Combination therapy of antibodies to human CSF-1R and antibodies to human PD-L1

Abstract

The present invention relates to combination therapy of specific antibodies that bind to human CSF-1R and specific antibodies that bind to human PD-L1. Wherein the antibody is administered in combination with an antibody that binds to human PD-L1 for treating cancer, for preventing or treating metastasis, for treating inflammatory diseases, for treating bone loss, for treating immune-related diseases such as tumor immunity or delaying the progression thereof, or for stimulating an immune response or function such as T cell activity.

Description

[0001] The present application is a divisional application of an invention application filed on September 11, 2014, with Chinese application number 201480045288.2 and invention name “Combination therapy of antibodies against human CSF-1R and antibodies against human PD-L1”. Technical Field

[0002] The present invention relates to a combination therapy of a specific antibody that binds to human CSF-1R and a specific antibody that binds to human PD-L1. Background Art

[0003] CSF-1R and CSF-1R antibodies

[0004] The human CSF-1 receptor (CSF-1R; colony stimulating factor 1 receptor; synonyms: M-CSF receptor; macrophage colony stimulating factor 1 receptor, Fms proto-oncogene, c-fms, SEQ ID NO: 62) has been known since 1986 (Coussens, L. et al., Nature 320 (1986) 277-280). CSF-1R is a growth factor and is encoded by the c-fms proto-oncogene (for review, see, e.g., Roth, P. and Stanley, ER, Curr. Top. Microbiol. Immunol. 181 (1992) 141-167).

[0005] CSF-1R is the receptor for CSF-1 (colony stimulating factor 1, also known as M-CSF, macrophage colony stimulating factor) and mediates the biological effects of this cytokine (Sherr, CJ et al., Cell 41 (1985) 665-676). The cloning of the colony stimulating factor-1 receptor (CSF-1R), also known as c-fms, was first described by Roussel, MF et al., Nature 325 (1987) 549-552. In this publication, it was shown that CSF-1R has a transforming potential that depends on changes in the C-terminal tail of the protein, including the loss of inhibitory tyrosine 969 phosphorylation, which binds Cbl and thereby mediates receptor downregulation (Lee, PS et al., Embo J. 18 (1999) 3616-3628). Recently, a second ligand for CSF-1R was identified, termed interleukin-34 (IL-34) (Lin, H. et al., Science 320 (2008) 807-811).

[0006] Currently, two CSF-1R ligands are known that bind to the extracellular domain of CSF-1R. The first is CSF-1 (colony stimulating factor 1, also known as M-CSF, macrophage; SEQ ID NO: 86), which is found as a disulfide-linked homodimer outside the cell (Stanley, ER et al., Journal of Cellular Biochemistry 21 (1983) 151-159; Stanley, ER et al., Stem Cells 12 Suppl. 1 (1995) 15-24). The second is IL-34 (human IL-34; SEQ ID NO: 87) (Hume, DA et al., Blood 119 (2012) 1810-1820). The main biological effects of CSF-1R signaling are differentiation, proliferation, migration, and survival of hematopoietic precursor cells to the macrophage lineage (including osteoclasts). The activation of CSF-1R is mediated by its CSF-1R ligands CSF-1 (M-CSF) and IL-34. The binding of CSF-1 (M-CSF) to CSF-1R induces the formation of homodimers and the activation of the kinase through tyrosine phosphorylation (Li, W. et al., EMBO Journal. 10 (1991) 277-288; Stanley, ER et al., Mol. Reprod. Dev. 46 (1997) 4-10).

[0007] Biologically active homodimeric CSF-1 binds to CSF-1R within subdomains D1 to D3 of the CSF-1 receptor extracellular domain (CSF-1R-ECD). CSF-1R-ECD contains five immunoglobulin-like subdomains (designated D1 to D5). Subdomains D4 to D5 of the extracellular domain (CSF-1R-ECD) are not involved in CSF-1 binding (Wang, Z. et al., Molecular and Cellular Biology 13 (1993) 5348-5359). Subdomain D4 is involved in dimerization (Yeung, YG. et al., Molecular & Cellular Proteomics 2 (2003) 1143-1155; Pixley, FJ et al., Trends Cell Biol 14 (2004) 628-638).

[0008] Additional signaling is mediated by the p85 subunit of PI3K and Grb2, which connect to the PI3K / AKT and Ras / MAPK pathways, respectively. These two important signaling pathways regulate proliferation, survival, and apoptosis. Other signaling molecules that bind to the phosphorylated CSF-1R intracellular domain include STAT1, STAT3, PLCy, and Cb1 (Bourette, RP and Rohrschneider, LR, Growth Factors 17 (2000) 155-166).

[0009] CSF-1R signaling has physiological roles in immune responses, in bone remodeling, and in the reproductive system. Animals knocked out for either CSF-1 (Pollard, JW, Mol. Reprod. Dev. 46 (1997) 54-61) or CSF-1R (Dai, XM et al., Blood 99 (2002) 111-120) have been shown to have osteopetrosis, hematopoiesis, tissue macrophages, and reproductive phenotypes, consistent with the role of CSF-1R in various cell types.

[0010] Sherr, CJ et al., Blood 73 (1989) 1786-1793, relates to certain antibodies against CSF-1R, which inhibit CSF-1 activity. Ashmun, RA et al., Blood 73 (1989) 827-837, relates to CSF-1R antibodies. Lenda, D. et al., Journal of Immunology 170 (2003) 3254-3262, relates to reduced macrophage recruitment, proliferation, and activation in CSF-1-deficient mice, leading to reduced tubular apoptosis during renal inflammation. Kitaura, H. et al., Journal of Dental Research 87 (2008) 396-400, refers to an anti-CSF-1 antibody, which inhibits orthodontic tooth movement. WO 2001 / 030381, refers to CSF-1 activity inhibitors, including antisense nucleotides and antibodies, although only CSF-1 antisense nucleotides are disclosed. WO 2004 / 045532 relates to the prevention of metastasis and bone loss and the treatment of metastatic cancer by CSF-1 antagonists, disclosing only antagonistic anti-CSF-1 antibodies. WO 2005 / 046657 relates to the treatment of inflammatory bowel disease by anti-CSF-1 antibodies. US2002 / 0141994 relates to inhibitors of colony stimulating factor. WO 2006 / 096489 relates to the treatment of rheumatoid arthritis by anti-CSF-1 antibodies. WO2009 / 026303 and WO 2009 / 112245 relate to certain anti-CSF-1R antibodies that bind to CSF-1R within the first three subdomains (D1 to D3) of the extracellular domain (CSF-1R-ECD). WO2011 / 123381 (A1) relates to antibodies against CSF-1R. WO2011 / 070024 relates to certain anti-CSF-1R antibodies that bind to CSF-1R within the dimerization domain (D4 to D5).

[0011] PD-L1 and PD-L1 Antibodies

[0012] Costimulation or the provision of two distinct signals to T cells is a widely accepted model for lymphocyte activation of resting T lymphocytes by antigen presenting cells (APCs). Lafferty et al., Aust. J. Exp. Biol. Med. Sci. 53:27-42 (1975).

[0013] This model further provides self- and non-self discrimination and immune tolerance. Bretscher et al., Science 169: 1042-1049 (1970); Bretscher, PA, PNAS USA 96: 185-190 (1999); Jenkins et al., J. Exp. Med. 165: 302-319 (1987). After recognition of foreign antigenic peptides presented in the context of the major histocompatibility complex (MHC), the first signal, or antigen-specific signal, is transduced via the T cell receptor (TCR). The second or co-stimulatory signal is delivered to T cells through co-stimulatory molecules expressed on antigen presenting cells (APCs), and induces T cells to promote clonal expansion, cytokine secretion, and effector function. Lenschow et al., Ann. Rev. Immunol. 14: 233 (1996). In the absence of co-stimulation, T cells can become refractory to antigenic stimulation, fail to mount an effective immune response, and further can lead to tolerance or exhaustion to foreign antigens.

[0014] The simple two-signal model may be an oversimplification, as the strength of the TCR signal actually has a quantitative effect on T cell activation and differentiation. Viola et al., Science 273: 104-106 (1996); Sloan-Lancaster, Nature 363: 156-159 (1993). Moreover, T cell activation can occur even in the absence of co-stimulatory signals if the TCR signal strength is high. More importantly, T cells receive both positive and negative second co-stimulatory signals. Regulation of such positive and negative signals is critical for maximizing the host's protective immune response while maintaining immune tolerance and preventing autoimmunity.

[0015] The negative second signal appears to be necessary for the induction of T cell tolerance, whereas the positive signal promotes T cell activation. Although the simple two-signal model still provides a valid explanation for unimmunized lymphocytes, the host immune response is a dynamic process and co-stimulatory signals can also be provided to antigen-exposed T cells.

[0016] The mechanism of costimulation is of therapeutic interest because it has been shown that the operation of costimulatory signals provides a means of enhancing or terminating a cell-based immune response. Recently, it has been found that T cell dysfunction or anergy occurs in parallel with the induction and sustained expression of inhibitory receptors (programmed death 1 polypeptide (PD-1)). Therefore, therapeutic agents targeting PD-1 and other molecules that signal via interaction with PD-1, such as programmed death ligand 1 (PD-L1) and programmed death ligand 2 (PD-L2) are areas of intense interest. It has been proposed that inhibition of PD-1 signaling is a means of enhancing T cell immunity to treat cancer (e.g., tumor immunity) and infection (including both acute and chronic infections, such as persistent). However, since the best therapeutic agent for targets in this approach is still to be commercialized, there is a significant unmet medical need. Antibodies for PD-L1 are described in, for example, WO 2010 / 077634. Summary of the invention

[0017] The present invention comprises a combination therapy of an antibody that binds to human CSF-1R and an antibody that binds to human PD-L1 for treating cancer, for preventing or treating metastasis, for treating inflammatory diseases, for treating bone loss, for treating immune-related diseases such as tumor immunity or delaying its progression, or for stimulating an immune response or function, such as T cell activity.

[0018] The invention further comprises the use of an antibody that binds to human CSF-1R in the manufacture of a medicament for treating cancer, for treating inflammatory diseases, for treating bone loss, for treating immune-related diseases such as tumor immunity or delaying its progression, or for stimulating an immune response or function, such as T cell activity, wherein the antibody is administered in combination with an antibody that binds to human PD-L1.

[0019] The antibody binding to human CSF-1R used in the combination therapy is characterized by comprising

[0020] a) a heavy chain variable domain VH of SEQ ID NO: 23 and a light chain variable domain VL of SEQ ID NO: 24, or

[0021] b) a heavy chain variable domain VH of SEQ ID NO: 31 and a light chain variable domain VL of SEQ ID NO: 32, or

[0022] c) a heavy chain variable domain VH of SEQ ID NO: 39 and a light chain variable domain VL of SEQ ID NO: 40, or

[0023] d) a heavy chain variable domain VH of SEQ ID NO: 47 and a light chain variable domain VL of SEQ ID NO: 48, or

[0024] e) a heavy chain variable domain VH of SEQ ID NO: 55 and a light chain variable domain VL of SEQ ID NO: 56; and the antibody binding to human PD-L1 used in the combination therapy is characterized by comprising

[0025] a) a heavy chain variable domain VH of SEQ ID NO: 89 and a light chain variable domain VL of SEQ ID NO: 92, or

[0026] b) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 93, or

[0027] c) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 94, or

[0028] d) a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:95, or

[0029] e) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 96, or

[0030] f) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 97, or

[0031] g) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 98, or

[0032] h) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 99, or

[0033] i) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 100, or

[0034] j) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 101, or

[0035] k) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 102, or

[0036] l) a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:103, or

[0037] m) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 104, or

[0038] n) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 105, or

[0039] o) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 106, or

[0040] p) the heavy chain variable domain VH of SEQ ID NO:91 and the light chain variable domain VL of SEQ ID NO:107.

[0041] In one embodiment, the antibody is used to treat cancer.

[0042] In one embodiment, the antibody is used to prevent or treat metastasis.

[0043] In one embodiment, the antibody is used to treat bone loss.

[0044] In one embodiment, the antibody is used to treat an inflammatory disease.

[0045] In one embodiment, the antibody is used to treat or delay progression of an immune-related disease such as tumor immunity.

[0046] In one embodiment, the antibody is used to stimulate an immune response or function, such as T cell activity.

[0047] The present invention further comprises an antibody that binds to human CSF-1R, wherein the antibody is administered in combination with an antibody that binds to human PD-L1 for

[0048] i) inhibiting cell proliferation in CSF-1R ligand-dependent and / or CSF-1 ligand-independent CSF-1R-expressing tumor cells;

[0049] ii) inhibiting cell proliferation in tumors infiltrated with CSF-1R ligand-dependent and / or CSF-1R ligand-independent CSF-1R-expressing macrophages;

[0050] iii) inhibiting cell survival in (CSF-1R ligand-dependent and / or CSF-1R ligand-independent) CSF-1R-expressing monocytes and macrophages; and / or

[0051] iv) inhibiting differentiation of CSF-1R-expressing monocytes into macrophages in (CSF-1R ligand-dependent and / or CSF-1R ligand-independent) CSF-1R-expressing monocytes;

[0052] wherein the antibody is administered in combination with an antibody that binds to human PD-L1;

[0053] The antibody binding to human CSF-1R used in the combination therapy is characterized by comprising

[0054] a) a heavy chain variable domain VH of SEQ ID NO: 23 and a light chain variable domain VL of SEQ ID NO: 24, or

[0055] b) a heavy chain variable domain VH of SEQ ID NO: 31 and a light chain variable domain VL of SEQ ID NO: 32, or

[0056] c) a heavy chain variable domain VH of SEQ ID NO: 39 and a light chain variable domain VL of SEQ ID NO: 40, or

[0057] d) a heavy chain variable domain VH of SEQ ID NO: 47 and a light chain variable domain VL of SEQ ID NO: 48, or

[0058] e) a heavy chain variable domain VH of SEQ ID NO: 55 and a light chain variable domain VL of SEQ ID NO: 56; and the antibody binding to human PD-L1 used in the combination therapy is characterized by comprising

[0059] a) a heavy chain variable domain VH of SEQ ID NO: 89 and a light chain variable domain VL of SEQ ID NO: 92, or

[0060] b) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 93, or

[0061] c) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 94, or

[0062] d) a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:95, or

[0063] e) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 96, or

[0064] f) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 97, or

[0065] g) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 98, or

[0066] h) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 99, or

[0067] i) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 100, or

[0068] j) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 101, or

[0069] k) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 102, or

[0070] l) a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:103, or

[0071] m) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 104, or

[0072] n) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 105, or

[0073] o) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 106, or

[0074] p) the heavy chain variable domain VH of SEQ ID NO:91 and the light chain variable domain VL of SEQ ID NO:107.

[0075] The invention further comprises an antibody that binds to human CSF-1R for use in treating a patient having a CSF-1R expressing tumor or a tumor with CSF-1R expressing macrophage infiltration, wherein the tumor is characterized by elevated CSF-1R ligand, and wherein the anti-CSF-1R antibody is administered in combination with an antibody that binds to human PD-L1,

[0076] The antibody binding to human CSF-1R used in the combination therapy is characterized by comprising

[0077] a) a heavy chain variable domain VH of SEQ ID NO: 23 and a light chain variable domain VL of SEQ ID NO: 24, or

[0078] b) a heavy chain variable domain VH of SEQ ID NO: 31 and a light chain variable domain VL of SEQ ID NO: 32, or

[0079] c) a heavy chain variable domain VH of SEQ ID NO: 39 and a light chain variable domain VL of SEQ ID NO: 40, or

[0080] d) a heavy chain variable domain VH of SEQ ID NO: 47 and a light chain variable domain VL of SEQ ID NO: 48, or

[0081] e) a heavy chain variable domain VH of SEQ ID NO: 55 and a light chain variable domain VL of SEQ ID NO: 56; and the antibody binding to human PD-L1 used in the combination therapy is characterized by comprising

[0082] a) a heavy chain variable domain VH of SEQ ID NO: 89 and a light chain variable domain VL of SEQ ID NO: 92, or

[0083] b) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 93, or

[0084] c) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 94, or

[0085] d) a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:95, or

[0086] e) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 96, or

[0087] f) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 97, or

[0088] g) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 98, or

[0089] h) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 99, or

[0090] i) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 100, or

[0091] j) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 101, or

[0092] k) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 102, or

[0093] l) a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:103, or

[0094] m) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 104, or

[0095] n) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 105, or

[0096] o) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 106, or

[0097] p) the heavy chain variable domain VH of SEQ ID NO:91 and the light chain variable domain VL of SEQ ID NO:107.

[0098] In one embodiment, the antibody is of human IgG1 subclass or human IgG4 subclass.

[0099] The present invention further comprises:

[0100] A) a method for

[0101] i) inhibiting cell proliferation in CSF-1R ligand-dependent and / or CSF-1R ligand-independent CSF-1R-expressing tumor cells;

[0102] ii) inhibiting cell proliferation in tumors infiltrated with CSF-1R ligand-dependent and / or CSF-1R ligand-independent CSF-1R-expressing macrophages;

[0103] iii) inhibiting cell survival in (CSF-1R ligand-dependent and / or CSF-1R ligand-independent) CSF-1R-expressing monocytes and macrophages; and / or

[0104] iv) a method for inhibiting differentiation of (CSF-1R ligand-dependent and / or CSF-1R ligand-independent) CSF-1R-expressing monocytes into macrophages,

[0105] wherein an antibody that binds to human CSF-1R is administered in combination with an antibody that binds to human PD-L1,

[0106] or

[0107] B) a method of treating a patient having a CSF-1R expressing tumor or a tumor with infiltration of CSF-1R expressing macrophages, wherein the tumor is characterized by elevated CSF-1R ligand, and wherein an antibody that binds human CSF-1R is administered in combination with an antibody that binds human PD-L1,

[0108] The antibody binding to human CSF-1R used in the combination therapy is characterized by comprising

[0109] a) a heavy chain variable domain VH of SEQ ID NO: 23 and a light chain variable domain VL of SEQ ID NO: 24, or

[0110] b) a heavy chain variable domain VH of SEQ ID NO: 31 and a light chain variable domain VL of SEQ ID NO: 32, or

[0111] c) a heavy chain variable domain VH of SEQ ID NO: 39 and a light chain variable domain VL of SEQ ID NO: 40, or

[0112] d) a heavy chain variable domain VH of SEQ ID NO: 47 and a light chain variable domain VL of SEQ ID NO: 48, or

[0113] e) a heavy chain variable domain VH of SEQ ID NO: 55 and a light chain variable domain VL of SEQ ID NO: 56; and the antibody binding to human PD-L1 used in the combination therapy is characterized by comprising

[0114] a) a heavy chain variable domain VH of SEQ ID NO: 89 and a light chain variable domain VL of SEQ ID NO: 92, or

[0115] b) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 93, or

[0116] c) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 94, or

[0117] d) a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:95, or

[0118] e) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 96, or

[0119] f) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 97, or

[0120] g) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 98, or

[0121] h) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 99, or

[0122] i) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 100, or

[0123] j) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 101, or

[0124] k) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 102, or

[0125] l) a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:103, or

[0126] m) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 104, or

[0127] n) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 105, or

[0128] o) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 106, or

[0129] p) the heavy chain variable domain VH of SEQ ID NO:91 and the light chain variable domain VL of SEQ ID NO:107.

