. FIG. 7 shows the alignment of the humanized heavy chain variable region against each of the humanized antibodies huAb1 to huAb16 described in Example 1. The residues in the box are the amino acids in the human receptor sequence that are reverted to the corresponding mouse residues. FIG. 7 shows the alignment of the humanized heavy chain variable region against each of the humanized antibodies huAb1 to huAb16 described in Example 1. The residues in the box are the amino acids in the human receptor sequence that are reverted to the corresponding mouse residues. FIG. 7 shows the alignment of the humanized light chain variable region against each of the humanized antibodies huAb1 to huAb16 described in Example 1. The amino acids in the box are the residues in the human receptor sequence that are reverted to the corresponding mouse residues. FIG. 7 shows the alignment of the humanized light chain variable region against each of the humanized antibodies huAb1 to huAb16 described in Example 1. The amino acids in the box are the residues in the human receptor sequence that are reverted to the corresponding mouse residues. FIG. 7 shows the alignment of the humanized light chain variable region against each of the humanized antibodies huAb1 to huAb16 described in Example 1. The amino acids in the box are the residues in the human receptor sequence that are reverted to the corresponding mouse residues. FIG. 10 shows the reduction of CD16 + monocytes in cynomolgus monkeys administered huAb1 as described in Example 2. 18 shows dose dependent binding of surrogate antibody cAb1 to mouse CSF1R, as described in Example 3. 18 shows dose dependent suppression of CSF1 and IL-34 induced proliferation in mNFS60 cells as described in Example 4. FIG. 16 shows suppression of clinical disease score in a mouse model of rheumatoid arthritis after administration of cAb1, as described in Example 5. FIG. 16 shows the suppression of bone loss indicated by plasma TRAP5b levels in a mouse model of rheumatoid arthritis after administration of cAb1, as described in Example 6. FIG. 16 shows the suppression of inflammation, pannus formation, cartilage damage, and bone damage in a mouse model of rheumatoid arthritis after administration of cAb1, as described in Example 7. 18 shows suppression of macrophage numbers in the ankle and knee joints in a mouse model of rheumatoid arthritis after administration of cAb1, as described in Example 8. FIG. 16 shows the suppression of autoantibody formation in a mouse model of rheumatoid arthritis after administration of cAb1, as described in Example 9. 18 shows the suppression of bone loss in a mouse model of established rheumatoid arthritis following administration of cAb1, as described in Example 10. FIG. 16 shows inhibition of pannus formation and bone destruction in a mouse model of established rheumatoid arthritis following administration of cAb1, as described in Example 11. FIG. 16 shows suppression of glomerulonephritis, interstitial nephritis, and perivascular infiltration in a mouse model of systemic lupus erythematosus after administration of cAb1, as described in Example 12. 18 shows the suppression of skin lesions in a mouse model of systemic lupus erythematosus following administration of cAb1, as described in Example 13. FIG. 16 shows the suppression of clinical disease score in a mouse model of multiple sclerosis after administration of cAb1, as described in Example 14.

Detailed Description A method of treating a condition is provided comprising administering an antibody that binds CSF1R and blocks ligand binding of CSF1 and IL-34. As discussed herein, antibodies that bind CSF1R and block CSF1 and IL-34 ligand binding are effective in treating rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis. is there. We show that administering such an antibody to cynomolgus monkeys reduces the number of CD16 + peripheral blood monocytes in cynomolgus monkeys, but does not affect the number of CD16-peripheral blood monocytes; The spheres were found to be highly inflammatory monocytes. For example, see Ziegler-Heitbrock, J. et al. Leukocyte Biol. , 2007, 81: 584-592. Furthermore, administering such an antibody in a mouse model of rheumatoid arthritis suppresses the clinical disease score, as measured by, for example, suppression of erythema and swelling, reduction in tartrate-resistant acid phosphatase 5b (TRAP 5b) levels. These include suppressed bone loss, suppressed inflammation, cartilage destruction, pannus formation, and bone destruction. In particular, bone loss (as measured by a decrease in TRAP 5b levels), pannus formation, and bone destruction were reduced when the antibody was administered after the appearance of clinical symptoms of rheumatoid arthritis. Also, in such a mouse model of rheumatoid arthritis, administering such antibodies reduced the number of joint macrophages and autoantibody formation.

  In a systemic lupus erythematosus mouse model, administering an antibody that binds CSF1R and blocks CSF1 and IL-34 ligand binding suppressed glomerulonephritis, interstitial nephritis, and perivascular infiltration. Furthermore, in the systemic lupus erythematosus mouse model, administering such antibodies suppressed skin lesions. Finally, in the mouse model of multiple sclerosis, administering this antibody suppressed the clinical disease score of MS.

  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 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.

  Exemplary techniques used in connection with recombinant DNA, oligonucleotide synthesis, tissue culture, and transformation (eg, electroporation, lipofection), enzymatic reactions, and purification methods are known in the art. There is. Many such techniques and procedures are described, for example, in Sambrook et al. Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). Additionally, exemplary techniques for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and treatment of patients are also known in the art.

  In this application, the use of "or" means "and / or" unless stated otherwise. In the context of several dependent claims, the use of "or" only refers back to two or more preceding independent or dependent claims. Also, terms such as "element" or "component" encompass both elements and components that include one unit and elements and components that include two or more subunits, unless otherwise specified.

  As used in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings.

  The terms "nucleic acid molecule" and "polynucleotide" may be used interchangeably and refer to a polymer of nucleotides. Such nucleotide polymers may include natural and / or non-natural nucleotides, including but not limited to DNA, RNA, and PNA. "Nucleic acid sequence" refers to a linear sequence of nucleotides comprising a nucleic acid molecule or polynucleotide.

  The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues and is not limited to the minimum length. Such polymers of amino acid residues may comprise naturally occurring or non-naturally occurring amino acid residues, including but not limited to peptides, oligopeptides, dimers, trimers and multimers of amino acid residues . Both full length proteins and fragments thereof are encompassed by this definition. These terms also include post expression modifications of the polypeptide, such as, for example, glycosylation, sialylation, acetylation, phosphorylation and the like. Furthermore, for the purposes of the present invention, "polypeptides" are modifications, eg, deletions, additions, and substitutions (generally conservative in nature) to the native sequence, as long as the protein retains the desired activity. Refers to a protein containing These modifications may be intentional, such as by site-directed mutagenesis, or accidental, such as by mutations in the host producing the protein or errors due to PCR amplification, etc. It is also good.

  The term "CSF1R" as used herein refers to full-length CSF1R comprising an N-terminal ECD, a transmembrane domain and an intracellular tyrosine kinase domain, with or without an N-terminal leader sequence. In some embodiments, CSF1R is human CSF1R having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

  As used herein, the term "CSF1R extracellular domain" ("CSF1R ECD") refers to a CSF1R polypeptide that lacks an intracellular and transmembrane domain. The CSF1R ECD includes full-length CSF1R ECD and CSF1R ECD fragments capable of binding CSF1R and / or IL-34. Human full-length CSF1R ECD, as used herein, comprises either amino acids 1-512 (ie, including the leader sequence) or amino acids 20-512 (ie, lacking the leader sequence) of SEQ ID NO: 2. It is defined as In some embodiments, the human CSF1R ECD fragment comprises amino acids 20-506 of SEQ ID NO: 2 (see SEQ ID NO: 5). In some embodiments, the human CSF1R fragment ends at amino acids 507, 508, 509, 510, or 511. In some embodiments, the monkey CSF1R ECD comprises the sequence of SEQ ID NO: 7 (including the leader sequence) or amino acids 20-506 (not including the leader sequence) of SEQ ID NO: 7.

  The terms "active" or "activity" or "function", and grammatical variants thereof, in the context of anti-CSF1R antibodies, suppress or block at least one of the foregoing activities Used to refer to the ability to Antibodies and antibody fragments termed "functional" are characterized as having such properties.

  "Immunological" activity refers only to the ability to induce the production of antibodies to an antigenic epitope possessed by a naturally occurring or naturally occurring CSF1R polypeptide.

As used herein, the term "antibody" is a molecule comprising at least complementarity determining region (CDR) 1, CDR2 and CDR3 of the heavy chain and at least CDR1, CDR2 and CDR3 of the light chain, , Refers to a molecule capable of binding an antigen. The term antibody includes, but is not limited to, fragments capable of binding an 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 various species of antibodies, such as mouse, human, cynomolgus monkeys, and the like.

  In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region. In some embodiments, the 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 a light chain variable region at least a portion of a light chain constant region It contains at least one light chain. In some embodiments, the antibody comprises two heavy chains, each heavy chain comprising a heavy chain variable region and at least a portion of a heavy chain constant region, and each light chain comprises a light chain variable region and a light chain. It includes two light chains, including at least a portion of chain constant regions. As used herein, any other antibody comprising a single chain Fv (scFv) or a single chain polypeptide comprising, for example, all six CDRs (three heavy chain CDRs and three light chain CDRs) Is believed to have heavy and light chains. In some such embodiments, the heavy chain is a region of an antibody that comprises three heavy chain CDRs, and the light chain is in the region of an antibody that comprises three light chain CDRs.

  The term "heavy chain variable region" as used herein refers to the region comprising heavy chain CDR1, framework (FR) 2, CDR2, FR3 and CDR3. In some embodiments, the heavy chain variable region also comprises at least a portion of FR1 and / or at least a portion of FR4. In some embodiments, heavy chain CDR1 corresponds to Kabat residues 26-35, heavy chain CDR2 corresponds to Kabat residues 50-65, and heavy chain CDR3 corresponds to Kabat residues 95-102. Do. See, for example, Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, Md.), And FIG. In some embodiments, heavy chain CDR1 corresponds to Kabat residues 31-35, heavy chain CDR2 corresponds to Kabat residues 50-65, and heavy chain CDR3 corresponds to Kabat residues 95-102. Do. See the same document.

The term "heavy chain constant regions", as used herein, refers to a region including at least three of the heavy chain constant domains, C H _1, C H _2, and C _{H 3.} Non-limiting exemplary heavy chain constant regions include gamma, delta, and alpha. Non-limiting exemplary heavy chain constant regions also include ε and μ. Each heavy constant region corresponds to the isotype of the antibody. For example, an antibody containing a γ constant region is an IgG antibody, an antibody containing a δ constant region is an IgD antibody, and an antibody containing an α constant region is an IgA antibody. Furthermore, the antibody containing the μ constant region is an IgM antibody, and the antibody containing the ε constant region is an IgE antibody. Certain isotypes can be further subdivided into subclasses. For example, IgG antibodies, IgG1 (gamma including ₁ constant region), IgG2 (gamma containing ₂ constant region), IgG3 (gamma including ₃ constant region), and (including gamma ₄ constant region) IgG4 including antibody , but are not limited to, IgA antibodies, including and IgA2 (alpha containing ₂ constant region) antibodies (including alpha ₁ constant region) IgAl, but are not limited to, IgM antibodies comprise IgM1 and IgM2 However, it is not limited to these.

In some embodiments, the heavy chain constant region comprises one or more mutations (or substitutions), additions or deletions that impart the desired characteristics to the antibody. A non-limiting exemplary mutation is the S241P mutation in the IgG4 hinge region (between the constant domains C _H 1 and C _H 2), which has a similar CPCPC to the corresponding motif of the IgG1 motif CPSCP to IgG1. change. In some embodiments, the mutation results in a more stable IgG4 antibody. For example, 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).

  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, the 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.

  The term "light chain variable region" as used herein refers to the region comprising light chain CDR1, framework (FR) 2, CDR2, FR3 and CDR3. In some embodiments, the light chain variable region also comprises FR1 and / or FR4. In some embodiments, light chain CDR1 corresponds to Kabat residues 24-34, light chain CDR2 corresponds to Kabat residues 50-56, and light chain CDR3 corresponds to Kabat residues 89-97. Do. See, for example, Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, Md.), And FIG.

The term "light chain constant regions", as used herein, refers to a region comprising the light chain constant domain C _L. Non-limiting exemplary light chain constant regions include λ and κ.

  The term "light chain" as used herein refers to a polypeptide comprising at least a light chain variable region and including or not including a leader sequence. In some embodiments, the 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.

  A "chimeric antibody" as used herein comprises at least one variable region derived from a first species (e.g. mouse, rat, cynomolgus monkey etc) and a second species (e.g. human, cynomolgus monkey etc) An antibody, which comprises at least one constant region derived therefrom. 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 monkey 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 variable regions of the chimeric antibody are from a first species, and all constant regions of the chimeric antibody are from a second species.

As used herein, "humanized antibody" refers to an antibody in which at least one amino acid within the non-human variable region framework 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 a fragment thereof. In some embodiments, the humanized antibody is Fab, scFv, (Fab ′) ₂ or the like.

  As used herein, a "CDR grafted antibody" is one in which complementarity determining regions (CDRs) of a first (non-human) species are grafted onto framework regions (FRs) of a second (human) species Refers to a humanized antibody.

  As used herein, “human antibodies” include human-produced antibodies, non-human animals comprising human immunoglobulin genes, eg, antibodies produced in Xenomouse®, and in vitro methods, eg, phage An antibody selected using a display, wherein the antibody repertoire is based on human immunoglobulin sequences.

  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 mammalian cells. The leader sequence may be cleaved upon export of the mammalian cell-derived polypeptide to form a mature protein. The leader sequences may be natural or synthetic and may be heterologous or homologous to the protein to which they are attached. Exemplary leader sequences include, but are not limited to, the antibody leader sequences, eg, the amino acid sequences of SEQ ID NOs: 3 and 4 that correspond to human light and heavy chain leader sequences, respectively. Non-limiting exemplary leader sequences also include leader sequences derived from heterologous proteins. In some embodiments, the antibody lacks a leader sequence. In some embodiments, the antibody comprises at least one leader sequence, which may be selected from natural antibody leader sequences and heterologous leader sequences.

  The term "vector" is used to denote a polynucleotide which can be engineered to contain a cloned polynucleotide or which can be propagated in a host cell. The vector may comprise an origin of replication, one or more regulatory sequences (eg, a promoter and / or an enhancer) that modulates expression of the polypeptide of interest, and / or one or more selectable marker genes (eg, antibiotic resistance). Sex genes and one or more of the elements of genes that can be used in colorimetric assays, such as β-galactosidase, may be included. The term "expression vector" refers to a vector used to express a polypeptide of interest in a host cell.

  "Host cell" refers to a cell that can be or is a recipient of a vector or isolated polynucleotide. Host cells can be prokaryotic or eukaryotic cells. Exemplary prokaryotic cells include mammalian cells, such as primate or non-primate animal cells, fungal cells such as yeast, plant cells, and insect cells. Non-limiting exemplary mammalian cells include NSO cells, PER. C6 cells (Crucell), 293 and CHO cells, and their derivatives, including but not limited to 293-6E and DG44 cells, respectively.

  The term "isolated" as used herein generally refers to a molecule that is separated from at least a portion of the components found in nature. For example, a polypeptide is said to be "isolated" if it is separated from at least a portion of the components of the cell in which it is produced. Where the polypeptide is secreted by cells after expression, physically separating the supernatant containing the polypeptide from the produced cells is considered as "isolated" the polypeptide. Similarly, the polynucleotide is usually not part of a larger polynucleotide (eg, genomic DNA or mitochondrial DNA in the case of DNA polynucleotides) found in nature, or, for example, in the case of RNA polynucleotides. When separated from at least a portion of the components of a cell, it is referred to as "isolated." Thus, a DNA polynucleotide contained in a vector in a host cell can be referred to as "isolated", as long as the polynucleotide is not naturally found in the vector.

  The terms "subject" and "patient" are used interchangeably herein to refer to human. In some embodiments, including, but not limited to, rodents, apes, cats, dogs, horses, cows, pigs, sheep, goats, mammalian laboratory animals, mammalian livestock, mammalian sport animals, and mammalian pets, for example. Methods of treating other mammals not being treated are also provided.

