HK1252860B - Antibodies against human csf-1r for use in inducing lymphocytosis in lymphomas or leukemias - Google Patents

Description

Antibodies against human CSF-1R for inducing lymphocytosis in lymphoma or leukemia

The present invention relates to antibodies against human CSF-1R that are useful for inducing lymphocytosis in lymphomas or leukemias. The present invention further relates to combination therapies using specific antibodies that bind to human CSF-1R and specific antibodies that bind to human CD20, and methods for targeting CD20-expressing cancers using anti-CD20 antibodies in combination with CSF-1R inhibitors that target macrophages to prevent escape from CD20-targeted therapies.

Background of the Invention

CSF-1R and CSF-1R antibodies

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, E.R., Curr. Top. Microbiol. Immunol. 181 (1992) 141-167).

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, C.J. 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, M.F. et al., Nature 325 (1987) 549-552. In this publication, it was shown that CSF-1R has 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, P.S. 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).

Currently, two CSF-1R ligands are known to 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, E.R. et al., Journal of Cellular Biochemistry 21 (1983) 151-159; Stanley, E.R. et al., Stem Cells 12 Suppl. 1 (1995) 15-24). The second is IL-34 (human IL-34; SEQ ID NO: 87) (Hume, D.A. et al., Blood 119 (2012) 1810-1820). 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). Activation of CSF-1R is mediated by its CSF-1R ligands, CSF-1 (M-CSF) and IL-34. Binding of CSF-1 (M-CSF) to CSF-1R induces homodimer formation and activation of the kinase through tyrosine phosphorylation (Li, W. et al., EMBO Journal. 10 (1991) 277-288; Stanley, E.R. et al., Mol. Reprod. Dev. 46 (1997) 4-10).

Biologically active homodimers of CSF-1 bind to CSF-1R within subdomains D1 to D3 of the CSF-1 receptor extracellular domain (CSF-1R-ECD). The 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, Y-G. et al., Molecular & Cellular Proteomics 2 (2003) 1143-1155; Pixley, F.J. et al., Trends Cell Biol 14 (2004) 628-638).

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 intracellular domain of CSF-1R include STAT1, STAT3, PLCy, and Cb1 (Bourette, R.P. and Rohrschneider, L.R., Growth Factors 17 (2000) 155-166).

CSF-1R signaling has physiological roles in immune responses, bone remodeling, and the reproductive system. Animals with either CSF-1 (Pollard, J.W., Mol. Reprod. Dev. 46 (1997) 54-61) or CSF-1R (Dai, X.M. et al., Blood 99 (2002) 111-120) knockouts have been shown to have osteopetrosis, hematopoiesis, tissue macrophage, and reproductive phenotypes, consistent with a role for CSF-1R in various cell types.

Sherr, C.J. et al., Blood 73 (1989) 1786-1793, relates to antibodies against CSF-1R that inhibit CSF-1 activity. Ashmun, R.A. 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, relates to an anti-CSF-1 antibody that inhibits orthodontic tooth movement. WO 2001/030381 relates 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. US 2002/0141994 relates to inhibitors of colony stimulating factor. WO 2006/096489 relates to the treatment of rheumatoid arthritis by anti-CSF-1 antibodies. WO 2009/026303 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). WO 2011/123381 (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).

CD20 and CD20 antibodies

The CD20 molecule (also known as human B lymphocyte-restricted differentiation antigen or Bp35) is a well-described hydrophobic transmembrane protein located on pre-B and mature B lymphocytes (Valentine, M.A. et al., J. Biol. Chem. 264 (1989) 11282-11287; and Einfeld, D.A. et al., EMBO J. 7 (1988) 711-717; Tedder, T.F. et al., Proc. Natl. Acad. Sci. U.S.A. 85 (1988) 208-212; Stamenkovic, I. et al., J. Exp. Med. 167 (1988) 1975-1980; Tedder, T.F. et al., J. Immunol. 142 (1989) 2560-2568). CD20 is expressed on greater than 90% of B-cell non-Hodgkin's lymphomas (NHL) (Anderson, K.C. et al., Blood 63 (1984) 1424-1433), but is not found on hematopoietic stem cells, pro-B cells, normal plasma cells, or other normal tissues (Tedder, T.F. et al., J. Immunol. 135 (1985) 973-979).

There are two different types of anti-CD20 antibodies that differ significantly in their CD20 binding patterns and biological activities (Cragg, M.S. et al., Blood 103 (2004) 2738-2743; and Cragg, M.S. et al., Blood 101 (2003) 1045-1052). Type I antibodies (such as rituximab, a non-afucosylated antibody with a fucose content of 85% or more) are potent in complement-mediated cytotoxicity.

Type II antibodies (such as, for example, Tositumomab (B1), 11B8, AT80 or humanized B-Ly1 antibody) effectively initiate target cell death via caspase-independent apoptosis and concomitant phosphatidylserine exposure.

Common features shared by type I and type II anti-CD20 antibodies are summarized in Table 1 .

Table 1: Characteristics of Type I and Type II anti-CD20 antibodies

SUMMARY OF THE INVENTION

The present invention relates to an antibody that binds to CSF-1R, which is used for inducing lymphocytosis of leukemic cells in lymphoma or leukemia.

The present invention relates to the use of antibodies that bind to CSF-1R for inducing lymphocytosis of leukemic cells in lymphoma or leukemia.

The present invention relates to a method of treatment comprising administering an effective amount of an antibody that binds to CSF-1R for inducing lymphocytosis of leukemic cells in lymphoma or leukemia.

One embodiment is the use of an antibody that binds to CSF-1R for the manufacture of a medicament for inducing lymphocytosis of leukemic cells in lymphoma or leukemia.

In one embodiment, the lymphocytosis increases the percentage of circulating leukemic cells expressing CD19 and/or expressing CD20 in the peripheral blood.

In one embodiment, the lymphocytosis increases the percentage of circulating leukemic cells expressing CD19 and/or expressing CD20 in the peripheral blood and renders the lymphoma or leukemia susceptible to treatment with an anti-CD19 antibody and/or an anti-CD20 antibody.

In one embodiment, the lymphocytosis increases circulating leukemic cells expressing CD20.

In one embodiment, the lymphocytosis increases the percentage of circulating leukemic cells that express CD20 and renders the lymphoma or leukemia susceptible to treatment with an anti-CD20 antibody.

The present invention further relates to an antibody that binds to human CSF-1R, wherein the antibody is used in combination with an antibody that binds to human CD20.

i) for the treatment of cancers expressing CD20; or

ii) for stimulating an immune response or function, such as T cell activity; or

iii) for stimulating a cell-mediated immune response, in particular stimulating cytotoxic T-lymphocytes, stimulating T-cell activity, or stimulating macrophage activity; or

iv) for delaying cancer progression; or

v) For prolonging the survival of patients suffering from cancer.

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 wherein the antibody binding to human CD20 used in the combination therapy is characterized by comprising

a) a heavy chain variable domain VH of SEQ ID NO: 69 and a light chain variable domain VL of SEQ ID NO: 76, or

b) a heavy chain variable domain VH of SEQ ID NO: 70 and a light chain variable domain VL of SEQ ID NO: 76, or

c) a heavy chain variable domain VH of SEQ ID NO: 71 and a light chain variable domain VL of SEQ ID NO: 76, or

d) a heavy chain variable domain VH of SEQ ID NO: 72 and a light chain variable domain VL of SEQ ID NO: 76, or

e) a heavy chain variable domain VH of SEQ ID NO: 73 and a light chain variable domain VL of SEQ ID NO: 76, or

f) a heavy chain variable domain VH of SEQ ID NO: 74 and a light chain variable domain VL of SEQ ID NO: 76, or

g) the heavy chain variable domain VH of SEQ ID NO: 75 and the light chain variable domain VL of SEQ ID NO: 76.

The present invention further relates to a method of targeting CD20-expressing cancers with anti-CD20 antibodies in combination with a CSF-1R inhibitor for preventing escape from CD20-targeted therapies by targeting macrophages.

In one embodiment, the macrophages are targeted with an anti-CSF1R antibody.

In one embodiment, in such methods, the antibody binding to human CSF-1R used in the combination therapy is characterized in that it comprises

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 wherein the antibody binding to human CD20 used in the combination therapy is characterized by comprising

a) a heavy chain variable domain VH of SEQ ID NO: 69 and a light chain variable domain VL of SEQ ID NO: 76, or

b) a heavy chain variable domain VH of SEQ ID NO: 70 and a light chain variable domain VL of SEQ ID NO: 76, or

c) a heavy chain variable domain VH of SEQ ID NO: 71 and a light chain variable domain VL of SEQ ID NO: 76, or

d) a heavy chain variable domain VH of SEQ ID NO: 72 and a light chain variable domain VL of SEQ ID NO: 76, or

e) a heavy chain variable domain VH of SEQ ID NO: 73 and a light chain variable domain VL of SEQ ID NO: 76, or

f) a heavy chain variable domain VH of SEQ ID NO: 74 and a light chain variable domain VL of SEQ ID NO: 76, or

g) the heavy chain variable domain VH of SEQ ID NO: 75 and the light chain variable domain VL of SEQ ID NO: 76.

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

In a preferred embodiment, the antibody is used to treat cancers that express CD20, such as lymphomas (preferably B-cell non-Hodgkin's lymphoma (NHL)) and lymphocytic leukemias.

In a preferred embodiment, the antibody is used to treat multiple myeloma.

In a preferred embodiment, the antibody is used to treat follicular lymphoma.

In a preferred embodiment, the antibody is used to treat Hodgkin's disease.

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

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

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

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

The inventors have discovered a set of molecular interactions that support the in vivo dependence of leukemic cells on monocytes/macrophages. The present invention relates to the discovery that CSF1R inhibition reduces leukemic cell load (particularly in the bone marrow) and increases circulating CD20+ leukemic cells, thus suggesting a therapeutic combination of two different antibodies, one targeting TAMs and the other targeting the CD20 molecule on leukemic cells.

Such combination therapies with specific antibodies described herein have shown benefit for patients in need of CSF-1R and CD20-targeted therapies. The inventors discovered that administration of anti-CSF1R antibodies induces significant lymphocytosis, which renders lymphomas and leukemias more susceptible to treatment with anti-CD20 and/or anti-CD19 antibodies. Furthermore, they show that the associated increase in macrophage depletion of circulating CD20+ leukemic cells represents a therapeutic opportunity in combination with anti-CD20 antibodies. Based on the increased percentage of CD19+CD20+ circulating leukemic cells, effective combination therapies with specific anti-CSF-1R and anti-CD20 antibodies can be administered to patients suffering from lymphomas and leukemias, particularly those expressing CD20. Even more strikingly, when an anti-human CSF1R monoclonal antibody is combined with GA101, leukemic cell depletion is significantly enhanced. This observation suggests a novel therapeutic strategy based on the combination of two different antibodies, one targeting TAMs and the other targeting CD20+ circulating cells.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1a-b 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. Cell viability was measured in a CTG viability assay (Promega) on day 7 of antibody treatment. % cell viability was calculated as the RLU signal from treated cells divided by the RLU signal from untreated controls without antibody (n=4).

1b: Human monocytes differentiated into macrophages after treatment with GM-CSF (M1) or M-CSF (M2) for 7 days. Phenotype was analyzed by indirect fluorescence analysis using 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 ratio between the mean fluorescence intensity (MFI) of cells stained with the selected antibody (open bars) and the corresponding isotype control (negative control; solid gray bars) was calculated (mean ± SD; n ≥ 5).

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

Figure 3. T cell expansion induced by CD3 and CD28 activation is suppressed in the presence of TAMs: TAMs were isolated from MC38 tumors and co-cultured with CFSE-labeled CD8+ T cells at the indicated ratios in the presence of CD3/CD28 stimulation. T cell proliferation was analyzed after 3 days using beads quantified for CFSE- ^low dividing cells. One representative experiment from two was depicted with the mean + SEM of triplicate wells.

Fig. 4 Anti-leukemic effect of anti-CSF1R mAb in CLL xenograft system.

(ABCDE) Rag2 ^−/− γ _c ^−/− mice challenged with ivMEC1 (day 0) were treated with 30 mg/kg anti-CSF1R mAb iv (days +11, +25) (n=3, white circles) or left untreated (n=4, black circles) and sacrificed on day 27 (A). The mean absolute number of CD11b+F4/80+ cells gated on CD45+ in the SP (B) and BM (C) is shown. The mean absolute number of human CD19+ cells in the BM (D) is shown. The mean absolute number of human CD19+ cells and Ann-PI+ cells gated on hCD19+ cells in the SP (E) is shown. Data are from one representative experiment out of three. Statistical analysis: *p<0.05, **p<0.01, Student's t-test. (FGHI) Rag2 ^-/- _γc ^-/- mice challenged with ivMEC1 (day 0) were treated with 30 mg/kg anti-CSF1R mAb iv (days +11, +25) (n=7, white circles) or left untreated (n=7, black circles) and killed on days 27 and 29, 48 and 96 hours after the last mAb injection (F). The mean absolute number of human CD19+ cells and Ann-PI+ cells gated on hCD19+ cells in the SP on days 27 and 29 are shown (G). The mean absolute number of CD11b+F4/80+ cells gated on CD45+ in the SP on day 27 are shown (H). The mean absolute number of CD11b+MRC1+ cells versus CD45+ gated CSF1R+ macrophages in the SP on day 27 are shown (I). Data are from one representative experiment out of two. Statistical analysis: *p < 0.05, Student's t-test. See also Figure S4.

Fig. 5 Anti-leukemic efficacy and survival impact of anti-CSF1R mAb in CLL xenograft system.

(ABC) Rag2 ^−/− γ _c ^−/− mice challenged with ivMEC1 (day 0) were treated with 30 mg/kg anti-CSF1R mAb iv (days +11, +25) (n=3, white circles) or left untreated (n=4, black circles) and sacrificed on day 27 (A). Mean values for the percentages of hCD19+ (B) and hCD19+CD20+ (C) cells in PB are shown. Data are from one representative experiment out of two. Statistically significant differences were calculated using the Student's t-test. **p<0.01, ***p<0.001. (D-E) Rag2 ^−/− γ _c ^−/− mice challenged with ivMEC1 (day 0) were left untreated (n=10, black circles) or treated with: 30 mg/kg i.v. anti-CSF1R mAb (n=11, white circles; day +11, +25); or 30 mg/kg ip anti-CD20 mAb GA101 (n=10, blue circles; day +11, +25); or 30 mg/kg ip anti-CD20 mAb GA101 (day +11, +25) + 30 mg/kg i.v. anti-CSF1R mAb (day +18, +32) (n=10, purple circles) and monitored for survival (D). Kaplan-Meier survival curves are presented (E), and statistical analysis was performed using the log-rank test. αCSF1R vs. control: p=0.01; αCD20 vs. control: p=0.006; αCD20+αCSF1R vs. control: p<0.0001; αCD20+αCSF1R vs. αCD20: p=0.0585.

Fig. 6 Effects of macrophage targeting on the microenvironment of CLL mouse models.

(ABCDEF) Rag2 ^-/- _γc ^-/- mice challenged with ivMEC1 (day 0) were treated with 30 mg/kg anti-CSF1R mAb iv (days +11, +25) (n=4, white circles) or left untreated (n=4, black circles) and sacrificed on day 27 or 29 (A). The mean relative contribution of CD11b+CSF1R+ cells gated on CD45+ in PB is shown (B). The mean relative contribution of CD11b+CSF1R-SSC-high neutrophils gated on CD45+ in PB is shown (C). The mean relative contribution of CD11b+Gr1+ cells gated on CD45+ in PB is shown (D). Data are from one representative experiment out of two. The graph shows the mean relative contribution of monocytes (Ly6C+Ly6G) gated on CD11b+F4/80 ^low (E) and granulocytes (Ly6ClowLy6Ghigh) gated ^{on CD11b+F4/80 low ( F} ) in PB. Statistical analysis: *p<0.05, **p<0.01, ***p<0.001, Student's t-test. (G, H, J, K) Rag2 ^-/- _γc ^-/- mice challenged with ivMEC1 (day 0) were treated with 60 μl clodrolip i.v. (days +11, +25) (n=4, white circles) or left untreated (n=4, black circles) and sacrificed on day 26 (G). The graph shows the mean relative contribution of CD11b+CSF1R- ^SSChigh neutrophils gated on CD45+ in PB (H). The graph shows the mean relative contribution of CD11b+Grl+ cells gated on CD45+ in PB (I). The graph shows the mean absolute number of CD11b+CSF1R-SSC- ^high neutrophils gated on CD45+ in BM (J). The graph shows the mean absolute number of CD11b+Grl+ cells gated on CD45+ in BM (K). Data are from one representative experiment out of two. Statistical analysis: *p<0.05, Student's t-test. (LMN) Rag2 ^-/- _γc ^-/- mice challenged with ivMEC1 (day 0) were treated with 30 mg/kg anti-CSF1R mAb iv (days +11, +25) (n=4, white circles) or left untreated (n=4, black circles) and sacrificed on day 27 or 29 (A). The graph shows the mean absolute number of CD11b+CSF1R+ cells gated on CD45+ in BM (L). Data are from one representative experiment out of two. The figure shows the mean absolute number of monocytes (Ly6C+Ly6G-) gated on CD11b+F4/80 ^low (M) and granulocytes (Ly6ClowLy6Ghigh) gated on CD11b+F4/80 ^{low ( N} ) in the BM. Statistical analysis: *p<0.05, Student's t-test. (OPQRSTU) C57BL/6 mice transplanted with leukemic B cells from Eμ-TCL1 transgenic mice were treated with 30 mg/kg anti-CSF1R mAb (days +17, +23) (n=3, white circles) or left untreated (n=3, black circles) and sacrificed on day 26 (O). The figure shows the mean relative contribution of CD44+CD62L+ centrifugal and CD44+CD62Llow/ ^negative effector memory CD8+ T cells in the PB (P). The figure shows the mean of the absolute number of CD44+CD62L+ central and CD44+CD62L ^low /negative effector memory CD8+ T cells in BM (Q) and SP (R). Intracellular IFNγ production was measured by flow cytometry, and the figure shows the mean of the absolute number of CD8+CD44+IFNγ+ T cells (S). The figure shows the mean of the absolute number of Ann+PI- cells gated on CD19+CD5+ cells versus the entire B cell pool in SP (T). The figure shows the mean of the absolute number of CD4+CD25+ T cells (U). Statistical analysis: *p<0.05, **p<0.01, ***p<0.001, Student's t-test. (VWX) C57BL/6 mice transplanted i.p. with leukemic B cells from Eμ-TCL1 transgenic mice were treated i.p. with 50 μl of clodrolip (starting on day 17, every 3 days) (n=4, white circles) or left untreated (n=4, black circles) and killed on day 24 (V). The mean absolute number of CD44+CD62L+ centrifugal and CD44+CD62L low/negative effector memory CD8+ T cells in SP is shown (W). The mean absolute number of CD4+CD25+ T cells in SP is shown (X).

Figure 7 BM microenvironment of xenograft mice: monocyte and macrophage populations

(ABCDEF) Rag2 ^-/- γc-/ _- mice that were not injected (UN, black circles) or injected iv with MEC1 (white circles) cells (day 0) were killed at the early (n=3) and late (n= ³⁾ stages of leukemia and analyzed by flow cytometry (A). The mean of the relative contribution of hCD19+ cells in the BM is shown (B). The mean of the absolute number of CD11b+CSF1R+ cells gated on CD45+ in the BM is shown (C). The mean of the absolute number of CD11b+F4/80+ cells gated on CD45+ in the BM is shown (D). The mean of the absolute number of CD11b+MRC1+ cells gated on CD45+ to the total macrophage population (CD11b+F4/80+) in the BM is shown (E). Data are from one representative experiment out of three. Statistical analysis: *p<0.05, **p<0.01, ***p<0.001, Student's t-test. The figure shows the mean absolute number of CD86+MRC1+ and CSF1R+MRC1+ cells relative to the entire CD45+CD11b+ population in BM (F). Statistical analysis: *p<0.05, Student's t-test.

FIG8 Cytokine production by BM stromal cells from CLL xenograft mice.

(ABC) Rag2 ^-/- _γc ^-/- mice that were not injected (UN, black circles, n=3) or injected IV with MEC1 (white circles) cells (day 0) were killed at the early stage of leukemia (n=5) and analyzed by flow cytometry. Intracellular cytokine production was measured by flow cytometry. Left: The graph shows the mean absolute number of IL-17a-producing CD11b+Gr1+ cells in the BM gated on CD45+; Right: Representative flow cytometric graph of IL-17a-producing CD11b+Gr1+ cells in the BM gated on CD45+ (A). Left: The mean absolute number of IL-17a-producing non-hematopoietic CD45- cells; Right: Representative flow cytometric graph of IL-17a-producing non-hematopoietic CD45- cells in the BM (B). Left: Mean absolute number of IL-2-producing non-hematopoietic CD45- cells; Right: Representative flow cytometry images of IL-2-producing non-hematopoietic CD45- cells in BM (C). Statistical analysis: *p<0.05, **p<0.01, Student's t-test.

Figure 9 Transcriptome analysis of monocytes/macrophages and MEC1 leukemia cells in xenograft mice

(ABCDEF) Rag2 ^-/- _γc ^-/- mice were injected iv with MEC1 cells and sacrificed at an early stage of leukemia along with age-matched untransplanted Rag2 ^-/- _γc ^-/- mice (n=3/group). RNA was isolated from BM murine monocytes/macrophages purified by magnetic separation and processed for Illumina whole-genome gene expression direct hybridization assays. A supervised analysis between monocytes/macrophages from xenotransplanted and untransplanted (UN) mice was performed. The heat map presents hierarchical clustering of differentially expressed genes (transplanted vs. UN) (adjusted P value < 0.05). The expression level of each gene was normalized by subtracting the mean expression of the gene and then dividing by the standard deviation across all samples. This ranked expression value (expressed as Row Z score) is plotted on a red-blue color scale, with red indicating high expression. Gene expression differences between groups were functionally categorized as follows: nuclear effectors, tumor suppressors, and translation-related (A); intracellular functions (B); membrane/extracellular proteins (C), apoptosis-related (D), and inflammatory mediators (E). RNA was isolated from BM-infiltrating human CD19+ MEC1 cells purified by magnetic separation and processed for Illumina whole-genome gene expression direct hybridization assays. A supervised analysis was performed between xenografted and pre-injected MEC1 cells in vitro. A heat map presents hierarchical clustering of differentially expressed genes (heterografted vs. pre-injection) (adjusted P value < 0.05). Gene expression differences between groups involving monocyte/macrophage and B cell interactions are depicted (F). (G) Flow cytometry analysis of Rag2 ^-/- γc-/ ^- mice (n=4, white circles) and age-matched uninjected (UN) Rag2 ^-/- _γc ^-/- mice (n=4, black circles) injected i.v. with MEC1 cells and _sacrificed on day 21. Left: Representative flow cytometric plots of CD11b+ICAM1+ cells gated on CD45+ versus the total macrophage population (CD11b+F4/80+) in SP, BM, and PE. Right: The graph shows the mean absolute number of CD11b+ICAM1+ cells versus the total macrophage population (CD11b+F4/80+) gated on CD45+ in SP, BM, and PE. Statistical significance was analyzed by Student's t-test. *p<0.05, **p<0.01, ***p<0.001.

Figure 10 Transcriptome analysis suggests a role for crosstalk between monocytes/macrophages and leukemic cells (related to Figure 9).

(A) Relative mRNA expression of Fcgr1, Saa3, Icam1, Dleu2, Apoe, and Tlr7 in monocytes/macrophages from the BM of Rag2 ^−/− γ _c ^−/− mice that were uninjected (UN) and injected IV with MEC1 cells and sacrificed at the early stage of leukemia. Expression was normalized to β-actin levels. Data are shown as mean ± SD (n = 3/group). (B) Relative mRNA expression of CCL2, CEBPB, CR2, IL10, IL23R, ADAM10, PTEN, and RNASET2 in MEC1 B cells from the BM and pre-injection cells from xenografted Rag2 ^−/− γ _c ^−/− mice (n = 3/group) sacrificed at the early stage of leukemia. Expression was normalized to β-actin levels. Data are shown as mean ± SD (n = 3/group). (C) Fresh peripheral blood samples were collected from 18 CLL patients and 4 healthy donors. Monocytes were analyzed for ICAM1 expression by flow cytometry. CD14+ monocytes were subdivided into CD14++CD16- classical monocytes, CD14++CD16+ intermediate monocytes, and CD14+CD16++ non-classical monocytes. The mean ± SD of the relative contribution of ICAM1+ cells to the CD14-gated monocyte population is shown. (D-E) Rag2 ^-/- _γc ^-/- mice challenged with ivMEC1 (day 0) were pretreated with 2 mg/kg i.v. ICAM1-blocking mAb clone YN1/1.7.4 every 3/4 days starting on day -1 (n=3, white triangles) or left untreated (n=3, black circles) and analyzed by flow cytometry (D). The mean of the percentage of human CD19+ cells in PB and the absolute number of human CD19+ cells in BM, SP, and PE are shown (E). Statistical analysis: *p<0.05, **p<0.01, ***p<0.001, Student's t-test.

Figure 11 Mechanism of cell death induced by macrophage targeting in leukemia cells

(A) Left: MEC1 cells were plated in 96-well plates with increasing concentrations of clodrolip (10 μM, 100 μM, 1000 μM) and PBS liposomes (v, 1000 μM) alone (c) and subjected to a luminescence assay at 24, 48, and 72 hours to assess the sensitivity of MEC1 cells to the drugs. Right: MEC1 cells were plated in 96-well plates with anti-human CSF1R mAb RG7155 (hMab 2F11-e7 IgG1 isotype) (1-10 μg/ml) alone (c) and subjected to a luminescence assay at 48 and 72 hours to assess the sensitivity of MEC1 cells to the drugs.

Figure 12 Mechanism of TNF-dependent macrophage-mediated leukemia cell death in vivo

(ABCDEFG) Rag2 ^-/- _γc ^-/- mice challenged with ivMEC1 (day 0) were pretreated with 200 μl ivclodrolip every 3 days starting on day -1 (n=4, white circles) or left untreated (n=3, black circles) and analyzed by flow cytometry (A). The mean percentage of human CD19+ cells in SP, BM, PB, and PE is shown (B). The mean relative contribution of CD11b+CSF1R+ cells gated on CD45 in PB is shown (C). The mean absolute number or percentage of CD11b+F4/80+ cells gated on CD45+ in SP, BM, PE (D), and PB (E) is shown. The mean relative contribution of Ann+PI+ cells gated on hCD19+ cells in SP, BM, and PE (F) and the mean relative contribution of Ann-PI+ cells gated on hCD19+ cells in PB (G) are shown. Statistical significance was analyzed by Student's t-test. *p<0.05, **p<0.01, ***p<0.001.