[0130] As used herein, the term "ligand-independent" refers to ligand-independent signaling via the extracellular ECD (and excludes ligand-independent signaling mediated by activating point mutations in the intracellular kinase domain). In one embodiment, the CSF-1R ligand in this context refers to a CSF-1R ligand selected from human CSF-1 (SEQ ID NO: 86) and human IL-34 (SEQ ID NO: 87); in one embodiment, the CSF-1R ligand is human CSF-1 (SEQ ID NO: 86); in one embodiment, the CSF-1R ligand is human IL-34 (SEQ ID NO: 87).

[0131] The invention comprises a combination treatment of patients having a CSF-1R expressing tumor or a tumor with CSF-1R expressing macrophage infiltration, wherein the tumor is characterized by elevated CSF-1R ligand (in one embodiment, the CSF-1R ligand is selected from human CSF-1 (SEQ ID NO: 86) and human IL-34 (SEQ ID NO: 87); in one embodiment, the CSF-1R ligand is human CSF-1 (SEQ ID NO: 86); in one embodiment, the CSF-1R ligand is human IL-34 (SEQ ID NO: 87)) (detected in serum, urine or tumor biopsy), wherein an antibody that binds to human CSF-1R as described herein is administered in combination with an anti-PD-L1 antibody as described herein. The term "elevated CSF-1R ligand" refers to overexpression of human CSF-1R ligand (in one embodiment, the CSF-1R ligand is selected from human CSF-1 (SEQ ID NO: 86) and human IL-34 (SEQ ID NO: 87); in one embodiment, the CSF-1R ligand is human CSF-1 (SEQ ID NO: 86); in one embodiment, the CSF-1R ligand is human IL-34 (SEQ ID NO: 87)) before treatment (compared to normal tissue) or overexpression of human CSF-1R ligand induced by anti-CSF-1R antibody treatment (and compared to the expression level before treatment). In certain embodiments, the term "elevated" or "exceeding" refers to a level that exceeds a reference level or an overall increase of 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100% or more in the level of CSF-1R ligand detected by the methods described herein compared to the level of CSF-1R ligand from a reference sample. In certain embodiments, the term elevated refers to an increase in the level of CSF-1R ligand, wherein the increase is at least about 1.5, 1.75, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, or 100 times higher than the level of CSF-1R ligand (e.g., previously determined from a reference sample). In a preferred embodiment, the term elevated level relates to a value that is at or above a reference level.

[0132] Combination therapy with the antibodies described herein shows benefit for patients in need of CSF-1R targeted therapy. Specific anti-CSF-1R antibodies according to the present invention show potent anti-proliferative activity against ligand-independent and ligand-dependent proliferation and are particularly useful in combination with specific anti-PD-L1 antibodies described herein for the treatment of cancer and metastasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0133] Figure 1a-1b Figure 1a: Human monocytes differentiated into macrophages in co-cultures with GM-CSF or CSF-1 (100 ng / ml ligand). hMab 2F11-e7 was added after 6 days of differentiation. On day 7 of antibody treatment, CTG viability assay ( Cell viability was measured in ELISA kit (Promega). Calculation of % cell viability: RLU signal from treated cells divided by RLU signal from untreated controls without antibody (n=4). Figure 1b : Human monocytes differentiated into macrophages after 7 days of GM-CSF (M1) or M-CSF (M2). Phenotype was analyzed by indirect fluorescence analysis – staining with anti-CD163-PE, anti-CD80-PE or anti-HLA-DR / DQ / DP-Zenon-Alexa647 markers. The values ​​in each bar correspond to the mean ratio fluorescence intensity (MRFI); the calculated ratio between the mean fluorescence intensity (MFI) of cells stained with the selected antibody (open bars) and the corresponding isotype control (negative control; gray solid bars) (mean ± SD; n ≥ 5).

[0134] Figure 2a-2d CSF-1 levels in macaques after application of different doses of the anti-CSF-1R antibody hMab 2F11-e7.

[0135] Figure 3 In the presence of TAM, T cell expansion induced by CD3 and CD28 activation is suppressed: TAM is isolated from MC38 tumors and co-cultured with CFSE-labeled CD8+T cells at the ratio shown in the presence of CD3 / CD28 stimulation. After 3 days, the beads of CFSElow split cells are used to quantify T cell proliferation. A representative experiment in two is depicted with the mean + SEM of triplicate wells.

[0136] Figure 4 <Mouse CSF1R> Antibody / <pd-l1>Antitumor efficacy of the antibody combination in the MC38 mouse CRC model in vivo (tumor volume progression >700mm 3 Kaplan-Meier curve plot).

[0137] Figure 5 <Mouse CSF1R> Antibody / <pd-l1>Antitumor efficacy of the antibody combination in a subcutaneous syngeneic CT26.WT colon cancer in vivo model (tumor volume progression >700mm 3 Kaplan-Meier curve plot). DETAILED DESCRIPTION

[0138] Many tumors are characterized by prominent immune cell infiltration, including macrophages. Initially, it was believed that immune cells were part of the defense mechanism against tumors, but recent data support the idea that several immune cell populations (including macrophages) may in fact promote tumor progression. Macrophages are characterized by their plasticity. Depending on the cytokine microenvironment, macrophages can exhibit so-called M1 or M2 subtypes. M2 macrophages participate in suppressing tumor immunity. They also play an important role in tissue repair functions such as angiogenesis and tissue remodeling (they are incorporated by tumors to support growth). In contrast to tumor-promoting M2 macrophages, M1 macrophages exhibit anti-tumor activity by secreting inflammatory cytokines and participating in antigen presentation and phagocytosis (Mantovani, A. et al., Curr. Opin. Immunol. 2 (2010) 231-237).

[0139] By secreting various cytokines, such as colony stimulating factor 1 (CSF-1) and IL-10, tumor cells can recruit and shape macrophages into M2 subtypes, while cytokines such as granulocyte macrophage colony stimulating factor (GM-CSF), IFN-γ program macrophages toward M1 subtypes. Using immunohistochemistry, it is possible to distinguish between macrophage subpopulations (which are likely to be enriched M2 macrophages) that co-express CD68 and CD163 and subsets (most likely including M1 macrophages) that display CD68+ / MHC II+ or CD68+ / CD80+ immunophenotypes. The cell shape, size, and spatial distribution of CD68 and CD163 positive macrophages are consistent with the hypothesis published on the tumor promotion effect of M2 macrophages (e.g., their preferential positioning in tumor cross matrix and vital tumor areas). On the contrary, CD68+ / MHC II class+ macrophages are found everywhere. Their putative role in phagocytosis is reflected by clusters of CD68+ / MHC class II+ but CD163- immunophenotype near apoptotic cells and necrotic tumor areas.

[0140] The subtypes and marker expression of different macrophage subsets are associated with their functional status. M2 macrophages can support tumorigenesis by:

[0141] a) enhance angiogenesis via secretion of angiogenic factors such as VEGF or bFGF,

[0142] b) Supporting metastasis formation by secreting matrix metalloproteinases (MMPs), growth factors and migration factors (guiding tumor cells to the bloodstream and establishing metastatic niches) (Wyckoff, J. et al., Cancer Res. 67 (2007) 2649-2656),

[0143] c) play a role in establishing an immunosuppressive environment by secreting immunosuppressive cytokines such as IL-4, 11-13, IL-1ra and IL-10, which in turn regulate T regulatory cell function. In contrast, CD4-positive T cells have been shown to enhance the activity of tumor-promoting macrophages in preclinical models (Mantovani, A. et al., Eur. J. Cancer 40 (2004) 1660-1667; DeNardo, D. et al., Cancer Cell 16 (2009) 91-102).

[0144] Thus, in several types of cancer (e.g., breast, ovarian, Hodgkin's lymphoma), the prevalence of M2 subtype tumor-associated macrophages (TAMs) has been associated with poor prognosis (Bingle, L. et al., J. Pathol. 3 (2002) 254-265; Orre, M., and Rogers, PA, Gynecol. Oncol. 1 (1999) 47-50; Steidl, C. et al., N. Engl. J. Med. 10 (2010) 875-885). Recent data show the correlation of CD163-positive macrophage infiltration in tumors and tumor grade (Kawamura, K. et al., Pathol. Int. 59 (2009) 300-305). TAMs isolated from patient tumors have a resistant phenotype and are not cytotoxic to tumor cells (Mantovani, A. et al., Eur. J. Cancer 40 (2004) 1660-1667). However, infiltration of TAMs in the presence of cytotoxic T cells is associated with improved survival in non-small cell lung cancer and thus reflects the more prominent M1 macrophage infiltration in this tumor type (Kawai, O. et al., Cancer 6 (2008) 1387-1395).

[0145] Recently, a so-called immune signature comprising a large number of macrophages and CD4-positive T cells but a small number of cytotoxic CD8-positive T cells was shown to be associated with shortened overall survival (OS) in breast cancer patients and to represent an independent prognostic factor (DeNardo, D. et al., Cancer Discovery 1 (2011) 54-67).

[0146] Consistent with the role of CSF-1 in driving the pro-tumorigenic function of M2 macrophages, high CSF-1 expression in rare sarcomas or locally aggressive connective tissue tumors such as pigmented villonodular synovitis (PVNS) and tenosynovial giant cell tumors (TGCT), partly due to CSF-1 gene translocation, leads to the accumulation of monocytes and macrophages expressing the receptor for CSF-1 (colony stimulating factor 1 receptor (CSF-1R)), forming the majority of the tumor mass (West, RB et al., Proc. Natl. Acad. Sci. USA 3 (2006) 690-695). These tumors were subsequently used to define a CSF-1-dependent macrophage signature by gene expression profiling. This CSF-1 responsive gene signature predicts poor prognosis in breast cancer and leiomyosarcoma patient tumors (Espinosa, I. et al., Am. J. Pathol. 6 (2009) 2347-2356; Beck, A. et al., Clin. Cancer Res. 3 (2009) 778-787).

[0147] CSF-1R belongs to the class III subfamily of receptor tyrosine kinases and is encoded by the c-fms proto-oncogene. Binding of CSF-1 or IL-34 induces receptor dimerization, followed by autophosphorylation and activation of downstream signaling cascades. Activation of CSF-1R regulates the survival, proliferation and differentiation of monocytes and macrophages (Xiong, Y. et al., J. Biol. Chem. 286 (2011) 952-960).

[0148] In addition to cells of the monocytic lineage and osteoclasts, which are derived from the same hematopoietic precursors as macrophages, CSF-1R / c-fms has also been found to be expressed by several human epithelial cancers such as ovarian and breast cancer, and in leiomyosarcoma and TGCT / PVNS, albeit at lower levels than in macrophages. For TGCT / PVNS, elevated levels of CSF-1 (a ligand for CSF-1R) in the ascites and serum of ovarian cancer patients are associated with a poor prognosis (Scholl, S. et al., Br. J. Cancer 62 (1994) 342-346; Price, F. et al., Am. J. Obstet. Gynecol. 168 (1993) 520-527). Furthermore, a constitutively active mutant form of CSF 1R is able to transform NIH3T3 cells, one of the characteristics of an oncogene (Chambers, S., Future Oncol 5 (2009) 1429-1440).

[0149] Preclinical models provide validation of CSF-1R as an oncology target. Blocking CSF-1 and CSF-1R activity results in reduced TAM recruitment. Chemotherapy results in elevated CSF-1 expression in tumor cells, leading to enhanced TAM recruitment. In a spontaneous transgenic breast cancer model, blocking CSF-1R in combination with paclitaxel results in activation of CD8-positive cytotoxic T cells, leading to reduced tumor growth and metastatic burden (DeNardo, D. et al., Cancer Discovery 1 (2011) 54-67).

[0150] Human CSF-1R (CSF-1 receptor; synonyms: M-CSF receptor; macrophage colony stimulating factor 1 receptor, Fms proto-oncogene, c-fms, SEQ ID NO: 22) has been known since 1986 (Coussens, L. et al., Nature 320 (1986) 277-280). CSF-1R is a growth factor and is encoded by the c-fms proto-oncogene (for review, see, e.g., Roth, P. and Stanley, ER, Curr. Top. Microbiol. Immunol. 181 (1992) 141-167).

[0151] CSF-1R is a receptor for the CSF1R ligands CSF-1 (macrophage colony stimulating factor, also known as M-CSF) (SEQ ID NO: 86) and IL-34 (SEQ ID NO: 87) and mediates the biological effects of these cytokines (Sherr, CJ et al., Cell 41 (1985) 665-676; Lin, H. et al., Science 320 (2008) 807-811). The cloning of colony stimulating factor-1 receptor (also known as c-fms) was first described in Roussel, MF et al., Nature 325 (1987) 549-552. In this publication, it was shown that CSF-1R has a transforming potential that depends on changes in the C-terminal tail of the protein, including loss of inhibitory tyrosine 969 phosphorylation, which binds Cbl and thereby mediates receptor downregulation (Lee, PS et al., Embo J. 18 (1999) 3616-3628).

[0152] CSF-1R is a single-chain, transmembrane receptor tyrosine kinase (RTK) and a member of the RTK family containing immunoglobulin (Ig) motifs, characterized by five repeats of Ig-like subdomains D1-D5 in the extracellular domain (ECD) of the receptor (Wang, Z. et al., Molecular and Cellular Biology 13 (1993) 5348-5359). The human CSF-1R extracellular domain (CSF-1R-ECD) (SEQ ID NO: 64) contains all five extracellular Ig-like subdomains D1-D5. The human CSF-1R fragment delD4 (SEQ ID NO: 65) contains extracellular Ig-like subdomains D1-D3 and D5, but lacks the D4 subdomain. The human CSF-1R fragment D1-D3 (SEQ ID NO: 66) contains each of the subdomains D1-D3. The sequence without the signal peptide MGSGPGVLLL LLVATAWHGQ G (SEQ ID NO: 67) is listed. The human CSF-1R fragment D4-D3 (SEQ ID NO: 85) comprises the corresponding subdomains D4-D3.

[0153] Currently, two CSF-1R ligands are known that bind to the extracellular domain of CSF-1R. The first is CSF-1 (colony stimulating factor 1, also known as M-CSF, macrophage; human CSF-1, SEQ ID NO: 86), which is found as a disulfide-linked homodimer outside the cell (Stanley, ER et al., Journal of Cellular Biochemistry 21 (1983) 151-159; Stanley, ER et al., Stem Cells 12 Suppl. 1 (1995) 15-24). The second is IL-34 (human IL-34; SEQ ID NO: 87) (Hume, DA et al., Blood 119 (2012) 1810-1820). Thus, in one embodiment, the term "CSF-1R ligand" refers to human CSF-1 (SEQ ID NO: 86) and / or human IL-34 (SEQ ID NO: 87).

[0154] For experiments, an active 149 amino acid (aa) fragment of human CSF-1 (aa33-181 of SEQ ID NO: 86) is often used. This active 149aa fragment of human CSF-1 (aa 33-181 of SEQ ID NO: 86) is contained in all three major forms of CSF-1 and is sufficient to mediate binding to CSF-1R (Hume, DA et al., Blood 119 (2012) 1810-1820).

[0155] The main biological effect of CSF-1R signaling is the differentiation, proliferation, migration, and survival of hematopoietic precursor cells to the macrophage lineage (including osteoclasts). The activation of CSF-1R is mediated by its CSF-1R ligands CSF-1 (M-CSF) and IL-34. The binding of CSF-1 (M-CSF) to CSF-1R induces the formation of homodimers and the activation of kinases through tyrosine phosphorylation (Li, W. et al., EMBO Journal. 10 (1991) 277-288; Stanley, ER et al., Mol. Reprod. Dev. 46 (1997) 4-10).

[0156] The intracellular protein tyrosine kinase domain is interrupted by a unique insertion domain that is also present in other related RTK class III family members, including platelet-derived growth factor receptor (PDGFR), stem cell growth factor receptor (c-Kit), and fins-like cytokine receptor (FLT3). Despite the structural homology within this family of growth factor receptors, they have distinct tissue-specific functions.

[0157] CSF-1R is expressed primarily on cells of the monocytic lineage and in the female reproductive tract and placenta. In addition, CSF-1R expression has been reported in Langerhans cells (a subset of smooth muscle cells) in the skin (Inaba, T. et al., J. Biol. Chem. 267 (1992) 5693-5699), B cells (Baker, AH et al., Oncogene 8 (1993) 371-378), and microglia (Sawada, M. et al., Brain Res. 509 (1990) 119-124). It is known that cells with mutant human CSF-1R (SEQ ID NO: 23) proliferate independently of ligand stimulation.

[0158] As used herein, "binds to human CSF-1R" or "specifically binds to human CSF-1R" or "anti-CSF-1R antibody" refers to an antibody that binds to human CSF-1R with D Value 1.0x 10 -8 mol / l or less (in one embodiment, K D Value 1.0x10 -9 The binding affinity of the protein is determined by a standard binding assay, such as surface plasmon resonance (SPR) technology. GE-Healthcare, Uppsala, Sweden). Thus, as used herein, an "antibody that binds to human CSF-1R" refers to an antibody that binds to human CSF-1R with a K D 1.0x 10 -8 mol / l or less (in one embodiment, 1.0 x 10 -8 mol / l-1.0x10 -13 mol / l), in one embodiment, K D 1.0x10 -9 mol / l or less (in one embodiment, 1.0 x 10 -9 mol / l-1.0x 10 -13 mol / l) of the antibody that specifically binds to the human CSF-1R antigen.

[0159] PD-1 / PD-L1 / PD-L2 pathway:

[0160] An important negative co-stimulatory signal regulating T cell activation is provided by the programmed death-1 receptor (PD-1) (CD279) and its ligand binding partners PD-L1 (B7-H1, CD274; SEQ ID NO: 88) and PD-L2 (B7-DC, CD273). The negative regulatory role of PD-1 was revealed by PD-1 knockout (Pdcd1- / -), which is prone to autoimmunity. Nishimura et al., Immunity 11: 141-51 (1999); Nishimura et al., Science 291: 319-22 (2001). PD-1 is related to CD28 and CTLA-4, but lacks membrane-proximal cysteines that allow homodimerization. The cytoplasmic domain of PD-1 contains an immunoreceptor tyrosine-based inhibitory motif (ITIM, V / IxYxxL / V). PD-1 binds only PD-L1 and PD-L2. Freeman et al., J. Exp. Med. 192: 1-9 (2000); Dong et al., Nature Med. 5: 1365-1369 (1999); Latchman et al., Nature Immunol. 2: 261-268 (2001); Tseng et al., J. Exp. Med. 193: 839-846 (2001).

[0161] PD-1 can be expressed on T cells, B cells, natural killer T cells, activated monocytes and dendritic cells (DC). PD-1 is expressed by activated but not by unstimulated human CD4+ and CD8+ T cells, B cells and myeloid cells. This is in contrast to the more restricted expression of CD28 and CTLA-4. Nishimura et al., Int. Immunol. 8:773-80 (1996); Boettler et al., J. Virol. 80:3532-40 (2006). At least 4 variants of PD-1 have been cloned from activated human T cells, including transcripts lacking (i) exon 2, (ii) exon 3, (iii) exons 2 and 3, or (iv) exons 2 to 4. Nielsen et al., Cell. Immunol. 235:109-16 (2005). Except for PD-1Δex3, all variants are expressed at levels similar to full-length PD-1 in resting peripheral blood mononuclear cells (PBMCs). Expression of all variants is significantly induced after activation of human T cells with anti-CD3 and anti-CD28. The PD-1Δex3 variant lacks the transmembrane domain and resembles soluble CTLA-4, which plays an important role in autoimmunity. Ueda et al., Nature 423:506-11 (2003). This variant is enriched in the synovial fluid and serum of patients with rheumatoid arthritis. Wan et al., J. Immunol. 177:8844-50 (2006).