  As used herein, "rheumatoid arthritis" or "RA" refers to recognized disease states that may be diagnosed according to the American Rheumatoid Association criteria or any similar criteria revised in 2000 for the classification of RA. Point to. In some embodiments, the term "rheumatoid arthritis" is a chronic autoimmunity characterized primarily by inflammation of the intima (synovial membrane) that causes joint damage resulting in chronic pain, loss of function, and disability. Refers to a disease. RA is referred to as a systemic disease because it affects multiple organs of the body, including the skin, lungs, and the eyes.

  The term "rheumatoid arthritis" includes not only active and early RA, but also early RA, as defined below. Physiological indicators of RA include symmetrical joint swelling that is characteristic of RA but not constant. The fusiform expansion of the proximal interphalangeal (PIP) joint of the hand, as well as the metacarpal segment (MCP), wrist, elbow, knee, ankle, and metatarsophalangeal (MTP) joint are generally affected The swelling is easily detected. Pain in passive exercise is the most sensitive test for joint inflammation, and inflammation and structural deformation often limit the range of movement of the affected joint. Typical visible changes include ulnar deflection of the finger at the MCP joint, hyperextension or hyperflexion of the MCP and PIP joints, flexion contracture of the elbow, and carpal and toe subluxation. Subjects having RA may be resistant to disease modifying anti-rheumatic drugs (DMARDs), and / or non-steroidal anti-inflammatory drugs (NSAIDs). Non-limiting exemplary "DMARDs" include hydroxychloroquine, sulfasalazine, methotrexate (MTX), leflunomide, etanercept, infliximab (also oral and subcutaneous MTX), azathioprine, D-penicillamine, gold salt (oral) , Gold salts (im injection), minocycline, cyclosporin A and cyclosporin including topical cyclosporin, and staphylococcal protein A (Goodyear and Silverman, J. Exp. Med., 197 (9): 1125-1139 (2003)). These also include salts and derivatives thereof and the like. Additional candidates for treatment according to the present invention may have an inadequate response to previous or current treatment with TNF inhibitors such as etanercept, infliximab, and / or adalimumab due to toxicity or inadequate efficacy. Includes those who have experienced it.

  A patient having "active rheumatoid arthritis" refers to a patient who is active and has symptoms of RA that is not latent. Patients with “early activity rheumatoid arthritis” are diagnosed as active RA for at least 8 weeks but not longer than 4 years according to the revised 1987 ACR criteria for RA classification Target.

  Subjects who have “early rheumatoid arthritis” are those who have been diagnosed with RA for at least 8 weeks, but not for 4 years or more, according to the revised 1987 ACR criteria for RA classification . RA includes, for example, juvenile onset RA, juvenile idiopathic arthritis (JIA), or juvenile RA (JRA).

  Patients with “early RA” have early polyarthritis that does not adequately meet the ACR criteria for diagnosis of RA in relation to the presence of RA-specific prognostic biomarkers such as anti-CCP and shared epitopes Suffer from They include patients with polyarthritis but who have not yet been diagnosed with RA and who are truly at risk for developing ACR criteria RA (95% chance) positive anti-CCP antibody .

  "Joint damage" is used in a broad sense and refers to damage or partial or complete destruction to any part of one or more joints, including connective tissue and cartilage, damage being structural and / or functional from any cause Injury, which may or may not cause joint pain / arthrgia. It includes, but is not limited to, inflammatory joint disease as well as joint damage associated with or obtained from non-inflammatory joint disease. This damage can be caused by any condition, for example autoimmune diseases, in particular arthritis, and even RA. Exemplary such conditions include acute and chronic arthritis, rheumatoid arthritis (including juvenile onset RA, juvenile idiopathic arthritis (JIA), and juvenile rheumatoid arthritis (JRA)), rheumatoid synovitis Gout or gouty arthritis, acute immunological arthritis, chronic inflammatory arthritis, degenerative arthritis, collagen type II induced arthritis, infectious arthritis, septic arthritis, Lyme disease arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, vertebral arthritis, osteoarthritis, progressive chronic arthritis (arthritis chronica progrediente), osteoarthritis, primary chronic polyarthritis (polyarthritis chronica primaria), reactive arthritis, menopausal arthritis, estrigen-depleted arthritis, and rigidity Spondylitis / Rheumatoid spine Etc., rheumatoid autoimmune diseases other than RA, as well as severe systemic lesions secondary to RA (including but not limited to vasculitis, pulmonary fibrosis, or Fertiti syndrome) Be For purposes herein, a joint is a point of contact between elements of the skeleton (of a vertebrate such as an animal) with the part surrounding and supporting it, eg, the buttocks, the spinal cord Intervertebral joint, joint between spinal cord and pelvis (sacroiliac joint), joint where tendon and ligament adhere to bone, joint between calcaneus and spinal cord, shoulder, knee, foot, elbow, hand, finger, ankle, And toes, particularly but not limited to the joints of the hands and feet.

  As used herein, the term "swelling" is generally an autoimmune disease or disorder resulting from an antibody that attacks connective tissue. The major forms of lupus are systemic and systemic lupus erythematosus (SLE), including skin SLE and subacute skin SLE, and other types of lupus (nephritis, extrarenal, encephalitis, childhood, kidney External, discoid, and alopecia). In certain embodiments, the term "systemic lupus erythematosus" includes skin lesions, joint pain and swelling, kidney disease (lupus nephritis), heart and / or fluid around the lungs, heart inflammation, and various others. Refers to a chronic autoimmune disease that can lead to a generalized condition. In one embodiment, the term "lupus nephritis" refers to the inflammation of the kidneys that occurs in patients with SLE. Lupus nephritis may include, for example, glomerulonephritis and / or interstitial nephritis, and may cause hypertension, proteinuria, and renal failure. Lupus nephritis may be classified based on the severity and degree of the disease, as defined, for example, by the International Society of Nephrology / Renal / Pathology Society. Classification of lupus nephritis includes type I (micro mesangial lupus nephritis), type II (mesangial proliferative lupus nephritis), type III (focal lupus nephritis), type IV (diffuse segmental (IV-S) or diffuse) All node (IV-G) lupus nephritis), type V (membranous lupus nephritis), and type VI (advanced sclerosing lupus nephritis) are included. The term "lupus nephritis" encompasses all of these categories.

  The term "multiple sclerosis" ("MS") refers to a chronic and often disabling disease of the central nervous system characterized by progressive destruction of myelin. "Demyelination" occurs when the myelin sheath becomes inflamed, damaged, and detached from nerve fibers. Four internationally recognized forms of MS: primary progressive multiple sclerosis (PPMS), relapsing-remitting multiple sclerosis (RRMS), secondary progressive multiple sclerosis (SPMS), and progressive relapse There is multiple sclerosis (PRMS).

  "Primary-progressive multiple sclerosis" or "PPMS" is characterized by a gradual progression of the disease from its onset with no overlap of relapses and remissions. There may be periods of steady state disease activity, and there may be good days or weeks and bad days or weeks. PPMS is typically in the late 30's or early 40's, with men as likely to develop as women, and early disease activity is often in the spinal cord rather than in the brain. Are different from RRMS and SPMS. PPMS often travels to the brain but has a lower probability of damaging the brain area than RRMS or SPMS, eg, those with PPMS have a lower probability of developing cognitive problems. PPMS is the subtype of MS that has the lowest probability of showing inflammatory (gadolium-enhanced) lesions on MRI scans. The primary progressive form of the disease affects 10-15% of all people with multiple sclerosis. PPMS is described by McDonald et al. It can be defined according to the criteria of Ann Neurol 50: 121-7 (2001). A subject having PPMS to be treated herein is usually a subject who has an estimated or definitive diagnosis of PPMS.

  "Relapsing-remitting multiple sclerosis" or "RRMS" is characterized by relapse (also known as exacerbation), a time when new symptoms appear and old symptoms may recur or worsen. After the relapse, a remission period follows, during which period one completely or partially recovers from the impairment obtained during the relapse. Relapses may last for days, weeks or months, and recovery may be slow and gradual or almost instantaneous. Most people who present with MS are first diagnosed with RRMS. This is generally the case when they are in their twenties or thirties, but the diagnosis of much earlier or later is also known. Twice as many women as men present this subtype of MS. During relapse, myelin, a protective insulating myelin sheath around nerve fibers (neurons) in the white matter region of the central nervous system (CNS), can be damaged by the inflammatory response by the body's own immune system. This results in a variety of neurological symptoms that vary significantly depending on which area of the CNS is damaged. Immediately after relapse, the inflammatory response goes away and supports the process by which special types of glial cells in the CNS (called oligodendrocytes) can remyelinate, that is, repair the myelin sheath around the axon. It is this remyelination that may be involved in remission. About 50% of patients with RRMS convert to SPMS within 10 years of disease onset. After 30 years, this figure will rise to 90%. At any point in time, the relapsing-remitting form of the disease accounts for approximately 55% of all people with MS.

  "Secondary progressive multiple sclerosis" or "SPMS" is characterized by steady progression of clinical neurological damage with or without superimposed relapses and mild remissions and plateaus. People who develop SPMS will have experienced a period of RRMS that may last from 2 to 40 years or more. Any overlapping relapses and remissions tend to fade over time. From the onset of the second progressive stage of the disease, disability starts and progresses faster than occurred during RRMS, but the progression may still be quite slow in some individuals. SPMS tends to have lower levels of inflammatory lesion formation than in RRMS, but the full burden of disease continues to progress. At any one time, SPMS accounts for approximately 30% of all people with multiple sclerosis.

  "Progressive relapsing multiple sclerosis" or "PRMS" is characterized by a steady progression of clinical neurological damage with overlapping relapses and remissions. There is a significant recovery immediately after relapse, but there is a gradual deterioration of symptoms during relapse. PRMS affects approximately 5% of all people with multiple sclerosis. Some neurologists consider PRMS to be a mutated form of PPMS.

  The term "CD16 + disease" means a disease in which mammalian CD16 + monocytes cause, mediate or otherwise result in morbidity in a mammal. Also included are diseases that have the effect of reducing CD16 + monocytes leading to an improvement in the progression of the disease. Included within this term are CD16 + inflammatory diseases, infections, immunodeficiency, neoplasia and the like. In one embodiment, the CD16 + inflammatory disease comprises an inflammatory disease that does not respond to methotrexate therapy. In certain embodiments, CD16 + inflammatory disease includes methotrexate resistant rheumatoid arthritis, methotrexate resistant multiple sclerosis, methotrexate resistant epilepsy, methotrexate resistant inflammatory bowel disease, methotrexate resistant Crohn's disease, It includes methotrexate resistant asthma and methotrexate resistant psoriasis. In certain embodiments, a patient having a methotrexate resistant disease, eg, methotrexate resistant rheumatoid arthritis, is referred to as a responder who is methotrexate deficient or a methotrexate deficient responder.

  Examples of CD16 + disorders which can be treated according to the invention include systemic lupus erythematosus, rheumatoid arthritis, juvenile chronic arthritis, spondyloarthritis, systemic sclerosis (scieroderma), spontaneous inflammatory Myopathy (dermomyositis, polymyositis), Sjögren's syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia (immune pancytopenia, paroxysmal nocturnal hemoglobinuria), autoimmune thrombocytopenia (spontaneous) Thrombocytopenic purpura, immune-mediated thrombocytopenia), thyroiditis (graves disease, Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis), diabetes mellitus, immune-mediated kidney disease (glomerulonephritis, Tubulointerstitial nephritis), demyelinating diseases of the central and peripheral nervous system, such as multiple sclerosis, idiopathic demyelinating polyneuropathy, or Guillain-Barre syndrome, and chronic Demyelinating polyneuropathy, hepatobiliary disease such as infectious hepatitis (type A, type B, type C, type D, type E hepatitis E and other non-hepatotropic viruses), autoimmune chronic active hepatitis, primary Biliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis, ulcerative colitis, inflammatory bowel disease (IBD) including Crohn's disease, gluten hypersensitivity enteropathy, and Whipple's disease, bullous disease, polymorphism Erythema and contact dermatitis, autoimmune or immune-mediated skin diseases including psoriasis, asthma, allergic rhinitis, atopic dermatitis, food hypersensitivity, allergic diseases including urticaria, eosinophilic pneumonia , Idiopathic pulmonary fibrosis, and lung immune diseases such as hypersensitivity pneumonia, graft related diseases including graft rejection and graft versus host disease, fibrosis including kidney fibrosis and lung fibrosis, atherosclerotic artery Sclerosis and crown Cardiovascular disease including cardiovascular disease, chronic kidney disease, cardiovascular events associated with myocardial infarction and congestive heart failure, diabetes including type II diabetes, bronchiolitis with organized pneumonia (BOOP), hemophagocytic syndrome, macrophages These include, but are not limited to, activation syndrome, sarcoidosis and periodontitis. Infectious diseases including viral diseases such as AIDS (HIV infection), A, B, C, D and E, bacterial infections, fungal infections, protozoal infections and parasitic infections.

  "Treatment," as used herein, refers to both therapeutic treatment and prophylactic or protective measures, the purpose of which is to prevent or retard (reduce) the target condition or disorder. It is. In certain embodiments, the term "treatment" covers any administration or application of a therapeutic agent for a disease in a mammal, including a human, to inhibit or slow down the disease or the progression of the disease, for example, causing regression. Or partially or completely ameliorate the disease by recovering or repairing lost, missing, or diminished function, stimulate an inefficient process, or bring about a reduction in severity during the stable phase of the disease Including. The term "treatment" also includes reducing the severity of, and / or reducing the occurrence, extent, or likelihood of any phenotypic feature. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in which the disorder is to be prevented.

  "Chronic" administration refers to administration of the drug in a continuous mode, as opposed to an acute mode, to maintain the initial therapeutic effect (activity) for an extended period of time. "Intermittent" administration is not continuous, but without interruption, but rather is actually a periodic treatment.

  The terms "effective amount" or "therapeutically effective amount" refer to an amount of drug effective to treat a disease or disorder in a subject. In certain embodiments, an effective amount refers to an amount effective at the required dose and duration to achieve the desired therapeutic or prophylactic result. The therapeutically effective amount of the anti-CSF1R antibodies of the invention may vary depending on factors such as the disease state, age, sex and weight of the individual, and the ability of the anti-CSF1R antibody to elicit the desired response in the individual. A therapeutically effective amount includes an amount that has a therapeutically beneficial effect over any toxic or detrimental effects of the anti-CSF1R antibody.

  A "prophylactically effective amount" refers to an amount effective at the dosage and for the duration required to achieve the desired prophylactic result. Typically, but not necessarily, the prophylactic dose will be less than the therapeutically effective amount to be used in the subject prior to or at an earlier stage of the disease.

  Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

  “Pharmaceutically acceptable carrier” is a non-toxic solid, semi-solid or liquid filler conventionally used in the art for use in conjunction with a therapeutic substance together with a “pharmaceutical composition” for administration to a subject Refers to a diluent, encapsulating material, formulation aid, or carrier. Pharmaceutically acceptable carriers are nontoxic to recipients at the dosages and concentrations employed and are compatible with the other ingredients of the formulation. Pharmaceutically acceptable carriers are suitable for the formulations used. For example, when the therapeutic substance is administered orally, the carrier may be a gel capsule. When the therapeutic substance is administered subcutaneously, this carrier is theoretically non-irritating to the skin and does not generate an injection site reaction.

Anti-CSF1R Antibodies Anti-CSF1R antibodies include, but are not limited to, humanized antibodies, chimeric antibodies, mouse antibodies, human antibodies, and antibodies comprising heavy and / or light chain CDRs as discussed herein.

Exemplary Humanized Antibodies In some embodiments, humanized antibodies that bind CSF1R are provided. Humanized antibodies can result in an immune response to antibody therapeutics, which can reduce the efficacy of the therapeutic, reduce or eliminate human immune responses (eg, human anti-mouse antibodies (HAMA) responses) to non-human antibodies , Useful as therapeutic molecules.

