JP2017125040A - Methods for treating conditions using antibodies that bind colony stimulating factor 1 receptor (csf1r) - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide methods for treating rheumatoid arthritis and related conditions, methods for treating systemic lupus erythematosus and related conditions, and methods for treating multiple sclerosis.SOLUTION: The invention provides a method for treating rheumatoid arthritis related conditions comprising a step of administering an antibody that bind colony stimulating factor 1 receptor (CSF1R) to a subject with rheumatoid arthritis, where the antibody blocks binding of colony stimulating factor 1 (CSF1) to CSF1R and binding of IL-34 to CSF1R, which elicits at least one effect selected from reduction of inflammation, reduction of pannus formation, reduction of cartilage damage, reduction of bone resorption, reduction of macrophage numbers in at least one joint suffering from rheumatoid arthritis, reduction of autoantibody level and reduction of bone loss.SELECTED DRAWING: 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 systemic lupus erythematosus.

Background Colony stimulating factor 1 receptor (referred to herein as CSF1R, also referred to in the art as FMS, FIM2, C-FMS, M-CSF receptor, and CD115) is N-terminal cell It is a single-transmembrane receptor having an outer domain (ECD) and a C-terminal intracellular domain and having tyrosine kinase activity. Ligand binding of CSF1 or interleukin 34 ligand (herein referred to as IL-34, Lin et al., Science 320: 807-11 (2008)) to CSF1R is 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 into macrophages, as well as differentiation into other monocyte cell lineages such as osteoclasts, dendritic cells, and microglia.

  Many tumor cells have been found to secrete CSF1, which activates monocyte / macrophage cells through CSF1R. It has been shown that the level of CSF1 in the tumor correlates with the level of tumor associated macrophages (TAM) in the tumor. It has been found that higher levels of TAM correlate with a patient's worse prognosis. 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 metastasis. For example, Ohno et al. Mol. Cancer Ther. 5: 2634-43 (2006) (Non-patent Document 3).

  CSF1 and its receptors 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 by rheumatoid arthritis have been found to produce CSF1, suggesting a role for CSF1 and its receptors in the disease. Blocking CSF1R activity by antibodies results in positive clinical effects in mice models of arthritis, including reduced bone and cartilage destruction and reduced macrophage numbers. 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 cells, 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 into differentiated bone marrow lineage cells such as macrophages, microglia cells, and osteoclasts . Therefore, CSF1R stimulation contributes to the proliferation, survival, activation, and maturation of differentiated myeloid lineage cells, and in pathological situations, CSF1R stimulation contributes to the ability of differentiated bone marrow 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, a method of treating a condition associated with rheumatoid arthritis is provided. In some embodiments, the method comprises administering to a subject having rheumatoid arthritis an antibody that binds a colony stimulating factor 1 receptor (CSF1R), the antibody stimulating colony to CSF1R. Blocks factor 1 (CSF1) binding and blocks IL-34 binding 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 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 includes reducing inflammation. In some embodiments, a method of reducing inflammation associated with rheumatoid arthritis includes determining a erythrocyte sedimentation rate, where the reduction in sedimentation rate indicates a decrease 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 includes reducing bone resorption. In some such embodiments, the level of at least one marker for bone resorption is decreased. In some embodiments, the bone resorption marker is tartrate resistant acid phosphatase 5b (TRAP5b), urinary total pyridinoline, urinary total deoxypyridinoline, urinary free pyridinoline, serum type I collagen cross-linked N-telopeptide Urinary type I collagen cross-linked N-telopeptide and serum type I collagen carboxy terminal telopeptide.

  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, the imaging techniques include x-ray imaging, magnetic resonance imaging, computed tomography (CT) scanning, arthroscopy, scintigraphy, ultrasonography, bone densitometry, simple imaging. Single 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 A method selected from: a duplex ultrasonography method, and a power Doppler imaging method.

  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 is 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, wherein the antibody is directed against CSF1R. Blocks binding of CSF1 and blocks binding of IL-34 to CSF1R, and the antibody reduces the number of CD16 + monocytes by at least 30% and does not decrease CD16− monocytes in the subject Or 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, anti-TNF agent, glucocorticoid, cyclosporine, leflunomide, azathioprine, JAK inhibitor, SYK inhibitor, anti-IL-6 antibody, 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 The method further includes administering a substance.

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

  In some embodiments, a method of treating lupus nephritis is provided. In some embodiments, the method comprises administering to a subject having lupus nephritis an antibody that binds CSF1R, wherein the antibody blocks CSF1 binding 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 reduced in the subject. In some embodiments, the glomerular filtration rate is increased in the subject.

  In some embodiments, a method of treating lupus is provided. In some embodiments, one method comprises administering to a subject with lupus an antibody that binds CSF1R, said antibody blocking CSF1 binding to CSF1R and to CSF1R. Blocks IL-34 binding. In some embodiments, the antibody reduces the number of CD16 + monocytes in the subject by at least 30% and CD16− monocytes are not decreased or decreased 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 for treating lupus, lupus nephritis, or skin lesions associated with lupus include hydroxychloroquine (Plaquenil), corticosteroids, cyclophosphamide (Cytoxan), azathioprine (Imuran, Azasan), The method further comprises administering at least one additional therapeutic agent selected from mycophenolate (Cellcept), leflunomide (Arav) 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, said antibody blocking CSF1 binding to CSF1R, and CSF1R. Blocks IL-34 binding to. In some embodiments, the antibody reduces the number of CD16 + monocytes by at least 30% and CD16− monocytes are not decreased or decreased 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 of treating a CD16 + disorder is provided. In some embodiments, one method comprises administering to a subject having a CD16 + disorder an antibody that binds CSF1R, said antibody blocking 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 CD16− monocytes are not decreased or decreased 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 treating discussed herein include treating a condition that does not respond to methotrexate.

  In some embodiments, a method for reducing the number of CD16 + monocytes is provided. In some embodiments, one method comprises administering to a subject an antibody that binds CSF1R, said antibody blocking CSF1 binding 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 CD16− monocytes are not decreased or decreased 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 of delaying the progression of kidney conditions associated with lupus is provided. In some embodiments, the method comprises administering to the subject an antibody that binds CSF1R, said antibody blocking CSF1 binding 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 whom the antibody is not administered. In some embodiments, glomerular filtration rate does not decrease in the subject or does not decrease in the subject at the same rate as a subject to whom the antibody is not administered.

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

  In some embodiments, a method of delaying the progression of bone loss in a subject having rheumatoid arthritis is provided. In some embodiments, one method comprises administering to a subject having rheumatoid arthritis an antibody that binds a colony stimulating factor 1 receptor (CSF1R), the antibody stimulating colony to CSF1R. Blocks factor 1 (CSF1) binding and blocks IL-34 binding 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. Includes 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 NOs: 10, 12, 14, and 46-52 Includes 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. The light chain comprises a sequence that is identical and 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 NOs: 10, 12, 14, and 46-52 Includes 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 includes a set of sequences selected from SEQ ID NOs: 27, 28, and 29. In some embodiments, LC CDR1, LC CDR2, and LC CDR3 are (a) SEQ ID NOs: 18, 19, and 20, (b) SEQ ID NOs: 24, 25, and 26, and (c) It includes a set of sequences selected from SEQ ID NOs: 30, 31, and 32.

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

  In some embodiments, an isolated antibody is provided, the antibody comprising a heavy chain and a light chain, the antibody comprising: (a) SEQ ID NO: 9 and at least 95%, at least 97%; A heavy chain comprising a sequence that is at least 99%, or 100% identical, and a light chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 10, (b) A heavy chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 11, and 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% with SEQ ID NO: 13 Or a heavy chain comprising a sequence that is 100% identical, and a light chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 14, (d) SEQ ID NO: A heavy chain comprising a sequence that is 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 that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 40, and SEQ ID NO: 46 to at least 95% A light chain comprising a sequence that 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 SEQ ID NO: 46 to at least 95%, at least 97%, at least 99%, or 100 A light chain comprising a sequence that is% identical, (g) a heavy chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 39, and SEQ ID NO: 47 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 SEQ ID NO: 47 and at least 95%, at least 97%, low A light chain comprising a sequence that is at least 99%, or 100% identical, (i) a heavy chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 41 A light chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 47, and (j) at least 95%, at least 97%, at least, SEQ ID NO: 42 A heavy chain comprising a sequence that is 99%, or 100% identical, and a light chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 48, (k) SEQ A sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to ID NO: 42 A light chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 49, (l) at least 95%, at least 97 with SEQ ID NO: 42 A heavy chain comprising a sequence that is%, at least 99%, or 100% identical, and a light chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 50, ( m) a heavy chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 43, and at least 95%, at least 97%, at least 99% SEQ ID NO: 48; Or a light chain comprising a sequence that is 100% identical, (n) SEQ ID NO: 43 and at least 95 A heavy chain comprising a sequence that is at least 97%, at least 99%, or 100% identical, and a light chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 49. (O) a heavy chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 43, and SEQ ID NO: 50 to at least 95%, at least 97%, A light chain comprising a sequence that is at least 99%, or 100% identical, (p) a heavy chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 44; SEQ ID NO: 51 and at least 95%, at least 97%, at least 99%, or 100% same A light chain comprising a sequence that is: (q) a heavy chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 44; and SEQ ID NO: 52 and at least 95 A light chain comprising a sequence that is%, at least 97%, at least 99%, or 100% identical, (r) a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 45 A light chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 51, or (s) 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, and SEQ ID N : 52 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, the antibody comprising a heavy chain and a light chain, the antibody comprising: (a) a heavy chain (HC) CDR1, SEQ ID having the sequence of 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, SEQ ID NO: 19 having the sequence of SEQ ID NO: 18 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, the sequence of SEQ ID NO: 22 HC CDR2 having, and heavy chain comprising HC CDR3 having the sequence of SEQ ID NO: 23, sequence of SEQ ID NO: 24 A light chain comprising a light chain (LC) CDR1, an LC CDR2 having a sequence of SEQ ID NO: 25, and an LC CDR3 having a sequence of SEQ ID NO: 26, or (c) a sequence of SEQ ID NO: 27 A heavy chain comprising a heavy chain (HC) CDR1, a HC CDR2 having a sequence of SEQ ID NO: 28, and a HC CDR3 having a sequence of SEQ ID NO: 29, and a light chain having a sequence of SEQ ID NO: 30 ( LC) CDR1, LC CDR2 having the sequence of SEQ ID NO: 31, and a light chain comprising LC CDR3 having the sequence of SEQ ID NO: 32.

  In some embodiments, the antibody comprises a heavy chain and a light chain, the 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 SEQ ID NO: 65 A light chain comprising the sequence. In some embodiments, the antibody comprises a heavy chain and a light chain, the antibody comprising: (a) a heavy chain consisting of the sequence of SEQ ID NO: 53 and a light chain consisting of the sequence of SEQ ID NO: 60 (B) a heavy chain consisting of the sequence of SEQ ID NO: 53 and a light chain consisting of the sequence of SEQ ID NO: 61, or (c) a heavy chain consisting of the sequence of SEQ ID NO: 58 And a light chain consisting of the sequence of SEQ ID NO: 65.

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 IgG. In some embodiments, the antibody is IgG4. In some embodiments, the antibody is an IgG4 comprising an S241P mutation in at least one IgG4 heavy chain constant region.

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

  In some embodiments, a pharmaceutical composition comprising an antibody that binds CSF1R is provided.