(HI) Rag2 ^-/- _γc ^-/- mice challenged with ivMEC1 (day 0) were left untreated (n=6, black circles) or treated iv with 60 μl clodrolip (n=4, white circles) or 60 μl clodrolip (days +11, +25) plus 10 mg/kg etanercept (ip, days +10, +12, +15, +18, +21, +24) (n=6, red triangles) (days +11, +25) and sacrificed on day 26 (H). The mean absolute number of human CD19+ cells in BM is shown (I). Statistical significance was analyzed by Student's t-test. *p<0.05, **p<0.01, ***p<0.001.

(JK) Rag2 ^-/- _γc ^-/- mice challenged with ivMEC1 (day 0) were left untreated (n=5, black circles) or treated iv with 30 mg/kg anti-CSF1R mAb (n=7, white circles) or 30 mg/kg anti-CSF1R mAb (days +11, +25) plus 10 mg/kg etanercept (ip, days +10, +12, +15, +18, +21, +24) (n=6, red triangles) (days +11, +25) and sacrificed on day 29 (J). The mean absolute number of human CD19+ cells in SP is shown (K). Statistical significance was analyzed by Student's t-test. *p<0.05.

Figure 13 Monocytes/macrophages and leukemia cells in humans

(G) hCD19+CD5+ (black circles) and hCD14+ (white circles) depletion after treatment with anti-CD20 mAb GA101 10 μg/ml, anti-human CSF1R mAb RG7155 (hMab 2F11-e7 IgG1 isotype) (1-10 μg/ml), anti-CD20 mAb GA101 10 μg/ml + anti-human CSF1R mAb RG7155 (hMab 2F11-e7 IgG1 isotype) (1-10 μg/ml) for 48 hours (n=4) was calculated by the following equation: 100-% remaining cells, where % remaining cells = (absolute number in treated samples/absolute number in untreated samples) x 100. Horizontal bars represent means (*p<0.05; **p<0.01), Student's t-test.

Figure 14. Mechanism of cell death induced by clodrolip in primary leukemic cells from CLL patients (related to Figure 13).

(A) Relative mRNA expression of FAS in human primary CLL cells purified by negative selection from PBMCs of CLL patients (n=3, Pt#24-26) and plated (in triplicate) in 6-well plates for 24 hours, either alone (C, black bars) or with 1000 μM clodrolip (Clo, white bars). Expression was normalized to β-actin levels. Data are shown as mean ± SD (n=3/group). Statistically significant differences were calculated using the Student's t-test. *p<0.05.

(B) Relative mRNA expression of TNFR1, FADD, BID, and TRAIL-R2 in human primary CLL cells purified from PBMCs of a CLL patient by negative selection and plated (in triplicate) in 6-well plates alone (C, black bars) or with 1000 μM clodrolip (Clo, white bars) for 24 hours. Expression was normalized to β-actin levels. Data are shown as mean ± SD (n = 3/group). Statistically significant differences were calculated using the Student's t-test. *p < 0.05

(C) hCD19+CD5+ depletion after 24 h treatment with 100 μM, 500 μM, or 1000 μM clodrolip in the presence (black bars) or absence (white bars) of etanercept (10 μg/ml) (n=3) was calculated using the following equation: 100 − % remaining cells, where % remaining cells = (absolute number in treated samples/absolute number in untreated samples) x 100. (*p<0.05), Student's t-test.

(D) hCD19+CD5+ depletion after 24 h (n=3) treatment with clodrolip 1000 μM in the presence (black bars) or absence (white bars) of a blocking anti-TRAIL-R2 mAb (1 μg/ml) was calculated using the following equation: 100 −% remaining cells, where % remaining cells = (absolute number in treated samples/absolute number in untreated samples) x 100. (*p<0.05), Student's t-test.

Detailed Description of the Invention

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 recruited 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).

By secreting a variety of cytokines, such as colony-stimulating factor 1 (CSF-1) and IL-10, tumor cells are able to recruit and shape macrophages into the M2 subtype, while cytokines such as granulocyte macrophage colony-stimulating factor (GM-CSF), IFN-γ, and others program macrophages toward the M1 subtype. Using immunohistochemistry, it is possible to distinguish between a subpopulation of macrophages that co-express CD68 and CD163 (which is likely to be enriched for M2 macrophages) and a subset that displays a CD68+/MHC II+ or CD68+/CD80+ immunophenotype (which is likely to include M1 macrophages). The cell shape, size, and spatial distribution of CD68 and CD163-positive macrophages are consistent with the published hypothesis about the tumor-promoting role of M2 macrophages (e.g., their preferential localization in tumor-cross-matrix and vital tumor areas). In contrast, CD68+/MHC class II+ 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.

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

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

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

c) play a role in establishing an immunosuppressive environment by secreting immunosuppressive cytokines such as IL-4, IL-13, IL-1ra, and IL-10, which in turn regulate T regulatory cell function. Conversely, 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).

Thus, in several types of cancer (e.g., breast, ovary, 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, P.A., Gynecol. Oncol. 1 (1999) 47-50; Steidl, C. et al., N. Engl. J. Med. 10 (2010) 875-885). Recent data show the correlation between 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).

Recently, a so-called immune signature comprising a high number of macrophages and CD4-positive T cells but a low 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).

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 CSF-1 receptor (colony stimulating factor 1 receptor (CSF-1R)), which form the majority of the tumor mass (West, R.B. 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).

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).

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, as well as in leiomyosarcomas and TGCT/PVNS, although at lower levels than in macrophages. In 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 poorer 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 oncogenes (Chambers, S., Future Oncol 5 (2009) 1429-1440).

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

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, E.R., Curr. Top. Microbiol. Immunol. 181 (1992) 141-167).

CSF-1R is a receptor for the CSF-1R 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, C.J. et al., Cell 41 (1985) 665-676; Lin, H. et al., Science 320 (2008) 807-811). The cloning of the colony-stimulating factor-1 receptor (also known as c-fms) was first described by Roussel, M.F. 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, P.S. et al., Embo J. 18 (1999) 3616-3628).

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 receptor's extracellular domain (ECD) (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 all five subdomains D1-D3. The sequence is listed without the signal peptide MGSGPGVLLLLLVATAWHGQ G (SEQ ID NO:67). Human CSF-1R fragment D4-D3 (SEQ ID NO: 85) comprises the corresponding subdomains D4-D3.

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, E.R. et al., Journal of Cellular Biochemistry 21 (1983) 151-159; Stanley, E.R. et al., Stem Cells 12 Suppl. 1 (1995) 15-24). The second is IL-34 (human IL-34; SEQ ID NO: 87) (Hume, D.A. 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).

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 149 aa fragment of human CSF-1 (aa33-181 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, D.A. et al., Blood 119 (2012) 1810-1820).

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). Activation of CSF-1R is mediated by its CSF-1R ligands CSF-1 (M-CSF) and IL-34. Binding of CSF-1 (M-CSF) to CSF-1R induces the formation of homodimers and activation of kinases through tyrosine phosphorylation (Li, W. et al., EMBO Journal. 10 (1991) 277-288; Stanley, E.R. et al., Mol. Reprod. Dev. 46 (1997) 4-10).

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.

CSF-1R is primarily expressed on cells of the monocytic lineage and in the female reproductive tract and placenta. Furthermore, 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, A.H. et al., Oncogene 8 (1993) 371-378), and microglia (Sawada, M. et al., Brain Res. 509 (1990) 119-124). It is known that cells harboring a mutant human CSF-1R (SEQ ID NO: 23) proliferate independently of ligand stimulation.

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 specifically binds to the human CSF-1R antigen with a binding affinity of 1.0 x ^10-8 mol/l or less (in one embodiment, with _{a KD} value of 1.0 x ^10-9 mol/l or less). 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 CSF-1R" refers to an antibody that specifically binds to the human CSF-1R antigen with a binding affinity of K _D 1.0 x 10 ^-8 mol/l or less (in one embodiment, 1.0 x 10 ^-8 mol/l to 1.0 x ^{10 -13} mol/l), and in one embodiment, with a binding affinity of K _D 1.0 x 10 ^-9 mol/l or less (in one embodiment, 1.0 ^x 10 -9 mol/l to 1.0 x 10 ^-13 mol/l).

CD20 (also known as B lymphocyte antigen CD20, B lymphocyte surface antigen B1, Leu-16, Bp35, BM5, and LF5; the sequence is characterized by SwissProt database entry P11836) is a hydrophobic transmembrane protein with a molecular weight of approximately 35 kD located on pre-B and mature B lymphocytes (Valentine, M.A. et al., J. Biol. Chem. 264 (1989) 11282-11287; Tedder, T.F. et al., Proc. Natl. Acad. Sci. U.S.A. 85 (1988) 208-212; Stamenkovic, I. et al., J. Exp. Med. 167 (1988) 1975-1980; Einfeld, D.A. et al., EMBO J. 7 (1988) 711-717; Tedder, T.F. et al., J. Immunol. 142 (1989) 2560-2568). The corresponding human gene is transmembrane 4 domain, subfamily A, member 1, also known as MS4A1. This gene encodes a member of the transmembrane 4A gene family. Members of this nascent protein family are characterized by common structural features and similar intron/exon splice boundaries and exhibit distinct expression patterns in hematopoietic cells and non-lymphoid tissues. This gene encodes a B lymphocyte surface molecule that plays a role in B cell development and differentiation into plasma cells. This family member is located in a cluster at 11q12. Alternative splicing of this gene produces two transcript variants encoding the same protein.

The terms "CD20" and "CD20 antigen" are used interchangeably herein and include any variants, isoforms, and species homologs of human CD20 that are naturally expressed by cells or expressed on cells transfected with the CD20 gene. Binding of the antibodies of the present invention to the CD20 antigen mediates killing of cells expressing CD20 (e.g., tumor cells) by inactivating CD20. Killing of cells expressing CD20 can occur through one or more of the following mechanisms: cell death/apoptosis induction, ADCC, and CDC.

As recognized in the art, synonyms for CD20 include B lymphocyte antigen CD20, B lymphocyte surface antigen Bl, Leu-16, Bp35, BM5, and LF5.

The term "anti-CD20 antibody" according to the present invention is an antibody that specifically binds to the human CD20 antigen. Based on their binding properties and biological activities toward the CD20 antigen, two types of anti-CD20 antibodies (type I and type II anti-CD20 antibodies) can be distinguished according to Cragg, M.S. et al., Blood 103 (2004) 2738-2743; and Cragg, M.S. et al., Blood 101 (2003) 1045-1052, see Table 2.

Table 1: Characteristics of Type I and Type II anti-CD20 antibodies

Examples of type II anti-CD20 antibodies include, for example, humanized B-Ly1 antibody IgG1 (a chimeric humanized IgG1 antibody, as disclosed in WO 2005/044859), 11B8 IgG1 (as disclosed in WO 2004/035607), and AT80 IgG1. Generally, type II anti-CD20 antibodies of the IgG1 isotype exhibit characteristic CDC characteristics. Compared to type I antibodies of the IgG1 isotype, type II anti-CD20 antibodies have reduced CDC (if of the IgG1 isotype).

Examples of type I anti-CD20 antibodies include, e.g., rituximab, HI47IgG3 (ECACC, hybridoma), 2C6IgG1 (as disclosed in WO 2005/103081), 2F2IgG1 (as disclosed in WO 2004/035607 and WO 2005/103081), and 2H7IgG1 (as disclosed in WO 2004/056312).

The "rituximab" antibody (reference antibody; an example of a type I anti-CD20 antibody) is a genetically engineered chimeric monoclonal antibody directed against the human CD20 antigen containing a human γ1 murine constant domain. This chimeric antibody contains a human γ1 constant domain and is identified by the designation "C2B8" in US Patent No. 5,736,137 (Andersen et al.), issued April 17, 1998, to IDEC Pharmaceuticals Corporation. Rituximab is approved for the treatment of patients with relapsed or refractory low-grade or follicular, CD20-positive, B-cell non-Hodgkin's lymphoma. In vitro mechanism of action studies have shown that rituximab exhibits human complement-dependent cytotoxicity (CDC) (Reff, M.E. et al., Blood 83 (1994) 435-445). In addition, it exhibits significant activity in an assay measuring antibody-dependent cellular cytotoxicity (ADCC). Rituximab is not afucosylated.

As used herein, "binds to human CD20" or "specifically binds to human CD20" or "anti-CD20 antibody" refers to an antibody that specifically binds to the human CD20 antigen with a _binding affinity of 1.0 x ^10-8 mol/l or less (in one embodiment, with a _KD value of 1.0 x ^10-9 mol/l or less). 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 CD20" refers to an antibody that specifically binds to the human CD20 antigen with a binding affinity of K _D 1.0 x 10 ^-8 mol/l or less (in one embodiment, 1.0 x 10 ^-8 mol/l-1.0 x 10 ^-13 mol/l), and in one embodiment, with a binding affinity of K _D 1.0 x ^{10 -9} mol/l or less (in one embodiment, 1.0 x 10 -9 mol/l-1.0 x 10 ^-13 mol/l).

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.

These antibodies are described in WO 2011/070024 and are characterized by comprising the following VH and VL sequences described therein:

Table 2

In a preferred embodiment, the antibody that binds to human CSF-1R used in the combination therapy described herein is hMab 2F11-e7 (VH, SEQ ID NO: 39; VL, SEQ ID NO: 40).

In one embodiment, the antibody that binds to human CD20 used in the combination therapy described herein is a humanized B-Ly1 antibody:

The term "humanized B-Ly1 antibody" refers to humanized B-Ly1 antibodies as disclosed in WO 2005/044859 and WO 2007/031875, which are obtained from the murine monoclonal anti-CD20 antibody B-Ly1 (see Poppema, S. and Visser, L., Biotest Bulletin 3 (1987) 131-139) by chimerization with human constant domains from IgG1 and subsequent humanization (see WO 2005/044859 and WO 2007/031875). These "humanized B-Ly1 antibodies" are disclosed in detail in WO 2005/044859 and WO 2007/031875.

In one embodiment, the "humanized B-Ly1 antibody" has a heavy chain variable region (VH) selected from the group consisting of SEQ ID NO. 69 to SEQ ID NO. 75 (B-HH2, B-HH3, B-HH6, B-HH8, B-HL8, B-HL11, and B-HL13 of WO 2005/044859 and WO 2007/031875). In one embodiment, the "humanized B-Ly1 antibody" has a light chain variable region (VL) of SEQ ID NO. 76 (B-KV1 of WO 2005/044859 and WO 2007/031875).

These antibodies are characterized by comprising the following VH and VL sequences described herein:

Table 3

In a preferred embodiment, the antibody that binds to human CD20 used in the combination therapy described herein is selected from the humanized B-Ly1 antibody B-HH6/B-KV1, which comprises the heavy chain variable region (VH) of SEQ ID No. 71 (B-HH6) and the light chain variable region (VL) of SEQ ID No. 76 (B-KV1).

Furthermore, in one embodiment, the humanized B-Ly1 antibody is an afucosylated antibody of the IgG1 isotype.

According to the present invention, such afucosylated humanized B-Ly1 antibody is glycoengineered (GE) in the Fc region according to the procedures described in WO 2005/044859, WO 2004/065540, WO 2007/031875, Umana, P. et al., Nature Biotechnol. 17 (1999) 176-180, and WO 99/154342. In one embodiment, the afucosylated glycoengineered humanized B-Ly1 is B-HH6-B-KV1 GE. Such glycoengineered humanized B-Ly1 antibodies have an altered glycosylation pattern in the Fc region, preferably with reduced levels of fucose residues. In one embodiment, the amount of fucose is 60% or less of the total amount of oligosaccharides at Asn297 (in one embodiment, the amount of fucose is 40% to 60%, in another embodiment, the amount of fucose is 50% or less, and in yet another embodiment, the amount of fucose is 30% or less). In another embodiment, preferably, the oligosaccharides in the Fc region are bisected. These glycoengineered humanized B-Ly1 antibodies have increased ADCC.

In a preferred embodiment, the human CD20-binding antibody used in the combination therapy described herein is selected from the humanized B-Ly1 antibody B-HH6/B-KV1, which comprises the heavy chain variable region (VH) of SEQ ID No. 71 (B-HH6) and the light chain variable region (VL) of SEQ ID No. 76 (B-KV1) and is an afucosylated antibody of the IgG1 isotype with an altered glycosylation pattern in the Fc region, wherein the amount of fucose-containing oligosaccharides is between 40% and 60% of the total amount of oligosaccharides at Asn297.

Unlike anti-CD20 antibodies without reduced fucose, the afucosylated anti-CD20 antibodies according to the present invention have increased antibody-dependent cellular cytotoxicity (ADCC).

By "an afucosylated anti-CD20 antibody with increased antibody-dependent cellular cytotoxicity (ADCC)" is meant an afucosylated anti-CD20 antibody with increased ADCC as determined by any suitable method known to one of ordinary skill in the art, as that term is defined herein. One generally accepted in vitro ADCC assay is as follows:

1) The assay uses target cells known to express the target antigen recognized by the antigen-binding region of the antibody;

2) The assay uses human peripheral blood mononuclear cells (PBMCs) isolated from the blood of randomly selected healthy donors as effector cells;

3) Perform the assay according to the following protocol:

i) PBMCs were isolated using a standard density centrifugation protocol and suspended in RPMI cell culture medium at 5 x 10 ⁶ cells/ml;

ii) target cells were cultured by standard tissue culture methods, harvested from the exponential growth phase with a viability greater than 90%, washed in RPMI cell culture medium, labeled with 100 microcuries ⁵¹ Cr, washed twice with cell culture medium, and resuspended in cell culture medium at a density of 10 ⁵ cells/ml;

iii) transferring 100 μl of the final target cell suspension to each well of a 96-well microtiter plate;

iv) serially diluting the antibody from 4000 ng/ml to 0.04 ng/ml in cell culture medium and adding 50 μl of the resulting antibody solution to target cells in a 96-well microtiter plate, testing multiple antibody concentrations covering the entire concentration range in triplicate;

v) For maximum release (MR) controls, three additional wells in the plate containing labeled target cells received 50 μl of 2% nonionic detergent (Nonidet, Sigma, St. Louis).

(VN) aqueous solution, instead of the antibody solution (point iv above);

vi) For spontaneous release (SR) controls, three additional wells of the plate containing labeled target cells received 50 μl of RPMI cell culture medium instead of the antibody solution (point iv above);

vii) The 96-well microtiter plate was then centrifuged at 50 x g for 1 minute and incubated at 4°C for 1 hour;

viii) 50 μl of the PBMC suspension (point i above) was added to each well to produce an effector cell:target cell ratio of 25:1, and the plate was placed in an incubator at 37°C in a 5% _CO2 atmosphere for 4 hours;

ix) harvesting the cell-free supernatant from each well and quantifying the experimentally released radioactivity (ER) using a gamma counter;

x) calculating the percentage of specific lysis for each antibody concentration according to the formula (ER-MR)/(MR-SR) x 100, where ER is the mean radioactivity quantified (see point ix above) for the antibody concentration, MR is the mean radioactivity quantified (see point ix above) for the MR control (see point v above), and SR is the mean radioactivity quantified (see point ix above) for the SR control (see point vi above);

4) "Increased ADCC" is defined as an increase in the maximum percentage of specific lysis observed within the antibody concentration range tested above and/or a decrease in the antibody concentration required to achieve half of the maximum percentage of specific lysis observed within the antibody concentration range tested above. The increase in ADCC is relative to ADCC mediated by the same antibody as measured by the above assay, produced by the same type of host cells using the same standard production, purification, formulation, and storage methods known to those skilled in the art, but not produced by host cells engineered to overexpress GnTIII.

Said "increased ADCC" can be obtained by glycoengineering of the antibody, which means enhancing the natural, cell-mediated effector function of a monoclonal antibody by engineering its oligosaccharide components, as described in Umana, P. et al., Nature Biotechnol. 17 (1999) 176-180 and US 6,602,684.

The term "complement-dependent cytotoxicity (CDC)" refers to the lysis of human tumor target cells by an antibody according to the present invention in the presence of complement. Preferably, CDC is measured by treating a preparation of CD20-expressing cells with an anti-CD20 antibody according to the present invention in the presence of complement. CDC is detected if the antibody induces 20% or more tumor cell lysis (cell death) after 4 hours at a concentration of 100 nM. Preferably, the assay is performed using tumor cells labeled with ⁵¹ Cr or Eu and measurement of released ⁵¹ Cr or Eu. A control comprises incubating the tumor target cells with complement but not with the antibody.

The term "afucosylated antibody" refers to an antibody of the IgG1 or IgG3 isotype (preferably of the IgG1 isotype) having an altered glycosylation pattern at Asn297 in the Fc region and having reduced levels of fucose residues. Glycosylation of human IgG1 or IgG3 occurs at Asn297 as a core fucosylated biantennary complex oligosaccharide glycosylation with up to two Gal residues at the end. Depending on the amount of terminal Gal residues, these structures are referred to as G0, G1 (α1,6 or α1,3) or G2 glycan residues (Raju, T.S., BioProcess Int. 1 (2003) 44-53). CHO-type glycosylation of the Fc portion of an antibody is described, for example, by Routier, F.H., Glycoconjugate J. 14 (1997) 201-207. Antibodies recombinantly expressed in non-glycomodified CHO host cells are typically fucosylated at Asn297 in an amount of at least 85%. It should be understood that, as used herein, the term afucosylated antibody includes antibodies that do not have fucose in their glycosylation pattern. It is generally known that the typical glycosylation residue position in antibodies is asparagine at position 297 according to the EU numbering system ("Asn297").

The “EU numbering system” or “EU index” is generally used when referring to residues in the constant region of an immunoglobulin heavy chain (e.g., the EU index as reported in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), which is expressly incorporated herein by reference).

Thus, an afucosylated antibody according to the present invention refers to an antibody of the IgG1 or IgG3 isotype (preferably of the IgG1 isotype) in which the amount of fucose is 60% or less of the total amount of oligosaccharides (sugars) at Asn297 (this means that at least 40% or more of the oligosaccharides at Asn297 in the Fc region are afucosylated). In one embodiment, the amount of fucose is 40% to 60% of the oligosaccharides at Asn297 in the Fc region. In another embodiment, the amount of fucose is 50% or less, and in yet another embodiment, the amount of fucose is 30% or less of the oligosaccharides at Asn297 in the Fc region. According to the present invention, "the amount of fucose" means the amount of oligosaccharide (fucose) within the oligosaccharide (sugar) chain at Asn297 relative to the sum of all oligosaccharides (sugars) (e.g., complex, hybrid, and high mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry and calculated as an average value (for detailed procedures for determining the amount of fucose, see, e.g., WO 2008/077546). Furthermore, in one embodiment, the oligosaccharides in the Fc region are bifurcated. The afucosylated antibody according to the present invention can be expressed in a glycomodified host cell engineered to express at least one nucleic acid encoding a polypeptide having GnTIII activity in an amount sufficient to partially fucosylate the oligosaccharides in the Fc region. In one embodiment, the polypeptide having GnTIII activity is a fusion polypeptide. Alternatively, the α1,6-fucosyltransferase activity of the host cell can be reduced or eliminated in accordance with US Pat. No. 6,946,292 to generate a glycomodified host cell. The amount of antibody fucosylation can be predetermined, for example, by fermentation conditions (eg, fermentation time) or by combining at least two antibodies with different amounts of fucosylation. Such afucosylated antibodies and corresponding glycoengineering methods are described in WO 2005/044859, WO 2004/065540, WO 2007/031875, Umana, P. et al., Nature Biotechnol. 17 (1999) 176-180, WO 99/154342, WO 2005/018572, WO 2006/116260, WO 2006/114700, WO 2005/011735, WO 2005/027966, WO 97/028267, US 2006/0134709, US 2005/0054048, US 2005/0152894, WO 2003/035835, WO 2000/061739. These glycoengineered antibodies have increased ADCC. Other glycoengineering methods for producing afucosylated antibodies according to the present invention are described, for example, in Niwa, R. et al., J. Immunol. Methods 306 (2005) 151-160; Shinkawa, T. et al., J. Biol. Chem. 278 (2003) 3466-3473; WO 03/055993 or US 2005/0249722.

Thus, one aspect of the present invention is an afucosylated anti-CD20 antibody of the IgG1 or IgG3 isotype (preferably the IgG1 isotype) that specifically binds to CD20 and has an amount of fucose that is 60% or less of the total amount of oligosaccharides (saccharides) at Asn297, for use in combination with the CSF1R antibodies described herein for treating cancer. Another aspect of the present invention is the use of an afucosylated anti-CD20 antibody of the IgG1 or IgG3 isotype (preferably the IgG1 isotype) that specifically binds to CD20 and has an amount of fucose that is 60% or less of the total amount of oligosaccharides (saccharides) at Asn297 for the manufacture of a medicament for treating cancer in combination with the CSF1R antibodies described herein. In one embodiment, the amount of fucose is between 60% and 20% of the total amount of oligosaccharides (saccharides) at Asn297. In one embodiment, the amount of fucose is between 60% and 40% of the total amount of oligosaccharides (saccharides) at Asn297. In one embodiment, the amount of fucose is between 0% and 10% of the total amount of oligosaccharides (sugars) at Asn297.

In one embodiment of the invention, the antibody binding to human CSF-1R used in the combination therapy described herein is characterized in that it comprises

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 CD20 used in the combination therapy is characterized by comprising

a) a heavy chain variable domain VH of SEQ ID NO: 69 and a light chain variable domain VL of SEQ ID NO: 76, or

b) a heavy chain variable domain VH of SEQ ID NO: 70 and a light chain variable domain VL of SEQ ID NO: 76, or

c) a heavy chain variable domain VH of SEQ ID NO: 71 and a light chain variable domain VL of SEQ ID NO: 76, or

d) a heavy chain variable domain VH of SEQ ID NO: 72 and a light chain variable domain VL of SEQ ID NO: 76, or

e) a heavy chain variable domain VH of SEQ ID NO: 73 and a light chain variable domain VL of SEQ ID NO: 76, or

f) a heavy chain variable domain VH of SEQ ID NO: 74 and a light chain variable domain VL of SEQ ID NO: 76, or

g) the heavy chain variable domain VH of SEQ ID NO: 75 and the light chain variable domain VL of SEQ ID NO: 76.