  Non-limiting exemplary humanized antibodies include the huAb1-huAb16 described herein. Non-limiting exemplary humanized antibodies also include antibodies comprising a heavy chain variable region of an antibody selected from huAb1 to huAb16 and / or a light chain variable region of an antibody selected from huAb1 to huAb16. Non-limiting exemplary humanized antibodies include antibodies comprising a heavy chain variable region selected from SEQ ID NO: 39-45 and / or a light chain variable region selected from SEQ ID NO: 46-52. Be Exemplary humanized antibodies also include 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. However, it is not limited to these.

  In some embodiments, the humanized anti-CSF1R antibody comprises heavy chain CDR1, CDR2 and CDR3 and / or light chain CDR1, CDR2 and CDR3 of an antibody selected from 0301, 0302 and 0311. Non-limiting exemplary humanized anti-CSF1R antibodies are selected from SEQ ID NOs: 15, 16, and 17, SEQ ID NOs: 21, 22, and 23, and SEQ ID NOs: 27, 28, and 29. Antibodies comprising a set of heavy chain CDR1, CDR2 and CDR3. Also, non-limiting exemplary humanized anti-CSF1R antibodies include SEQ ID NOs: 18, 19 and 20, SEQ ID NOs: 24, 25 and 26, and SEQ ID NOs: 30, 31 and 32. Also included are antibodies comprising the selected light chain CDR1, CDR2 and CDR3 pair.

  For the non-limiting exemplary humanized anti-CSF1R antibodies, the combination of heavy chain CDR1, CDR2, and CDR3 with light chain CDR1, CDR2, and CDR3 of Table 1 (SEQ ID NOs: is provided and refer to Table 8 for sequence) Containing antibodies. Each row of Table 1 shows the heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3 of an exemplary antibody.

TABLE 1 Heavy Chain CDRs and Light Chain CDRs

Additional Exemplary Humanized Antibodies In some embodiments, the humanized anti-CSF1R antibody is at least 90%, at least 91%, at least 92 with a sequence selected from SEQ ID NO: 9, 11, 13, and 39-45. %, At least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical heavy chain comprising a variable region sequence, the antibody binds CSF1R . In some embodiments, the humanized anti-CSF1R antibody is at least 90%, at least 91%, at least 92%, at least 93%, with a sequence selected from SEQ ID NO: 10, 12, 14, and 46-52. A light chain comprising a variable region sequence that is at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the antibody binds CSF1R. In some embodiments, the humanized anti-CSF1R antibody is at least 90%, at least 91%, at least 92%, at least 93%, with a sequence selected from SEQ ID NOs: 9, 11, 13, and 39-45 A heavy chain comprising a variable region sequence that is at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical; SEQ ID NOS: 10, 12, 14, and 46-52 A variable 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 the sequence selected from A light chain containing the domain sequence, and the antibody binds CSF1R Let

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

  In some embodiments, a humanized anti-CSF1R antibody comprises at least one of the CDRs discussed herein. That is, in some embodiments, the humanized anti-CSF1R antibody comprises the heavy chain CDR1 discussed herein, the heavy chain CDR2 discussed herein, the heavy chain CDR3 discussed herein, The light chain CDR1 discussed, the light chain CDR2 discussed herein, and the at least one CDR selected from the light chain CDR3 discussed herein. Further, in some embodiments, the humanized anti-CSF1R antibody comprises at least one mutant CDR based on the CDRs discussed herein, wherein the mutant CDRs are directed against the CDRs discussed herein. , 1, 2, 3 or 4 amino acid substitutions. 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 appropriate conservative amino acid substitutions for a particular CDR sequence, which suitably alters the binding properties of the antibody containing the mutated CDRs. It is not predicted.

  Exemplary humanized anti-CSF1R antibodies also include antibodies that compete with the antibodies described herein for binding to CSF1R. Thus, in some embodiments, antibodies selected from Fab0301, 0302, and 0311, and bivalent (ie, having two heavy chains and two light chains) antibody forms of these Fabs, and to CSF1R Humanized anti-CSF1R antibodies that compete for binding are provided.

Exemplary Humanized Antibody Constant Regions In some embodiments, the humanized antibodies described herein comprise one or more human constant regions. In some embodiments, the human heavy chain constant region consists of an isotype selected from IgA, IgG, and IgD. In some embodiments, the human light chain constant region consists of an isotype selected from kappa and lambda. In some embodiments, the humanized antibodies described herein comprise human IgG constant regions. In some embodiments, the humanized antibodies described herein comprise a human IgG4 heavy chain constant region. In some such embodiments, the humanized antibodies described herein comprise an S241P mutation in a human IgG4 constant region. In some embodiments, the humanized antibodies described herein comprise a human IgG4 constant region and a human kappa light chain.

  Selection of the heavy chain constant region can determine whether the antibody has effector function in vivo. Such effector functions, in some embodiments, include antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), and kill the cells to which the antibody binds. Can bring In some therapeutic methods, including methods of treating some cancers, cell killing may be desirable, for example, when the antibody binds to cells that support tumor maintenance or growth. Exemplary cells that can support tumor maintenance or growth include the tumor cells themselves, cells that support tumor angiogenesis, cells that provide a ligand, growth factors, or counter acceptance to support or promote tumor growth or tumor survival. Body included, but not limited to. In some embodiments, when effector function is desired, anti-CSF1R antibodies comprising human IgG1 heavy chain or human IgG3 heavy chain are selected.

  In some therapeutic methods, effector function may be undesirable. 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-CSF1R antibodies developed for the treatment of cancer may not be suitable for use in the treatment of lupus and / or MS and / or RA and / or osteolysis. Thus, in some embodiments, anti-CSF1R antibodies lacking significant effector function are used in the treatment of lupus and / or MS and / or RA and / or osteolysis. In some embodiments, anti-CSF1 R antibodies for the treatment of lupus and / or MS and / or RA and / or osteolysis comprise human IgG4 or IgG2 heavy chain constant regions. In some embodiments, the IgG4 constant region comprises an S241P mutation.

  The antibodies may be humanized by any method. Non-limiting exemplary methods of humanization include, for example, US Patent Nos. 5,530,101, 5,585,089, 5,693,761, 5,693,762. No. 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. Patent Application Publication No. 2009/0136500.

  As described above, a humanized antibody is an antibody in which at least one amino acid in the framework region of the non-human variable region is substituted with an amino acid at the corresponding position in the 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 within the framework region of the non-human variable region Ten, at least 11, at least 12, at least 15, or at least 20 amino acids are substituted for amino acids at one or more corresponding positions within one or more human framework regions.

  In some embodiments, some of the corresponding human amino acids used for substitution are derived from framework regions of different human immunoglobulin genes. That is, in some such embodiments, one or more of the non-human amino acids are derived from a human framework region of a first human antibody, or the corresponding human immunoglobulin gene encoded by the first human immunoglobulin gene The amino acids may be substituted, and one or more of the non-human amino acids are derived from the human framework region of the second human antibody or to the corresponding amino acids encoded by the second human immunoglobulin gene Optionally substituted, one or more of the non-human amino acids are derived from the human framework region of the third human antibody or substituted with the corresponding amino acid encoded by the third human immunoglobulin gene May be Furthermore, in some embodiments, not all of the single framework regions, eg, corresponding human amino acids used for substitution in FR2, need to be from the same human framework. However, in some embodiments, all of the corresponding human amino acids used for substitution are derived from the same human antibody or encoded by the same human immunoglobulin gene.

  In some embodiments, antibodies are humanized by replacing one or more entire framework regions with corresponding human framework regions. In some embodiments, human framework regions with the highest level of homology to the non-human framework regions to be substituted are selected. In some embodiments, such humanized antibodies are CDR grafted antibodies.

  In some embodiments, after CDR grafting, one or more framework amino acids are changed back to the corresponding amino acids in the mouse framework region. Such "back mutations" may, in some embodiments, be attributed to the structure of one or more CDRs, and / or may be involved in antigen adhesion, and / or It is done to retain one or more mouse framework amino acids that appear to be involved in the overall structural integrity of the antibody. In some embodiments, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, 1 or 0 Back mutations are generated in the framework regions of the antibody after CDR grafting.

  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 In some embodiments, the anti-CSF1R antibody is a chimeric antibody. In some embodiments, the anti-CSF1R antibody comprises at least one non-human variable region and at least one human constant region. In some such embodiments, the variable regions of the anti-CSF1R antibody are all non-human variable regions, and the constant regions of the anti-CSF1R antibody are all human constant regions. In some embodiments, one or more variable regions of the chimeric antibody are mouse variable regions. The human constant regions of a chimeric antibody need not be of the same isotype as the non-human constant regions, even if the non-human constant regions it replaces are present. Chimeric antibodies are described, for example, in US Pat. No. 4,816,567, and Morrison et al. Proc. Natl. Acad. Sci. USA 81: 6851-55 (1984).

  Non-limiting exemplary chimeric antibodies include chimeric antibodies comprising heavy and / or light chain variable regions of antibodies selected from 0301, 0302, and 0311. Additional non-limiting exemplary chimeric antibodies include chimeric antibodies comprising heavy chain CDR1, CDR2, and CDR3 of the antibody selected from 0301, 0302, and 0311, and / or light chain CDR1, CDR2, and CDR3. Be

  Non-limiting exemplary chimeric anti-CSF1R 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 NO: : 13 and 14.

  Non-limiting exemplary anti-CSF1R antibodies include antibodies comprising the heavy chain CDR1, CDR2, and CDR3 pairs shown in Table 1 above, and the light chain CDR1, CDR2, and CDR3 pairs.

Additional Exemplary Chimeric Antibodies In some embodiments, the chimeric anti-CSF1R antibody is at least 90%, at least 91%, at least 92%, with a sequence selected from SEQ ID NO: 9, 11, 13, and 39-45. A heavy chain comprising a variable region sequence that is at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the antibody binds CSF1R. In some embodiments, the chimeric anti-CSF1R antibody is at least 90%, at least 91%, at least 92%, at least 93%, at least 90% with a sequence selected from SEQ ID NO: 10, 12, 14, and 46-52. The antibody comprises a light chain comprising a variable region sequence that is 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the antibody binds CSF1R. In some embodiments, the chimeric anti-CSF1R antibody is at least 90%, at least 91%, at least 92%, at least 93%, at least 90% with a sequence selected from SEQ ID NO: 9, 11, 13, and 39-45 A heavy chain comprising a variable region sequence that is 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and SEQ ID NO: 10, 12, 14, and 46-52 Variable regions which are 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 the sequence selected. A light chain containing the sequence, and the antibody binds CSF1R Let

  In some embodiments, a chimeric anti-CSF1R antibody comprises at least one of the CDRs discussed herein. That is, in some embodiments, the chimeric anti-CSF1R antibody comprises the heavy chain CDR1 discussed herein, the heavy chain CDR2 discussed herein, the heavy chain CDR3 discussed herein, And at least one CDR selected from light chain CDR2 as discussed herein and light chain CDR3 as discussed herein. Further, in some embodiments, the chimeric anti-CSF1R antibody comprises at least one mutant CDR based on the CDRs discussed herein, wherein the mutant CDRs are relative to the CDRs discussed herein: It contains one, two, three or four amino acid substitutions. 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 appropriate conservative amino acid substitutions for a particular CDR sequence, which suitably alters the binding properties of the antibody containing the mutated CDRs. It is not predicted.

  Exemplary chimeric anti-CSF1R antibodies also include chimeric antibodies that compete with the antibodies described herein for binding to CSF1R. Thus, in some embodiments, antibodies selected from Fab0301, 0302, and 0311, and bivalent (ie, having two heavy chains and two light chains) antibody forms of these Fabs, and to CSF1R Chimeric anti-CSF1R antibodies that compete for binding are provided.

Exemplary Chimeric Antibody Constant Regions In some embodiments, the chimeric antibodies described herein comprise one or more human constant regions. In some embodiments, the human heavy chain constant region consists of an isotype selected from IgA, IgG, and IgD. In some embodiments, the human light chain constant region consists of an isotype selected from kappa and lambda. 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, the chimeric antibodies described herein comprise an S241P mutation in a human IgG4 constant region. In some embodiments, a chimeric antibody described herein comprises a human IgG4 constant region and a human kappa light chain.

  As mentioned above, whether effector function is desired may vary depending on the particular therapeutic method being for the antibody. Thus, in some embodiments, where effector function is desired, a chimeric anti-CSF1R antibody comprising human IgG1 heavy chain constant region or human IgG3 heavy chain constant region is selected. In some embodiments, where effector function is desired, a chimeric anti-CSF1R antibody comprising human IgG4 or IgG2 heavy chain constant region is selected.

Exemplary Human Antibodies Human antibodies can be made by any suitable method. A non-limiting exemplary method involves producing human antibodies in transgenic mice containing human immunoglobulin loci. For example, 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, and the like. Nos. 5,545,807, 6,300,129, 6,255,458, 5,877,397, 5,874,299, and 5,545 , 806.

  Non-limiting exemplary methods also include producing human antibodies using phage display libraries. For example, Hoogenboom et al. , J. Mol. Biol. 227: 381-8 (1992), Marks et al. , J. Mol. Biol. 222: 581-97 (1991), and also PCT Publication No. WO 99/10494.

  In some embodiments, a human anti-CSF1R antibody binds to a polypeptide having the sequence of SEQ ID NO: 1. Exemplary human anti-CSF1R antibodies also include antibodies that compete with the antibodies described herein for binding to CSF1R. Thus, in some embodiments, antibodies selected from Fab0301, 0302, and 0311, and bivalent (ie, having two heavy chains and two light chains) antibody forms of these Fabs, and CSF1R There is provided a human anti-CSF1R antibody that competes for the binding of

  In some embodiments, the human anti-CSF1R antibody comprises one or more human constant regions. In some embodiments, the human heavy chain constant region consists of an isotype selected from IgA, IgG, and IgD. In some embodiments, the human light chain constant region consists of an isotype selected from kappa and lambda. In some embodiments, the human antibodies described herein comprise human IgG constant regions. In some embodiments, the human antibodies described herein comprise a human IgG4 heavy chain constant region. In some such embodiments, the human antibodies described herein comprise an S241P mutation in a human IgG4 constant region. In some embodiments, the human antibodies described herein comprise a human IgG4 constant region and a human kappa light chain.

  In some embodiments, where effector function is desired, a human anti-CSF1 R antibody is selected which comprises a human IgG1 heavy chain constant region or a human IgG3 heavy chain constant region. In some embodiments, where effector function is not desired, a human anti-CSF1 R antibody comprising human IgG4 or IgG2 heavy chain constant region is selected.

Additional Exemplary Anti-CSF1R Antibodies Exemplary anti-CSF1R antibodies also include, for example, murine, humanized, human, chimeric, and engineered antibodies that include one or more of the CDR sequences described herein. But not limited to. In some embodiments, the anti-CSF1R antibody comprises a heavy chain variable region as described herein. In some embodiments, the anti-CSF1R antibody comprises a light chain variable region as described herein. In some embodiments, an anti-CSF1R antibody comprises a heavy chain variable region as described herein and a light chain variable region as described herein. In some embodiments, the anti-CSF1R antibody comprises heavy chain CDR1s, CDR2s and CDR3s described herein. In some embodiments, an anti-CSF1R antibody comprises light chain CDR1s, CDR2s and CDR3s described herein. In some embodiments, the anti-CSF1R antibody comprises heavy chain CDR1s, CDR2s and CDR3s described herein and light chain CDR1s, CDR2s and CDR3s described herein.

  In some embodiments, the anti-CSF1R antibody comprises the heavy chain variable region of an antibody selected from Fab0301, 0302, and 0311. Non-limiting exemplary anti-CSF1 R antibodies also include antibodies comprising the heavy chain variable region of an antibody selected from humanized antibodies huAb1 to huAb16. Non-limiting exemplary anti-CSF1R antibodies include antibodies comprising a heavy chain variable region comprising a sequence selected from SEQ ID NO: 9, 11, 13, and 39-45.

