antibodies binding to human csf-1r, pharmaceutical composition and uses of the antibody

专利摘要:
antibodies binding to human csf-1r, pharmaceutical composition and uses of the antibody the present invention relates to antibodies against human csf-1r (anti-csf-1r antibodies), methods for producing these, pharmaceutical compositions containing said antibodies , and their uses.
公开号:BR112012013717B1
申请号:R112012013717
申请日:2010-12-07
公开日:2020-01-28
发明作者:Fidler Alexander；Ries Carola；Fertig Georg；Kaluza Klaus；Pickl Marlene；Lanzendoerfer Martin (Falecido)；Dimoudis Nikolaos；Seeber Stefan
申请人:Hoffmann La Roche；
IPC主号:

专利说明:

“HUMAN CSF-1R BINDING ANTIBODIES, PHARMACEUTICAL COMPOSITION AND USES OF THE ANTIBODY” [001] The present invention relates to antibodies against human CSF-1R (anti-CSF-1 R antibodies), methods for producing these antibodies, pharmaceutical compositions containing said antibodies, and their uses.
Background of the Invention [002] The human CSF-1 receptor (CSF-1R; type 1 colony stimulating factor receptor; synonyms: M-CSF receptor; type 1 macrophage colony stimulating factor 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 encoded by the protooncogene c-fms (reviewed, for example, in Roth, P., and Stanley, E.R., Curr. Top.Microbiol. Immunol. 181 (1992) 141-67).
[003] CSF-1R is the receptor for CSF-1 (colony stimulating factor 1, also called M-CSF, macrophage colony stimulating factor) and mediates the biological effects of this cytokine (Sherr, CJ, et al. , Cell 41 (1985) 665-676). The cloning of the type 1 colony stimulating factor receptor (CSF-1R) (also called c-fms) was first described in Roussel, M.F., et al, Nature 325 (1987) 549-552. In that publication, it was shown that CSF-1R had a potential for transformation dependent on changes in the C-terminal tail of the protein, including the loss of inhibitory tyrosine 969 phosphorylation that binds Cbl and thus regulates down regulation of the recipient (Lee, PS, et al., EMBO J. 18 (1999) 3616-3628). Recently, a second ligand for CSF-1R called interleukin-34 (IL-34) has been identified (Lin, H., et al, Science 320 (2008) 807-811).
[004] The CSF-1 cytokine (type 1 colony stimulating factor,
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2/91 also called M-CSF, macrophage) is found extracellularly as a disulfide-bound homodimer (Stanley, E.R. et al, Journal of Cellular Biochemistry 21 (1983) 151-159 ;. Stanley, E.R. et al., Stem Cells
Suppl.12. 1 (1995) 15-24).
[005] The main biological effects of CSF-1R signaling are the differentiation, proliferation, migration and survival of hematopoietic precursor cells to the macrophage lineage (including osteoclasts). Activation of CSF-1R is mediated by its ligands, CSF-1 (M-CSF) and IL-34. Binding of CSF-1 (M-CSF) to CSF-1R induces homodimer formation and kinase activation by tyrosine phosphorylation (Li, W. et al, EMBO Journal.10 (1991) 277-288; Stanley, ER, et al., Mol.Reprod. Dev. 46 (1997) 4-10).
[006] The biologically active CSF-1 homodimer binds to CSF1R within subdomains D1 to D3 of the extracellular domain of the CSF-1 receptor (CSF-1R-ECD). CSF-1R-ECD comprises five immunoglobulin-like subdomains (immunoglobulin-like) (designated D1 to D5). The 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). The 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).
[007] Additional signaling is mediated by the p85 subunit of PI3K and Grb2 binding to the PI3K / AKT and Ras / MAPK pathways, respectively. These two important signaling pathways can regulate proliferation, survival and apoptosis. Other signaling molecules that bind to the CSF-1R phosphorylated intracellular domain include STAT1, STAT3, PLCy and Cbl (Bourette, R.P. and Rohrschneider, L.R., Growth Factor 17 (2000) 155-166).
[008] CSF-1R signaling has a physiological role in immune responses, bone remodeling and the reproductive system. Was
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3/91 demonstrated that knockout animals for CSF-1 (Pollard, JW, Mol.Reprod. Dev. 46 (1997) 54-6 1) or CSF-1R (Dai, XM, et al., Blood 99 (2002) 111120 ) have osteopetrotic, hematopoietic, macrophage and reproductive phenotypes consistent with a role for CSF-1R in the respective cell types.
[009] Sherr, CJ, et al., Blood 73 (1989) 1786-1793 refer to some antibodies against CSF-1R that inhibit CSF-1 activity (see Sherr, CJ et al., Blood 73 (1989) 1786-1793). Ashmun, R.A., et al., Blood 73 (1989) 827-837 refer to antibodies to CSF-1R. Lenda, D., et al., Journal of Immunology 170 (2003) 3254-3262 describe that the reduction in the recruitment, proliferation and activation of macrophages in CSF-1 deficient mice results in the decrease in tubular apoptosis during renal inflammation. Kitaura, H., et al., Journal of Dental Research 87 (2008) 396-400 refer to an anti-CSF-1 antibody that inhibits orthodontic tooth movement. WO 2001/030381 mentions inhibitors of CSF-1 activity, including antisense nucleotides and antibodies, although it discloses only antisense nucleotides for CSF-1. WO 2004/045532 concerns the prevention of metastasis and bone loss and treatment of metastatic cancer by the use of a CSF-1 antagonist disclosing only anti-CSF-1 antibodies as an antagonist. WO 2005/046657 relates to the treatment of inflammatory bowel disease with anti-CSF-1 antibodies. US application 2002/0141994 relates to inhibitors of colony stimulating factors. WO 2006/096489 concerns the treatment of rheumatoid arthritis with anti-CSF-1 antibodies. WO 2009/026303 and WO 2009/112245 are related to certain anti-CSF-1R antibodies that bind CSF-1R within the first three subdomains (D1 to D3) of the extracellular domain (CSF-1R-ECD).
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4/91
Brief Description of the Invention [010] The invention comprises a human CSF1R binding antibody, characterized by the fact that the antibody binds to the delD4 fragment of human CSF-1R (SEQ ID NO: 65) and to the extracellular domain of human CSF1R ( SEQ ID NO: 64) with a ratio of 1:50 or less.
[011] The invention further comprises an antibody according to the invention characterized by the fact that:
a) the heavy chain variable domain is SEQ ID NO: 7 and the light chain variable domain is SEQ ID NO: 8,
b) the heavy chain variable domain is SEQ ID NO: 15 and the light chain variable domain is SEQ ID NO: 16,
c) the heavy chain variable domain is SEQ ID NO: 75 and the light chain variable domain is SEQ ID NO: 76,
d) the heavy chain variable domain is SEQ ID NO: 83 and the light chain variable domain is SEQ ID NO: 84, or a humanized version of it.
[012] The invention further comprises an antibody according to the invention characterized by the fact that:
a) the heavy chain variable domain is SEQ ID NO: 7 and the light chain variable domain is SEQ ID NO: 8,
b) the variable domain of the heavy chain is SEQ ID NO: 15 and the variable domain of the light chain is SEQ ID NO: 16, or a humanized version of it.
[013] In an example of an embodiment, the antibody according to the invention is characterized by the fact that:
a) the heavy chain variable domain is SEQ ID NO: 23 and the light chain variable domain is SEQ ID NO: 24, or
b) the heavy chain variable domain is SEQ ID NO: 31 and the
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5/91 light chain variable domain is SEQ ID NO: 32, or
c) the heavy chain variable domain is SEQ ID NO: 39 and the light chain variable domain is SEQ ID NO: 40, or
d) the heavy chain variable domain is SEQ ID NO: 47 and the light chain variable domain is SEQ ID NO: 48, or
e) the heavy chain variable domain is SEQ ID NO: 55 and the light chain variable domain is SEQ ID NO: 56.
[014] The invention further comprises an antibody according to the invention, characterized by the fact that:
a) the heavy chain variable domain comprises a region
CDR3 of SEQ ID NO: 1, a CDR2 region of SEQ ID NO: 2, and a region
CDR1 of SEQ ID NO: 3, and the light chain variable domain comprises a CDR3 region of SEQ ID NO: 4, a CDR2 region of SEQ ID NO: 5, and a CDR1 region of SEQ ID NO: 6, or
b) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 9, a CDR2 region of SEQ ID NO: 10, and a CDR1 region of SEQ ID NO: 11, and the light chain variable domain comprises a region CDR3 of SEQ ID NO: 12, a CDR2 region of SEQ ID NO: 13, and a CDR1 region of SEQ ID NO: 14, or
c) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 17, a CDR2 region of SEQ ID NO: 18, and a CDR1 region of SEQ ID NO: 19, and the variable domain of the light chain comprises a region CDR3 of SEQ ID NO: 20, a CDR2 region of SEQ ID NO: 21, and a CDR1 region of SEQ ID NO: 22, or
d) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 25, a CDR2 region of SEQ ID NO: 26, and a CDR1 region of SEQ ID NO: 27, and the variable domain of the light chain comprises a region CDR3 of SEQ ID NO: 28, a CDR2 region of SEQ ID NO: 29, and
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6/91 a CDR1 region of SEQ ID NO: 30, or
e) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 33, a CDR2 region of SEQ ID NO: 34, and a CDR1 region of SEQ ID NO: 35, and the light chain variable domain comprises a region CDR3 of SEQ ID NO: 36, a CDR2 region of SEQ ID NO: 37, and a CDR1 region of SEQ ID NO: 38, or
f) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 41, a CDR2 region of SEQ ID NO: 42, and a CDR1 region of SEQ ID NO: 43, and the light chain variable domain comprises a region CDR3 of SEQ ID NO: 44, a CDR2 region of SEQ ID NO: 45, and a CDR1 region of SEQ ID NO: 46, or
g) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 49, a CDR2 region of SEQ ID NO: 50, and a CDR1 region of SEQ ID NO: 51, and the light chain variable domain comprises a region CDR3 of SEQ ID NO: 52, a CDR2 region of SEQ ID NO: 53, and a CDR1 region of SEQ ID NO: 54, or
h) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 69, a CDR2 region of SEQ ID NO: 70, and a CDR1 region of SEQ ID NO: 71, and the light chain variable domain comprises a region CDR3 of SEQ ID NO: 72, a CDR2 region of SEQ ID NO: 73, and a CDR1 region of SEQ ID NO: 74, or
i) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 77, a CDR2 region of SEQ ID NO: 78, and a CDR1 region of SEQ ID NO: 79, and the variable domain of the light chain comprises a region CDR3 of SEQ ID NO: 80, a CDR2 region of SEQ ID NO: 81, and a CDR1 region of SEQ ID NO: 82.
[015] Preferably, the antibody according to the invention is from the human IgG1 subclass or the human IgG4 subclass.
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7/91 [016] An additional embodiment of the invention is a pharmaceutical composition comprising an antibody according to the invention.
[017] The invention further comprises the use of an antibody according to the invention for the manufacture of a medicament for the treatment of a disease mediated by CSF-1R.
[018] The invention further comprises the use of an antibody according to the invention for the manufacture of a medicament for the treatment of cancer.
[019] The invention further comprises the use of an antibody according to the invention for the manufacture of a medicament for the treatment of bone loss.
[020] The invention further comprises the use of an antibody according to the invention for the manufacture of a medicament for the treatment of metastases.
[021] The invention further comprises the use of an antibody according to the invention for the manufacture of a medicament for the treatment of inflammatory diseases.
[022] The invention further comprises an antibody according to the invention for the treatment of a disease mediated by CSF-1R.
[023] The invention further comprises an antibody according to the invention for the treatment of cancer.
[024] The invention further comprises an antibody according to the invention for the treatment of bone loss.
[025] The invention further comprises an antibody according to the invention for the treatment of metastases.
[026] The invention further comprises an antibody according to the invention for the treatment of inflammatory diseases.