[0162] The two PD-1 ligands differ in their expression patterns. PD-L1 is constitutively expressed on mouse T and B cells, CD, macrophages, mesenchymal stem cells, and bone marrow-derived mast cells. Yamazaki et al., J. Immunol. 169: 5538-45 (2002). PD-L1 is expressed on a wide range of non-hematopoietic cells (e.g., cornea, lung, vascular epithelium, liver non-parenchymal cells, mesenchymal stem cells, pancreatic islets, placental syncytiotrophoblast, keratinocytes, etc.) [Keir et al., Annu. Rev. Immunol. 26: 677-704 (2008)] and is upregulated on a variety of cell types after activation. Both type I and type II interferons IFN upregulate PD-L1. Eppihimer et al., Microcirculation 9:133-45 (2002); Schreiner et al., J. Neuroimmunol. 155:172-82 (2004). PD-L1 expression in cell lines is reduced upon inhibition of MyD88, TRAF6, and MEK. Liu et al., Blood 110:296-304 (2007). JAK2 has also been linked to PD-L1 induction. Lee et al., FEBS Lett. 580:755-62 (2006); Liu et al., Blood 110:296-304 (2007). Loss or inhibition of phosphatase and tensin homolog (PTEN), a cellular phosphatase that modifies phosphatidylinositol 3-kinase (PI3K) and Akt signaling, increases posttranscriptional PD-L1 expression in cancer. Parsa et al., Nat. Med. 13:84-88 (2007).

[0163] PD-L2 expression is more restricted than PD-L1. PD-L2 is inducibly expressed on DCs, macrophages, and bone marrow-derived mast cells. PD-L2 is also expressed on about one-half to two-thirds of resting peritoneal B1 cells, but not on conventional B2 B cells. Zhong et al., Eur. J. Immunol. 37: 2405-10 (2007). PD-L2+B1 cells bind phosphatidylcholine and may be important for innate immune responses to bacterial antigens. IFN-γ induction of PD-L2 is partially dependent on NF-κB. Liang et al., Eur. J. Immunol. 33: 2706-16 (2003). PD-L2 can also be induced by GM-CF, IL-4, and IFN-γ on monocytes and macrophages. Yamazaki et al., J. Immunol. 169:5538-45 (2002); Loke et al., PNAS 100:5336-41 (2003).

[0164] PD-1 signaling generally has a greater effect on cytokine production than on cell proliferation, with significant effects on IFN-γ, TNF-α, and IL-2 production. PD-1-mediated inhibitory signaling also depends on the strength of TCR signaling, delivering greater inhibition at low levels of TCR stimulation. This reduction can be overcome by costimulation via CD28 [Freeman et al., J. Exp. Med. 192: 1027-34 (2000)] or the presence of IL-2 [Carter et al., Eur. J. Immunol. 32: 634-43 (2002)].

[0165] There is increasing evidence that signaling through PD-L1 and PD-L2 may be bidirectional. That is, in addition to modifying TCR or BCR signaling, signaling can also be delivered back to cells expressing PD-L1 and PD-L2. Although treatment of dendritic cells with natural human anti-PD-L2 antibodies isolated from patients with Waldenstrom's macroglobulinemia did not find upregulation of MHC II or B7 co-stimulatory molecules, such cells did produce significant amounts of proinflammatory cytokines, particularly TNF-α and IL-6, and stimulated T cell proliferation. Nguyen et al., J. Exp. Med. 196: 1393-98 (2002). Treatment of mice with this antibody also (1) enhanced resistance to transplanted b16 melanoma and rapidly induced tumor-specific CTLs. Radhakrishnan et al., J. Immunol. 170: 1830-38 (2003); Radhakrishnan et al., Cancer Res. 64: 4965-72 (2004); Heckman et al., Eur. J. Immunol. 37: 1827-35 (2007); (2) Blocking the occurrence of airway inflammatory disease in a mouse model of allergic asthma. Radhakrishnan et al., J. Immunol. 173: 1360-65 (2004); Radhakrishnan et al., J. Allergy Clin. Immunol. 116: 668-74 (2005).

[0166] Further evidence for reverse signaling into dendritic cells ("DC") comes from studies of bone marrow-derived DC cultured with soluble PD-1 (PD-1 EC domain fused to Ig constant region - "s-PD-1"). Kuipers et al., Eur. J. Immunol. 36:2472-82 (2006). This sPD-1 inhibits DC activation and increases IL-10 production in a manner that is reversible by administration of anti-PD-1.

[0167] In addition, several studies have shown a PD-1-independent receptor for PD-L1 or PD-L2. B7.1 has long been identified as a binding partner of PD-L1. Butte et al., Immunity 27: 111-22 (2007). Chemical cross-linking studies suggest that PD-L1 and B7.1 can interact via their IgV-like domains. B7.1:PD-L1 interactions can induce inhibitory signals into T cells. Inhibitory signals are delivered by B7.1 ligating PD-L1 on CD4+T cells or by PD-L1 ligating B7.1 on CD4+T cells. T cells lacking CD28 and CTLA-4 show reduced proliferation and cytokine production when stimulated by anti-CD3 plus B7.1 coated beads. In T cells lacking all receptors for B7.1 (i.e., CD28, CTLA-4, and PD-L1), T cell proliferation and cytokine production are no longer inhibited by anti-CD3 plus B7.1 coated beads. This indicates that B7.1 specifically acts via PD-L1 on T cells in the absence of CD28 and CTLA-4. Similarly, T cells lacking PD-1 show reduced proliferation and cytokine production when stimulated in the presence of anti-CD3 plus PD-L1 coated beads, demonstrating the inhibitory effect of PD-L1 on B7.1 on T cells. When T cells lack all known receptors for PD-L1 (ie, without PD-1 and B7.1), T cell proliferation is no longer weakened by anti-CD3 plus PD-L1 coated beads. In this way, PD-L1 can exert an inhibitory effect on T cells via either B7.1 or PD-1.

[0168] The direct interaction between B7.1 and PD-L1 suggests that the current understanding of co-stimulation is incomplete and emphasizes the significance of the expression of these molecules on T cells. Studies on PD-L1- / - T cells indicate that PD-L1 on T cells can downregulate cytokine production by T cells. Latchman et al., Proc. Natl. Acad. Sci. USA 101:10691-96 (2004). Because both PD-L1 and B7.1 are expressed on T cells, B cells, DCs, and macrophages, a directional interaction between B7.1 and PD-L1 on these cell types is possible. In addition, PD-L1 on non-hematopoietic cells may interact with B7.1 as well as PD-1 on T cells, raising the question of whether PD-L1 is involved in their regulation. One possible explanation for the inhibitory effect of the B7.1:PD-L1 interaction is that T cell PD-L1 may trap or sequester APC B7.1 from interacting with CD28.

[0169] As a result, antagonizing signaling via PD-L1 (including blocking the interaction of PD-L1 with either or both of PD-1, B7.1, thereby preventing PD-L1 from sending negative co-stimulatory signals to T cells and other antigen presenting cells) is likely to enhance immunity in response to infection (e.g., acute and chronic) and tumor immunity. In addition, the anti-PD-L1 antibodies of the present invention can be combined with antagonists of other components of PD-1:PD-L1 signaling (e.g., antagonistic anti-PD-1 and anti-PD-L2 antibodies).

[0170] The term "human PD-L1" refers to the human protein PD-L1 (SEQ ID NO: 88, PD-1 signaling, generally). As used herein, "binding to human PD-L1" or "specifically binding to human PD-L1" or "anti-PD-L1 antibody" refers to an antibody that binds to human PD-L1 with a K D Value 1.0x 10 - 8 mol / l or less (in one embodiment, K D Value 1.0x10 -9 mol / l or less) specifically binds to human PD-L1 antigen. The binding affinity is determined using standard binding assays, such as surface plasmon resonance technology ( GE-Healthcare, Uppsala, Sweden). Thus, as used herein, "an antibody that binds to human PD-L1" refers to an antibody that binds to human PD-L1 with a K D 1.0x 10 -8 mol / l or less (in one embodiment, 1.0 x 10 -8 mol / l-1.0x 10 -13 mol / l), in one embodiment, K D 1.0x10 -9 mol / l or less (in one embodiment, 1.0 x 10 -9 mol / l-1.0x10 -13 mol / l) of the antibody that specifically binds to the human PD-L1 antigen.

[0171] In one embodiment, the antibody that binds to human CSF-1R used in the combination therapy described herein is selected from the group consisting of hMab 2F11-c11, hMab 2F11-d8, hMab 2F11-e7, hMab 2F11-f12, and hMab 2F11-g1.

[0172] These antibodies are described in WO 2011 / 070024 and are characterized in comprising the following VH and VL sequences as described herein:

[0173] Table 1

[0174]

[0175] In one embodiment, the antibody that binds to human PD-L1 used in the combination therapy described herein is selected from the group consisting of 243.55.S70, 243.55.H1, 243.55.H12, 243.55.H37, 243.55.H70, 243.55.H89, 243.55.S1, 243.55.5, 243.55.8, 243.55.30, 243.55.34, 243.55.S37, 243.55.49, 243.55.51, 243.55.62, and 243.55.84.

[0176] These antibodies are described in WO 2010 / 77634 (sequences are shown in Figure 11 of WO 2010 / 77634) and are characterized in comprising the following VH and VL sequences as described herein:

[0177] Table 2

[0178]

[0179]

[0180] In one embodiment of the invention, the antibody binding to human CSF-1R used in the combination therapy described herein is characterized in comprising

[0181] a) a heavy chain variable domain VH of SEQ ID NO: 23 and a light chain variable domain VL of SEQ ID NO: 24, or

[0182] b) a heavy chain variable domain VH of SEQ ID NO: 31 and a light chain variable domain VL of SEQ ID NO: 32, or

[0183] c) a heavy chain variable domain VH of SEQ ID NO: 39 and a light chain variable domain VL of SEQ ID NO: 40, or

[0184] d) a heavy chain variable domain VH of SEQ ID NO: 47 and a light chain variable domain VL of SEQ ID NO: 48, or

[0185] e) a heavy chain variable domain VH of SEQ ID NO: 55 and a light chain variable domain VL of SEQ ID NO: 56; and the antibody binding to human PD-L1 used in the combination therapy is characterized by comprising

[0186] a) a heavy chain variable domain VH of SEQ ID NO: 89 and a light chain variable domain VL of SEQ ID NO: 92, or

[0187] b) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 93, or

[0188] c) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 94, or

[0189] d) a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:95, or

[0190] e) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 96, or

[0191] f) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 97, or

[0192] g) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 98, or

[0193] h) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 99, or

[0194] i) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 100, or

[0195] j) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 101, or

[0196] k) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 102, or

[0197] l) a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:103, or

[0198] m) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 104, or

[0199] n) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 105, or

[0200] o) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 106, or

[0201] p) the heavy chain variable domain VH of SEQ ID NO:91 and the light chain variable domain VL of SEQ ID NO:107.

[0202] In one embodiment, the antibody binding to human CSF-1R used in the combination therapy is characterized in comprising a heavy chain variable domain VH of SEQ ID NO:23 and a light chain variable domain VL of SEQ ID NO:24.

[0203] In one embodiment, the antibody binding to human CSF-1R used in the combination therapy is characterized in comprising a heavy chain variable domain VH of SEQ ID NO:31 and a light chain variable domain VL of SEQ ID NO:32.

[0204] In one embodiment, the antibody binding to human CSF-1R used in the combination therapy is characterized in comprising a heavy chain variable domain VH of SEQ ID NO:39 and a light chain variable domain VL of SEQ ID NO:40.

[0205] In one embodiment, the antibody binding to human CSF-1R used in the combination therapy is characterized in comprising a heavy chain variable domain VH of SEQ ID NO:47 and a light chain variable domain VL of SEQ ID NO:48.

[0206] In one embodiment, the antibody binding to human PD-L1 used in the combination therapy is characterized in comprising a heavy chain variable domain VH of SEQ ID NO:89 and a light chain variable domain VL of SEQ ID NO:92.

[0207] In one embodiment, the antibody binding to human PD-L1 used in the combination therapy is characterized in comprising a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:93.

[0208] In one embodiment, the antibody binding to human PD-L1 used in the combination therapy is characterized in comprising a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:94.

[0209] In one embodiment, the antibody binding to human PD-L1 used in the combination therapy is characterized in comprising a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:95.

[0210] In one embodiment, the antibody binding to human PD-L1 used in the combination therapy is characterized in comprising a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:96.

[0211] In one embodiment, the antibody binding to human PD-L1 used in the combination therapy is characterized in comprising a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:97.

[0212] In one embodiment, the antibody binding to human PD-L1 used in the combination therapy is characterized in comprising a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:98.

[0213] In one embodiment, the antibody binding to human PD-L1 used in the combination therapy is characterized in comprising a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:99.

[0214] In one embodiment, the antibody binding to human PD-L1 used in the combination therapy is characterized in comprising a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:100.

[0215] In one embodiment, the antibody binding to human PD-L1 used in the combination therapy is characterized in comprising a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:101.

[0216] In one embodiment, the antibody binding to human PD-L1 used in the combination therapy is characterized in comprising a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:102.

[0217] In one embodiment, the antibody binding to human PD-L1 used in the combination therapy is characterized in comprising a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:103.

[0218] In one embodiment, the antibody binding to human PD-L1 used in the combination therapy is characterized in comprising a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:104.

[0219] In one embodiment, the antibody binding to human PD-L1 used in the combination therapy is characterized in comprising a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:105.

[0220] In one embodiment, the antibody binding to human PD-L1 used in the combination therapy is characterized in comprising a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:106.

[0221] In one embodiment, the antibody binding to human PD-L1 used in the combination therapy is characterized in comprising a heavy chain variable domain VH of SEQ ID NO:91 and a light chain variable domain VL of SEQ ID NO:107.

[0222] In a preferred embodiment of the present invention, the antibody binding to human CSF-1R used in the combination therapy described herein is characterized by comprising a heavy chain variable domain VH of SEQ ID NO: 39 and a light chain variable domain VL of SEQ ID NO: 40, and the antibody binding to human PD-L1 used in the combination therapy is characterized by comprising a heavy chain variable domain VH of SEQ ID NO: 89 and a light chain variable domain VL of SEQ ID NO: 92.

[0223] The term "epitope" refers to a protein determinant in human CSF-1R or PD-L1 that is capable of specific binding to an antibody. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains, and epitopes usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that binding to the former, but not the latter, is lost in the presence of denaturing solvents.

[0224] As used herein, a "variable domain" (light chain variable domain (V L ), heavy chain variable domain (V H )) represents each of the light and heavy chain domain pairs that are directly involved in the binding of the antibody to the antigen. The variable light and heavy chain domains have the same overall structure, and each domain comprises four framework (FR) regions, whose sequences are widely conserved, connected by three "hypervariable regions" (or complementarity determining regions, CDRs). The framework regions adopt a β-sheet conformation, and the CDRs can form loops connecting the β-sheet structure. The CDRs in each chain maintain their three-dimensional structure by the framework regions, and together with the CDRs from the other chain form an antigen binding site. The heavy and light chain CDR3 regions of the antibody play a particularly important role in the binding specificity / affinity of the antibodies according to the invention, and therefore provide a further object of the invention.

[0225] The term "antigen-binding portion of an antibody" as used herein refers to the amino acid residues in an antibody that are responsible for antigen binding. The antigen-binding portion of an antibody comprises amino acid residues from "complementarity determining regions" or "CDRs". "Framework" or "FR" regions are those regions of the variable domain that differ from the hypervariable region residues as defined herein. Thus, the light and heavy chain variable domains of an antibody comprise, from N to C-terminus, the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In particular, CDR3 of the heavy chain is the region that contributes most to antigen binding and defines the properties of the antibody. CDR and FR regions are determined according to the standard definitions of Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) and / or those residues from the "hypervariable loops".

[0226] As used herein, the term "nucleic acid" or "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA.

[0227] As used within this application, the term "amino acid" refers to the group of naturally occurring carboxyl α-amino acids, including alanine (three letter code: ala, single letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine ​​(cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

[0228] The "Fc portion" of an antibody is not directly involved in the binding of the antibody to an antigen, but exhibits a variety of effector functions. "Fc portion of an antibody" is a term well known to the skilled artisan and is defined based on the cleavage of an antibody by papain. According to the amino acid sequence of its heavy chain constant region, antibodies or immunoglobulins are divided into classes: IgA, IgD, IgE, IgG and IgM, and several of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, and IgG4, IgA1, and IgA2. According to the heavy chain constant region, different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The Fc portion of an antibody is directly involved in ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity) based on complement activation, C1q binding and Fc receptor binding. Complement activation (CDC) is initiated by complement factor C1q binding to the Fc portion of most IgG antibody subclasses. While the effect of antibodies on the complement system is dependent on certain conditions, binding to CIq is caused by a defined binding site in the Fc part. Such binding sites are known in the art and are described, for example, in Boackle, RJ et al., Nature 282 (1979) 742-743; Lukas, TJ et al., J. Immunol. 127 (1981) 2555-2560; Brunhouse, R. and Cebra, JJ, Mol. Immunol. 16 (1979) 907-917; Burton, DR et al., Nature 288 (1980) 338-344; Thommesen, JE et al., Mol. Immunol. 37 (2000) 995-1004; Idusogie, EE et al., J. Immunol. 164 (2000) 4178-4184; Hezareh, M. et al., J. Virology 75 (2001) 12161-12168; Morgan, A. et al., Immunology 86 (1995) 319-324; EP 0307434. Such binding sites are, for example, L234, L235, D270, N297, E318, K320, K322, P331 and P329 (numbering according to the EU index of Kabat, EA, see below). Antibodies of the subclasses IgG1, IgG2 and IgG3 usually show complement activation and binding to C1q and C3, whereas IgG4 does not activate the complement system and does not bind to C1q and C3.

[0229] In one embodiment, the antibody according to the invention comprises an Fc part derived from human origin and preferably all other parts of human constant regions. As used herein, the term "Fc part derived from human origin" means an Fc part that is an Fc part of a human antibody belonging to the subclass IgG1, IgG2, IgG3 or IgG4, preferably an Fc part from the human IgG1 subclass, a mutant Fc part from the human IgG1 subclass (in one embodiment with mutations on L234A + L235A), an Fc part from the human IgG4 subclass or a mutant Fc part from the human IgG4 subclass (in one embodiment with mutations on S228P). In a preferred embodiment, the human heavy chain constant region is SEQ ID NO: 58 (human IgG1 subclass), in another preferred embodiment, the human heavy chain constant region is SEQ ID NO: 59 (human IgG1 subclass with mutations L234A and L235A), in another preferred embodiment, the human heavy chain constant region is SEQ ID NO: 60 (human IgG4 subclass), and in another preferred embodiment, the human heavy chain constant region is SEQ ID NO: 61 (human IgG4 subclass with mutation S228P). In one embodiment, the antibody has reduced or minimal effector function. In one embodiment, the minimal effector function is derived from an effector-reducing Fc mutation. In one embodiment, the effector-reducing Fc mutation is L234A / L235A or L234A / L235A / P329G or N297A or D265A / N297A. In one embodiment, the effector reducing Fc mutations selected for each antibody are independently selected from the group comprising L234A / L235A, L234A / L235A / P329G, N297A and D265A / N297A.