  In some embodiments, the anti-CSF1R antibody comprises the light chain variable region of an antibody selected from Fabs 0301, 0302, and 311. Non-limiting exemplary anti-CSF1 R antibodies also include antibodies comprising the light chain variable region of an antibody selected from humanized antibodies huAb1 to huAb16. Non-limiting exemplary anti-CSF1 R antibodies include antibodies comprising a light chain variable region comprising a sequence selected from SEQ ID NO: 10, 12, 14, and 46-52.

  In some embodiments, the anti-CSF1R antibody comprises the heavy chain variable region and the light chain variable region of an antibody selected from Fab0301, 0302, and 0311. Non-limiting exemplary anti-CSF1 R antibodies also include antibodies comprising heavy and light chain variable regions of an antibody selected from humanized antibodies huAb1 to huAb16. Non-limiting exemplary anti-CSF1R 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, SEQ ID NO: 13 And 14, SEQ ID NO: 39 and 40, SEQ ID NO: 41 and 42, SEQ ID NO: 43 and 44, SEQ ID NO: 45 and 46, SEQ ID NO: 47 and 48, SEQ ID NO: 49 and 50 , And SEQ ID NOs: 51 and 52. Non-limiting exemplary anti-CSF1R antibodies also include antibodies comprising the following pairs of heavy and light chains: SEQ ID NO: 33 and 34, SEQ ID NO: 35 and 36, and SEQ ID NO: 37 and 38.

  In some embodiments, the anti-CSF1R antibody comprises heavy chain CDR1, CDR2 and CDR3 of an antibody selected from Fab0301, 0302 and 0311. Non-limiting exemplary anti-CSF1R antibodies comprise a heavy chain selected from SEQ ID NOs: 15, 16 and 17, SEQ ID NOs: 21, 22 and 23 and SEQ ID NOs: 27, 28 and 29. Included are antibodies comprising a set of chain CDRl, CDR2 and CDR3.

  In some embodiments, the anti-CSF1R antibody comprises light chains CDR1, CDR2 and CDR3 of an antibody selected from Fab0301, 0302 and 0311. Non-limiting exemplary anti-CSF1R antibodies include light selected from SEQ ID NOs: 18, 19 and 20, SEQ ID NOs: 24, 25 and 26, and SEQ ID NOs: 30, 31 and 32. Included are antibodies comprising a set of chain CDRl, CDR2 and CDR3.

  In some embodiments, the anti-CSF1R antibody comprises heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3 of an antibody selected from Fab0301, 0302, and 0311.

  Non-limiting exemplary anti-CSF1R antibodies include antibodies comprising the heavy chain CDR1, CDR2, and CDR3 pairs shown in Table 1 above, and the light chain CDR1, CDR2, and CDR3 pairs.

Additional Exemplary Antibodies In some embodiments, the anti-CSF1R antibody is at least 90%, at least 91%, at least 92%, at least 93 with a sequence selected from SEQ ID NOs: 9, 11, 13, and 39-45. %, At least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical heavy chain comprising a variable region sequence, the antibody binds CSF1R. In some embodiments, the anti-CSF1R antibody is at least 90%, at least 91%, at least 92%, at least 93%, at least 94 with a sequence selected from SEQ ID NO: 10, 12, 14, and 46-52. A light chain comprising a variable region sequence that is%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the antibody binds CSF1R. In some embodiments, the anti-CSF1R antibody is at least 90%, at least 91%, at least 92%, at least 93%, at least 94 with a sequence selected from SEQ ID NO: 9, 11, 13, and 39-45 A heavy chain comprising a variable region sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and selected from SEQ ID NOs: 10, 12, 14, and 46-52 Variable region sequences that are 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 the sequence And an antibody that binds CSF1R. .

  In some embodiments, the anti-CSF1R antibody comprises at least one of the CDRs discussed herein. Thus, in some embodiments, the anti-CSF1R antibody is a heavy chain CDR1 discussed herein, a heavy chain CDR2 discussed herein, a heavy chain CDR3 discussed herein, And at least one CDR selected from light chain CDR1, light chain CDR2 discussed herein, and light chain CDR3 discussed herein. Furthermore, in some embodiments, the anti-CSF1R antibody comprises at least one mutant CDR based on the CDRs discussed herein, wherein the mutant CDRs are 1 relative to the CDRs discussed herein. It contains one, two, three or four amino acid substitutions. 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 appropriate conservative amino acid substitutions for a particular CDR sequence, which suitably alters the binding properties of the antibody containing the mutated CDRs. It is not predicted.

  Exemplary anti-CSF1R antibodies also include antibodies that compete with the antibodies described herein for binding to CSF1R. Thus, in some embodiments, antibodies selected from Fab0301, 0302, and 0311, and bivalent (ie, having two heavy chains and two light chains) antibody forms of these Fabs, and to CSF1R An anti-CSF1R antibody that competes for binding is provided.

Exemplary Antibody Constant Regions In some embodiments, the antibodies described herein comprise one or more human constant regions. In some embodiments, the human heavy chain constant region consists of an isotype selected from IgA, IgG, and IgD. In some embodiments, the human light chain constant region consists of an isotype selected from kappa and lambda. In some embodiments, the antibodies described herein comprise human IgG constant regions. In some embodiments, the antibodies described herein comprise a human IgG4 heavy chain constant region. In some such embodiments, the antibodies described herein comprise an S241P mutation in a human IgG4 constant region. In some embodiments, the antibodies described herein comprise a human IgG4 constant region and a human kappa light chain.

  As mentioned above, whether effector function is desired may vary depending on the particular therapeutic method being for the antibody. Thus, in some embodiments, when effector function is desired, an anti-CSF1R antibody comprising human IgG1 heavy chain constant region or human IgG3 heavy chain constant region is selected. In some embodiments, where effector function is not desired, anti-CSF1R antibodies comprising human IgG4 or IgG2 heavy chain constant regions are selected.

Exemplary Anti-CSF1R Heavy Chain Variable Region In some embodiments, an anti -CSF1R antibody heavy chain variable region is provided. In some embodiments, the anti-CSF1R antibody heavy chain variable region is a mouse variable region, a human variable region, or a humanized variable region.

  The anti-CSF1R antibody heavy chain variable region comprises heavy chain CDR1, FR2, CDR2, FR3 and CDR3. In some embodiments, the anti-CSF1R antibody heavy chain variable region further comprises heavy chains FR1 and / or FR4. Non-limiting exemplary heavy chain variable regions include, but are not limited to, heavy chain variable regions having an amino acid sequence selected from SEQ ID NO: 9, 11, 13, and 39-45.

  In some embodiments, the anti-CSF1R antibody heavy chain variable region comprises a CDR1 comprising a sequence selected from SEQ ID NOs: 15, 21 and 27.

  In some embodiments, the anti-CSF1R antibody heavy chain variable region comprises CDR2 comprising a sequence selected from SEQ ID NOs: 16, 22 and 28.

  In some embodiments, the anti-CSF1R antibody heavy chain variable region comprises a CDR3 comprising a sequence selected from SEQ ID NO: 17, 23 and 29.

  Non-limiting exemplary heavy chain variable regions are selected from SEQ ID NOs: 15, 16 and 17, SEQ ID NOs: 21, 22 and 23 and SEQ ID NOs: 27, 28 and 29 Including, but not limited to heavy chain variable regions comprising the set of CDRl, CDR2 and CDR3.

  In some embodiments, the anti-CSF1R antibody heavy chain is at least 90%, at least 91%, at least 92%, at least 93%, with a sequence selected from SEQ ID NO: 9, 11, 13, and 39-45 An antibody that binds CSF1R together with a light chain, comprising a variable region sequence that is at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical. It is possible to form

  In some embodiments, the anti-CSF1R antibody heavy chain comprises at least one of the CDRs discussed herein. Thus, in some embodiments, the anti-CSF1R antibody heavy chain is selected from the heavy chain CDR1s discussed herein, the heavy chain CDR2s discussed herein, and the heavy chain CDR3s discussed herein At least one CDR. Furthermore, in some embodiments, the anti-CSF1R antibody heavy chain comprises at least one mutant CDR based on the CDRs discussed herein, wherein the mutant CDRs are to the CDRs discussed herein. , 1, 2, 3 or 4 amino acid substitutions. 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 appropriate conservative amino acid substitutions for a particular CDR sequence, which suitably alters the binding characteristics of the heavy chain containing the mutated CDRs. It is not expected to do.

  In some embodiments, the heavy chain comprises a heavy chain constant region. In some embodiments, the heavy chain comprises a human heavy chain constant region. In some embodiments, the human heavy chain constant region consists 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, the heavy chain comprises a human IgG4 heavy chain constant region. In some such embodiments, the human IgG4 heavy chain constant region comprises a S241P mutation.

  In some embodiments, where effector function is desired, the heavy chain comprises a human IgG1 or IgG3 heavy chain constant region. In some embodiments, where effector function is less desirable, the heavy chain comprises a human IgG4 or IgG2 heavy chain constant region.

Exemplary Anti-CSF1R Light Chain Variable Regions In some embodiments, anti-CSF1R antibody light chain variable regions are provided. In some embodiments, the anti-CSF1R antibody light chain variable region is a mouse variable region, a human variable region, or a humanized variable region.

  Anti-CSF1R antibody light chain variable regions include light chain CDR1, FR2, CDR2, FR3 and CDR3. In some embodiments, the anti-CSF1R antibody light chain variable region further comprises light chains FR1 and / or FR4. Non-limiting exemplary light chain variable regions include light chain variable regions having an amino acid sequence selected from SEQ ID NOs: 10, 12, 14, and 46-52.

  In some embodiments, the anti-CSF1R antibody light chain variable region comprises a CDR1 comprising a sequence selected from SEQ ID NO: 18, 24 and 30.

  In some embodiments, the anti-CSF1R antibody light chain variable region comprises CDR2 comprising a sequence selected from SEQ ID NOs: 19, 25 and 31.

  In some embodiments, the anti-CSF1R antibody light chain variable region comprises a CDR3 comprising a sequence selected from SEQ ID NO: 20, 26, and 32.

  Non-limiting exemplary light chain variable regions are selected from SEQ ID NOs: 18, 19 and 20, SEQ ID NOs: 24, 25 and 26, and SEQ ID NOs: 30, 31 and 32 Included are, but are not limited to, light chain variable regions comprising the set of CDRl, CDR2 and CDR3.

  In some embodiments, the anti-CSF1R antibody light chain is at least 90%, at least 91%, at least 92%, at least 93%, with a sequence selected from SEQ ID NO: 10, 12, 14, and 46-52. An antibody that binds CSF1R together with a heavy chain, comprising a variable region sequence that is at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical. It is possible to form

  In some embodiments, the anti-CSF1R antibody light chain comprises at least one of the CDRs discussed herein. Thus, in some embodiments, the anti-CSF1R antibody light chain is selected from the light chain CDR1s discussed herein, the light chain CDR2s discussed herein, and the light chain CDR3s discussed herein At least one CDR. Further, in some embodiments, the anti-CSF1R antibody light chain comprises at least one mutant CDR based on the CDRs discussed herein, wherein the mutant CDRs are to the CDRs discussed herein. , 1, 2, 3 or 4 amino acid substitutions. 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 appropriate conservative amino acid substitutions for a particular CDR sequence, which suitably alters the binding properties of the light chain containing the mutated CDRs. It is not expected to do.

  In some embodiments, the light chain comprises a human light chain constant region. In some embodiments, the human light chain constant region is selected from human kappa and human lambda light chain constant regions.

Exemplary Additional CSF1R Binding Molecules In some embodiments, additional molecules that bind CSF1R are provided. Such molecules include, but are not limited to, non-standard scaffolds, such as anticalins, adnectins, ankyrin repeats, and the like. For example, Hosse et al. , Prot. Sci. 15: 14 (2006), Fiedler, M. et al. 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-CSF1R Antibodies In some embodiments, an antibody having the above-described structure binds CSF1R with a binding affinity (K _D ) of less than 1 nM and is capable of binding CSF1 and / or IL-34 to CSF1R. It blocks binding and suppresses CSF1 and / or IL-34 induced phosphorylation of CSF1R.

In some embodiments, the anti-CSF1R antibody binds to the extracellular domain of CSF1R (CSF1R-ECD). In some embodiments, the anti-CSF1R antibody has a binding affinity (K _D ) of less than 1 nM, less than 0.5 nM, less than 0.1 nM, or less than 0.05 nM for CSF1R. In some embodiments, the anti-CSF1R antibody is 0.01-1 nM, 0.01-0.5 nM, 0.01-0.1 nM, 0.01-0.05 nM, or 0.02-0.05 nM. Have a _KD of

  In some embodiments, the anti-CSF1R antibody blocks ligand binding to CSF1R. In some embodiments, the anti-CSF1R antibody blocks CSF1 binding to CSF1R. In some embodiments, the anti-CSF1R antibody blocks binding of IL-34 to CSF1R. In some embodiments, the anti-CSF1R antibody blocks both CSF1 and IL-34 binding to CSF1R. In some embodiments, the antibody that blocks ligand binding binds to the extracellular domain of CSF1R. The antibody is considered to "block ligand binding to CSF1R" if it reduces the amount of detectable binding of the ligand to CSF1R by at least 50% using the assay described in Example 7 . In some embodiments, the antibody has at least 60%, at least 70%, at least 80%, or at least the amount of detectable binding of ligand to CSF1R using the assay described in Example 7. Decrease by 90%. In some such embodiments, the antibody blocks ligand binding by at least 50%, at least 60%, at least 70%, etc.

  In some embodiments, the anti-CSF1R antibody inhibits ligand-induced phosphorylation of CSF1R. In some embodiments, the anti-CSF1R antibody inhibits CSF1-induced phosphorylation of CSF1R. In some embodiments, the anti-CSF1R antibody suppresses IL-34 induced CSF1R phosphorylation. In some embodiments, the anti-CSF1R antibody suppresses both CSF1-induced and IL-34-induced CSF1R phosphorylation. The antibody "suppresses ligand-induced phosphorylation of CSF1R when reducing the amount of detectable ligand-induced CSF1R phosphorylation by at least 50% using the assay described in Example 6 It is considered that In some embodiments, the antibody has at least 60%, at least 70%, at least 80% of the amount of detectable ligand-induced CSF1R phosphorylation using the assay described in Example 6. Or reduce by at least 90%. In some such embodiments, the antibody inhibits ligand induced CSF1R phosphorylation by at least at least 50%, at least 60%, at least 70%, etc.

  In some embodiments, the antibody suppresses monocyte proliferation and / or survival response in the presence of CSF1 and / or IL-34. The antibody inhibits the amount of proliferation and / or survival response of monocytes in the presence of CSF1 and / or IL-34 by at least 50% using the assay described in Example 10, It is considered as “suppressing the proliferation and / or survival response of monocytes”. In some embodiments, the antibody has an amount of proliferation and / or survival response of monocytes in the presence of CSF1 and / or IL-34 using the assay described in Example 10 of at least 60 %, At least 70%, at least 80%, or at least 90% inhibition. In some such embodiments, the antibody inhibits monocyte proliferation and / or survival response by at least at least 50%, at least 60%, at least 70%, etc.

Exemplary Antibody Conjugates In some embodiments, an anti-CSF1R 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 detection of the molecule to which the antibody binds. Non-limiting exemplary labels include, but are not limited to, radioactive isotopes, fluorescent groups, enzyme groups, chemiluminescent groups, biotin, epitope tags, metal binding tags, and the like. One skilled in the art can select an appropriate label according to the intended use.

  As used herein, a cytotoxic agent is a moiety that reduces the growth potential of one or more cells. A cell is proliferated because, for example, the cell undergoes apoptosis or otherwise dies, the cell can not advance the cell cycle, and / or can not divide, the cell differentiates. It has reduced proliferative capacity when it becomes difficult to do so. Non-limiting exemplary cytotoxic agents include, but are not limited to, radioactive isotopes, toxins, and chemotherapeutic agents. One skilled in the art can select appropriate cytotoxicity according to the intended use.