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8/91 [027] An example of a further embodiment of the invention is a nucleic acid encoding an antibody according to the invention characterized by the fact that:
a) the heavy chain variable domain comprises a region
CDR3 of SEQ ID NO: 1, a CDR2 region of SEQ ID NO: 2, and a region
CDR1 of SEQ ID NO: 3, and the light chain variable domain comprises a CDR3 region of SEQ ID NO: 4, a CDR2 region of SEQ ID NO: 5, and a CDR1 region of SEQ ID NO: 6, or
b) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 9, a CDR2 region of SEQ ID NO: 10, and a CDR1 region of SEQ ID NO: 11, and the light chain variable domain comprises a region CDR3 of SEQ ID NO: 12, a CDR2 region of SEQ ID NO: 13, and a CDR1 region of SEQ ID NO: 14, or
c) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 17, a CDR2 region of SEQ ID NO: 18, and a CDR1 region of SEQ ID NO: 19, and the variable domain of the light chain comprises a region CDR3 of SEQ ID NO: 20, a CDR2 region of SEQ ID NO: 21, and a CDR1 region of SEQ ID NO: 22, or
d) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 25, a CDR2 region of SEQ ID NO: 26, and a CDR1 region of SEQ ID NO: 27, and the variable domain of the light chain comprises a region CDR3 of SEQ ID NO: 28, a CDR2 region of SEQ ID NO: 29, and a CDR1 region of SEQ ID NO: 30, or
e) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 33, a CDR2 region of SEQ ID NO: 34, and a CDR1 region of SEQ ID NO: 35, and the light chain variable domain comprises a region CDR3 of SEQ ID NO: 36, a CDR2 region of SEQ ID NO: 37, and a CDR1 region of SEQ ID NO: 38, or
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9/91
f) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 41, a CDR2 region of SEQ ID NO: 42, and a CDR1 region of SEQ ID NO: 43, and the light chain variable domain comprises a region CDR3 of SEQ ID NO: 44, a CDR2 region of SEQ ID NO: 45, and a CDR1 region of SEQ ID NO: 46, or
g) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 49, a CDR2 region of SEQ ID NO: 50, and a CDR1 region of SEQ ID NO: 51, and the light chain variable domain comprises a region CDR3 of SEQ ID NO: 52, a CDR2 region of SEQ ID NO: 53, and a CDR1 region of SEQ ID NO: 54, or
h) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 69, a CDR2 region of SEQ ID NO: 70, and a CDR1 region of SEQ ID NO: 71, and the light chain variable domain comprises a region CDR3 of SEQ ID NO: 72, a CDR2 region of SEQ ID NO: 73, and a CDR1 region of SEQ ID NO: 74, or
i) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 77, a CDR2 region of SEQ ID NO: 78, and a CDR1 region of SEQ ID NO: 79, and the variable domain of the light chain comprises a region CDR3 of SEQ ID NO: 80, a CDR2 region of SEQ ID NO: 81, and a CDR1 region of SEQ ID NO: 82.
[028] An additional embodiment of the invention is a nucleic acid encoding an antibody according to the invention characterized by the fact that:
a) the heavy chain variable domain is SEQ ID NO: 7 and the light chain variable domain is SEQ ID NO: 8,
b) the heavy chain variable domain is SEQ ID NO: 15 and the light chain variable domain is SEQ ID NO: 16,
c) the heavy chain variable domain is SEQ ID NO: 75 and the
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10/91 light chain variable domain is SEQ ID NO: 76,
d) the heavy chain variable domain is SEQ ID NO: 83 and the light chain variable domain is SEQ ID NO: 84, or a humanized version of it.
[029] An additional embodiment of the invention is a nucleic acid encoding an antibody according to the invention characterized by the fact that:
a) the heavy chain variable domain is SEQ ID NO: 23 and the light chain variable domain is SEQ ID NO: 24, or
b) the heavy chain variable domain is SEQ ID NO: 31 and the light chain variable domain is SEQ ID NO: 32, or
c) the heavy chain variable domain is SEQ ID NO: 39 and the light chain variable domain is SEQ ID NO: 40, or
d) the heavy chain variable domain is SEQ ID NO: 47 and the light chain variable domain is SEQ ID NO: 48, or
e) the heavy chain variable domain is SEQ ID NO: 55 and the light chain variable domain is SEQ ID NO: 56.
[030] The invention further provides expression vectors containing the nucleic acid according to the invention capable of expressing said nucleic acid in a prokaryotic or eukaryotic host cell, and the host cells containing such vectors for recombinant production of an antibody according to with the invention.
[031] The invention further comprises a prokaryotic or eukaryotic host cell that comprises a vector according to the invention.
[032] Additionally the invention comprises a method for the production of a human or humanized recombinant antibody according to the invention, characterized by the expression of a nucleic acid according to
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11/91 with the invention in a prokaryotic or eukaryotic host cell and recovering said antibody from the mentioned cell or cell culture supernatant. The invention further comprises the antibody obtained by the mentioned recombinant method.
[033] The antibodies according to the invention exhibit benefits for patients who need therapy targeted at CSF-1R. The antibodies according to the invention show efficient antiproliferative activity against ligand-independent and ligand-dependent proliferation and are therefore especially useful in the treatment of cancer and metastases.
[034] The invention further provides a method for treating a cancer patient, comprising administering to a patient diagnosed as having such a disease (and therefore in need of such therapy) an effective amount of an antibody according to the invention. The antibody is preferably administered in a pharmaceutical composition.
[035] An example of a further embodiment of the invention is a method for the treatment of a cancer patient characterized by the administration of an antibody according to the invention to said patient.
[036] Surprisingly, it was found that, using a delD4 fragment of human CSF-1R in which subdomain D4 of human CSF-1RECD was deleted (SEQ ID NO: 65), the new anti-CSF-1R antibodies according to the invention can be selected. These antibodies exhibit valuable properties such as excellent inhibition of ligand-dependent cell growth and at the same time inhibition of ligand-independent cell growth in NIH 3T3 cells infected with retrovirus with an expression vector for full-length wild-type CSF-1R (SEQ ID NO : 62) or CSF-1R mutant L301S Y969F (SEQ ID NO: 63), through which
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12/91 recombinant cells with mutant CSF-1R are able to form spheroids independent of the CSF-1 ligand. In addition, the antibodies according to the invention inhibit the differentiation of (both) macrophages in humans and cinomolgos, since they inhibit the survival of human monocytes and cinomolgos.
Detailed Description of the Invention [037] The invention comprises a human CSF1R-binding antibody, characterized by the fact that the antibody binds to the delD4 fragment of human CSF-1R (comprising the extracellular subdomains D1D3 and D5) (SEQ ID NO: 65 ) and the human CSF-1R Extracellular Domain (CSF-1R-ECD) (comprising extracellular subdomains D1-D5) (SEQ ID NO: 64) with a ratio of 1:50 or less.
[038] The invention further comprises an antibody according to the invention characterized by the fact that it comprises as a CDR3 region of the heavy chain variable domain the SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 23, SEQ ID NO : 31, SEQ ID NO: 39, SEQ ID NO: 47 or SEQ ID NO: 55.
[039] The invention further comprises an antibody according to the invention characterized by the fact that:
a) the heavy chain variable domain is SEQ ID NO: 7 and the light chain variable domain is SEQ ID NO: 8,
b) the variable domain of the heavy chain is SEQ ID NO: 15 and the variable domain of the light chain is SEQ ID NO: 16, or a humanized version of it.
[040] The invention further comprises an antibody according to the invention characterized by the fact that:
a) the heavy chain variable domain is SEQ ID NO: 7 and the light chain variable domain is SEQ ID NO: 8,
b) the heavy chain variable domain is SEQ ID NO: 15 and the
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13/91 light chain variable domain is SEQ ID NO: 16,
c) the heavy chain variable domain is SEQ ID NO: 75 and the light chain variable domain is SEQ ID NO: 76,
d) the heavy chain variable domain is SEQ ID NO: 83 and the light chain variable domain is SEQ ID NO: 84, or a humanized version of it.
[041] The invention further comprises an antibody according to the invention characterized by the fact that:
a) the variable domain of the heavy chain is SEQ ID NO: 7 and the variable domain of the light chain is SEQ ID NO: 8, or a humanized version of it.
[042] In an example of an embodiment, the antibody according to the invention is characterized by the fact that:
a) the heavy chain variable domain is SEQ ID NO: 23 and the light chain variable domain is SEQ ID NO: 24, or
b) the heavy chain variable domain is SEQ ID NO: 31 and the light chain variable domain is SEQ ID NO: 32, or
c) the heavy chain variable domain is SEQ ID NO: 39 and the light chain variable domain is SEQ ID NO: 40, or
d) the heavy chain variable domain is SEQ ID NO: 47 and the light chain variable domain is SEQ ID NO: 48, or
e) the heavy chain variable domain is SEQ ID NO: 55 and the light chain variable domain is SEQ ID NO: 56.
[043] In an example of an embodiment, the antibody according to the invention is characterized by the fact that:
a) the heavy chain variable domain is SEQ ID NO: 23 and the light chain variable domain is SEQ ID NO: 24, or
b) the heavy chain variable domain is SEQ ID NO: 31 and the
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14/91 variable domain of the light chain is SEQ ID NO: 32, or
c) the heavy chain variable domain is SEQ ID NO: 39 and the light chain variable domain is SEQ ID NO: 40, or
d) the heavy chain variable domain is SEQ ID NO: 47 and the light chain variable domain is SEQ ID NO: 48.
[044] In an example of an embodiment, the antibody according to the invention is characterized by the fact that:
the variable domain of the heavy chain is SEQ ID NO: 23 and the variable domain of the light chain is SEQ ID NO: 24, or [045] In one embodiment, the antibody according to the invention is characterized by the fact that what:
the heavy chain variable domain is SEQ ID NO: 31 and the light chain variable domain is SEQ ID NO: 32.
[046] In an example of an embodiment, the antibody according to the invention is characterized by the fact that:
the heavy chain variable domain is SEQ ID NO: 39 and the light chain variable domain is SEQ ID NO: 40.
[047] In an example of an embodiment, the antibody according to the invention is characterized by the fact that:
the heavy chain variable domain is SEQ ID NO: 47 and the light chain variable domain is SEQ ID NO: 48.
[048] The invention further comprises an antibody according to the invention characterized by the fact that:
a) the variable domain of the heavy chain is SEQ ID NO: 15 and the variable domain of the light chain is SEQ ID NO: 16, or a humanized version of it.
[049] The invention further comprises an antibody according to the invention characterized by the fact that:
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15/91 the heavy chain variable domain is SEQ ID NO: 75 and the light chain variable domain is SEQ ID NO: 76;
or a humanized version of this.
[050] The invention further comprises an antibody according to the invention characterized by the fact that:
the heavy chain variable domain is SEQ ID NO: 83 and the light chain variable domain is SEQ ID NO: 84;
or a humanized version of this.
[051] The invention further comprises an antibody according to the invention, characterized by the fact that:
a) the heavy chain variable domain comprises a region
CDR3 of SEQ ID NO: 1, a CDR2 region of SEQ ID NO: 2, and a region
CDR1 of SEQ ID NO: 3, and the light chain variable domain comprises a CDR3 region of SEQ ID NO: 4, a CDR2 region of SEQ ID NO: 5, and a CDR1 region of SEQ ID NO: 6, or
b) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 9, a CDR2 region of SEQ ID NO: 10, and a CDR1 region of SEQ ID NO: 11, and the light chain variable domain comprises a region CDR3 of SEQ ID NO: 12, a CDR2 region of SEQ ID NO: 13, and a CDR1 region of SEQ ID NO: 14, or
c) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 17, a CDR2 region of SEQ ID NO: 18, and a CDR1 region of SEQ ID NO: 19, and the variable domain of the light chain comprises a region CDR3 of SEQ ID NO: 20, a CDR2 region of SEQ ID NO: 21, and a CDR1 region of SEQ ID NO: 22, or
d) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 25, a CDR2 region of SEQ ID NO: 26, and a CDR1 region of SEQ ID NO: 27, and the variable domain of the light chain comprises
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16/91 a CDR3 region of SEQ ID NO: 28, a CDR2 region of SEQ ID NO: 29, and a CDR1 region of SEQ ID NO: 30, or
e) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 33, a CDR2 region of SEQ ID NO: 34, and a CDR1 region of SEQ ID NO: 35, and the light chain variable domain comprises a region CDR3 of SEQ ID NO: 36, a CDR2 region of SEQ ID NO: 37, and a CDR1 region of SEQ ID NO: 38, or
f) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 41, a CDR2 region of SEQ ID NO: 42, and a CDR1 region of SEQ ID NO: 43, and the light chain variable domain comprises a region CDR3 of SEQ ID NO: 44, a CDR2 region of SEQ ID NO: 45, and a CDR1 region of SEQ ID NO: 46, or
g) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 49, a CDR2 region of SEQ ID NO: 50, and a CDR1 region of SEQ ID NO: 51, and the light chain variable domain comprises a region CDR3 of SEQ ID NO: 52, a CDR2 region of SEQ ID NO: 53, and a CDR1 region of SEQ ID NO: 54.
[052] The invention further comprises an antibody according to the invention, characterized by the fact that:
a) the heavy chain variable domain comprises a region
CDR3 of SEQ ID NO: 1, a CDR2 region of SEQ ID NO: 2, and a region
CDR1 of SEQ ID NO: 3, and the light chain variable domain comprises a CDR3 region of SEQ ID NO: 4, a CDR2 region of SEQ ID NO: 5, and a CDR1 region of SEQ ID NO: 6, or
b) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 9, a CDR2 region of SEQ ID NO: 10, and a CDR1 region of SEQ ID NO: 11, and the light chain variable domain comprises a region CDR3 of SEQ ID NO: 12, a CDR2 region of SEQ ID NO: 13, and
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17/91 a CDR1 region of SEQ ID NO: 14, or
c) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 17, a CDR2 region of SEQ ID NO: 18, and a CDR1 region of SEQ ID NO: 19, and the variable domain of the light chain comprises a region CDR3 of SEQ ID NO: 20, a CDR2 region of SEQ ID NO: 21, and a CDR1 region of SEQ ID NO: 22, or
d) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 25, a CDR2 region of SEQ ID NO: 26, and a CDR1 region of SEQ ID NO: 27, and the variable domain of the light chain comprises a region CDR3 of SEQ ID NO: 28, a CDR2 region of SEQ ID NO: 29, and a CDR1 region of SEQ ID NO: 30, or
e) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 33, a CDR2 region of SEQ ID NO: 34, and a CDR1 region of SEQ ID NO: 35, and the light chain variable domain comprises a region CDR3 of SEQ ID NO: 36, a CDR2 region of SEQ ID NO: 37, and a CDR1 region of SEQ ID NO: 38, or
f) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 41, a CDR2 region of SEQ ID NO: 42, and a CDR1 region of SEQ ID NO: 43, and the light chain variable domain comprises a region CDR3 of SEQ ID NO: 44, a CDR2 region of SEQ ID NO: 45, and a CDR1 region of SEQ ID NO: 46,
g) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 49, a CDR2 region of SEQ ID NO: 50, and a CDR1 region of SEQ ID NO: 51, and the light chain variable domain comprises a region CDR3 of SEQ ID NO: 52, a CDR2 region of SEQ ID NO: 53, and a CDR1 region of SEQ ID NO: 54,
h) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 69, a CDR2 region of SEQ ID NO: 70, and a region
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CDR1 of SEQ ID NO: 71, and the light chain variable domain comprises a CDR3 region of SEQ ID NO: 72, a CDR2 region of SEQ ID NO: 73, and a CDR1 region of SEQ ID NO: 74, or
i) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 77, a CDR2 region of SEQ ID NO: 78, and a CDR1 region of SEQ ID NO: 79, and the variable domain of the light chain comprises a region CDR3 of SEQ ID NO: 80, a CDR2 region of SEQ ID NO: 81, and a CDR1 region of SEQ ID NO: 82.