In some embodiments, a composition comprising an antibody that binds CSF1R is provided for use in a method of human or animal therapy. In some embodiments, an antibody that binds CSF1R and a composition comprising an antibody that binds CSF1R are provided for use in a method of treating a condition associated with rheumatoid arthritis in a human or animal. In some embodiments, an antibody that binds CSF1R and a composition comprising an antibody that binds CSF1R are provided for use in a method of treating multiple sclerosis in a human or animal. In some embodiments, an antibody that binds CSF1R and a composition comprising an antibody that binds CSF1R 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 a colony stimulating factor 1 receptor (CSF1R), wherein the antibody is directed to CSF1R. Blocking colony-stimulating factor 1 (CSF1) binding and blocking IL-34 binding to CSF1R and treating conditions associated with rheumatoid arthritis reduce inflammation, pannus formation Reducing, reducing cartilage damage, reducing bone resorption, reducing the number of macrophages in at least one joint affected by rheumatoid arthritis, reducing autoantibody levels, and reducing bone loss A method comprising at least one effect selected from:
[Invention 1002]
The method of the present invention 1001, wherein treating a condition associated with rheumatoid arthritis comprises reducing inflammation.
[Invention 1003]
The method of the present invention 1002, wherein the number of CD16 + monocytes is reduced by at least 30%.
[Invention 1004]
CD16—The method of the invention 1003, wherein the number of monocytes is not reduced or is reduced by less than 20%.
[Invention 1005]
The method of the present invention 1002, further comprising the step of determining an erythrocyte sedimentation rate, wherein the decrease in sedimentation rate indicates a decrease in inflammation.
[Invention 1006]
The method of the present 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 present invention 1006, wherein treating a condition associated with rheumatoid arthritis comprises reducing bone resorption.
[Invention 1008]
The method of the present invention 1007, wherein the level of at least one marker for bone resorption is decreased.
[Invention 1009]
The bone resorption marker is tartrate resistant acid phosphatase 5b (TRAP5b), urinary total pyridinoline, urinary total deoxypyridinoline, urinary free pyridinoline, serum type I collagen cross-linked N-telopeptide, urinary type I collagen The method of the present invention 1008 selected from a cross-linked N-telopeptide and a carboxy-terminal telopeptide of serum type I collagen.
[Invention 1010]
The method of 1006 of this invention, 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 the present invention 1010, wherein the at least one effect is measured using an imaging technique.
[Invention 1012]
The imaging techniques include X-ray imaging, magnetic resonance imaging, computed tomography (CT) scanning, arthroscopy, scintigraphy, ultrasonography, bone density measurement, single photon absorption measurement (SPA). ), Double photon absorption measurement (DPA), single energy X-ray absorption measurement (SXA), dual energy X-ray absorption measurement (DXA), scintigraphy, ultrasonic inspection, duplex ultrasonic inspection, 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 wherein the binding of IL-34 is blocked and the antibody reduces the number of CD16 + monocytes in the subject by at least 30% and CD16-monocytes are not reduced or less than 20%.
[Invention 1014]
Methotrexate, anti-TNF substance, glucocorticoid, cyclosporine, 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 the invention 1001-1013 further comprising 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]
A method of treating a skin lesion associated with lupus comprising administering to a subject having lupus an antibody that binds CSF1R, wherein the antibody blocks CSF1 binding to CSF1R, and Blocking IL-34 binding to CSF1R and treating skin lesions associated with lupus 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 binds to CSF1R. A method of blocking the binding of IL-34.
[Invention 1017]
The method of the present invention 1016, wherein renal function is improved in said subject.
[Invention 1018]
The method of the present invention 1016 or the present invention 1017, wherein proteinuria is reduced in the subject.
[Invention 1019]
The method of any of the present invention 1016-1018, wherein glomerular filtration rate is increased in the subject.
[Invention 1020]
A method of treating lupus comprising administering to a subject having lupus an antibody that binds CSF1R, wherein the antibody blocks binding of CSF1 to CSF1R and IL- to CSF1R. 34. The method of blocking 34 binding, and wherein the antibody reduces the number of CD16 + monocytes by at least 30% and the CD16− monocytes are not decreased or decreased by less than 20% in the subject.
[Invention 1021]
The method of the present invention 1020, wherein CD16 + monocytes are reduced by at least 50%.
[Invention 1022]
Selected 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 present invention 1015-1021 further comprising administering at least one additional therapeutic agent to be administered.
[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 CSF1 binding to CSF1R and to CSF1R. A method wherein the binding of IL-34 is blocked and the antibody reduces the number of CD16 + monocytes by at least 30% and the CD16− monocytes are not reduced or less than 20%.
[Invention 1024]
The method of the present invention 1023, wherein CD16 + monocytes are reduced by at least 50%.
[Invention 1025]
A method of treating a CD16 + disorder comprising administering to a subject having a CD16 + disorder an antibody that binds CSF1R, wherein the antibody blocks binding of CSF1 to CSF1R and binds to CSF1R. A method wherein the binding of IL-34 is blocked and the antibody reduces the number of CD16 + monocytes by at least 30% and the CD16− monocytes are not reduced or less than 20%.
[Invention 1026]
The method of the present invention 1025 wherein CD16 + monocytes are reduced by at least 50%.
[Invention 1027]
The method of any of claims 1010 to 126, wherein said lupus, said inflammatory condition, or said CD16 + disorder does 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, said antibody blocking CSF1 binding to CSF1R and IL-34 to CSF1R. A method wherein the binding is blocked and the antibody reduces the number of CD16 + monocytes by at least 30% and CD16− monocytes are not reduced or less than 20%.
[Invention 1029]
The method of the present invention 1028, wherein CD16 + monocytes are reduced by at least 50%.
[Invention 1030]
The method according to any of 1020 to 1029 of the present invention, wherein the CD16 + monocytes are CD16 + peripheral blood monocytes.
[Invention 1031]
A method of delaying progression of a renal condition associated with lupus 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 the binding of −34.
[Invention 1032]
The method of the invention 1031 wherein proteinuria does not increase in the subject, or does not increase in the subject at the same rate as a subject to whom the antibody is not administered.
[Invention 1033]
The method of the invention 1031 or the invention 1032 wherein the glomerular filtration rate does not decrease in the subject or does not decrease in the subject at the same rate as a subject to whom the antibody is not administered.
[Invention 1034]
A method of delaying the progression of pannus formation in a subject having rheumatoid arthritis, comprising administering to the subject having rheumatoid arthritis an antibody that binds colony stimulating factor 1 receptor (CSF1R). A method wherein the antibody blocks colony stimulating factor 1 (CSF1) binding to CSF1R and blocks IL-34 binding to CSF1R.
[Invention 1035]
A method of delaying the progression of bone loss in a subject having rheumatoid arthritis, comprising administering to the subject having rheumatoid arthritis an antibody that binds a colony stimulating factor 1 receptor (CSF1R). A method wherein the antibody blocks colony stimulating factor 1 (CSF1) binding to CSF1R and blocks IL-34 binding to CSF1R.
[Invention 1036]
The 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) a heavy chain comprising a heavy chain (HC) CDR1 having the sequence of SEQ ID NO: 15, an HC CDR2 having the sequence of SEQ ID NO: 16, and an HC CDR3 having the sequence of SEQ ID NO: 17; An antibody comprising: a light chain (LC) CDR1 having a sequence of ID NO: 18; an LC CDR2 having a sequence of SEQ ID NO: 19; and a light chain comprising an LC CDR3 having a sequence of SEQ ID NO: 20;
(c) The method according to any of the invention 1001 to 1035, wherein the method is 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 present invention 1036, wherein said 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 .

The alignment of the humanized heavy chain variable region with respect to each of the humanized antibodies huAb1 to huAb16 described in Example 1 is shown. The residues in the box are the amino acids in the human receptor sequence that have been converted back to the corresponding mouse residues. The alignment of the humanized heavy chain variable region with respect to each of the humanized antibodies huAb1 to huAb16 described in Example 1 is shown. The residues in the box are the amino acids in the human receptor sequence that have been converted back to the corresponding mouse residues. The alignment of the humanized heavy chain variable region with respect to each of the humanized antibodies huAb1 to huAb16 described in Example 1 is shown. The residues in the box are the amino acids in the human receptor sequence that have been converted back to the corresponding mouse residues. The alignment of the humanized light chain variable region with respect to each of the humanized antibodies huAb1 to huAb16 described in Example 1 is shown. The amino acids in the boxes are the residues in the human receptor sequence that have been converted back to the corresponding mouse residues. The alignment of the humanized light chain variable region with respect to each of the humanized antibodies huAb1 to huAb16 described in Example 1 is shown. The amino acids in the boxes are the residues in the human receptor sequence that have been converted back to the corresponding mouse residues. The alignment of the humanized light chain variable region with respect to each of the humanized antibodies huAb1 to huAb16 described in Example 1 is shown. The amino acids in the boxes are the residues in the human receptor sequence that have been converted back to the corresponding mouse residues. FIG. 3 shows CD16 + monocyte reduction in cynomolgus monkeys administered huAb1, as described in Example 2. FIG. 2 shows dose-dependent binding of surrogate antibody cAb1 to mouse CSF1R, as described in Example 3. FIG. 3 shows dose-dependent suppression of CSF1 and IL-34 induced proliferation in mNFS60 cells as described in Example 4. FIG. 5 shows the suppression of clinical disease scores in a mouse model of rheumatoid arthritis after administration of cAb1, as described in Example 5. FIG. 4 shows the suppression of bone loss as shown by plasma TRAP5b levels in a mouse model of rheumatoid arthritis after administration of cAb1, as described in Example 6. FIG. 5 shows 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. FIG. 9 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. FIG. 5 shows the suppression of autoantibody formation in a mouse model of rheumatoid arthritis after administration of cAb1, as described in Example 9. FIG. 4 shows the suppression of bone loss in a mouse model of established rheumatoid arthritis after administration of cAb1 as described in Example 10. FIG. FIG. 5 shows the inhibition of pannus formation and bone destruction in a mouse model of established rheumatoid arthritis after administration of cAb1, as described in Example 11. FIG. 5 shows suppression of glomerulonephritis, interstitial nephritis, and perivascular invasion in a mouse model of systemic lupus erythematosus after administration of cAb1 as described in Example 12. FIG. 4 shows the suppression of skin lesions in a mouse model of systemic lupus erythematosus after administration of cAb1, as described in Example 13. FIG. 6 shows the suppression of clinical disease scores 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 ligand binding of CSF1 and IL-34 are effective in treating rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis. is there. The inventors have administered such antibodies to cynomolgus monkeys, which reduces the number of CD16 + peripheral blood monocytes in cynomolgus monkeys, but does not affect the number of CD16-peripheral blood monocytes; We found that the sphere is a highly inflammatory monocyte. For example, Ziegler-Heitblock, J. et al. Leukocyte Biol. , 2007, 81: 584-592. Furthermore, in a mouse model of rheumatoid arthritis, administration of such antibodies suppresses clinical disease scores, as measured by, for example, suppression of erythema and swelling, reduction of tartrate-resistant acid phosphatase 5b (TRAP5b) levels Inhibited bone loss, suppressed inflammation, cartilage destruction, pannus formation, and bone destruction. In particular, bone loss (as measured by a decrease in TRAP5b levels), pannus formation, and bone destruction was reduced when the antibody was administered after the appearance of clinical symptoms of rheumatoid arthritis. Also, in a mouse model of rheumatoid arthritis, administration of such an antibody reduced the number of joint macrophages and autoantibody formation.

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

  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 defined otherwise, 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. Yes. Many such techniques and procedures are described, for example, by Sambrook et al. Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)). In addition, 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 multiple dependent claims, the use of “or” refers back to more than one preceding independent claim or dependent claim only. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise two or more subunits, unless otherwise specified.

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

  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 and refer to a polymer of amino acid residues and are not limited to a minimum length. Such polymers of amino acid residues may include natural or unnatural 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 glycosylation, sialylation, acetylation, phosphorylation and the like. Further, for purposes of the present invention, a “polypeptide” is a modification, eg, deletion, addition, and substitution (generally conservative in nature) to a native sequence, so long as the protein retains the desired activity. ). 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. Also good.

  The term “CSF1R” as used herein refers to a full-length CSF1R that includes an N-terminal ECD, a transmembrane domain, and an intracellular tyrosine kinase domain, with or without an N-terminal leader sequence. In some embodiments, the CSF1R is a human CSF1R having an 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 intracellular and transmembrane domains. The CSF1R ECD includes full-length CSF1R ECD and CSF1R ECD fragments capable of binding CSF1R and / or IL-34. The human full length CSF1R ECD herein includes either amino acids 1 to 512 of SEQ ID NO: 2 (ie, including the leader sequence) or amino acids 20 to 512 (ie, lacking the leader sequence). 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 with 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 of SEQ ID NO: 7 (excluding the leader sequence).

  In connection with anti-CSF1R antibodies, the term “active” or “activity” or “function”, and grammatical variations thereof, suppress (block or antagonist) or mimic at least one of the aforementioned activities. Used to refer to the ability to (acting antibody). Antibodies and antibody fragments termed “functional” are characterized by having such properties.

  “Immunological” activity refers only to the ability to induce the production of antibodies against an antigenic epitope carried by a natural or naturally occurring CSF1R polypeptide.

As used herein, the term “antibody” is a molecule comprising at least the complementarity determining regions (CDRs) 1, CDR2 and CDR3 of a heavy chain and at least CDR1, CDR2 and CDR3 of a light chain. Refers to a molecule capable of binding to an antigen. The term antibody includes, but is not limited to, fragments capable of binding an antigen, eg, 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 monkey.

  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 and at least a portion of a light chain constant region. Contains at least one light chain. In some embodiments, the antibody has 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 is light with a light chain variable region. It contains two light chains, including at least part of the chain constant region. 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) Are thought to have heavy and light chains. In some such embodiments, the heavy chain is a region of an antibody comprising three heavy chain CDRs and the light chain is in a region of the antibody comprising three light chain CDRs.

  As used herein, the term “heavy chain variable region” refers to a region comprising heavy chain CDR1, framework (FR) 2, CDR2, FR3, and CDR3. In some embodiments, the heavy chain variable region also includes 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. To 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. To do. See the same document.

As used herein, the term “heavy chain constant region” refers to a region comprising at least three heavy chain constant domains, C _H 1, C _H 2 and C _H 3. Non-limiting exemplary heavy chain constant regions include γ, δ, and α. Non-limiting exemplary heavy chain constant regions also include ε and μ. Each heavy constant region corresponds to an antibody isotype. 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 include IgG1 (including γ ₁ constant region), IgG2 (including γ ₂ constant region), IgG3 (including γ ₃ constant region), and IgG4 (including γ ₄ constant region) antibodies. , 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 confer desired characteristics on 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 changes from the IgG4 motif CPSCP to a CPPCP similar to the corresponding motif in 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).

  As used herein, the term “heavy chain” refers to a polypeptide that includes at least the heavy chain variable region and may or may not include a leader sequence. In some embodiments, the heavy chain comprises at least a portion of a heavy chain constant region. As used herein, the term “full-length heavy chain” refers to a polypeptide comprising a heavy chain variable region and a heavy chain constant region, with or without a leader sequence.

  As used herein, the term “light chain variable region” refers to a region comprising light chain CDR1, framework (FR) 2, CDR2, FR3, and CDR3. In some embodiments, the light chain variable region also includes FR1 and / or FR4. In some embodiments, the light chain CDR1 corresponds to Kabat residues 24-34, the light chain CDR2 corresponds to Kabat residues 50-56, and the light chain CDR3 corresponds to Kabat residues 89-97. To 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.