  In some embodiments, in vitro chemical methods are used to conjugate labels and / or cytotoxic agents to the antibody. Exemplary chemical methods of non-limiting conjugation are known in the art, eg, Thermo Scientific Life Science Research Produces (formerly Pierce, Rockford, Ill.), Prozyme (Hayward, Calif.), SACRI Antibody Services (Calgary, Canada), AbD Serotec (Raleigh, NC), etc., including commercially available services, methods, and / or reagents. In some embodiments, where the label and / or cytotoxic agent is a polypeptide, the label and / or cytotoxic agent is expressed from the same expression vector having at least one antibody chain and fused to the antibody chain And / or polypeptides comprising a cytotoxic agent can be produced. One skilled in the art can select an appropriate method for conjugating the label and / or cytotoxic substance to the antibody according to the intended use.

Exemplary Leader Sequences In order to express and secrete large amounts of some secreted proteins, heterologous protein derived leader sequences may be desirable. In some embodiments, the leader sequence is selected from light and heavy chain leader sequences SEQ ID NO: 3 and 4, respectively. In some embodiments, using heterologous leader sequences may be advantageous in that the resulting mature polypeptide remains unchanged as the leader sequence is removed in the ER during the secretory process. Addition of heterologous leader sequences may be necessary to express and secrete some proteins.

  The sequences of certain exemplary leader sequences are described, for example, in the on-line Leader sequence Database maintained by the Department of Biochemistry, National University of Singapore. Choo et al. , BMC Bioinformatics, 6: 249 (2005) and PCT Publication No. WO 2006/081430.

Nucleic Acid Molecules Encoding Anti-CSF1R Antibodies Nucleic acid molecules comprising a polynucleotide encoding one or more strands of an anti-CSF1R antibody are provided. In some embodiments, the nucleic acid molecule comprises a polynucleotide encoding a heavy or light chain of an anti-CSF1R antibody. In some embodiments, the nucleic acid molecule comprises both a polynucleotide encoding the heavy chain of the anti-CSF1R antibody and a polynucleotide encoding the light chain. In some embodiments, the first nucleic acid molecule comprises a first polynucleotide encoding a heavy chain and the second nucleic acid molecule comprises a second polynucleotide encoding a light chain.

  In some such embodiments, the heavy and light chains are expressed as two separate polypeptides from one nucleic acid molecule or from two separate nucleic acid molecules. In some embodiments, for example, where the antibody is a scFv, a single polynucleotide encodes a single polypeptide comprising both heavy and light chains linked together.

  In some embodiments, the polynucleotide encoding the heavy or light chain of the anti-CSF1R antibody comprises a nucleotide sequence encoding a leader sequence located at the N-terminus of the heavy or light chain when translated. As discussed above, the leader sequence may be a natural heavy or light chain leader sequence or may be another heterologous leader sequence.

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

Expression and Production Vectors for Anti-CSF1R Antibodies A vector comprising a polynucleotide encoding an anti-CSF1R heavy chain and / or an anti-CSF1R light chain is provided. Also provided is a vector comprising a polynucleotide encoding an anti-CSF1R heavy chain and / or an anti-CSF1R light chain. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors and the like. In some embodiments, the vector comprises a first polynucleotide sequence encoding a heavy chain and a second polynucleotide sequence encoding a light chain. In some embodiments, the heavy and light chains are expressed from the vector as two separate polypeptides. In some embodiments, the heavy and light chains are expressed as part of a single polypeptide, eg, when the antibody is a scFv.

  In some embodiments, the first vector comprises a polynucleotide encoding a heavy chain and the second vector comprises a polynucleotide encoding a light chain. In some embodiments, the first vector and the second vector are transfected into host cells in similar amounts (eg, similar molar amounts or similar masses). In some embodiments, the host cell is transfected with a first vector and a second vector in molar or mass ratios 5: 1 to 1: 5. In some embodiments, a weight ratio of 1: 1 to 1: 5 is used for heavy chain encoding vectors and light chain encoding vectors. In some embodiments, for the heavy chain encoding vector and the light chain encoding vector, a mass ratio of 1: 2 is used.

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

  In some embodiments, vectors for in vivo expression of anti-CSF1R heavy chain and / or anti-CSF1R light chain are selected 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, a liver-specific promoter is described, for example, in PCT Publication No. WO 2006/076288.

Host Cells In various embodiments, the anti-CSF1R heavy chain and / or anti-CSF1R light chain is a prokaryotic cell, such as a bacterial cell, or a eukaryotic cell, such as a fungal cell (such as yeast), a plant cell, an insect cell, and a mammal It can be expressed in cells. Such expression can be performed, for example, according to procedures known in the art. Exemplary eukaryotic cells that can be used to express the polypeptide include COS cells, including COS7 cells, 293 cells, including 293-6 E cells, CHO cells, including CHO-S and DG44 cells, PER. C6 cells (Crucell), as well as NSO cells are included, but not limited thereto. In some embodiments, the anti-CSF1R heavy chain and / or the anti-CSF1R light chain can be expressed in yeast. See, for example, U.S. Patent Application Publication No. 2006/0270045 Al. In some embodiments, particular eukaryotic host cells are selected based on their ability to make the desired post-translational modifications to the anti-CSF1R heavy chain and / or anti-CSF1R light chain. For example, in some embodiments, CHO cells produce a polypeptide with higher levels of sialylation than the same polypeptide produced in 293 cells.

Introduction of one or more nucleic acids into the desired host cell includes, but is not limited to, calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection etc. Not, can be achieved by any method. Non-limiting exemplary methods are described, for example, in Sambrook et al. ^{, Molecular Cloning, A Laboratory Manual,} 3 rd ed. Cold Spring Harbor Laboratory Press (2001). The nucleic acid may be transiently or stably transfected into the desired host cell according to any suitable method.

  In some embodiments, one or more polypeptides can be produced in vivo in an animal engineered or transfected with one or more nucleic acid molecules encoding the polypeptide according to any suitable method.

Purification of Anti-CSF1R Antibodies Anti-CSF1R 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, and the like. Suitable affinity ligands include ligands that bind CSF1R ECD and antibody constant regions. For example, protein A, protein G, protein A / G or antibody affinity columns may be used to bind constant regions and purify anti-CSF1R antibodies. Hydrophobic interaction chromatography, such as butyl or phenyl columns, may also be suitable for purifying some polypeptides. Many methods of purifying polypeptides are known in the art.

Cell-Free Production of Anti-CSF1R Antibodies In some embodiments, anti-CSF1R antibodies are produced in a cell-free system. Non-limiting exemplary cell-free systems are described, for example, by 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-CSF1R Antibodies Systemic lupus erythematosus (SLE), rheumatoid arthritis, and antibodies that bind CSF1R and block CSF1 and IL-34 ligand binding Provided herein are methods of treating multiple sclerosis.

  In some embodiments, methods of treating SLE are provided. In some such embodiments, the method administers to a subject having SLE an antibody that binds CSF1R and blocks CSF1 and IL-34 ligand binding, eg, an antibody selected from huAb1 to huAb16 Including the step of In some embodiments, treating SLE reduces the incidence and / or severity of skin lesions associated with SLE, and / or from glomerulonephritis, interstitial nephritis, and perivascular infiltration. It means reducing the selected kidney condition. In some embodiments, a method of treating lupus nephritis is provided, wherein the method comprises binding to CSF1R and blocking CSF1 and IL-34 ligand binding to a subject having lupus nephritis, eg, administering an antibody selected from huAb1-huAb16. In some embodiments, provided is a method of treating one type of lupus nephritis selected from type I, II, III, IV, V, and VI lupus nephritis, the method comprising Administering to a subject having nephritis an antibody that binds CSF1R and blocks CSF1 and IL-34 ligand binding, eg, an antibody selected from huAb1 to huAb16. Type III and IV lupus nephritis have a significantly higher CD16 + monocyte count than other types of lupus nephritis. Yoshimoto et al. , Am. J. Kidney Dis. 50: 47-58 (2007). Thus, in some embodiments, methods of treating type III or type IV lupus nephritis are provided, wherein the method comprises binding CSF1R to CSF1 and IL-34 ligand binding to a subject having lupus nephritis. Administering an antibody that blocks B. for example an antibody selected from huAb1 to huAb16. In some such embodiments, the method comprises administering to the subject huAb1.

  In some embodiments, a method of treating a skin lesion associated with SLE is provided, the method comprising an antibody that binds CSF1R to a subject having SLE, eg, an antibody selected from huAb1 to huAb16 Including the step of administering In some embodiments, a method of treating a skin lesion associated with SLE comprises administering huAb1 to a subject having SLE. In some embodiments, provided are methods of reducing the number, severity, and / or rate of formation of skin lesions associated with SLE, the methods comprise antibodies that bind CSF1R to a subject having SLE. For example, administering an antibody selected from huAb1 to huAb16. In some embodiments, a method of reducing the number, severity and / or rate of formation of skin lesions associated with SLE comprises administering huAb1 to a subject having SLE. In some embodiments, the number, severity and / or rate of formation of skin lesions can be determined by visual inspection. Such visual inspection may be performed, for example, by the clinician or the subject (eg, self-reported visual inspection).

  In some embodiments, methods of treating a renal condition associated with SLE are provided. In some such embodiments, the method selects an antibody that binds CSF1R and blocks CSF1 and IL-34 ligand binding to a subject having a renal condition associated with SLE, eg, from huAb1-huAb16 Administering the antibody. In some embodiments, antibody huAb1 is administered to a subject. In some embodiments, the renal condition is selected from glomerulonephritis, interstitial nephritis, and perivascular infiltration. In some embodiments, a method of treating lupus nephritis is provided, wherein the method comprises binding to CSF1R and blocking CSF1 and IL-34 ligand binding to a subject having lupus nephritis, eg, administering an antibody selected from huAb1-huAb16. In some embodiments, the method of treating lupus nephritis comprises administering an antibody huAb1 to a subject having lupus nephritis. In some embodiments, the degree of kidney damage in lupus nephritis can be determined using ultrasonography, scintigraphy, pyelography, or needle biopsy. In some embodiments, the degree of kidney damage in lupus nephritis can also be determined by measuring renal function, eg, measuring proteinuria and / or glomerular filtration rate.

  In some embodiments, a method is provided for reducing proteinuria associated with lupus nephritis, the method comprising: an antibody that binds CSF1R and blocks CSF1 and IL-34 ligand binding to a subject exhibiting proteinuria. For example, administering an antibody selected from huAb1 to huAb16. Proteinuria can be detected by any method in the art including, but not limited to, deep bar test, protein / creatinine ratio, serum albumin / creatinine ratio, and protein electrophoresis. In some embodiments, urinary protein levels are trace (5-20 mg / dl), 1+ (30 mg / dl), 2+ (100 mg / dl), 3+ (300 mg / dl), and 4+ (> 2000 mg / dl) It can be shown using the class dl). Protein / creatinine ratios less than 200 mg / g are considered normal. Serum albumin / creatinine ratios less than 17 mg / g and less than 25 mg / g, respectively, in men and women are considered normal. Proteinuria is the total protein amount of more than 300 mg / day or the presence of albumin in 24-hour urine excretion, the total protein amount of more than 30 mg / dL for the measurement result of the spot urine gauge bar (ie, in the above class 1+ or more) as a total protein amount / creatinine ratio of more than 200 mg / g, an albumin / creatinine ratio of more than 300 mg / g for spot urine measurement results (or 250 mg / g for men for urine results) As the albumin / creatinine ratio in excess, and the albumin / creatinine ratio in women over 355 mg / g for the results of measurement of spot urine, the National Kidney Foundation Kidney Disease Outcome Measures are taken as measures by the National Kidney Foundation. It is defined by uality Initiative).

  In some embodiments, administration of an antibody that binds CSF1R and blocks CSF1 and IL-34 ligand binding to a subject comprises at least one of the classes of "trace, 1+, 2+, 3+, 4+" proteinuria. Decrease step or at least two steps. That is, in some embodiments, administration of an antibody that binds CSF1R and blocks CSF1 and IL-34 ligand binding to a subject with 3+ proteinuria does not exceed 2+ or 1+ levels of proteinuria. Reduce to. In some embodiments, administering to the subject an antibody that binds CSF1R and blocks CSF1 and IL-34 ligand binding has a protein / creatinine ratio and / or an albumin / creatinine ratio of 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, administering to the subject an antibody that binds CSF1R and blocks CSF1 and IL-34 ligand binding has a protein / creatinine ratio and / or an albumin / creatinine ratio of less than 250 mg / g, 200 mg / g. Reduce to less than g, less than 150 mg / g, or less than 100 mg / g. In some embodiments, administration of an antibody that binds CSF1R and blocks CSF1 and IL-34 ligand binding to a subject produces a protein / creatinine ratio and / or an albumin / creatinine ratio within 2 times the normal ratio, Reduce to within 3 times, within 4 times, within 5 times. In some embodiments, the reduction in proteinuria is from the start of treatment for the first week, the first two weeks, the first month, the first two months, the first three months, the first four months, or the first It is judged in 6 months. In some embodiments, the subject is monitored for sustained suppression of proteinuria while being treated with an antibody that binds CSF1R and blocks CSF1 and IL-34 ligand binding.

In some embodiments, provided is a method of improving glomerular filtration rate in a subject having lupus nephritis, the method comprising binding CSF1R to CSF1 and IL to a subject having lupus nephritis. -34 comprising administering an antibody that blocks ligand binding, eg, an antibody selected from huAb1-huAb16. Glomerular filtration rate is determined by any method in the art, including serum or urine levels of insulin or sininstrin (in some embodiments, insulin or sininstrin is injected prior to analysis), These include, but are not limited to, 24 urine collections and creatinine clearance estimated from creatinine concentration in serum, as well as serum cystatin C levels. Normal glomerular filtration rate in adults is in the range of 90-130 ml / min / 1.73 m ² . The glomerular filtration rate of less than 90 ml / min / 1.73 m ² , 80 ml / min / 1.73 m ² , 70 ml / min / 1.73 m ² or less than 60 ml / min / 1.73 m ² In embodiments, chronic kidney disease is indicated, but rates less than 15 ml / min / 1.73 m ² indicate renal failure. In some embodiments, provided is a method of improving glomerular filtration rate in a subject having lupus nephritis, the method comprising binding CSF1R to CSF1 and IL to a subject having lupus nephritis. -34 comprising administering an antibody that blocks ligand binding, eg, an antibody selected from huAb1-huAb16. In some embodiments, the subject's glomerular filtration rate following administration of an antibody that binds CSF1R and blocks CSF1 and IL-34 ligand binding is 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 30ml / min 1.73 m ^2, at least 40ml / min 1.73 m ^2, or at least 50ml / min, /1.73m ² to improve. In some embodiments, the subject's glomerular filtration rate following administration of an antibody that binds CSF1R and blocks CSF1 and IL-34 ligand binding 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 from the start of treatment for the first week, the first two weeks, the first month, the first two months, the first three months, the first four months, or It is judged in the first six months. In some embodiments, the subject is monitored for sustained improvement in glomerular filtration rate while treating with an antibody that binds CSF1R and blocks CSF1 and IL-34 ligand binding.

  In some embodiments, a method of treating lupus nephritis is provided, wherein the method comprises binding to CSF1R and blocking CSF1 and IL-34 ligand binding to a subject having lupus nephritis, eg, administering an antibody selected from huAb1 to huAb16, wherein administering the antibody delays or blocks the progression of the renal condition associated with lupus nephritis. In some such embodiments, the proteinuria and / or glomerular filtration rate in the subject does not deteriorate after administration of the antibody and / or during a specific time interval being treated with the antibody (ie, proteinuria) Does not increase, and / or the glomerular filtration rate does not decrease). The treatment may be a single dose or multiple doses.