[0230] In one embodiment, the antibodies described herein are of the human IgG class (ie, of the IgGl subclass, IgG2 subclass, IgG3 subclass, or IgG4 subclass).

[0231] In a preferred embodiment, the antibodies described herein are of human IgG1 subclass or human IgG4 subclass. In one embodiment, the antibodies described herein are of human IgG1 subclass. In one embodiment, the antibodies described herein are of human IgG4 subclass.

[0232] In one embodiment, the antibodies described herein are characterized in that the constant chain is of human origin. Such constant chains are well known in the art and are described, for example, in Kabat, EA, (see, for example, Johnson, G. and Wu, TT, Nucleic Acids Res. 28 (2000) 214-218). For example, a useful human heavy chain constant region comprises the amino acid sequence SEQ ID NO: 58. For example, a useful human light chain constant region comprises the kappa light chain constant region amino acid sequence SEQ ID NO: 57.

[0233] The invention comprises a method for treating a patient in need of treatment, characterized in that a therapeutically effective amount of an antibody according to the invention is administered to the patient.

[0234] The invention comprises the use of an antibody according to the invention for such treatment.

[0235] A preferred embodiment of the present invention is the CSF-1R antibody of the present invention for use in treating a "CSF-1R mediated disease", or the CSF-1R antibody of the present invention for use in preparing a medicament for treating a "CSF-1R mediated disease", which can be described as follows:

[0236] CSF-1R signaling may be involved in tumor growth and metastasis through three distinct mechanisms. The first is that expression of CSF ligands and receptors is found in tumor cells derived from the female reproductive system (breast / mammary gland, ovary, endometrium, cervix) (Scholl, SM et al., J. Natl. Cancer Inst. 86 (1994) 120-126; Kacinski, BM, Mol. Reprod. Dev. 46 (1997) 71-74; Ngan, HY et al., Eur. J. Cancer 35 (1999) 1546-1550; Kirma, N. et al., Cancer Res 67 (2007) 1918-1926), and this expression is associated with breast cancer xenograft growth and poor prognosis in breast cancer patients. Two point mutations in CSF-1R were seen in approximately 10-20% of patients with acute myeloid leukemia, chronic myeloid leukemia, and myelodysplasia tested in one study, and one of the mutations was found to disrupt receptor turnover (Ridge, SA et al., Proc. Natl. Acad. Sci USA 87 (1990) 1377-1380). However, the incidence of the mutations could not be confirmed in a later study (Abu-Duhier, FM et al., Br. J. Haematol. 120 (2003) 464-470). Mutations have also been found in some cases of hepatocellular carcinoma (Yang, DH et al., Hepatobiliary Pancreat. Dis. Int. 3 (2004) 86-89) and idiopathic myelofibrosis (Abu-Duhier, FM et al., Br. J. Haematol. 120 (2003) 464-470). Recently, the Y571D mutation in CSF-1R was identified in the GDM-1 cell line derived from a patient with myelomonocytic leukemia (Chase, A. et al., Leukemia 23 (2009) 358-364).

[0237] Pigmented villonodular synovitis (PVNS) and tenosynovial giant cell tumor (TGCT) can occur due to a translocation that fuses the M-CSF gene to the collagen gene COL6A3 and results in overexpression of M-CSF (West, RB et al., Proc. Natl. Acad. Sci. USA 103 (2006) 690-695). It has been proposed that the landscape effect is responsible for the resulting tumor mass composed of mononuclear cells attracted to cells expressing M-CSF. TGCT is a smaller tumor that can be removed relatively easily from the site of its primary occurrence. PVNS is more aggressive because it can recur in large joints and is not easily controlled by surgery.

[0238] The second mechanism is based on blocking signal transduction via M-CSF / CSF-1R at metastatic sites in the bone, which induces osteoclastogenesis, bone resorption and osteolytic bone damage. Breast cancer, multiple myeloma and lung cancer are examples of cancers that have been found to metastasize to the bone and cause osteolytic bone disease leading to skeletal complications. M-CSF released by tumor cells and matrix cooperates with the receptor activator nuclear factor kappa-B ligand-RANKL to induce hematopoietic myeloid monocytic progenitors to differentiate into mature osteoclasts. In this process, M-CSF acts as a permissive factor by giving osteoclasts a survival signal (Tanaka, S. et al., J. Clin. Invest. 91 (1993) 257-263). Inhibiting CSF-1R activity with anti-CSF-1R antibodies during osteoclast differentiation and maturation is likely to prevent the imbalance of osteoclast activity that causes osteolytic disease and related skeletal related events in metastatic diseases. Although breast cancer, lung cancer and multiple myeloma usually lead to osteolytic lesions, in prostate cancer, metastasis to bone initially has an osteoblastic appearance, in which elevated bone formation activity produces "woven bone" different from the typical lamellar structure of normal bone. During disease progression, bone lesions exhibit significant osteolytic components and high serum levels of bone resorption, and suggest that antiresorptive therapy may be useful. Bisphosphonates have been shown to inhibit osteolytic lesion formation and reduce the number of skeletal-related events (only in men with hormone-refractory metastatic prostate cancer), but in this regard, their effect on osteoblastic lesions is controversial, and bisphosphonates have not been shown to be useful in preventing bone metastasis or hormone-responsive prostate cancer. The effects of antiresorptive agents in mixed osteolytic / osteoblastic prostate cancer are still under clinical investigation (Choueiri, MB et al., Cancer Metastasis Rev. 25 (2006) 601-609; Vessella, RL and Corey, E., Clin. Cancer Res. 12 (20 Pt 2) (2006) 6285s-6290s).

[0239] The third mechanism is based on recent observations that tumor-associated macrophages (TAMs) found in solid tumors of breast, prostate, ovarian, and cervical cancers are associated with poor prognosis (Bingle, L. et al., J. Pathol. 196 (2002) 254-265; Pollard, JW, Nat. Rev. Cancer 4 (2004) 71-78). Macrophages are recruited to tumors by M-CSF and other chemokines. Macrophages can then promote tumor progression by secreting angiogenic factors, proteases, and other growth factors and cytokines, and can be blocked by inhibiting CSF-1R signaling. Recently, Zins et al. (Zins, K. et al., Cancer Res. 67 (2007) 1038-1045) showed that expression of siRNA for tumor necrosis factor alpha (TNFα), M-CSF, or a combination of both reduced tumor growth by 34% to 50% in a mouse xenograft model after intratumoral injection of the corresponding siRNA. siRNA targeting TNFα secreted by human SW620 cells reduced mouse M-CSF levels and resulted in a decrease in macrophages in tumors. In addition, treatment of MCF7 tumor xenografts with antigen-binding fragments directed against M-CSF did result in 40% tumor growth inhibition, reversal of resistance to chemotherapeutic agents, and improved mouse survival when given in combination with chemotherapeutic agents (Paulus, P. et al., Cancer Res. 66 (2006) 4349-4356).

[0240] TAMs are the only example of a link between chronic inflammation and cancer. There is other evidence for the link between inflammation and cancer, such as many chronic diseases are associated with an increased risk of cancer, cancer occurs at sites of chronic inflammation, and chemical mediators of inflammation are found in many cancers; cells or chemical mediators that eliminate inflammation inhibit the occurrence of experimental cancers, and long-term use of anti-inflammatory agents reduces the risk of some cancers. Many inflammatory conditions are linked to cancer, including Helicobacter pylori (H.pylori)-induced gastritis for gastric cancer, schistosomiasis for bladder cancer, HHVX for Kaposi's sarcoma, endometriosis for ovarian cancer, and prostatitis for prostate cancer (Balkwill, F. et al., Cancer Cell 7 (2005) 211-217). Macrophages are key cells in chronic inflammation and respond differently to their microenvironment. There are two types of macrophages that are considered to be the extremes of a continuous functional state: M1 macrophages involve type 1 responses. These responses involve activation caused by microbial products and thus killing of pathogenic microorganisms, producing reactive oxygen mediators. At the other extreme are M2 macrophages, which are involved in type 2 responses, promote cell proliferation, regulate inflammation and adaptive immunity, and promote tissue remodeling, angiogenesis, and repair (Mantovani, A. et al., Trends Immunol. 25 (2004) 677-686). Chronic inflammation leading to the establishment of neoplasia is often associated with M2 macrophages. A key cytokine mediating inflammatory responses is TNFα, which, as its name suggests, can stimulate anti-tumor immunity and hemorrhagic necrosis at high doses, but has recently been found to be expressed by tumor cells and act as a tumor promoter (Zins, K. et al., Cancer Res. 67 (2007) 1038-1045; Balkwill, F., Cancer Metastasis Rev. 25 (2006) 409-416). There is still a need to better understand the specific role of macrophages on tumors, including the potential spatial and temporal dependence of their functions and their association with specific tumor types.

[0241] Thus, one embodiment of the present invention is a CSF-1R antibody as described herein, in combination with an anti-PD-L1 antibody as described herein, for use in treating cancer. As used herein, the term "cancer" can be, for example, lung cancer, non-small cell lung (NSCL) cancer, bronchoalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, cancer of the stomach, colon cancer, breast cancer, cancer of the uterus, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, kidney cancer, In some embodiments, the present invention relates to cancer of the adrenal gland, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic cancer, mesothelioma, hepatocellular carcinoma, gallbladder cancer, central nervous system (CNS) neoplasms, spinal axis tumors, brain stem gliomas, glioblastoma multiforme, astrocytomas, Schwannoma, ependymomas, medulloblastomas, meningiomas, squamous cell carcinomas, pituitary adenomas, lymphomas, lymphocytic leukemias, including refractory forms of any of the above cancers, or a combination of one or more of the above cancers. In a preferred embodiment, such cancer is breast cancer, colorectal cancer, melanoma, head and neck cancer, lung cancer, or prostate cancer. In a preferred embodiment, such cancer is breast cancer, ovarian cancer, cervical cancer, lung cancer, or prostate cancer. In a preferred embodiment, such cancer is breast cancer, lung cancer, colon cancer, ovarian cancer, melanoma cancer, bladder cancer, renal cancer, kidney cancer, liver cancer, head and neck cancer, colorectal cancer, pancreatic cancer, gastric cancer, esophageal cancer, mesothelioma, prostate cancer, leukemia, lymphoma, myeloma. In a preferred embodiment, such cancer is further characterized by CSF-1 or CSF-1R expression or overexpression. Yet another embodiment of the present invention is a CSF-1R antibody of the present invention for simultaneous treatment of primary tumors and new metastases. Thus, another embodiment of the present invention is a CSF-1R antibody of the present invention for the treatment of periodontitis, histiocytosis X, osteoporosis, Paget's disease of bone (PDB), bone loss due to cancer treatment, periprosthetic osteolysis, glucocorticoid-induced osteoporosis, rheumatoid arthritis, psoriatic arthritis, osteoarthritis, inflammatory arthritis, and inflammation.

[0242] Rabello, D. et al., Biochem. Biophys. Res. Commun. 347 (2006) 791-796 demonstrated that a SNP in the CSF1 gene exhibited a positive association with aggressive periodontitis, an inflammatory disease of the periodontium causing tooth loss due to resorption of alveolar bone.

[0243] Histiocytosis X (also known as Langerhans cell histiocytosis, LCH) is a proliferative disease of Langerhans dendritic cells (which show differentiation into osteoclasts in bone and extra-bone LCH lesions). Langerhans cells are derived from circulating monocytes. Elevated M-CSF levels measured in serum and lesions were found to correlate with disease severity (da Costa, CE et al., J. Exp. Med. 201 (2005) 687-693). The disease occurs primarily in pediatric patient populations and has to be treated with chemotherapy when the disease becomes systemic or relapses.

[0244] The pathophysiology of osteoporosis is mediated by the loss of bone-forming osteoblasts and increased osteoclast-dependent bone resorption. Supporting data are described in Cenci et al., showing that anti-M-CSF antibody injections preserved bone density and inhibited bone resorption in ovariectomized mice (Cenci, S. et al., J. Clin. Invest. 105 (2000) 1279-1287). Recently, a potential link between postmenopausal bone loss caused by estrogen deficiency was identified, and the presence of TNFα-producing T cells was found to affect bone metabolism (Roggia, C. et al., Minerva Med. 95 (2004) 125-132). One possible mechanism is the induction of M-CSF by TNFα in vivo. The important role of M-CSF in TNF-α-induced osteoclastogenesis is confirmed by the following effect: antibodies against M-CSF blocked TNFα-induced osteolysis in mice, thus making inhibitors of CSF-1R signaling a potential target for inflammatory arthritis (Kitaura, H. et al., J. Clin. Invest. 115 (2005) 3418-3427).

[0245] Paget's disease of bone (PDB) is the second most common bone metabolic disorder after osteoporosis, in which focal abnormalities of increased bone turnover lead to complications such as bone pain, deformity, pathological fractures, and deafness. Mutations in four genes have been identified that regulate normal osteoclast function and predispose individuals to PDB and related disorders: insertion mutations in TNFRSF11A encoding receptor activator of nuclear factor (NF)κB (RANK)-a key regulator of osteoclast function, inactivating mutations in TNFRSF11B encoding osteoprotegerin, a decoy receptor for RANK ligands, mutations in the sequestosome 1 gene (SQSTM1), encoding an important scaffolding protein in the NFκB pathway, and mutations in the valosin-containing protein (VCP) gene. This gene encodes VCP, which plays a role in targeting NFκB inhibitors for degradation by the proteasome (Daroszewska, A. and Ralston, SH, Nat. Clin. Pract. Rheumatol. 2 (2006) 270-277). Targeted CSF-1R inhibitors offer the opportunity to indirectly block dysregulation of RANKL signaling and add additional therapeutic options to currently used bisphosphonates.

[0246] Cancer therapy-induced bone loss, especially in patients with breast and prostate cancer, is another indication for which targeted CSF-1R inhibitors can prevent bone loss (Lester, JE et al., Br. J. Cancer 94 (2006) 30-35). As the prognosis of early breast cancer improves, the long-term consequences of adjuvant therapy become more important because some therapies (including chemotherapy, irradiation, aromatase inhibitors, and oophorectomy) affect bone metabolism by reducing bone mineral density, leading to osteoporosis and an increased risk of associated fractures (Lester, JE et al., Br. J. Cancer 94 (2006) 30-35). Equivalent to adjuvant aromatase inhibitor therapy in breast cancer is androgen ablation therapy in prostate cancer, which results in a significant increase in bone mineral density loss and osteoporosis-related fracture risk (Stoch, SA et al., J. Clin. Endocrinol. Metab. 86 (2001) 2787-2791).

[0247] Targeted inhibition of CSF-1R signaling may be beneficial in other indications and when targeting cell types including osteoclasts and macrophages (e.g., treatment response to specific complications of joint replacement due to rheumatoid arthritis). Periprosthetic bone loss and therefore implant failure due to prosthesis loosening is a major complication of joint replacement and requires repeated surgeries, placing a high socioeconomic burden on individual patients and the healthcare system. To date, there are no approved drug therapies to prevent or inhibit periprosthetic osteolysis (Drees, P. et al., Nat. Clin. Pract. Rheumatol. 3 (2007) 165-171).

[0248] Glucocorticoid-induced osteoporosis (GIOP) is another indication in which CSF-1R inhibitors can prevent bone loss following long-term glucocorticoid administration for various conditions such as chronic obstructive pulmonary disease, asthma, and rheumatoid arthritis (Guzman-Clark, JR et al., Arthritis Rheum. 57 (2007) 140-146; Feldstein, AC et al., Osteoporos. Int. 16 (2005) 2168-2174).

[0249] Rheumatoid arthritis, psoriatic arthritis and inflammatory joint diseases are potential indications for CSF-1R signaling inhibitors because they are composed of macrophage components and have varying degrees of bone destruction (Ritchlin, CT et al., J. Clin. Invest. 111 (2003) 821-831). Osteoarthritis and rheumatoid arthritis are inflammatory autoimmune diseases caused by the accumulation of macrophages in connective tissue and the infiltration of macrophages into the synovial fluid, which is at least partly mediated by M-CSF. Campbell, IK et al., J. Leukoc. Biol. 68 (2000) 144-150 demonstrated that M-CSF is produced in vitro by human joint tissue cells (chondrocytes, synovial fibroblasts) and is found in the synovial fluid of patients with rheumatoid arthritis, suggesting that it contributes to synovial tissue proliferation and macrophage infiltration, which is related to the pathogenesis of the disease. Inhibition of CSF-1R signaling has the potential to control the number of macrophages in the joints and alleviate pain from the associated bone destruction. To minimize adverse effects and further understand the impact of CSF-1R signaling in these indications, one approach is to specifically inhibit CSF-1R without targeting the myriad other kinases, such as Raf kinase.

[0250] Recent literature reports associate elevated circulating M-CSF with progression of atherosclerosis and poor prognosis in chronic coronary artery disease (Saitoh, T. et al., J. Am. Coll. Cardiol. 35 (2000) 655-665; Ikonomidis, I. et al., Eur. Heart. J. 26 (2005) p. 1618-1624); M-CSF affects the atherosclerotic process by helping to form foam cells (macrophages that have taken up oxidized LDL) that express CSF-1R and represent the initial plaque (Murayama, T. et al., Circulation 99 (1999) 1740-1746).

[0251] Expression and signaling of M-CSF and CSF-1R are found in activated microglia. Microglia (i.e., resident macrophages of the central nervous system) can be activated by a variety of injuries, including infection and trauma. M-CSF is considered to be a key regulator of inflammatory responses in the brain, and M-CSF levels are elevated in HIV-1, encephalitis, Alzheimer's disease (AD), and brain tumors. Microgliosis (microgliosis) (as a consequence of autocrine signaling through M-CSF / CSF-1R) leads to the induction of inflammatory cytokines and nitric oxide release, as demonstrated, for example, using experimental neuronal damage models (Hao, AJ et al., Neuroscience 112 (2002) 889-900; Murphy, GM, Jr. et al., J. Biol. Chem. 273 (1998) 20967-20971). It was found that microglia with increased CSF-1R expression surrounded plaques in AD and in the amyloid precursor protein V717F transgenic mouse model of AD (Murphy, GM, Jr. et al., Am. J. Pathol. 157 (2000) 895-904). On the other hand, op / op mice with fewer microglia in the brain led to small fiber deposition of A-β and neuronal loss compared with normal controls, suggesting that microglia do have a neuroprotective function in the occurrence of AD loss in op / op mice (Kaku, M. et al., Brain Res. Brain Res. Protoc. 12 (2003) 104-108).

[0252] Expression and signaling of M-CSF and CSF-1R are associated with inflammatory bowel disease (IBD) (WO2005 / 046657). The term "inflammatory bowel disease" refers to severe, chronic intestinal disorders characterized by chronic inflammation at multiple sites in the gastrointestinal tract, and specifically includes ulcerative colitis (UC) and Crohn's disease.