  As used herein, the term “light chain” refers to a polypeptide comprising at least the light chain variable region, with or without 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.

  As used herein, a “chimeric antibody” refers to at least one variable region derived from a first species (eg, mouse, rat, cynomolgus monkey, etc.) and a second species (eg, human, cynomolgus monkey, etc.). An antibody comprising at least one constant region from which it is derived. In some embodiments, the chimeric antibody comprises at least one mouse variable region and at least one human constant region. In some embodiments, the chimeric antibody comprises at least one cynomolgus variable region and at least one human constant region. In some embodiments, the 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 derived from the first species and all constant regions of the chimeric antibody are derived from the second species.

As used herein, “humanized antibody” refers to an antibody in which at least one amino acid in the framework region of the non-human variable region is replaced with a corresponding amino acid from a human variable region. In some embodiments, the humanized antibody comprises at least one human constant region or fragment thereof. In some embodiments, the humanized antibody is a Fab, scFv, (Fab ′) ₂ or the like.

  As used herein, a “CDR grafted antibody” has a first (non-human) species complementarity determining region (CDR) grafted to a second (human) species framework region (FR). Refers to a humanized antibody.

  As used herein, “human antibody” refers to antibodies produced in humans, non-human animals containing human immunoglobulin genes, such as those produced in Xeno mice®, and in vitro methods such as phage An antibody selected using 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 the polypeptide from mammalian cells. The leader sequence may be cleaved to form the mature protein upon export of the polypeptide from the mammalian cell. 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, antibody leader sequences, eg, the amino acid sequences of SEQ ID NOs: 3 and 4, corresponding 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 a natural antibody leader sequence and a heterologous leader sequence.

  The term “vector” is used to describe a polynucleotide that can be engineered to contain a cloned polynucleotide or that can be propagated in a host cell. A vector can be an origin of replication, one or more regulatory sequences that regulate expression of a polypeptide of interest (eg, a promoter and / or enhancer), and / or one or more selectable marker genes (eg, antibiotic resistance). It may contain one or more elements of sex genes and genes that can be used in colorimetric assays, eg, β-galactosidase. The term “expression vector” refers to a vector used to express a polypeptide of interest in a host cell.

  A “host cell” refers to a cell that can be or is the recipient of a vector or isolated polynucleotide. The host cell can be a prokaryotic cell or a eukaryotic cell. 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 derivatives thereof, such as, but not limited to, 293-6E and DG44 cells, respectively.

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

  The terms “subject” and “patient” are used interchangeably herein to refer to a human. In some embodiments, including, but not limited to, rodents, apes, cats, dogs, horses, cows, pigs, sheep, goats, mammalian laboratory animals, mammalian livestock, mammalian sports animals, and mammalian pets. Also provided are methods of treating other mammals that are not.

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

  The term “rheumatoid arthritis” includes not only activity and early RA, but also early RA, as defined below. Physiological indicators of RA include symmetric joint expansion, which is a feature in RA but not invariant. Spindle-like swelling of the proximal interphalangeal (PIP) joint of the hand, as well as the metacarpal (MCP), wrist, elbow, knee, ankle, and metatarsal (MTP) joints is generally affected The expansion 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 motion of the affected joint. Typical visual changes include finger ulnar deviation at the MCP joint, hyperextension or hyperflexion of the MCP and PIP joints, flexion contracture of the elbow, and carpal and toe subluxation. A subject with RA may be resistant to a disease modifying anti-rheumatic drug (DMARD) and / or a non-steroidal anti-inflammatory drug (NSAID). Non-limiting exemplary “DMARD” includes hydroxychloroquine, sulfasalazine, methotrexate (MTX), leflunomide, etanercept, infliximab (also oral and subcutaneous MTX), azathioprine, D-penicillamine, gold salt (oral) , Gold salt (muscular injection), minocycline, cyclosporine including cyclosporin A and topical cyclosporine, staphylococcal protein A (Goodyear and Silverman, J. Exp. Med., 197 (9): 1125-1139 (2003)) These include salts and derivatives thereof. Further candidates for treatment according to the present invention may have insufficient response to past or current treatment with TNF inhibitors such as etanercept, infliximab, and / or adalimumab due to toxicity or insufficient efficacy. Includes those who have experienced.

  A patient having "active rheumatoid arthritis" means a patient who is active and has symptoms of non-potential RA. Patients with “early active rheumatoid arthritis” are diagnosed with active RA for a period of at least 8 weeks but not more than 4 years, according to the revised 1987 ACR criteria for RA classification It is a target.

  A subject with “early rheumatoid arthritis” is a subject diagnosed with RA for a period of at least 8 weeks but not more than 4 years 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” may have early polyarthritis that does not fully 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 have a high risk (95% chance) of developing a true ACR-based RA .

  “Joint injury” is used in a broad sense and refers to damage or partial or complete destruction of any part of one or more joints, including connective tissue and cartilage, where damage is structural and / or functional due to any cause May or may not cause joint pain / arthalgia. It includes, but is not limited to, joint damage associated with or resulting from inflammatory joint disease as well as non-inflammatory joint disease. This damage can be caused by any condition, such as autoimmune diseases, especially 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, type II collagen-induced arthritis, infectious arthritis, septic arthritis, Lyme disease arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, vertebral arthritis, osteoarthritis, progressive chronic arthritis, osteoarthritis, primary chronic polyarthritis, reactive arthritis, menopausal arthritis, estrogenic degenerative arthritis Spondylitis / rheumatoid spine ), Rheumatoid autoimmune diseases other than RA, and severe systemic lesions secondary to RA (including but not limited to vasculitis, pulmonary fibrosis, or Ferti syndrome) It is. For purposes herein, a joint is a point of contact between skeletal elements (of a vertebrate such as an animal) along with the part that surrounds and supports it, eg, the hip, spinal cord Joints between vertebrae, joints between spinal cord and pelvis (sacral joint), joints where tendons and ligaments adhere to bone, joints between ribs and spinal cord, shoulders, knees, feet, elbows, hands, fingers, ankles, And toes, including but not limited to hand and foot joints.

  As used herein, the term “lupus” is generally an autoimmune disease or disorder caused by antibodies that attack connective tissue. The major form of lupus is systemic, systemic lupus erythematosus (SLE), including cutaneous SLE and subacute cutaneous SLE, and other types of lupus (nephritis, extrarenal, encephalitis, childhood, renal Including exogenous, discoid, and alopecia). In certain embodiments, the term “systemic lupus erythematosus” refers to skin lesions, joint pain and swelling, kidney disease (lupus nephritis), heart and / or peripulmonary fluid, heart inflammation, and various other Refers to a chronic autoimmune disease that can lead to a generalized condition. In certain embodiments, the term “lupus nephritis” refers to inflammation of the kidney that occurs in a patient having SLE. Lupus nephritis may include, for example, glomerulonephritis and / or qualitative nephritis and can cause hypertension, proteinuria, and renal failure. Lupus nephritis can be classified based on the severity and extent of the disease, as defined by, for example, International Society of Nephrology / Renal / Pathology Society. For the classification of lupus nephritis, type I (micromesangial lupus nephritis), type II (mesangial proliferative lupus nephritis), type III (focal lupus nephritis), type IV (diffuse segmental (IV-S) or diffuse) Global nodal (IV-G) lupus nephritis), type V (membrane lupus nephritis), and type VI (advanced sclerosing lupus nephritis). The term “lupus nephritis” encompasses all of these classifications.

  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 separated from nerve fibers. Four types of internationally recognized MS: primary progressive multiple sclerosis (PPMS), relapsing-remitting multiple sclerosis (RRMS), secondary progressive multiple sclerosis (SPMS), and advanced relapse There is type multiple sclerosis (PRMS).

  “Primary progressive multiple sclerosis” or “PPMS” is characterized by the slow progression of the disease from its onset without any overlap between relapse and remission. There may be periods of steady state of disease activity, there may be good days or weeks and bad days or weeks. PPMS has onset typically in the late 30s or early 40s, and men are more likely to develop it as women, and early disease activity is often in the spinal cord rather than in the brain. Is different from RRMS and SPMS. PPMS often moves to the brain, but has a lower probability of damaging brain regions than RRMS or SPMS, for example, those with PPMS are less likely to develop cognitive problems. PPMS is a subtype of MS with the lowest probability of showing inflammatory (gadolinium-enhanced) lesions on MRI scans. The primary progressive form of the disease affects 10-15% of all individuals with multiple sclerosis. PPMS is described in 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 undergone an estimated or definite diagnosis of PPMS.

  “Relapsing-remitting multiple sclerosis” or “RRMS” is characterized by recurrence (also known as exacerbation), a period in which new symptoms appear and old symptoms can come back or worsen. The relapse is followed by a period of remission during which the person recovers completely or partially from the injury gained during the relapse. Relapses can last for days, weeks, or months, and recovery can be slow and gradual, or almost instantaneous. The vast majority of people presenting with MS are first diagnosed with RRMS. This is generally the case when they are in their twenties or thirties, but diagnoses that are much earlier or later are also known. Twice as many women as men present with this subtype of MS. During recurrence, 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 an inflammatory response by the body's own immune system. This causes a variety of neurological symptoms that vary greatly depending on which area of the CNS is damaged. Immediately after recurrence, the inflammatory response subsides, supporting a special type of glial cell (called oligodendrocytes) in the CNS that 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 the onset of the disease. 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 the steady progression of clinical neurological damage with or without overlapping relapses, mild remissions, and plateaus. People who develop SPMS will have experienced a period of RRMS in the past that may last from 2 years to over 40 years. Any overlapping recurrences and remissions tend to fade over time. From the onset of the second progressive stage of the disease, disability begins and progresses faster than it occurred during RRMS, but progression may still be considerably slower in some individuals. SPMS tends to involve lower levels of inflammatory lesion formation than in RRMS, but the full burden of disease continues to progress. At any time, SPMS accounts for approximately 30% of all people with multiple sclerosis.

  “Progressive relapsing multiple sclerosis” or “PRMS” is characterized by the steady progression of clinical neurological damage with overlapping relapses and remissions. There is a significant recovery immediately after recurrence, but there is a gradual worsening of symptoms during the recurrence. PRMS affects approximately 5% of all people with multiple sclerosis. Some neurologists believe that PRMS is a variant of PPMS.

  The term “CD16 + disease” refers to a disease in which mammalian CD16 + monocytes cause, mediate or otherwise cause morbidity in a mammal. Also included are diseases in which a decrease in CD16 + monocytes has the effect of leading to improvement in disease progression. CD16 + inflammatory disease, infection, immunodeficiency, neoplasia, etc. are included within this term. In certain embodiments, CD16 + inflammatory diseases include inflammatory diseases that do not respond to methotrexate therapy. In certain embodiments, CD16 + inflammatory diseases include methotrexate resistant rheumatoid arthritis, methotrexate resistant multiple sclerosis, methotrexate resistant lupus, methotrexate resistant inflammatory bowel disease, methotrexate resistant Crohn's disease, Methotrexate resistant asthma and methotrexate resistant psoriasis are included. In certain embodiments, a patient having a methotrexate-resistant disease, such as methotrexate-resistant rheumatoid arthritis, is referred to as a responder with incomplete methotrexate or a poor responder with methotrexate.

  Examples of CD16 + disorders that can be treated in accordance with the present invention include systemic lupus erythematosus, rheumatoid arthritis, juvenile chronic arthritis, spondyloarthropathy, systemic sclerosis (scleroderma), idiopathic inflammatory Myopathy (dermatomyositis, polymyositis), Sjogren's syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia (immunopancytopenia, paroxysmal nocturnal hemoglobinuria), autoimmune thrombocytopenia (idiopathic) 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 systems, such as multiple sclerosis, idiopathic demyelinating polyneuropathy, or Guillain-Barre syndrome, and chronic Demyelinating polyneuropathy, hepatobiliary diseases such as infectious hepatitis (types A, B, C, D, hepatitis E and other non-hepatotropic viruses), autoimmune chronic active hepatitis, primary Biliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis, ulcerative colitis, inflammatory bowel disease including Crohn's disease (IBD), gluten-sensitive bowel disease, 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, and urticaria, eosinophilic pneumonia Pulmonary immune diseases such as idiopathic pulmonary fibrosis, and hypersensitivity pneumonia, transplant-related diseases including graft rejection and graft-versus-host disease, fibrosis including renal fibrosis and pulmonary fibrosis, atheromatous arteries Sclerosis and coronary Cardiovascular disease including pulse disease, chronic kidney disease, myocardial infarction, and cardiovascular events associated with congestive heart failure, diabetes including type II diabetes, bronchiolitis with organizing pneumonia (BOOP), hemophagocytic syndrome, macrophages Includes but is not limited to activation syndrome, sarcoidosis, and periodontitis. Infectious diseases including AIDS (HIV infection), viral diseases such as A, B, C, D and hepatitis 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 delay (reduce) the targeted pathological condition or disorder. It is. In certain embodiments, the term “treatment” encompasses 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 disease progression, eg, to cause regression. Or partially or completely remove disease by restoring or repairing lost, missing, or reduced function, stimulate inefficient processes, or reduce severity during stable disease Including that. The term “treatment” also includes reducing the severity of a feature of any phenotype and / or reducing the occurrence, extent, or likelihood of the feature. Those in need of treatment include those already with the disease as well as those prone to have the disease or those in which the disease is to be prevented.