  In some embodiments, a method of treating rheumatoid arthritis is provided, wherein the method comprises binding to CSF1R and blocking CSF1 and IL-34 ligand binding to a subject having rheumatoid arthritis, eg, administering an antibody selected from huAb1-huAb16. In some embodiments, a method of treating rheumatoid arthritis is provided, the method comprising administering an antibody huAb1 to a subject having rheumatoid arthritis. In some embodiments, treating rheumatoid arthritis means reducing a clinical disease score that can be measured, for example, by measuring erythema and swelling in a joint afflicted with rheumatoid arthritis. In some embodiments, treating rheumatoid arthritis reduces inflammation, reduces pannus formation, reduces cartilage damage, reduces bone resorption, and reduces the number of macrophages in a joint. , Reducing autoantibody formation, and / or reducing bone loss.

  In some embodiments, methods of reducing inflammation associated with rheumatoid arthritis are provided, wherein the method binds CSF1R and blocks CSF1 and IL-34 ligand binding to a subject having rheumatoid arthritis Administering an antibody, eg, an antibody selected from huAb1-huAb16. In some embodiments, provided is a method of reducing inflammation associated with rheumatoid arthritis, the method comprising administering antibody huAb1 to a subject having rheumatoid arthritis. In some embodiments, reducing inflammation comprises reducing the erythrocyte sedimentation rate and / or reducing the level of C-reactive protein in blood. If inflammation is found in the subject, the erythrocyte sedimentation rate is increased, probably due to the increase in fibrinogen levels in the blood. Erythrocyte sedimentation rate can be determined by any method in the art, including calculating the rate by measuring the change in height of anticoagulated red blood cells for one hour in a Westergren tube However, it is not limited to this. Procedures for the Erythrocyte Sedimentation Rate Test; Approved Standard-Fifth Edition. CLSI document H02-A5. See also Wayne, PA: Clinical and Laboratory Standards Institute; 2011. Blood C-reactive protein levels can be determined by any method in the art including, but not limited to, using RAPITEX® CRP test kit (Siemens).

  In some embodiments, reducing inflammation comprises reducing peripheral edema, which is tissue swelling due to fluid accumulation. In some cases, peripheral edema can occur in the ankles, feet, legs, and / or calf of a subject having rheumatoid arthritis. In some embodiments, reducing inflammation comprises reducing infiltration of inflammatory cells in the synovium of one or more affected joints. In some embodiments, synovial fluid may be collected by arthrocentesis.

  In some embodiments, provided is a method of reducing pannus formation associated with rheumatoid arthritis, the method comprises binding CSF1R to block CSF1 and IL-34 ligand binding to a subject having rheumatoid arthritis (E.g., an antibody selected from huAb1 to huAb16). In some embodiments, provided is a method of reducing pannus formation associated with rheumatoid arthritis, the method comprising administering antibody huAb1 to a subject having rheumatoid arthritis. In some embodiments, reducing pannus formation reduces pannus infiltration into cartilage and / or subchondral bone and / or reduces destruction of hard tissue resulting from pannus infiltration including. Pannus formation can be measured by any method in the art, including, but not limited to imaging one or more affected joints. Non-limiting exemplary imaging techniques for detecting pannus formation include magnetic resonance imaging (MRI), computed tomography (CT) scanning, arthroscopy, sonography, duplex echography , And power Doppler imaging.

  In some embodiments, provided is a method of delaying the progression of pannus formation associated with rheumatoid arthritis, the method comprises binding CSF1R to CSF1 and IL-34 ligand binding to a subject having rheumatoid arthritis Administering an antibody that blocks B. for example an antibody selected from huAb1 to huAb16. In some embodiments, the antibody is huAb1. In some embodiments, the formation of pannus is delayed after administration of the antibody and / or for a specific time interval during which the subject is being treated with the antibody. The treatment may be a single dose or multiple doses.

  In some embodiments, methods of reducing cartilage damage associated with rheumatoid arthritis are provided, wherein the method binds CSF1R and blocks CSF1 and IL-34 ligand binding to a subject having rheumatoid arthritis (E.g., an antibody selected from huAb1 to huAb16). In some embodiments, provided is a method of reducing cartilage damage associated with rheumatoid arthritis, the method comprising administering antibody huAb1 to a subject having rheumatoid arthritis. In some embodiments, reducing cartilage damage comprises reducing cartilage cell loss, reducing collagen breakdown, 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. Non-limiting exemplary imaging techniques for detecting cartilage damage include MRI, CT scan, arthroscopy, and x-ray imaging.

  In some embodiments, provided is a method of delaying the progression of cartilage damage associated with rheumatoid arthritis, the method comprises binding CSF1R to CSF1 and IL-34 ligand binding to a subject having rheumatoid arthritis Administering an antibody that blocks B. for example an antibody selected from huAb1 to huAb16. In some embodiments, the antibody is huAb1. In some embodiments, the progression of cartilage damage is delayed after administration of the antibody and / or for a specific time interval during which the subject is being treated with the antibody. The treatment may be a single dose or multiple doses.

  In some embodiments, provided is a method of reducing bone resorption associated with rheumatoid arthritis, the method comprises binding CSF1R to block CSF1 and IL-34 ligand binding to a subject having rheumatoid arthritis (E.g., an antibody selected from huAb1 to huAb16). In some embodiments, a method of reducing bone resorption associated with rheumatoid arthritis is provided, the method comprising administering to a subject having rheumatoid arthritis the antibody huAb1. In some embodiments, reducing bone resorption comprises reducing the number of osteoclasts in a joint afflicted with rheumatoid arthritis.

  In some embodiments, bone resorption can be measured by determining the level of TRAP5b in plasma from the subject, wherein an increase in the level of TRAP5b is indicative of an increase in bone resorption in the subject. Thus, in some embodiments, a decrease in the level of TRAP 5b is indicative of a decrease in bone resorption. TRAP 5b levels may optionally be determined before and after treatment with an antibody that binds CSF1R, and / or may be determined periodically throughout the course of treatment monitoring the effectiveness of treatment in reducing bone loss . TRAP 5b levels can be determined using any method in the art, including ELISA (FAICEA, or an immunocapture enzyme assay in which a fragment is absorbed, eg, Quidel® TRAP 5b assay Law, see, but not limited to, TECOmedical Group, Sissach, Switzerland).

  In some embodiments, provided is a method of reducing bone loss associated with rheumatoid arthritis, the method comprises binding CSF1R to block CSF1 and IL-34 ligand binding to a subject having rheumatoid arthritis (E.g., an antibody selected from huAb1 to huAb16). In some embodiments, provided is a method of reducing bone loss associated with rheumatoid arthritis, the method comprising administering antibody huAb1 to a subject having rheumatoid arthritis. Bone loss can be determined using any method in the art, including x-ray imaging, MRI, CT, bone densitometry, single and double photon absorptiometry (SPA, DPA ), Single and dual energy X-ray absorptiometry (SXA, DXA), sonography, scintigraphy, and by measuring levels of serum markers of bone formation and resorption etc. It is not limited to. Non-limiting exemplary bone formation and bone resorption serum markers are shown in Table 2.

TABLE 2 Serum markers for bone formation and resorption

  In some embodiments, provided is a method of delaying the progression of bone loss associated with rheumatoid arthritis, the method comprises binding CSF1R to CSF1 and IL-34 ligand binding to a subject having rheumatoid arthritis Administering an antibody that blocks B. for example an antibody selected from huAb1 to huAb16. In some embodiments, the antibody is huAb1. In some embodiments, bone loss is delayed after administration of the antibody and / or for a specific time interval during which the subject is being treated with the antibody. The treatment may be a single dose or multiple doses.

  In some embodiments, provided is a method of reducing the number of monocytic cells, eg, macrophages and / or CD16 + monocytes in a joint afflicted with rheumatoid arthritis, the method comprising a subject having rheumatoid arthritis Comprising administering an antibody that binds CSF1R and blocks CSF1 and IL-34 ligand binding, eg, an antibody selected from huAb1 to huAb16. In some embodiments, provided is a method of reducing the number of monocytic cells, eg, macrophages and / or CD16 + monocytes in a joint afflicted with rheumatoid arthritis, the method comprising a subject having rheumatoid arthritis , Administering the antibody huAb1. In some embodiments, provided is a method of reducing the number of monocytic cells, eg, macrophages and / or CD16 + monocytes in synovial fluid of a joint affected with rheumatoid arthritis, the method comprising treating rheumatoid arthritis Administering to said subject an antibody that binds CSF1R and blocks CSF1 and IL-34 ligand binding, for example an antibody selected from huAb1 to huAb16. In some embodiments, provided is a method of reducing the number of monocytic cells, eg, macrophages and / or CD16 + monocytes in synovial fluid of a joint affected with rheumatoid arthritis, the method comprising treating rheumatoid arthritis Administering to the subject a subject antibody huAb1.

  In some embodiments, provided is a method of reducing autoantibody levels in a subject having rheumatoid arthritis, the method comprising binding CSF1R to a subject having rheumatoid arthritis to bind CSF1 and IL- 34. administering an antibody that blocks ligand binding, eg, an antibody selected from huAb1-huAb16. In some embodiments, a method of reducing autoantibody levels in a subject having rheumatoid arthritis is provided, the method comprising administering antibody huAb1 to a subject having rheumatoid arthritis.

  In some embodiments, provided is a method of reducing autoantibody levels in a subject having rheumatoid arthritis, the method comprising binding CSF1R to a subject having rheumatoid arthritis to bind CSF1 and IL- 34. administering an antibody that blocks ligand binding, eg, an antibody selected from huAb1-huAb16. In some such embodiments, the method comprises administering huAb1 to a subject having rheumatoid arthritis. Levels of autoantibodies can be determined by any method in the art. In some embodiments, autoantibody levels are determined by the levels of rheumatoid factor (RF) and / or anti-citrullinated protein antibody (ACPA) and / or antinuclear antibody (ANA).

  In some embodiments, a method of treating multiple sclerosis is provided, wherein the method binds CSF1R and blocks CSF1 and IL-34 ligand binding to a subject having multiple sclerosis. Administering an antibody, eg, an antibody selected from huAb1-huAb16. In some embodiments, treating multiple sclerosis means reducing a clinical disease score.

  In some embodiments, a method is provided for reducing CD16 + monocytes, the method comprises an antibody that binds CSF1R and blocks CSF1 and IL-34 ligand binding to a subject with an increase in CD16 + monocytes, such as Administering an antibody selected from huAb1 to huAb16. 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 reduce the number of CD16-monocytes. In some embodiments, CD16 + monocytes that reduce to a greater extent than CD16-monocytes are reduced when an antibody that binds CSF1R and blocks CSF1 and IL-34 ligand binding is administered to the subject. In some embodiments, CD16 + 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, CD16-monocytes are reduced by less than 30%, less than 20%, or less than 10%. In some embodiments, the CD16 + monocytes are CD16 + peripheral blood monocytes. In some embodiments, the CD16-monocytes are CD16-peripheral blood monocytes.

Routes of Administration and Carriers In various embodiments, anti-CSF1R antibodies can be administered orally, intraarterially, parenterally, intranasally, intramuscularly, in the heart cavity, intraventricularly, intratracheally, intraorally, rectally, intraperitoneally, intracutaneous It can be administered in vivo by various routes including, but not limited to, topical, transdermal, and intrathecal. The composition may be formulated into a preparation in solid, semisolid, liquid or gaseous form, including tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections Agents, including but not limited to: agents, inhalants, and aerosols. Nucleic acid molecules encoding anti-CSF1R antibodies are coated on gold microparticles and particulates as described in the literature (see, eg, Tang et al., Nature 356: 152-154 (1992)). It can be delivered intradermally by a collision device or "gene gun". Appropriate formulations and routes of administration can be selected according to the intended application.

In various embodiments, compositions comprising anti-CSF1R antibodies are provided in formulations comprising various pharmaceutically acceptable carriers (eg, Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 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. , Pharmaceu See tical Press (2000)). Various pharmaceutically acceptable carriers can be used, including vehicles, adjuvants, and diluents. In addition, various pharmaceutically acceptable auxiliary substances such as pH adjusters and buffers, tonicity adjusters, stabilizers, wetting agents and the like can be used. Non-limiting exemplary carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.

  In various embodiments, the composition comprising the anti-CSF1R antibody can be aqueous or aqueous, optionally with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives. Formulated for injection, including subcutaneous administration, by dissolving, suspending, or emulsifying them in non-aqueous solvents such as plants or other oils, synthetic fatty acid glycerides, esters of higher aliphatic alcohols, or propylene glycol It can be done. In various embodiments, the composition can be formulated for inhalation using, for example, a pressurizable propellant such as dichlorodifluoromethane, propane, nitrogen and the like. The compositions may also be formulated in sustained release microcapsules, in various embodiments, for example, with biodegradable or non-degradable polymers. Nonlimiting exemplary biodegradable formulations include polylactic acid-glycolic acid polymers. Nonlimiting exemplary non-biodegradable formulations include polyglycerin fatty acid esters. Certain methods of making such formulations are described, for example, in EP 1 125 584 A1.

  Also provided are pharmaceutical packs and kits, each containing one or more doses of anti-CSF1R antibodies, and including one or more containers. In some embodiments, a unit dose is provided, the unit dose comprising a composition comprising a predetermined amount of an anti-CSF1R antibody, with or without one or more additives. In some embodiments, such unit doses are provided in disposable, prefilled syringes for injection. In various embodiments, the composition contained in the unit dose may comprise a buffer such as saline, sucrose etc., phosphate etc and / or may be formulated within a stable and effective pH range . Alternatively, in some embodiments, the composition can be provided as a suitable liquid, for example, a lyophilized powder that can be reconstituted upon addition of 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, the compositions of the present invention comprise heparin and / or proteoglucan.

  The pharmaceutical composition is administered in an amount effective to treat or prevent a particular indication. The therapeutically effective amount will typically vary depending on the weight of the subject being treated, the physical or physical condition of the subject, the extent of the condition being treated, or the age of the subject being treated. In general, anti-CSF1 R antibodies can be administered in an amount ranging from about 10 μg / kg body weight to about 100 mg / kg body weight per dose. In some embodiments, anti-CSF1R antibodies can be administered in an amount ranging from about 50 μg / kg body weight to about 5 mg / kg body weight per dose. In some embodiments, anti-CSF1R antibodies can be administered in an amount ranging from about 100 μg / kg body weight to about 10 mg / kg body weight per dose. In some embodiments, anti-CSF1R antibodies can be administered in an amount ranging from about 100 μg / kg body weight to about 20 mg / kg body weight per dose. In some embodiments, anti-CSF1R antibodies can be administered in an amount ranging from about 0.5 mg / kg body weight to about 20 mg / kg body weight per dose.

  An anti-CSF1R antibody composition can be administered to a subject as needed. The frequency of administration should be determined by the person skilled in the art, such as the treating physician, the age of the subject being treated, the severity of the condition being treated, the general health of the subject being treated, etc. It can be done. In some embodiments, an effective amount of anti-CSF1R antibody is administered to the subject one or more times. In various embodiments, an effective amount of anti-CSF1R antibody is administered to the subject once monthly, less than once monthly, eg, once every two months, or once every three months. In other embodiments, an effective amount of anti-CSF1R antibody is administered more than once a month, such as once every three weeks, once every two weeks, or weekly. An effective amount of anti-CSF1R antibody is administered to the subject at least once. In some embodiments, an effective amount of anti-CSF1R antibody can be administered multiple times for at least one month, at least six months, or at least one year.