In one embodiment, the antibody that binds to human CSF-1R used in the combination therapy 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 wherein the antibody that binds to human CD20 used in the combination therapy is characterized by comprising a heavy chain variable domain, VH, of SEQ ID NO: 71 and a light chain variable domain, VL, of SEQ ID NO: 76.

The term "epitope" refers to a protein determinant within human CSF-1R or CD20 that is capable of specific antibody binding. Epitopes typically consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and generally possess specific three-dimensional structural features, as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that binding to the former, but not the latter, is lost in the presence of denaturing solvents.

As used herein, "variable domain" (light chain variable domain ( _VL ), heavy chain variable domain ( _VH )) refers to each of the light and heavy chain domain pairs that are directly involved in antibody binding to antigen. The variable light and heavy chain domains have the same overall structure, and each domain comprises four framework (FR) regions, whose sequences are extensively 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 are maintained in their three-dimensional structure by the framework regions and, together with the CDRs from the other chain, form the antigen binding site. The heavy and light chain CDR3 regions of antibodies 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.

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 the "complementarity determining regions" or "CDRs". The "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, the CDR3 of the heavy chain is the region that contributes most to antigen binding and defines the properties of the antibody. The 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".

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.

As used within this application, the term "amino acid" refers to the group of naturally occurring carboxy α-amino acids, including alanine (three-letter code: ala, one-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).

The "Fc portion" of an antibody is not directly involved in the binding of the antibody to the antigen, but exhibits various effector functions. "Fc portion of an antibody" is a term well known to those skilled in the art and is defined based on the cleavage of an antibody by papain. Antibodies or immunoglobulins are divided into classes based on the amino acid sequence of their heavy chain constant regions: 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. Depending on the heavy chain constant region, the 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 the binding of complement factor C1q to the Fc portion of most IgG antibody subclasses. While the effect of antibodies on the complement system depends on certain conditions, binding to C1q is caused by a defined binding site in the Fc portion. Such binding sites are known in the art and are described, for example, in Boackle, R.J. et al., Nature 282 (1979) 742-743; Lukas, T.J. et al., J. Immunol. 127 (1981) 2555-2560; Brunhouse, R. and Cebra, J.J., Mol. Immunol. 16 (1979) 907-917; Burton, D.R. et al., Nature 288 (1980) 338-344; Thommesen, J.E. et al., Mol. Immunol. 37 (2000) 995-1004; Idusogie, E.E. 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, E.A., see below). Antibodies of the subclasses IgG1, IgG2 and IgG3 generally 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.

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 one 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 (the group consisting of L234A/L235A, L234A/L235A/P329G, N297A and D265A/N297A).

In one embodiment, the antibodies described herein are of the human IgG class (ie, of the IgG1 subclass, IgG2 subclass, IgG3 subclass, or IgG4 subclass).

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

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, E.A. (see, for example, Johnson, G. and Wu, T.T., Nucleic Acids Res. 28 (2000) 214-218). For example, a useful human heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 58. For example, a useful human light chain constant region comprises the kappa light chain constant region amino acid sequence of SEQ ID NO: 57.

The present 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.

The present invention comprises the use of an antibody according to the invention for such treatment.

A preferred embodiment of the present invention is a CSF-1R antibody of the present invention for use in treating a "CSF-1R-mediated disease", or a 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:

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, S.M. et al., J. Natl. Cancer Inst. 86 (1994) 120-126; Kacinski, B.M., Mol. Reprod. Dev. 46 (1997) 71-74; Ngan, H.Y. 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 found 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 impair receptor turnover (Ridge, S.A. 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, F.M. et al., Br. J. Haematol. 120 (2003) 464-470). Mutations have also been found in some cases of hepatocellular carcinoma (Yang, D.H. et al., Hepatobiliary Pancreat. Dis. Int. 3 (2004) 86-89) and idiopathic myelofibrosis (Abu-Duhier, F.M. 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).

The second mechanism is based on blocking signaling via M-CSF/CSF-1R at metastatic sites in the bone, which induces osteoclastogenesis, bone resorption, and osteolytic bone lesions. 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 stroma cooperates with the receptor activator nuclear factor kappa-B ligand, RANKL, to induce the differentiation of hematopoietic myeloid monocytic progenitors into mature osteoclasts. In this process, M-CSF acts as a permissive factor by providing survival signals to osteoclasts (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 has the potential to prevent the imbalance in osteoclast activity that causes osteolytic disease and related skeletal events in metastatic disease. While breast cancer, lung cancer, and multiple myeloma typically result in osteolytic lesions, in prostate cancer, metastases to the bone initially have an osteoblastic appearance, with elevated bone-forming activity producing "woven bone" that differs from the typical lamellar structure of normal bone. During disease progression, bone lesions exhibit a significant osteolytic component as well as high serum levels of bone resorption, suggesting 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 their effect on osteoblastic lesions is controversial at this point, and bisphosphonates have not yet been shown to be beneficial in preventing bone metastases or hormone-responsive prostate cancer. The effects of antiresorptive agents in mixed osteolytic/osteoblastic prostate cancer are still under clinical investigation (Choueiri, M.B. et al., Cancer Metastasis Rev. 25 (2006) 601-609; Vessella, R.L. and Corey, E., Clin. Cancer Res. 12 (20 Pt 2) (2006) 6285s-6290s).

A third mechanism is based on recent observations that tumor-associated macrophages (TAMs) are associated with poor prognosis in solid tumors of breast, prostate, ovarian, and cervical cancers (Bingle, L. et al., J. Pathol. 196 (2002) 254-265; Pollard, J. W., 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 this 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 against 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 M-CSF levels in mice and led to a decrease in macrophages in tumors. In addition, treatment of MCF7 tumor xenografts with an antigen-binding fragment directed against M-CSF resulted in 40% tumor growth inhibition, reversal of resistance to chemotherapy, and improved mouse survival when given in combination with chemotherapy (Paulus, P. et al., Cancer Res. 66 (2006) 4349-4356).

TAMs are the only example of an emerging link between chronic inflammation and cancer. Evidence for a link between inflammation and cancer is also available, including the association of many chronic diseases with an increased risk of cancer, the development of cancer at sites of chronic inflammation, the discovery of chemical mediators of inflammation in many cancers, the suppression of cellular or chemical mediators of inflammation in experimental cancers, and the reduction of the risk of some cancers with long-term use of anti-inflammatory agents. Numerous inflammatory conditions have been linked to cancer, including Helicobacter 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 differentially to their microenvironment. Two types of macrophages are considered to be at the extreme ends of a continuum of functional states: M1 macrophages are involved in type 1 responses. These responses involve activation by microbial products and, consequently, the killing of pathogenic microorganisms, generating reactive oxygen species. At the other extreme are M2 macrophages, which are involved in type 2 responses, promoting cell proliferation, regulating inflammation and adaptive immunity, and promoting tissue remodeling, angiogenesis, and repair (Mantovani, A. et al., Trends Immunol. 25 (2004) 677-686). Chronic inflammation that leads 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 in tumors, including the potential spatial and temporal dependence of their functions and their association with specific tumor types.

Thus, one embodiment of the present invention is a CSF-1R antibody as described herein, in combination with an anti-CD20 antibody as described herein, for use in treating cancer.

Detailed description of embodiments of the present invention

In one embodiment, the invention relates to an antibody that binds CSF-1R for use in inducing lymphocytosis of leukemic cells in lymphoma or leukemia.

As used herein, "lymphocytosis of leukemic cells in lymphoma or leukemia" refers to an absolute or relative increase in circulating leukemic cells in the peripheral blood of a patient suffering from/suffering from lymphoma or leukemia. In one embodiment, the lymphocytosis increases the percentage of circulating leukemic cells expressing CD19 and/or expressing CD20 in the peripheral blood. In a preferred embodiment, the lymphocytosis increases the percentage of circulating leukemic cells expressing CD19 and/or expressing CD20 in the peripheral blood (see also Hallek M, Cheson BD, Catovsky D, et al. Response assessment in chronic lymphocytic leukemia treated with novel agents causing an increase of peripheral blood lymphocytes. Blood. Epub June 4, 2012). Examples of such lymphocytosis are described, for example, below, in which two administrations of anti-CSF1R monoclonal antibodies ( FIG. 6A ) induced significant lymphocytosis ( FIG. 6B ), a therapeutically relevant compartment shift of infiltrating tissue into peripheral blood (PB). Similar lymphocytosis has been described in CLL patients treated with agents targeting BCR signaling, such as ibrutinib (Byrd et al., N Engl J Med. 2013 Jul 4; 369(1):32-42.), but has never been seen before with CSF-1R targeting agents.

In yet another embodiment, the invention relates to the use of an antibody that binds to CSF-1R for inducing lymphocytosis of leukemic cells in lymphoma or leukemia.

In another embodiment, the present invention relates to a method of treatment comprising administering an effective amount of an antibody that binds to CSF-1R for inducing lymphocytosis of leukemic cells in a lymphoma or leukemia or the use of an antibody that binds to CSF-1R for the manufacture of a medicament for inducing lymphocytosis of leukemic cells in a lymphoma or leukemia.

The present invention further relates to the antibodies, uses or methods as described above, wherein the lymphocytosis increases the percentage of circulating leukemic cells expressing CD19 and/or CD20 in the peripheral blood. In yet another embodiment, the present invention relates to the antibodies, uses or methods as described herein, wherein the lymphocytosis increases the percentage of circulating leukemic cells expressing CD19 and/or CD20 in the peripheral blood and renders the lymphoma or leukemia susceptible to treatment with anti-CD19 antibodies and/or anti-CD20 antibodies.

The present invention also relates to the antibody, use or method according to any preceding embodiment, wherein the lymphocytosis increases circulating leukemic cells expressing CD20, in particular, wherein the lymphocytosis increases the percentage of circulating leukemic cells expressing CD20 and renders the lymphoma or leukemia susceptible to treatment with an anti-CD20 antibody.

In another embodiment, the present invention relates to a combination therapy encompassing the co-administration of an antibody that binds to CSF-1R and an antibody that binds to CD20. This combination therapy can be applied to

a) Treat cancer,

b) delay cancer progression,

c) prolong the survival of patients with cancer, or

d) stimulation of a cell-mediated immune response, in particular stimulation of cytotoxic T-lymphocytes, stimulation of T cell activity, or stimulation of macrophage activity. In one embodiment of the invention, stimulation of a cell-mediated immune response occurs during the treatment of cancer.

In one embodiment, the stimulation of a cell-mediated immune response under d) encompasses stimulation of the activity of cytotoxic T-lymphocytes, or stimulation of the activity of macrophages.

The combination therapy has been shown to significantly improve CSF-1R-based cancer therapies in individuals with cancer. The inventors discovered that CSF-1R antibodies enhance the efficacy of CD20 antibodies in treating cancer, delaying tumor progression, or prolonging survival in patients with cancers such as lymphoma or leukemia. Delayed cancer progression and longer overall survival represent major benefits for patients.

Antibodies for use

One aspect of the invention relates to an antibody that binds to CSF-1R, or an antibody that binds to CSF-1R for use in a combination therapy according to the invention.

Anti-CSF-1R antibodies for use

In one embodiment, the invention relates to an antibody that binds to CSF-1R, wherein the antibody is administered in combination therapy with an antibody that binds to CD20, for use in

a) Treat cancer,

b) delay cancer progression,

c) prolong the survival of patients with cancer, or

d) stimulation of a cell-mediated immune response, in particular stimulation of cytotoxic T-lymphocytes, stimulation of T-cell activity, or stimulation of macrophage activity.

In one embodiment, the invention relates to an antibody that binds to CSF-1R, wherein the antibody is administered in combination therapy with an antibody that binds to CD20, for use in

a) Treat cancer,

b) delay cancer progression, or

c) Prolonging the survival of patients suffering from cancer.

In one embodiment, the invention relates to an antibody that binds to CSF-1R, wherein the antibody is administered in combination therapy with an antibody that binds to CD20 for the treatment of cancer. In one embodiment, the invention relates to an antibody that binds to CSF-1R, wherein the antibody is administered in combination therapy with an antibody that binds to CD20 for prolonging survival of a patient suffering from cancer.

Anti-CD20 antibodies for use

In one embodiment, the invention relates to an antibody that binds CD20, wherein the antibody is administered in combination therapy with an antibody that binds CSF-1R for use in

a) Treat cancer,

b) delay cancer progression,

c) prolong the survival of patients with cancer, or

d) stimulation of a cell-mediated immune response, in particular stimulation of cytotoxic T-lymphocytes, stimulation of T cell activity, or stimulation of macrophage activity. In one embodiment of the invention, stimulation of a cell-mediated immune response occurs during the treatment of cancer.

In one embodiment, the invention relates to an antibody that binds CD20, wherein the antibody is administered in combination therapy with an antibody that binds CSF-1R for use in

a) Treat cancer,

b) delay cancer progression, or

c) Prolonging the survival of patients suffering from cancer.

In one embodiment, the invention relates to an antibody that binds CD20, wherein the antibody is administered in combination therapy with an antibody that binds CSF-1R for the treatment of cancer. In one embodiment, the invention relates to an antibody that binds CD20, wherein the antibody is administered in combination therapy with an antibody that binds CSF-1R for prolonging survival of a patient suffering from cancer.

Treatment

In one embodiment, the present invention relates to a method for

a) Treat cancer,

b) delay cancer progression,

c) prolong the survival of patients with cancer, or

d) stimulation of a cell-mediated immune response, in particular stimulation of cytotoxic T-lymphocytes, stimulation of T-cell activity, or stimulation of macrophage activity,

The method comprises the step of administering to a patient in need thereof an effective amount of an antibody that binds to CSF-1R and an effective amount of an antibody that binds to CD20.

In one embodiment, the present invention relates to a method for

a) Treat cancer,

b) delay cancer progression, or

c) prolonging the survival of patients with cancer,

The method comprises the step of administering to a patient in need thereof an effective amount of an antibody that binds to CSF-1R and an effective amount of an antibody that binds to CD20.

In one embodiment, the present invention relates to a method for treating cancer, wherein the method comprises the step of administering to a patient in need thereof an effective amount of an antibody that binds to CSF-1R and an effective amount of an antibody that binds to CD20. In one embodiment, the present invention relates to a method for prolonging the survival of a patient suffering from cancer, wherein the method comprises the step of administering to a patient in need thereof an effective amount of an antibody that binds to CSF-1R and an effective amount of an antibody that binds to CD20.

Use of antibodies in the manufacture of drugs

One aspect of the invention relates to an antibody that binds to CSF-1R, or an antibody that binds to CD20, for use in the manufacture of a medicament for use in a combination therapy according to the invention.

Uses of anti-CSF-1R antibodies

In one embodiment, the invention relates to the use of an antibody that binds to CSF-1R for the manufacture of a medicament for:

a) Treat cancer,

b) delay cancer progression,

c) prolong the survival of patients with cancer, or

d) stimulation of a cell-mediated immune response, in particular stimulation of cytotoxic T-lymphocytes, stimulation of T-cell activity, or stimulation of macrophage activity,

wherein the antibody is for administration in combination therapy with an antibody that binds CD20.

In one embodiment, the invention relates to the use of an antibody that binds to CSF-1R for the manufacture of a medicament for:

a) Treat cancer,

b) delay cancer progression, or

c) prolonging the survival of patients with cancer,

wherein the antibody is for administration in combination therapy with an antibody that binds CD20.

In one embodiment, the invention relates to the use of an antibody that binds to CSF-1R for the manufacture of a medicament for the treatment of cancer, wherein the antibody is for administration in combination therapy with an antibody that binds to CD20. In one embodiment, the invention relates to the use of an antibody that binds to CSF-1R for the manufacture of a medicament for prolonging survival of a patient suffering from cancer, wherein the antibody is for administration in combination therapy with an antibody that binds to CD20.

Uses of anti-CD20 antibodies

In one embodiment, the invention relates to the use of an antibody that binds CD20 for the manufacture of a medicament for:

a) Treat cancer,

b) delay cancer progression,

c) prolonging the survival of patients with cancer, or

d) stimulation of a cell-mediated immune response, in particular stimulation of cytotoxic T-lymphocytes, stimulation of T-cell activity, or stimulation of macrophage activity,

wherein the antibody is for administration in combination therapy with an antibody that binds CSF-1R.

In one embodiment, the invention relates to the use of an antibody that binds CD20 for the manufacture of a medicament for:

a) Treat cancer,

b) delay cancer progression, or

c) prolonging the survival of patients with cancer,

wherein the antibody is for administration in combination therapy with an antibody that binds CSF-1R.

In one embodiment, the invention relates to the use of an antibody that binds to CD20 for the manufacture of a medicament for the treatment of cancer, wherein the antibody is for administration in combination therapy with an antibody that binds to CSF-1R. In one embodiment, the invention relates to the use of an antibody that binds to CD20 for the manufacture of a medicament for prolonging survival of a patient suffering from cancer, wherein the antibody is for administration in combination therapy with an antibody that binds to CSF-1R.

Uses of anti-CSF-1R antibodies and anti-CD20 antibodies

In one embodiment, the present invention relates to the use of an antibody that binds to CSF-1R and an antibody that binds to CD20 for the manufacture of a medicament for:

a) Treat cancer,

b) delay cancer progression,

c) prolonging the survival of patients with cancer, or

d) stimulation of a cell-mediated immune response, in particular stimulation of cytotoxic T-lymphocytes, stimulation of T-cell activity, or stimulation of macrophage activity.

In one embodiment, the present invention relates to the use of an antibody that binds to CSF-1R and an antibody that binds to CD20 for the manufacture of a medicament for:

a) Treat cancer,

b) delay cancer progression, or

c) Prolonging the survival of patients suffering from cancer.

In one embodiment, the invention relates to the use of an antibody that binds to CSF-1R and an antibody that binds to CD20 for the manufacture of a medicament for the treatment of cancer, wherein the antibody is for administration in combination therapy with an antibody that binds to CD20. In one embodiment, the invention relates to the use of an antibody that binds to CSF-1R and an antibody that binds to CD20 for the manufacture of a medicament for prolonging survival of a patient suffering from cancer, wherein the antibody is for administration in combination therapy with an antibody that binds to CD20.

Products

One aspect of the invention relates to an article of manufacture comprising a container, a composition comprising an antibody that binds CSF-1R or an antibody that binds CD20 within the container, and a package insert instructing the user of the composition to administer each antibody in a combination therapy according to the invention.

Preparations containing anti-CSF-1R antibodies

In one embodiment, the invention relates to an article of manufacture comprising (a) a container containing a composition comprising an antibody that binds to CSF-1R, and (b) a package insert instructing the user of the composition to administer the antibody that binds to CSF-1R to a patient.

a) Treat cancer,

b) delay cancer progression,

c) prolong the survival of patients with cancer, or

d) stimulation of a cell-mediated immune response, in particular stimulation of cytotoxic T-lymphocytes, stimulation of T-cell activity, or stimulation of macrophage activity,

wherein the antibody that binds CSF-1R is administered in combination therapy with an antibody that binds CD20.

In one embodiment, the invention relates to an article of manufacture comprising (a) a container containing a composition comprising an antibody that binds to CSF-1R, and (b) a package insert instructing the user of the composition to administer the antibody that binds to CD20 to a patient.

a) Treat cancer,

b) delay cancer progression, or

c) prolonging the survival of patients with cancer,

wherein the antibody that binds CSF-1R is administered in combination therapy with an antibody that binds CD20.

In one embodiment, the present invention relates to an article of manufacture comprising (a) a container, a composition comprising an antibody that binds to CSF-1R within the container, and (b) a package insert instructing the user of the composition to administer the antibody that binds to CSF-1R to a patient for the treatment of cancer, wherein the antibody that binds to CSF-1R is administered in combination therapy with an antibody that binds to CD20. In one embodiment, the present invention relates to an article of manufacture comprising (a) a container, a composition comprising an antibody that binds to CSF-1R within the container, and (b) a package insert instructing the user of the composition to administer the antibody that binds to CSF-1R to a patient to prolong survival of the patient suffering from cancer, wherein the antibody that binds to CSF-1R is administered in combination therapy with an antibody that binds to CD20.

Preparations containing anti-CD20 antibodies

In one embodiment, the invention relates to an article of manufacture comprising (a) a container containing a composition comprising an antibody that binds CD20, and (b) a package insert instructing the user of the composition to administer the antibody that binds CD20 to a patient.

a) Treat cancer,

b) delay cancer progression,

c) prolonging the survival of patients with cancer, or

d) stimulation of a cell-mediated immune response, in particular stimulation of cytotoxic T-lymphocytes, stimulation of T-cell activity, or stimulation of macrophage activity,

wherein the CD20-binding antibody is administered in combination therapy with an antibody that binds CSF-1R.

In one embodiment, the invention relates to an article of manufacture comprising (a) a container containing a composition comprising an antibody that binds CD20, and (b) a package insert instructing the user of the composition to administer the antibody that binds CD20 to a patient.

a) Treat cancer,

b) delay cancer progression, or

c) prolonging the survival of patients with cancer,

wherein the CD20-binding antibody is administered in combination therapy with an antibody that binds CSF-1R.

In one embodiment, the invention relates to an article of manufacture comprising (a) a container, a composition comprising an antibody that binds to CD20 within the container, and (b) a package insert instructing the user of the composition to administer the antibody that binds to CD20 to a patient for treating cancer, wherein the antibody that binds to CD20 is administered in combination therapy with an antibody that binds to CSF-1R. In one embodiment, the invention relates to an article of manufacture comprising (a) a container, a composition comprising an antibody that binds to CD20 within the container, and (b) a package insert instructing the user of the composition to administer the antibody that binds to CD20 to a patient to prolong survival of the patient suffering from cancer, wherein the antibody that binds to PD-1 is administered in combination therapy with an antibody that binds to CSF-1R.

Preparations containing anti-CSF-1R antibodies and anti-CD20 antibodies

In one embodiment, the invention relates to an article of manufacture comprising (a) a container containing a composition comprising an antibody that binds to CSF-1R, and (b) a container containing a composition comprising an antibody that binds to CD20, and (c) a package insert instructing the user of the composition to administer the antibody that binds to CSF-1R and the antibody that binds to CD20 to a patient.

a) Treat cancer,

b) delay cancer progression,

c) prolonging the survival of patients with cancer, or

d) stimulation of a cell-mediated immune response, in particular stimulation of cytotoxic T-lymphocytes, stimulation of T-cell activity, or stimulation of macrophage activity.

Pharmaceutical composition

In one embodiment, the invention relates to a pharmaceutical composition comprising an antibody that binds to CSF-1R, and an antibody that binds to CD20, wherein each antibody is formulated together with a pharmaceutically acceptable carrier.

In one embodiment of the pharmaceutical composition, the antibody that binds to CSF-1R and the antibody that binds to CD20 are formulated separately. In another embodiment of the pharmaceutical composition, the antibody that binds to CSF-1R and the antibody that binds to CD20 are formulated together.

Method for manufacturing a pharmaceutical composition

In one embodiment, the invention relates to a method for manufacturing a pharmaceutical composition comprising (a) formulating an antibody that binds to CSF-1R with a pharmaceutically acceptable carrier, and (b) separately formulating an antibody that binds to CD20 with a pharmaceutically acceptable carrier.

In one embodiment, the invention relates to a method for manufacturing a pharmaceutical composition comprising formulating an antibody that binds CSF-1R in combination with an antibody that binds CD20 in combination with a pharmaceutically acceptable carrier.

Specific antibodies used in the above embodiments

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.

These antibodies are described in WO 2011/070024 and are characterized by comprising the following VH and VL sequences described therein:

Table 2

In a preferred embodiment, the antibody that binds to human CSF-1R used in the combination therapy described herein is hMab 2F11-e7 (VH, SEQ ID NO: 39; VL, SEQ ID NO: 40).

In one embodiment, the antibody that binds to human CD20 used in the combination therapy described herein is a humanized B-Ly1 antibody:

The term "humanized B-Ly1 antibody" refers to humanized B-Ly1 antibodies as disclosed in WO 2005/044859 and WO 2007/031875, which are obtained from the murine monoclonal anti-CD20 antibody B-Ly1 (see Poppema, S. and Visser, L., Biotest Bulletin 3 (1987) 131-139) by chimerization with human constant domains from IgG1 and subsequent humanization (see WO 2005/044859 and WO 2007/031875). These "humanized B-Ly1 antibodies" are disclosed in detail in WO 2005/044859 and WO 2007/031875.

In one embodiment, the "humanized B-Ly1 antibody" has a heavy chain variable region (VH) selected from the group consisting of SEQ ID NO. 69 to SEQ ID NO. 75 (B-HH2, B-HH3, B-HH6, B-HH8, B-HL8, B-HL11, and B-HL13 of WO 2005/044859 and WO 2007/031875). In one embodiment, the "humanized B-Ly1 antibody" has a light chain variable region (VL) of SEQ ID NO. 76 (B-KV1 of WO 2005/044859 and WO 2007/031875).

These antibodies are characterized by comprising the following VH and VL sequences described herein:

Table 3

In a preferred embodiment, the antibody that binds to human CD20 used in the combination therapy described herein is selected from the humanized B-Ly1 antibody B-HH6/B-KV1, which comprises the heavy chain variable region (VH) of SEQ ID No. 71 (B-HH6) and the light chain variable region (VL) of SEQ ID No. 76 (B-KV1).

Furthermore, in one embodiment, the humanized B-Ly1 antibody is an afucosylated antibody of the IgG1 isotype.

According to the present invention, such afucosylated humanized B-Ly1 antibody is glycoengineered (GE) in the Fc region according to the procedures described in WO 2005/044859, WO 2004/065540, WO 2007/031875, Umana, P. et al., Nature Biotechnol. 17 (1999) 176-180, and WO 99/154342. In one embodiment, the afucosylated glycoengineered humanized B-Ly1 is B-HH6-B-KV1 GE. Such glycoengineered humanized B-Ly1 antibodies have an altered glycosylation pattern in the Fc region, preferably with reduced levels of fucose residues. In one embodiment, the amount of fucose is 60% or less of the total amount of oligosaccharides at Asn297 (in one embodiment, the amount of fucose is 40% to 60%, in another embodiment, the amount of fucose is 50% or less, and in yet another embodiment, the amount of fucose is 30% or less). In another embodiment, preferably, the oligosaccharides in the Fc region are bisected. These glycoengineered humanized B-Ly1 antibodies have increased ADCC.