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

  The term “effective amount” or “therapeutically effective amount” refers to an amount of a drug effective to treat a disease or disorder in a subject. In certain embodiments, an effective amount refers to an amount that is effective at the dosage and duration required to achieve the desired therapeutic or prophylactic result. A therapeutically effective amount of an anti-CSF1R antibody of the invention can 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 a desired response in the individual. A therapeutically effective amount includes an amount where the therapeutically beneficial effect exceeds any toxic or deleterious effect of the anti-CSF1R antibody.

  A “prophylactically effective amount” refers to an amount that is effective at the dosage and duration required to achieve the desired prophylactic result. Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

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

  “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semi-solid, or liquid filler that is conventional in the art for use with therapeutic agents that together include a “pharmaceutical composition” for administration to a subject, Refers to a diluent, encapsulant, compounding aid, or carrier. Pharmaceutically acceptable carriers are non-toxic to recipients at the dosages and concentrations used and are compatible with the other ingredients of the formulation. Pharmaceutically acceptable carriers are suitable for the formulation used. For example, when the therapeutic substance is administered orally, the carrier may be a gel capsule. When the therapeutic agent is administered subcutaneously, the 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, murine antibodies, human antibodies, and antibodies that include the heavy and / or light chain CDRs discussed herein.

Exemplary Humanized Antibodies In some embodiments, humanized antibodies that bind CSF1R are provided. Humanized antibodies reduce or eliminate a human immune response (eg, a human anti-mouse antibody (HAMA) response) to a non-human antibody that can result in an immune response to the antibody therapeutic and reduce the effectiveness of the therapeutic. It is useful as a therapeutic molecule.

  Non-limiting exemplary humanized antibodies include huAb1-huAb16 as described herein. Non-limiting exemplary humanized antibodies also include antibodies comprising a heavy chain variable region of an antibody selected from huAb1-huAb16 and / or a light chain variable region of an antibody selected from huAb1-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 It is. Exemplary humanized antibodies also include humanized antibodies comprising heavy chain CDR1, CDR2, and CDR3 of antibodies selected from 0301, 0302, and 0311, and / or light chain CDR1, CDR2, and CDR3 However, it is not limited to these.

  In some embodiments, the humanized anti-CSF1R antibody comprises the heavy chain CDR1, CDR2, and CDR3 of an antibody selected from 0301, 0302, and 0311, and / or the light chain CDR1, CDR2, and CDR3. 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 Antibody 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 a set of selected light chain CDR1, CDR2, and CDR3.

  Non-limiting exemplary humanized anti-CSF1R antibodies include heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3 pairs of Table 1 (SEQ ID NOs: see Table 8 for sequences) Including the antibody. Each column in 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 and light chain CDRs

Further Exemplary Humanized Antibodies In some embodiments, the humanized anti-CSF1R antibody has at least 90%, at least 91%, at least 92 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 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, the antibody binds CSF1R . In some embodiments, the humanized anti-CSF1R antibody has at least 90%, at least 91%, at least 92%, at least 93% with a sequence selected from SEQ ID NOs: 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, the antibody binds CSF1R. In some embodiments, the humanized anti-CSF1R antibody has 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; and 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 a sequence selected from A light chain comprising a region sequence, wherein the antibody binds CSF1R Let me.

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

  In some embodiments, the humanized anti-CSF1R antibody comprises at least one of the CDRs discussed herein. That is, in some embodiments, a humanized anti-CSF1R antibody is a heavy chain CDR1 as discussed herein, a heavy chain CDR2 as discussed herein, a heavy chain CDR3 as discussed herein, It comprises at least one CDR selected from light chain CDR1 as discussed, light chain CDR2 as discussed herein, and light chain CDR3 as discussed herein. Further, in some embodiments, the humanized anti-CSF1R antibody comprises at least one mutated CDR based on the CDRs discussed herein, wherein the mutated CDRs are against the CDRs discussed herein. Includes 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 of ordinary skill in the art can select one or more appropriate conservative amino acid substitutions for a particular CDR sequence, and this appropriate conservative amino acid substitution significantly alters the binding properties of an antibody containing a mutated CDR. It is not predicted.

  Exemplary humanized anti-CSF1R antibodies also include antibodies that compete for binding to CSF1R with the antibodies described herein. Thus, in some embodiments, an antibody selected from Fab 0301, 0302, and 0311, and a bivalent (ie, having two heavy and two light chains) antibody form of these Fabs, and 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 κ and λ. In some embodiments, the humanized antibody described herein comprises a human IgG constant region. In some embodiments, a humanized antibody described herein comprises a human IgG4 heavy chain constant region. In some such embodiments, the humanized antibody described herein comprises an S241P mutation within the human IgG4 constant region. In some embodiments, a humanized antibody described herein comprises 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 functions in vivo. Such effector functions include, in some embodiments, antibody dependent cell mediated cytotoxicity (ADCC) and / or complement dependent cytotoxicity (CDC), killing cells to which the antibody binds. Can bring. In some methods of treatment, including methods of treating some cancers, cell killing may be desirable, for example, when antibodies bind to cells that support tumor maintenance or growth. Exemplary cells that can support tumor maintenance or growth include tumor cells themselves, cells that help tumor angiogenesis, cells that provide ligands, growth factors, or counter-receptors that support or promote tumor growth or tumor survival Including but not limited to the body. In some embodiments, if effector function is desired, an anti-CSF1R antibody comprising a human IgG1 heavy chain or a human IgG3 heavy chain is selected.

  In some treatment 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, the anti-CSF1R antibody for the treatment of lupus and / or MS and / or RA and / or osteolysis comprises a human IgG4 or IgG2 heavy chain constant region. In some embodiments, the IgG4 constant region comprises an S241P mutation.

  The antibody may be humanized by any method. Non-limiting exemplary methods of humanization include, for example, US Pat. 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 US 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 replaced with an amino acid at a corresponding position in the human framework region. In some embodiments, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 9, 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 replaced with one or more corresponding amino acids in 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 the human framework regions of the first human antibody or correspondingly encoded by the first human immunoglobulin gene. An amino acid 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 acid encoded by the second human immunoglobulin gene. One or more of the non-human amino acids may be substituted and replaced with the corresponding amino acid derived from the human framework region of the third human antibody or encoded by the third human immunoglobulin gene. May be. Further, in some embodiments, not all of the corresponding human amino acids used for substitution in a single framework region, eg, FR2, need 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, the antibody is humanized by replacing one or more entire framework regions with corresponding human framework regions. In some embodiments, a human framework region is selected that has the highest level of homology to the non-human framework region to be replaced. In some embodiments, such a humanized antibody is a CDR grafted antibody.

  In some embodiments, after CDR grafting, one or more framework amino acids are returned to the corresponding amino acids in the mouse framework regions. Such “backmutations” may in some embodiments be attributed to the structure of one or more CDRs and / or may be involved in antigen adhesion, and / or This 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 fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, 1 or 0 Back mutations are made in the framework region of the antibody after CDR grafting.

  In some embodiments, the humanized antibody also comprises a human heavy chain constant region and / or a human light chain constant region.

Exemplary Chimeric Antibody 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, the one or more variable regions of the chimeric antibody are mouse variable regions. The human constant region of the chimeric antibody need not be the same isotype as the non-human constant region, even if a non-human constant region it replaces is 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 an antibody selected from 0301, 0302, and 0311. Further non-limiting exemplary chimeric antibodies include chimeric antibodies comprising heavy chain CDR1, CDR2, and CDR3 of an antibody selected from 0301, 0302, and 0311, and / or light chain CDR1, CDR2, and CDR3. It is.

  Non-limiting exemplary chimeric anti-CSF1R antibodies include antibodies comprising the following pairs of heavy and light chain variable regions: SEQ ID NO: 9 and 10, SEQ ID NO: 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 and light chain CDR1, CDR2, and CDR3 pairs shown in Table 1 above.

Further 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 NOs: 9, 11, 13, and 39-45. Comprising 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, wherein the antibody binds CSF1R. In some embodiments, the chimeric anti-CSF1R antibody has at least 90%, at least 91%, at least 92%, at least 93%, at least a sequence selected from SEQ ID NOs: 10, 12, 14, and 46-52 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, the antibody binds CSF1R. In some embodiments, the chimeric anti-CSF1R antibody has at least 90%, at least 91%, at least 92%, at least 93%, at least with a sequence selected from SEQ ID NOs: 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 to SEQ ID NOs: 10, 12, 14, and 46-52 Variable regions 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 selected sequence A light chain comprising a sequence, wherein the antibody binds CSF1R Let me.

  In some embodiments, the chimeric anti-CSF1R antibody comprises at least one of the CDRs discussed herein. That is, in some embodiments, a chimeric anti-CSF1R antibody is a heavy chain CDR1 as discussed herein, a heavy chain CDR2 as discussed herein, a heavy chain CDR3 as discussed herein, as discussed herein. Comprising at least one CDR selected from light chain CDR1 as discussed herein, 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. 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 of ordinary skill in the art can select one or more appropriate conservative amino acid substitutions for a particular CDR sequence, and this appropriate conservative amino acid substitution significantly alters the binding properties of an antibody containing a mutated CDR. 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, an antibody selected from Fab 0301, 0302, and 0311, and a bivalent (ie, having two heavy and two light chains) antibody form of these Fabs, and 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 κ and λ. In some embodiments, the chimeric antibodies described herein comprise a human IgG constant region. In some embodiments, the chimeric antibodies described herein comprise a human IgG4 heavy chain constant region. In some such embodiments, the chimeric antibodies described herein comprise an S241P mutation within the 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 described above, whether effector function is desirable may depend on the particular treatment method that is intended for the antibody. Thus, in some embodiments, a chimeric anti-CSF1R antibody comprising a human IgG1 heavy chain constant region or a human IgG3 heavy chain constant region is selected when effector function is desired. In some embodiments, a chimeric anti-CSF1R antibody comprising a human IgG4 or IgG2 heavy chain constant region is selected if effector function is desired.

Exemplary human antibodies Human antibodies can be made by any suitable method. A non-limiting exemplary method involves generating a human antibody in a transgenic mouse comprising a human immunoglobulin locus. See, 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. Pat. Nos. 5,545,807, 6,713,610, 6,673,986, 6,162,963, 5,545,807, 6,300,129, 6,255,458, 5,877,397, 5,874,299, and 5,545. 806.

  Non-limiting exemplary methods also include generating human antibodies using a phage display library. For example, Hoogenboom et al. , J .; Mol. Biol. 227: 381-8 (1992), Marks et al. , J .; Mol. Biol. 222: 581-97 (1991) and PCT Publication No. WO99 / 10494.

  In some embodiments, the 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 for binding to CSF1R with the antibodies described herein. Thus, in some embodiments, an antibody selected from Fab 0301, 0302, and 0311, and a bivalent (ie, having two heavy and two light chains) antibody form of these Fabs and to CSF1R Human anti-CSF1R antibodies that compete for the binding of are provided.

  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 κ and λ. In some embodiments, the human antibodies described herein comprise a human IgG constant region. 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 within the 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, if effector function is desired, a human anti-CSF1R antibody comprising a human IgG1 heavy chain constant region or a human IgG3 heavy chain constant region is selected. In some embodiments, a human anti-CSF1R antibody comprising a human IgG4 or IgG2 heavy chain constant region is selected if effector function is not desired.

Further exemplary anti-CSF1R antibodies Exemplary anti-CSF1R antibodies also include, for example, murine, humanized, human, chimeric, and engineered antibodies that comprise one or more of the CDR sequences described herein. However, it is not limited to these. In some embodiments, the anti-CSF1R antibody comprises a heavy chain variable region described herein. In some embodiments, the anti-CSF1R antibody comprises a light chain variable region described herein. In some embodiments, the anti-CSF1R antibody comprises a heavy chain variable region described herein and a light chain variable region described herein. In some embodiments, the anti-CSF1R antibody comprises a heavy chain CDR1, CDR2, and CDR3 as described herein. In some embodiments, the anti-CSF1R antibody comprises a light chain CDR1, CDR2, and CDR3 described herein. In some embodiments, the anti-CSF1R antibody comprises a heavy chain CDR1, CDR2, and CDR3 described herein and a light chain CDR1, CDR2, and CDR3 described herein.

  In some embodiments, the anti-CSF1R antibody comprises a heavy chain variable region of an antibody selected from Fab0301, 0302, and 0311. Non-limiting exemplary anti-CSF1R antibodies also include antibodies comprising the heavy chain variable region of an antibody selected from humanized antibodies huAb1-huAb16. Non-limiting exemplary anti-CSF1R antibodies include antibodies comprising a heavy chain variable region comprising a sequence selected from SEQ ID NOs: 9, 11, 13, and 39-45.

  In some embodiments, the anti-CSF1R antibody comprises a light chain variable region of an antibody selected from Fab0301, 0302, and 311. Non-limiting exemplary anti-CSF1R antibodies also include antibodies comprising a light chain variable region of an antibody selected from humanized antibodies huAb1-huAb16. Non-limiting exemplary anti-CSF1R antibodies include antibodies comprising a light chain variable region comprising a sequence selected from SEQ ID NOs: 10, 12, 14, and 46-52.

  In some embodiments, the anti-CSF1R antibody comprises a heavy chain variable region and a light chain variable region of an antibody selected from Fab0301, 0302, and 0311. Non-limiting exemplary anti-CSF1R antibodies also include antibodies comprising a heavy chain variable region and a light chain variable region of an antibody selected from humanized antibodies huAb1-huAb16. Non-limiting exemplary anti-CSF1R antibodies include antibodies comprising the following pairs of heavy and light chain variable regions: SEQ ID NO: 9 and 10, SEQ ID NO: 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 NO: 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 a heavy chain CDR1, CDR2, and CDR3 of an antibody selected from Fab0301, 0302, and 0311. Non-limiting exemplary anti-CSF1R antibodies include heavy weights 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 sets of chains CDR1, CDR2, and CDR3 are included.