Combination Therapy The anti-CSF1R antibodies may be administered alone or in conjunction with other therapeutic modalities. These may be provided prior to, substantially simultaneously with, or after administration of a biologic, such as, for example, surgery, chemotherapy, radiation therapy, or another therapeutic antibody. For the treatment of rheumatoid arthritis, anti-CSF1R antibodies may be used as other therapeutic agents, such as methotrexate, anti-TNF agents such as Remicade (infliximab), Humira (adalimumab), Simponi (golimumab), and Enbrel (etanercept), prednisone , Glucocorticoids such as leflunomide, azothioprine, JAK inhibitors such as CP590690, SYK inhibitors such as R788, anti-IL-6 antibodies, anti-IL-6R antibodies such as tocilizumab, anti-CD-20 antibodies such as rituximab, and anti-CD19 antibodies It may be administered together with an anti-GM-CSF antibody, an anti-GM-CSF-R antibody, an IL-1 receptor antagonist such as anakinra, a CTLA-4 antagonist such as abatacept, an immunosuppressant such as cyclosporin.

  For the treatment of systemic lupus erythematosus, anti-CSF1R antibodies may be other therapeutic agents, for example hydroxychloroquine (Plaquenil); corticosteroids such as prednisone, methylprednisone, and prednisolone; cyclophosphamide (Cytoxan), It may be administered with an immunosuppressant such as azathioprine (Imuran, Azasan), mycophenolate (Cellcept), leflunomide (Arava) and methotrexate (Trexall), and belimumab (Benlysta).

  For the treatment of multiple sclerosis, anti-CSF1R antibodies may be used as other therapeutic agents, eg, interferon alpha, interferon beta, prednisone, anti-alpha 4 integrin antibodies such as Tysabri, anti-CD20 antibodies such as Rituxan, FTY 720 (Fingolimod) And with cladribine (Leustatin).

  The examples discussed below are purely for the purpose of illustrating the invention and are not to be considered as limiting the invention in any way. This example is not intended to indicate that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to the numbers used (eg, amounts, temperatures, etc.) but some errors and deviations should be taken into account. 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 pressure.

Example 1 Humanized Anti-CSF1R Antibodies Various humanized anti-CSF1R antibodies have been previously developed. See, for example, PCT Publication No. WO 2011/140249.

  The sequences of a humanized heavy chain variable region and a humanized light chain variable region aligned with the sequences of the parent chimeric antibody variable region and the human receptor variable framework region are shown in FIGS. 1 (heavy chain) and 2 (light chain). Chain). Boxed changes in humanized variable region sequence to human receptor variable framework region sequence. Each of the CDRs for each of the variable regions is shown in the boxed region and displayed as "CDR" above the boxed sequence.

  Table 5 below shows the complete sequences of the humanized heavy and light chains of the antibodies huAb1 to huAb16. The names and SEQ ID NOs of the humanized heavy and light chains of each of these antibodies are shown in Table 3.

TABLE 3 Humanized heavy and light chains of huAb1 to huAb16

  Sixteen humanized antibodies were tested for binding to human, cynomolgus monkey and mouse CSF1R ECD as described above. See, for example, PCT Publication No. WO 2011/140249. The antibody was found to bind to both human and cynomolgus monkey CSF1R ECD, but not to mouse CSF1R ECD. The humanized antibody also blocks the binding of CSF1 and IL-34 to both human and mouse CSF1R, and suppresses the phosphorylation of CSF1- and IL-34-induced human CSF1R expressed in CHO cells It was also found that See, for example, PCT Publication No. WO 2011/140249.

_K a, _{k d,} and _{K D} for binding to human CSFlR ECD is predetermined, it is shown in Table 4. See, for example, PCT Publication No. WO 2011/140249.

TABLE 4 Binding affinity of humanized antibodies to human CSF1R

Example 2: HuAb1 reduces CD16 + peripheral blood monocytes but not CD16-peripheral blood monocytes in cynomolgus monkeys 0 mg / kg, 3 mg / kg, 10 mg / kg or 30 mg / kg huAb1 Two monkeys per group were administered intravenously. Peripheral blood was collected 14 days later and analyzed by flow cytometry for the number of CD16 + or CD16- monocytes. Briefly, 0.2 ml of peripheral blood was labeled with 20 μl of anti-CD16-PE (Becton-Dickenson) for 30 minutes at room temperature in the dark. Erythrocytes were lysed by incubation for 20 minutes with 2 ml of 1 × BD Pharmalyse (Becton-Dickenson) and cells were washed with 1 ml of FACS buffer (PBS / 0.1% BSA) prior to analysis. Data were collected on a LSRII flow cytometer using the Diva software program (Becton-Dickenson) and results were analyzed using the FloJo data analysis program (Tree Star).

  FIG. 3 shows that in monkeys, treatment with huAb1 resulted in a dose-dependent reduction of CD16 + peripheral blood monocytes compared to untreated animals, but that CD16-peripheral blood monocytes were dose-dependent Indicates that no reduction occurred. These data indicate that huAb1 primarily reduces monocytes that respond to autoimmune disease, but that other subsets of monocytes involved in pathogen clearance can remain intact. This suggests that an antibody that binds CSF1R and blocks ligand binding, such as huAb1, may be effective in treating autoimmune disease while maintaining a good safety profile against infection .

Example 3: Characterization of Surrogate Anti-Mouse CSF1R Antibodies Because the humanized antibodies huAb1 to huAb16 do not bind to mouse CSF1R, a chimeric anti-mouse CSF1R antibody (“cAb1”) is selected as a surrogate antibody for use in mouse models The This antibody contains a rat variable region and a mouse constant region. To generate cAb1, a rat is immunized with a mouse CSF1R-ECD-Fc fusion protein, and a hybridoma that secretes an antibody that blocks both mouse CSF1 and mouse IL-34 binding to mouse CSF1R is assayed by ELISA assay Identified. The variable regions of one such blocking antibody were generated using standard molecular biology techniques (Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Third Edition, CSH Press, Cold Spring Harbor, New York, 2001). Then, they were grafted onto mouse IgG constant region. The recombinant chimeric antibody cAb1 was then expressed in CHO cells using standard techniques (Sambrook and Russell, ibid.).

  Binding of cAb1 to mouse CSF1R was determined as follows. 96 well clear bottom ELISA plates were coated overnight with 1 μg / ml of recombinant mouse CSF1 R ECD-Fc chimera (R & D Systems) in PBS. The next morning, wells were washed 4 times with 0.05% Tween 20 in PBS (PBST) and blocked with Blocker-Blotto (Pierce). Starting with a concentration of 2.6 μg / ml, 50 μl of 0.5 fold serial dilutions of cAb1 diluted 1: 1 in Blocker-Blotto were added to the CSF1R coated wells. After incubation for 90 minutes at room temperature (RT), the wells were washed 4 times with PBST and a 1: 5000 dilution of peroxidase conjugated goat anti-mouse IgG (Sigma) in Blocker-Blotto was added to each well. After incubation for 60 minutes at room temperature, the wells were washed 4 times with PBST and 50 μl of o-phenylenediamine dihydrochloride peroxidase substrate (Sigma) was added to each well. After incubation for 30 minutes at room temperature, A450 values for each well were read directly on a SpectraMax Plus spectrophotometer using SoftMaxPro software (Molecular Devices).

  The experimental results are shown in FIG. cAb1 bound to mouse CSF1R ECD in a dose dependent manner.

Example 4: cAb1 blocks ligand-induced proliferation of factor-dependent cell lines cAb1 was tested for its ability to block proliferation of the ligand-induced 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 available from R & D Systems) in the presence or absence of serial dilutions of cAb1. After incubation for 48 hours at 37 ° C., the relative cellular ATP content of the cultures of each individual was determined 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, thus reflecting the proliferative response of mNFS60 cells.

  The experimental results are shown in FIG. cAb1 blocked CSF1 or IL-34 induced proliferation of mNFS60 cells in a dose dependent manner.

Example 5: cAb1 Inhibits Clinical Disease Score in a Mouse Model of Rheumatoid Arthritis In male DBA / 1 mice, on study day 0 and again on study day 21, 2 mg / ml in 150 μl of complete Freund's adjuvant (Sigma) Arthritis was induced by intradermal injection of bovine type II collagen (Elastin Products, Owensville, MO). On study day 0, mice were randomized into treatment groups and dosing started. Mice are dosed intraperitoneally with vehicle or cAb1 (3, 6, 10, or 30 mg / kg) three times weekly (3 times / week) up to day 32 of the study, study day 18 to day 35 The clinical disease score for each foot was measured. Clinical disease scores were assigned as follows.
0 = normal
1 = 1 posterior or forefoot joint affected or minimal diffuse erythema and swelling.
2 = 2 posterior or forefoot joints affected, or mild diffuse erythema and swelling.
3 = 3 posterior or forefoot joints affected or moderate diffuse erythema and swelling.
4 = 4 posterior or forefoot joints affected or marked diffuse erythema and swelling.
5 = all feet affected, severe diffuse erythema, and severe swelling, unable to move fingers.

  FIG. 6 shows the results of that experiment. cAb1 suppressed the clinical disease score in a collagen-induced arthritis model of rheumatoid arthritis. These data suggest that in human patients, antibodies that bind CSF1R and block ligand binding, such as huAb1, may be effective in reducing the signs and symptoms of rheumatoid arthritis.

Example 6 cAb1 Inhibits Bone Loss in a Mouse Model of Rheumatoid Arthritis High Levels of Tartrate-Resistant Acid Phosphatase 5b (TRAP 5b) are Associated with Bone Remodeling and Various Features Are Induced by Increased Bone Resorption It has been observed in Thus, TRAP5b levels can be used as a marker of bone resorption (ie, bone loss).

  In mice, arthritis was induced and mice were treated with cAb1 as described in Example 5. Blood is collected on day 35 and using a commercially available solid phase immunofixation enzyme activity assay for determination of osteoclast-derived TRAP 5b in mouse serum (Immunodiagnostics Systems) according to the manufacturer's instructions , Plasma levels of TRAP 5b (tartaric acid resistant acid phosphatase 5b) were measured. TRAP5b is a bone turnover marker.

  FIG. 7 shows the results of the experiment. cAb1 suppressed bone loss in the collagen-induced arthritis mode of rheumatoid arthritis as measured by suppression of TRAP5b's bone turnover marker. These data suggest that in human patients, an antibody that binds CSF1R and blocks ligand binding, such as huAb1, can inhibit bone erosion and joint destruction in rheumatoid arthritis.

Example 7: cAb1 Inhibits Inflammation, Cartilage Damage, Pannus Formation, and Bone Destruction in a Mouse Model of Rheumatoid Arthritis As described in Example 5, arthritis was induced in mice and mice were treated with cAb1 . At the end of the study, the paws and knees are collected and placed in neutral buffered formalin (NBF; available from Sigma-Aldrich, catalog number # HT-501128) overnight, then for histopathology and immunohistochemistry To 70% ethanol.

Six joints from each animal were processed for histopathologic evaluation. These joints were then scored as follows.
0 inflammation = normal.
1 = minimal infiltration of inflammatory cells in synovial and periarticular tissues of affected joints.
2 = mild infiltration of inflammatory cells. When referring to the foot, it is generally limited to the affected joint (1-3 affected areas).
3 = moderate infiltration with moderate edema. When referring to the foot, it is generally limited to the affected joints of three to four joints, including at least one wrist or ankle joint.
4 = There may be significant infiltration, most of the areas with significant edema, one or two unaffected joints.
5 = Severe diffuse infiltration with severe edema affecting all joints and periarticular tissues.
Pannus 0 = normal.
1 = minimal infiltration of cartilage and subchondral bone, pannus in marginal zone.
2 = mild infiltration with marginal zone destruction of hard tissue in affected joints.
3 = Moderate infiltration with moderate hard tissue destruction in the affected joint.
4 = significant infiltration with significant destruction of the joint structure, affecting most joints.
5 = severe infiltration with destruction of all or nearly all joint structures affecting all joints.
Cartilage damage 0 = normal.
1 = minimal = apparent minimal chondrocyte loss in affected joints or general minimal to mild loss of toluidine blue staining without collagen degradation.
2 = mild = generally mild loss of toluidine blue staining with loss of cartilage and / or collagen breakdown in focal areas in some affected joints.
3 = moderate = generally moderate loss of toluidine blue staining with multifocal chondrocyte loss and / or collagen breakdown in the afflicted joint, some matrices have any diseased area with severe matrix loss area Stay on the surface.
4 = Notable = With multifocal, prominent (to deep depth) chondrocyte loss and / or collagen breakdown in most joints if all or nearly all of the cartilage is lost on one surface of the knee Marked loss of toluidine blue staining.
5 = Severe = multifocal severe (to the depth of the tide mark) chondrocytes in all joints if all or nearly all of the cartilage is lost on two or more surfaces of the knee Severe diffuse loss of toluidine blue staining with loss and / or collagen breakdown.
Bone resorption 0 = normal.
1 = minimal = small area resorption, not readily apparent at low magnification, osteoclasts in affected joints are rare and limited to marginal zones.
2 = Mild = resorption of more areas, limited to more osteoclasts in the affected joint, marginal zone not readily apparent at low magnification.
3 = Moderate = no apparent loss of the meningeal and cortical bone, loss of some meninged trabeculae, loss of obvious at low magnification, more in the affected joints, without loss of all layers in the cortex Osteoclasts.
4 = notable = loss of all layers in cortical bone, often with deformation of profile staying on cortical surface, significant loss of medullary bone, many osteoclasts, affecting most joints.
5 = severe = loss of all layers in cortical bone, and destruction of the joint structure of all joints.
The sum of histologic scores for each parameter of all six joints was calculated.

  FIG. 8 shows the results of the experiment. cAb1 inhibited inflammation, cartilage damage, pannus formation, and bone destruction in a collagen-induced arthritis model of rheumatoid arthritis.

Example 8 cAb1 Suppresses Ankle and Knee Joint Macrophage Numbers in a Mouse Model of Rheumatoid Arthritis As described in Example 5, arthritis is induced in mice and mice are treated with cAb1, Example 7 Foot and knee joints were prepared as described in The number of macrophages was counted by staining the foot and knee joints with peroxidase conjugated anti-F4 / 80 antibody and counting positively stained cells under a microscope. Tissue sectioning and staining was performed by Histotox Labs, Boulder, CO. F4 / 80 is a mouse macrophage marker (Leenen et al., J. Immunol. Methods, 1994, 174: 5-19).

  The experimental results are shown in FIG. cAb1 reduced the number of macrophages in both the foot and knee joints in a collagen-induced arthritis model of rheumatoid arthritis.

Example 9 cAb1 Inhibits Autoantibody Formation in a Mouse Model of Rheumatoid Arthritis In mice, arthritis was induced and mice were treated with cAb1 as described in Example 5. On day 35, blood was collected and plasma levels of anti-mouse type II collagen antibody were measured using a mouse anti-type 1 and 2 collagen IgG assay kit (Chondrex) according to the manufacturer's instructions.

  The experimental results are shown in FIG. cAb1 inhibited the formation of anti-mouse type II collagen 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 huAb1, may reduce an important trigger of joint inflammation, ie, autoantibodies. Additionally, in some embodiments, autoantibodies, such as RF and ACPA, can be used to monitor drug effects.

Example 10 cAb1 Inhibits Bone Loss in an Established Mouse Model of Rheumatoid Arthritis As described in Example 5, arthritis was induced in mice. Mice were treated intraperitoneally 3 times a week starting with 30 mg / kg cAb1 when the clinical disease score reached 0.05-1.0. Twenty-three days after initial treatment with cAb1, blood is collected and commercially available solid phase immunofixation enzyme activity for determination of osteoclast-derived TRAP5b in mouse serum (Immunodiagnostics Systems) according to the manufacturer's instructions Assays were used to measure plasma levels of TRAP 5b (tartaric acid resistant acid phosphatase 5b). TRAP5b is a bone turnover marker.

  The experimental results are shown in FIG. CAb1 administered after development of the clinical disease score inhibited bone loss in the collagen-induced joint mode of rheumatoid arthritis as measured by suppression of TRAP 5b bone turnover markers. These data suggest that in human patients, an antibody that binds CSF1R and blocks ligand binding, such as huAb1, can inhibit bone erosion and joint destruction in rheumatoid arthritis.