In a preferred embodiment, the human CD20-binding antibody used in the combination therapy described herein is selected from the humanized B-Ly1 antibody B-HH6/B-KV1, which comprises the heavy chain variable region (VH) of SEQ ID No. 71 (B-HH6) and the light chain variable region (VL) of SEQ ID No. 76 (B-KV1) and is an afucosylated antibody of the IgG1 isotype with an altered glycosylation pattern in the Fc region, wherein the amount of fucose-containing oligosaccharides is between 40% and 60% of the total amount of oligosaccharides at Asn297.

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, cutaneous 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, cancer of the endocrine system, thyroid cancer, parathyroid cancer, kidney cancer, or Cancer of the upper gland, soft tissue sarcoma, urethra cancer, penile cancer, prostate cancer, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvis cancer, mesothelioma, hepatocellular carcinoma, gallbladder cancer, central nervous system (CNS) neoplasms, spinal axis tumors, brain stem gliomas, glioblastoma multiforme, astrocytoma, Schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, lymphoma, lymphocytic leukemia, including refractory forms of any of the above cancers, or a combination of one or more of the above cancers. In a preferred embodiment, the cancer refers to lymphoma (preferably B cell non-Hodgkin's lymphoma (NHL)) and lymphocytic leukemia. Such lymphomas and lymphocytic leukemias include, for example, a) follicular lymphoma, b) small non-cleaved cell lymphoma, c) leukemia, and d) leukemia. Lymphoma)/Burkitt's lymphoma (including endemic Burkitt's lymphoma, sporadic Burkitt's lymphoma and non-Burkitt's lymphoma), c) marginal zone lymphoma (including extranodal marginal zone B-cell lymphoma (mucosa-associated lymphoid tissue lymphoma, MALT), nodal marginal zone B-cell lymphoma and splenic marginal zone lymphoma), d) mantle cell lymphoma (MCL), e) large cell lymphoma (including B-cell diffuse large cell lymphoma (DLCL), diffuse mixed cell lymphoma, immunoblastic leukemia (IPL), and leukemia. B-cell lymphoma, primary mediastinal B-cell lymphoma, angiocentric lymphoma-pulmonary B-cell lymphoma), f) hairy cell leukemia, g) lymphocytic lymphoma, Waldenstrom's macroglobulinemia, h) acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL), B-cell prolymphocytic leukemia, i) plasma cell neoplasms, plasma cell myeloma, multiple myeloma, plasmacytoma, j) Hodgkin's disease.

In another preferred embodiment, the term cancer refers to a "CD20-expressing cancer," wherein the cancer cells show expression of the CD20 antigen. In another preferred embodiment, as used herein, the term "CD20-expressing cancer" refers to lymphomas (preferably B-cell non-Hodgkin's lymphoma (NHL)) and lymphocytic leukemias. Such lymphomas and lymphocytic leukemias include, for example, a) follicular lymphoma, b) small non-cleaved cell lymphoma, c) leukemia, and d) follicular lymphoma. B-cell lymphoma (including endemic Burkitt's lymphoma, sporadic Burkitt's lymphoma and non-Burkitt's lymphoma), c) marginal zone lymphoma (including extranodal marginal zone B-cell lymphoma (mucosa-associated lymphoid tissue lymphoma, MALT), nodal marginal zone B-cell lymphoma and splenic marginal zone lymphoma), d) mantle cell lymphoma (MCL), e) large cell lymphoma (including B-cell diffuse large cell lymphoma (DLCL), diffuse mixed cell lymphoma, immune f) hairy cell leukemia, g) lymphocytic lymphoma, Waldenstrom's macroglobulinemia, h) acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL), B-cell prolymphocytic leukemia, i) plasma cell neoplasms, plasma cell myeloma, multiple myeloma, plasmacytoma, j) Hodgkin's disease.

In another preferred embodiment, the cancer expressing CD20 is B-cell non-Hodgkin's lymphoma (NHL). In another embodiment, the cancer expressing CD20 is mantle cell lymphoma (MCL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma, hairy cell leukemia, follicular lymphoma, multiple myeloma, marginal zone lymphoma, post-transplant lymphoproliferative disorder (PTLD), HIV-associated lymphoma, Waldenstrom's macroglobulinemia, or primary CNS lymphoma.

In a preferred embodiment, the cancer or cancer expressing CD20 is B-cell non-Hodgkin's lymphoma (NHL). In another preferred embodiment, the cancer or cancer expressing CD20 is chronic lymphocytic leukemia (CLL), follicular lymphoma, multiple myeloma. In another preferred embodiment, the cancer or cancer expressing CD20 is Hodgkin's disease.

Rheumatoid arthritis, psoriatic arthritis, and inflammatory joint diseases are potential indications for CSF-1R signaling inhibitors because they are composed of macrophages and have varying degrees of bone destruction (Ritchlin, C.T. 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 mediated at least in part by M-CSF. Campbell, I.K. 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 rheumatoid arthritis patients, suggesting that it contributes to synovial tissue proliferation and macrophage infiltration, which is involved in the pathogenesis of the disease. Inhibition of CSF-1R signaling has the potential to control the number of macrophages in the joints and alleviate the pain caused by 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.

Thus, another embodiment of the present invention is a CSF-1R antibody characterized by the amino acid sequence and amino acid sequence fragments described above, in combination with an anti-CD20 antibody characterized by the amino acid sequence and amino acid sequence fragments described above, for use in the treatment of rheumatoid arthritis, psoriatic arthritis, osteoarthritis, inflammatory joint diseases, and inflammation.

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-CD20 antibody (characterized by the amino acid sequence and amino acid sequence fragments described above) for treating cancer.

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-CD20 antibody (characterized by the amino acid sequence and amino acid sequence fragments described above) for preventing or treating metastasis.

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-CD20 antibody (characterized by the amino acid sequence and amino acid sequence fragments described above) for treating inflammatory diseases.

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-CD20 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.

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-CD20 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.

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 treating cancer in combination with anti-CD20 antibodies or for preparing a medicament for treating cancer in combination with the anti-CD20 antibodies described herein.

The present invention comprises the use of antibodies (characterized in that they comprise antibodies that bind to human CSF-1R, characterized in that they comprise the amino acid sequences and amino acid sequence fragments described above) for preventing or treating metastasis in combination with the anti-CD20 antibodies described herein or for the preparation of a medicament for preventing or treating metastasis in combination with the anti-CD20 antibodies described herein.

The present invention comprises the use of antibodies (characterized in that they comprise antibodies that bind to human CSF-1R, characterized in that they comprise the amino acid sequences and amino acid sequence fragments described above) for treating inflammatory diseases in combination with the anti-CD20 antibodies described herein or for preparing a medicament for treating inflammatory diseases in combination with the anti-CD20 antibodies described herein.

The present invention comprises the use of antibodies (characterized in that they comprise antibodies that bind to human CSF-1R, characterized in that they comprise the amino acid sequences and amino acid sequence fragments described above) for treating immune-related diseases such as tumor immunity or delaying their progression in combination with the anti-CD20 antibodies described herein, or for preparing a medicament for treating immune-related diseases such as tumor immunity or delaying their progression in combination with the anti-CD20 antibodies described herein.

The present invention comprises the use of antibodies (characterized in that they comprise 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-CD20 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-CD20 antibodies described herein.

In a preferred embodiment of the 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 wherein the antibody binding to CD20 used in such combined treatment is characterized by comprising a heavy chain variable domain VH of SEQ ID NO: 71 and a light chain variable domain VL of SEQ ID NO: 76.

The antibodies described herein are preferably produced by recombinant means. Such methods are widely known in the art and include protein expression in prokaryotic and eukaryotic cells, followed by isolation of the antibody polypeptide and typically 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 antibody is recovered from the cells (from the supernatant or after cell lysis).

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

Antibodies can be present in intact cells, in cell lysates, or in partially purified or substantially pure form. Purification is performed to eliminate other cellular components or other contaminants, such as other cellular nucleic acids or proteins, by standard techniques, 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).

Expression in NS0 cells is described, for example, in Barnes, L.M. et al., Cytotechnology 32 (2000) 109-123; Barnes, L.M. 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.

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 transcription termination to form an expression vector construct. The heavy and light chain expression constructs can be combined into a single vector and co-transfected, sequentially transfected, or transfected separately into host cells and then fused to form a single host cell expressing both chains.

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.

A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, if a presequence or secretory leader DNA is expressed as a preprotein that participates in the secretion of the 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 contiguous, and in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking is achieved by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice.

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 are easily separated and sequenced. 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 otherwise generate immunoglobulin proteins, to obtain the synthesis of recombinant monoclonal antibodies in the host cell.

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, due to intentional or inadvertent mutations, the DNA content of all progeny may not be precisely identical. Variant progeny that have the same function or biological activity as screened for in the initially transformed cell are included.

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

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.

The compositions of the present invention can 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.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the preparation of 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.

Regardless of the route of administration selected, the compounds of the present invention, which can be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

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 that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient (an effective amount). 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 rate of excretion 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 arts.

The term "treatment 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 "treatment 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 light of medical history and estimated patient survival expectations will be implemented.

The terms "administered in combination with," "co-administered," "combination therapy," or "combination treatment" refer to the administration of an anti-CSF-1R antibody described herein and an anti-CD20 antibody described herein, for example, as separate formulations/applications (or as a single formulation/application). Co-administration can be simultaneous or sequential in either 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 (i.v.) 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; while the term "simultaneously" means at the same time. The term "co-administered" with respect to maintenance doses of anti-CSF-1R antibody and/or anti-CD20 antibody means that if the treatment cycle is suitable for both agents, for example, weekly, the maintenance doses can be co-administered simultaneously. Alternatively, for example, the other agent can be administered every first to third day, while the antibody can be administered weekly. Alternatively, the maintenance doses can be co-administered sequentially within a single day or over several days.

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 will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician.

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 can be appropriately co-administered to the patient at one time or over a series of treatments, for example, on the same day or the following day.

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-CD20 antibody is an initial candidate dose for co-administration of both drugs to a patient. The present invention includes the use of an antibody according to the present invention for treating a patient suffering from cancer, particularly colon, lung or pancreatic cancer.

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-CD20 antibody is an initial candidate dose for co-administration of both drugs to a patient. The present invention includes the use of an antibody according to the present invention for treating a patient suffering from cancer, particularly colon, lung or prostate cancer.

In addition to the anti-CSF-1R antibody in combination with the anti-CD20 antibody, a chemotherapeutic agent or targeted therapy may also be administered.

In one embodiment, such additional chemotherapeutic agents that can be administered with the anti-CSF-1R antibodies described herein and the anti-CD20 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 ^™ (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), 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, cytosine arabinoside (AraC, cytarabine), cytosine arabinoside (AraC, cytarabine), 5-azacytidine, 2,2'-difluorodeoxycytidine, purine analogs such as 6-mercaptopurine, 6-thioguanine, azathioprine, 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. D), daunomycin (rubidomycin), doxorubicin, mitoxantrone, idarubicin, bleomycin, plicamycin (mithramycin), mitomycin C 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 ^™ (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, a gonadotropin-releasing hormone analog, and leuprolide; and nonsteroidal antiandrogens such as flutamide. Targeted epigenetic therapies 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, Such as paclitaxel (Taxol), docetaxel (Taxotere), modified paclitaxel (e.g., 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).

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; and therapy with Taxol and/or carboplatin for the treatment of lung cancer.

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).

In one embodiment, the CSF-1R antibody/CD20 antibody combination therapy is not administered with a chemotherapeutic agent or targeted therapy.

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

Targeted therapies can also be administered in addition to anti-CSF-1R antibodies in combination with anti-CD20 antibodies.

Anti-PD-L1 or anti-PD1 antibodies may be administered in addition to the anti-CSF-1R antibody in combination with the anti-CD20 antibody.

PD-1/PD-L1/PD-L2 pathway:

An important negative co-stimulatory signal that regulates 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 the 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 only binds 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).

PD-1 is expressed on T cells, B cells, natural killer T cells, activated monocytes, and dendritic cells (DCs). PD-1 is expressed by activated, but not unstimulated, human CD4+ and CD8+ T cells, B cells, and myeloid cells. This contrasts with 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 four 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). The 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).

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 syncytiotrophoblasts, keratinocytes, etc.) [Keir et al., Annu. Rev. Immunol. 26: 677-704 (2008)] and is upregulated on multiple cell types after activation. Both type I and type II interferons (IFNs) 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).

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 approximately one-half to two-thirds of resting peritoneal B1 cells, but not on conventional B2B 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).

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)].

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 reveal 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 stimulate 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 development 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).

Further evidence for reverse signaling into dendritic cells ("DCs") comes from studies of bone marrow-derived DCs cultured with soluble PD-1 (the PD-1 EC domain fused to an 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.

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. The B7.1:PD-L1 interaction 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 showed 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, no 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.

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 T cell cytokine production. 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 directed 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, preventing it from interacting with CD28.

As a result, antagonizing signaling through PD-L1 (including blocking the interaction of PD-L1 with either or both 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).

The mechanism of PD-L1/PD-1-mediated CD8 T-cell dysfunction in the context of age-related immunodeficiency in the Eμ-TCL1 CLL mouse model is described in McClanahan F, et al. Blood. 2015 May 15. blood-2015-02-626754 (epub ahead of print - PMID: 25979947). McClanahan F, et al. Blood. 2015 Mar 23. (pii: blood-2015-01-622936) mentioned that PD-L1 checkpoint blockade prevented immune dysfunction and leukemia in a mouse model of chronic lymphocytic leukemia.

The term "human PD-L1" refers to the human protein PD-L1 (SEQ ID NO: 97, 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 specifically binds to the human PD-L1 antigen with a binding affinity of 1.0 ^{x 10-8} mol/l or less (in one embodiment, with _{a KD} value of 1.0 x ^10-9 mol/l or less). 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 specifically binds to the human PD-L1 antigen with a binding affinity of K _D 1.0 x 10 ^-8 mol/l or less (in one embodiment, 1.0 x 10 ^-8 mol/l-1.0 x 10 ^-13 mol/l), and in one embodiment, with a binding affinity of K _D 1.0 x 10 ^-9 mol/l or less (in one embodiment, 1.0 x 10 ^-9 mol/l-1.0 x 10 ^-13 mol/l).

In a preferred embodiment, an anti-PD-L1 antibody may be administered in addition to the anti-CSF-1R antibody in combination with the anti-CD20 antibody.

In one embodiment, the anti-PD-L1 antibody (an 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.

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

Table 4

In one embodiment of the invention, the PD-L1 binding antibody used in the combination therapy according to the invention comprises a variable domain amino acid sequence selected from the group consisting of:

- the variable heavy chain domain VH of SEQ ID NO: 78, and the variable light chain domain VL of SEQ ID NO: 81 (corresponding to the VH and VL domains of <PD-L1> "243.55.S70" disclosed herein);

- the variable heavy chain domain VH of SEQ ID NO: 79, and the variable light chain domain VL of SEQ ID NO: 82 (corresponding to the VH and VL domains of <PD-L1> "243.55.H1" disclosed herein);

- the variable heavy chain domain VH of SEQ ID NO: 79, and the variable light chain domain VL of SEQ ID NO: 83 (corresponding to the VH and VL domains of <PD-L1> "243.55.H12" disclosed herein);

- the variable heavy chain domain VH of SEQ ID NO: 79, and the variable light chain domain VL of SEQ ID NO: 84 (corresponding to the VH and VL domains of <PD-L1> "243.55.H37" disclosed herein);

- the variable heavy chain domain VH of SEQ ID NO: 79, and the variable light chain domain VL of SEQ ID NO: 85 (corresponding to the VH and VL domains of <PD-L1> "243.55.H70" disclosed herein);

- the variable heavy chain domain VH of SEQ ID NO: 79, and the variable light chain domain VL of SEQ ID NO: 86 (corresponding to the VH and VL domains of <PD-L1> "243.55.H89" disclosed herein);

- the variable heavy chain domain VH of SEQ ID NO: 79, and the variable light chain domain VL of SEQ ID NO: 87 (corresponding to the VH and VL domains of <PD-L1> "243.55.S1" disclosed herein);

- the variable heavy chain domain VH of SEQ ID NO: 79, and the variable light chain domain VL of SEQ ID NO: 88 (corresponding to the VH and VL domains of <PD-L1> "243.55.5" disclosed herein);

- the variable heavy chain domain VH of SEQ ID NO: 79, and the variable light chain domain VL of SEQ ID NO: 89 (corresponding to the VH and VL domains of <PD-L1> "243.55.8" disclosed herein);

- the variable heavy chain domain VH of SEQ ID NO: 79, and the variable light chain domain VL of SEQ ID NO: 90 (corresponding to the VH and VL domains of <PD-L1> "243.55.30" disclosed herein);

- the variable heavy chain domain VH of SEQ ID NO: 79, and the variable light chain domain VL of SEQ ID NO: 91 (corresponding to the VH and VL domains of <PD-L1> "243.55.34" disclosed herein);

- the variable heavy chain domain VH of SEQ ID NO: 79, and the variable light chain domain VL of SEQ ID NO: 92 (corresponding to the VH and VL domains of <PD-L1> "243.55.S37" disclosed herein);

- the variable heavy chain domain VH of SEQ ID NO: 79, and the variable light chain domain VL of SEQ ID NO: 93 (corresponding to the VH and VL domains of <PD-L1> "243.55.49" disclosed herein);

- the variable heavy chain domain VH of SEQ ID NO: 79, and the variable light chain domain VL of SEQ ID NO: 94 (corresponding to the VH and VL domains of <PD-L1> "243.55.51" disclosed herein);

- the variable heavy chain domain VH of SEQ ID NO: 79, and the variable light chain domain VL of SEQ ID NO: 95 (corresponding to the VH and VL domains of <PD-L1> "243.55.62" disclosed herein); and

- the variable heavy chain domain VH of SEQ ID NO: 80, and the variable light chain domain VL of SEQ ID NO: 96 (corresponding to the VH and VL domains of <PD-L1> "243.55.84" disclosed herein).

In a preferred embodiment of the present invention, the PD-L1 binding antibody used in the combination therapy according to the present invention comprises the following variable domain amino acid sequences: the variable heavy chain domain VH of SEQ ID NO: 78, and the variable light chain domain VL of SEQ ID NO: 81 (corresponding to the VH and VL domains of <PD-L1> "243.55.S70" disclosed herein).

In one embodiment, the anti-PD-L1 antibody (antibody that binds to human PD-L1) used in the combination therapy described herein is atezolizumab (CAS No.: 1380723-44-3). In a preferred embodiment of the present invention, the PD-L1-binding antibody used in the combination therapy according to the present invention comprises the variable domain amino acid sequence of the variable heavy chain domain VH and the variable light chain domain VL of atezolizumab (CAS No.: 1380723-44-3).

In one embodiment, an anti-PD1 antibody may be administered in addition to the anti-CSF-1R antibody in combination with the anti-CD20 antibody.In one embodiment of the invention, the PD-1 binding antibody used in the combination therapy according to the invention binds to human PD-1.

In one embodiment of the present invention, the PD-1 binding antibody used in the combination therapy according to the present invention comprises the following variable domain amino acid sequences: the variable heavy chain domain VH of SEQ ID NO: 98, and the variable light chain domain VL of SEQ ID NO: 99 (corresponding to the VH and VL domains of <PD-1> nivolumab disclosed herein).

In one embodiment of the present invention, the PD-1 binding antibody used in the combination therapy according to the present invention comprises the following variable domain amino acid sequences: a variable heavy chain domain VH of SEQ ID NO: 100, and a variable light chain domain VL of SEQ ID NO: 101 (corresponding to the VH and VL domains of <PD-1> pembrolizumab disclosed herein).

In another preferred embodiment, ibrutinib may be administered in addition to the anti-CSF-1R antibody in combination with the anti-CD20 antibody.

Ibrutinib (1-[(3R)-3-[4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]piperidin-1-yl]prop-2-en-1-one) (USAN, [1] also known as PCI-32765 and sold under the name Imbruvica) is an anticancer drug that targets B-cell malignancies.

Thus, in one embodiment, ibrutinib is administered and/or used in combination with an anti-CSF-1R antibody in the methods and medical uses described above (particularly of CSF1R and CD20 antibodies) in addition to an anti-CD20 antibody (triple combination).

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.

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

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 formulation, preferably together with a pharmaceutically acceptable carrier, for treating a patient suffering from cancer.

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 invention.

Sequence Description

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

SEQ ID NO:2 Heavy chain CDR2, Mab 2F11

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

SEQ ID NO: 57 Human kappa light chain constant region

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

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

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

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

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

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

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

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

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

SEQ ID NO:67 Signal peptide

SEQ ID NO:68 Primer

SEQ ID NO:69 Heavy chain variable domain, humanized B-Ly1 variant B-HH2

SEQ ID NO:70 Heavy chain variable domain, humanized B-Ly1 variant B-HH3

SEQ ID NO:71 Heavy chain variable domain, humanized B-Ly1 variant B-HH6

SEQ ID NO:72 Heavy chain variable domain, humanized B-Ly1 variant B-HH8

SEQ ID NO:73 Heavy chain variable domain, humanized B-Ly1 variant B-HL8

SEQ ID NO:74 Heavy chain variable domain, humanized B-Ly1 variant B-HL11

SEQ ID NO:75 Heavy chain variable domain, humanized B-Ly1 variant B-HL13

SEQ ID NO:76 Light chain variable domain (VL), humanized B-Ly1 variant B-KV1

SEQ ID NO: 77 human CD20

SEQ ID NO:78 <PD-L1> Variable heavy chain domain VH of 243.55 variant 1

SEQ ID NO:79 <PD-L1> Variable heavy chain domain VH of 243.55 variant 2

SEQ ID NO:80 <PD-L1> Variable heavy chain domain VH of 243.55 variant 3

SEQ ID NO:81 <PD-L1> Variable light chain domain VL of 243.55 variant 1

SEQ ID NO:82 <PD-L1> Variable light chain domain VL of 243.55 variant 2

SEQ ID NO:83 <PD-L1> Variable light chain domain VL of 243.55 variant 3

SEQ ID NO:84 <PD-L1> Variable light chain domain VL of 243.55 variant 4

SEQ ID NO:85 <PD-L1> 243.55 variable light chain domain VL of variant 5

SEQ ID NO:86 <PD-L1> Variable light chain domain VL of 243.55 variant 6

SEQ ID NO:87 <PD-L1> Variable light chain domain VL of 243.55 variant 7

SEQ ID NO:88 <PD-L1> Variable light chain domain VL of 243.55 variant 8

SEQ ID NO:89 <PD-L1> Variable light chain domain VL of 243.55 variant 9

SEQ ID NO:90 <PD-L1> 243.55 variable light chain domain VL of variant 10

SEQ ID NO:91 <PD-L1> Variable light chain domain VL of 243.55 variant 11

SEQ ID NO:92 <PD-L1> Variable light chain domain VL of 243.55 variant 12

SEQ ID NO:93 <PD-L1> Variable light chain domain VL of 243.55 variant 13

SEQ ID NO:94 <PD-L1> 243.55 variable light chain domain VL of variant 14

SEQ ID NO:95 <PD-L1> 243.55 Variable light chain domain VL of variant 15

SEQ ID NO:96 <PD-L1> Variable light chain domain VL of 243.55 variant 16

SEQ ID NO:97 Human programmed death ligand 1

SEQ ID NO:98 <PD-1> variable heavy chain domain VH of nivolumab

SEQ ID NO:99 <PD-1> variable light chain domain VL of nivolumab

SEQ ID NO: 100 <PD-1> Variable heavy chain domain VH of pembrolizumab

SEQ ID NO: 101 <PD-1> Variable light chain domain VL of pembrolizumab

The specific embodiments of the present invention are described below:

An antibody that binds to CSF-1R for use in inducing lymphocytosis of leukemic cells in lymphoma or leukemia.

2. Use of an antibody that binds to CSF-1R for inducing lymphocytosis of leukemic cells in lymphoma or leukemia.

3. A method of treatment comprising administering an effective amount of an antibody that binds to CSF-1R for inducing lymphocytosis of leukemic cells in lymphoma or leukemia.

4. Use of an antibody that binds to CSF-1R for the manufacture of a medicament for inducing lymphocytosis of leukemic cells in lymphoma or leukemia.

5. The antibody, use or method according to any preceding embodiment, wherein the lymphocytosis increases the percentage of circulating leukemic cells expressing CD19 and/or expressing CD20 in the peripheral blood.

6. The antibody, use or method according to any preceding embodiment, wherein the lymphocytosis increases the percentage of circulating CD19-expressing and/or CD20-expressing leukemic cells in the peripheral blood and renders the lymphoma or leukemia susceptible to treatment with an anti-CD19 antibody and/or an anti-CD20 antibody.

7. The antibody, use or method according to any preceding embodiment, wherein the lymphocytosis increases circulating leukemic cells expressing CD20.

8. The antibody, use or method according to any preceding embodiment, wherein the lymphocytosis increases the percentage of circulating leukemic cells expressing CD20 and renders the lymphoma or leukemia susceptible to treatment with an anti-CD20 antibody.

9. The antibody, use or method according to any preceding embodiment,

The antibody binding to human CSF-1R used in the combination therapy comprises

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 CD20 used in the combination therapy comprises

a) a heavy chain variable domain VH of SEQ ID NO: 69 and a light chain variable domain VL of SEQ ID NO: 76, or

b) a heavy chain variable domain VH of SEQ ID NO: 70 and a light chain variable domain VL of SEQ ID NO: 76, or

c) a heavy chain variable domain VH of SEQ ID NO: 71 and a light chain variable domain VL of SEQ ID NO: 76, or

d) a heavy chain variable domain VH of SEQ ID NO: 72 and a light chain variable domain VL of SEQ ID NO: 76, or

e) a heavy chain variable domain VH of SEQ ID NO: 73 and a light chain variable domain VL of SEQ ID NO: 76, or

f) a heavy chain variable domain VH of SEQ ID NO: 74 and a light chain variable domain VL of SEQ ID NO: 76, or

g) the heavy chain variable domain VH of SEQ ID NO: 75 and the light chain variable domain VL of SEQ ID NO: 76.

10. The antibody, use or method according to any preceding embodiment,

The antibody binding to human CSF-1R used in the combination therapy comprises

The heavy chain variable domain VH of SEQ ID NO:39 and the light chain variable domain VL of SEQ ID NO:40, and

The antibody binding to human CD20 used in the combination therapy comprises

The heavy chain variable domain VH of SEQ ID NO: 71 and the light chain variable domain VL of SEQ ID NO: 76

11. The antibody, use or method according to any preceding embodiment,

The antibody binding to human CSF-1R used in the combination therapy comprises

The heavy chain variable domain VH of SEQ ID NO:39 and the light chain variable domain VL of SEQ ID NO:40, and

The human CD20-binding antibody used in the combination therapy is an afucosylated antibody of the IgG1 isotype with an altered glycosylation pattern in the Fc region, wherein the amount of fucose-containing oligosaccharides is between 40% and 60% of the total oligosaccharides at Asn297; and comprises a heavy chain variable domain VH of SEQ ID NO: 71 and a light chain variable domain VL of SEQ ID NO: 76.