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

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

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

Further 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. 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, the antibody binds CSF1R. In some embodiments, the anti-CSF1R antibody has at least 90%, at least 91%, at least 92%, at least 93%, at least 94 with a sequence selected from SEQ ID NOs: 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, the antibody binds CSF1R. In some embodiments, the anti-CSF1R antibody has at least 90%, at least 91%, at least 92%, at least 93%, at least 94 with a sequence selected from SEQ ID NOs: 9, 11, 13, and 39-45. %, At least 95%, at least 96%, at least 97%, at least 98%, or at least 99% selected from SEQ ID NOs: 10, 12, 14, and 46-52 A variable region sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence being And the antibody binds CSF1R .

  In some embodiments, the anti-CSF1R antibody comprises at least one of the CDRs discussed herein. That is, in some embodiments, an anti-CSF1R antibody is a heavy chain CDR1 as discussed herein, a heavy chain CDR2 as discussed herein, a heavy chain CDR3 as discussed herein, as discussed herein. It comprises at least one CDR selected from light chain CDR1, light chain CDR2 as discussed herein, and light chain CDR3 as discussed herein. Further, 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 to the CDRs discussed herein. Contains 2, 2, 3 or 4 amino acid substitutions. In some embodiments, one or more of the amino acid substitutions are conservative amino acid substitutions. One of ordinary skill in the art can select one or more appropriate conservative amino acid substitutions for a particular CDR sequence, and this appropriate conservative amino acid substitution significantly alters the binding properties of an antibody containing a mutated CDR. It is not predicted.

  Exemplary anti-CSF1R antibodies also include antibodies that compete for binding to CSF1R with the antibodies described herein. Thus, in some embodiments, an antibody selected from Fab 0301, 0302, and 0311, and a bivalent (ie, having two heavy and two light chains) antibody form of these Fabs, and CSF1R Anti-CSF1R antibodies that compete for binding are 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 κ and λ. In some embodiments, the antibodies described herein comprise a human IgG constant region. 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 within the human IgG4 constant region. In some embodiments, the antibodies described herein comprise a human IgG4 constant region and a human kappa light chain.

  As described above, whether effector function is desirable may depend on the particular treatment method that is intended for the antibody. Thus, in some embodiments, if effector function is desired, an anti-CSF1R antibody comprising a human IgG1 heavy chain constant region or a human IgG3 heavy chain constant region is selected. In some embodiments, if effector function is not desired, an anti-CSF1R antibody comprising a human IgG4 or IgG2 heavy chain constant region is 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 murine variable region, a human variable region, or a humanized variable region.

  Anti-CSF1R antibody heavy chain variable regions include heavy chain CDR1, FR2, CDR2, FR3, and CDR3. In some embodiments, the anti-CSF1R antibody heavy chain variable region further comprises heavy chain 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 NOs: 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 a 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 NOs: 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. A heavy chain variable region including, but not limited to, a set of CDR1, CDR2, and CDR3 is included.

  In some embodiments, the anti-CSF1R antibody heavy chain has 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. An antibody 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, the heavy chain together with the light chain binding CSF1R Can be formed.

  In some embodiments, the anti-CSF1R antibody heavy chain comprises at least one of the CDRs discussed herein. That is, in some embodiments, the anti-CSF1R antibody heavy chain is selected from the heavy chain CDR1, discussed herein, the heavy chain CDR2, discussed herein, and the heavy chain CDR3, discussed herein. Contains at least one CDR. Further, 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 against the CDRs discussed herein. Includes 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 suitable conservative amino acid substitutions for a particular CDR sequence, which significantly alters the binding properties of the heavy chain containing the mutated CDR. 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 an 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 Region In some embodiments, an anti-CSF1R antibody light chain variable region is provided. In some embodiments, the anti-CSF1R antibody light chain variable region is a murine 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 NOs: 18, 24, and 30.

  In some embodiments, the anti-CSF1R antibody light chain variable region comprises a 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 NOs: 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 A light chain variable region comprising, but not limited to, a set of CDR1, CDR2, and CDR3 is included.

  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 NOs: 10, 12, 14, and 46-52, An antibody 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, wherein the light chain together with the heavy chain binds CSF1R Can be formed.

  In some embodiments, the anti-CSF1R antibody light chain comprises at least one of the CDRs discussed herein. That is, in some embodiments, the anti-CSF1R antibody light chain is selected from the light chain CDR1, discussed herein, the light chain CDR2, discussed herein, and the light chain CDR3, discussed herein. Contains 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 against the CDRs discussed herein. Includes 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 suitable conservative amino acid substitutions for a particular CDR sequence, which significantly alters the binding properties of the light chain containing the mutated CDR. 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 κ and human λ 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 backbones such as anticalin, adnectin, ankyrin repeats, and the like. For example, Hosse et al. , Prot. Sci. 15:14 (2006), Fiedler, M .; and Skerra, A .; , "Non-Antibody Scaffolds," pp. 467-499 in Handbook of Therapeutic Antibodies, Dubel, S .; , Ed. , Wiley-VCH, Weinheim, Germany, 2007.

Exemplary Properties of Anti-CSF1R Antibodies In some embodiments, an antibody having the structure described above binds to CSF1R with a binding affinity (K _D ) of less than 1 nM and CSF1 and / or IL-34 to CSF1R. Blocks binding and suppresses CSF1 and / or IL-34-induced CSF1R phosphorylation.

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 ) for CSF1R of less than 1 nM, less than 0.5 nM, less than 0.1 nM, or less than 0.05 nM. 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. with a _{K D.}

  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 IL-34 binding 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. An 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 uses the assay described in Example 7 to at least 60%, at least 70%, at least 80%, or at least the amount of detectable binding of the ligand to CSF1R. Reduce by 90%. In some such embodiments, the antibody is said to block ligand binding by at least 50%, at least 60%, at least 70%, etc.

  In some embodiments, the anti-CSF1R antibody inhibits ligand-induced CSF1R phosphorylation. In some embodiments, the anti-CSF1R antibody inhibits CSF1-induced CSF1R phosphorylation. In some embodiments, the anti-CSF1R antibody inhibits IL-34-induced CSF1R phosphorylation. In some embodiments, the anti-CSF1R antibody inhibits both CSF1-induced and IL-34-induced CSF1R phosphorylation. An antibody "inhibits ligand-induced CSF1R phosphorylation if it reduces the amount of detectable ligand-induced CSF1R phosphorylation by at least 50% using the assay described in Example 6. Is considered. In some embodiments, the antibody uses the assay described in Example 6 to at least 60%, at least 70%, at least 80%, the amount of detectable ligand-induced CSF1R phosphorylation, Or reduce by at least 90%. In some such embodiments, the antibody is said to inhibit ligand-induced CSF1R phosphorylation by 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. An antibody uses the assay described in Example 10 if it inhibits the amount of monocyte proliferation and / or survival response in the presence of CSF1 and / or IL-34 by at least 50%. Inhibits monocyte proliferation and / or survival response ”. In some embodiments, the antibody uses the assay described in Example 10 to produce an amount of monocyte proliferation and / or survival response of at least 60 in the presence of CSF1 and / or IL-34. %, At least 70%, at least 80%, or at least 90%. In some such embodiments, the antibody is said to inhibit monocyte proliferation and / or survival response by at least 50%, at least 60%, at least 70%, etc.

Exemplary Antibody Conjugates In some embodiments, the 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 an antibody and / or facilitates detection of a molecule to which the antibody binds. Non-limiting exemplary labels include, but are not limited to, radioisotopes, fluorescent groups, enzyme groups, chemiluminescent groups, biotin, epitope tags, metal binding tags, and the like. A person 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 proliferative ability of one or more cells. A cell is proliferated because, for example, the cell undergoes apoptosis or otherwise dies, the cell cannot progress through the cell cycle, and / or cannot divide, the cell differentiates When it becomes difficult to do, it has a reduced ability to grow. Non-limiting exemplary cytotoxic agents include, but are not limited to, radioisotopes, toxins, and chemotherapeutic agents. One skilled in the art can select appropriate cytotoxicity according to the intended application.

  In some embodiments, in vitro chemical methods are used to conjugate the label and / or cytotoxic agent to the antibody. Exemplary chemical methods of non-limiting conjugation are known in the art, for example, Thermo Scientific Life Science Research Products (formerly Pierce, Rockford, IL), Prozyme (Hayward, CA), SACRI Antibic. (Calgary, Canada), AbD Serotec (Raleigh, NC), and other services, methods, and / or reagents that are commercially available. 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. Polypeptides can be produced that contain labels and / or cytotoxic agents. One skilled in the art can select an appropriate method for conjugating the label and / or cytotoxic agent to the antibody according to the intended application.

Exemplary leader sequences For some secreted proteins to be expressed and secreted in large quantities, leader sequences from heterologous proteins may be desirable. In some embodiments, the leader sequence is selected from SEQ ID NOs: 3 and 4, which are light and heavy chain leader sequences, respectively. In some embodiments, the use of a heterologous leader sequence can be advantageous in that the resulting mature polypeptide remains unchanged as the leader sequence is removed at the ER during the secretion process. The addition of a heterologous leader sequence may be necessary to express and secrete some proteins.

  The sequence of one exemplary leader sequence is described, for example, in the online leader sequence database maintained by Department of Biochemistry, National University of Singapore. Choo et al. , BMC Bioinformatics, 6: 249 (2005) and PCT Publication No. WO2006 / 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 that encodes the 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 an anti-CSF1R antibody and a polynucleotide encoding the light chain. In some embodiments, the first nucleic acid molecule includes a first polynucleotide that encodes a heavy chain, and the second nucleic acid molecule includes a second polynucleotide that encodes 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 an scFv, a single polynucleotide encodes a single polypeptide that includes 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 that, when translated, encodes a leader sequence located at the N-terminus of the heavy or light chain. 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 recombinant DNA technology conventional in the art. In some embodiments, the nucleic acid molecule is an expression vector suitable for expression in the selected host cell.

Expression and Production Vectors for Anti-CSF1R Antibodies Vectors comprising polynucleotides that encode anti-CSF1R heavy chains and / or anti-CSF1R light chains are provided. Also provided are vectors 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 an scFv.

  In some embodiments, the first vector includes a polynucleotide that encodes a heavy chain, and the second vector includes a polynucleotide that encodes 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, a first or second vector in a molar or mass ratio of 5: 1 to 1: 5 is transfected into a host cell. In some embodiments, for a vector encoding a heavy chain and a vector encoding a light chain, a mass ratio of 1: 1 to 1: 5 is used. In some embodiments, a mass ratio of 1: 2 is used for the vector encoding the heavy chain and the vector encoding the light chain.

  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, a vector is selected for in vivo expression of anti-CSF1R heavy chain and / or anti-CSF1R light chain in animals, including humans. In some such embodiments, polypeptide expression is under the control of a promoter that functions in a tissue-specific manner. For example, a liver-specific promoter is described in, for example, PCT Publication No. WO2006 / 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 a polypeptide include COS cells including COS7 cells, 293 cells including 293-6E cells, CHO cells including CHO-S and DG44 cells, PER. Including, but not limited to, C6® cells (Crucell), as well as NSO cells. In some embodiments, the anti-CSF1R heavy chain and / or anti-CSF1R light chain can be expressed in yeast. See, for example, US Patent Application Publication No. 2006/0270045 A1. In some embodiments, a particular eukaryotic host cell is selected based on its 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 having a higher level of sialylation than the same polypeptide produced in 293 cells.

Introduction of one or more nucleic acids into a desired host cell includes, but is not limited to, calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection, etc. It can be achieved by any method that is not. 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 antibody The anti-CSF1R antibody may be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrices or hydrophobic interaction chromatography. Suitable affinity ligands include those that bind the CSF1R ECD and antibody constant regions. For example, protein A, protein G, protein A / G, or antibody affinity columns may be used to bind the constant regions and purify the anti-CSF1R antibody. Hydrophobic interaction chromatography, such as butyl or phenyl columns, may also be suitable for purifying some polypeptides. Many methods for purifying polypeptides are known in the art.

Cell-free production of anti-CSF1R antibodies In some embodiments, anti-CSF1R antibodies are produced in cell-free systems. Non-limiting exemplary cell-free systems are described, for example, in Sitaraman et al. , Methods Mol. Biol. 498: 229-44 (2009), Spirin, Trends Biotechnol. 22: 538-45 (2004), Endo et al. Biotechnol. Adv. 21: 695-713 (2003).

Therapeutic Compositions and Methods Methods of Treating Diseases Using Anti-CSF1R Antibodies Using antibodies that bind CSF1R and block CSF1 and IL-34 ligand binding, systemic lupus erythematosus (SLE), rheumatoid arthritis, and Provided herein are methods for treating multiple sclerosis.