[053] In an example of an embodiment, the antibody according to the invention is characterized by the fact that:
a) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 69, a CDR2 region of SEQ ID NO: 70, and a CDR1 region of SEQ ID NO: 71, and the light chain variable domain comprises a region CDR3 of SEQ ID NO: 72, a CDR2 region of SEQ ID NO: 73, and a CDR1 region of SEQ ID NO: 74, or
b) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 77, a CDR2 region of SEQ ID NO: 78, and a CDR1 region of SEQ ID NO: 79, and the variable domain of the light chain comprises a region CDR3 of SEQ ID NO: 80, a CDR2 region of SEQ ID NO: 81, and a CDR1 region of SEQ ID NO: 82.
[054] In one embodiment, the antibody according to the invention is characterized by the fact that:
a) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 17, a CDR2 region of SEQ ID NO: 18, and a CDR1 region of SEQ ID NO: 19, and the variable domain of the light chain comprises a region CDR3 of SEQ ID NO: 20, a CDR2 region of SEQ ID NO: 21, and a CDR1 region of SEQ ID NO: 22, or
b) the heavy chain variable domain comprises a region
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CDR3 of SEQ ID NO: 25, a CDR2 region of SEQ ID NO: 26, and a CDR1 region of SEQ ID NO: 27, and the light chain variable domain comprises a CDR3 region of SEQ ID NO: 28, a CDR2 region of SEQ ID NO: 29, and a CDR1 region of SEQ ID NO: 30, or
c) the variable domain of the heavy chain comprises a CDR3 region of SEQ ID NO: 33, a CDR2 region of SEQ ID NO: 34, and a CDR1 region of SEQ ID NO: 35, and the variable domain of the light chain comprises a region CDR3 of SEQ ID NO: 36, a CDR2 region of SEQ ID NO: 37, and a CDR1 region of SEQ ID NO: 38, or
d) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 41, a CDR2 region of SEQ ID NO: 42, and a CDR1 region of SEQ ID NO: 43, and the light chain variable domain comprises a region CDR3 of SEQ ID NO: 44, a CDR2 region of SEQ ID NO: 45, and a CDR1 region of SEQ ID NO: 46, or
e) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 49, a CDR2 region of SEQ ID NO: 50, and a CDR1 region of SEQ ID NO: 51, and the variable domain of the light chain comprises a region CDR3 of SEQ ID NO: 52, a CDR2 region of SEQ ID NO: 53, and a CDR1 region of SEQ ID NO: 54.
[055] In an example of an embodiment, the antibody according to the invention is characterized by the fact that:
a) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 17, a CDR2 region of SEQ ID NO: 18, and a CDR1 region of SEQ ID NO: 19, and the variable domain of the light chain comprises a region CDR3 of SEQ ID NO: 20, a CDR2 region of SEQ ID NO: 21, and a CDR1 region of SEQ ID NO: 22, or
b) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 25, a CDR2 region of SEQ ID NO: 26, and a region
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CDR1 of SEQ ID NO: 27, and the light chain variable domain comprises a CDR3 region of SEQ ID NO: 28, a CDR2 region of SEQ ID NO: 29, and a CDR1 region of SEQ ID NO: 30, or
c) the variable domain of the heavy chain comprises a CDR3 region of SEQ ID NO: 33, a CDR2 region of SEQ ID NO: 34, and a CDR1 region of SEQ ID NO: 35, and the variable domain of the light chain comprises a region CDR3 of SEQ ID NO: 36, a CDR2 region of SEQ ID NO: 37, and a CDR1 region of SEQ ID NO: 38, or
d) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 41, a CDR2 region of SEQ ID NO: 42, and a CDR1 region of SEQ ID NO: 43, and the light chain variable domain comprises a region CDR3 of SEQ ID NO: 44, a CDR2 region of SEQ ID NO: 45, and a CDR1 region of SEQ ID NO: 46.
[056] In an example of an embodiment, the antibody according to the invention is characterized by the fact that:
the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 17, a CDR2 region of SEQ ID NO: 18, and a CDR1 region of SEQ ID NO: 19, and the light chain variable domain comprises a CDR3 region of SEQ ID NO: 20, a CDR2 region of SEQ ID NO: 21, and a CDR1 region of SEQ ID NO: 22.
[057] In an example of an embodiment, the antibody according to the invention is characterized by the fact that:
the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 25, a CDR2 region of SEQ ID NO: 26, and a CDR1 region of SEQ ID NO: 27, and the light chain variable domain comprises a CDR3 region of SEQ ID NO: 28, a CDR2 region of SEQ ID NO: 29, and a CDR1 region of SEQ ID NO: 30.
[058] In one embodiment, the antibody according to
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21/91 the invention is characterized by the fact that:
the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 33, a CDR2 region of SEQ ID NO: 34, and a CDR1 region of SEQ ID NO: 35, and the light chain variable domain comprises a CDR3 region of SEQ ID NO: 36, a CDR2 region of SEQ ID NO: 37, and a CDR1 region of SEQ ID NO: 38.
[059] In an example of an embodiment, the antibody according to the invention is characterized by the fact that:
the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 41, a CDR2 region of SEQ ID NO: 42, and a CDR1 region of SEQ ID NO: 43, and the light chain variable domain comprises a CDR3 region of SEQ ID NO: 44, a CDR2 region of SEQ ID NO: 45, and a CDR1 region of SEQ ID NO: 46.
[060] In one embodiment, the human CSF-1R binding antibody is characterized by the fact that the antibody binds to the delD4 fragment of human CSF-1R (SEQ ID NO: 65) and CSF-1R-ECD human (SEQ ID NO: 64) with a ratio of 1:50 or less, and is further characterized by the fact that it does not bind to the human CSF-1R D1-D3 fragment (SEQ ID NO: 66).
[061] The term "antibody" encompasses the various forms of antibodies including, but not limited to: complete antibodies, antibody fragments, human antibodies, humanized antibodies, chimeric antibodies, T cell epitope depletion antibodies, and additionally antibodies genetically modified, as long as the characteristic properties according to the invention are maintained. The "antibody fragments" comprise a portion of a full-length antibody, preferably its variable domain, or at least the antigen-binding site thereof. Examples of antibody fragments include diabody,
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22/91 single chain antibody molecules, and multispecific antibodies formed from antibody fragments. ScFv antibodies are, for example, described in Houston, J.S., Methods in Enzymol. 203 (1991) 46-88). In addition, antibody fragments comprise single chain polypeptides having the characteristics of a VH binding domain for CSF1R, which is known to be able to assemble together with a VL domain, or a VL binding domain for CSF-1R, which is known to be able to assemble together with a VH domain a functional antigen binding site, thus providing the property.
[062] The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of a single amino acid composition.
[063] The term "chimeric antibody" refers to a monoclonal antibody that comprises a variable region, that is, a mouse binding region and at least a portion of a constant region derived from a different source or species, usually prepared by recombinant DNA techniques. Chimeric antibodies comprising a mouse variable region and a human constant region are especially preferred. Such mouse / human chimeric antibodies are the product of expressed immunoglobulin genes that comprise segments of DNA that encode the variable regions of mouse immunoglobulin and DNA segments that encode constant regions of human immunoglobulin. Other forms of "chimeric antibodies" encompassed by the present invention are those in which the class or subclass has been modified or altered from that found in the original antibody. Such "chimeric" antibodies are also referred to as "class-switched antibodies". Methods for producing chimeric antibodies involve techniques
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23/91 conventional methods of recombinant DNA and gene transfection already well known in the art. See, for example, Morrison, S.L., et al., Proc. Natl. Acad, Sci. USA 81 (1984) 6851-6855; US 5,202,238 and US 5,204,244.
[064] The term “humanized antibody” refers to antibodies in which the framework region or “complementarity determining region” (CDR) has been modified to include the CDR of an immunoglobulin of different specificity when compared to that of parental immunoglobulin (original). In an example of a preferred embodiment, a murine CDR is grafted into the framework region of a human antibody to prepare the "humanized antibody". See, for example, Riechmann, L., et al., Nature 332 (1988) 323-327; and Neuberger, M.S., et al., Nature 314 (1985) 268-270. Optionally, the framework region can be modified by additional mutations. In addition, CDRs can be modified by one or more mutations to generate antibodies according to the invention, for example by molecular modeling based mutagenesis, as described by Riechmann, L., et al., Nature 332 (1988) 323- 327 and Queen, C., et al., Proc. Natl. Acad. Sci. USA 86 (1989) 10029-10033, or others. Particularly the preferred CDRs correspond to those representing the antigen recognition sequences referred to above for chimeric antibodies. A “humanized version of an antibody according to the invention” (which is, for example, of mouse origin) refers to an antibody, which is based on the mouse antibody sequences in which VH and VL are humanized by standard techniques (including CDR grafting and, optionally, subsequent mutagenesis of certain amino acids in the regions of the framework and CDRs). Preferably such a humanized version is chimerized with a human constant region (see, for example, Sequences SEQ ID NO: 57-61).
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24/91 [065] Other forms of "humanized antibodies" encompassed by the present invention are those in which the constant region has been further modified or altered from the original antibody to generate the properties according to the invention, especially with respect to binding to the C1q and / or connection to the Fc receptor (FCR).
[066] In the following examples, the terms "Mab" or "muMab" refer to murine monoclonal antibodies, such as Mab 2F11 or Mab 2E10, while the term "hMAb" refers to humanized monoclonal versions of such murine antibodies, such as hMab 2F11-C11, hMab 2F11-d8, hMab 2F11e7, hMab 2F11-f12, etc.
[067] The term "human antibody" as used herein, is intended to include antibodies having variable and constant regions derived from human germline from immunoglobulin sequences. Human antibodies are well known in the art (van Dijk, M.A., and van de Winkel, J.G., Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human antibodies can also be produced in transgenic animals (for example, mice) that are capable, through immunization, of producing a complete repertoire or selection of human antibodies in the absence of endogenous immunoglobulin production. The transfer of the immunoglobulin gene array from human germline in such mice of mutant germline will result in the production of human antibodies after the antigen challenge (see, for example, Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90 (1993) 2551-2555; Jakobovits, A., et al, Nature 362 (1993) 255-258; Brueggemann, M., et al, Tear Immunol.7 (1993) 33-40). Human antibodies can also be produced in phage display libraries (Hoogenboom, HR, and Winter, GJ Mol. Biol. 227 (1992) 381388; Marks, JD, et al, J. Mol .. Biol. 222 (1991) 581-597). The techniques of Cole, et al., And Boerner et al., Are also available for the preparation of
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25/91 human monoclonal antibodies (Cole, SPC, et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); and Boerner, P., et al, J. Immunol.147 (1991) 86-95). As already mentioned for chimeric and humanized antibodies according to the invention, the term "human antibody" as used herein also comprises such antibodies that are modified in the constant region to generate the properties according to the invention, especially with respect to binding to the C1q and / or binding to the FcR, for example, by "class change", ie change or mutation of Fc portions (for example, from IgG1 to IgG4 and / or IgG1 / IgG4 mutation).
[068] The term "recombinant human antibody" as used herein is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from a host cell, such as an NS0 cell or CHO, or from an animal (for example, a mouse), which is transgenic for human immunoglobulin genes or antibodies expressed using a recombinant expression vector transfected in a host cell. Such recombinant human antibodies have variable and constant regions in a rearranged manner. The recombinant human antibodies according to the invention were subjected to somatic hypermutation in vivo. Thus, the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, although derived from the human germline VH and VL sequences, may not naturally exist within the human germline antibody repertoire in vivo.
[069] The antibodies according to the invention additionally include such antibodies having "conservative changes in the sequence", changes in the sequence of amino acids and nucleotides that do not affect or alter the characteristics mentioned above of the antibody of
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26/91 according to the present invention. Modifications can be made using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include those in which the amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the prior art. These families include amino acids with basic side chains (for example, lysine, arginine, histidine), acidic side chains (for example, aspartic acid, glutamic acid) and uncharged polar side chains (for example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and the chains aromatic sides (eg tyrosine, phenylalanine, tryptophan, histidine). Thus, a non-essential amino acid residue predicted on a human anti-CSF-1R antibody can preferably be replaced by another amino acid residue from the same side chain family.