12.A) An antibody that binds to human CSF-1R, wherein the antibody is used in combination with an antibody that binds to human CD20; or

B) use of an antibody that binds to human CSF-1R for combination therapy with an antibody that binds to human CD20; or

C) a method of treatment comprising administering an effective amount of an antibody that binds to human CSF-1R, wherein the antibody is used in combination with an antibody that binds to human CD20; or

D) use of an antibody that binds to human CSF-1R in the manufacture of a medicament for combination with an antibody that binds to human CD20;

The antibodies, uses or methods under A), B), C) and D)

i) for the treatment of cancers expressing CD20; or

ii) for stimulating an immune response or function, such as T cell activity; or

iii) for stimulating a cell-mediated immune response, in particular stimulating cytotoxic T-lymphocytes, stimulating T-cell activity, or stimulating macrophage activity; or

iv) for delaying cancer progression; or

v) for prolonging the survival of patients suffering from cancer,

And wherein 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 wherein the antibody binding to human CD20 used in the combination therapy is characterized by comprising

a) a heavy chain variable domain VH of SEQ ID NO: 69 and a light chain variable domain VL of SEQ ID NO: 76, or

b) a heavy chain variable domain VH of SEQ ID NO: 70 and a light chain variable domain VL of SEQ ID NO: 76, or

c) a heavy chain variable domain VH of SEQ ID NO: 71 and a light chain variable domain VL of SEQ ID NO: 76, or

d) a heavy chain variable domain VH of SEQ ID NO: 72 and a light chain variable domain VL of SEQ ID NO: 76, or

e) a heavy chain variable domain VH of SEQ ID NO: 73 and a light chain variable domain VL of SEQ ID NO: 76, or

f) a heavy chain variable domain VH of SEQ ID NO: 74 and a light chain variable domain VL of SEQ ID NO: 76, or

g) the heavy chain variable domain VH of SEQ ID NO: 75 and the light chain variable domain VL of SEQ ID NO: 76.

13. The antibody, use or method according to embodiment 12,

The antibody binding to human CSF-1R used in the combination therapy comprises

The heavy chain variable domain VH of SEQ ID NO:39 and the light chain variable domain VL of SEQ ID NO:40, and

The antibody binding to human CD20 used in the combination therapy comprises

The heavy chain variable domain VH is SEQ ID NO: 71 and the light chain variable domain VL is SEQ ID NO: 76.

14. The antibody, use or method according to embodiment 12,

The antibody binding to human CSF-1R used in the combination therapy comprises

The heavy chain variable domain VH of SEQ ID NO:39 and the light chain variable domain VL of SEQ ID NO:40, and

The human CD20-binding antibody used in the combination therapy is an afucosylated antibody of the IgG1 isotype with an altered glycosylation pattern in the Fc region, wherein the amount of fucose-containing oligosaccharides is between 40% and 60% of the total oligosaccharides at Asn297; and comprises a heavy chain variable domain VH of SEQ ID NO: 71 and a light chain variable domain VL of SEQ ID NO: 76.

15. A method of targeting CD20-expressing cancers with an anti-CD20 antibody in combination with a CSF-1R inhibitor for preventing escape from CD20-targeted therapies by targeting macrophages.

16. The method of embodiment 15, wherein macrophages are targeted with an anti-CSF1R antibody.

17. The method according to claim 15 or 16,

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 wherein the antibody binding to human CD20 used in the combination therapy is characterized by comprising

a) a heavy chain variable domain VH of SEQ ID NO: 69 and a light chain variable domain VL of SEQ ID NO: 76, or

b) a heavy chain variable domain VH of SEQ ID NO: 70 and a light chain variable domain VL of SEQ ID NO: 76, or

c) a heavy chain variable domain VH of SEQ ID NO: 71 and a light chain variable domain VL of SEQ ID NO: 76, or

d) a heavy chain variable domain VH of SEQ ID NO: 72 and a light chain variable domain VL of SEQ ID NO: 76, or

e) a heavy chain variable domain VH of SEQ ID NO: 73 and a light chain variable domain VL of SEQ ID NO: 76, or

f) a heavy chain variable domain VH of SEQ ID NO: 74 and a light chain variable domain VL of SEQ ID NO: 76, or

g) the heavy chain variable domain VH of SEQ ID NO: 75 and the light chain variable domain VL of SEQ ID NO: 76.

18. The method according to claim 15 or 16,

The antibody binding to human CSF-1R used in the combination therapy comprises

The heavy chain variable domain VH of SEQ ID NO:39 and the light chain variable domain VL of SEQ ID NO:40, and

The antibody binding to human CD20 used in the combination therapy comprises

The heavy chain variable domain VH of SEQ ID NO: 71 and the light chain variable domain VL of SEQ ID NO: 76

19. The method according to claim 15 or 16,

The antibody binding to human CSF-1R used in the combination therapy comprises

The heavy chain variable domain VH of SEQ ID NO:39 and the light chain variable domain VL of SEQ ID NO:40, and

The human CD20-binding antibody used in the combination therapy is an afucosylated antibody of the IgG1 isotype with an altered glycosylation pattern in the Fc region, wherein the amount of fucose-containing oligosaccharides is between 40% and 60% of the total oligosaccharides at Asn297; and comprises a heavy chain variable domain VH of SEQ ID NO: 71 and a light chain variable domain VL of SEQ ID NO: 76.

20. The antibody, use or method according to any one of the preceding embodiments for the treatment of lymphomas and lymphocytic leukemias.

21. The antibody, use or method according to any one of the preceding embodiments for use in the treatment of B-cell non-Hodgkin's lymphoma (NHL).

22. The antibody, use or method according to any one of the preceding embodiments for use in the treatment of multiple myeloma, follicular lymphoma, or Hodgkin's disease.

23. The antibody, use or method according to any one of the preceding embodiments, wherein the cancer, lymphoma or leukemia expresses CD20.

24. The antibody, use or method according to any one of the preceding embodiments for treating an immune related disease such as tumor immunity or delaying its progression.

25. The antibody, use or method according to any preceding embodiment for stimulating an immune response or function, such as T cell activity.

26. The antibody, use or method according to any one of the preceding embodiments for preventing or treating metastasis.

27. The antibody, use or method according to any one of the preceding embodiments for use in the treatment of an inflammatory disease.

28. The antibody, use or method according to any one of the preceding embodiments, wherein the antibody that binds to human CSF-R and the antibody that binds to human CD20 are of human IgG1 subclass.

29. The antibody, use or method according to any one of the preceding embodiments, wherein no additional chemotherapeutic agents and/or targeted therapies are administered in addition to the anti-CSF-1R antibody and anti-CD20 antibody combination therapy.

30. The antibody, use or method according to any one of the preceding embodiments, wherein the antibody that binds to CSF-1R and the antibody that binds to human CD20 are co-administered simultaneously.

31. The antibody, use or method according to any one of the preceding embodiments, wherein the antibody that binds to CSF-1R and the antibody that binds to human CD20 are co-administered sequentially.

32. The antibody, use or method according to any one of the preceding embodiments, wherein an anti-PD-L1 antibody is administered in addition to the anti-CSF-1R antibody in combination with the anti-CD20 antibody.

33. The antibody, use or method according to embodiment 32, wherein the PD-L1 binding antibody used comprises a variable domain amino acid sequence selected from the group consisting of:

- the variable heavy chain domain VH of SEQ ID NO: 78, and the variable light chain domain VL of SEQ ID NO: 81 (corresponding to the VH and VL domains of <PD-L1> "243.55.S70" disclosed herein);

- the variable heavy chain domain VH of SEQ ID NO: 79, and the variable light chain domain VL of SEQ ID NO: 82 (corresponding to the VH and VL domains of <PD-L1> "243.55.H1" disclosed herein);

- the variable heavy chain domain VH of SEQ ID NO: 79, and the variable light chain domain VL of SEQ ID NO: 83 (corresponding to the VH and VL domains of <PD-L1> "243.55.H12" disclosed herein);

- the variable heavy chain domain VH of SEQ ID NO: 79, and the variable light chain domain VL of SEQ ID NO: 84 (corresponding to the VH and VL domains of <PD-L1> "243.55.H37" disclosed herein);

- the variable heavy chain domain VH of SEQ ID NO: 79, and the variable light chain domain VL of SEQ ID NO: 85 (corresponding to the VH and VL domains of <PD-L1> "243.55.H70" disclosed herein);

- the variable heavy chain domain VH of SEQ ID NO: 79, and the variable light chain domain VL of SEQ ID NO: 86 (corresponding to the VH and VL domains of <PD-L1> "243.55.H89" disclosed herein);

- the variable heavy chain domain VH of SEQ ID NO: 79, and the variable light chain domain VL of SEQ ID NO: 87 (corresponding to the VH and VL domains of <PD-L1> "243.55.S1" disclosed herein);

- the variable heavy chain domain VH of SEQ ID NO: 79, and the variable light chain domain VL of SEQ ID NO: 88 (corresponding to the VH and VL domains of <PD-L1> "243.55.5" disclosed herein);

- the variable heavy chain domain VH of SEQ ID NO: 79, and the variable light chain domain VL of SEQ ID NO: 89 (corresponding to the VH and VL domains of <PD-L1> "243.55.8" disclosed herein);

- the variable heavy chain domain VH of SEQ ID NO: 79, and the variable light chain domain VL of SEQ ID NO: 90 (corresponding to the VH and VL domains of <PD-L1> "243.55.30" disclosed herein);

- the variable heavy chain domain VH of SEQ ID NO: 79, and the variable light chain domain VL of SEQ ID NO: 91 (corresponding to the VH and VL domains of <PD-L1> "243.55.34" disclosed herein);

- the variable heavy chain domain VH of SEQ ID NO: 79, and the variable light chain domain VL of SEQ ID NO: 92 (corresponding to the VH and VL domains of <PD-L1> "243.55.S37" disclosed herein);

- the variable heavy chain domain VH of SEQ ID NO: 79, and the variable light chain domain VL of SEQ ID NO: 93 (corresponding to the VH and VL domains of <PD-L1> "243.55.49" disclosed herein);

- the variable heavy chain domain VH of SEQ ID NO: 79, and the variable light chain domain VL of SEQ ID NO: 94 (corresponding to the VH and VL domains of <PD-L1> "243.55.51" disclosed herein);

- the variable heavy chain domain VH of SEQ ID NO: 79, and the variable light chain domain VL of SEQ ID NO: 95 (corresponding to the VH and VL domains of <PD-L1> "243.55.62" disclosed herein); and

- the variable heavy chain domain VH of SEQ ID NO: 80, and the variable light chain domain VL of SEQ ID NO: 96 (corresponding to the VH and VL domains of <PD-L1> "243.55.84" disclosed herein).

34. The antibody, use or method according to embodiment 32, wherein the antibody that binds to PD-L1 is atezolizumab.

35. The antibody, use or method according to any one of embodiments 32 to 34, wherein the antibody that binds to CSF-1R, the antibody that binds to human CD20, and the antibody that binds to human PD-L1 are co-administered simultaneously.

36. The antibody, use or method according to any one of embodiments 32 to 34, wherein the antibody that binds to CSF-1R, the antibody that binds to human CD20, and the antibody that binds to human PD-L1 are co-administered sequentially.

37. The antibody, use or method according to any one of the preceding embodiments, wherein an anti-PD-1 antibody is administered in addition to the anti-CSF-1R antibody in combination with the anti-CD20 antibody.

38. The antibody, use or method according to embodiment 37, wherein the antibody that binds to PD-1 comprises the following variable domain amino acid sequence:

The variable heavy chain domain VH of SEQ ID NO: 98 and the variable light chain domain VL of SEQ ID NO: 99 (corresponding to the VH and VL domains of <PD-1> nivolumab disclosed herein).

39. The antibody, use or method according to embodiment 38, wherein the PD-1 binding antibody comprises the following variable domain amino acid sequence:

The variable heavy chain domain VH of SEQ ID NO: 100 and the variable light chain domain VL of SEQ ID NO: 101 (corresponding to the VH and VL domains of <PD-1> pembrolizumab disclosed herein).

40. The antibody, use or method according to embodiments 37 to 39, wherein the antibody that binds to CSF-1R, the antibody that binds to human CD20, and the antibody that binds to human PD-1 are co-administered simultaneously.

41. The antibody, use or method according to embodiments 37 to 39, wherein the antibody that binds to CSF-1R, the antibody that binds to human CD20, and the antibody that binds to human PD-1 are co-administered sequentially.

42. The antibody, use or method according to any one of the preceding embodiments, wherein ibrutinib is administered in addition to the anti-CSF-1R antibody in combination with the anti-CD20 antibody.

Experimental procedures

mice

All mice were housed and bred in a specific pathogen-free animal facility and handled according to European Union guidelines and with approval from the Institutional Ethics Committee of the San Raffaele Scientific Institute. Rag2 ^−/− γc ^−/− mice on a BALB/c background were generously provided by CIEA and Taconic, Eμ-TCL1 transgenic mice on a C57BL/6 background were generously provided by Dr. Byrd, and wild-type C57BL/6 mice were supplied by Charles River Laboratories.

Cells and reagents

Human primary samples were obtained from patients with RAI stage 0-1 CLL after informed consent, as approved by the Institutional Ethics Committee of the San Raffaele Scientific Institute (Milan, Italy) (Protocol VIVI-CLL), in accordance with the Declaration of Helsinki. The MEC1 CLL cell line was obtained from the German Collection of Microorganisms and Cell Cultures (DMSZ) and cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum and gentamicin (15 μg/mL; Sigma-Aldrich). Clodrolip and phosphate-buffered saline (PBS) liposomes were purchased from ClodLip B.V. The anti-mouse CSF1R antibody 2G2 and the anti-human CSF1R antibody huMab 2F11-2e 7 were provided by the Roche Innovation Center, Penzberg, Germany. Anti-human CD20 GA101 (glycoengineered humanized B-Ly1 antibody BHH6-B-KV1 GE with fucose content between 40 and 60%) was provided by the Roche Innovation Center, Schlieren, Switzerland.

In vivo studies

For xenograft studies, 8-week-old Rag2 ^-/- γc ^-/- mice were challenged with ^10x106 MEC1 cells in 0.1 mL of saline via a 27-gauge needle either intravenously or subcutaneously. Depending on the experiment, mice were injected with clodrolip, anti-mouse CSF1R mAb, or anti-human CD20GA101 mAb. For transgenic transplantation studies, 16 (day 0) C57BL/6 male mice were challenged intravenously with ^10x106 cells purified from the spleens of leukemic male Eμ-TCL1 transgenic mice. Depending on the experiment, mice were treated with clodrolip or anti-CSF1R mAb at various doses and schedules.

Mouse cell preparation

PB, PE, SP, and femur were collected from mice and cells were isolated. Red blood cells from BM, PE, SP, and PB samples were lysed by incubation in ammonium chloride solution (ACK) lysis buffer ( _NH4Cl 0.15M, KHCO3 _10mM , _Na2EDTA 0.1mM, pH 7.2-7.4) at room temperature for 5 minutes. After blocking the crystallizable fragment (Fc) receptor with Fc blocking reagent (BD Biosciences) at room temperature for 10 minutes, cells from PB, BM, PE, and SP were stained with the antibodies listed below (15 minutes at 4°C) and analyzed using a Beckman Coulter FC500 flow cytometer. For intracellular cytokine detection, see below. Absolute numbers were obtained by multiplying the percentage of cells by the total number of splenocytes, peritoneal cells, or BM cells flushed from one femur (except Figure 7 (Figure 1 of the paper) BCDE, where absolute numbers refer to the total BM cells flushed from both the femur and tibia).

Mouse cell flow cytometry

Phenotypic analysis was performed using the following antibodies: PE-Cy7 or PE rat anti-mouse CD11b (M1/70), APC rat anti-mouse CD5 (53-7.3), PE-Cy7 rat anti-mouse CD19 (1D3), APC or FITC rat anti-mouse CD45 (30-F11), FITC rat anti-mouse CD206 (MR5D3), FITC hamster anti-mouse CD54 (3E2), PE rat anti-mouse CD86 (GL1), FITC or PE-Cy7 rat anti-mouse CD8 (53-6.7), FITC rat anti-mouse CD44 (IM7), PE rat anti-mouse CD62L (MEL-14), PE-Cy7 rat anti-mouse CD4 (GK1.5), APC rat anti-mouse CD25 (PC61), FITC rat anti-mouse Gr1 (RB6-8C5), FITC Annexin V apoptosis detection kit by BD Biosciences. Biosciences; FITC rat anti-mouse κ (187.1), PE rat anti-mouse λ (JC5-1), purchased from Beckman Coulter; PE anti-mouse F4/80 (BM8), FITC rat anti-mouse Ly6C (HK1.4), APC rat anti-mouse Ly6G (1A8), obtained from BioLegend; APC anti-mouse CSF1R (AFS98), purchased from eBioscience; PE rat anti-mouse CSF1R (604B5 2E11), purchased from AbD Serotec.

Intracellular cytokine detection

For IFNγ detection on CD8 T cells, splenocytes were stimulated in vitro with ionomycin and brefeldin A and stained with FITC anti-CD44, PE-Cy7 anti-CD8 and APC anti-mouse IFNγ (XMG1.2, BD Biosciences). For IL17a and IL2 detection on stromal cells, cell surface was labeled with anti-CD45, anti-Gr1, and anti-CD11b, then the cells were washed, fixed and permeabilized with Cytofix/Cytoperm-Perm/Wash (BD Biosciences) and labeled with biotin-streptavidin-PE anti-mouse IL2 (JES6-5H4) or PE anti-mouse IL17a (TC11-18H10) purchased from BD Biosciences.

In vitro culture and cell depletion assays from CLL samples

Fresh PBMCs from untreated CLL patients were seeded in triplicate at 3x10 ⁶ cells/ml in culture medium and treated with clodrolip or PBS liposomes (100, 500, 1000 μM) for 30 minutes and 24 hours in the presence or absence of the TNF-α antagonist etanercept (10 μg/ml) from Pfizer or anti-TRAIL-R2 (human, 1 μg/ml) monoclonal antibody (HS201) from Adipogen AG. For CSF1R inhibition studies, PBMCs from CLL patients were treated for 48 hours with anti-human CSF1R monoclonal antibody (1-10 μg/ml) or anti-human CSF1R monoclonal antibody + anti-CD20 monoclonal antibody GA101 (10 μg/ml). The specific percentage of remaining leukemic CD19+CD5+ or CD14+ cells in the treated samples was calculated as (absolute number in treated samples/absolute number in control samples) x 100. For each condition, the absolute number of remaining cells was calculated as the total number of viable cells (trypan blue exclusion assay) x % viable cells (flow cytometry assay). Specific cell depletion was then calculated as follows: 100% specific remaining cells, as described in {Laprevotte, 2013 #46}.

Gene expression profile analysis

Following the manufacturer's instructions, hCD19- cells from mouse BM were secondary enriched for monocytes/macrophages by depleting T, NK, dendritic cells, progenitor cells, granulocytes, and erythrocytes using the EasySep Negative Selection Monocyte Enrichment Kit on an EasySep Magnet (StemCell Technologies). RNA extraction was performed using the RNeasy Mini Kit (QIAGEN). 200 ng of total RNA sample was processed using the Illumina TotalPrep Amplification Kit (Ambion-Lifetechnologies) in strict accordance with the manufacturer's protocol. Subsequently, biotinylated cRNA was hybridized on a mouse WG-6v2.0 expression BeadChip (containing probes for ~45,000 transcripts) or a human HT-12v4 BeadChip (containing probes for ~47,000 transcripts) for 16 hours, followed by Cy3 staining. After hybridization, the BeadChip underwent a wash and streptavidin-Cy3 (GE Healthcare Bio-Sciences) conjugate staining protocol. Once the BeadChip is processed, it is scanned using the Illumina BeadArray Reader. Briefly, the array is scanned on a BeadScan instrument and the fluorescence intensities are extracted and summarized using BeadStudio software (Illumina), generating a set of summarized fluorescence measurements.

Statistical analysis

Statistical analysis was performed using the Student's t-test. Data were expressed as mean ± SD, and comparisons of growth curves with a p value less than 0.05 were considered statistically significant. Comparisons of survival curves were performed using the log-rank test.

Mouse genotype determination

Eμ-TCL1 transgenic mice were genotyped for the hTCL1 transgene by a PCR-based screening assay as previously described {Bertilaccio, 2011 #1}.

Xenograft studies

(Day 0) 8-week-old Rag2 ^-/- γc ^-/- mice were iv attacked with ^10x106 MEC1 cells in 0.1 mL saline via a 27-gauge needle as described in (Bertilaccio MTS, Blood, 2010). In some experiments, mice were injected iv with clodrolip (200 μl) or PBS liposomes (200 μl) (as a control) every 3 days starting on day -1 of the leukemia attack. Depending on the experiment, mice were monitored for weight once a week and killed in the early stages of leukemia (days 18-21, 7 clodrolip injections) or the late stages of leukemia (days 28-31, 5 clodrolip injections). PE, PB, SP and femoral BM were analyzed. In selected experiments, BM cells were flushed out from the mouse femur for magnetic separation of leukemia cells and monocytes/macrophages. In preclinical experiments, Rag2 ^-/- γc ^-/- mice were sc challenged with ^10x106 MEC1 cells in 0.1mL saline on the left side (day 0). Once mice bearing subcutaneous tumors developed palpable tumor-draining axillary LN (TDAL), they were sc injected with clodrolip (60 μl) (days +39, +45, +52, +56, +60, +63, +66) or PBS liposomes (60 μl) (as a control) at the TDAL site. Mice were monitored for weight, tumor, and LN growth (three perpendicular diameters were measured with calipers) once a week and killed when the mean LN volume reached 1000 ^mm3 or greater, before clinical signs and symptoms were achieved, in accordance with ethical guidelines to avoid unnecessary pain and discomfort. For preclinical purposes, Rag2 ^-/- γc ^-/- mice transplanted with ^10x106 MEC1 cells were also injected iv with clodrolip (60 μl) starting on day 11 of leukemia challenge (day 0) and sacrificed on days 26-32 (after two weeks of treatment with biweekly clodrolip injections; after two clodrolip injections, every other week) or monitored for survival (after two clodrolip injections, every other week). For in vivo CSF1R inhibition studies, xenograft mice were injected iv with anti-CSF1R mAb on days +11, +25 and sacrificed on days 27-29. For survival experiments, xenograft mice were injected iv with anti-CSF1R or GA101 mAb (monoclonal antibody) on days +11, +25 as single agents or in a combination setting. For functional studies, xenograft mice injected iv with clodrolip or anti-CSF1R mAb (at days +11, +25) were given repeated ip administration of etanercept (Pfizer, 10 mg/kg) and sacrificed on day 26 or 29, respectively. In one experiment, xenograft mice were pretreated with 2 mg/kg anti-ICAM1 blocking mAb (YN1/1.7.4, Biolegend) every 3/4 days starting on day -1 and sacrificed on day +19. For transgenic studies, 8-week-old syngeneic immunocompetent C57BL/ ⁶ male mice were challenged ip with 10x10 cells purified from the spleens of leukemic male Eμ-TCL1 transgenic mice using the EasySep Mouse B-Cell Enrichment Kit (day 0). The purity of the transplanted CD19+CD5+Igk+ cells was assessed by flow cytometry. Mice were injected with clodrolip (200 μl) ip every 3 days starting on day -1 of leukemia attack (days -1, +2, +5, +8, +11, +14, +18) or PBS liposomes (200 μl) (as a control). In preclinical studies, transplanted mice were administered clodrolip (50 μl) or PBS liposomes (50 μl) every 3/4 days starting on day +16 or +17 of leukemia attack (as a control). Mice were monitored for weight and leukemia (by flow cytometry analysis of PB samples) once a week and killed on days 24-28. PE, PB, and organs (SP, LN, femur BM) were analyzed. For in vivo CSF1R inhibition studies, transplanted mice were treated with 30 mg/kg anti-CSF1R mAb ip (days +17, +23) and monitored as described above.

Example

Example 1

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

4.5 x 10 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), 1× 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 (the 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 of 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 1 M NaF was added. After 30 minutes of ice storage, the plate was shaken vigorously on a plate shaker for 3 minutes and then centrifuged at 2200 rpm for 10 minutes (Heraeus Megafuge 10).

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. To detect total CSF-1R, 10 μl of lysate was immobilized on a plate using the capture antibody included in the kit. A 1:750 dilution of biotinylated anti-CSF-1R antibody, BAF329 (R&D Systems), and a 1:1000 dilution of streptavidin-HRP conjugate were then added. After 60 minutes, the plates were developed with freshly prepared solution and the absorbance was measured. Data were calculated as a percentage of the positive control without antibody and as the ratio of phosphorylated to total receptor expression. A negative control was defined as the absence of M-CSF-1. As a reference control, anti-CSF-1R SC 2-4A5 (Santa Cruz Biotechnology, US, see also Sherr, C.J. et al., Blood 73 (1989) 1786-1793) which inhibits ligand-receptor interaction was used.

Table 3: Calculated IC50 values for inhibition of CSF-1 receptor phosphorylation.

Example 2

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

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 plates to prevent adhesion to plastic surfaces in DMEM high glucose medium (PAA, Pasching, Austria) supplemented with 2 mM L-glutamine, 2 mM sodium pyruvate, non-essential amino acids, and 10% fetal bovine serum (Sigma, Taufkirchen, Germany). Cells were plated in serum-replaced medium supplemented 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 No. 60530), cells expressing wtCSF-1R formed dense, three-dimensional spheroids, a property known as anchorage independence. These spheroids resemble the three-dimensional structure and organization of orthotopic solid tumors. Cells recombinant with the mutant CSF-1R were able to form spheroids independently of the CSF-1 ligand. The anti-CSF-1R antibody hMab 2F11-e7 according to the present invention and the anti-CSF-1R antibodies 1.2.SM (a ligand-displacing CSF-1R antibody described in WO 2009/026303), CXIIG6 (a 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, C.J. et al., Blood 73 (1989) 1786-1793) and Mab R&D-Systems 3291 were investigated. The reference control Mab R&D-Systems 3291 did not show inhibition of the proliferation of cells recombinant with mutant CSF-1R.