  In some embodiments, a method of treating SLE is 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-huAb16. Including the steps of: In some embodiments, treating SLE reduces the occurrence and / or severity of skin lesions associated with SLE, and / or glomerulonephritis, interstitial nephritis, and perivascular invasion. It means reducing the selected kidney condition. In some embodiments, a method of treating lupus nephritis is provided, wherein the method binds CSF1R to a subject having lupus nephritis and blocks CSF1 and IL-34 ligand binding, eg, administering an antibody selected from huAb1-huAb16. In some embodiments, there is provided a method of treating one type of lupus nephritis selected from type I, type II, type III, type IV, type V, and type 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-huAb16. Type III and IV lupus nephritis has a significantly higher number of CD16 + monocytes than other types of lupus nephritis. Yoshimoto et al. , Am. J. et al. Kidney Dis. 50: 47-58 (2007). Accordingly, in some embodiments, a method of treating type III or type IV lupus nephritis is provided, the method comprising binding CSF1R to a subject having lupus nephritis to bind CSF1 and IL-34 ligand. Administering an antibody that blocks, such as an antibody selected from huAb1-huAb16. In some such embodiments, the method comprises administering huAb1 to the subject.

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

  In some embodiments, a method of treating a renal condition associated with SLE is provided. In some such embodiments, the method is selected from an antibody that binds CSF1R and blocks CSF1 and IL-34 ligand binding to a subject having a renal condition associated with SLE, eg, huAb1-huAb16. Administering the antibody to be administered. In some embodiments, antibody huAb1 is administered to the subject. In some embodiments, the renal condition is selected from glomerulonephritis, interstitial nephritis, and perivascular invasion. In some embodiments, a method of treating lupus nephritis is provided, wherein the method binds CSF1R to a subject having lupus nephritis and blocks CSF1 and IL-34 ligand binding, eg, administering an antibody selected from huAb1-huAb16. In some embodiments, a method of treating lupus nephritis comprises administering antibody huAb1 to a subject having lupus nephritis. In some embodiments, the extent of kidney damage in lupus nephritis can be determined using ultrasonography, scintigraphy, pyelography, or needle biopsy. In some embodiments, the extent 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 of reducing proteinuria associated with lupus nephritis is provided, wherein the method binds CSF1R to a subject exhibiting proteinuria and blocks CSF1 and IL-34 ligand binding; For example, the method includes a step of administering an antibody selected from huAb1 to huAb16. Proteinuria can be detected by any method in the art including, but not limited to, dipstick test, protein / creatinine ratio, serum albumin / creatinine ratio, and protein electrophoresis. In some embodiments, protein levels in urine are traces (5-20 mg / dl), 1+ (30 mg / dl), 2+ (100 mg / dl), 3+ (300 mg / dl), and 4+ (> 2000 mg / dl). dl) can be used to indicate. A protein / creatinine ratio of less than 200 mg / g is considered normal. In men and women, serum albumin / creatinine ratios below 17 mg / g and 25 mg / g, respectively, are considered normal. Proteinuria is defined as the presence of total protein or albumin in excess of 300 mg / day in 24 hour urinary excretion, and the total protein content in excess of 30 mg / dL (ie, in the above class for the results of spot urine dipstick measurements). 1+ or more) as total protein / creatinine ratio above 200 mg / g, albumin / creatinine ratio above 300 mg / g for spot urine measurement results (or 250 mg / g for men for spot urine measurement results) As for the measurement result of albumin / creatinine ratio and spot urine exceeding 355 mg / g, the kidney kidney prognosis improvement measure (National Kidney Foundation Kidney Outcome) 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 has at least 1 proteinuria in the “trace, 1+, 2+, 3+, 4+” class. Decrease stage or at least two stages. 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 results in proteinuria below 2+ levels, or below 1+ levels. To decrease. In some embodiments, administration of an antibody that binds CSF1R and blocks CSF1 and IL-34 ligand binding to a subject has a protein / creatinine ratio and / or 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, administration of an antibody to a subject that binds CSF1R and blocks CSF1 and IL-34 ligand binding results in a protein / creatinine ratio and / or 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 to a subject that binds CSF1R and blocks CSF1 and IL-34 ligand binding results in a protein / creatinine ratio and / or albumin / creatinine ratio within two times the normal ratio, Decrease within 3 times, within 4 times, within 5 times. In some embodiments, the decrease in proteinuria is from the first week of treatment, the first two weeks, the first two months, the first one month, the first two months, the first three months, the first four months, or the first Judged at 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, a method is provided for increasing glomerular filtration rate in a subject having lupus nephritis, the method comprising binding CSF1R to a subject having lupus nephritis to bind CSF1 and IL. Administering an antibody that blocks -34 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 sinistrin (in some embodiments, insulin or sinistrin is injected prior to analysis), Includes, but is not limited to, 24 urine collections and creatinine clearance estimated from serum creatinine concentration, and serum cystatin C values. Normal glomerular filtration rate in adults is in the range of 90-130 ml / min / 1.73 m ² . Glomerular filtration rates of 90 ml / min / 1.73 m ² , 80 ml / min / 1.73 m ² , 70 ml / min / 1.73 m ² , or 60 ml / min / 1.73 m ² In embodiments, indicating chronic kidney disease, a rate of less than 15 ml / min / 1.73 m ² indicates renal failure. In some embodiments, a method is provided for increasing glomerular filtration rate in a subject having lupus nephritis, the method comprising binding CSF1R to a subject having lupus nephritis to bind CSF1 and IL. Administering an antibody that blocks -34 ligand binding, eg, an antibody selected from huAb1-huAb16. In some embodiments, the subject's glomerular filtration rate after 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 improvement. In some embodiments, the subject's glomerular filtration rate after 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 decrease in glomerular filtration rate is from the start of treatment, first week, first two weeks, first one month, first two months, first three months, first four months, or Judged in the first 6 months. In some embodiments, the subject is monitored for sustained improvement in glomerular filtration rate while being treated 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 binds CSF1R to a subject having lupus nephritis and blocks CSF1 and IL-34 ligand binding, eg, administering an antibody selected from huAb1-huAb16, wherein the administration of the antibody delays or prevents progression of a 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 particular time interval receiving treatment with the antibody (ie, proteinuria). Does not increase and / or glomerular filtration rate does not decrease). Treatment may be a single dose or multiple doses.

  In some embodiments, a method of treating rheumatoid arthritis is provided, the method comprising, in a subject having rheumatoid arthritis, an antibody that binds CSF1R and blocks CSF1 and IL-34 ligand binding, such as 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 affected by rheumatoid arthritis. In some embodiments, treating rheumatoid arthritis reduces inflammation, reduces pannus formation, reduces cartilage damage, reduces bone resorption, reduces the number of macrophages in the joint Means reducing autoantibody formation and / or reducing bone loss.

  In some embodiments, a method of reducing inflammation associated with rheumatoid arthritis is provided, wherein the method binds CSF1R to a subject having rheumatoid arthritis and blocks CSF1 and IL-34 ligand binding. Administering an antibody, eg, an antibody selected from huAb1-huAb16. In some embodiments, a method of reducing inflammation associated with rheumatoid arthritis is provided, the method comprising administering antibody huAb1 to a subject having rheumatoid arthritis. In some embodiments, reducing inflammation includes reducing erythrocyte sedimentation rate and / or reducing C-reactive protein levels in the blood. If inflammation is seen in a subject, the rate of erythrocyte sedimentation increases, probably due to increased levels of fibrinogen in the blood. The erythrocyte sedimentation rate may be determined by any method in the art, including calculating the rate by measuring the change in height of the 1 hour anticoagulated erythrocytes in the Westergen tube. However, it is not limited to this. Procedures for the Erythrocyte Sedimentation Rate Test; Appended 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 the RAPITEX® CRP test kit (Siemens).

  In some embodiments, reducing inflammation includes reducing peripheral edema, which is tissue swelling due to fluid accumulation. In some cases, peripheral edema can occur in the ankle, foot, leg, and / or calf of a subject with rheumatoid arthritis. In some embodiments, reducing inflammation includes reducing inflammatory cell infiltration of synovium of one or more affected joints. In some embodiments, synovial fluid can be collected by joint puncture.

  In some embodiments, a method of reducing pannus formation associated with rheumatoid arthritis is provided, wherein the method binds CSF1R to block CSF1 and IL-34 ligand binding to a subject having rheumatoid arthritis. Administering, for example, an antibody selected from huAb1-huAb16. In some embodiments, a method of reducing pannus formation associated with rheumatoid arthritis is provided, the method comprising administering an 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 hard tissue destruction 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) scan, arthroscopy, ultrasonography, duplex ultrasonography. , And power Doppler imaging.

  In some embodiments, a method of delaying the progression of pannus formation associated with rheumatoid arthritis is provided, wherein the method binds CSF1R to a subject having rheumatoid arthritis to bind CSF1 and IL-34 ligand. Administering an antibody that blocks, such as an antibody selected from huAb1-huAb16. In some embodiments, the antibody is huAb1. In some embodiments, the formation of pannus is delayed after administration of the antibody and / or during a particular time interval in which the subject is being treated with the antibody. Treatment may be a single dose or multiple doses.

  In some embodiments, a method of reducing cartilage damage associated with rheumatoid arthritis is provided, wherein the method binds CSF1R to block CSF1 and IL-34 ligand binding to a subject having rheumatoid arthritis. Administering, for example, an antibody selected from huAb1-huAb16. In some embodiments, a method of reducing cartilage damage associated with rheumatoid arthritis is provided, the method comprising administering an antibody huAb1 to a subject having rheumatoid arthritis. In some embodiments, reducing cartilage damage includes reducing chondrocyte 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, a method of delaying the progression of cartilage damage associated with rheumatoid arthritis is provided, wherein the method binds CSF1R to a subject having rheumatoid arthritis to bind CSF1 and IL-34 ligand. Administering an antibody that blocks, such as an antibody selected from huAb1-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 during a particular time interval in which the subject is being treated with the antibody. Treatment may be a single dose or multiple doses.

  In some embodiments, a method of reducing bone resorption associated with rheumatoid arthritis is provided, wherein the method binds CSF1R to block CSF1 and IL-34 ligand binding in a subject having rheumatoid arthritis. Administering, for example, an antibody selected from huAb1-huAb16. In some embodiments, a method of reducing bone resorption associated with rheumatoid arthritis is provided, the method comprising administering the antibody huAb1 to a subject having rheumatoid arthritis. In some embodiments, reducing bone resorption includes reducing the number of osteoclasts in a joint affected by rheumatoid arthritis.

  In some embodiments, bone resorption can be measured by determining the level of TRAP5b in plasma from the subject, and an increase in the level of TRAP5b indicates an increase in bone resorption in the subject. Thus, in some embodiments, a decrease in the level of TRAP5b indicates a decrease in bone resorption. TRAP5b levels can optionally be determined before and after treatment with an antibody that binds CSF1R, and / or periodically throughout the course of therapy monitoring the effectiveness of the treatment in reducing bone loss. . TRAP5b levels can be determined using any method in the art, including ELISA (FAICEA, or immunocapture enzyme assays in which fragments are absorbed, eg, Quidel® TRAP5b assay Method, TEC Medical Group, Sissach, Switzerland)), but is not limited thereto.

  In some embodiments, a method of reducing bone loss associated with rheumatoid arthritis is provided, wherein the method binds CSF1R to block CSF1 and IL-34 ligand binding in a subject having rheumatoid arthritis. Administering, for example, an antibody selected from huAb1-huAb16. In some embodiments, a method of reducing bone loss associated with rheumatoid arthritis is provided, 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 absorption measurements (SPA, DPA) ), Single and dual energy X-ray absorptiometry (SXA, DXA), ultrasonography, scintigraphy, and by measuring the level of serum markers of bone formation and resorption, etc. It is not limited to. Non-limiting example serum markers for bone formation and bone resorption are shown in Table 2.

Table 2 Serum markers for bone formation and resorption

  In some embodiments, a method of delaying the progression of bone loss associated with rheumatoid arthritis is provided, wherein the method binds CSF1R to a subject having rheumatoid arthritis to bind CSF1 and IL-34 ligand. Administering an antibody that blocks, such as an antibody selected from huAb1-huAb16. In some embodiments, the antibody is huAb1. In some embodiments, bone loss is delayed after administration of the antibody and / or during a particular time interval in which the subject is being treated with the antibody. Treatment may be a single dose or multiple doses.

  In some embodiments, provided is a method of reducing the number of monocyte cells, eg, macrophages and / or CD16 + monocytes, in a joint affected with rheumatoid arthritis, wherein the method comprises a subject with rheumatoid arthritis And administering an antibody that binds CSF1R and blocks CSF1 and IL-34 ligand binding, eg, an antibody selected from huAb1-huAb16. In some embodiments, provided is a method of reducing the number of monocyte cells, eg, macrophages and / or CD16 + monocytes, in a joint affected with rheumatoid arthritis, wherein the method comprises a subject with rheumatoid arthritis The step of administering antibody huAb1. In some embodiments, there is provided a method of reducing the number of monocytic cells, such as macrophages and / or CD16 + monocytes, in the synovial fluid of joints affected by rheumatoid arthritis, the method comprising rheumatoid arthritis. Administering to the subject an antibody that binds CSF1R and blocks CSF1 and IL-34 ligand binding, eg, an antibody selected from huAb1-huAb16. In some embodiments, there is provided a method of reducing the number of monocytic cells, such as macrophages and / or CD16 + monocytes, in the synovial fluid of joints affected by rheumatoid arthritis, the method comprising rheumatoid arthritis. Administering the antibody huAb1 to the subject.

  In some embodiments, a method of reducing autoantibody levels in a subject having rheumatoid arthritis is provided, wherein the method binds CSF1R to a subject having rheumatoid arthritis to bind CSF1 and IL- Administering an antibody that blocks 34 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 an antibody huAb1 to a subject having rheumatoid arthritis.