[070] Amino acid substitutions can be performed by mutagenesis based on molecular modeling, as described by Riechmann, L., et al., Nature 332 (1988) 323-327 and Queen, C., et al., Proc. Natl.Acad. Sci. USA 86 (1989) 10029-10033.
[071] Human CSF-1R (CSF-1 receptor; synonyms: M-CSF receptor; type 1 macrophage colony stimulating factor receptor, Fms proto-oncogene, c-fms, SEQ ID NO: 22) is known since 1986 (Coussens, L., et al., Nature 320 (1986) 277-280). CSF-1R is a growth factor encoded by the proto-oncogene c-fms (reviewed, for example, in Roth, P., and Stanley, ER, Curr. Top.Microbiol. Immunol. 181 (1992) 141-67) .
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27/91 [072] CSF-1R is the receptor for CSF-1 (macrophage colony stimulating factor, also called M-CSF) and IL-34 and mediates the biological effects of this cytokine Sherr, CJ, et al. , Cell 41 (1985) 665-676 (Lin, H., et al., Science 320 (2008) 807-811). The cloning of the type 1 colony stimulating factor receptor (also called c-fms) was first described in Roussel, M.F., et al., Nature 325 (1987) 549-552. In that publication, it was shown that CSF-1R had a potential for transformation dependent on changes in the C-terminal tail of the protein, including the loss of inhibitory phosphorylation of tyrosine 969 that binds Cbl and thus regulates down regulation of the recipient (Lee, PS, et al., EMBO J. 18 (1999) 3616-3628).
[073] CSF-1R is a single-chain transmembrane tyrosine kinase (RTK) receptor and a member of the immunoglobulin (Ig) motif family containing RTKs characterized by 5 repeated Ig-like subdomains D1-D5 in the extracellular domain (ECD ) of the recipient (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) comprises all five extracellular Ig-like subdomains D1-D5. The delD4 fragment of human CSF-1R (SEQ ID NO: 65) comprises the extracellular Ig-like subdomains D1-D3 and D5, but lacks the subdomain D4. The human CSF-1R fragment D1D3 (SEQ ID NO: 66) comprises the respective subdomains D1-D3. The sequences are listed without the MGSGPGVLLL LLVATAWHGQ G signal peptide (SEQ ID NO: 67).
[074] The intracellular domain of protein tyrosine kinase is disrupted by a single insertion domain that is also present in the other class III members of the related RTK family including platelet-derived growth factor (PDGFR) receptors and receptor of stem cell growth factor (c-Kit) and similar cytokine receptor
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28/91 a (fms-like) (FLT3). Despite the structural homology between this family of growth factor receptors, they have distinct tissue-specific functions.
[075] CSF-1R is expressed mainly in cells of the monocytic lineage and in the female reproductive tract and placenta. In addition, CSF-1R expression in Langerhans cells in the skin, a subset of smooth muscle cells (Inaba, T., et al., J. Biol. Chem. 267 (1992) 5693-5699), cells B (Baker, AH, et al, Oncogene 8 (1993) 371-378) and microglia (Sawada, M., et al., Brain Res. 509 (1990) 119-124). Cells with mutant human CSF-1R ((SEQ ID NO: 23) are known to proliferate, regardless of ligand stimulation.
[076] As used herein, the terms "binding to human CSF-1R" or "specifically binding to human CSF-1R" refer to an antibody that specifically binds to the human CSF-1R antigen with an affinity for binding with a Kd value of 1.0 x 10 ^-8 mol / l or less at 35 ° C, in an example of an embodiment with a Kd value of 1.0 x 10 ^-9 mol / l or less at 35 ° C. The binding affinity is determined with a standard 35 ° C binding assay, such as by the surface plasmon resonance assay (BIAcore®, GE-Healthcare Uppsala, Sweden). A method for determining the Kd value of binding affinity is described in Example 9. Thus, a "human CSF-1R binding antibody" as used herein refers to an antibody that specifically binds to the CSF-1R antigen. with a binding affinity of Kd 1.0 x 10 ^-8 mol / l or less (preferably 1.0 x 10 ^-8 mol / l - 1.0 x 10 ^-9 mol / l) at 35 ° C, preferably with a Kd value of 1.0 x 10 ^-9 mol / l, or less at 35 ° C (preferably 1.0 x 10 ^-9 mol / l - 1.0 x 10 ^-12 mol / l).
[077] The “binding to the delD4 fragment of human CSF-1R (SEQ ID NO: 65) and to the extracellular domain of human CSF-1R (SEQ ID NO: 64)
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29/91 as used herein is measured by the surface plasmon resonance assay (BIAcore assay) as described in Example 4. The delD4 fragment of human CSF-1R (SEQ ID NO: 65) or Extracellular Domain of human CSF-1R ( SEQ ID NO: 64), respectively, are captured on the surface (each on a separate surface) and the test antibodies are added (each on a separate measurement) and the respective binding signals (response units (RU)) are determined. Reference signals (white surface) are subtracted. If the signs of unbound test antibodies are slightly below 0, then the values are set to 0. Then the ratio between the respective binding signals (binding signal (RU) of the human CSF-1R / delD4 fragment) binding signal (RU) of the human CSF-1R Extracellular Domain (CSF-1R-ECD)) is determined. The antibodies according to the invention have a ratio between the binding signals (RU (delD4) / RU (CSF-1R-ECD) of 1:50 or less, preferably 1: 100 or less (the lower limit included is 0 (for example if the UK is 0, then the ratio is 0:50 or 0: 100)).
[078] This means that such anti-CSF-1R antibodies according to the invention do not bind to the delD4 fragment of human CSF-1R (like the anti-CCR5 m <CCR5> Pz03.1C5 antibody (deposited as DSM ACC 2683 in 18/08/2004 in DSMZ) and have binding signals for binding to the human CSF-1R delD4 fragment in the anti-CCR5 m <CCR5> Pz03.1C5 antibody range, which is below 20 RU (Response Units) , preferably below 10 RU in a surface plasmon resonance assay (BIAcore), as shown in Example 4.
[079] The term "binding to the human CSF-1R D1-D3 fragment" refers to a determination of binding affinity by a surface plasmon resonance assay (BIAcore assay). The test antibody is captured on the surface and the human CSF-1R fragment D1-D3 (SEQ ID NO:
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66) was added and the respective binding affinities were determined. The term "non-binding to the human CSF-1R fragment D1-D3" denotes that, in such an assay the detected signal was in the area of no more than 1.2 times the background signal and, consequently, no significant binding could be detected and no binding affinity could be determined (see Example 10).
[080] An example of an embodiment of the invention is a screening method for the selection of antibodies according to the invention comprising the following steps:
a) determination of the binding signal (response units (RU)) of anti-CSF-1R antibodies to the delD4 fragment of human CSF-1R (SEQ ID NO: 65) and to the extracellular domain of human CSF-1R (CSF-1R -ECD) (SEQ ID NO: 64) by the surface plasmon resonance assay (Biacore assay).
b) selection of antibodies with a ratio of the binding signals (delD4 fragment of human CSF-1R / Extracellular Domain of human CSF-1R (CSF-1R-ECD)) of 50: 1 or less.
[081] In one example, the determination is carried out at 25 ° C.
[082] In one embodiment, the screening method comprises the additional steps of measuring the binding of antiCSF-1R antibodies to the human CSF-1R fragment D1-D3 (SEQ ID NO: 66) (D1-D3) and selection of antibodies that do not show binding to said fragment.
[083] The term "epitope" denotes a protein determinant of human CSF-1R capable of binding specifically to an antibody. Epitopes generally consist of chemically active surface clusters of molecules, such as amino acids or sugar side chains, and epitopes generally have three-dimensional structural characteristics.
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31/91, as well as specific load characteristics. Conformational and non-conformational epitopes are distinguished by the fact that the bond to the former, but not to the latter, is lost in the presence of denaturing solvents. Preferably, an antibody according to the invention specifically binds to native and denatured CSF-1R.
[084] The term "variable domain" (variable domain of a light chain (Vl), variable domain of a heavy chain (Vh)), as used herein denotes each of the pairs of light and heavy chain domains that are directly involved in binding the antibody to the antigen. The variable light and heavy chain domains have the same general structure and each domain comprises four framework regions (FR) whose sequences are largely conserved, linked by three “hypervariable regions” (or complementarity determining regions, CDRs). The framework regions (framework or FR) adopt a β-sheet conformation and the CDRs can form loops linking the β-sheet structure. The CDRs in each chain are maintained in their three-dimensional structures by the framework regions and form, together with the CDRs in the other chain, the antigen binding site. The light and heavy chain CDR3 regions of the antibody play an especially important role in the binding / affinity specificity of the antibodies according to the invention and therefore provide an additional object of the invention.
[085] The term "antigen-binding portion of an antibody" is used here to refer to the amino acid residues of an antibody that are responsible for binding to the antigen. The antigen-binding portion of an antibody comprises amino acid residues from "complementarity determining regions" or "CDRs". The “framework” (structural region or framework) or “FR” regions are those regions of the variable domain that are not residues of the hypervariable region as
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32/91 defined herein. Therefore, the light and heavy chain variable domains of an antibody comprise from the N- to C-terminal end the FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 domains. Especially, the heavy chain CDR3 is the region that contributes the most to binding the antigen and defines the properties of the antibody. The CDR and FR regions are determined according to the standard definition 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 of a “hypervariable loop”.
[086] The terms "nucleic acid" or "nucleic acid molecule", as used herein, are intended to include DNA molecules and RNA molecules. A nucleic acid molecule can be single-stranded or double-stranded, but is preferably double-stranded DNA.
[087] The term “amino acid” as used in this application indicates the naturally occurring group of α-amino acids carboxy comprising alanine (three letter code: Ala, one letter code: A), arginine (Arg, R), asparagine ( Asn, N), aspartic acid (Asp, D), cysteine (Cis, 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 (Fen, F), proline (Pro, P), serine (Ser, S), threonine ( Tre, T), tryptophan (Trp, W), tyrosine (Tir, Y) and valine (Val, V).
[088] In one embodiment, the antibodies according to the invention inhibit the binding of CSF-1 to CSF-1R. In an example of an embodiment with an IC50 of 200 ng / ml or less, in an example of an embodiment with an IC50 of 50 ng / ml or less. The IC50 of inhibition of CSF-1 binding to CSF-1R can be determined as indicated in Example 2.
[089] In one embodiment, the antibodies according to the invention inhibit CSF-1R-induced phosphorylation of CSF-1 (in cells
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NIH3T3-CSF-1R recombinants).
[090] In an example of an embodiment with an IC50 of 800 ng / ml or less, in an example of an embodiment with an IC50 of 600 ng / ml or less, in an example of an embodiment with an IC50 of 250 ng / ml or less . The IC50 value of CSF-1R-induced phosphorylation of CSF-1 can be determined as indicated in Example 3.
[091] In one embodiment, the antibodies according to the invention inhibit the growth of recombinant NIH3T3 cells expressing human CSF-1R (SEQ ID NO: 62). 62). In an example of an embodiment with an IC50 of 10 pg / ml or less, in an example of an embodiment with an IC50 of 5 pg / ml or less, in an example of an embodiment with an IC50 of 2 pg / ml or less. In an example of an embodiment with an IC30 of 10 pg / ml or less, in an example of an embodiment with an IC30 of 5 pg / ml or less, in an example of an embodiment with an IC30 of 2 pg / ml or less. The IC50 value, the IC30 value or the% growth inhibition are determined as shown in Example 5.
[092] In one embodiment, the antibodies according to the invention inhibit the growth of recombinant NIH3T3 cells that express human CSF-1R with the L301S Y969F mutations (SEQ ID NO: 62). In an example of an embodiment with an IC50 of 15 pg / ml or less, in an example of an embodiment with an IC50 of 10 pg / ml or less. In an example of an embodiment with an IC30 of 10 pg / ml or less, in an example of an embodiment with an IC50 of 5 pg / ml or less, in an example of an embodiment with an IC50 of 2 pg / ml or less. The IC50 value, the IC30 value or the% growth inhibition are determined as shown in Example 5.
[093] In one embodiment, the antibodies according to the invention inhibit the growth of BeWo tumor cells (ATCC CCL-98) by 65% or more (at an antibody concentration of 10 pg / ml; and in
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34/91 compared to the absence of antibody). The% growth inhibition is determined as shown in Example 8. Mab 2F11 shows a 70% growth inhibition of BeWo tumor cells.
[094] In an example of an embodiment the antibodies according to the invention inhibit (both) the differentiation of human macrophages and cinomolgos (which is indicated by inhibiting the survival of human monocytes and cinomolgos as shown in Examples 7 and 8). In one embodiment, the antibodies according to the invention inhibit the survival of human monocytes with an IC 50 of 0.15 pg / ml or less, and in an embodiment with an IC 50 of 0.10 pg / ml or less. Inhibition of human monocyte survival is determined as shown in Example 7. In one embodiment the antibodies according to the invention inhibit the survival of cinomolgy monocytes by 80% or more, in an example embodiment by 90% or more (at an antibody concentration of 5 μ / ml, and compared to the absence of antibody). Inhibition of human monocyte survival is determined as shown in Example 8.