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

Table 4

Example 3

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

Human monocytes were isolated from peripheral blood using RosetteSep ^™ Human Monocyte Enrichment Mixture (Stemcell Technologies, Catalog No. 15028). The enriched monocyte population was seeded into 96-well microtiter plates (2.5 x ¹⁰ cells/well) in 100 μl RPMI ₁₆₄₀ (Gibco, Catalog No. 31870) supplemented with 10% FCS (GIBCO, Catalog No. 011-090014M), 4 mM L-glutamine (GIBCO, Catalog No. 25030), and 1 x PenStrep (Roche, Catalog No. 1074440) at 37°C in a humidified atmosphere with 5% CO₂. When 150 ng/ml of huCSF-1 was added to the culture medium, clear differentiation into adherent macrophages was observed. This differentiation could be inhibited by the addition of anti-CSF-1R antibodies. Furthermore, monocyte survival was affected and could be analyzed by CellTiterGlo (CTG) assay. From the concentration-dependent inhibition of monocyte survival by antibody treatment, _{IC50 values} were calculated (see table below).

Table 5

CSF-1R Mab Mab 2F11 0.08

In separate tests, a series of humanized forms of Mab 2F11, such as hMab 2F11-c11, hMab 2F11-d8, hMab 2F11-e7, and 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).

Example 4

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

Human monocytes were isolated from peripheral blood using RosetteSep ^™ Human Monocyte Enrichment Mixture (Stemcell Technologies, Catalog No. 15028). The enriched monocyte population was seeded into 96-well microtiter plates (2.5 x ¹⁰ cells/well) in 100 μl RPMI ₁₆₄₀ (Gibco, Catalog No. 31870) supplemented with 10% FCS (GIBCO, Catalog No. 011-090014M), 4 mM L-glutamine (GIBCO, Catalog No. 25030), and 1 x PenStrep (Roche, Catalog No. 1074440) at 37°C in a humidified atmosphere with 5% CO₂. When 150 ng/ml of huCSF-1 was added to the culture medium, clear differentiation into adherent macrophages was observed. This differentiation could be inhibited by the addition of anti-CSF-1R antibodies. In addition, monocyte survival was affected and could be analyzed by CellTiterGlo (CTG) assay. From the concentration-dependent inhibition of monocyte survival by antibody treatment, _{IC50 values} were calculated. Humanized forms of Mab 2F11, such as hMab 2F11-c11, hMab 2F11-d8, hMab 2F11-e7, and 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).

Example 5

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

Human monocytes were isolated from peripheral blood using RosetteSep ^™ Human Monocyte Enrichment Mix (StemCell Technologies, catalog number 15028). The enriched monocyte population was seeded into 96-well microtiter plates (2.5 x 10 cells/well ₎ in 100 μl of 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 % CO2 in a humidified atmosphere. When 100 ng/ml huCSF-1 was added to the culture medium for 6 days, differentiation into adherent, elongated M2 macrophages was 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 was 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 of staining buffer and the cells were counted in a Neubauer trough. Approximately 1 x 10e5 cells were transferred to each FACS tube, centrifuged at 300 x g for 5 minutes, and resuspended in staining buffer. Fc γ receptors are blocked by incubation on ice for 20 minutes in staining buffer with 1 μg human IgG/2.5x10e4 individual cell (JIR catalog number 009-000-003).Then for CD80 and CD163 detection, cell is mixed with 1.5 μl antibody/2.5x10e4 individual cell, and for II class MHC detection, 5 μl antibody/2.5x10e4 individual antibody is used: the mouse anti-human CD163 (BD Bioscience catalog number 556018) of PE labeling, the mouse anti-human CD80 (BD Bioscience catalog number 557227) of PE labeling and the mouse anti-human II class MHC (Dako catalog number M0775) of Alexa 647 labeling.By using Zenon Alexa 647 mouse IgG labeling kit (Invitrogen catalog number Z25008), Alexa 647 labeling is conjugated to antibody. After incubation on ice for 1 hour, cells were washed twice with staining buffer, resuspended, and measured on a FACS Canto II.

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

Example 6

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

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

TAM inhibition assay

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 removal 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 mononuclear cells from TAM preparations. Final cell purity was analyzed, typically >90%. Subsequently, TAMs were titrated with the ratio of the total CD3+T cells labeled with CFSE as shown in a 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).

Example 7

Targeting monocytes/macrophages through CSF-1R inhibition may be used as a therapeutic strategy in CLL

To evaluate the therapeutic potential of macrophage targeting, we investigated the antileukemic effects of a monoclonal antibody that inhibits CSF1R signaling and blocks macrophage differentiation from monocyte precursors {Ries, 2014 #16}. Rag2 ^−/− γc ^−/− mice transplanted with MEC1 cells were injected intravenously with an anti-CSF1R mAb (30 mg/kg, days +11 and +25) and sacrificed on day 27, 48 hours after the last mAb injection (Figure 4A). Macrophage depletion (Figures 4B–C) significantly reduced the number of leukemic B cells in the BM (Figure 4D) and induced the appearance of CD19+ AnnV-PI+ necrotic leukemic cells in the SP (Figure 4E). To confirm that CSF1R blockade reduces disease severity in the SP, we performed a time-course experiment in which mice were sacrificed on days 27 and 29, 48 and 96 hours after the last mAb injection, respectively (Figure 4F). CSF1R blocking can stabilize the disease and induce necrosis of spleen leukemia cells over time (Fig. 4G). This anti-leukemia effect is related to the notable and selective elimination of CSF1R+MRC1+M2-like TAMs (Fig. 4H-I). As shown in solid tumors {Ries, 2014#16}, CSF1R blocking together with monocyte elimination (Fig. 6A-B) induces the relative increase (Fig. 6D) of SSC ^high neutrophils (Fig. S4C) and Gr1+ myeloid cells in PB, covering both monocytes (Ly6C+) and granulocytes (Ly6G+) subsets (Fig. 6E-F). It is worth noting that this effect was not observed for clodrolip (Fig. 6G-HIJK). In BM, CSF1R blocking reduces monocytes identified as CD11b+CSF1R+ (Fig. 6L) or Ly6C+ (Fig. 6M-N). In the Eμ-TCL1tg transplant model (Figure 6O), we observed a significant increase in CD8+ effector T cells in PB (Figure 6P), BM (Figure 6Q), and SP (Figure 6R-S), accompanied by a significant increase in apoptotic leukemic cells (Figure 6T) and a decrease in splenic CD4+CD25+ T cells (Figure 6U). In contrast, clodrolip did not affect T cells (Figure 6V-WX). Two administrations of anti-CSF1R mAb (Figure 5A) induced significant lymphocytosis (Figure 5B-C), a treatment-related process that has long been seen in CLL patients treated with agents targeting BCR signaling (such as ibrutinib) and is not associated with disease progression (Byrd JCNEJM 2013). Because mAb treatment increases the percentage of CD19+CD20+ circulating leukemic cells (Figure 5C), we tested a two-pronged approach by combining anti-CSF1R mAbs with a glycoengineered type II CD20 mAb, GA101 (Moessner E Blood, 2010) (Figure 5D). Notably, anti-mouse CSF1R mAbs affected mouse survival as single agents (p = 0.01, αCSF1R mAb vs. untreated), and this was even more significant in the combined setting (p < 0.0001, αCD20 + αCSF1R mAb vs. untreated) (Figure 5E). Taken together, these findings indicate that CSF1R blockade provides therapeutic benefit in mouse models of CLL. Furthermore, they show that the increase in circulating CD20+ leukemic cells associated with macrophage depletion represents a therapeutic opportunity for combination with anti-CD20 antibodies.

Characterization of BM monocytes and macrophages in CLL xenograft-bearing mice

We asked whether monocytes/macrophages influence CLL growth in the BM. Eight-week-old Rag2 ^-/- γc ^-/- mice were injected intravenously (iv; day 0) with MEC1 cells (a CLL cell line) {Stacchini, 1999 #50} and killed at the early (day 21) or overt (day 31) stages of leukemic growth (Figure 7A), at which point the percentages of human CD19+ leukemic cells in the BM were 33.83±15.03 and 86±9.13, respectively (Figure 7B). We found fewer CD11b+CSF1R+ monocytes and CD11b+F4/80+ macrophages in the BM of overt leukemic Rag2 ^-/- γc ^-/- mice compared to mice with early leukemia (Figures 7C-D). Notably, the abundance of CD11b+ cells co-expressing the macrophage mannose receptor (MRC1/CD206), CSF1R, and CD86 increased with leukemia progression (Figure 7E-F), a feature associated with solid tumor progression {De Palma, 2013#48} (Noy & Pollard Immunity 2014).

We next evaluated the expression of chemokines, cytokines, and growth factors in non-leukemic cells in the BM microenvironment of Rag2 ^-/- γc ^-/- mice with early leukemia, compared to uninjected mice. Magnetic beads were used to deplete human CD19+ leukemic cells from the BM cells of xenograft mice to perform RNA extraction and qPCR array analysis (RT2 Profiler ^™ ). As shown in Figure 7G, the presence of leukemic cells induced a unique inflammatory profile. We observed a significant upregulation of cytokines with leukemia growth-promoting ability (Yan XJ Blood 2011), such as Il6, Cd401g, Il2, and Il17a. ^Gr1 + myeloid-derived suppressor cells (MDSCs) were identified as the source of IL-17a (Figure 8A), while non-hematopoietic CD45- cells were identified as the source of IL-17a (Figure 8B) and IL-2 (Figure 8C) protein production. Also upregulated are Il10, a cytokine with immunosuppressive activity; Csf1, which promotes monocyte/macrophage differentiation and survival; and multiple chemokines/stromal cell chemoattractants, such as Ccl1, Ccl3, and Ccl4 {De Palma, 2013 #48} (Noy & Pollard Immunity 2014). This cytokine/chemokine profile suggests a BM microenvironment that is conducive to the growth of leukemic cells.

To specifically characterize the molecular profile of CLL-associated monocytes/macrophages ( FIG. 7C-D ), genome-wide transcriptional profiling of monocytes/macrophages isolated from the BM of Rag2 ^−/− γc ^−/− mouse xenograft mice sacrificed at day 21 (early leukemia) was performed using the Illumina hybridization system.

Compared to age-matched, uninjected mice, a complex gene regulatory network involving the regulation of 164 transcripts (84 upregulated and 80 downregulated; 1.6% of all transcripts; adjusted P value < 0.05) distinguished monocytes/macrophages from mice harboring leukemia. The differentially expressed genes were organized into five putative functional categories, including inflammation and intracellular/extracellular functions, as shown in Figures 9A-E. In parallel, we also analyzed transcriptional changes in leukemic MEC1 cells purified from the same mice. In addition to genes implicated in CLL progression (such as NOTCH1, BIRC3, and PTEN), differential expression of genes supporting the presence of monocyte/macrophage-leukemic cell crosstalk was particularly relevant, including IL10, RNASET2, and CCL2, a potent monocyte chemoattractant (Noy & Pollard Immunity 2014) (Figure 9F). We also confirmed the differential expression of a panel of selected genes in mouse monocytes/macrophages (Figure 10A) and human MEC1 cells (Figure 10B) by qPCR. Due to its role in B cell adhesion, antigen presentation, and activation (Batista F Nat Rev Immunol 2009), we verified the upregulation of ICAM1 at the protein level in mouse macrophages from spleen (SP), BM, or peritoneal exudate (PE) (Figure 9G) and human classical monocytes from CLL patients (Figure 10C). Xenograft studies using an ICAM1 blocking mAb administered every 3/4 days starting from day -1 (Figure 10D) demonstrated that its inhibition increased the number of leukemic cells in PB, SP, and PE, but not in BM (Figure 10E), suggesting the functional relevance of ICAM1 upregulation in different tissues.

Together, these findings indicate that CLL cells profoundly sculpt the BM microenvironment and regulate the expression of multiple gene transcripts involved in CLL cell-monocyte/macrophage interactions.

Leukemic cell death induced by macrophage targeting is TNF-dependent

As a next step, we evaluated how leukemic cells are affected by macrophage targeting. Clodrolip and anti-human CSF-1R monoclonal antibody (hMab 2F11-e7) were not directly toxic to leukemic B cells in vitro, as shown by cytotoxicity assays performed on MEC1 cells (Figure 11A). We therefore investigated alternative mechanisms by which clodrolip and anti-CSF1R might induce leukemic cell death in vivo. To address this question, Rag2 ^-/- γc ^-/- mice transplanted with MEC1 cells were depleted of macrophages by intravenous injection of clodrolip every 3 days starting on day -1 (7 consecutive injections) and sacrificed 1 day after the last treatment (Figure 12A). The reduction in leukemia (Figure 12B) and monocyte/macrophage (Figure 12C-DE) cell load induced by clodrolip in all tissues was paralleled by the induction of leukemia cell apoptosis, as shown by a significant increase in the frequency of CD19+AnnV+PI+ late apoptotic cells in SP and BM and CD19+AnnV-PI+ necrotic cells in PB (Figure 12F-G). These results confirm the increase in CD19+AnnV-PI+ necrotic cells observed in the SP of mice treated with anti-CSF1R monoclonal antibodies (Figure 4G). The induction of leukemia B cell apoptosis after clodrolip and the interaction between leukemia B cells and monocytes/macrophages were also shown in time course experiments, in which GFP-labeled monocytes/macrophages were added to leukemia cells from Eμ-TCL1 transgenic mice in the absence or presence of clodrolip. Interestingly, AnnV/PI staining and flow cytometric analysis showed that clodrolip induced apoptosis in leukemia cells only in the presence of monocytes and macrophages ( Figure 11B ).

To investigate the cell death pathway induced by clodrolip-induced macrophage killing in leukemia cells, we purified CD19+MEC1 cells from the BM of transplanted Rag2 ^-/- γc ^-/- mice and evaluated the expression of key effector molecules involved in the cell death pathway regulated by TNF, TRAIL, ROS, and FAS/FASL at the RNA level by qPCR. For the TNF pathway, we observed up-regulated expression of TNFR1, FADD, BID, BAX, and CASP3 (Figure S5C). Interestingly, we also found up-regulated levels of TRAIL-R2 and AIFM1 (Figure S5C), which are involved in ROS-mediated cell death {Joza, 2009#49}.

To confirm the mechanism by which TNF (Figure 11C) is involved in macrophage-targeted leukemic cell death, we utilized etanercept, a soluble TNF receptor fusion protein {Deeg, 2002 #44}, in xenografted Rag2-/-γc ^-/- mice treated with clodrolip or CSF1R mAb. Rag2 ^-/- γc-/- mice transplanted with MEC1 cells iv and injected with clodrolip (iv, days +11, +25) and etanercept (ip, starting on day ⁺¹⁰ ) were sacrificed 24 hours after the last clodrolip ^injection (Figure 12H). Etanercept attenuated clodrolip-induced leukemic cell depletion in the BM (Figure 12I). In xenograft mice treated iv with CSF1R mAb (days +11, +25) and sacrificed 96 h after the last mAb injection ( FIG7J ), the anti-leukemic effect in SP was abolished when the TNF pathway was blocked in vivo by etanercept ( FIG12K ).

Furthermore, we functionally inactivated FAS/FASL signaling in the TCL1 transplantation system using a blocking antibody (clone Kay10, Biolegend). FASL blockade did not abrogate the reduction of leukemic cells in transplanted mice treated with clodrolip (Figures 11D-E-F-G) or aCSF1R (hMab2F11-e7) (Figures 11H-I-J). The same clodrolip results were confirmed by transplanting leukemic B cells into Faslgld mice carrying a loss-of-function mutation in the Fas1 gene (Figures 11E-F-G).

These findings led us to conclude that macrophage killing primarily sensitizes leukemic cells to apoptosis through induction of TNF signaling.

Targeting primary human CLL cells through monocyte/macrophage killing

Finally, we applied the relevant molecular and functional information collected in the mouse model to human samples. We first analyzed LN sections from CLL patients by immunohistochemistry and observed that CD68+ macrophages were close to the proliferating (Ki67+) CLL cells in the proliferation center (Figure 13A). As a next step, we evaluated whether human primary leukemia cells could be affected by clodrolip. Clodrolip has no direct toxic effect on leukemia B cells in vitro, as shown by the cytotoxicity studies implemented on MEC1 cells (Figure 11A) and purified human primary CLL cells (Figure 13B). However, when unfractionated peripheral blood mononuclear cells (PBMCs) (which contain both leukemia and normal hematopoietic cells) of CLL patients were treated with different doses of clodrolip, we observed a significant reduction in both CD14+ monocytes and leukemia B cells, as early as 30 minutes after treatment (Figure 13C), and even more significant after 24 hours (Figure 13D). A large number of leukemia cell deaths induced by monocyte killing are shown in Figure 13E. Transwell experiments implemented by inoculating monocyte-depleted PBMCs from CLL patients showed that cell-cell contact does not necessarily induce CLL death (Figure 13F). We then evaluated the expression of key effector molecules involved in the cell death mechanism in human primary leukemia cells. For this purpose, we purified leukemia cells from PBMCs of 24-hour clodrolip cultures and untreated control cultures by magnetic negative selection. We observed a significant increase in FAS in all processed samples analyzed (Figure 14A, n=3). In addition to FAS, we observed an increase in TNFR1, FADD, BID, TRAIL-R2 in a patient's sample (Figure 14B), suggesting that several pathways of cell death (such as FAS/FASL, TRAIL and TNF) may also be involved in the killing of human primary CLL cells induced by monocytes/macrophages.

In order to investigate the involvement of TNF and TRAIL in the mechanism of leukemic cell death, we utilized etanercept and a blocking anti-human TRAIL-R2 monoclonal antibody (Germano G CC 2013). As shown in Figure 14C-D, in 3 of 4 samples, the leukemic cell elimination induced by clodrolip was reduced when blocking TNF (Figure S6C) and TRAIL signal conduction (Figure 14D). In order to finally test the efficacy of macrophage targeting strategy on primary cells from CLL patients, PBMC (Figure 13G) was processed with anti-human CSF1R monoclonal antibody (hMab 2F11-e7). We observed the relevant elimination of CD14+ monocytes and leukemic B cells after 48 hours. More surprisingly, leukemic cell elimination was significantly increased (Figure 13G) when anti-human CSF1R monoclonal antibody (hMab 2F11-e7) was combined with GA101.

Together, these findings indicate that macrophage killing either restores CLL sensitivity to apoptosis or directly induces their death. They further support the rationale for translating these findings into novel combination therapies.

Our data using an anti-CSF1R mAb, known to block the formation of new macrophages by inducing apoptosis or inhibiting monocyte differentiation, confirm the dependence of leukemic clones on monocytes/macrophages, particularly in the BM niche, where CSF1 normally induces monocyte differentiation and maturation {Ries, 2014 #16; MacDonald, 2010 #36}. Proof of principle that monocyte/macrophage depletion leads to antileukemic effects was obtained by clodrolip killing. Results obtained with the therapeutically relevant anti-CSF1R mAb paralleled those obtained with clodrolip. In different mouse models, macrophage targeting impaired CLL cell engraftment and, even more interestingly, was associated with striking antileukemic effects and significantly improved mouse survival.

The depletion of monocyte/macrophage populations is accompanied by apoptosis of leukemic cells. This suggests that the molecular mechanism of leukemic cell death in vivo appears to require RNA upregulation of key molecules of the TNF pathway. Macrophage targeting, through the induction of TNF signaling, sensitizes leukemic cells to apoptosis and triggers their death via a TNF-dependent mechanism. Our in vitro findings on primary human CLL cells and improved survival of xenograft mice following combination therapy confirm the strong potential of this innovative strategy.

Sequence Listing

<110> F. Hoffmann-La Roche AG

<120> Antibodies against human CSF-1R for inducing lymphocytosis in lymphoma or leukemia

<130> P32943 FT

<150> EP15173638

<151> 2015-06-24

<150> EP16151129

<151> 2016-01-13

<160> 101

<170> PatentIn version 3.5

<210> 1

<211> 8

<212> PRT

<213> Mouse (Mus musculus)

<400> 1

Asp Gln Arg Leu Tyr Phe Asp Val

1 5

<210> 2

<211> 16

<212> PRT

<213> Mouse (Mus musculus)

<400> 2

Val Ile Trp Thr Asp Gly Gly Thr Asn Tyr Asn Ser Pro Phe Met Ser

1 5 10 15

<210> 3

<211> 5

<212> PRT

<213> Mouse (Mus musculus)

<400> 3

Thr Tyr Asp Ile Ser

1 5

<210> 4

<211> 8

<212> PRT

<213> Mouse (Mus musculus)

<400> 4

Gly Gln Ser Phe Ser Tyr Pro Thr

1 5

<210> 5

<211> 7

<212> PRT

<213> Mouse (Mus musculus)

<400> 5

Gly Ala Ser Asn Arg Tyr Thr

1 5

<210> 6

<211> 11

<212> PRT

<213> Mouse (Mus musculus)

<400> 6

Lys Ala Ser Glu Asp Val Asn Thr Tyr Val Ser

1 5 10

<210> 7

<211> 116

<212> PRT

<213> Mouse (Mus musculus)

<400> 7

Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Thr Tyr

20 25 30

Asp Ile Ser Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Thr Asp Gly Gly Thr Asn Tyr Asn Ser Pro Phe Met

50 55 60

Ser Arg Leu Ser Ile Arg Lys Asp Asn Ser Lys Ser Gln Val Phe Leu

65 70 75 80

Lys Met Asn Arg Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Val

85 90 95

Arg Asp Gln Arg Leu Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val

100 105 110

Thr Val Ser Ser

115

<210> 8

<211> 106

<212> PRT

<213> Mouse (Mus musculus)

<400> 8

Asn Ile Val Met Thr Gln Ser Pro Lys Ser Met Ser Met Ser Val Gly

1 5 10 15

Glu Arg Val Thr Leu Asn Cys Lys Ala Ser Glu Asp Val Asn Thr Tyr

20 25 30

Val Ser Trp Tyr Gln Gln Gln Pro Glu Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Gly Gly Ser Thr Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala

65 70 75 80

Glu Asp Leu Ala Asp Tyr Phe Cys Gly Gln Ser Phe Ser Tyr Pro Thr

85 90 95

Phe Gly Thr Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 9

<211> 7

<212> PRT

<213> Mouse (Mus musculus)

<400> 9

Asp Pro Arg Leu Tyr Phe Asp

1 5

<210> 10

<211> 16

<212> PRT

<213> Mouse (Mus musculus)

<400> 10

Val Ile Trp Thr Gly Gly Gly Thr Asn Tyr Asn Ser Gly Phe Met Ser

1 5 10 15

<210> 11

<211> 5

<212> PRT

<213> Mouse (Mus musculus)

<400> 11

Ser Phe Asp Ile Ser

1 5

<210> 12

<211> 8

<212> PRT

<213> Mouse (Mus musculus)

<400> 12

Gly Gln Thr Phe Ser Tyr Pro Thr

1 5

<210> 13

<211> 7

<212> PRT

<213> Mouse (Mus musculus)

<400> 13

Gly Ala Ser Asn Arg Tyr Thr

1 5

<210> 14

<211> 11

<212> PRT

<213> Mouse (Mus musculus)

<400> 14

Lys Ala Ser Glu Asp Val Val Thr Tyr Val Ser

1 5 10

<210> 15

<211> 116

<212> PRT

<213> Mouse (Mus musculus)

<400> 15

Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Lys

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Ser Ser Leu Asp Ser Phe

20 25 30

Asp Ile Ser Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Thr Gly Gly Gly Thr Asn Tyr Asn Ser Gly Phe Met

50 55 60

Ser Arg Leu Arg Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Leu Leu

65 70 75 80

Lys Met Asn Ser Leu Gln Ser Asp Asp Thr Ala Ile Tyr Tyr Cys Val

85 90 95

Arg Asp Pro Arg Leu Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val

100 105 110

Thr Val Ser Ser

115

<210> 16

<211> 106

<212> PRT

<213> Mouse (Mus musculus)

<400> 16

Asn Ile Val Met Thr Gln Ser Pro Lys Ser Met Ser Met Ser Val Gly

1 5 10 15

Glu Arg Val Thr Leu Ser Cys Lys Ala Ser Glu Asp Val Val Thr Tyr

20 25 30

Val Ser Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala

65 70 75 80

Glu Asp Leu Ala Asp Tyr Tyr Cys Gly Gln Thr Phe Ser Tyr Pro Thr

85 90 95

Phe Gly Thr Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 17

<211> 8

<212> PRT

<213> Artificial

<220>

<223> Heavy chain CDR3, hMab 2F11-c11

<400> 17

Asp Gln Arg Leu Tyr Phe Asp Val

1 5

<210> 18

<211> 16

<212> PRT

<213> Artificial

<220>

<223> Heavy chain CDR2, hMab 2F11-c11

<400> 18

Val Ile Trp Thr Asp Gly Gly Thr Asn Tyr Asn Ser Pro Phe Met Ser

1 5 10 15

<210> 19

<211> 5

<212> PRT

<213> Artificial

<220>

<223> Heavy chain CDR1, hMab 2F11-c11

<400> 19

Thr Tyr Asp Ile Ser

1 5

<210> 20

<211> 8

<212> PRT

<213> Artificial

<220>

<223> Light chain CDR3, hMab 2F11-c11

<400> 20

Gly Gln Ser Phe Ser Tyr Pro Thr

1 5

<210> 21

<211> 7

<212> PRT

<213> Artificial

<220>

<223> Light chain CDR2, hMab 2F11-c11

<400> 21

Gly Ala Ser Asn Arg Tyr Thr

1 5

<210> 22

<211> 11

<212> PRT

<213> Artificial

<220>

<223> Light chain CDR1, hMab 2F11-c11

<400> 22

Arg Ala Ser Glu Asp Val Asn Thr Tyr Val Ser

1 5 10

<210> 23

<211> 116

<212> PRT

<213> Artificial

<220>

<223> Heavy chain variable domain, hMab 2F11-c11

<400> 23

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Ser Leu Thr Thr Tyr

20 25 30

Asp Ile Ser Trp Ile Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Trp Thr Asp Gly Gly Thr Asn Tyr Asn Ser Pro Phe Met

50 55 60

Ser Arg Val Thr Ile Thr Lys Asp Glu Ser Thr Ser Thr Ala Tyr Met

65 70 75 80

Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Val

85 90 95

Arg Asp Gln Arg Leu Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser

115

<210> 24

<211> 106

<212> PRT

<213> Artificial

<220>

<223> Light chain variable domain, hMab 2F11-c11

<400> 24

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asp Val Asn Thr Tyr

20 25 30

Val Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gly Gln Ser Phe Ser Tyr Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 25