Example 11 cAb1 Inhibits Pannus Formation and Bone Destruction in the Established Mouse Model of Rheumatoid Arthritis As described in Example 10, arthritis was induced in mice and mice were treated with cAb1. At the end of the study, paws and knees were harvested, placed in neutral buffered formalin (NBF) overnight and then transferred to 70% ethanol for histopathology and immunohistochemistry.

  Six joints from each animal were processed for histopathological evaluation and the sum of histologic scores of all six joints was calculated. These joints were scored as described in Example 7.

  The experimental results are shown in FIG. CAb1 administered after development of the clinical disease score suppressed pannus formation and bone destruction in the collagen-induced arthritis model of rheumatoid arthritis. Histological scores of inflammation and cartilage damage were not affected. These data also suggest that in human patients, antibodies that bind CSF1R and block ligand binding, such as huAb1, may inhibit bone erosion and joint destruction in rheumatoid arthritis.

Example 12 cAb1 Inhibits Glomerulonephritis, Interstitial Nephritis, and Perivascular Invasion in a Mouse Model of Systemic Lupus Erythematosus A subset of lupus patients develop lupus nephritis, which is associated with 1 in the kidney. It can cause inflammation of one or more structures, leading to renal failure. Lupus nephritis can be detected in patients, and the severity of the disease measures, for example, proteinuria or protein in the urine, and / or measurement results indicating how much fluid is filtered through the kidney per unit time Is evaluated by determining the glomerular filtration rate. Lupus nephritis can 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) .

Mouse MRL / MpJ-Fas ^lpr / J strains spontaneously develop lupus-like symptoms including glomerular, tubulointerstitial, and perivascular renal disease, lymph node swelling, splenomegaly, circulating autoantibodies, and skin lesions It develops in. Vehicle or 50 mg / kg cAb1 was intraperitoneally administered daily for 10 weeks to female MRL / MpJ-Fas ^lpr / J mice at 10 to 20 weeks of age. At the end of the study (mouse 20 weeks old), kidneys were collected, weighed and placed in 10% neutral buffered formalin (NBF) for histological evaluation. The kidneys were scored according to the following system.
Glomerulonephritis 0 = normal.
1 = focal or multifocal, minimal to mild, early growth.
2 = multifocal mild to moderate or mild diffuse growth.
3 = Moderate diffuse growth with or without multifocal severe areas.
4 = Marked diffuse growth with crescents / sclerosis, multifocal protein cast.
5 = severe diffuse growth with crescents / sclerosis, diffuse protein casts present.
Protein cast severity 0 = normal tubular.
1 = minimal = profile of 1-5 tubules with protein cast.
2 = mild = multifocal tubules with protein cast (6-20).
3 = moderate = multifocal tubules with protein cast (21-40).
4 = Notable = Multifocal tubules with protein casts (more than 40 of all tubules but less than 50% diseased).
5 = Serious = Diffuse tubules with protein casts, more than 50% of all tubules are affected.
Interstitial nephritis (inflammation not clearly associated with blood vessels)
0 = normal
0.5 = very minimal = small focal area of MNC with pelvis only.
1 = minimal = small amount of focal MNC may accumulate and suffers less than 10% of the total stroma and is generally localized around the pelvis.
2 = Mild = multifocal small to higher infiltration dispersed around the pelvis and cortex: affecting 10-25% of the cortical area.
3 = moderate = multifocal small to extensive infiltration in pelvis and cortex: affecting 26-50% of the cortical area.
4 = notable = multifocal to diffuse infiltration: affecting 51-75% of pelvic and cortical areas.
5 = severe = diffuse infiltration: affecting 76-100% of pelvic and cortical areas.
Vessel 0 = normal.
0.5 = Very minimal = 1 vessel has minimal perivascular infiltration.
1 = minimal = small amount but define perivascular infiltration (1-2).
2 = Mild = There are several (3-4) foci of perivascular infiltration but no necrosis.
3 = moderate = multifocal (5-6) foci of perivascular infiltration may be more extensive and may have necrosis of part of the vessel wall.
4 = Prominent = Multifocal (7-8) foci of perivascular infiltrates are widespread with necrosis.
5 = Severe = multifocal (> 8) foci of perivascular infiltration, widespread with necrosis.

  The experimental results are shown in FIG. cAb1 suppressed glomerulonephritis, intersitital nephritis, and perivascular infiltration in the MRL / lupus lpr model. Histological scores of the severity of protein casts were not affected. These data suggest that in human patients, an antibody that binds CSF1R and blocks ligand binding, such as huAb1, may reduce renal inflammation and restore kidney function associated with systemic lupus erythematosus. ing.

Example 13: cAb1 suppresses skin lesions in a mouse model of systemic lupus erythematosus Female MRL / MpJ-Fas ^lpr / J mice were dosed with cAb1 described in Example 12. 10-20 week old mice were scored for the presence and severity of skin lesions. Skin lesions were scored as follows.
0 = none
1 = Mild (brows and ears).
2 = Moderate (in the mouth less than 2 cm, ears, and in the scapula).
3 = severe (in the mouth, ear and scapula more than 2 cm).

  The experimental results are shown in FIG. cAb1 suppressed the development and severity of skin lesions in the MRL / lupus lpr model. These results suggest that in human patients, an antibody that binds CSF1R and blocks ligand binding, such as huAb1, can reduce systemic symptoms of SLE, including skin inflammation, rashes, and discoid lesions doing.

Example 14: cAb1 suppresses clinical disease score in a mouse model of multiple sclerosis Experimental autoimmune encephalitis (EAE) is 300 ng in Complete Freund's Adjuvant on Study Day 0 and again on Study Day 7 Were induced in female C57B1 / 6 mice by subcutaneous injection of the myelin oligodendrocyte glycoprotein peptide 35-55 (MOG 35-55, Sigma-Aldrich, Catalog No. # M4939). To enhance EAE induction and consistency, 500 ng of pertussis toxin (List Biological Laboratories, Catalog No. # 180) was administered intraperitoneally on days 0 and 2. EAE is a multiple sclerosis model. On study day 0, mice were randomized into treatment groups and dosing started. Mice were dosed ip with vehicle or 30 mg / kg cAb1 three times weekly (3 times / week) until study day 15. 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 dragging the tail.
1.0 = The tail is paralyzed.
2.0 = Loss of coordination, hind limb paralysis.
2.5 = One of the hind limbs was paralyzed.
3.0 = both hind limbs paralyzed.
3.5 = The hind limbs become paralyzed and the forelimbs become fragile.
4.0 = The forelimbs and hindlimbs were paralyzed.
5.0 = dying.

  The experimental results are shown in FIG. cAb1 suppressed the clinical disease score in an experimental autoimmune encephalitis model of multiple sclerosis. These data show that in human patients, antibodies that bind CSF1R and block ligand binding, such as huAb1, can reduce inflammation in the central nervous system associated with multiple sclerosis, and MS-related demyelination and nerve damage Suggest that it can reverse the paralysis caused by

SEQUENCE LISTING Table 5 provides the specific sequences discussed herein. All polypeptide and antibody sequences are shown without the leader sequence unless otherwise indicated.

TABLE 5 Sequence and Description

Claims (38)

  A method of treating a condition associated with rheumatoid arthritis comprising administering to a subject having rheumatoid arthritis an antibody that binds colony stimulating factor 1 receptor (CSF1R), said antibody to CSF1R. Blocks binding of colony-stimulating factor 1 (CSF1) and blocks binding of IL-34 to CSF1 R and treating conditions associated with rheumatoid arthritis reduces inflammation, pannus formation Reducing, reducing cartilage damage, reducing bone resorption, reducing the number of macrophages in at least one joint affected with rheumatoid arthritis, reducing autoantibody levels, and reducing bone loss A method comprising at least one effect selected from:   5. The method of claim 1, wherein treating a condition associated with rheumatoid arthritis comprises reducing inflammation.   3. The method of claim 2, wherein the number of CD16 + monocytes is reduced by at least 30%.   4. The method of claim 3, wherein the number of CD16-monocytes is not reduced or reduced by less than 20%.   3. The method of claim 2, further comprising the step of determining the erythrocyte sedimentation rate, wherein a reduction in sedimentation rate indicates a reduction in inflammation.   2. The method of claim 1, wherein treating a condition associated with rheumatoid arthritis comprises at least one effect selected from reducing pannus formation, reducing bone resorption, and reducing bone loss. Method.   7. The method of claim 6, wherein treating a condition associated with rheumatoid arthritis comprises reducing bone resorption.   8. The method of claim 7, wherein the level of at least one marker for bone resorption is reduced.   The bone resorption marker includes tartaric acid-resistant acid phosphatase 5b (TRAP 5b), total pyridinoline in urine, total deoxypyridinoline in urine, free pyridinoline in urine, type I collagen cross-linked N-telopeptide in serum, type I collagen in urine 9. The method of claim 8, wherein the method is selected from cross-linked N-telopeptides and carboxy-terminal telopeptides of serum type I collagen.   7. The method of claim 6, wherein treating a condition associated with rheumatoid arthritis comprises at least one effect selected from reducing pannus formation and reducing bone loss.   11. The method of claim 10, wherein the at least one effect is measured using an imaging technique.   Said imaging techniques include X-ray imaging, magnetic resonance imaging, computed tomography (CT) scanning, arthroscopy, scintigraphy, sonography, bone densitometry, single photon absorptiometry (SPA) ), Double photon absorption measurement (DPA), single energy X-ray absorption measurement (SXA), dual energy X-ray absorption measurement (DXA), scintigraphy, ultrasonography, duplex ultrasonography, The method according to claim 11, comprising a method selected from: and power doppler imaging.   A method of treating rheumatoid arthritis comprising administering to a subject having rheumatoid arthritis an antibody that binds CSF1R, wherein the antibody blocks binding of CSF1 to CSF1R and to CSF1R. A method which blocks binding of IL-34 and wherein said antibody reduces the number of CD16 + monocytes by at least 30% and said CD16-monocytes are not reduced or reduced by less than 20% in said subject.   Methotrexate, anti-TNF substance, glucocorticoid, cyclosporin, leflunomide, azathioprine, JAK inhibitor, SYK inhibitor, anti-IL-6 antibody, anti-IL-6R antibody, anti-CD-20 antibody, anti-CD19 antibody, anti-GM-CSF antibody 14. The method of any one of claims 1-13, further comprising the step of administering at least one additional therapeutic agent selected from an IL-1 receptor antagonist, a CTLA-4 antagonist, and an anti-GM-CSF-R antibody. Method described in Section.   CLAIMS: 1. A method of treating skin lesions associated with lupus comprising administering to a subject having lupus, an antibody that binds CSF1 R, wherein the antibody blocks binding of CSF1 to CSF1 R, and Blocking the binding of IL-34 to CSF1R and treating acne-related skin lesions reduces the number of skin lesions, reduces the rate of formation of skin lesions, and the severity of skin lesions A method comprising at least one effect selected from reducing the degree.   A method of treating lupus nephritis comprising administering to a subject having lupus nephritis an antibody that binds CSF1R, wherein the antibody blocks binding of CSF1 to CSF1R, and to CSF1R. A method of blocking IL-34 binding.   17. The method of claim 16, wherein renal function is improved in the subject.   18. The method of claim 16 or claim 17, wherein proteinuria is reduced in said subject.   19. The method of any one of claims 16-18, wherein glomerular filtration rate is improved in the subject.   A method of treating lupus comprising administering an antibody that binds CSF1R to a subject having lupus, said antibody blocking binding of CSF1 to CSF1R and IL-to CSF1R. 34. A method which blocks 34 binding and wherein said antibody reduces the number of CD16 + monocytes by at least 30% and said CD16-monocytes are not reduced or reduced by less than 20% in said subject.   21. The method of claim 20, wherein the CD16 + monocytes are reduced by at least 50%.   Selection from Hydroxychloroquine (Plaquenil), Corticosteroids, Cyclophosphamide (Cytoxan), Azathioprine (Imuran, Azasan), Mycophenolate (Cellcept), Leflunomide (Arava), and Methotrexate (Trexall), and Belimumab (Benlysta) 22. The method of any one of claims 15-21, further comprising administering at least one additional therapeutic agent to be administered.   A method of treating an inflammatory condition comprising administering to a subject having an inflammatory condition an antibody that binds CSF1R, wherein the antibody blocks binding of CSF1 to CSF1R and to CSF1R. A method which blocks the binding of IL-34 and wherein said antibody reduces the number of CD16 + monocytes by at least 30% and does not reduce or less than 20% of CD16-monocytes.   24. The method of claim 23, wherein CD16 + monocytes are reduced by at least 50%.   A method of treating a CD16 + disorder comprising administering to the subject having a CD16 + disorder an antibody that binds CSF1R, wherein the antibody blocks binding of CSF1 to CSF1R, and to CSF1R. A method which blocks the binding of IL-34 and wherein said antibody reduces the number of CD16 + monocytes by at least 30% and does not reduce or less than 20% of CD16-monocytes.   26. The method of claim 25, wherein the CD16 + monocytes are reduced by at least 50%.   27. The method of any of claims 20-26, wherein the lupus, the inflammatory condition, or the CD16 + disorder do not respond to methotrexate.   A method of reducing the number of CD16 + monocytes, comprising administering to a subject an antibody that binds CSF1R, wherein the antibody blocks binding of CSF1 to CSF1R and IL-34 to CSF1R. A method that blocks binding and wherein the antibody reduces the number of CD16 + monocytes by at least 30% and does not reduce or reduce CD16-monocytes by less than 20%.   29. The method of claim 28, wherein the CD16 + monocytes are reduced by at least 50%.   30. The method of any one of claims 20-29, wherein the CD16 + monocytes are CD16 + peripheral blood monocytes.   A method of delaying the progression of a lupus related renal condition comprising administering to a subject an antibody that binds CSF1R, wherein the antibody blocks binding of CSF1 to CSF1R and IL to CSF1R. A method of blocking -34 binding.   32. The method of claim 31, wherein proteinuria does not increase in the subject or does not increase in the subject at the same rate as a subject to which the antibody is not administered.   33. The method of claim 31 or 32, wherein the glomerular filtration rate does not decrease in the subject or does not decrease in the subject at the same rate as the subject to which the antibody is not administered.   A method of delaying the progression of pannus formation in a subject having rheumatoid arthritis comprising administering to a subject having rheumatoid arthritis an antibody that binds colony stimulating factor 1 receptor (CSF1R). , Wherein the antibody blocks binding of colony stimulating factor 1 (CSF1) to CSF1R and blocks binding of IL-34 to CSF1R.   A method of delaying the progression of bone loss in a subject having rheumatoid arthritis comprising administering to a subject having rheumatoid arthritis an antibody that binds colony stimulating factor 1 receptor (CSF1R) , Wherein the antibody blocks binding of colony stimulating factor 1 (CSF1) to CSF1R and blocks binding of IL-34 to CSF1R. Said antibody is
(a) an antibody comprising a heavy chain comprising the sequence of SEQ ID NO: 39 and a light chain comprising the sequence of SEQ ID NO: 46,
(b) Heavy chain (HC) CDR1 having the sequence of SEQ ID NO: 15, HC CDR2 having the sequence of SEQ ID NO: 16, and a heavy chain comprising HC CDR3 having the sequence of SEQ ID NO: 17 An antibody comprising a light chain (LC) CDR1 having the sequence of ID NO: 18, an LC CDR2 having the sequence of SEQ ID NO: 19, and a light chain comprising an LC CDR3 having the sequence of SEQ ID NO: 20,
(c) selected from antibodies comprising a heavy chain comprising the sequence of SEQ ID NO: 53 and a light chain comprising the sequence of SEQ ID NO: 60 Method.   37. The method of claim 36, wherein the antibody is a humanized antibody. 38. The method of claim 36 or 37, wherein the antibody is selected from Fab, Fv, scFv, Fab 'and (Fab') ₂ .

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