[095] An example of a further embodiment of the invention is a method for the production of an antibody against CSF-1R characterized by the fact that the nucleic acid sequence encoding the heavy chain of a CSF-binding human IgG1 class antibody Human 1R according to the invention, said modified nucleic acid and the nucleic acid encoding the light chain of said antibody are inserted into an expression vector, said vector is inserted into a eukaryotic host cell, the encoded protein is expressed and recovered from the host cell or supernatant.
[096] The antibodies according to the invention are preferably produced by recombinant means. Consequently, the
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35/91 antibody is preferably an isolated monoclonal antibody. Such recombinant methods are widely known in the art and comprise expression of the protein in prokaryotic and eukaryotic cells, with subsequent isolation of the antibody polypeptide and usually purification and to a pharmaceutically acceptable degree of 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 on suitable prokaryotic or eukaryotic host cells, such as CHO cells, cells, NS0, SP2 / 0 cells, HEK293 cells and COS cells, yeasts or E. coli cells, and the antibody is recovered from the cells (from the supernatant or the cells themselves after lysis).
[097] Recombinant antibody production is well known in the art and described, for example, in the review articles by 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.
[098] Antibodies can be present in whole cells, in a cell lysate, or in a partially purified, or substantially pure form. Purification is performed in order to eliminate other cellular components or other contaminants, for example, other nucleic acids or cellular proteins, by standard techniques, including alkaline / SDS treatment, CsCl bandage, column chromatography, agarose gel electrophoresis , and other methods well known in the art. See, Ausubel, F., et al., Ed. Current Protocols in Molecular Biology, Greene Publishing & Wiley Interscience, New York (1987).
[099] Expression in NS0 cells is described, for example, by Barnes, L.M., et al., Cytotechnology 32 (2000) 109-123; and Barnes, L.M., et al.,
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Biotech. Bioeng. 73 (2001) 261-270. Transient expression is described, for example, by Durocher, Y., et al., Nucl. Acids. Res. 30 (2002) E9. The cloning of variable domains is described by 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; and Norderhaug, L., et al, J. Immunol. Methods 204 (1997) 7787. A preferred transient expression system (HEK 293) is described by Schlaeger, E.-J., and Christensen, K., in Cytotechnology 30 (1999) 71-83 and by Schlaeger, E.- J., in J. Immunol.Methods 194 (1996) 191-199.
[0100] Control sequences that are appropriate for prokaryotes include, for example, a promoter, optionally an operator sequence, a ribosome binding site. Eukaryotic cells are known to use promoters, enhancers and polyadenylation signals.
[0101] A nucleic acid is "operationally linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, the DNA of a pre-sequence or secretion leader is operationally linked to the DNA encoding a polypeptide if it is expressed in the form of a pre-protein that participates in the secretion of the polypeptide; a promoter or amplifier is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operationally linked to a coding sequence if it is positioned to facilitate translation. Generally, "operationally linked" indicates that the linked DNA sequences are contiguous and, in the case of the secretory leader, is contiguous and is within the reading frame. Enhancers, however, do not need to be contiguous. The connection is carried out by means of connection at convenient restriction sites. If these sites do not exist, synthetic oligonucleotide adapters or ligands are used according to conventional practice.
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37/91 [0102] Monoclonal antibodies are appropriately separated from the culture medium by conventional immunoglobulin purification procedures, such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography . The DNA and RNA encoding the monoclonal antibodies are readily isolated and sequenced using conventional procedures. Hybridoma cells can serve as a source of such DNA and RNA. Once isolated, DNA can be inserted into expression vectors, which are then transfected into host cells, such as HEK 293 cells, CHO cells or myeloma cells that would otherwise not produce the immunoglobulin protein to obtain antibody synthesis monoclonal cells in recombinant host cells.
[0103] As used herein, the expressions "cell", "cell line" and "cell culture" are used interchangeably for all designations and include their progenies. Thus, the words "transformants" and "transformed cells" include the primary cell in question and the cultures derived from it, without taking into account the number of transfers. It should also be understood that every progeny may not be exactly identical in terms of DNA content, due to deliberate or accidental mutations. Variant progenies that have the same biological function or activity are included, as selected in the initial transformed cell.
[0104] The "Fc portion" of an antibody is not directly involved in the binding of an antibody to an antigen, but exhibits several effector functions. An "Fc portion of an antibody" is a term well known to those skilled in the art and defined based on the cleavage of antibodies with papain. Depending on the amino acid sequences of the heavy chain constant region, the antibodies or immunoglobulins
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38/91 can be divided into classes: IgA, IgD, IgE, IgG and IgM, and several of these classes can be further divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3 and IgG4, IgA1 and IgA2. According to the heavy chain constant regions, the different classes of antibodies 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, binding to C1q and binding to the Fc receptor. Complement activation (CDC) is initiated by binding the complement factor C1q to the Fc portion of most IgG antibody subclasses. Although the influence of an antibody on the complement system is dependent on certain conditions, binding to C1q is caused by binding sites defined on the Fc portion. Such binding sites are known in the art and described, for example, by 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, EE, et al., J. Immunol.164 (2000) 4178-4184, Hezareh, M., et al., J. Virology 75 (2001) 12161-12168, Morgan, A., et al., Immunology 86 (1995) 319324, EP 0307434. Such binding sites are, for example, L234, L235, D270, N297, E318, K320, K322, P331 and P329 (numbering system according to the EU Kabat index, EA, see below). The antibodies of the subclass IgG1, IgG2 and IgG3 generally show complement activation and binding of C1q and C3, while IgG4 does not activate the complement system and does not bind to C1q and C3.
[0105] In an example of an embodiment, the antibody according to the invention comprises an Fc portion derived from human origin and preferably all other portions of the constant human regions. As used herein, the term “Fc portion derived from
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39/91 human ”denotes an Fc portion that is an Fc portion of a human antibody of the IgG1, IgG2, IgG3 or IgG4 subclass, preferably an Fc portion of the human IgG1 subclass, or a mutated Fc portion of the human IgG1 subclass (preferably with an L234A + L235A mutation), an Fc portion of the human IgG4 subclass or a mutated Fc portion of the human IgG4 subclass (preferably with an S228P mutation). The most preferred human heavy chain constant regions are SEQ ID NO: 58 (human IgG1 subclass), SEQ ID NO: 59 (human IgG1 subclass with L234A and L235A mutations), SEQ ID NO: 60 (human IgG4 subclass), or SEQ ID NO: 61 (human IgG4 subclass with S228P mutation).
[0106] Preferably, the antibody according to the invention is from the human IgG1 subclass or the human IgG4 subclass. In one embodiment, the antibody according to the invention is from the human IgG1 subclass: In one embodiment, the antibody according to the invention is from the human IgG4 subclass:
[0107] In one embodiment, the antibody according to the invention is characterized by the fact that the constant chains are of human origin. Such constant chains are well known in the art and described, for example, by Kabat, E.A., (see, for example, Johnson, G. and Wu, TT, Nucleic Acids Res. 28 (2000) 214-218). For example, a useful human constant heavy chain region comprises an amino acid sequence of SEQ ID NO: 58. For example, a useful human light chain constant region comprises an amino acid sequence of the kappa light chain constant region of SEQ ID NO: : 57.
[0108] Another aspect of the invention is a human CSF-1R binding antibody, characterized by the fact that:
a) the heavy chain variable domain is SEQ ID NO: 7 and the light chain variable domain is SEQ ID NO: 8,
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b) the variable domain of the heavy chain is SEQ ID NO: 15 and the variable domain of the light chain is SEQ ID NO: 16, or a humanized version of it.
[0109] Another aspect of the invention is an antibody binding to
Human CSF-1R, characterized by the fact that:
a) the heavy chain variable domain is SEQ ID NO: 7 and the light chain variable domain is SEQ ID NO: 8,
b) the heavy chain variable domain is SEQ ID NO: 15 and the light chain variable domain is SEQ ID NO: 16,
c) the heavy chain variable domain is SEQ ID NO: 75 and the light chain variable domain is SEQ ID NO: 76,
d) the heavy chain variable domain is SEQ ID NO: 83 and the light chain variable domain is SEQ ID NO: 84, or a humanized version of it.
[0110] Another aspect of the invention is an antibody binding to
Human CSF-1R, characterized by the fact that:
a) the variable domain of the heavy chain is SEQ ID NO: 7 and the variable domain of the light chain is SEQ ID NO: 8, or a humanized version of it.
[0111] Another aspect of the invention is a human CSF-1 R binding antibody, characterized by the fact that:
a) the heavy chain variable domain is SEQ ID NO: 23 and the light chain variable domain is SEQ ID NO: 24, or
b) the heavy chain variable domain is SEQ ID NO: 31 and the light chain variable domain is SEQ ID NO: 32, or
c) the heavy chain variable domain is SEQ ID NO: 39 and the light chain variable domain is SEQ ID NO: 40, or
d) the heavy chain variable domain is SEQ ID NO: 47 and the
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41/91 light chain variable domain is SEQ ID NO: 48, or
e) the heavy chain variable domain is SEQ ID NO: 55 and the light chain variable domain is SEQ ID NO: 56.
[0112] Another aspect of the invention is an antibody binding to
Human CSF-1R, characterized by the fact that:
a) the heavy chain variable domain is SEQ ID NO: 23 and the light chain variable domain is SEQ ID NO: 24, or
b) the heavy chain variable domain is SEQ ID NO: 31 and the light chain variable domain is SEQ ID NO: 32, or
c) the heavy chain variable domain is SEQ ID NO: 39 and the light chain variable domain is SEQ ID NO: 40, or
d) the heavy chain variable domain is SEQ ID NO: 47 and the light chain variable domain is SEQ ID NO: 48.
[0113] Another aspect of the invention is a human CSF-1R binding antibody, characterized by the fact that:
the heavy chain variable domain is SEQ ID NO: 23 and the light chain variable domain is SEQ ID NO: 24, or
Another aspect of the invention is a human CSF-1R binding antibody, characterized by the fact that:
the heavy chain variable domain is SEQ ID NO: 31 and the light chain variable domain is SEQ ID NO: 32.
[0114] Another aspect of the invention is a human CSF-1R binding antibody, characterized by the fact that:
the heavy chain variable domain is SEQ ID NO: 39 and the light chain variable domain is SEQ ID NO: 40.
[0115] Another aspect of the invention is an antibody binding to
Human CSF-1R, characterized by the fact that:
the heavy chain variable domain is SEQ ID NO: 47 and the
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42/91 variable domain of the light chain is SEQ ID NO: 48.
[0116] Another aspect of the invention is a human CSF-1R binding antibody, characterized by the fact that:
a) the variable domain of the heavy chain is SEQ ID NO: 15 and the variable domain of the light chain is SEQ ID NO: 16, or a humanized version of it.
[0117] Another aspect of the invention is a human CSF-1R binding antibody, characterized by the fact that:
the heavy chain variable domain is SEQ ID NO: 75 and the light chain variable domain is SEQ ID NO: 76;
or a humanized version of this.
[0118] Another aspect of the invention is a human CSF-1R binding antibody, characterized by the fact that:
the heavy chain variable domain is SEQ ID NO: 83 and the light chain variable domain is SEQ ID NO: 84;
or a humanized version of this.
[0119] Another aspect of the invention is a human CSF-1 R binding antibody, characterized by the fact that:
a) the heavy chain variable domain comprises a region
CDR3 of SEQ ID NO: 1, a CDR2 region of SEQ ID NO: 2, and a region
CDR1 of SEQ ID NO: 3, and the light chain variable domain comprises a CDR3 region of SEQ ID NO: 4, a CDR2 region of SEQ ID NO: 5, and a CDR1 region of SEQ ID NO: 6, or
b) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 9, a CDR2 region of SEQ ID NO: 10, and a CDR1 region of SEQ ID NO: 11, and the light chain variable domain comprises a region CDR3 of SEQ ID NO: 12, a CDR2 region of SEQ ID NO: 13, and a CDR1 region of SEQ ID NO: 14, or
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c) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 17, a CDR2 region of SEQ ID NO: 18, and a CDR1 region of SEQ ID NO: 19, and the variable domain of the light chain comprises a region CDR3 of SEQ ID NO: 20, a CDR2 region of SEQ ID NO: 21, and a CDR1 region of SEQ ID NO: 22, or
d) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 25, a CDR2 region of SEQ ID NO: 26, and a CDR1 region of SEQ ID NO: 27, and the variable domain of the light chain comprises a region CDR3 of SEQ ID NO: 28, a CDR2 region of SEQ ID NO: 29, and a CDR1 region of SEQ ID NO: 30, or
e) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 33, a CDR2 region of SEQ ID NO: 34, and a CDR1 region of SEQ ID NO: 35, and the light chain variable domain comprises a region CDR3 of SEQ ID NO: 36, a CDR2 region of SEQ ID NO: 37, and a CDR1 region of SEQ ID NO: 38, or
f) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 41, a CDR2 region of SEQ ID NO: 42, and a CDR1 region of SEQ ID NO: 43, and the light chain variable domain comprises a region CDR3 of SEQ ID NO: 44, a CDR2 region of SEQ ID NO: 45, and a CDR1 region of SEQ ID NO: 46,
g) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 49, a CDR2 region of SEQ ID NO: 50, and a CDR1 region of SEQ ID NO: 51, and the light chain variable domain comprises a region CDR3 of SEQ ID NO: 52, a CDR2 region of SEQ ID NO: 53, and a CDR1 region of SEQ ID NO: 54,
h) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 69, a CDR2 region of SEQ ID NO: 70, and a CDR1 region of SEQ ID NO: 71, and the variable domain of the light chain comprises
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44/91 a CDR3 region of SEQ ID NO: 72, a CDR2 region of SEQ ID NO: 73, and a CDR1 region of SEQ ID NO: 74, or
i) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 77, a CDR2 region of SEQ ID NO: 78, and a CDR1 region of SEQ ID NO: 79, and the variable domain of the light chain comprises a region CDR3 of SEQ ID NO: 80, a CDR2 region of SEQ ID NO: 81, and a CDR1 region of SEQ ID NO: 82.