  In some embodiments, a method of reducing autoantibody levels in a subject having rheumatoid arthritis is provided, wherein the method binds CSF1R to a subject having rheumatoid arthritis to bind CSF1 and IL- Administering an antibody that blocks 34 ligand binding, eg, an antibody selected from huAb1-huAb16. In some such embodiments, the method comprises administering huAb1 to a subject having rheumatoid arthritis. The level 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 anti-nuclear antibody (ANA).

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

  In some embodiments, a method of reducing CD16 + monocytes is provided, wherein the method binds CSF1R to a subject having an increase in CD16 + monocytes and blocks CSF1 and IL-34 ligand binding, such as Administering an antibody selected from huAb1-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 decrease 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, the anti-CSF1R antibody is oral, intraarterial, parenteral, intranasal, intramuscular, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, intradermal, It can be administered in vivo by various routes, including but not limited to, topical, transdermal, and intrathecal, or by implantation or inhalation. The composition may be formulated into a preparation in the form of a solid, semi-solid, liquid, or gas, including tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections Including, but not limited to, agents, inhalants, and aerosols. Nucleic acid molecules encoding anti-CSF1R antibodies can be coated onto gold microparticles and particles 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 administration routes can be selected according to the intended use.

In various embodiments, a composition comprising an anti-CSF1R antibody is provided in a formulation comprising various pharmaceutically acceptable carriers (eg, Gennaro, Remington: The Science and Practice of Pharmaceuticals 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)). A variety of pharmaceutically acceptable carriers can be used including vehicles, adjuvants, and diluents. In addition, various pharmaceutically acceptable auxiliary substances such as pH adjusting and buffering agents, isotonicity adjusting agents, 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 an anti-CSF1R antibody can be aqueous or optionally with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers, and preservatives. Formulated for injection including subcutaneous administration by dissolving, suspending or emulsifying them in non-aqueous solvents such as vegetable or other oils, synthetic fatty acid glycerides, esters of higher fatty alcohols, or propylene glycol Can be done. In various embodiments, the composition can be formulated for inhalation using, for example, a pressurizable propellant such as dichlorodifluoromethane, propane, nitrogen. The composition can also be formulated in sustained release microcapsules in various embodiments, for example, with biodegradable or non-degradable polymers. Non-limiting exemplary biodegradable formulations include polylactic acid-glycolic acid polymers. Non-limiting exemplary non-biodegradable formulations include polyglycerol fatty acid esters. One particular method of making such a formulation is 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 antibody and comprising one or more containers. In some embodiments, a unit dose is provided, the unit dose containing a composition comprising a predetermined amount of an anti-CSF1R antibody, with or without one or more additives. In some embodiments, such unit dose is provided in a disposable prefilled syringe for injection. In various embodiments, the composition contained in a unit dose can include a buffer, such as saline, sucrose, phosphate, and / or can be formulated within a stable and effective pH range. . Alternatively, in some embodiments, the composition can be provided as a lyophilized powder that can be reconstituted upon addition of a suitable liquid, eg, 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 composition of the invention comprises heparin and / or proteoglucan.

  The pharmaceutical composition is administered in an amount effective to treat or prevent a particular indication. A therapeutically effective amount typically varies depending on the weight of the subject being treated, the subject's body or health, the extent of the condition being treated, or the age of the subject being treated. In general, anti-CSF1R 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, the anti-CSF1R antibody 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, the anti-CSF1R antibody 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, the anti-CSF1R antibody 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, the anti-CSF1R antibody can be administered in an amount ranging from about 0.5 mg / kg body weight to about 20 mg / kg body weight per dose.

  The anti-CSF1R antibody composition can be administered to the subject as needed. The frequency of administration should be determined by those skilled in the art, such as the attending physician, taking into account the condition being treated, the age of the subject being treated, the severity of the condition being treated, the general health of the subject being treated, etc. Can be done. In some embodiments, an effective amount of an anti-CSF1R antibody is administered to the subject one or more times. In various embodiments, an effective amount of an anti-CSF1R antibody is administered to a subject once a month, less than once a month, eg, once every two months, or once every three months. In other embodiments, an effective amount of an anti-CSF1R antibody is administered more than once a month, eg, once every three weeks, once every two weeks, or weekly. An effective amount of an anti-CSF1R antibody is administered to the subject at least once. In some embodiments, an effective amount of an anti-CSF1R antibody may be administered multiple times over at least 1 month, at least 6 months, or at least 1 year.

Combination Therapy Anti-CSF1R antibodies may be administered alone or in conjunction with other treatment modalities. These can be provided, for example, prior to, substantially simultaneously with, or after other treatment modalities such as surgery, chemotherapy, radiation therapy, or administration of a biologic such as another therapeutic antibody. For the treatment of rheumatoid arthritis, anti-CSF1R antibodies may be used with other therapeutic agents such as methotrexate, anti-TNF agents such as Remicade (infliximab), Humira (adalimumab), Simponi (golimumab), and Enbrel (etanercept), prednisone. Such as glucocorticoid, leflunomide, azothiopurine, JAK inhibitor such as CP590690, SYK inhibitor such as R788, anti-IL-6 antibody, anti-IL-6R antibody such as tocilizumab, anti-CD-20 antibody such as rituximab, anti-CD19 antibody , 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, and an immunosuppressive agent such as cyclosporine.

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

  For the treatment of multiple sclerosis, anti-CSF1R antibodies may be used as other therapeutic agents, for example, anti-alpha4 integrin antibodies such as interferon alpha, interferon beta, prednisone, Tysabri, anti-CD20 antibodies such as Rituxan, FTY720 (fingolimod). And Cladribine (Leustatin).

  The examples discussed below are purely illustrative of the invention and should not be construed as limiting the invention in any way. This example is not intended to indicate that the following experiment is all or the only experiment performed. Efforts have been made to ensure accuracy with respect to numbers used (eg amounts, temperature, etc.) but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

Example 1: Humanized anti-CSF1R antibodies Various humanized anti-CSF1R antibodies have been previously developed. For example, see PCT Publication No. WO2011 / 140249.

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

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

Table 3 Humanized heavy and light chain of huAb1-huAb16

  Sixteen humanized antibodies were tested for binding to human, cynomolgus monkey, and mouse CSF1R ECD as described above. For example, see PCT Publication No. WO2011 / 140249. The antibody was found to bind to both human and cynomolgus CSF1R ECD but not to mouse CSF1R ECD. Humanized antibodies also block CSF1 and IL-34 binding to both human and mouse CSF1R, and inhibit CSF1-induced and IL-34-induced human CSF1R phosphorylation expressed in CHO cells It was also found to do. For example, see PCT Publication No. WO2011 / 140249.

_K a, _{k d,} and _{K D} for binding to human CSFlR ECD is predetermined, it is shown in Table 4. For example, see PCT Publication No. WO2011 / 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 of huAb1 in cynomolgus monkeys ( 2 monkeys per group) were administered intravenously. Peripheral blood was collected after 14 days and the number of CD16 + or CD16− monocytes was analyzed by flow cytometry. 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. Red blood cells were lysed by incubation with 2 ml 1X BD Pharmlyse (Becton-Dickenson) for 20 minutes and cells were washed with 1 ml FACS buffer (PBS / 0.1% BSA) prior to analysis. Data was collected on an LSRII flow cytometer using the Diva software program (Becton-Dickenson) and the results analyzed using the FloJo data analysis program (Tree Star).

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

Example 3: Characterization of surrogate anti-mouse CSF1R antibody Since the humanized antibodies huAb1-huAb16 do not bind to mouse CSF1R, a chimeric anti-mouse CSF1R antibody ("cAb1") was selected as the surrogate antibody for use in the mouse model. It was. This antibody comprises a rat variable region and a mouse constant region. To generate cAb1, hybridomas that immunize rats with mouse CSF1R-ECD-Fc fusion protein and secrete antibodies that block the binding of both mouse CSF1 and mouse IL-34 to mouse CSF1R are obtained by ELISA assay. Identified. The variable region of one such blocking antibody is obtained using standard molecular biology techniques (Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Third Edition, CSH Press, Cold Spring Harbor, New York, 2001). And grafted onto the 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. A 96 well clear bottom ELISA plate was coated overnight with 1 μg / ml recombinant mouse CSF1R ECD-Fc chimera (R & D Systems) in PBS. The next morning, the wells were washed 4 times with 0.05% Tween 20 in PBS (PBST) and blocked with Blocker-Blotto (Pierce). Starting from a concentration of 2.6 μg / ml, 50 μl of a 0.5-fold serial dilution of cAb1 diluted 1: 1 in Blocker-Blotto was added to wells coated with CSF1R. After 90 minutes incubation 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 60 minutes incubation at room temperature, the wells were washed 4 times with PBST and 50 μl o-phenylenediamine dihydrochloride peroxider substrate (Sigma) was added to each well. After 30 minutes incubation at room temperature, the A450 value of each well was read directly on a SpectraMaxPlus 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 Growth of Factor-Dependent Cell Lines cAb1 was tested for its ability to block the growth of 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 commercially 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 each individual culture was determined using Cell Titer Glo reagent (Promega) according to the manufacturer's instructions. In this assay, the relative cellular ATP content is directly proportional to the number of viable cells in the culture and thus reflects the proliferative response of mNFS60 cells.

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

Example 5: cAb1 Suppresses Clinical Disease Score in a Mouse Model of Rheumatoid Arthritis In Male DBA / 1 Mice, 2 mg / ml in 150 μl Complete Freund's Adjuvant (Sigma) on Study Day 0 and again on Study Day 21 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 was initiated. Mice were dosed intraperitoneally with vehicle or cAb1 (3, 6, 10, or 30 mg / kg) three times a week (3 times / week) until study day 32, studies days 18-35 In addition, the clinical disease score for each paw was measured. Clinical disease scores were assigned as follows:
0 = normal.
1 = one hind or forefoot joint affected or minimal diffuse erythema and swelling.
2 = Two hind or forefoot joints were affected or mild diffuse erythema and swelling.
3 = 3 hind or forefoot joints affected or moderate diffuse erythema and swelling.
4 = 4 hind 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 the experiment. cAb1 suppressed the clinical disease score in a collagen-induced arthritis model of rheumatoid arthritis. These data suggest that antibodies that bind CSF1R and block ligand binding, such as huAb1, in human patients may be effective in reducing the signs and symptoms of rheumatoid arthritis.

Example 6: cAb1 Suppresses Bone Loss in a Mouse Model of Rheumatoid Arthritis High levels of tartrate-resistant acid phosphatase 5b (TRAP5b) are associated with bone remodeling and are characterized by increased bone resorption Has been observed. Thus, TRAP5b levels can be used as a marker for bone resorption (ie, bone loss).

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

  FIG. 7 shows the results of the experiment. cAb1 inhibited bone loss in the arthritis mode induced by collagen of rheumatoid arthritis, as measured by inhibition of TRAP5b bone turnover marker. These data suggest that antibodies, such as huAb1, that bind CSF1R and block ligand binding in human patients can suppress bone erosion and joint destruction in rheumatoid arthritis.

Example 7: cAb1 Suppresses Inflammation, Cartilage Injury, Pannus Formation, and Bone Destruction in a Mouse Model of Rheumatoid Arthritis Inducing Arthritis in Mice and Treating Mice with cAb1 as described in Example 5 . At the end of the study, the feet and knees were 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 histopathological evaluation. These joints were then scored as follows.
Inflammation 0 = normal.
1 = Minimal infiltration of inflammatory cells in the synovium and periarticular tissue of the affected joint.
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 3 to 4 affected joints, including at least one wrist or ankle joint.
4 = There may be significant invasion affecting most 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 invasion of pannus in cartilage and subchondral bone, marginal zone.
2 = Mild infiltration with hard tissue marginal zone destruction in affected joints.
3 = moderate infiltration with moderate hard tissue destruction in affected joints.
4 = Significant infiltration with significant destruction of joint structure, affecting most joints.
5 = Severe infiltration with destruction of the entire joint structure or nearly the whole, affecting all joints.
Cartilage damage 0 = normal.
1 = minimal = approximately minimal to mild loss of toluidine blue staining without obvious chondrocyte loss or collagen disruption in affected joints.
2 = Mild = Generally mild loss of toluidine blue staining with focal cartilage loss and / or collagen disruption in some affected joints.
3 = moderate = multifocal chondrocyte loss in affected joints and / or generally moderate loss of toluidine blue staining with collagen disruption, some matrices may have any affected area with severe matrix loss area Stay on the surface.
4 = prominent = when all or nearly all of the cartilage is lost on one surface of the knee, with multifocal prominent (up to deep depth) chondrocyte loss and / or collagen collapse in most joints Significant loss of toluidine blue staining.
5 = severe = multifocal severe (up to tide mark depth) chondrocytes in all joints if all or nearly all of the cartilage is lost on more than one surface of the knee Severe diffuse loss of toluidine blue staining with loss and / or collagen breakdown.
Bone resorption 0 = normal.
1 = minimum = small area resorption, osteoclasts in affected joints, which are not readily apparent at low magnification, are rare and limited to the marginal zone.
2 = Mild = Resorption of a larger number of areas, limited to a greater number of osteoclasts in the affected joint, marginal zone, not readily apparent at low magnification.
3 = moderate = apparent resorption of medullary and cortical bone without loss of full thickness in the cortex, loss of some medullary trabeculae, apparent lesions at low magnification, more in affected joints Osteoclasts.
4 = Prominent = Defects in all layers within cortical bone, often affecting significant loss of medullary bone, deformed profiles remaining on the cortical surface, numerous osteoclasts, and most joints.
5 = severe = depletion of all layers in cortical bone and destruction of the joint structure of all joints.
The sum of the histological 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 rheumatoid arthritis collagen-induced arthritis model.