<211> 8

<212> PRT

<213> Artificial

<220>

<223> Heavy chain CDR3, hMab 2F11-d8

<400> 25

Asp Gln Arg Leu Tyr Phe Asp Val

1 5

<210> 26

<211> 16

<212> PRT

<213> Artificial

<220>

<223> Heavy chain CDR2, hMab 2F11-d8

<400> 26

Val Ile Trp Thr Asp Gly Gly Ala Asn Tyr Ala Gln Lys Phe Gln Gly

1 5 10 15

<210> 27

<211> 5

<212> PRT

<213> Artificial

<220>

<223> Heavy chain CDR1, hMab 2F11-d8

<400> 27

Thr Tyr Asp Ile Ser

1 5

<210> 28

<211> 8

<212> PRT

<213> Artificial

<220>

<223> Light chain CDR3, hMab 2F11-d8

<400> 28

Gly Gln Ser Phe Ser Tyr Pro Thr

1 5

<210> 29

<211> 7

<212> PRT

<213> Artificial

<220>

<223> Light chain CDR2, hMab 2F11-d8

<400> 29

Gly Ala Ser Asn Arg Tyr Thr

1 5

<210> 30

<211> 11

<212> PRT

<213> Artificial

<220>

<223> Light chain CDR1, hMab 2F11-d8

<400> 30

Lys Ala Ser Glu Asp Val Asn Thr Tyr Val Ser

1 5 10

<210> 31

<211> 116

<212> PRT

<213> Artificial

<220>

<223> Heavy chain variable domain, hMab 2F11-d8

<400> 31

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Ser Leu Thr Thr Tyr

20 25 30

Asp Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Trp Thr Asp Gly Gly Ala Asn Tyr Ala Gln Lys Phe Gln

50 55 60

Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met

65 70 75 80

Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Gln Arg Leu Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser

115

<210> 32

<211> 106

<212> PRT

<213> Artificial

<220>

<223> Light chain variable domain, hMab 2F11-d8

<400> 32

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asp Val Asn Thr Tyr

20 25 30

Val Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gly Gln Ser Phe Ser Tyr Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 33

<211> 8

<212> PRT

<213> Artificial

<220>

<223> Heavy chain CDR3, hMab 2F11-e7

<400> 33

Asp Gln Arg Leu Tyr Phe Asp Val

1 5

<210> 34

<211> 16

<212> PRT

<213> Artificial

<220>

<223> Heavy chain CDR2, hMab 2F11-e7

<400> 34

Val Ile Trp Thr Asp Gly Gly Thr Asn Tyr Ala Gln Lys Leu Gln Gly

1 5 10 15

<210> 35

<211> 5

<212> PRT

<213> Artificial

<220>

<223> Heavy chain CDR1, hMab 2F11-e7

<400> 35

Ser Tyr Asp Ile Ser

1 5

<210> 36

<211> 8

<212> PRT

<213> Artificial

<220>

<223> Light chain CDR3, hMab 2F11-e7

<400> 36

Gln Gln Ser Phe Ser Tyr Pro Thr

1 5

<210> 37

<211> 7

<212> PRT

<213> Artificial

<220>

<223> Light chain CDR2, hMab 2F11-e7

<400> 37

Ala Ala Ser Asn Arg Tyr Thr

1 5

<210> 38

<211> 11

<212> PRT

<213> Artificial

<220>

<223> Light chain CDR1, hMab 2F11-e7

<400> 38

Arg Ala Ser Glu Asp Val Asn Thr Tyr Val Ser

1 5 10

<210> 39

<211> 116

<212> PRT

<213> Artificial

<220>

<223> Heavy chain variable domain, hMab 2F11-e7

<400> 39

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asp Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Trp Thr Asp Gly Gly Thr Asn Tyr Ala Gln Lys Leu Gln

50 55 60

Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr Met

65 70 75 80

Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Gln Arg Leu Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser

115

<210> 40

<211> 106

<212> PRT

<213> Artificial

<220>

<223> Light chain variable domain, hMab 2F11-e7

<400> 40

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asp Val Asn Thr Tyr

20 25 30

Val Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Phe Ser Tyr Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 41

<211> 8

<212> PRT

<213> Artificial

<220>

<223> Heavy chain CDR3, hMab 2F11-f12

<400> 41

Asp Gln Arg Leu Tyr Phe Asp Val

1 5

<210> 42

<211> 16

<212> PRT

<213> Artificial

<220>

<223> Heavy chain CDR2, hMab 2F11-f12

<400> 42

Val Ile Trp Thr Asp Gly Gly Thr Asn Tyr Asn Ser Pro Phe Met Ser

1 5 10 15

<210> 43

<211> 5

<212> PRT

<213> Artificial

<220>

<223> Heavy chain CDR1, hMab 2F11-f12

<400> 43

Thr Tyr Asp Ile Ser

1 5

<210> 44

<211> 8

<212> PRT

<213> Artificial

<220>

<223> Light chain CDR3, hMab 2F11-f12

<400> 44

Gly Gln Ser Phe Ser Tyr Pro Thr

1 5

<210> 45

<211> 7

<212> PRT

<213> Artificial

<220>

<223> Light chain CDR2, hMab 2F11-f12

<400> 45

Gly Ala Ser Ser Leu Gln Ser

1 5

<210> 46

<211> 11

<212> PRT

<213> Artificial

<220>

<223> Light chain CDR1, hMab 2F11-f12

<400> 46

Arg Ala Ser Glu Asp Val Asn Thr Tyr Val Ser

1 5 10

<210> 47

<211> 116

<212> PRT

<213> Artificial

<220>

<223> Heavy chain variable domain, hMab 2F11-f12

<400> 47

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Ser Leu Thr Thr Tyr

20 25 30

Asp Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Trp Thr Asp Gly Gly Thr Asn Tyr Asn Ser Pro Phe Met

50 55 60

Ser Arg Val Thr Ile Thr Lys Asp Glu Ser Thr Ser Thr Ala Tyr Met

65 70 75 80

Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Val

85 90 95

Arg Asp Gln Arg Leu Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser

115

<210> 48

<211> 106

<212> PRT

<213> Artificial

<220>

<223> Light chain variable domain, hMab 2F11-f12

<400> 48

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asp Val Asn Thr Tyr

20 25 30

Val Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gly Gln Ser Phe Ser Tyr Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 49

<211> 8

<212> PRT

<213> Artificial

<220>

<223> Heavy chain CDR3, hMab 2F11-g1

<400> 49

Asp Gln Arg Leu Tyr Phe Asp Val

1 5

<210> 50

<211> 16

<212> PRT

<213> Artificial

<220>

<223> Heavy chain CDR2, hMab 2F11-g1

<400> 50

Val Ile Trp Thr Asp Gly Gly Thr Asn Tyr Asn Ser Pro Leu Lys Ser

1 5 10 15

<210> 51

<211> 5

<212> PRT

<213> Artificial

<220>

<223> Heavy chain CDR1, hMab 2F11-g1

<400> 51

Thr Tyr Asp Ile Ser

1 5

<210> 52

<211> 8

<212> PRT

<213> Artificial

<220>

<223> Light chain CDR3, hMab 2F11-g1

<400> 52

Gly Gln Ser Phe Ser Tyr Pro Thr

1 5

<210> 53

<211> 7

<212> PRT

<213> Artificial

<220>

<223> Light chain CDR2, hMab 2F11-g1

<400> 53

Gly Ala Ser Ser Arg Ala Thr

1 5

<210> 54

<211> 11

<212> PRT

<213> Artificial

<220>

<223> Light chain CDR1, hMab 2F11-g1

<400> 54

Arg Ala Ser Glu Asp Val Asn Thr Tyr Leu Ala

1 5 10

<210> 55

<211> 116

<212> PRT

<213> Artificial

<220>

<223> Heavy chain variable domain, hMab 2F11-g1

<400> 55

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Thr Tyr

20 25 30

Asp Ile Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Thr Asp Gly Gly Thr Asn Tyr Asn Ser Pro Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Gln Arg Leu Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser

115

<210> 56

<211> 106

<212> PRT

<213> Artificial

<220>

<223> Light chain variable domain, hMab 2F11-g1

<400> 56

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Glu Asp Val Asn Thr Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gly Gln Ser Phe Ser Tyr Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 57

<211> 107

<212> PRT

<213> Homo sapiens

<400> 57

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 58

<211> 330

<212> PRT

<213> Homo sapiens

<400> 58

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 59

<211> 330

<212> PRT

<213> Artificial

<220>

<223> Human heavy chain constant region derived from IgG1 with mutations at L234A and L235A