[0120] Another aspect of the invention is a human CSF-1R binding antibody, characterized by the fact that:
a) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 17, a CDR2 region of SEQ ID NO: 18, and a CDR1 region of SEQ ID NO: 19, and the variable domain of the light chain comprises a region CDR3 of SEQ ID NO: 20, a CDR2 region of SEQ ID NO: 21, and a CDR1 region of SEQ ID NO: 22, or
b) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 25, a CDR2 region of SEQ ID NO: 26, and a CDR1 region of SEQ ID NO: 27, and the variable domain of the light chain comprises a region CDR3 of SEQ ID NO: 28, a CDR2 region of SEQ ID NO: 29, and a CDR1 region of SEQ ID NO: 30, or
c) the variable domain of the heavy chain comprises a CDR3 region of SEQ ID NO: 33, a CDR2 region of SEQ ID NO: 34, and a CDR1 region of SEQ ID NO: 35, and the variable domain of the light chain comprises a region CDR3 of SEQ ID NO: 36, a CDR2 region of SEQ ID NO: 37, and a CDR1 region of SEQ ID NO: 38, or
d) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 41, a CDR2 region of SEQ ID NO: 42, and a CDR1 region of SEQ ID NO: 43, and the light chain variable domain comprises a region CDR3 of SEQ ID NO: 44, a CDR2 region of SEQ ID NO: 45, and
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45/91 a CDR1 region of SEQ ID NO: 46, or
e) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 49, a CDR2 region of SEQ ID NO: 50, and a CDR1 region of SEQ ID NO: 51, and the variable domain of the light chain comprises a region CDR3 of SEQ ID NO: 52, a CDR2 region of SEQ ID NO: 53, and a CDR1 region of SEQ ID NO: 54.
[0121] Another aspect of the invention is a human CSF-1R binding antibody, characterized by the fact that:
a) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 17, a CDR2 region of SEQ ID NO: 18, and a CDR1 region of SEQ ID NO: 19, and the variable domain of the light chain comprises a region CDR3 of SEQ ID NO: 20, a CDR2 region of SEQ ID NO: 21, and a CDR1 region of SEQ ID NO: 22, or
b) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 25, a CDR2 region of SEQ ID NO: 26, and a CDR1 region of SEQ ID NO: 27, and the variable domain of the light chain comprises a region CDR3 of SEQ ID NO: 28, a CDR2 region of SEQ ID NO: 29, and a CDR1 region of SEQ ID NO: 30, or
c) the variable domain of the heavy chain comprises a CDR3 region of SEQ ID NO: 33, a CDR2 region of SEQ ID NO: 34, and a CDR1 region of SEQ ID NO: 35, and the variable domain of the light chain comprises a region CDR3 of SEQ ID NO: 36, a CDR2 region of SEQ ID NO: 37, and a CDR1 region of SEQ ID NO: 38, or
d) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 41, a CDR2 region of SEQ ID NO: 42, and a CDR1 region of SEQ ID NO: 43, and the light chain variable domain comprises a region CDR3 of SEQ ID NO: 44, a CDR2 region of SEQ ID NO: 45, and a CDR1 region of SEQ ID NO: 46.
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46/91 [0122] Another aspect of the invention is a human CSF-1R binding antibody, characterized by the fact that:
the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 17, a CDR2 region of SEQ ID NO: 18, and a CDR1 region of SEQ ID NO: 19, and the light chain variable domain comprises a CDR3 region of SEQ ID NO: 20, a CDR2 region of SEQ ID NO: 21, and a CDR1 region of SEQ ID NO: 22.
[0123] Another aspect of the invention is a human CSF-1R binding antibody, characterized by the fact that:
the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 25, a CDR2 region of SEQ ID NO: 26, and a CDR1 region of SEQ ID NO: 27, and the light chain variable domain comprises a CDR3 region of SEQ ID NO: 28, a CDR2 region of SEQ ID NO: 29, and a CDR1 region of SEQ ID NO: 30.
[0124] Another aspect of the invention is a human CSF-1R binding antibody, characterized by the fact that:
the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 33, a CDR2 region of SEQ ID NO: 34, and a CDR1 region of SEQ ID NO: 35, and the light chain variable domain comprises a CDR3 region of SEQ ID NO: 36, a CDR2 region of SEQ ID NO: 37, and a CDR1 region of SEQ ID NO: 38.
[0125] Another aspect of the invention is a human CSF-1R binding antibody, characterized by the fact that:
the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 41, a CDR2 region of SEQ ID NO: 42, and a CDR1 region of SEQ ID NO: 43, and the light chain variable domain comprises a CDR3 region of SEQ ID NO: 44, a CDR2 region of SEQ ID NO: 45, and a CDR1 region of SEQ ID NO: 46.
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47/91 [0126] The invention comprises a method for treating a patient in need of therapy, characterized by administering to the patient a therapeutically effective amount of an antibody according to the invention.
[0127] The invention comprises the use of an antibody according to the invention for therapy.
[0128] A preferred embodiment of the invention is the CSF-1R antibodies of the present invention for use in the treatment of "CSF-1R-mediated diseases" or the CSF-1R antibodies of the present invention for use in the manufacture of a medicament in the treatment of “CSF-1R-mediated diseases”, which can be described as follows:
[0129] There are 3 distinct mechanisms through which CSF-1R signaling is likely to be involved in the growth of tumors and metastases. The first is that the expression of the CSF ligand and the receptor was found in tumor cells from the female reproductive system (ovary, breast, endometrium, cervix) (Scholl, SM, et al., J. Natl. Cancer Inst. 86 (1994 ) 120-126; Kacinski, BM, Mol. Reprod. Dev. 46 (1997) 71-74 ;. Ngan, HY, et al, Eur. J. Cancer 35 (1999) 1546-1550 ;. Kirma, N., et al, Cancer Res. 67 (2007) 1918-1926) and the expression was associated with the growth of breast cancer xenograft, as well as with the poor prognosis in patients with breast cancer. Two point mutations were observed in CSF-1R in about 10-20% of cases of acute myelocytic leukemia, chronic myelocytic leukemia and myelodysplasias evaluated in one study, and one of the mutations was found to disrupt receptor turnover (Ridge, SA, et al., Proc. Natl. Acad. Sci. USA 87 (1990) 1377-1380). However, the incidence of mutations could not be confirmed in further studies (Abu-Duhier, F.M., et al., Br. J. Haematol. 120 (2003) 464-470). Mutations have also been found in some cases of carcinoma
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48/91 hepatocellular (Yang, DH, et al., Pancreat hepatobiliary. Dis. Int. 3 (2004) 86-89) and idiopathic myelofibrosis (Abu-Duhier, FM, et al., Br. J. Haematol. 120 ( 2003) 464-470). Recently, the Y571D mutation in CSF-1R was identified in a GDM-1 cell line derived from a patient with myelomonoblastic leukemia (Chase, A., et al., Leukemia 23 (2009) 358-364).
[0130] Pigmented villonodular synovitis (SVNP) and tenosynovial giant cell tumors (TGCT) can occur as a result of a translocation that fuses the M-CSF gene to a COL6A3 collagen gene and results in the overexpression of M-CSF (West , RB, et al., Proc. Natl. Acad. Sci. USA 103 (2006) 690-695). A landscape effect is proposed to be responsible for the resulting tumor mass that consists of monocytic cells attracted by cells that express M-CSF. TGCTs are smaller tumors that can be removed relatively easily from the fingers where they usually occur. SVNP is more aggressive, as it can resort to large joints and is not so easily controlled by surgery.
[0131] The second mechanism is based on blocking signaling through M-CSF / CSF-1R at metastatic sites in the bone that induce osteoclastogenesis, bone resorption and osteolytic bone lesions. Breast cancers, multiple myeloma and lung cancers are examples of the types of cancers that have been found to metastasize to bones and cause osteolytic bone disease resulting in skeletal complications. The M-CSF released by tumor cells and stroma induces the differentiation of myeloid monocytic hematopoietic progenitors to mature osteoclasts in collaboration with the nuclear factor kappa-B ligand activating receptor, RANKL. During this process, M-CSF acts as a permissive factor giving the survival signal to osteoclasts (Tanaka, S., et al., J. Clin. Invest. 91 (1993) 257-263). Inhibition of CSF-1R activity during osteoclast differentiation and maturation with an anti-CSF-1R antibody is
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49/91 likely to prevent the unbalanced activity of osteoclasts that cause osteolytic disease and associated bone complications in metastatic disease. Although breast, lung and multiple myeloma cancer usually result in osteolytic lesions, bone metastases in prostate cancer initially have an osteoblastic appearance in which increased bone-forming activity results in 'braided bone' (or not- lamellar woven bone) which is different from the typical lamellar structure of normal bone. During the progression of the disease, bone lesions exhibit a significant osteolytic component, as well as high serum levels of bone resorption suggesting that anti-resorption therapy may be useful. Bisphosphonates have been shown to inhibit the formation of osteolytic lesions and reduce the number of events related to the skeletal system only in men with hormone-refractory prostate cancer, but at this point their effect on osteoblastic lesions is controversial and to date bisphosphonates were not beneficial in preventing bone metastasis or hormone-responsive prostate cancer. The effect of anti-resorption agents on osteolytic / osteoblastic mixed prostate cancer is still being studied in the clinic (Choueiri, MB, et al., Cancer Metastasis Rev. 25 (2006) 601-609 ;. Vessella, RL and Corey, E ., Clin Cancer Cancer 12 (20 Pt 2) (2006) 6285s-6290s).
[0132] The third mechanism is based on the recent observation that tumor-associated macrophages (MAT) found in solid tumors of the breast, prostate, ovary and cervical cancers correlate with the prognosis (Bingle, L., et al. , J. Pathol.196 (2002) 254-265; Pollard, JW, Nat. Rev. Cancer 4 (2004) 71-78). Macrophages are recruited to the tumor by M-CSF and other chemokines. Macrophages can then contribute to tumor progression by secreting angiogenic factors, proteases and other growth factors and cytokines and can be blocked by inhibiting
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50/91 CSF-1R signaling. It was recently demonstrated by Zins et al. (Zins, K., et al., Cancer Res. 67 (2007) 1038-1045) that siRNA expression of tumor necrosis factor alpha (TNF-alpha), M-CSF or the combination of both reduces tumor growth in a mouse xenograft model between 34% and 50% after intratumoral injection of the respective siRNA. TNF-alpha targeted siRNA secreted by human SW620 cells reduced the levels of mouse M-CSF and led to a reduction of macrophages in the tumor. The treatment of MCF7 tumor xenografts with an antigen-binding fragment directed against M-CSF in addition to resulting in tumor growth inhibition by 40%, it reversed resistance to chemotherapeutic agents and improved the survival of mice when administered in combination with chemotherapeutic agents. (Paulus, P., et al., Cancer Res. 66 (2006) 4349-4356).
[0133] MATs are just one example of an emerging link between chronic inflammation and cancer. There is additional evidence for the link between inflammation and cancer, many chronic diseases are associated with an increased risk of developing cancer, the appearance of cancers at the sites of chronic inflammation, and chemical mediators of inflammation are found in many cancers; the suppression of cellular or chemical mediators of inflammation inhibits the development of experimental cancers and the long-term use of anti-inflammatory agents reduces the risk of some cancers. A link with cancer exists for a number of inflammatory conditions including H. pylori-induced gastritis for gastric cancer, schistosomiasis for bladder cancer, HHVX for Kaposi's sarcoma, endometriosis for ovarian cancer and prostatitis for prostate cancer (Balkwill, F., et al., Cancer Cell 7 (2005) 211-217). Macrophages are important cells in chronic inflammation and respond differentially to their microenvironment. There are two types of
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51/91 macrophages that are considered extreme in series of functional states: M1 macrophages are involved in Type 1 reactions. These reactions involve the activation of microbial products and the consequent death of pathogenic microorganisms that results in reactive oxygen intermediates (ROIs). At the other end are the M2 macrophages involved in Type 2 reactions that promote cell proliferation, adjust inflammation and adaptive immunity and promote tissue remodeling, angiogenesis and repair (Mantovani, A., et al., Trends Immunol. 25 (2004 ) 677-686). Chronic inflammation, resulting in established neoplasia, is usually associated with M2 macrophages. A central cytokine that mediates inflammatory reactions is TNF alpha which, by the true meaning of the term, can stimulate anti-tumor immunity and hemorrhagic necrosis in high doses, but has also recently been found to be expressed by tumor cells and acting as a tumor promoter (Zins , K., et al, Cancer Res. 67 (2007) 1038-1045; Balkwill, F., Cancer Metastasis Rev. 25 (2006) 409416). The specific role of macrophages in relation to the tumor still needs to be better understood, including the spatial potential and the temporal dependence of its function and relevance to specific tumor types.