Example 8 cAb1 Inhibits Ankle and Knee Macrophage Counts in a Mouse Model of Rheumatoid Arthritis Induction of Arthritis in Mice and Treatment of Mice with cAb1 as described in Example 5 Foot and knee joints were prepared as described in Macrophage numbers were 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 were performed by Histox 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 macrophage numbers in both the foot and knee joints in an arthritis model induced by rheumatoid arthritis collagen.

Example 9: cAb1 Suppresses Autoantibody Formation in a Rheumatoid Arthritis Mouse Model Arthritis was induced in mice 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 antibodies were measured using a mouse anti-type 1 and type 2 collagen IgG assay kit (Chondrex) according to the manufacturer's instructions.

  The experimental results are shown in FIG. cAb1 suppressed the formation of anti-mouse type II collagen autoantibodies in the arthritic mode induced by rheumatoid arthritis collagen. These data suggest that in human patients, antibodies that bind CSF1R and block ligand binding, such as huAb1, can reduce an important trigger of joint inflammation, ie, autoantibodies. Further, 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 Induced arthritis in mice as described in Example 5. When the clinical disease score reached 0.05-1.0, mice were treated intraperitoneally 3 times / week starting with 30 mg / kg cAb1. 23 days after initial treatment with cAb1, blood was collected and commercially available solid-phase immunofixing enzyme activity for determination of osteoclast-derived TRAP5b in mouse serum (Immunodiagnostics Systems) according to manufacturer's instructions An assay was used to measure plasma levels of TRAP5b (tartrate resistant acid phosphatase 5b). TRAP5b is a bone turnover marker.

  The experimental results are shown in FIG. CAb1 administered after the development of the clinical disease score was in joint mode induced by rheumatoid arthritis collagen as measured by suppression of TRAP5b bone turnover marker and suppressed bone loss. These data suggest that antibodies, such as huAb1, that bind CSF1R and block ligand binding in human patients can suppress bone erosion and joint destruction in rheumatoid arthritis.

Example 11 cAb1 Inhibits Pannus Formation and Bone Destruction in an Established Rheumatoid Arthritis Mouse Model As described in Example 10, arthritis was induced in mice and mice were treated with cAb1. At the end of the study, the feet and knees were collected and 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 the histological 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 the development of the clinical disease score suppressed pannus formation and bone destruction in a rheumatoid arthritis collagen-induced arthritis model. The histological scores of inflammation and cartilage damage were not affected. These data also suggest that antibodies, such as huAb1, that bind CSF1R and block ligand binding in human patients can suppress 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 develops lupus nephritis, which It can cause inflammation of more than one structure, resulting in renal failure. Lupus nephritis can be detected in a patient and the severity of the disease is measured, for example, measuring proteinuria, or urinary protein, and / or 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 renal biopsy testing or through imaging techniques such as ultrasonography (Hahn et al., Arthr. Care Res., 2012, 64: 797-808). .

The MRL / MpJ-Fas ^lpr / J strain of mice spontaneously develops lupus-like symptoms including glomerular, tubulointerstitial, and perivascular renal disease, lymph node swelling, splenomegaly, circulating autoantibodies, and skin lesions Develops. Female or 50 mg / kg cAb1 were intraperitoneally administered daily for 10 weeks to 10 to 20 week old female MRL / MpJ-Fas ^lpr / J mice. At the end of the study (mouse 20 weeks of age), kidneys were collected, weighed and placed in 10% neutral buffered formalin (NBF) for histological evaluation. The kidney was 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 area.
4 = prominent diffuse growth with crescent / sclerosis, multifocal protein casts present.
5 = severe crescent / severe diffuse growth with sclerosis, diffuse protein cast is present.
Protein cast severity 0 = normal tubule.
1 = minimal = profile of 1-5 tubules with protein casts.
2 = mild = multifocal tubules with protein casts (6-20).
3 = medium = multifocal tubules with protein casts (21-40).
4 = prominent = multifocal tubule with protein cast (over 40 of all tubules but suffering less than 50%).
5 = severe = diffuse tubule with protein cast, more than 50% of all tubules affected.
Interstitial nephritis (inflammation not clearly associated with blood vessels)
0 = normal.
0.5 = ultra-minimal = small focal area of MNC with pelvic only.
1 = minimal = small amounts of focal MNC may accumulate, affect less than 10% of the total stroma, and are generally localized around the pelvis.
2 = Mild = Multifocal small to large infiltrations distributed around the pelvis and cortex: affects 10-25% of cortical areas.
3 = moderate = multifocal small to widespread infiltration in the pelvis and cortex: affects 26-50% of cortical areas.
4 = prominent = multifocal to diffuse infiltration: affects 51-75% of pelvic and cortical areas.
5 = severe = diffuse infiltration: affects 76-100% of pelvic and cortical areas.
Blood vessel 0 = normal.
0.5 = ultra-minimum = one blood vessel has minimal perivascular invasion.
1 = minimal = small but defines perivascular invasion (1-2).
2 = Mild = Several (3-4) foci of perivascular invasion but no necrosis.
3 = Moderate = Multifocal (5-6) lesions with perivascular invasion may be more extensive and have some necrosis of the vessel wall.
4 = Remarkable = Multifocal (7-8) lesions with perivascular invasion are extensive with necrosis.
5 = severe = multifocal (> 8) lesions with perivascular invasion are extensive with necrosis.

  The experimental results are shown in FIG. cAb1 suppressed glomerulonephritis, interstitial nephritis, and perivascular invasion in the MRL / lupus lpr model. The histological score of protein cast severity was not affected. These data suggest that antibodies that bind CSF1R and block ligand binding, such as huAb1, in human patients can reduce renal inflammation associated with systemic lupus erythematosus and restore renal function. ing.

Example 13: cAb1 inhibits 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. Mice between 10 and 20 weeks of age were scored for the presence and severity of skin lesions. Skin lesions were scored as follows.
0 = None.
1 = mild (mouth and ear).
2 = Moderate (less than 2 cm mouth, ear, and scapula).
3 = severe (over 2 cm mouth, ear, and scapula).

  The experimental results are shown in FIG. cAb1 suppressed the occurrence and severity of skin lesions in the MRL / lupus lpr model. These results suggest that antibodies that bind CSF1R and block ligand binding, such as huAb1, in human patients may reduce systemic symptoms of SLE, including skin inflammation, rash, and discoid lesions. doing.

Example 14: cAb1 Suppresses Clinical Disease Score in a Mouse Model of Multiple Sclerosis Experimental autoimmune encephalitis (EAE) was observed at 300 ng in complete Freund's adjuvant on study day 0 and again on study day 7. Of myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55, Sigma-Aldrich, catalog number # M4939) was induced in female C57B1 / 6 mice. To enhance the induction and consistency of EAE, 500 ng pertussis toxin (List Biological Laboratories, catalog number # 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 was initiated. Mice were administered ip or vehicle or 30 mg / kg cAb1 three times a week (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 = the tail is partially dragged.
1.0 = Tail is paralyzed.
2.0 = Loss of coordination, hindlimb paralysis.
2.5 = One hind limb was paralyzed.
3.0 = Both hind limbs were paralyzed.
3.5 = The hind limbs are paralyzed and the fore limbs become fragile.
4.0 = The forelimbs and hind limbs were paralyzed.
5.0 = drowning.

  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 injury Suggests that the paralysis caused by can be reversed.

Sequence Listing Table 5 provides 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), wherein the antibody is directed to CSF1R. Blocking colony-stimulating factor 1 (CSF1) binding and blocking IL-34 binding to CSF1R and treating conditions associated with rheumatoid arthritis reduce inflammation, pannus formation Reducing, reducing cartilage damage, reducing bone resorption, reducing the number of macrophages in at least one joint affected by rheumatoid arthritis, reducing autoantibody levels, and reducing bone loss A method comprising at least one effect selected from:   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 is reduced by less than 20%.   3. The method of claim 2, further comprising determining an erythrocyte sedimentation rate, wherein the decrease in sedimentation rate indicates a decrease in inflammation.   The treatment 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 is tartrate-resistant acid phosphatase 5b (TRAP5b), urinary total pyridinoline, urinary total deoxypyridinoline, urinary free pyridinoline, serum type I collagen cross-linked N-telopeptide, urinary type I collagen 9. The method of claim 8, wherein the method is selected from a cross-linked N-telopeptide and a carboxy-terminal telopeptide 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.   The method of claim 10, wherein the at least one effect is measured using an imaging technique.   The imaging techniques include X-ray imaging, magnetic resonance imaging, computed tomography (CT) scanning, arthroscopy, scintigraphy, ultrasonography, bone density measurement, single photon absorption measurement (SPA). ), Double photon absorption measurement (DPA), single energy X-ray absorption measurement (SXA), dual energy X-ray absorption measurement (DXA), scintigraphy, ultrasonic inspection, duplex ultrasonic inspection, 12. The method of 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 wherein the binding of IL-34 is blocked and the antibody reduces the number of CD16 + monocytes in the subject by at least 30% and CD16- monocytes are not reduced or less than 20%.   Methotrexate, anti-TNF substance, glucocorticoid, cyclosporine, 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 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. The method according to item.   A method of treating a skin lesion associated with lupus comprising administering to a subject having lupus an antibody that binds CSF1R, wherein the antibody blocks binding of CSF1 to CSF1R, and Blocking IL-34 binding to CSF1R and treating skin lesions associated with lupus reduce the number of skin lesions, reduce the rate of formation of skin lesions, and 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 the binding of IL-34.   The method of claim 16, wherein renal function is improved in the subject.   18. A method according to claim 16 or claim 17, wherein proteinuria is reduced in the subject.   19. A method according to any one of claims 16-18, wherein glomerular filtration rate is improved in the subject.   A method of treating lupus comprising administering to a subject having lupus an antibody that binds CSF1R, wherein the antibody blocks binding of CSF1 to CSF1R and IL- to CSF1R. 34. The method blocks 34 binding and the antibody reduces the number of CD16 + monocytes by at least 30% and the CD16− monocytes are not decreased or decreased by less than 20% in the subject.   21. The method of claim 20, wherein CD16 + monocytes are reduced by at least 50%.   Selected from hydroxychloroquine (Plaquenil), corticosteroids, cyclophosphamide (Cytoxan), azathioprine (Imuran, Azasan), mycophenolate (Cellcept), leflunomide (Arava), and methotrexate (Trexall) and belimumab (Benlysta) 23. 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 CSF1 binding to CSF1R and to CSF1R. A method wherein the binding of IL-34 is blocked and the antibody reduces the number of CD16 + monocytes by at least 30% and the CD16− monocytes are not reduced or less than 20%.   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 a subject having a CD16 + disorder an antibody that binds CSF1R, wherein the antibody blocks CSF1 binding to CSF1R and to CSF1R. A method wherein the binding of IL-34 is blocked and the antibody reduces the number of CD16 + monocytes by at least 30% and the CD16− monocytes are not reduced or less than 20%.   26. The method of claim 25, wherein CD16 + monocytes are reduced by at least 50%.   27. The method of any one of claims 20 to 26, wherein the lupus, the inflammatory condition, or the CD16 + disorder does not respond to methotrexate.   A method of reducing the number of CD16 + monocytes, comprising administering to a subject an antibody that binds CSF1R, said antibody blocking CSF1 binding to CSF1R and IL-34 to CSF1R. A method wherein the binding is blocked and the antibody reduces the number of CD16 + monocytes by at least 30% and CD16− monocytes are not reduced or less than 20%.   29. The method of claim 28, wherein CD16 + monocytes are reduced by at least 50%.   30. The method according to any one of claims 20 to 29, wherein the CD16 + monocytes are CD16 + peripheral blood monocytes.   A method of delaying progression of a renal condition associated with lupus comprising administering to a subject an antibody that binds CSF1R, wherein the antibody blocks CSF1 binding to CSF1R and IL to CSF1R A method of blocking the binding of -34.   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 whom the antibody is not administered.   33. The method of claim 31 or claim 32, wherein glomerular filtration rate does not decrease in the subject or does not decrease in the subject at the same rate as a subject to whom the antibody is not administered.   A method of delaying the progression of pannus formation in a subject having rheumatoid arthritis, comprising administering to the subject having rheumatoid arthritis an antibody that binds colony stimulating factor 1 receptor (CSF1R). The antibody blocks colony stimulating factor 1 (CSF1) binding to CSF1R and blocks IL-34 binding to CSF1R.   A method of delaying the progression of bone loss in a subject having rheumatoid arthritis, comprising administering to the subject having rheumatoid arthritis an antibody that binds a colony stimulating factor 1 receptor (CSF1R). The antibody blocks colony stimulating factor 1 (CSF1) binding to CSF1R and blocks IL-34 binding to CSF1R. The 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) a heavy chain comprising a heavy chain (HC) CDR1 having the sequence of SEQ ID NO: 15, an HC CDR2 having the sequence of SEQ ID NO: 16, and an HC CDR3 having the sequence of SEQ ID NO: 17; An antibody comprising a light chain (LC) CDR1 having a sequence of ID NO: 18, an LC CDR2 having a sequence of SEQ ID NO: 19, and a light chain comprising LC CDR3 having a sequence of SEQ ID NO: 20, and
36. (c) The antibody according to any one of claims 1 to 35, which is selected from an antibody 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.   38. The method of claim 36, wherein the antibody is a humanized antibody. 38. The method of claim 36 or claim 37, wherein the antibody is selected from Fab, Fv, scFv, Fab ', and (Fab') ₂ .

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