[0253] Thus, another embodiment of the present invention is a CSF-1R antibody characterized by the amino acid sequences and amino acid sequence fragments described above, in combination with an anti-PD-L1 antibody characterized by the amino acid sequences and amino acid sequence fragments described above, for the treatment of periodontitis, histiocytosis X, osteoporosis, Paget's disease of bone (PDB), bone loss caused by cancer therapy, periprosthetic osteolysis, glucocorticoid-induced osteoporosis, rheumatoid arthritis, psoriatic arthritis, osteoarthritis, inflammatory joint diseases, and inflammation.

[0254] The present invention comprises a combination therapy of an antibody that binds to human CSF-1R (characterized by the amino acid sequence and amino acid sequence fragments described above) and an anti-PD-L1 antibody (characterized by the amino acid sequence and amino acid sequence fragments described above) for treating cancer.

[0255] The present invention comprises a combination therapy of an antibody that binds to human CSF-1R (characterized by the amino acid sequence and amino acid sequence fragments described above) and an anti-PD-L1 antibody (characterized by the amino acid sequence and amino acid sequence fragments described above) for treating bone loss.

[0256] The present invention comprises a combination therapy of an antibody that binds to human CSF-1R (characterized by the amino acid sequence and amino acid sequence fragments described above) and an anti-PD-L1 antibody (characterized by the amino acid sequence and amino acid sequence fragments described above) for preventing or treating metastasis.

[0257] The present invention comprises a combination therapy of an antibody that binds to human CSF-1R (characterized by the amino acid sequence and amino acid sequence fragments described above) and an anti-PD-L1 antibody (characterized by the amino acid sequence and amino acid sequence fragments described above) for treating inflammatory diseases.

[0258] The present invention comprises a combination therapy of an antibody that binds to human CSF-1R (characterized by the amino acid sequence and amino acid sequence fragments described above) and an anti-PD-L1 antibody (characterized by the amino acid sequence and amino acid sequence fragments described above) for treating immune-related diseases such as tumor immunity or delaying its progression.

[0259] The present invention comprises a combination therapy of an antibody that binds to human CSF-1R (characterized by the amino acid sequence and amino acid sequence fragments described above) and an anti-PD-L1 antibody (characterized by the amino acid sequence and amino acid sequence fragments described above) for stimulating an immune response or function, such as T cell activity.

[0260] The present invention comprises the use of antibodies (characterized in comprising antibodies that bind to human CSF-1R, characterized in that they comprise the amino acid sequence and amino acid sequence fragments described above) for treating cancer in combination with the anti-PD-L1 antibodies described herein or for preparing a medicament for treating cancer in combination with the anti-PD-L1 antibodies described herein.

[0261] The present invention comprises the use of an antibody (characterized in comprising an antibody that binds to human CSF-1R, characterized in that it comprises the amino acid sequence and the amino acid sequence fragment described above) for treating bone loss in combination with the anti-PD-L1 antibody described herein or for preparing a medicament for treating bone loss in combination with the anti-PD-L1 antibody described herein.

[0262] The present invention comprises the use of antibodies (characterized in comprising antibodies that bind to human CSF-1R, characterized in that the amino acid sequence and amino acid sequence fragment described above) for preventing or treating metastasis in combination with the anti-PD-L1 antibodies described herein or for preparing a medicament for preventing or treating metastasis in combination with the anti-PD-L1 antibodies described herein.

[0263] The present invention comprises the use of antibodies (characterized in comprising antibodies that bind to human CSF-1R, characterized in that they comprise the amino acid sequence and amino acid sequence fragments described above) for treating inflammatory diseases in combination with the anti-PD-L1 antibodies described herein or for preparing a medicament for treating inflammatory diseases in combination with the anti-PD-L1 antibodies described herein.

[0264] The present invention comprises an antibody (characterized in that it comprises an antibody that binds to human CSF-1R, characterized in that it comprises an amino acid sequence and an amino acid sequence fragment as described above) for treating immune-related diseases such as tumor immunity or delaying its progression in combination with the anti-PD-L1 antibody as described herein or for preparing a medicament for treating immune-related diseases such as tumor immunity or delaying its progression in combination with the anti-PD-L1 antibody as described herein.

[0265] The present invention comprises the use of antibodies (characterized in comprising antibodies that bind to human CSF-1R, characterized in that they comprise the amino acid sequences and amino acid sequence fragments described above) for stimulating an immune response or function, such as T cell activity, in combination with the anti-PD-L1 antibodies described herein or for the preparation of a medicament for stimulating an immune response or function, such as T cell activity, in combination with the anti-PD-L1 antibodies described herein.

[0266] In a preferred embodiment of the present invention, the antibody binding to human CSF-1R used in the combined treatment and medical use in the different diseases described above is characterized by comprising a heavy chain variable domain VH of SEQ ID NO: 39 and a light chain variable domain VL of SEQ ID NO: 40, and the antibody binding to human PD-L1 used in such combined treatment is characterized by comprising a heavy chain variable domain VH of SEQ ID NO: 89 and a light chain variable domain VL of SEQ ID NO: 92.

[0267] The antibodies described herein are preferably produced by recombinant means. Such methods are widely known in the prior art and include protein expression in prokaryotic and eukaryotic cells, followed by separation of antibody polypeptides and usually purification to a pharmaceutically acceptable purity. For protein expression, nucleic acids encoding light and heavy chains or fragments thereof are inserted into expression vectors by standard methods. Expression is performed in suitable prokaryotic or eukaryotic host cells, such as CHO cells, NSO cells, SP2 / 0 cells, HEK293 cells, COS cells, yeast, or E. coli cells, and the antibodies are recovered from the cells (from the supernatant or after cell lysis).

[0268] The recombinant production of antibodies is well known in the art and is described in, for example, review articles Makrides, SC, Protein Expr. Purif. 17 (1999) 183-202; Geisse, S. et al., Protein Expr. Purif. 8 (1996) 271-282; Kaufman, RJ, Mol. Biotechnol. 16 (2000) 151-161; Werner, RG, Drug Res. 48 (1998) 870-880.

[0269] The antibody can be present in intact cells, in cell lysates, or in partially purified or substantially pure form. Purification is performed by standard techniques to eliminate other cellular components or other contaminants, such as other cellular nucleic acids or proteins, including alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques known in the art. See Ausubel, F. et al., eds., Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

[0270] Expression in NS0 cells is described, for example, in Barnes, LM et al., Cytotechnology 32 (2000) 109-123; Barnes, LM et al., Biotech. Bioeng. 73 (2001) 261-270. Transient expression is described, for example, in Durocher, Y. et al., Nucl. Acids. Res. 30 (2002) E9. Cloning of variable domains is described in Orlandi, R. et al., Proc. Natl. Acad. Sci. USA 86 (1989) 3833-3837; Carter, P. et al., Proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289; Norderhaug, L. et al., J. Immunol. Methods 204 (1997) 77-87. A preferred transient expression system (HEK 293) is described by Schlaeger, E.-J. and Christensen, K., Cytotechnology 30 (1999) 71-83, and Schlaeger, E.-J., J. Immunol. Methods 194 (1996) 191-199.

[0271] The heavy and light chain variable domains according to the present invention are combined with sequences for promoter, translation initiation, constant region, 3' untranslated region, polyadenylation, and translation termination to form an expression vector construct. The heavy and light chain expression constructs can be combined into a single vector, co-transfected, sequentially transfected, or transfected separately into host cells and then fused to form a single host cell expressing both chains.

[0272] Control sequences suitable for prokaryotes include, for example, a promoter, an optional operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, enhancers, and polyadenylation signals.

[0273] A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, if the DNA of a presequence or secretory leader is expressed as a preprotein that participates in the secretion of a polypeptide, it is operably linked to the DNA of the polypeptide; if a promoter or enhancer affects the transcription of the sequence, it is operably linked to a coding sequence; or if a ribosome binding site is positioned so as to facilitate translation, it is operably linked to a coding sequence. In general, "operably linked" means that the DNA sequences being linked are continuous, and in the case of a secretory leader, continuous and in the reading frame. However, enhancers do not have to be continuous. Connection is achieved by connection at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or joints are used in accordance with conventional practice.

[0274] Monoclonal antibodies are suitably separated from culture fluid by conventional immunoglobulin purification procedures, such as, for example, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography. Using conventional procedures, DNA and RNA encoding monoclonal antibodies and sequencing are easily separated. Hybridoma cells can serve as the source of such DNA and RNA. Once separated, the DNA can be inserted into an expression vector, which is then transfected into a host cell (such as HEK 293 cells, CHO cells or myeloma cells) that does not generate immunoglobulin proteins in addition to obtain the synthesis of recombinant monoclonal antibodies in the host cell.

[0275] As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the terms "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, regardless of the number of transfers. It is also understood that the DNA content of all progeny may not be precisely identical, due to intentional or unintentional mutations. Variant progeny that have the same function or biological activity as screened for in the initial transformed cell are included.

[0276] In another aspect, the invention provides a composition, such as a pharmaceutical composition, comprising one or a group of monoclonal antibodies or antigen-binding portions thereof of the invention, formulated together with a pharmaceutically acceptable carrier.

[0277] As used herein, "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption / resorption delaying agents, etc. Preferably, the carrier is suitable for injection or infusion.

[0278] The compositions of the present invention may be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and / or mode of administration will vary depending on the desired results.

[0279] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for preparing sterile injectable solutions or dispersions. The use of such media and medicaments for pharmaceutically active substances is known in the art. In addition to water, the carrier can be, for example, an isotonic buffered saline solution.

[0280] Regardless of the route of administration selected, the compound of the present invention and / or the pharmaceutical composition of the present invention, which may be used in a suitable hydrated form, is formulated into a pharmaceutically acceptable dosage form by conventional methods known to those skilled in the art.

[0281] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of the active ingredient (effective amount) that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient. The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the particular composition of the present invention employed, or its ester, salt or amide, the route of administration, the time of administration, the excretion rate of the particular compound employed, other drugs, compounds and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and similar factors well known in the medical field.

[0282] The term "therapeutic method" or its equivalent, when applied to, for example, cancer, refers to a procedure or course of action designed to reduce or eliminate the number of cancer cells in a patient, or to alleviate the symptoms of cancer. A "therapeutic method" for cancer or another proliferative disorder does not necessarily mean that the cancer cells or other disorder will actually be eliminated, the number of cells or the disorder will actually be reduced, or the symptoms of the cancer or other disorder will actually be alleviated. Often, even methods of treating cancer that have a lower probability of success but are nevertheless believed to induce an overall beneficial course of action in view of the medical history and estimated patient survival expectations will be implemented.

[0283] The terms "administered in combination with", "co-administration", "combination therapy" or "combination treatment" refer to the administration of an anti-CSF-1R described herein and an anti-PD-L1 antibody described herein, for example, as separate formulations / applications (or as one single formulation / application). Co-administration can be simultaneous or sequential in any order, wherein preferably there is a period during which all active agents exert their biological activities simultaneously. The antibody and the additional agent are co-administered simultaneously or sequentially (e.g., intravenously (iv) via continuous infusion). When the two therapeutic agents are co-administered sequentially, the doses are administered on the same day in two separate administrations, or one of the agents is administered on day 1 and the second is co-administered on day 2 to day 7, preferably on day 2 to day 4. Thus, in one embodiment, the term "sequentially" means within 7 days after the dose of the first component, preferably within 4 days after the dose of the first component; and the term "simultaneously" means the same time. The term "co-administered" with respect to maintenance doses of anti-CSF-1R antibody and / or anti-PD-L1 antibody means that if the treatment cycle is suitable for both drugs, for example weekly, the maintenance doses can be co-administered at the same time. Alternatively, for example, the other agent is administered every first to third day, while the antibody is administered weekly. Alternatively, the maintenance doses are co-administered sequentially within one day or over several days.

[0284] It is self-evident that an antibody is administered to a patient in a "therapeutically effective amount" (or just "effective amount"), which is that amount of the corresponding compound or combination that elicits the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical physician or other clinician.

[0285] The amount of co-administration and the timing of co-administration will depend on the type (species, sex, age, weight, etc.) and condition of the patient being treated and the severity of the disease or condition being treated. The anti-CSF-1R antibody and the additional agent may be appropriately co-administered to the patient at one time or over a series of treatments, e.g., on the same day or on the following day.

[0286] Depending on the type and severity of the disease, about 0.1 mg / kg to 50 mg / kg (e.g. 0.1-20 mg / kg) of the anti-CSF-1R antibody and / or anti-PD-L1 antibody is an initial candidate dose for co-administering both drugs to the patient. The invention includes the use of an antibody according to the invention for treating a patient suffering from cancer, in particular colon, lung or pancreatic cancer.

[0287] Depending on the type and severity of the disease, about 0.1 mg / kg to 50 mg / kg (e.g. 0.1-20 mg / kg) of the anti-CSF-1R antibody and / or anti-PD-L1 antibody is an initial candidate dose for co-administering both drugs to a patient. The invention includes the use of an antibody according to the invention for treating a patient suffering from cancer, in particular colon, lung or prostate cancer.

[0288] In addition to the anti-CSF-1R antibody in combination with the anti-PD-L1 antibody, a chemotherapeutic agent may also be administered.

[0289] In one embodiment, such additional chemotherapeutic agents that can be administered with the anti-CSF-1R antibodies described herein and the anti-PD-L1 antibodies described herein include, but are not limited to, anti-tumor agents, including alkylating agents, including: nitrogen mustards, such as mechlorethamine, cyclophosphamide, ifosfamide, melphalan, and chlorambucil; nitrosoureas, such as carmustine (BCNU), lomustine (CCNU), and semustine (methyl-CCNU); Temodal TM (temozolamide), ethyleneimine / methylmelamines such as triethylenemelamine (TEM), triethylenethiophosphoramide (thiotepa), hexamethylmelamine (HMM, altretamine); alkyl sulfonates such as busulfan; triazines such as dacarbazine (DTIC); antimetabolites including folic acid analogs such as methotrexate and trimetrexate, pyrimidine analogs such as Such as 5-fluorouracil (5FU), fluorodeoxyuridine, gemcitabine, cytosine arabinoside (AraC, cytarabine), 5-azacytidine, 2,2'-difluorodeoxycytidine, purine analogs such as 6-mercaptopurine, 6-thioguanine, azathioprine, T-deoxycoformycin (pentostatin), erythrohydroxynonyladenine (EHNA), fludarabine phosphate (fludarabine phosphate), and 2-chlorodeoxyadenosine (cladribine, 2-CdA); natural products, including antimitotic drugs such as paclitaxel, vinca alkaloids (including vinblastine (VLB), vincristine, and vinorelbine), taxotere, estramustine, and estramustine phosphate; epipodophyllotoxins such as etoposide and teniposide; antibiotics such as actinomycin D (actinomycin D), daunomycin (rubidomycin), doxorubicin, mitoxantrone, idarubicin, bleomycin, plicamycin (mithramycin), mitomycin C, and actinomycin; enzymes such as L-asparaginase; biological response modifiers such as interferon-α, IL-2, G-CSF and GM-CSF;Miscellaneous agents including platinum coordination complexes such as oxaliplatin, cisplatin and carboplatin, anthracenediones such as mitoxantrone, substituted ureas such as hydroxyurea, methylhydrazine derivatives including N-methylhydrazine (MIH) and procarbazine, adrenocortical suppressants such as mitotane (o, p-DDD) and aminoglutethimide; hormones and antagonists including adrenocortical steroid antagonists such as prednisone and equivalents, dexamethasone and aminoglutethimide; Gemzar; TM (gemcitabine), progestogens such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrolacetate; estrogens such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogens such as tamoxifen; androgens including testosterone propionate and fluoxymesterone / equivalents; antiandrogens such as flutamide, gonadotropin-releasing hormone analogs, and leuprolide; and nonsteroidal antiandrogens such as flutamide. Therapies targeting epigenetic mechanisms include, but are not limited to, histone deacetylase inhibitors, demethylating agents (e.g., Vidaza), and release transcriptional repression (ATRA) therapy can also be combined with antigen binding proteins. In one embodiment, the chemotherapeutic agent is selected from the group consisting of taxanes (such as, for example, paclitaxel (Taxol), docetaxel (Taxotere), modified paclitaxel (such as Abraxane and Opaxio), doxorubicin, sunitinib (Sutent), sorafenib (Nexavar), and other multikinase inhibitors, oxaliplatin, cisplatin and carboplatin, etoposide, gemcitabine, and vinblastine. In one embodiment, the chemotherapeutic agent is selected from the group consisting of taxanes (such as, for example, paclitaxel (Taxol), docetaxel (Taxotere), modified paclitaxel (such as Abraxane and Opaxio). In one embodiment, the additional chemotherapeutic agent is selected from 5-fluorouracil (5-FU), folinic acid, irinotecan, or oxaliplatin. In one embodiment, the chemotherapeutic agent is 5-fluorouracil, folinic acid, and irinotecan (FOLFIRI). In one embodiment, the chemotherapeutic agent is 5-fluorouracil and oxaliplatin (FOLFOX).

[0290] Specific examples of combination therapies with other chemotherapeutic agents include, for example, therapy with a taxane (e.g., docetaxel or paclitaxel) or a modified paclitaxel (e.g., Abraxane or Opaxio), doxorubicin, capecitabine, and / or bevacizumab (Avastin) for the treatment of breast cancer; therapy with carboplatin, oxaliplatin, cisplatin, paclitaxel, doxorubicin (or modified doxorubicin (Caelyx or Doxil)), or topotecan (Hycamtin) for the treatment of ovarian cancer; therapy with a multikinase inhibitor MKI (Sutent, Nexavar, or 706) and / or doxorubicin for the treatment of renal cancer; therapy with oxaliplatin, cisplatin, and / or radiation for the treatment of squamous cell carcinoma; therapy with Taxol and / or carboplatin for the treatment of lung cancer.

[0291] Thus, in one embodiment, the additional chemotherapeutic agent is selected from the group consisting of a taxane (docetaxel or paclitaxel) or modified paclitaxel (Abraxane or Opaxio), doxorubicin, capecitabine, and / or bevacizumab (for the treatment of breast cancer).

[0292] In one embodiment, the CSF-1R antibody / PD-L1 antibody combination therapy is not administered with a chemotherapeutic agent.

[0293] The invention also encompasses a method for treating a patient suffering from such a disease.

[0294] The present invention further provides a method for preparing a pharmaceutical composition comprising an effective amount of an antibody according to the present invention and a pharmaceutically acceptable carrier and the use of an antibody according to the present invention for such a method.

[0295] The invention further provides the use of an effective amount of an antibody according to the invention for the preparation of a pharmaceutical formulation, preferably together with a pharmaceutically acceptable carrier, for treating a patient suffering from cancer.

[0296] The present invention also provides the use of an effective amount of an antibody according to the present invention for the preparation of a pharmaceutical preparation, preferably together with a pharmaceutically acceptable carrier, for treating a patient suffering from cancer.

[0297] The following examples, sequence listing and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims.It is understood that changes can be made in the procedures set forth without departing from the spirit of the present invention.