[0134] Thus an example of an embodiment of the invention are the CSF-1R antibodies of the present invention for use in the treatment of cancer. The term "cancer" as used herein may be, for example, lung cancer, non-small cell lung cancer (NSCL), bronchiolarveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin cancer or intraocular melanoma, uterine cancer carcinoma, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colon cancer, breast cancer, uterine cancer , fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma,
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52/91 vagina carcinoma, vulvar carcinoma, Hodgkin's disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, soft tissue sarcoma, cancer urethra, penis cancer, prostate cancer, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvis carcinoma, mesothelioma, hepatocellular cancer, bile duct cancer, CNS neoplasms, tumors in the spinal axis, brain stem glioma, glioblastoma multiforme, astrocytoma, schwannomas, ependimonas, medulloblastomas, meningiomas, squamous cell carcinomas, pituitary adenoma, lymphoma, lymphocytic leukemia, including refractory versions of any of the above cancers, or a combination of one or more of the cancers described above. Preferably, cancer is breast cancer, ovarian cancer, cervical cancer, lung cancer or prostate cancer. Preferably, said cancers are further characterized by the expression or overexpression of CSF-1 or CSF-1R. An additional embodiment of the invention are CSF-1R antibodies of the present invention for use in the simultaneous treatment of primary tumors and new metastases.
[0135] Thus, another example of carrying out the invention are the CSF-1R antibodies of the present invention for use in the treatment of periodontitis, histiocytosis X, osteoporosis, Paget's disease of the bone (PDB), bone loss due to cancer therapy, periprosthetic osteolysis and glucocorticoid-induced osteoporosis, rheumatoid arthritis, psoriatic arthritis, osteoarthritis, inflammatory arthritis, and inflammation.
[0136] Rabello, D., et al., Biochem. Biophys. Commun. 347 (2006) 791-796 demonstrated that SNPs in the CSF1 gene had a positive association with aggressive periodontitis: an inflammatory disease of the periodontal tissues that causes tooth loss due to bone resorption
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53/91 alveolar.
[0137] Histiocytosis X (also called Langerhans cell histiocytosis, LCH) is a proliferative disease of dendritic Langerhans cells that appear to differentiate into bone osteoclasts and extra-bony LCH lesions. Langerhans cells are derived from circulating monocytes. The increased levels of M-CSF that were measured in serum and lesions correlated with disease severity (da Costa, C.E., et al., J. Exp. Med. 201 (2005) 687-693). The disease occurs mainly in a population of pediatric patients and has to be treated with chemotherapy when the disease becomes systemic or when it recurs.
[0138] The pathophysiology of osteoporosis is mediated by the loss of bone-forming osteoblasts and increased osteoclast-dependent bone resorption. Data that support this mechanism have been described by Cenci et al. showing that an injection of anti-M-CSF antibody preserves bone density and inhibits bone resorption in ovariectomized mice (Cenci, S., et al., J. Clin. Invest. 105 (2000) 1279-1287). Recently, a potential link between postmenopausal bone loss due to estrogen deficiency has been identified and the presence of TNF-alpha producing T cells has been found to affect bone metabolism (Roggia, C., et al., Minerva Med. 95 (2004) 125-132). A possible mechanism could be the induction of M-CSF by TNF alpha in vivo. An important role for M-CSF in induced TNF-alpha-induced osteoclastogenesis was confirmed by the effect of an antibody directed against M-CSF that blocked TNF-alpha-induced osteolysis in mice, thereby making CSF signaling inhibitors -1R potential targets for inflammatory arthritis (Kitaura, H., etal., J. Clin. Invest. 115 (2005) 3418-3427).
[0139] Paget's disease of the bone (PDB) is the second disorder
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54/91 of the most common bone metabolism after osteoporosis in which focal abnormalities of increased bone metabolism lead to complications such as bone pain, deformities, pathological fractures and deafness. Mutations have been identified in four genes that regulate the function of normal osteoclasts and predispose individuals to PDB and related disorders: insertion mutations in TNFRSF11A, which encodes nuclear factor activating receptor (NF) kappaB (RANK) - a critical regulator of the function of osteoclasts, inactivating mutations of TNFRSF11B encoding osteoprotegerin (a decoy receptor (decoy receptor for the RANK ligand), mutations of the sequestosome 1 gene (SQSTM1), which encodes an important scaffold protein in the NFkappaB pathway and mutations in the protein gene containing valosine (PCV). This gene encodes VCP, which plays a role in guiding the NFkappaB inhibitor for proteasome degradation (Daroszewska, A. and Ralston, SH, Nat. Clin. Pract. Rheumatol. 2 (2006) 270277) Inhibitors targeting CSF-1R provide an opportunity to block deregulation of RANKL signaling indirectly and add a treatment option to bisphosphonates currently used osphonates.
[0140] Bone loss induced by cancer therapy especially in patients with breast and prostate cancer is an additional indication that an inhibitor targeting CSF-1R could prevent bone loss (Lester, JE, et al., Br. J. Cancer 94 (2006) 30-35). As the prognosis of early breast cancer improves, the long-term consequences of adjuvant therapies become more important for some therapies, including chemotherapy, irradiation, aromatase inhibitors and ovarian ablation, which affect bone metabolism by decreasing bone mineral density, resulting in an increased risk of osteoporosis and associated fractures (Lester, JE, et al., Br. J. Cancer 94 (2006) 30-35). The equivalent for adjuvant inhibitor therapy
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55/91 aromatase in breast cancer is androgenic ablation therapy in prostate cancer that leads to loss of bone mineral density and significantly increases the risk of osteoporosis-related fractures (Stoch, SA, et al., J. Clin. Endocrinol, Metab. 86 (2001) 2787-2791).
[0141] Inhibition of CSF-1R signaling is likely to be beneficial in other indications, as well as when target cell types include osteoclasts and macrophages, for example, in the treatment of specific complications in response to joint replacement as a consequence rheumatoid arthritis. Implant failure due to periprosthetic bone loss and the consequent loosening of the prostheses is an important complication in joint replacement and requires repeated surgeries with high socioeconomic burdens for the patient and the health system. To date, there is no approved drug therapy to prevent or inhibit periprosthetic osteolysis (Drees, P., et al., Nat. Clin. Pract. Rheumatol. 3 (2007) 165-171).
[0142] Glucocorticoid-induced osteoporosis (GIOP) is another indication that a CSF-1R inhibitor could prevent bone loss after long-term use of glucocorticoids, which is given as a result of several conditions, including disease chronic obstructive pulmonary disease, asthma and rheumatoid attrition (Guzman-Clark, JR, et al., Arthritis Rheum.57 (2007) 140-146; Feldstein, CA, et al, Osteoporos. Int. 16 (2005) 2168-2174).
[0143] Rheumatoid arthritis, psoriatic arthritis and inflammatory arthritis are potential indications for inhibitors of CSF-1R signaling in that they consist of a macrophage component and varying degrees of bone destruction (Ritchlin, CT, et al. , J. Clin. Invest. 111 (2003) 821-831). Osteoarthritis and rheumatoid arthritis are autoimmune inflammatory diseases caused by the accumulation of macrophages in connective tissue and
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56/91 macrophage infiltration into synovial fluid, which is at least partially mediated by M-CSF. Campbell, I., K., et al., J. Leukoc.Biol. 68 (2000) 144 150, demonstrated that M-CSF is produced by cells of human articular tissue (chondrocytes, synovial fibroblasts) in vitro and is found in the synovial fluid of patients with rheumatoid arthritis, suggesting that it contributes to the proliferation of tissue synovial and macrophage infiltration and that is associated with the pathogenesis of the disease. Inhibition of CSF-1R signaling is likely to control the number of macrophages in the joint and relieve the pain of associated bone destruction. In order to minimize adverse effects and to better understand the impact of CSF-1R signaling on these indications, one method is to specifically inhibit CSF-1R without targeting a large number of other kinases, such as Raf kinase.
[0144] Recent literature reports have correlated the increase in circulating M-CSF with the poor prognosis and progression of atherosclerosis in chronic coronary artery disease (Saitoh, T., et al., J. Am. Coll. Cardiol. 35 (2000) 655-665 ;. Ikonomidis, I., et al, Eur. Heart. J. 26 (2005) pages 1618-1624); M-CSF influences the atherosclerotic process, helping the formation of foam cells (macrophages that ingest oxidized LDL) that express CSF-1R and represent the initial plaque (Murayama, T., et al, Circulation 99 (1999) 1740-1746) .
[0145] M-CSF and CSF-1R expression and signaling are found in the activated microglia. Microglia, which are macrophages residing in the central nervous system, can be activated by various insults, including infection and traumatic injury. M-CSF is considered a key regulator of inflammatory responses in the brain and M-CSF levels increase in HIV-1 infection, encephalitis, Alzheimer's disease (AD) and brain tumors. Microgliosis as a consequence of autocrine signaling by MCSF / CSF-1R results in the induction of inflammatory cytokines and nitric oxides
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57/91 being released as demonstrated, for example, by using an experimental neuronal damage model (Hao, AJ, et al., Neuroscience 112 (2002) 889900; Murphy, GM, Jr., et al, J. Biol. Chem. 273 (1998) 20.967-20.971). Microglia that have an increased expression of CSF-1R are found in surrounding plaques in AD and in the amyloid precursor protein in a model of AD in a transgenic mouse V717F (Murphy, GM, Jr., et al., Am. J. Pathol. 157 (2000) 895-904). In contrast, in op / op mice with less microglia in the brain it resulted in A-beta fibrillar deposition and neuronal loss compared to normal control, suggesting that the microglia have a neuroprotective function in the development of absent AD in op / op mice. (Kaku, M., et al., Brain Res. Brain Res. Protoc. 12 (2003) 104-108).
[0146] M-CSF and CSF-1R expression and signaling is associated with inflammatory bowel disease (DII) (WO 2005/046657). The term "inflammatory bowel disease" refers to severe chronic disorders of the intestinal tract, characterized by chronic inflammation at various locations in the gastrointestinal tract, and specifically includes ulcerative colitis (UC) and Crohn's disease.
[0147] The invention comprises a human CSF1R-binding antibody being characterized by the epitope-binding properties mentioned above or alternatively by the amino acid sequences and fragments of amino acid sequences mentioned above for the treatment of cancer.
[0148] The invention comprises a human CSF1R binding antibody being characterized by the epitope binding properties mentioned above or alternatively by the amino acid sequences and fragments of amino acid sequences mentioned above for the treatment of bone loss.
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58/91 [0149] The invention comprises a human CSF1R binding antibody being characterized by the epitope binding properties mentioned above or alternatively by the amino acid sequences and fragments of amino acid sequences mentioned above for the prevention or treatment of metastases.
[0150] The invention comprises a human CSF1R binding antibody being characterized by the epitope-binding properties mentioned above or alternatively by the amino acid sequences and fragments of amino acid sequences mentioned above for the treatment of inflammatory diseases.
[0151] The invention comprises the use of an antibody characterized by comprising the binding of said antibody to human CSF-1 R, being characterized by the binding properties to the epitopes mentioned above or alternatively by the amino acid sequences and fragments of amino acid sequences mentioned above for the treatment of cancer or alternatively for the manufacture of a medicine for the treatment of cancer.
[0152] The invention comprises the use of an antibody characterized by comprising the binding of said antibody to human CSF-1R, being characterized by the binding properties to the epitopes mentioned above or alternatively by the amino acid sequences and fragments of amino acid sequences mentioned above for the treatment of bone loss or alternatively for the manufacture of a medicine for the treatment of bone loss.
[0153] The invention comprises the use of an antibody characterized by comprising the binding of said antibody to human CSF-1R, being characterized by the binding properties to the epitopes mentioned above or alternatively by the amino acid sequences and fragments of
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59/91 amino acid sequences mentioned above for the prevention or treatment of metastases or alternatively for the manufacture of a medicament for the prevention or treatment of metastases.
[0154] The invention comprises the use of an antibody characterized by comprising the binding of said antibody to human CSF-1R, being characterized by the binding properties to the epitopes mentioned above or alternatively by the amino acid sequences and fragments of amino acid sequences mentioned above for the treatment of inflammatory diseases or alternatively for the manufacture of a medicine for the treatment of inflammatory diseases.
[0155] An example of a further embodiment of the invention is a method for the production of an antibody against CSF-1R characterized by the fact that the nucleic acid sequence encoding the heavy chain of a CSF-binding human IgG1 class antibody Human 1R according to the invention, said modified nucleic acid and the nucleic acid encoding the light chain of said antibody are inserted into an expression vector, said vector is inserted into a eukaryotic host cell, the encoded protein is expressed and recovered from the host cell or supernatant.
[0156] The antibodies according to the invention are preferably produced by recombinant means. Such methods are widely known in the art and comprise expression of the protein in prokaryotic and eukaryotic cells, with subsequent isolation of the antibody polypeptide and usually purification and to a pharmaceutically acceptable degree of purity. For protein expression, nucleic acids encoding light and heavy chains or fragments thereof are inserted into expression vectors by standard methods. Expression is carried out in suitable prokaryotic or eukaryotic host cells, such as
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60/91 CHO cells, cells, NS0, SP2 / 0 cells, HEK293 cells, COS cells, yeasts or E. coli cells, and the antibody is recovered from the cells (from the supernatant or from the cells themselves after lysis) ).