<400> 59

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 60

<211> 327

<212> PRT

<213> Homo sapiens

<400> 60

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210> 61

<211> 327

<212> PRT

<213> Artificial

<220>

<223> Human heavy chain constant region derived from IgG4 mutated at S228P

<400> 61

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210> 62

<211> 972

<212> PRT

<213> Homo sapiens

<400> 62

Met Gly Pro Gly Val Leu Leu Leu Leu Leu Val Ala Thr Ala Trp His

1 5 10 15

Gly Gln Gly Ile Pro Val Ile Glu Pro Ser Val Pro Glu Leu Val Val

20 25 30

Lys Pro Gly Ala Thr Val Thr Leu Arg Cys Val Gly Asn Gly Ser Val

35 40 45

Glu Trp Asp Gly Pro Pro Ser Pro His Trp Thr Leu Tyr Ser Asp Gly

50 55 60

Ser Ser Ser Ile Leu Ser Thr Asn Asn Ala Thr Phe Gln Asn Thr Gly

65 70 75 80

Thr Tyr Arg Cys Thr Glu Pro Gly Asp Pro Leu Gly Gly Ser Ala Ala

85 90 95

Ile His Leu Tyr Val Lys Asp Pro Ala Arg Pro Trp Asn Val Leu Ala

100 105 110

Gln Glu Val Val Val Phe Glu Asp Gln Asp Ala Leu Leu Pro Cys Leu

115 120 125

Leu Thr Asp Pro Val Leu Glu Ala Gly Val Ser Leu Val Arg Val Arg

130 135 140

Gly Arg Pro Leu Met Arg His Thr Asn Tyr Ser Phe Ser Pro Trp His

145 150 155 160

Gly Phe Thr Ile His Arg Ala Lys Phe Ile Gln Ser Gln Asp Tyr Gln

165 170 175

Cys Ser Ala Leu Met Gly Gly Arg Lys Val Met Ser Ile Ser Ile Arg

180 185 190

Leu Lys Val Gln Lys Val Ile Pro Gly Pro Pro Ala Leu Thr Leu Val

195 200 205

Pro Ala Glu Leu Val Arg Ile Arg Gly Glu Ala Ala Gln Ile Val Cys

210 215 220

Ser Ala Ser Ser Val Asp Val Asn Phe Asp Val Phe Leu Gln His Asn

225 230 235 240

Asn Thr Lys Leu Ala Ile Pro Gln Gln Ser Asp Phe His Asn Asn Arg

245 250 255

Tyr Gln Lys Val Leu Thr Leu Asn Leu Asp Gln Val Asp Phe Gln His

260 265 270

Ala Gly Asn Tyr Ser Cys Val Ala Ser Asn Val Gln Gly Lys His Ser

275 280 285

Thr Ser Met Phe Phe Arg Val Val Glu Ser Ala Tyr Leu Asn Leu Ser

290 295 300

Ser Glu Gln Asn Leu Ile Gln Glu Val Thr Val Gly Glu Gly Leu Asn

305 310 315 320

Leu Lys Val Met Val Glu Ala Tyr Pro Gly Leu Gln Gly Phe Asn Trp

325 330 335

Thr Tyr Leu Gly Pro Phe Ser Asp His Gln Pro Glu Pro Lys Leu Ala

340 345 350

Asn Ala Thr Thr Lys Asp Thr Tyr Arg His Thr Phe Thr Leu Ser Leu

355 360 365

Pro Arg Leu Lys Pro Ser Glu Ala Gly Arg Tyr Ser Phe Leu Ala Arg

370 375 380

Asn Pro Gly Gly Trp Arg Ala Leu Thr Phe Glu Leu Thr Leu Arg Tyr

385 390 395 400

Pro Pro Glu Val Ser Val Ile Trp Thr Phe Ile Asn Gly Ser Gly Thr

405 410 415

Leu Leu Cys Ala Ala Ser Gly Tyr Pro Gln Pro Asn Val Thr Trp Leu

420 425 430

Gln Cys Ser Gly His Thr Asp Arg Cys Asp Glu Ala Gln Val Leu Gln

435 440 445

Val Trp Asp Asp Pro Tyr Pro Glu Val Leu Ser Gln Glu Pro Phe His

450 455 460

Lys Val Thr Val Gln Ser Leu Leu Thr Val Glu Thr Leu Glu His Asn

465 470 475 480

Gln Thr Tyr Glu Cys Arg Ala His Asn Ser Val Gly Ser Gly Ser Trp

485 490 495

Ala Phe Ile Pro Ile Ser Ala Gly Ala His Thr His Pro Pro Asp Glu

500 505 510

Phe Leu Phe Thr Pro Val Val Val Ala Cys Met Ser Ile Met Ala Leu

515 520 525

Leu Leu Leu Leu Leu Leu Leu Leu Leu Tyr Lys Tyr Lys Gln Lys Pro

530 535 540

Lys Tyr Gln Val Arg Trp Lys Ile Ile Glu Ser Tyr Glu Gly Asn Ser

545 550 555 560

Tyr Thr Phe Ile Asp Pro Thr Gln Leu Pro Tyr Asn Glu Lys Trp Glu

565 570 575

Phe Pro Arg Asn Asn Leu Gln Phe Gly Lys Thr Leu Gly Ala Gly Ala

580 585 590

Phe Gly Lys Val Val Glu Ala Thr Ala Phe Gly Leu Gly Lys Glu Asp

595 600 605

Ala Val Leu Lys Val Ala Val Lys Met Leu Lys Ser Thr Ala His Ala

610 615 620

Asp Glu Lys Glu Ala Leu Met Ser Glu Leu Lys Ile Met Ser His Leu

625 630 635 640

Gly Gln His Glu Asn Ile Val Asn Leu Leu Gly Ala Cys Thr His Gly

645 650 655

Gly Pro Val Leu Val Ile Thr Glu Tyr Cys Cys Tyr Gly Asp Leu Leu

660 665 670

Asn Phe Leu Arg Arg Lys Ala Glu Ala Met Leu Gly Pro Ser Leu Ser

675 680 685

Pro Gly Gln Asp Pro Glu Gly Gly Val Asp Tyr Lys Asn Ile His Leu

690 695 700

Glu Lys Lys Tyr Val Arg Arg Asp Ser Gly Phe Ser Ser Gln Gly Val

705 710 715 720

Asp Thr Tyr Val Glu Met Arg Pro Val Ser Thr Ser Ser Asn Asp Ser

725 730 735

Phe Ser Glu Gln Asp Leu Asp Lys Glu Asp Gly Arg Pro Leu Glu Leu

740 745 750

Arg Asp Leu Leu His Phe Ser Ser Gln Val Ala Gln Gly Met Ala Phe

755 760 765

Leu Ala Ser Lys Asn Cys Ile His Arg Asp Val Ala Ala Arg Asn Val

770 775 780

Leu Leu Thr Asn Gly His Val Ala Lys Ile Gly Asp Phe Gly Leu Ala

785 790 795 800

Arg Asp Ile Met Asn Asp Ser Asn Tyr Ile Val Lys Gly Asn Ala Arg

805 810 815

Leu Pro Val Lys Trp Met Ala Pro Glu Ser Ile Phe Asp Cys Val Tyr

820 825 830

Thr Val Gln Ser Asp Val Trp Ser Tyr Gly Ile Leu Leu Trp Glu Ile

835 840 845

Phe Ser Leu Gly Leu Asn Pro Tyr Pro Gly Ile Leu Val Asn Ser Lys

850 855 860

Phe Tyr Lys Leu Val Lys Asp Gly Tyr Gln Met Ala Gln Pro Ala Phe

865 870 875 880

Ala Pro Lys Asn Ile Tyr Ser Ile Met Gln Ala Cys Trp Ala Leu Glu

885 890 895

Pro Thr His Arg Pro Thr Phe Gln Gln Ile Cys Ser Phe Leu Gln Glu

900 905 910

Gln Ala Gln Glu Asp Arg Arg Glu Arg Asp Tyr Thr Asn Leu Pro Ser

915 920 925

Ser Ser Arg Ser Gly Gly Ser Gly Ser Ser Ser Ser Ser Glu Leu Glu Glu

930 935 940

Glu Ser Ser Ser Glu His Leu Thr Cys Cys Glu Gln Gly Asp Ile Ala

945 950 955 960

Gln Pro Leu Leu Gln Pro Asn Asn Tyr Gln Phe Cys

965 970

<210> 63

<211> 972

<212> PRT

<213> Artificial

<220>

<223> Mutant CSF-1R L301S Y969F

<400> 63

Met Gly Pro Gly Val Leu Leu Leu Leu Leu Val Ala Thr Ala Trp His

1 5 10 15

Gly Gln Gly Ile Pro Val Ile Glu Pro Ser Val Pro Glu Leu Val Val

20 25 30

Lys Pro Gly Ala Thr Val Thr Leu Arg Cys Val Gly Asn Gly Ser Val

35 40 45

Glu Trp Asp Gly Pro Pro Ser Pro His Trp Thr Leu Tyr Ser Asp Gly

50 55 60

Ser Ser Ser Ile Leu Ser Thr Asn Asn Ala Thr Phe Gln Asn Thr Gly

65 70 75 80

Thr Tyr Arg Cys Thr Glu Pro Gly Asp Pro Leu Gly Gly Ser Ala Ala

85 90 95

Ile His Leu Tyr Val Lys Asp Pro Ala Arg Pro Trp Asn Val Leu Ala

100 105 110

Gln Glu Val Val Val Phe Glu Asp Gln Asp Ala Leu Leu Pro Cys Leu

115 120 125

Leu Thr Asp Pro Val Leu Glu Ala Gly Val Ser Leu Val Arg Val Arg

130 135 140

Gly Arg Pro Leu Met Arg His Thr Asn Tyr Ser Phe Ser Pro Trp His

145 150 155 160

Gly Phe Thr Ile His Arg Ala Lys Phe Ile Gln Ser Gln Asp Tyr Gln

165 170 175

Cys Ser Ala Leu Met Gly Gly Arg Lys Val Met Ser Ile Ser Ile Arg

180 185 190

Leu Lys Val Gln Lys Val Ile Pro Gly Pro Pro Ala Leu Thr Leu Val

195 200 205

Pro Ala Glu Leu Val Arg Ile Arg Gly Glu Ala Ala Gln Ile Val Cys

210 215 220

Ser Ala Ser Ser Val Asp Val Asn Phe Asp Val Phe Leu Gln His Asn

225 230 235 240

Asn Thr Lys Leu Ala Ile Pro Gln Gln Ser Asp Phe His Asn Asn Arg

245 250 255

Tyr Gln Lys Val Leu Thr Leu Asn Leu Asp Gln Val Asp Phe Gln His

260 265 270

Ala Gly Asn Tyr Ser Cys Val Ala Ser Asn Val Gln Gly Lys His Ser

275 280 285

Thr Ser Met Phe Phe Arg Val Val Glu Ser Ala Tyr Ser Asn Leu Ser

290 295 300

Ser Glu Gln Asn Leu Ile Gln Glu Val Thr Val Gly Glu Gly Leu Asn

305 310 315 320

Leu Lys Val Met Val Glu Ala Tyr Pro Gly Leu Gln Gly Phe Asn Trp

325 330 335

Thr Tyr Leu Gly Pro Phe Ser Asp His Gln Pro Glu Pro Lys Leu Ala

340 345 350

Asn Ala Thr Thr Lys Asp Thr Tyr Arg His Thr Phe Thr Leu Ser Leu

355 360 365

Pro Arg Leu Lys Pro Ser Glu Ala Gly Arg Tyr Ser Phe Leu Ala Arg

370 375 380

Asn Pro Gly Gly Trp Arg Ala Leu Thr Phe Glu Leu Thr Leu Arg Tyr

385 390 395 400

Pro Pro Glu Val Ser Val Ile Trp Thr Phe Ile Asn Gly Ser Gly Thr

405 410 415

Leu Leu Cys Ala Ala Ser Gly Tyr Pro Gln Pro Asn Val Thr Trp Leu

420 425 430

Gln Cys Ser Gly His Thr Asp Arg Cys Asp Glu Ala Gln Val Leu Gln

435 440 445

Val Trp Asp Asp Pro Tyr Pro Glu Val Leu Ser Gln Glu Pro Phe His

450 455 460

Lys Val Thr Val Gln Ser Leu Leu Thr Val Glu Thr Leu Glu His Asn

465 470 475 480

Gln Thr Tyr Glu Cys Arg Ala His Asn Ser Val Gly Ser Gly Ser Trp

485 490 495

Ala Phe Ile Pro Ile Ser Ala Gly Ala His Thr His Pro Pro Asp Glu

500 505 510

Phe Leu Phe Thr Pro Val Val Val Ala Cys Met Ser Ile Met Ala Leu

515 520 525

Leu Leu Leu Leu Leu Leu Leu Leu Leu Tyr Lys Tyr Lys Gln Lys Pro

530 535 540

Lys Tyr Gln Val Arg Trp Lys Ile Ile Glu Ser Tyr Glu Gly Asn Ser

545 550 555 560

Tyr Thr Phe Ile Asp Pro Thr Gln Leu Pro Tyr Asn Glu Lys Trp Glu

565 570 575

Phe Pro Arg Asn Asn Leu Gln Phe Gly Lys Thr Leu Gly Ala Gly Ala

580 585 590

Phe Gly Lys Val Val Glu Ala Thr Ala Phe Gly Leu Gly Lys Glu Asp

595 600 605

Ala Val Leu Lys Val Ala Val Lys Met Leu Lys Ser Thr Ala His Ala

610 615 620

Asp Glu Lys Glu Ala Leu Met Ser Glu Leu Lys Ile Met Ser His Leu

625 630 635 640

Gly Gln His Glu Asn Ile Val Asn Leu Leu Gly Ala Cys Thr His Gly

645 650 655

Gly Pro Val Leu Val Ile Thr Glu Tyr Cys Cys Tyr Gly Asp Leu Leu

660 665 670

Asn Phe Leu Arg Arg Lys Ala Glu Ala Met Leu Gly Pro Ser Leu Ser

675 680 685

Pro Gly Gln Asp Pro Glu Gly Gly Val Asp Tyr Lys Asn Ile His Leu

690 695 700

Glu Lys Lys Tyr Val Arg Arg Asp Ser Gly Phe Ser Ser Gln Gly Val

705 710 715 720

Asp Thr Tyr Val Glu Met Arg Pro Val Ser Thr Ser Ser Asn Asp Ser

725 730 735

Phe Ser Glu Gln Asp Leu Asp Lys Glu Asp Gly Arg Pro Leu Glu Leu

740 745 750

Arg Asp Leu Leu His Phe Ser Ser Gln Val Ala Gln Gly Met Ala Phe

755 760 765

Leu Ala Ser Lys Asn Cys Ile His Arg Asp Val Ala Ala Arg Asn Val

770 775 780

Leu Leu Thr Asn Gly His Val Ala Lys Ile Gly Asp Phe Gly Leu Ala

785 790 795 800

Arg Asp Ile Met Asn Asp Ser Asn Tyr Ile Val Lys Gly Asn Ala Arg

805 810 815

Leu Pro Val Lys Trp Met Ala Pro Glu Ser Ile Phe Asp Cys Val Tyr

820 825 830

Thr Val Gln Ser Asp Val Trp Ser Tyr Gly Ile Leu Leu Trp Glu Ile

835 840 845

Phe Ser Leu Gly Leu Asn Pro Tyr Pro Gly Ile Leu Val Asn Ser Lys

850 855 860

Phe Tyr Lys Leu Val Lys Asp Gly Tyr Gln Met Ala Gln Pro Ala Phe

865 870 875 880

Ala Pro Lys Asn Ile Tyr Ser Ile Met Gln Ala Cys Trp Ala Leu Glu

885 890 895

Pro Thr His Arg Pro Thr Phe Gln Gln Ile Cys Ser Phe Leu Gln Glu

900 905 910

Gln Ala Gln Glu Asp Arg Arg Glu Arg Asp Tyr Thr Asn Leu Pro Ser

915 920 925

Ser Ser Arg Ser Gly Gly Ser Gly Ser Ser Ser Ser Ser Glu Leu Glu Glu

930 935 940

Glu Ser Ser Ser Glu His Leu Thr Cys Cys Glu Gln Gly Asp Ile Ala

945 950 955 960

Gln Pro Leu Leu Gln Pro Asn Asn Phe Gln Phe Cys

965 970

<210> 64

<211> 493

<212> PRT

<213> Artificial

<220>

<223> Human CSF-1R extracellular domain

<400> 64

Ile Pro Val Ile Glu Pro Ser Val Pro Glu Leu Val Val Lys Pro Gly

1 5 10 15

Ala Thr Val Thr Leu Arg Cys Val Gly Asn Gly Ser Val Glu Trp Asp

20 25 30

Gly Pro Pro Ser Pro His Trp Thr Leu Tyr Ser Asp Gly Ser Ser Ser

35 40 45

Ile Leu Ser Thr Asn Asn Ala Thr Phe Gln Asn Thr Gly Thr Tyr Arg

50 55 60

Cys Thr Glu Pro Gly Asp Pro Leu Gly Gly Ser Ala Ala Ile His Leu

65 70 75 80

Tyr Val Lys Asp Pro Ala Arg Pro Trp Asn Val Leu Ala Gln Glu Val

85 90 95

Val Val Phe Glu Asp Gln Asp Ala Leu Leu Pro Cys Leu Leu Thr Asp

100 105 110

Pro Val Leu Glu Ala Gly Val Ser Leu Val Arg Val Arg Gly Arg Pro

115 120 125

Leu Met Arg His Thr Asn Tyr Ser Phe Ser Pro Trp His Gly Phe Thr

130 135 140

Ile His Arg Ala Lys Phe Ile Gln Ser Gln Asp Tyr Gln Cys Ser Ala

145 150 155 160

Leu Met Gly Gly Arg Lys Val Met Ser Ile Ser Ile Arg Leu Lys Val

165 170 175

Gln Lys Val Ile Pro Gly Pro Pro Ala Leu Thr Leu Val Pro Ala Glu

180 185 190

Leu Val Arg Ile Arg Gly Glu Ala Ala Gln Ile Val Cys Ser Ala Ser

195 200 205

Ser Val Asp Val Asn Phe Asp Val Phe Leu Gln His Asn Asn Thr Lys

210 215 220

Leu Ala Ile Pro Gln Gln Ser Asp Phe His Asn Asn Arg Tyr Gln Lys

225 230 235 240

Val Leu Thr Leu Asn Leu Asp Gln Val Asp Phe Gln His Ala Gly Asn

245 250 255

Tyr Ser Cys Val Ala Ser Asn Val Gln Gly Lys His Ser Thr Ser Met

260 265 270

Phe Phe Arg Val Val Glu Ser Ala Tyr Leu Asn Leu Ser Ser Glu Gln

275 280 285

Asn Leu Ile Gln Glu Val Thr Val Gly Glu Gly Leu Asn Leu Lys Val

290 295 300

Met Val Glu Ala Tyr Pro Gly Leu Gln Gly Phe Asn Trp Thr Tyr Leu

305 310 315 320

Gly Pro Phe Ser Asp His Gln Pro Glu Pro Lys Leu Ala Asn Ala Thr

325 330 335

Thr Lys Asp Thr Tyr Arg His Thr Phe Thr Leu Ser Leu Pro Arg Leu

340 345 350

Lys Pro Ser Glu Ala Gly Arg Tyr Ser Phe Leu Ala Arg Asn Pro Gly

355 360 365

Gly Trp Arg Ala Leu Thr Phe Glu Leu Thr Leu Arg Tyr Pro Pro Glu

370 375 380

Val Ser Val Ile Trp Thr Phe Ile Asn Gly Ser Gly Thr Leu Leu Cys

385 390 395 400

Ala Ala Ser Gly Tyr Pro Gln Pro Asn Val Thr Trp Leu Gln Cys Ser

405 410 415

Gly His Thr Asp Arg Cys Asp Glu Ala Gln Val Leu Gln Val Trp Asp

420 425 430

Asp Pro Tyr Pro Glu Val Leu Ser Gln Glu Pro Phe His Lys Val Thr

435 440 445

Val Gln Ser Leu Leu Thr Val Glu Thr Leu Glu His Asn Gln Thr Tyr

450 455 460

Glu Cys Arg Ala His Asn Ser Val Gly Ser Gly Ser Trp Ala Phe Ile

465 470 475 480

Pro Ile Ser Ala Gly Ala His Thr His Pro Pro Asp Glu

485 490

<210> 65

<211> 388

<212> PRT

<213> Artificial

<220>

<223> Human CSF-1R fragment delD4

<400> 65

Ile Pro Val Ile Glu Pro Ser Val Pro Glu Leu Val Val Lys Pro Gly

1 5 10 15

Ala Thr Val Thr Leu Arg Cys Val Gly Asn Gly Ser Val Glu Trp Asp

20 25 30

Gly Pro Pro Ser Pro His Trp Thr Leu Tyr Ser Asp Gly Ser Ser Ser

35 40 45

Ile Leu Ser Thr Asn Asn Ala Thr Phe Gln Asn Thr Gly Thr Tyr Arg

50 55 60

Cys Thr Glu Pro Gly Asp Pro Leu Gly Gly Ser Ala Ala Ile His Leu

65 70 75 80

Tyr Val Lys Asp Pro Ala Arg Pro Trp Asn Val Leu Ala Gln Glu Val

85 90 95

Val Val Phe Glu Asp Gln Asp Ala Leu Leu Pro Cys Leu Leu Thr Asp

100 105 110

Pro Val Leu Glu Ala Gly Val Ser Leu Val Arg Val Arg Gly Arg Pro

115 120 125

Leu Met Arg His Thr Asn Tyr Ser Phe Ser Pro Trp His Gly Phe Thr

130 135 140

Ile His Arg Ala Lys Phe Ile Gln Ser Gln Asp Tyr Gln Cys Ser Ala

145 150 155 160

Leu Met Gly Gly Arg Lys Val Met Ser Ile Ser Ile Arg Leu Lys Val

165 170 175

Gln Lys Val Ile Pro Gly Pro Pro Ala Leu Thr Leu Val Pro Ala Glu

180 185 190

Leu Val Arg Ile Arg Gly Glu Ala Ala Gln Ile Val Cys Ser Ala Ser

195 200 205

Ser Val Asp Val Asn Phe Asp Val Phe Leu Gln His Asn Asn Thr Lys

210 215 220

Leu Ala Ile Pro Gln Gln Ser Asp Phe His Asn Asn Arg Tyr Gln Lys

225 230 235 240

Val Leu Thr Leu Asn Leu Asp Gln Val Asp Phe Gln His Ala Gly Asn

245 250 255

Tyr Ser Cys Val Ala Ser Asn Val Gln Gly Lys His Ser Thr Ser Met

260 265 270

Phe Phe Arg Tyr Pro Pro Glu Val Ser Val Ile Trp Thr Phe Ile Asn

275 280 285

Gly Ser Gly Thr Leu Leu Cys Ala Ala Ser Gly Tyr Pro Gln Pro Asn

290 295 300

Val Thr Trp Leu Gln Cys Ser Gly His Thr Asp Arg Cys Asp Glu Ala

305 310 315 320

Gln Val Leu Gln Val Trp Asp Asp Pro Tyr Pro Glu Val Leu Ser Gln

325 330 335

Glu Pro Phe His Lys Val Thr Val Gln Ser Leu Leu Thr Val Glu Thr

340 345 350

Leu Glu His Asn Gln Thr Tyr Glu Cys Arg Ala His Asn Ser Val Gly

355 360 365

Ser Gly Ser Trp Ala Phe Ile Pro Ile Ser Ala Gly Ala His Thr His

370 375 380

Pro Pro Asp Glu

385

<210> 66

<211> 292

<212> PRT

<213> Artificial

<220>

<223> Human CSF-1R fragment D1-D3

<400> 66

Ile Pro Val Ile Glu Pro Ser Val Pro Glu Leu Val Val Lys Pro Gly

1 5 10 15

Ala Thr Val Thr Leu Arg Cys Val Gly Asn Gly Ser Val Glu Trp Asp

20 25 30

Gly Pro Pro Ser Pro His Trp Thr Leu Tyr Ser Asp Gly Ser Ser Ser

35 40 45

Ile Leu Ser Thr Asn Asn Ala Thr Phe Gln Asn Thr Gly Thr Tyr Arg

50 55 60

Cys Thr Glu Pro Gly Asp Pro Leu Gly Gly Ser Ala Ala Ile His Leu

65 70 75 80

Tyr Val Lys Asp Pro Ala Arg Pro Trp Asn Val Leu Ala Gln Glu Val

85 90 95

Val Val Phe Glu Asp Gln Asp Ala Leu Leu Pro Cys Leu Leu Thr Asp

100 105 110

Pro Val Leu Glu Ala Gly Val Ser Leu Val Arg Val Arg Gly Arg Pro

115 120 125

Leu Met Arg His Thr Asn Tyr Ser Phe Ser Pro Trp His Gly Phe Thr

130 135 140

Ile His Arg Ala Lys Phe Ile Gln Ser Gln Asp Tyr Gln Cys Ser Ala

145 150 155 160

Leu Met Gly Gly Arg Lys Val Met Ser Ile Ser Ile Arg Leu Lys Val

165 170 175

Gln Lys Val Ile Pro Gly Pro Pro Ala Leu Thr Leu Val Pro Ala Glu

180 185 190

Leu Val Arg Ile Arg Gly Glu Ala Ala Gln Ile Val Cys Ser Ala Ser

195 200 205

Ser Val Asp Val Asn Phe Asp Val Phe Leu Gln His Asn Asn Thr Lys

210 215 220

Leu Ala Ile Pro Gln Gln Ser Asp Phe His Asn Asn Arg Tyr Gln Lys

225 230 235 240

Val Leu Thr Leu Asn Leu Asp Gln Val Asp Phe Gln His Ala Gly Asn

245 250 255

Tyr Ser Cys Val Ala Ser Asn Val Gln Gly Lys His Ser Thr Ser Met

260 265 270

Phe Phe Arg Val Val Glu Ser Ala Tyr Leu Asn Leu Ser Ser Glu Gln

275 280 285

Asn Leu Ile Gln

290

<210> 67

<211> 21

<212> PRT

<213> Artificial

<220>

<223> Signal peptide

<400> 67

Met Gly Ser Gly Pro Gly Val Leu Leu Leu Leu Leu Val Ala Thr Ala

1 5 10 15

Trp His Gly Gln Gly

20

<210> 68

<211> 36

<212> DNA

<213> Artificial

<220>

<223> Primer

<400> 68

cacctccatg ttcttccggt accccccaga ggtaag 36

<210> 69

<211> 119

<212> PRT

<213> Artificial

<220>

<223> Amino acid sequence of the heavy chain variable region (VH) of the humanized B-Ly1 antibody (B-HH2)

<400> 69

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 70

<211> 119

<212> PRT

<213> Artificial

<220>

<223> Amino acid sequence of the heavy chain variable region (VH) of the humanized B-Ly1 antibody (B-HH3)

<400> 70

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Leu Cys

85 90 95

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 71

<211> 119

<212> PRT

<213> Artificial

<220>

<223> Amino acid sequence of the heavy chain variable region (VH) of the humanized B-Ly1 antibody (B-HH6)

<400> 71

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser

20 25 30

Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 72

<211> 119

<212> PRT

<213> Artificial

<220>

<223> Amino acid sequence of the heavy chain variable region (VH) of the humanized B-Ly1 antibody (B-HH8)

<400> 72

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Tyr Ser

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 73

<211> 119

<212> PRT

<213> Artificial

<220>

<223> Amino acid sequence of the heavy chain variable region (VH) of the humanized B-Ly1 antibody (B-HL8)

<400> 73

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Ser

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 74

<211> 119

<212> PRT

<213> Artificial

<220>

<223> Amino acid sequence of the heavy chain variable region (VH) of the humanized B-Ly1 antibody (B-HL11)

<400> 74

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Ser

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 75

<211> 119

<212> PRT

<213> Artificial

<220>

<223> Amino acid sequence of the heavy chain variable region (VH) of the humanized B-Ly1 antibody (B-HL13)

<400> 75

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Ser

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 76

<211> 115

<212> PRT

<213> Artificial

<220>

<223> Amino acid sequence of the light chain variable region (VL) of the humanized B-Ly1 antibody B-KV1

<400> 76

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser

20 25 30

Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Val Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn

85 90 95

Leu Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val

115

<210> 77

<211> 297

<212> PRT

<213> Homo sapiens

<400> 77

Met Thr Thr Pro Arg Asn Ser Val Asn Gly Thr Phe Pro Ala Glu Pro

1 5 10 15

Met Lys Gly Pro Ile Ala Met Gln Ser Gly Pro Lys Pro Leu Phe Arg

20 25 30

Arg Met Ser Ser Leu Val Gly Pro Thr Gln Ser Phe Phe Met Arg Glu

35 40 45

Ser Lys Thr Leu Gly Ala Val Gln Ile Met Asn Gly Leu Phe His Ile

50 55 60

Ala Leu Gly Gly Leu Leu Met Ile Pro Ala Gly Ile Tyr Ala Pro Ile

65 70 75 80

Cys Val Thr Val Trp Tyr Pro Leu Trp Gly Gly Ile Met Tyr Ile Ile

85 90 95

Ser Gly Ser Leu Leu Ala Ala Thr Glu Lys Asn Ser Arg Lys Cys Leu

100 105 110

Val Lys Gly Lys Met Ile Met Asn Ser Leu Ser Leu Phe Ala Ala Ile

115 120 125

Ser Gly Met Ile Leu Ser Ile Met Asp Ile Leu Asn Ile Lys Ile Ser

130 135 140

His Phe Leu Lys Met Glu Ser Leu Asn Phe Ile Arg Ala His Thr Pro

145 150 155 160

Tyr Ile Asn Ile Tyr Asn Cys Glu Pro Ala Asn Pro Ser Glu Lys Asn

165 170 175

Ser Pro Ser Thr Gln Tyr Cys Tyr Ser Ile Gln Ser Leu Phe Leu Gly

180 185 190

Ile Leu Ser Val Met Leu Ile Phe Ala Phe Phe Gln Glu Leu Val Ile

195 200 205

Ala Gly Ile Val Glu Asn Glu Trp Lys Arg Thr Cys Ser Arg Pro Lys

210 215 220

Ser Asn Ile Val Leu Leu Ser Ala Glu Glu Lys Lys Glu Gln Thr Ile

225 230 235 240

Glu Ile Lys Glu Glu Val Val Gly Leu Thr Glu Thr Ser Ser Gln Pro

245 250 255

Lys Asn Glu Glu Asp Ile Glu Ile Ile Pro Ile Gln Glu Glu Glu Glu

260 265 270

Glu Glu Thr Glu Thr Asn Phe Pro Glu Pro Pro Gln Asp Gln Glu Ser

275 280 285

Ser Pro Ile Glu Asn Asp Ser Ser Pro

290 295

<210> 78

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> <PD-L1> 243.55 variable heavy chain domain VH of variant 1

<400> 78

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ala

115

<210> 79

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> <PD-L1> 243.55 variable heavy chain domain VH of variant 2

<400> 79

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ala

115

<210> 80

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> <PD-L1> 243.55 variable heavy chain domain VH of variant 3

<400> 80

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Ser

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Trp Ile Leu Pro Tyr Gly Gly Ser Ser Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ala

115

<210> 81

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> <PD-L1> 243.55 variable light chain domain VL of variant 1

<400> 81

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 82

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> <PD-L1> 243.55 variable light chain domain VL of variant 2

<400> 82

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asn Val Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 83

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> <PD-L1> 243.55 variable light chain domain VL of variant 3

<400> 83

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ala Pro Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 84

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> <PD-L1> 243.55 variable light chain domain VL of variant 4

<400> 84

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Thr Val Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 85

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> <PD-L1> 243.55 variable light chain domain VL of variant 5

<400> 85

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Val Ile Asn Thr Phe

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Thr Val Pro Arg

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 86

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> <PD-L1> 243.55 variable light chain domain VL of variant 6

<400> 86

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Gly Val Pro Arg

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 87

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> <PD-L1> 243.55 variable light chain domain VL of variant 7

<400> 87

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Phe Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 88

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> <PD-L1> 243.55 variable light chain domain VL of variant 8

<400> 88

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Phe Ile Thr Pro Thr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 89

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> <PD-L1> 243.55 variable light chain domain VL of variant 9

<400> 89

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Tyr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 90

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> <PD-L1> 243.55 variable light chain domain VL of variant 10

<400> 90

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Phe Tyr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 91

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> <PD-L1> 243.55 variable light chain domain VL of variant 11

<400> 91

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Leu Phe Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 92

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> <PD-L1> 243.55 variable light chain domain VL of variant 12

<400> 92

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Leu Tyr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 93

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> <PD-L1> 243.55 variable light chain domain VL of variant 13

<400> 93

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Trp Tyr His Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 94

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> <PD-L1> 243.55 variable light chain domain VL of variant 14

<400> 94

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Phe Tyr Ile Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 95

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> <PD-L1> 243.55 variable light chain domain VL of variant 15

<400> 95

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Tyr Thr Pro Thr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 96

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> <PD-L1> 243.55 variable light chain domain VL of variant 16

<400> 96

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Phe Ile Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 97

<211> 290

<212> PRT

<213> Homo sapiens

<400> 97

Met Arg Ile Phe Ala Val Phe Ile Phe Met Thr Tyr Trp His Leu Leu

1 5 10 15

Asn Ala Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr

20 25 30

Gly Ser Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu

35 40 45

Asp Leu Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile

50 55 60

Ile Gln Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser

65 70 75 80

Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn

85 90 95

Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr

100 105 110

Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val

115 120 125

Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val

130 135 140

Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr

145 150 155 160

Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser

165 170 175

Gly Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn

180 185 190

Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr

195 200 205

Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu

210 215 220

Val Ile Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Thr His

225 230 235 240

Leu Val Ile Leu Gly Ala Ile Leu Leu Cys Leu Gly Val Ala Leu Thr

245 250 255

Phe Ile Phe Arg Leu Arg Lys Gly Arg Met Met Asp Val Lys Lys Cys

260 265 270

Gly Ile Gln Asp Thr Asn Ser Lys Lys Gln Ser Asp Thr His Leu Glu

275 280 285

Glu Thr

290

<210> 98

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> <PD-1> nivolumab variable heavy chain domain VH

<400> 98

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

100 105 110

Ser

<210> 99

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> <PD-1> nivolumab variable light chain domain VL

<400> 99

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 100

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> <PD-1> pembrolizumab variable heavy chain domain VH

<400> 100

Gln Val Gln Leu Val Gln Ser Gly Val Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr

65 70 75 80

Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 101

<211> 111

<212> PRT

<213> Artificial sequence

<220>

<223> <PD-1> pembrolizumab variable light chain domain VL

<400> 101

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser

20 25 30

Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

Arg Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg

85 90 95

Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Claims (37)

1. The use of an antibody binding to human CSF-1R and an antibody binding to human CD20 to prepare a drug for inducing lymphocytosis in leukemia cells in lymphoma or leukemia, wherein the antibody binding to human CSF-1R comprises the heavy chain variable domain VH of SEQ ID NO:39 and the light chain variable domain VL of SEQ ID NO:40; and wherein the antibody binding to human CD20 comprises the heavy chain variable domain VH of SEQ ID NO:71 and the light chain variable domain VL of SEQ ID NO:76. 2. The use according to claim 1, wherein the antibody binding human CD20 is a fucosylated antibody of the IgG1 isotype having a modified glycosylation pattern in the Fc region, wherein the amount of the fucose-containing oligosaccharide is between 40% and 60% of the total oligosaccharide at Asn297. 3. The use according to claim 1 or 2, wherein the lymphocyte increase increases the percentage of circulating leukemia cells expressing CD20 in peripheral blood. 4. The use according to claim 1 or 2, wherein the lymphocyte increase increases the percentage of circulating leukemia cells expressing CD20 in peripheral blood and makes the lymphoma or leukemia susceptible to treatment with anti-CD20 antibodies. 5. The use according to claim 1 or 2, wherein the lymphocyte increase enhances the expression of CD20 in circulating leukemia cells. 6. The use according to claim 1 or 2, wherein the lymphocyte increase increases the percentage of circulating leukemia cells expressing CD20 and makes the lymphoma or leukemia susceptible to treatment with anti-CD20 antibodies. 7. The use according to claim 1 or 2, wherein the lymphoma or leukemia expresses CD20. 8. Preparation of antibodies conjugated to human CSF-1R and human CD20 for use in the following pharmaceutical applications: i) Used to treat cancers expressing CD20; or ii) Used to stimulate immune response or function; or iii) Used to stimulate cell-mediated immune responses; or iv) Used to delay the progression of cancers expressing CD20; or v) Used to prolong the survival of patients with cancers that express CD20. The antibody that binds to human CSF-1R is characterized by containing The heavy chain variable domain VH of SEQ ID NO:39 and the light chain variable domain VL of SEQ ID NO:40; Furthermore, the antibody that binds to human CD20 is characterized by containing The heavy chain variable domain VH of SEQ ID NO:71 and the light chain variable domain VL of SEQ ID NO:76. 9. The use according to claim 8, The antibody that binds to human CD20 is an IgG1 isotype without fucoidanization, which has a modified glycosylation pattern in the Fc region, wherein the amount of fucose-containing oligosaccharides is between 40% and 60% of the total oligosaccharides at Asn297. 10. The use according to claim 8 or 9, wherein stimulating an immune response or function is stimulating T cell activity. 11. The use according to claim 8 or 9, wherein stimulating cell-mediated immune responses is stimulating cytotoxic T-lymphocytes, stimulating T-cell activity, or stimulating macrophage activity. 12. The use according to claim 8 or 9, wherein the drug is used to treat lymphoma and lymphocytic leukemia. 13. The use according to claim 8 or 9, wherein the drug is used to treat B-cell non-Hodgkin's lymphoma. 14. The use according to claim 8 or 9, wherein the drug is used to treat multiple myeloma, follicular lymphoma, or Hodgkin's disease. 15. The use according to claim 8 or 9, wherein the drug is used to treat immune-related diseases or to delay their progression. 16. The use according to claim 15, wherein the immune-related disease is tumor immunity. 17. The use according to claim 8 or 9, wherein the drug is used for the prevention or treatment of metastasis. 18. The use according to claim 8 or 9, wherein the drug is used to treat inflammatory diseases. 19. Use of an antibody binding to human CSF-1R and an antibody binding to human CD20 for the preparation of a medicament for use in a method comprising combining an antibody binding to human CSF-1R with an antibody binding to human CD20 for preventing escape from CD20-targeted therapy via targeting macrophages, and a method of targeting CD20-expressing cancers with an antibody binding to human CD20, wherein the antibody binding to human CSF-1R comprises the heavy chain variable domain VH of SEQ ID NO:39 and the light chain variable domain VL of SEQ ID NO:40; and wherein the antibody binding to human CD20 comprises the heavy chain variable domain VH of SEQ ID NO:71 and the light chain variable domain VL of SEQ ID NO:76. 20. The use according to claim 19, wherein the antibody binding human CD20 is a fucosylated antibody of the IgG1 isotype having a modified glycosylation pattern in the Fc region, wherein the amount of the oligosaccharide containing fucose is between 40% and 60% of the total oligosaccharide at Asn297. 21. The use according to claim 19 or 20, wherein the antibody binding to human CSF-1R is used to target macrophages. 22. The use according to any one of claims 1, 2, 8, 9, 19 and 20, wherein the antibody binding human CSF-R and the antibody binding human CD20 are human IgG1 subclasses. 23. The use according to any one of claims 1, 2, 8, 9, 19 and 20, wherein no additional chemotherapeutic agents and/or targeted therapies are administered other than the antibody binding human CSF-R and the antibody binding human CD20. 24. The use according to any one of claims 1, 2, 8, 9, 19 and 20, wherein the antibody binding human CSF-1R and the antibody binding human CD20 are administered simultaneously. 25. The use according to any one of claims 1, 2, 8, 9, 19 and 20, wherein the antibody binding human CSF-1R and the antibody binding human CD20 are administered sequentially. 26. The use according to any one of claims 1, 2, 8, 9, 19 and 20, wherein an anti-PD-L1 antibody is administered in addition to the antibody binding human CSF-1R and the antibody binding human CD20. 27. The use according to claim 26, wherein the PD-L1-binding antibody comprises a variable domain amino acid sequence selected from the group consisting of: -The variable heavy chain domain VH of SEQ ID NO:78 and the variable light chain domain VL of SEQ ID NO:81; -The variable heavy chain domain VH of SEQ ID NO:79 and the variable light chain domain VL of SEQ ID NO:82; -The variable heavy chain domain VH of SEQ ID NO:79 and the variable light chain domain VL of SEQ ID NO:83; -The variable heavy chain domain VH of SEQ ID NO:79 and the variable light chain domain VL of SEQ ID NO:84; -The variable heavy chain domain VH of SEQ ID NO:79 and the variable light chain domain VL of SEQ ID NO:85; -The variable heavy chain domain VH of SEQ ID NO:79 and the variable light chain domain VL of SEQ ID NO:86; -The variable heavy chain domain VH of SEQ ID NO:79 and the variable light chain domain VL of SEQ ID NO:87; -The variable heavy chain domain VH of SEQ ID NO:79 and the variable light chain domain VL of SEQ ID NO:88; -The variable heavy chain domain VH of SEQ ID NO:79 and the variable light chain domain VL of SEQ ID NO:89; -The variable heavy chain domain VH of SEQ ID NO:79 and the variable light chain domain VL of SEQ ID NO:90; -The variable heavy chain domain VH of SEQ ID NO:79 and the variable light chain domain VL of SEQ ID NO:91; -The variable heavy chain domain VH of SEQ ID NO:79 and the variable light chain domain VL of SEQ ID NO:92; -The variable heavy chain domain VH of SEQ ID NO:79 and the variable light chain domain VL of SEQ ID NO:93; -The variable heavy chain domain VH of SEQ ID NO:79 and the variable light chain domain VL of SEQ ID NO:94; -The variable heavy chain domain VH of SEQ ID NO:79, and the variable light chain domain VL of SEQ ID NO:95; and -The variable heavy chain domain VH of SEQ ID NO:80 and the variable light chain domain VL of SEQ ID NO:96. 28. The use according to claim 26, wherein the antibody binding PD-L1 is atezolizumab. 29. The use according to claim 26, wherein the antibody binding human CSF-1R, the antibody binding human CD20, and the antibody binding human PD-L1 are simultaneously co-administered. 30. The use according to claim 26, wherein the antibody binding human CSF-1R, the antibody binding human CD20, and the antibody binding human PD-L1 are administered sequentially. 31. The use according to any one of claims 1, 2, 8, 9, 19 and 20, wherein an anti-PD-1 antibody is administered in addition to the antibody binding human CSF-1R and the antibody binding human CD20. 32. The use according to claim 31, wherein the antibody binding human CSF-1R, the antibody binding human CD20, and the antibody binding human PD-1 are administered simultaneously. 33. The use according to claim 31, wherein the antibody binding human CSF-1R, the antibody binding human CD20, and the antibody binding human PD-1 are administered sequentially. 34. A formulation comprising an antibody binding to human CSF-1R and an antibody binding to human CD20, wherein the antibody binding to human CSF-1R comprises the heavy chain variable domain VH of SEQ ID NO:39 and the light chain variable domain VL of SEQ ID NO:40; and wherein the antibody binding to human CD20 comprises the heavy chain variable domain VH of SEQ ID NO:71 and the light chain variable domain VL of SEQ ID NO:76. 35. The formulation according to claim 34, The antibody that binds to human CD20 is an IgG1 isotype without fucoidanization, which has a modified glycosylation pattern in the Fc region, wherein the amount of fucose-containing oligosaccharides is between 40% and 60% of the total oligosaccharides at Asn297. 36. An article comprising: a pharmaceutical composition comprising an antibody binding to human CSF-1R and a pharmaceutical composition comprising an antibody binding to human CD20, wherein the antibody binding to human CSF-1R comprises the heavy chain variable domain VH of SEQ ID NO:39 and the light chain variable domain VL of SEQ ID NO:40; and wherein the antibody binding to human CD20 comprises the heavy chain variable domain VH of SEQ ID NO:71 and the light chain variable domain VL of SEQ ID NO:76. 37. The article of manufacture according to claim 36, The antibody that binds to human CD20 is an IgG1 isotype without fucoidanization, which has a modified glycosylation pattern in the Fc region, wherein the amount of fucose-containing oligosaccharides is between 40% and 60% of the total oligosaccharides at Asn297.