[0298] Sequence Description

[0299] SEQ ID NO: 1 Heavy chain CDR3, Mab 2F11

[0300] SEQ ID NO: 2 Heavy chain CDR2, Mab 2F11

[0301] SEQ ID NO: 3 Heavy chain CDR1, Mab 2F11

[0302] SEQ ID NO: 4 Light chain CDR3, Mab 2F11

[0303] SEQ ID NO: 5 Light chain CDR2, Mab 2F11

[0304] SEQ ID NO:6 Light chain CDR1, Mab 2F11

[0305] SEQ ID NO: 7 Heavy chain variable domain, Mab 2F11

[0306] SEQ ID NO: 8 Light chain variable domain, Mab 2F11

[0307] SEQ ID NO:9 Heavy chain CDR3, Mab 2E10

[0308] SEQ ID NO: 10 Heavy chain CDR2, Mab 2E10

[0309] SEQ ID NO: 11 Heavy chain CDR1, Mab 2E10

[0310] SEQ ID NO: 12 Light chain CDR3, Mab 2E10

[0311] SEQ ID NO: 13 Light chain CDR2, Mab 2E10

[0312] SEQ ID NO: 14 Light chain CDR1, Mab 2E10

[0313] SEQ ID NO: 15 Heavy chain variable domain, Mab 2E10

[0314] SEQ ID NO: 16 Light chain variable domain, Mab 2E10

[0315] SEQ ID NO: 17 Heavy chain CDR3, hMab 2F11-c11

[0316] SEQ ID NO: 18 Heavy chain CDR2, hMab 2F11-c11

[0317] SEQ ID NO: 19 Heavy chain CDR1, hMab 2F11-c11

[0318] SEQ ID NO: 20 Light chain CDR3, hMab 2F11-c11

[0319] SEQ ID NO: 21 Light chain CDR2, hMab 2F11-c11

[0320] SEQ ID NO: 22 Light chain CDR1, hMab 2F11-c11

[0321] SEQ ID NO: 23 Heavy chain variable domain, hMab 2F11-c11

[0322] SEQ ID NO: 24 Light chain variable domain, hMab 2F11-c11

[0323] SEQ ID NO: 25 Heavy chain CDR3, hMab 2F11-d8

[0324] SEQ ID NO: 26 Heavy chain CDR2, hMab 2F11-d8

[0325] SEQ ID NO: 27 Heavy chain CDR1, hMab 2F11-d8

[0326] SEQ ID NO: 28 Light chain CDR3, hMab 2F11-d8

[0327] SEQ ID NO: 29 Light chain CDR2, hMab 2F11-d8

[0328] SEQ ID NO: 30 Light chain CDR1, hMab 2F11-d8

[0329] SEQ ID NO: 31 Heavy chain variable domain, hMab 2F11-d8

[0330] SEQ ID NO: 32 Light chain variable domain, hMab 2F11-d8

[0331] SEQ ID NO: 33 Heavy chain CDR3, hMab 2F11-e7

[0332] SEQ ID NO: 34 Heavy chain CDR2, hMab 2F11-e7

[0333] SEQ ID NO: 35 Heavy chain CDR1, hMab 2F11-e7

[0334] SEQ ID NO: 36 Light chain CDR3, hMab 2F11-e7

[0335] SEQ ID NO: 37 Light chain CDR2, hMab 2F11-e7

[0336] SEQ ID NO: 38 Light chain CDR1, hMab 2F11-e7

[0337] SEQ ID NO: 39 Heavy chain variable domain, hMab 2F11-e7

[0338] SEQ ID NO: 40 Light chain variable domain, hMab 2F11-e7

[0339] SEQ ID NO: 41 Heavy chain CDR3, hMab 2F11-f12

[0340] SEQ ID NO: 42 Heavy chain CDR2, hMab 2F11-f12

[0341] SEQ ID NO: 43 Heavy chain CDR1, hMab 2F11-f12

[0342] SEQ ID NO: 44 Light chain CDR3, hMab 2F11-f12

[0343] SEQ ID NO: 45 Light chain CDR2, hMab 2F11-f12

[0344] SEQ ID NO: 46 Light chain CDR1, hMab 2F11-f12

[0345] SEQ ID NO: 47 Heavy chain variable domain, hMab 2F11-f12

[0346] SEQ ID NO: 48 Light chain variable domain, hMab 2F11-f12

[0347] SEQ ID NO: 49 Heavy chain CDR3, hMab 2F11-g1

[0348] SEQ ID NO: 50 Heavy chain CDR2, hMab 2F11-g1

[0349] SEQ ID NO: 51 Heavy chain CDR1, hMab 2F11-g1

[0350] SEQ ID NO: 52 Light chain CDR3, hMab 2F11-g1

[0351] SEQ ID NO: 53 Light chain CDR2, hMab 2F11-g1

[0352] SEQ ID NO: 54 Light chain CDR1, hMab 2F11-g1

[0353] SEQ ID NO: 55 Heavy chain variable domain, hMab 2F11-g1

[0354] SEQ ID NO: 56 Light chain variable domain, hMab 2F11-g1

[0355] SEQ ID NO: 57 Human kappa light chain constant region

[0356] SEQ ID NO: 58 Human heavy chain constant region derived from IgG1

[0357] SEQ ID NO: 59 Human heavy chain constant region derived from IgG1 with mutations at L234A and L235A

[0358] SEQ ID NO: 60 Human heavy chain constant region derived from IgG4

[0359] SEQ ID NO: 61 Human heavy chain constant region derived from IgG4 with mutation at S228P

[0360] SEQ ID NO: 62 Human wild-type CSF-1R (wt CSF-1R) (including signal sequence)

[0361] SEQ ID NO: 63 Human mutant CSF-1R L301S Y969F (including signal sequence)

[0362] SEQ ID NO: 64 Human CSF-1R extracellular domain (domains D1-D5)

[0363] SEQ ID NO: 65 Human CSF-1R fragment delD4

[0364] SEQ ID NO: 66 Human CSF-1R fragment domains D1-D3

[0365] SEQ ID NO: 67 Signal peptide

[0366] SEQ ID NO: 68 Primer

[0367] SEQ ID NO: 69 Heavy chain CDR3, Mab 1G10

[0368] SEQ ID NO: 70 Heavy chain CDR2, Mab 1G10

[0369] SEQ ID NO: 71 Heavy chain CDR1, Mab 1G10

[0370] SEQ ID NO: 72 Light chain CDR3, Mab 1G10

[0371] SEQ ID NO: 73 Light chain CDR2, Mab 1G10

[0372] SEQ ID NO: 74 Light chain CDR1, Mab 1G10

[0373] SEQ ID NO: 75 Heavy chain variable domain, Mab 1G10

[0374] SEQ ID NO: 76 Light chain variable domain, Mab 1G10

[0375] SEQ ID NO: 77 Heavy chain CDR3, Mab 2H7

[0376] SEQ ID NO: 78 Heavy chain CDR2, Mab 2H7

[0377] SEQ ID NO: 79 Heavy chain CDR1, Mab 2H7

[0378] SEQ ID NO: 80 Light chain CDR3, Mab 2H7

[0379] SEQ ID NO: 81 Light chain CDR2, Mab 2H7

[0380] SEQ ID NO: 82 Light chain CDR1, Mab 2H7

[0381] SEQ ID NO: 83 Heavy chain variable domain, Mab 2H7

[0382] SEQ ID NO: 84 Light chain variable domain, Mab 2H7

[0383] SEQ ID NO: 85 Human CSF-1R fragment domains D4-D5

[0384] SEQ ID NO: 86 Human CSF-1 (including signal sequence)

[0385] SEQ ID NO: 87 Human IL-34 (including signal sequence)

[0386] SEQ ID NO: 88 Human PD-L1 (including signal sequence)

[0387] SEQ ID NO: 89 Heavy chain variable domain VH variant 1, anti-PD-L1 243.55

[0388] SEQ ID NO: 90 Heavy chain variable domain VH variant 2, anti-PD-L1 243.55

[0389] SEQ ID NO: 91 Heavy chain variable domain VH variant 3, anti-PD-L1 243.55

[0390] SEQ ID NO: 92 Light chain variable domain VL variant 1, anti-PD-L1 243.55

[0391] SEQ ID NO: 93 Light chain variable domain VL variant 2, anti-PD-L1 243.55

[0392] SEQ ID NO: 94 Light chain variable domain VL variant 3, anti-PD-L1 243.55

[0393] SEQ ID NO: 95 Light chain variable domain VL variant 4, anti-PD-L1 243.55

[0394] SEQ ID NO: 96 Light chain variable domain VL variant 5, anti-PD-L1 243.55

[0395] SEQ ID NO: 97 Light chain variable domain VL variant 6, anti-PD-L1 243.55

[0396] SEQ ID NO: 98 Light chain variable domain VL variant 7, anti-PD-L1 243.55

[0397] SEQ ID NO: 99 Light chain variable domain VL variant 8, anti-PD-L1 243.55

[0398] SEQ ID NO: 100 Light chain variable domain VL variant 9, anti-PD-L1 243.55

[0399] SEQ ID NO: 101 Light chain variable domain VL variant 10, anti-PD-L1 243.55

[0400] SEQ ID NO: 102 Light chain variable domain VL variant 11, anti-PD-L1 243.55

[0401] SEQ ID NO: 103 Light chain variable domain VL variant 12, anti-PD-L1 243.55

[0402] SEQ ID NO: 104 Light chain variable domain VL variant 13, anti-PD-L1 243.55

[0403] SEQ ID NO: 105 Light chain variable domain VL variant 14, anti-PD-L1 243.55

[0404] SEQ ID NO: 106 Light chain variable domain VL variant 15, anti-PD-L1 243.55

[0405] SEQ ID NO: 107 Light chain variable domain VL variant 16, anti-PD-L1 243.55

[0406] In the following embodiments of the present invention, it is described:

[0407] 1.A) an antibody that binds to human CSF-1R, wherein the antibody is administered in combination with an antibody that binds to human PD-L1, for treating cancer, for preventing or treating metastasis, for treating inflammatory diseases, for treating bone loss, for treating immune-related diseases such as tumor immunity or delaying its progression, or for stimulating an immune response or function, such as T cell activity; or

[0408] B) use of an antibody that binds to human CSF-1R for the manufacture of a medicament for treating cancer, for preventing or treating metastasis, for treating inflammatory diseases, for treating bone loss, for treating immune-related diseases such as tumor immunity or delaying its progression, or for stimulating an immune response or function, such as T cell activity, wherein the antibody is administered in combination with an antibody that binds to human PD-L1;

[0409] The antibody binding to human CSF-1R used in the combination therapy is characterized by comprising

[0410] a) a heavy chain variable domain VH of SEQ ID NO: 23 and a light chain variable domain VL of SEQ ID NO: 24, or

[0411] b) a heavy chain variable domain VH of SEQ ID NO: 31 and a light chain variable domain VL of SEQ ID NO: 32, or

[0412] c) a heavy chain variable domain VH of SEQ ID NO: 39 and a light chain variable domain VL of SEQ ID NO: 40, or

[0413] d) a heavy chain variable domain VH of SEQ ID NO: 47 and a light chain variable domain VL of SEQ ID NO: 48, or

[0414] e) a heavy chain variable domain VH of SEQ ID NO: 55 and a light chain variable domain VL of SEQ ID NO: 56; and the antibody binding to human PD-L1 used in the combination therapy is characterized by comprising

[0415] a) a heavy chain variable domain VH of SEQ ID NO: 89 and a light chain variable domain VL of SEQ ID NO: 92, or

[0416] b) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 93, or

[0417] c) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 94, or

[0418] d) a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:95, or

[0419] e) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 96, or

[0420] f) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 97, or

[0421] g) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 98, or

[0422] h) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 99, or

[0423] i) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 100, or

[0424] j) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 101, or

[0425] k) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 102, or

[0426] l) a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:103, or

[0427] m) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 104, or

[0428] n) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 105, or

[0429] o) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 106, or

[0430] p) the heavy chain variable domain VH of SEQ ID NO:91 and the light chain variable domain VL of SEQ ID NO:107.

[0431] 2.A) Include

[0432] a) a heavy chain variable domain VH of SEQ ID NO: 23 and a light chain variable domain VL of SEQ ID NO: 24, or

[0433] b) a heavy chain variable domain VH of SEQ ID NO: 31 and a light chain variable domain VL of SEQ ID NO: 32, or

[0434] c) a heavy chain variable domain VH of SEQ ID NO: 39 and a light chain variable domain VL of SEQ ID NO: 40, or

[0435] d) a heavy chain variable domain VH of SEQ ID NO: 47 and a light chain variable domain VL of SEQ ID NO: 48, or

[0436] e) an antibody that binds to human CSF-1R comprising a heavy chain variable domain VH of SEQ ID NO: 55 and a light chain variable domain VL of SEQ ID NO: 56

[0437] and

[0438] B) Include

[0439] a) a heavy chain variable domain VH of SEQ ID NO: 89 and a light chain variable domain VL of SEQ ID NO: 92, or

[0440] b) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 93, or

[0441] c) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 94, or

[0442] d) a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:95, or

[0443] e) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 96, or

[0444] f) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 97, or

[0445] g) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 98, or

[0446] h) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 99, or

[0447] i) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 100, or

[0448] j) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 101, or

[0449] k) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 102, or

[0450] l) a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:103, or

[0451] m) the heavy chain variable domain VH of SEQ ID NO: 90 and the light chain variable domain of SEQ ID NO: 104

[0452] VL, or

[0453] n) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain of SEQ ID NO: 105

[0454] VL, or

[0455] o) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain of SEQ ID NO: 106

[0456] VL, or

[0457] p) Use of a combination of an antibody that binds to human PD-L1 comprising a heavy chain variable domain VH of SEQ ID NO: 91 and a light chain variable domain VL of SEQ ID NO: 107 in the manufacture of a medicament for treating cancer, for preventing or treating metastasis, for treating inflammatory diseases, for treating bone loss, for treating immune-related diseases such as tumor immunity or delaying its progression, or for stimulating an immune response or function, such as T cell activity, wherein the antibody that binds to human CSF-1R is administered in combination with an antibody that binds to human PD-L1.

[0458] 3. The antibody or use according to any one of embodiments 1 or 2, for treating cancer.

[0459] 4. The antibody or use according to embodiment 3, for treating breast cancer, lung cancer, colon cancer, ovarian cancer, melanoma cancer, bladder cancer, renal cancer, kidney cancer, liver cancer, head and neck cancer, colorectal cancer, pancreatic cancer, gastric cancer, esophageal cancer, mesothelioma, prostate cancer, leukemia, lymphoma, myeloma.

[0460] 5. The antibody or the use according to any one of embodiments 1 or 2, for preventing or treating metastasis.

[0461] 6. The antibody or use according to any one of embodiments 1 or 2, for treating bone loss.

[0462] 7. The antibody or use according to any one of embodiments 1 or 2, for treating an inflammatory disease.

[0463] 8. The antibody or use according to any one of embodiments 1 or 2, for treating an immune-related disease such as tumor immunity or delaying its progression.

[0464] 9. The antibody or use according to any one of embodiments 1 or 2, for stimulating an immune response or function, such as T cell activity.

[0465] 10.A) An antibody that binds to human CSF-1R, wherein the antibody is administered in combination with an antibody that binds to human PD-L1, for

[0466] i) inhibiting cell proliferation in CSF-1R ligand-dependent and / or CSF-1 ligand-independent CSF-1R-expressing tumor cells;

[0467] ii) inhibiting cell proliferation in tumors infiltrated with CSF-1R ligand-dependent and / or CSF-1R ligand-independent CSF-1R-expressing macrophages;

[0468] iii) inhibiting cell survival in (CSF-1R ligand-dependent and / or CSF-1R ligand-independent) CSF-1R-expressing monocytes and macrophages; and / or

[0469] iv) inhibiting differentiation of CSF-1R-expressing monocytes into macrophages in (CSF-1R ligand-dependent and / or CSF-1R ligand-independent) CSF-1R-expressing monocytes;

[0470] or

[0471] B) Production of antibodies binding to human CSF-1R for

[0472] i) inhibiting cell proliferation in CSF-1R ligand-dependent and / or CSF-1 ligand-independent CSF-1R-expressing tumor cells;

[0473] ii) inhibiting cell proliferation in tumors infiltrated with CSF-1R ligand-dependent and / or CSF-1R ligand-independent CSF-1R-expressing macrophages;

[0474] iii) inhibiting cell survival in (CSF-1R ligand-dependent and / or CSF-1R ligand-independent) CSF-1R-expressing monocytes and macrophages; and / or

[0475] iv) Use of a medicament for inhibiting differentiation of CSF-1R-expressing monocytes into macrophages (CSF-1R ligand-dependent and / or CSF-1R ligand-independent), wherein the antibody is administered in combination with an antibody that binds to human PD-L1;

[0476] The antibody binding to human CSF-1R used in the combination therapy is characterized by comprising

[0477] a) a heavy chain variable domain VH of SEQ ID NO: 23 and a light chain variable domain VL of SEQ ID NO: 24, or

[0478] b) a heavy chain variable domain VH of SEQ ID NO: 31 and a light chain variable domain VL of SEQ ID NO: 32, or

[0479] c) a heavy chain variable domain VH of SEQ ID NO: 39 and a light chain variable domain VL of SEQ ID NO: 40, or

[0480] d) a heavy chain variable domain VH of SEQ ID NO: 47 and a light chain variable domain VL of SEQ ID NO: 48, or

[0481] e) a heavy chain variable domain VH of SEQ ID NO: 55 and a light chain variable domain VL of SEQ ID NO: 56; and the antibody binding to human PD-L1 used in the combination therapy is characterized by comprising

[0482] a) a heavy chain variable domain VH of SEQ ID NO: 89 and a light chain variable domain VL of SEQ ID NO: 92, or

[0483] b) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 93, or

[0484] c) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 94, or

[0485] d) a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:95, or

[0486] e) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 96, or

[0487] f) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 97, or

[0488] g) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 98, or

[0489] h) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 99, or

[0490] i) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 100, or

[0491] j) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 101, or

[0492] k) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 102, or

[0493] l) a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:103, or

[0494] m) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 104, or

[0495] n) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 105, or

[0496] o) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 106, or

[0497] p) the heavy chain variable domain VH of SEQ ID NO: 91 and the light chain variable domain of SEQ ID NO: 107

[0498] VL.

[0499] 11.A) An antibody that binds to human CSF-1R for use in treating a patient having a CSF-1R expressing tumor or a tumor with CSF-1R expressing macrophage infiltration, wherein the tumor is characterized by elevated CSF-1R ligand, and wherein the anti-CSF-1R antibody is administered in combination with an antibody that binds to human PD-L1,

[0500] or

[0501] B) use of an antibody that binds to human CSF-1R for the manufacture of a medicament for treating a patient having a CSF-1R expressing tumor or a tumor with infiltration of CSF-1R expressing macrophages, wherein the tumor is characterized by elevated CSF-1R ligand, and wherein the anti-CSF-1R antibody is administered in combination with an antibody that binds to human PD-L1,

[0502] The antibody binding to human CSF-1R used in the combination therapy is characterized by comprising

[0503] a) a heavy chain variable domain VH of SEQ ID NO: 23 and a light chain variable domain VL of SEQ ID NO: 24, or

[0504] b) a heavy chain variable domain VH of SEQ ID NO: 31 and a light chain variable domain VL of SEQ ID NO: 32, or

[0505] c) a heavy chain variable domain VH of SEQ ID NO: 39 and a light chain variable domain VL of SEQ ID NO: 40, or

[0506] d) a heavy chain variable domain VH of SEQ ID NO: 47 and a light chain variable domain VL of SEQ ID NO: 48, or

[0507] e) a heavy chain variable domain VH of SEQ ID NO: 55 and a light chain variable domain VL of SEQ ID NO: 56; and the antibody binding to human PD-L1 used in the combination therapy is characterized by comprising

[0508] a) a heavy chain variable domain VH of SEQ ID NO: 89 and a light chain variable domain VL of SEQ ID NO: 92, or

[0509] b) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 93, or

[0510] c) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 94, or

[0511] d) a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:95, or

[0512] e) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 96, or

[0513] f) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 97, or

[0514] g) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 98, or

[0515] h) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 99, or

[0516] i) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 100, or

[0517] j) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 101, or

[0518] k) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 102, or

[0519] l) a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:103, or

[0520] m) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 104, or

[0521] n) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 105, or

[0522] o) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 106, or

[0523] p) the heavy chain variable domain VH of SEQ ID NO: 91 and the light chain variable domain of SEQ ID NO: 107

[0524] VL.

[0525] 12. The antibody or use according to any one of the preceding embodiments,

[0526] The antibody binding to human CSF-1R used in the combination therapy is characterized by comprising

[0527] c) a heavy chain variable domain VH of SEQ ID NO: 39 and a light chain variable domain VL of SEQ ID NO: 40, wherein the antibody binding to human PD-L1 used in the combination therapy is characterized by comprising

[0528] a) The heavy chain variable domain VH of SEQ ID NO:89 and the light chain variable domain VL of SEQ ID NO:92.

[0529] 13. The antibody or use according to any one of the preceding embodiments, characterized in that the antibody is of human IgG1

[0530] subclass or human IgG4 subclass.

[0531] 14. The antibody or use according to any one of the preceding embodiments, characterized in that the antibody has reduced or minimal effector function.

[0532] 15. The antibody or use according to any one of the preceding embodiments, wherein said minimal effector function results from an effector-reducing Fc mutation.

[0533] 16. The antibody or use according to any one of the preceding embodiments, wherein the effector reducing Fc mutation is L234A / L235A or L234A / L235A / P329G or N297A or D265A / N297A.

[0534] 17.A) A method for

[0535] i) In CSF-1R ligand-dependent and / or CSF-1 ligand-independent CSF-1R-expressing tumors

[0536] Inhibit cell proliferation in cells;

[0537] ii) inhibiting cell proliferation in tumors infiltrated with CSF-1R ligand-dependent and / or CSF-1R ligand-independent CSF-1R-expressing macrophages;

[0538] iii) inhibiting cell survival in (CSF-1R ligand-dependent and / or CSF-1R ligand-independent) CSF-1R-expressing monocytes and macrophages; and / or

[0539] iv) a method for inhibiting differentiation of (CSF-1R ligand-dependent and / or CSF-1R ligand-independent) CSF-1R-expressing monocytes into macrophages,

[0540] wherein an antibody that binds to human CSF-1R is administered in combination with an antibody that binds to human PD-L1, or

[0541] B) a method of treating a patient having a CSF-1R expressing tumor or a tumor with infiltration of CSF-1R expressing macrophages, wherein the tumor is characterized by elevated CSF-1R ligand, and wherein an antibody that binds human CSF-1R is administered in combination with an antibody that binds human PD-L1,

[0542] The antibody binding to human CSF-1R used in the combination therapy is characterized by comprising

[0543] a) a heavy chain variable domain VH of SEQ ID NO: 23 and a light chain variable domain VL of SEQ ID NO: 24, or

[0544] b) a heavy chain variable domain VH of SEQ ID NO: 31 and a light chain variable domain VL of SEQ ID NO: 32, or

[0545] c) a heavy chain variable domain VH of SEQ ID NO: 39 and a light chain variable domain VL of SEQ ID NO: 40, or

[0546] d) a heavy chain variable domain VH of SEQ ID NO: 47 and a light chain variable domain VL of SEQ ID NO: 48, or

[0547] e) a heavy chain variable domain VH of SEQ ID NO: 55 and a light chain variable domain VL of SEQ ID NO: 56; and the antibody binding to human PD-L1 used in the combination therapy is characterized by comprising

[0548] a) a heavy chain variable domain VH of SEQ ID NO: 89 and a light chain variable domain VL of SEQ ID NO: 92, or

[0549] b) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 93, or

[0550] c) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 94, or

[0551] d) a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:95, or

[0552] e) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 96, or

[0553] f) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 97, or

[0554] g) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 98, or

[0555] h) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 99, or

[0556] i) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 100, or

[0557] j) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 101, or

[0558] k) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 102, or

[0559] l) a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:103, or

[0560] m) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 104, or

[0561] n) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 105, or

[0562] o) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 106, or

[0563] p) the heavy chain variable domain VH of SEQ ID NO:91 and the light chain variable domain VL of SEQ ID NO:107.

[0564] Example

[0565] Example 1

[0566] Inhibition of CSF-1R phosphorylation induced by CSF-1 in NIH3T3-CSF-1R recombinant cells

[0567] 4.5x10 3 NIH 3T3 cells retrovirally infected with an expression vector for full-length CSF-1R were cultured in DMEM (PAA, catalog number E15-011), 2 mM L-glutamine (Sigma, catalog number G7513), 2 mM sodium pyruvate, 1x non-essential amino acids, 10% FKS (PAA, catalog number A15-649), and 100 μg / ml PenStrep (Sigma, catalog number P4333 [10 mg / ml]) until they reached confluence. Thereafter, the cells were washed with serum-free DMEM medium (PAA, catalog number E15-011) supplemented with sodium selenite [5 ng / ml] (Sigma, catalog number S9133), transferrin [10 μg / ml] (Sigma, catalog number T8158), BSA [400 μg / ml] (Roche Diagnostics GmbH, catalog number 10735078), 4 mM L-glutamine (Sigma, catalog number G7513), 2 mM sodium pyruvate (Gibco, catalog number 11360), 1x non-essential amino acids (Gibco, catalog number 11140-035), 2-mercaptoethanol [0.05 mM] (Merck, catalog number M7522), 100 μg / ml and PenStrep (Sigma, catalog number P4333) and incubated in 30 μl of the same medium for 16 hours to allow receptor upregulation. 10 μl of diluted anti-CSR-1R antibody was added to the cells for 1.5 hours. The cells were then stimulated with 10 μl of 100 ng / ml huCSF-1 (active 149 aa fragment of human CSF-1 (aa33-181 of SEQ ID NO: 86); Biomol, DE, catalog number 60530) for 5 minutes. After incubation, the supernatant was removed, the cells were washed twice with 80 μl of ice-cold PBS and 50 μl of freshly prepared ice-cold lysis buffer (150 mM NaCl / 20 mM Tris pH 7.5 / 1 mM EDTA / 1 mM EGTA / 1% Triton X-100 / 1 protease inhibitor tablet (Roche Diagnostics GmbH, catalog number 1836170) per 10 ml buffer / 10 μl / ml phosphatase inhibitor cocktail 1 (Sigma, catalog number P-2850, 100x stock) / 10 μl / ml protease inhibitor 1 (Sigma, catalog number P-5726, 100x stock) / 10 μl / ml 1M NaF was added. After 30 minutes on ice, the plate was shaken vigorously on a plate shaker for 3 minutes and then centrifuged at 2200 rpm for 10 minutes (Heraeus Megafuge 10).

[0568] Cell lysates were analyzed for the presence of phosphorylated and total CSF-1 receptor using ELISA. Phosphorylated receptors were detected using a kit from R&D Systems (Cat. No. DYC3268-2) according to the supplier's instructions. For detection of total CSF-1R, 10 μl of lysate were immobilized on a plate using the capture antibody included in the kit. Biotinylated anti-CSF-1R antibody BAF329 (R&D Systems) at a dilution of 1:750 and streptavidin-HRP conjugate at a dilution of 1:1000 were then added. After 60 minutes, the plates were washed with freshly prepared The solution was developed and the absorbance was measured. The data were calculated as the percentage of the positive control without antibody and the ratio of phosphorylated / total receptor expressed. The negative control was defined as the absence of M-CSF-1. Anti-CSF-1R SC 2-4A5 (Santa Cruz Biotechnology, US, see also Sherr, CJ et al., Blood 73 (1989) 1786-1793) which inhibits ligand-receptor interaction was used as a reference control.

[0569] Table 3: Calculated IC50 values ​​for inhibition of CSF-1 receptor phosphorylation.

[0570]

[0571]

[0572] Example 2

[0573] Growth inhibition of NIH3T3-CSF-1R recombinant cells in 3D culture upon treatment with anti-CSF-1R monoclonal antibodies (CellTiterGlo assay)

[0574] NIH 3T3 cells retrovirally infected with expression vectors for full-length wild-type CSF-1R (SEQ ID NO: 62) or mutant CSF-1R L301S Y969F (SEQ ID NO: 63) were cultured on poly-HEMA (poly(2-hydroxyethylmethacrylate)) (Polysciences, Warrington, PA, USA) coated (to prevent adhesion to plastic surfaces) plates in DMEM high glucose medium (PAA, Pasching, Austria) supplemented with 2 mM L-glutamine, 2 mM sodium pyruvate and non-essential amino acids and 10% fetal bovine serum (Sigma, Taufkirchen, Germany). Cells were seeded in medium in which serum was replaced with 5 ng / ml sodium selenite, 10 mg / ml transferrin, 400 μg / ml BSA and 0.05 mM 2-mercaptoethanol. When treated with 100 ng / ml huCSF-1 (the active 149 aa fragment of human CSF-1 (aa 33-181 of SEQ ID NO: 86); Biomol, DE, catalog number 60530), cells expressing wtCSF-1R formed dense spheroids that grew in three dimensions, a property known as anchorage independence. These spheroids approximated the three-dimensional structure and organization of solid tumors in situ. Mutant CSF-1R recombinant cells were able to form spheroids independent of CSF-1 ligand. The anti-CSF-1R antibody hMab 2F11-e7 according to the invention and the anti-CSF-1R antibodies 1.2.SM (ligand-displacing CSF-1R antibody described in WO 2009 / 026303), CXIIG6 (ligand-displacing CSF-1R antibody described in WO 2009 / 112245), goat polyclonal anti-CSF-1R antibodies ab10676 (abcam), and SC 2-4A5 (Santa Cruz Biotechnology, US - see also Sherr, CJ et al., Blood 73 (1989) 1786-1793) and Mab R&D-Systems 3291 were investigated. The reference control Mab R&D-Systems 3291 showed no inhibition of mutant CSF-1R recombinant cell proliferation.

[0575] Spheroid cultures were incubated for 3 days in the presence of different concentrations of antibodies to determine the IC30 (concentration at which cell viability is inhibited by 30%). The maximum concentration was 20 μg / ml. Cell viability was measured by measuring the ATP content of the cells using the CellTiterGlo assay.

[0576] Table 4

[0577]

[0578]

[0579] Example 3

[0580] Inhibition of human macrophage differentiation upon treatment with anti-CSF-1R monoclonal antibodies (CellTiterGlo assay)

[0581] Using RosetteSep TM Human monocytes were isolated from peripheral blood using human monocyte enrichment cocktail (Stemcell Tech. - Catalog No. 15028). The enriched monocyte population was seeded in 96-well microtiter plates (2.5 x 10 4 The cells were cultured in 100 μl RPMI 1640 (Gibco - Cat. No. 31870) supplemented with 10% FCS (GIBCO - Cat. No. 011-090014M), 4 mM L-glutamine (GIBCO - Cat. No. 25030) and 1x PenStrep (Roche Cat. No. 1074440) in 4% 4% 4% 2 ... 50 (See table below).

[0582] Table 5

[0583] CSF-1R Mab <![CDATA[IC 50 [μg / ml]]]> Mab 2F11 0.08 Mab 2E10 0.06 Mab 2H7 0.03 Mab 1G10 0.06 SC 2-4A5 0.36

[0584] In separate tests, a series of humanized forms of Mab 2F11, such as hMab 2F11-c11, hMab 2F11-d8, hMab 2F11-e7, hMab 2F11-f12, showed IC50 values ​​of 0.07 μg / ml (hMab2F11-c11), 0.07 μg / ml (hMab2F11-d8), 0.04 μg / ml (hMab 2F11-e7) and 0.09 μg / ml (hMab 2F11-f12).

[0585] Example 4

[0586] Inhibition of human macrophage differentiation upon treatment with anti-CSF-1R monoclonal antibodies (CellTiterGlo assay)

[0587] Using RosetteSep TM Human monocytes were isolated from peripheral blood using human monocyte enrichment cocktail (Stemcell Tech. - Catalog No. 15028). The enriched monocyte population was seeded in 96-well microtiter plates (2.5 x 10 4 The cells were cultured in 100 μl RPMI 1640 (Gibco - Cat. No. 31870) supplemented with 10% FCS (GIBCO - Cat. No. 011-090014M), 4 mM L-glutamine (GIBCO - Cat. No. 25030) and 1x PenStrep (Roche Cat. No. 1074440) in 4% 4% 4% 2 ... 50 Humanized forms of Mab 2F11, such as hMab 2F11-c11, hMab 2F11-d8, hMab 2F11-e7, hMab 2F11-f12, showed IC50 values ​​of 0.07 μg / ml (hMab 2F11-c11), 0.07 μg / ml (hMab 2F11-d8), 0.04 μg / ml (hMab 2F11-e7), and 0.09 μg / ml (hMab 2F11-f12).

[0588] Example 5

[0589] Inhibition of human M1 and M2 macrophage differentiation upon anti-CSF-1R monoclonal antibody treatment (CellTiterGlo assay)

[0590] Using RosetteSep TM Human monocytes were isolated from peripheral blood using human monocyte enrichment cocktail (StemCell Tech. - catalog number 15028). The enriched monocyte population was seeded into 96-well microtiter plates (2.5 x 10 ) in 100 μl RPMI 1640 (Gibco - catalog number 31870) supplemented with 10% FCS (GIBCO - catalog number 011-090014M), 4 mM L-glutamine (GIBCO - catalog number 25030) and 1 x PenStrep (Roche - catalog number 1 074 440) at 37°C and 5% CO in a humidified atmosphere. 4 Cells / well). When 100 ng / ml huCSF-1 was added to the culture medium for 6 days, differentiation into adherent M2 macrophages with an elongated morphology could be clearly observed. When 100 ng / ml huGM-CSF was added to the culture medium for 6 days, differentiation into adherent M1 macrophages with a rounded morphology could be clearly observed. This differentiation was associated with the expression of certain markers, such as CD163 for M2 macrophages and CD80 or high class II MHC for M1 macrophages, as assessed by flow cytometry. The cells were washed with PBS and, if adherent, dissociated using a 5 mM EDTA solution in PBS (at 37°C for 20 minutes). The cells were then resuspended well, washed with staining buffer (PBS containing 5% FCS), and centrifuged at 300 x g for 5 minutes. The pellet was resuspended in 1 ml staining buffer and the cells were counted in a Neubauer trough. About 1x10e5 cells were transferred in each FACS tube, centrifuged at 300xg for 5 minutes, and resuspended in staining buffer. Fcγ receptors were blocked by incubation on ice in staining buffer for 20 minutes with 1 μg human IgG / 2.5x10e4 cells (JIR catalog number 009-000-003). Then for CD80 and CD163 detection, cells were mixed with 1.5 μl antibodies / 2.5x10e4 cells, and for class II MHC detection, 5 μl antibodies / 2.5x10e4 antibodies were used: mouse anti-human CD163 (BD ​​Bioscience catalog number 556018) marked with PE, mouse anti-human CD80 (BD Bioscience catalog number 557227) marked with PE, and mouse anti-human class II MHC (Dako catalog number M0775) marked with Alexa 647. Alexa 647 label was conjugated to the antibody by using Zenon Alexa 647 Mouse IgG Labeling Kit (Invitrogen Cat. No. Z25008).After incubation on ice for 1 hour, cells were washed twice with staining buffer, resuspended, and measured on FACS Canto II.

[0591] Uniquely, M2 macrophage differentiation, characterized by CD163 expression, CD80 loss, and low MHC class II expression, could be inhibited by the addition of humanized anti-CSF-1R antibody hMab 2F11-e7. Moreover, M2 but not M1 macrophage survival was affected and could be analyzed by CellTiterGlo (CTG) analysis. Concentration-dependent inhibition of macrophage survival by antibody treatment for 7 days is depicted in Figure 1a The expression of M1 and M2 macrophage markers assessed by flow cytometry is shown in Figure 1b .

[0592] Example 6

[0593] CSF-1 levels increase during CSF-1R inhibition in macaques

[0594] Serum CSF-1 levels provide a pharmacodynamic marker for the CSF-1R neutralizing activity of the anti-human CSF-1R dimerization inhibitor hMab 2F11-e7. One male and one female macaque were intravenously administered anti-CSF1R antibody hMab 2F11-e7 to each dose group (1 and 10 mg / kg). Blood samples were collected 1 week before treatment (pre-drug), 2, 24, 48, 72, 96, 168 hours after treatment, and weekly for another two weeks for analysis of CSF-1 levels. The serum CSF-1 levels were analyzed using a commercial ELISA kit ( CSF-1 levels were determined using the human M-CSF (R&D Systems, UK) assay according to the manufacturer's instructions. Monkey CSF-1 levels were determined by comparison with the CSF-1 standard curve samples provided in the kit.

[0595] Administration of hMab 2F11-e7 induced a significant increase in CSF-1 by approximately 1000-fold, depending on the dose administered for 48 hours (1 mg / kg) or 15 days (10 mg / kg). Thus, in contrast to ligand-displacing antibodies, dimerization inhibitors against CSF-1R offer the advantage of not directly competing with the significantly upregulated ligand for binding to the receptor (results are shown in Figure 2).

[0596] Example 7

[0597] Correlation between M2 subtype tumor-associated macrophages (TAMs) and T cells - Principle of combining anti-CSF-R1 antibodies and T cell engagers

[0598] To investigate the functional correlation between TAMs and T cells, we isolated TAMs from MC38 tumors and co-cultured them with CD8+ T cells.

[0599] TAM repression assay

[0600] After enzymatic digestion, TAMs were enriched from the single cell suspension of MC38 tumors using a two-step protocol: single cells were stained with CD11b-FITC (clone M1 / 70) and positively enriched on MACS columns by anti-FITC beads (Miltenyi). After being taken out from the column, the anti-FITC beads were dissociated using the release buffer solution provided by the manufacturer. Finally, TAMs were separated by adding anti-Ly6G and anti-Ly6C positive selection beads to remove granulocytes and monocytes from TAM preparations. Final cell purity was analyzed, usually >90%. Subsequently, TAMs were titrated with the ratio of total CD3+T cells labeled with CFSE as shown in the U bottom plate coated with anti-CD3, and soluble anti-CD28 was added. After 3 days of incubation, cell proliferation was determined from CFSE low cells using blank Sphero beads as previously described (Hoves, S. et al., Monocyte-derived human macrophages mediate anergy in allogeneic T cells and induce regulatory T cells. J. Immunol. 177, 2691-2698 (2006)). In the presence of TAM, T cell expansion induced by CD3 and CD28 activation was suppressed (see Figure 3 ).

[0601] Example 8

[0602] Inhibition of tumor growth under anti-CSF-1R monoclonal antibody treatment in combination with PD-L1 antibody in a subcutaneous syngeneic MC38 colon cancer model

[0603] Cells of the murine colorectal adenocarcinoma cell line MC-38 (obtained from Beckman Research Institute of the City of Hope, California, USA) were cultured in Dulbecco's Modified Eagle Medium (DMEM, PAN Biotech) supplemented with 10% FCS and 2 mM L-glutamine at 37°C and 5% CO2 in a water-saturated atmosphere. On the day of inoculation, MC38 tumor cells were harvested from culture flasks with PBS and transferred into culture medium, centrifuged, washed once, and resuspended in PBS. For cell injection, the final titer was adjusted to 1×10 7 Then 100 μl of this suspension (1x10 6 100 cells) were inoculated subcutaneously into 7-9 week old female C57BL / 6N mice (obtained from Charles River, Sulzfeld, Germany). 3 After the average size of 100 μg / kg, the tumor volume was measured twice a week and the weight of the animals was monitored.

[0604] In the first experiment, monotherapy with the <mouse CSF1R> antibody did not inhibit primary tumor growth compared with control antibody treatment (TGI: 0%, TCR: 1.07, CI: 0.80-1.43, median >700 mm 3 Time to progression: 21 days). Anti-PD-L1 monotherapy had an effect on MC38 primary tumor growth (TGI: 83%, TCR: 0.27, CI: 0.09-0.49, median >700 mm). 3 Time to progression: 32 days). Addition of the <mouse CSF1R> antibody to anti-PD-L1 therapy resulted in a slight improvement in anti-tumor efficacy compared with anti-PD-L1 treatment alone (TGI: 83%, TCR: 0.28, CI: 0.19-0.51, median >700 mm 3 Time to progression: 37 days) (see table below).

[0605] Table 6:

[0606] In vivo MC38 mouse CRC model

[0607] Anti-tumor efficacy of the <mouse anti-CSF1R> antibody / <anti-PD-L1> antibody combination

[0608]

[0609] Median values ​​in animals treated with control (mouse IgG1) were >700 mm 3 The time to progression was 21 days. Monotherapy with the <mouse CSF1R> antibody did not inhibit primary tumor growth (median >700 mm) compared with control antibody treatment. 3 Time to progression: 21 days). Anti-PD-L1 monotherapy had an effect on MC38 primary tumor growth (median >700mm 3 Time to progression: 32 days). Addition of <mouse CSF1R> antibody to anti-PD-L1 therapy resulted in slightly improved anti-tumor efficacy compared with PD-L1 treatment alone (median >700 mm 3 Time before progression: 37 days) (see table below and Figure 4 ).

[0610] Table 7:

[0611] In vivo MC38 mouse CRC model

[0612] Anti-tumor efficacy of the <mouse anti-CSF1R> antibody / <anti-PD-L1> antibody combination

[0613] (Median value>700mm 3 Time before progression)

[0614]

[0615]

[0616] Initiate treatment at similar but different tumor sizes (e.g., when tumors reach 100 mm 3 Inhibition of tumor growth under combined treatment with anti-CSF-1R monoclonal antibody and anti-PD-L1 antibody was evaluated in a subcutaneous syngeneic MC38 colon cancer model in two experiments (different groups were evaluated) and in another experiment also using different PD-L1 antibodies as described in Table 2.

[0617] Example 9

[0618] Inhibition of tumor growth under anti-CSF-1R monoclonal antibody treatment in combination with PD-L1 antibody in a subcutaneous syngeneic CT26.WT colon cancer model

[0619] Cells of the murine colorectal adenocarcinoma cell line CT26.WT tumor cells (obtained from ATCC) were cultured in Dulbecco's Modified Eagle's Medium (DMEM, PAN Biotech) supplemented with 10% FCS and 2 mM L-glutamine at 37°C and 5% CO2 in a water-saturated atmosphere. On the day of inoculation, CT26.WT tumor cells were harvested from the culture flask with PBS and transferred into the culture medium, centrifuged, washed once, and resuspended in PBS. For cell injection, the final titer was adjusted to 1×10 7 Then 100 μl of this suspension (1x10 6 11-13 week old female Balb / c mice (obtained from Charles River, Sulzfeld, Germany) were inoculated subcutaneously. 3 The average size of the tumor cells was then treated with a control antibody (MOPC-21; Bio X Cell, West Lebanon), anti-mouse CSF-1R monoclonal antibody <mouse CSF1R> antibody, at a weekly dose of 30 mg / kg, intraperitoneally, alone or in combination with a mouse cross-reactive PD-L1 antibody (10 mg / kg, intraperitoneally, 6x q3d). Although treatment in the monotherapy group started on day 9 after tumor cell inoculation, treatment in the combination group was sequential (day 9: start of treatment with anti-mouse CSF-1R monoclonal antibody; day 11: start of treatment with anti-PD-L1 antibody). Tumor volume was measured twice a week in parallel and animal weight was monitored. The results are shown in Figure

[0620] The median value of the IgG control treatment group was ≥700 mm 3 The time to progression was 17 days, compared with 16 days in the <mouse anti-CSF1R> antibody monotherapy group, 18 days in the <anti-PD-L1> antibody monotherapy group, and 18 days in the <mouse anti-CSF1R> / <anti-PD-L1> antibody combination group.

[0621] While all animals in the control or monotherapy groups required termination due to progressive tumor burden, one animal in the <mouse anti-CSF1R> / <anti-PD-L1> antibody combination group experienced tumor shrinkage and remained tumor-free by the end of the study at day 79 after tumor inoculation.

[0622] The present disclosure relates to the following embodiments:

[0623] 1. An antibody that binds to human CSF-1R, wherein the antibody is administered in combination with an antibody that binds to human PD-L1, for use in treating cancer, for preventing or treating metastasis, for treating inflammatory diseases, for treating bone loss, for treating immune-related diseases such as tumor immunity or delaying its progression, or for stimulating an immune response or function, such as T cell activity,

[0624] The antibody binding to human CSF-1R used in the combination therapy is characterized by comprising

[0625] a) a heavy chain variable domain VH of SEQ ID NO: 23 and a light chain variable domain VL of SEQ ID NO: 24, or

[0626] b) a heavy chain variable domain VH of SEQ ID NO: 31 and a light chain variable domain VL of SEQ ID NO: 32, or

[0627] c) a heavy chain variable domain VH of SEQ ID NO: 39 and a light chain variable domain VL of SEQ ID NO: 40, or

[0628] d) a heavy chain variable domain VH of SEQ ID NO: 47 and a light chain variable domain VL of SEQ ID NO: 48, or

[0629] e) a heavy chain variable domain VH of SEQ ID NO: 55 and a light chain variable domain VL of SEQ ID NO: 56; and the antibody binding to human PD-L1 used in the combination therapy is characterized by comprising

[0630] a) a heavy chain variable domain VH of SEQ ID NO: 89 and a light chain variable domain VL of SEQ ID NO: 92, or

[0631] b) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 93, or

[0632] c) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 94, or

[0633] d) a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:95, or

[0634] e) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 96, or

[0635] f) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 97, or

[0636] g) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 98, or

[0637] h) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 99, or

[0638] i) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 100, or

[0639] j) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 101, or

[0640] k) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 102, or

[0641] l) a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:103, or

[0642] m) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 104, or

[0643] n) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 105, or

[0644] o) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 106, or

[0645] p) the heavy chain variable domain VH of SEQ ID NO:91 and the light chain variable domain VL of SEQ ID NO:107.

[0646] 2. The antibody according to item 1, for use in treating cancer.

[0647] 3. The antibody according to item 2, for use in the treatment of breast cancer, lung cancer, colon cancer, ovarian cancer, melanoma cancer, bladder cancer, renal cancer, kidney cancer, liver cancer, head and neck cancer, colorectal cancer, pancreatic cancer, gastric cancer, esophageal cancer, mesothelioma, prostate cancer, leukemia, lymphoma, myeloma.

[0648] 4. The antibody according to item 1, for use in preventing or treating metastasis.

[0649] 5. The antibody according to item 1, for use in treating bone loss.

[0650] 6. The antibody according to item 1, for use in treating an inflammatory disease.

[0651] 7. The antibody according to item 1, for use in treating immune-related diseases such as tumor immunity or delaying its progression.

[0652] 8. The antibody according to item 1, for use in stimulating an immune response or function, such as T cell activity.

[0653] 9. An antibody that binds to human CSF-1R, wherein the antibody is administered in combination with an antibody that binds to human PD-L1, for

[0654] i) inhibiting cell proliferation in CSF-1R ligand-dependent and / or CSF-1 ligand-independent CSF-1R-expressing tumor cells;

[0655] ii) inhibiting cell proliferation in tumors infiltrated with CSF-1R ligand-dependent and / or CSF-1R ligand-independent CSF-1R-expressing macrophages;

[0656] iii) inhibiting cell survival in (CSF-1R ligand-dependent and / or CSF-1R ligand-independent) CSF-1R-expressing monocytes and macrophages; and / or

[0657] iv) inhibiting differentiation of CSF-1R-expressing monocytes into macrophages in (CSF-1R ligand-dependent and / or CSF-1R ligand-independent) monocytes,

[0658] The antibody binding to human CSF-1R used in the combination therapy is characterized by comprising

[0659] a) a heavy chain variable domain VH of SEQ ID NO: 23 and a light chain variable domain VL of SEQ ID NO: 24, or

[0660] b) a heavy chain variable domain VH of SEQ ID NO: 31 and a light chain variable domain VL of SEQ ID NO: 32, or

[0661] c) a heavy chain variable domain VH of SEQ ID NO: 39 and a light chain variable domain VL of SEQ ID NO: 40, or

[0662] d) a heavy chain variable domain VH of SEQ ID NO: 47 and a light chain variable domain VL of SEQ ID NO: 48, or

[0663] e) a heavy chain variable domain VH of SEQ ID NO: 55 and a light chain variable domain VL of SEQ ID NO: 56; and the antibody binding to human PD-L1 used in the combination therapy is characterized by comprising

[0664] a) a heavy chain variable domain VH of SEQ ID NO: 89 and a light chain variable domain VL of SEQ ID NO: 92, or

[0665] b) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 93, or

[0666] c) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 94, or

[0667] d) a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:95, or

[0668] e) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 96, or

[0669] f) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 97, or

[0670] g) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 98, or

[0671] h) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 99, or

[0672] i) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 100, or

[0673] j) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 101, or

[0674] k) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 102, or

[0675] l) a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:103, or

[0676] m) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 104, or

[0677] n) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 105, or

[0678] o) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 106, or

[0679] p) the heavy chain variable domain VH of SEQ ID NO:91 and the light chain variable domain VL of SEQ ID NO:107.

[0680] 10. An antibody that binds to human CSF-1R for use in treating a patient having a CSF-1R expressing tumor or a tumor with CSF-1R expressing macrophage infiltration, wherein the tumor is characterized by elevated CSF-1R ligand, and wherein the anti-CSF-1R antibody is administered in combination with an antibody that binds to human PD-L1,

[0681] The antibody binding to human CSF-1R used in the combination therapy is characterized by comprising

[0682] a) a heavy chain variable domain VH of SEQ ID NO: 23 and a light chain variable domain VL of SEQ ID NO: 24, or

[0683] b) a heavy chain variable domain VH of SEQ ID NO: 31 and a light chain variable domain VL of SEQ ID NO: 32, or

[0684] c) a heavy chain variable domain VH of SEQ ID NO: 39 and a light chain variable domain VL of SEQ ID NO: 40, or

[0685] d) a heavy chain variable domain VH of SEQ ID NO: 47 and a light chain variable domain VL of SEQ ID NO: 48, or

[0686] e) a heavy chain variable domain VH of SEQ ID NO: 55 and a light chain variable domain VL of SEQ ID NO: 56; and the antibody binding to human PD-L1 used in the combination therapy is characterized by comprising

[0687] a) a heavy chain variable domain VH of SEQ ID NO: 89 and a light chain variable domain VL of SEQ ID NO: 92, or

[0688] b) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 93, or

[0689] c) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 94, or

[0690] d) a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:95, or

[0691] e) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 96, or

[0692] f) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 97, or

[0693] g) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 98, or

[0694] h) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 99, or

[0695] i) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 100, or

[0696] j) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 101, or

[0697] k) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 102, or

[0698] l) a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:103, or

[0699] m) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 104, or

[0700] n) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 105, or

[0701] o) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 106, or

[0702] p) the heavy chain variable domain VH of SEQ ID NO:91 and the light chain variable domain VL of SEQ ID NO:107.

[0703] 11. The antibody according to any of the preceding items, characterized in that the antibody is of human IgG1 subclass or human IgG4 subclass.

[0704] 12. The antibody according to any of the preceding items, characterized in that the antibody has reduced or minimal effector function.

[0705] 13. The antibody according to any of the preceding items, characterized in that said minimal effector function results from an effector-reducing Fc mutation.

[0706] 14. The antibody according to any of the preceding items, characterized in that the effector reducing Fc mutation is L234A / L235A or L234A / L235A / P329G or N297A or D265A / N297A.

Claims

1. An antibody that binds to human CSF-1R, wherein the antibody is administered in combination with an antibody that binds to human PD-L1, for use in treating cancer, for preventing or treating metastasis, for treating inflammatory diseases, for treating bone loss, for treating immune-related diseases such as tumor immunity or delaying its progression, or for stimulating an immune response or function, such as T cell activity, The antibody binding to human CSF-1R used in the combination therapy is characterized by comprising a) a heavy chain variable domain VH of SEQ ID NO: 23 and a light chain variable domain VL of SEQ ID NO: 24, or b) a heavy chain variable domain VH of SEQ ID NO: 31 and a light chain variable domain VL of SEQ ID NO: 32, or c) a heavy chain variable domain VH of SEQ ID NO: 39 and a light chain variable domain VL of SEQ ID NO: 40, or d) a heavy chain variable domain VH of SEQ ID NO: 47 and a light chain variable domain VL of SEQ ID NO: 48, or e) a heavy chain variable domain VH of SEQ ID NO: 55 and a light chain variable domain VL of SEQ ID NO: 56; and the antibody binding to human PD-L1 used in the combination therapy is characterized by comprising a) a heavy chain variable domain VH of SEQ ID NO: 89 and a light chain variable domain VL of SEQ ID NO: 92, or b) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 93, or c) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 94, or d) a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:95, or e) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 96, or f) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 97, or g) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 98, or h) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 99, or i) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 100, or j) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 101, or k) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 102, or l) a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:103, or m) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 104, or n) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 105, or o) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 106, or p) the heavy chain variable domain VH of SEQ ID NO:91 and the light chain variable domain VL of SEQ ID NO:

107.

2. The antibody according to claim 1, for use in the treatment of cancer.

3. The antibody according to claim 2 for use in the treatment of breast cancer, lung cancer, colon cancer, ovarian cancer, melanoma cancer, bladder cancer, renal cancer, kidney cancer, liver cancer, head and neck cancer, colorectal cancer, pancreatic cancer, gastric cancer, esophageal cancer, mesothelioma, prostate cancer, leukemia, lymphoma, myeloma.

4. The antibody according to claim 1, for use in preventing or treating metastasis.

5. The antibody according to claim 1, for use in treating bone loss.

6. The antibody according to claim 1 for use in the treatment of an inflammatory disease.

7. The antibody according to claim 1, for use in treating immune related diseases such as tumor immunity or delaying its progression.

8. The antibody according to claim 1, for use in stimulating an immune response or function, such as T cell activity.

9. An antibody that binds to human CSF-1R, wherein the antibody is administered in combination with an antibody that binds to human PD-L1, for i) inhibiting cell proliferation in CSF-1R ligand-dependent and / or CSF-1 ligand-independent CSF-1R-expressing tumor cells; ii) inhibiting cell proliferation in tumors infiltrated with CSF-1R ligand-dependent and / or CSF-1R ligand-independent CSF-1R-expressing macrophages; iii) inhibiting cell survival in (CSF-1R ligand-dependent and / or CSF-1R ligand-independent) CSF-1R-expressing monocytes and macrophages; and / or iv) inhibiting differentiation of CSF-1R-expressing monocytes into macrophages in (CSF-1R ligand-dependent and / or CSF-1R ligand-independent) monocytes, The antibody binding to human CSF-1R used in the combination therapy is characterized by comprising a) a heavy chain variable domain VH of SEQ ID NO: 23 and a light chain variable domain VL of SEQ ID NO: 24, or b) a heavy chain variable domain VH of SEQ ID NO: 31 and a light chain variable domain VL of SEQ ID NO: 32, or c) a heavy chain variable domain VH of SEQ ID NO: 39 and a light chain variable domain VL of SEQ ID NO: 40, or d) a heavy chain variable domain VH of SEQ ID NO: 47 and a light chain variable domain VL of SEQ ID NO: 48, or e) a heavy chain variable domain VH of SEQ ID NO: 55 and a light chain variable domain VL of SEQ ID NO: 56; and the antibody binding to human PD-L1 used in the combination therapy is characterized by comprising a) a heavy chain variable domain VH of SEQ ID NO: 89 and a light chain variable domain VL of SEQ ID NO: 92, or b) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 93, or c) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 94, or d) a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:95, or e) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 96, or f) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 97, or g) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 98, or h) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 99, or i) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 100, or j) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 101, or k) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 102, or l) a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:103, or m) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 104, or n) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 105, or o) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 106, or p) the heavy chain variable domain VH of SEQ ID NO:91 and the light chain variable domain VL of SEQ ID NO:

107.

10. An antibody that binds to human CSF-1R for use in treating a patient having a CSF-1R expressing tumor or a tumor with CSF-1R expressing macrophage infiltration, wherein the tumor is characterized by elevated CSF-1R ligand, and wherein the anti-CSF-1R antibody is administered in combination with an antibody that binds to human PD-L1, The antibody binding to human CSF-1R used in the combination therapy is characterized by comprising a) a heavy chain variable domain VH of SEQ ID NO: 23 and a light chain variable domain VL of SEQ ID NO: 24, or b) a heavy chain variable domain VH of SEQ ID NO: 31 and a light chain variable domain VL of SEQ ID NO: 32, or c) a heavy chain variable domain VH of SEQ ID NO: 39 and a light chain variable domain VL of SEQ ID NO: 40, or d) a heavy chain variable domain VH of SEQ ID NO: 47 and a light chain variable domain VL of SEQ ID NO: 48, or e) a heavy chain variable domain VH of SEQ ID NO: 55 and a light chain variable domain VL of SEQ ID NO: 56; and the antibody binding to human PD-L1 used in the combination therapy is characterized by comprising a) a heavy chain variable domain VH of SEQ ID NO: 89 and a light chain variable domain VL of SEQ ID NO: 92, or b) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 93, or c) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 94, or d) a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:95, or e) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 96, or f) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 97, or g) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 98, or h) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 99, or i) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 100, or j) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 101, or k) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 102, or l) a heavy chain variable domain VH of SEQ ID NO:90 and a light chain variable domain VL of SEQ ID NO:103, or m) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 104, or n) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 105, or o) a heavy chain variable domain VH of SEQ ID NO: 90 and a light chain variable domain VL of SEQ ID NO: 106, or p) the heavy chain variable domain VH of SEQ ID NO:91 and the light chain variable domain VL of SEQ ID NO:107.

Images