[0157] Recombinant antibody production is well known in the art and described, for example, in the review articles by 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.
[0158] Antibodies can be present in whole cells, in a cell lysate, or in a partially purified, or substantially pure form. Purification is performed in order to eliminate other cellular components or other contaminants, for example, other nucleic acids or cellular proteins, by standard techniques, including alkaline / SDS treatment, CsCl bandage, column chromatography, agarose gel electrophoresis , and other methods well known in the art. See, Ausubel, F., et al., Ed. Current Protocols in Molecular Biology, Greene Publishing & Wiley Interscience, New York (1987).
[0159] Expression in NS0 cells is described, for example, by 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, by Durocher, Y., et al., Nucl. Acids. Res. 30 (2002) E9. The cloning of variable domains is described by 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) 7787. A preferred transient expression system (HEK 293) is described by Schlaeger, E.-J., and Christensen, K., in Cytotechnology 30 (1999) 71-83 and by Schlaeger, E.- J., in J. Immunol.Methods 194 (1996) 191-199.
[0160] The nucleic acid molecules that encode variants
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61/91 of the anti-CSF-1R antibody amino acid sequence are prepared by a series of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring variant amino acid sequences) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR and cassette mutagenesis mutagenesis of a previously prepared variant or a non-variant version of the humanized anti-CSF-1R antibody.
[0161] The heavy and light chain variable domains according to the invention are combined with the promoter sequences, translation initiation, constant region, 3 'untranslated region, polyadenylation and transcription termination to form the expression vector construct . The heavy and light chain expression constructs can be combined into a single vector, co-transfected, transfected in series, or transfected separately into host cells that are then fused to form a single host cell expressing both chains.
[0162] In another aspect, the present invention provides a composition, for example, a pharmaceutical composition, containing one or a combination of monoclonal antibodies, or the antigen-binding portion thereof of the present invention, formulated together with a pharmaceutically carrier acceptable.
[0163] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonics and absorption / retarding agents, and the like that are physiologically compatible. Preferably, the vehicle is suitable for injection or infusion.
[0164] A composition of the present invention can be
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62/91 administered by a variety of methods known in the art. As will be appreciated by the person skilled in the art, the route and / or method of administration will vary, depending on the desired results.
[0165] Pharmaceutically acceptable vehicles include sterile aqueous solutions or dispersions and sterile powder for the preparation of sterile injectable solutions or dispersions. The use of such means and agents for pharmaceutically active substances is known in the art. In addition to water, the vehicle can be, for example, an isotonic buffered saline solution.
[0166] 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 in pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art. skilled in the matter.
[0167] The actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention can be modified so as to obtain an amount of active ingredient that is effective in achieving the desired therapeutic response in a specific patient, composition and mode of administration, without being toxic to the patient (effective amount). The dosage level selected will depend on several factors including the pharmacokinetic activity of the specific compositions of the present invention being used, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the specific compound being used, other drugs, compounds and / or materials used in combination with the specific compositions being used, the age, sex, weight, condition, general health and previous medical history of the patient to be treated, and similar factors well known in the arts medical.
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63/91 [0168] The invention comprises the use of the antibodies according to the invention for the treatment of a patient suffering from cancer, especially colon, lung or pancreatic cancer.
[0169] The invention also comprises a method for treating a patient suffering from such diseases.
[0170] The invention further provides a method for the manufacture of a pharmaceutical composition comprising an effective amount of an antibody according to the invention, together with a pharmaceutically acceptable carrier and the use of the antibody according to the invention for such a method.
[0171] The invention further provides the use of an antibody according to the invention in an amount effective for the manufacture of a pharmaceutical agent, preferably together with a pharmaceutically acceptable carrier, for the treatment of a patient suffering from cancer.
[0172] The invention also provides the use of an antibody according to the invention in an amount effective for the manufacture of a pharmaceutical agent, preferably together with a pharmaceutically acceptable carrier, for the treatment of a patient suffering from cancer.
[0173] The following examples, listing of sequences and figures are provided to aid the understanding of the present invention, the true scope of which is set out in the attached claims. It should be understood that modifications can be made to established procedures without departing from the spirit of the present invention.
Strings Description
SEQ ID NO: 1 CDR3 of the heavy chain, Mab2F11
SEQ ID NO: 2 CDR2 heavy chain, Mab2F11
SEQ ID NO: 3 CDR1 of the heavy chain, Mab2F11
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2F11-c11
SEQ ID NO: 4
SEQ ID NO: 5
SEQ ID NO: 6
SEQ ID NO: 7
SEQ ID NO: 8
SEQ ID NO: 9
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
SEQ ID NO: 20
SEQ ID NO: 21
SEQ ID NO: 22
SEQ ID NO: 23
Light chain CDR3, Mab 2F11
Light chain CDR2, Mab 2F11
Light chain CDR1, Mab 2F11 heavy chain variable domain, Mab 2F11 light chain variable domain, Mab 2F11
Heavy chain CDR3, Mab 2E10
Heavy chain CDR2, Mab 2E10
Heavy chain CDR1, Mab 2E10
Light chain CDR3, Mab 2E10
Light chain CDR2, Mab 2E10
Light chain CDR1, Mab 2E10 heavy chain variable domain, Mab 2E10 light chain variable domain, Mab 2E10
Heavy chain CDR3, hMab 2F11-c11
Heavy chain CDR2, hMab 2F11-c11
Heavy chain CDR1, hMab 2F11-c11
Light chain CDR3, hMab 2F11-c11
Light chain CDR2, hMab 2F11-c11
Light chain CDR1, hMab 2F11-c11 heavy chain variable domain, hMab
SEQ ID NO: 24 light chain variable domain, hMab 2F11c11
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
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SEQ ID NO: 30
SEQ ID NO: 31
2F11-d8
SEQ ID NO: 32 d8
SEQ ID NO: 33
SEQ ID NO: 34
SEQ ID NO: 35
SEQ ID NO: 36
SEQ ID NO: 37
SEQ ID NO: 38
SEQ ID NO: 39
2F11-e7
SEQ ID NO: 40 e7
SEQ ID NO: 41
SEQ ID NO: 42
SEQ ID NO: 43
SEQ ID NO: 44
SEQ ID NO: 45
SEQ ID NO: 46
SEQ ID NO: 47
2F11-f12
SEQ ID NO: 48 f12
SEQ ID NO: 49
SEQ ID NO: 50
SEQ ID NO: 51
Light chain CDR1, hMab 2F11 -d8 heavy chain variable domain, hMab light chain variable domain, hMab 2F11CDR3 heavy chain, hMab 2F11-e7
Heavy chain CDR2, hMab 2F11-e7
Heavy chain CDR1, hMab 2F11-e7
Light chain CDR3, hMab 2F11-e7
Light chain CDR2, hMab 2F11-e7
Light chain CDR1, hMab 2F11-e7 heavy chain variable domain, hMab light chain variable domain, hMab 2F11CDR3 heavy chain, hMab 2F11-f12
Heavy chain CDR2, hMab 2F11-f12
Heavy chain CDR1, hMab 2F11-f12
Light chain CDR3, hMab 2F11 -f12
Light chain CDR2, hMab 2F11 -f12
Light chain CDR1, hMab 2F11-f12 heavy chain variable domain, hMab light chain variable domain, hMab 2F11CDR3 heavy chain, hMab 2F11-g1
Heavy chain CDR2, hMab 2F11-g1
Heavy chain CDR1, hMab 2F11-g1
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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 IgG1 derived human heavy chain constant region
SEQ ID NO: 59 IgG1-derived human heavy chain constant region mutated in L234A and L235A
SEQ ID NO: 60 IgG4 derived human heavy chain constant region
SEQ ID NO: 61 S228P mutated IgG4 derived human heavy chain constant region
SEQ ID NO: 62 CSF-1 R wild-type human (wt CSF-1 R) SEQ ID NO: 63 CSF-1 R human mutant L301S Y969F SEQ ID NO: 64 Extracellular domain of human CSF-1 R SEQ ID NO: 65 delD4 fragment of human CSF-1 R SEQ ID NO: 66 fragment D1-D3 of human CSF-1R SEQ ID NO: 67 signal peptide SEQ ID NO: 68 Primer SEQ ID NO: 69 Heavy chain CDR3, Mab 1G10 SEQ ID NO: 70 Heavy chain CDR2, Mab 1G10 SEQ ID NO: 71 Heavy chain CDR1, Mab 1G10 SEQ ID NO: 72 Light chain CDR3, Mab 1G10
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1G10
SEQ ID NO: 73
SEQ ID NO: 74
SEQ ID NO: 75
SEQ ID NO: 76
SEQ ID NO: 77
SEQ ID NO: 78
SEQ ID NO: 79
SEQ ID NO: 80
SEQ ID NO: 81
SEQ ID NO: 82
SEQ ID NO: 83
SEQ ID NO: 84
Light chain CDR2, Mab 1G10
Light chain CDR1, Mab 1G10 heavy chain variable domain, Mab light chain variable domain, Mab 1G10
Heavy chain CDR3, Mab 2H7
Heavy chain CDR2, Mab 2H7
Heavy chain CDR1, Mab 2H7
Light chain CDR3, Mab 2H7
Light chain CDR2, Mab 2H7
Light chain CDR1, Mab 2H7 heavy chain variable domain, Mab 2H7 light chain variable domain, Mab 2H7 [0174] The following examples, sequence listing and figures are provided to aid the understanding of the present invention, the true scope is established in the appended claims. It should be understood that modifications can be made to established procedures without departing from the spirit of the present invention.
Description of the Figures [0175] Figure 1- Inhibition of the growth of BeWo tumor cells in 3D culture under treatment with different monoclonal antibodies anti-CSF-1R at a concentration of 10pg / ml. X-axis: average relative light units (RLU) normalized by the viability corresponding to the ATP content of the cells (CellTiterGlo assay). Y-axis: probes tested: Minimum Medium (0.5% FBS), mouse IgG1 (mIgG1, 10pg / ml), mouse IgG2a (mIgG2a 10pg / ml), CSF-1 only, Mab 2F11, Mab 2E10, Mab2H7, Mab1G10 and SC 2 -4a5. The highest inhibition of CSF-1-induced growth was observed with the anti-CSF-1R antibodies according to the invention.
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68/91 [0176] Figure 2a Biacore sensogram of the binding of different anti-CSF-1R antibodies to the delD4 fragment of immobilized human CSF-1R (comprising extracellular subdomains D1-D3 and D5) (SEQ ID NO: 65) (y axis : Binding signal in Response Units (RU), baseline = 0 RU, x-axis: time in seconds (s)): Although antibodies Mab 3291 and sc 2-4a5 clearly show binding to this delD4 fragment, antibodies according to the invention, for example Mab 2F11, and Mab 2E10, did not bind to the delD4 fragment of CSF-1R. The anti-CCR5 m <CCR5> Pz03.1 C5 control antibody also does not bind to the delD4 fragment of CSF-1R.
[0177] Figure 2b Biacore sensogram of the binding of different anti-CSF-1R antibodies to the extracellular domain of the immobilized human CSF-1R (CSF-1R-ECD) (comprising the extracellular subdomains D1-D5) (SEQ ID NO: 64) ( y axis: link signal in Response Units (RU), baseline = 0 RU, x axis: time in seconds (s)):
[0178] All anti-CSF-1R antibodies show binding to CSF1R-ECD. The anti-CCR5 m <CCR5> control antibody Pz03.1C5 does not bind to CSF-1R-ECD.
[0179] Figure 2c Biacore sensogram of the binding of different anti-CSF-1R antibodies to the delD4 fragment of immobilized human CSF-1R (comprising the extracellular subdomains D1-D3 and D5) (SEQ ID NO: 65) (y-axis: binding in Response Units (RU), baseline = 0 RU, x-axis: time in seconds (s)): Mab 1G10, Mab 2H7 and humanized hMab 2F11-e7 did not bind to the delD4 fragment of CSF-1R. The anti-CCR5 m <CCR5> control antibody Pz03.1C5 also does not bind to the delD4 fragment of CSF-1R.
[0180] Figure 2d Biacore Sensogram of the binding of different anti-CSF-1R antibodies to the Extracellular Domain of the immobilized human CSF-1R (CSF-1R-ECD) (comprising the extracellular subdomains
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D1-D5) (SEQ ID NO: 64) (y axis: binding signal in Response Units (RU), baseline = 0 RU, x axis: time in seconds (s)): All anti-CSF- 1R - Mab 1G10, Mab 2H7 and humanized hMab 2F11-e7 exhibited binding to CSF-1R -ECD. The control anti-CCR5 m antibody <CCR5> Pz03.1C5 did not bind to CSF-1R-ECD.
[0181] Figure 2e Biacore Sensogram of the binding of different anti-CSF-1R antibodies to the delD4 fragment of immobilized human CSF-1R (comprising the extracellular subdomains D1-D3 and D5) (SEQ ID NO: 65) (y-axis: Response Unit (RU) binding, baseline = 0 RU, x-axis: time in seconds (s)): All anti-CSF-1R antibodies - 1.2.SM, CXIIG6, ab10676 and MAB3291 exhibited binding to the delD4 fragment of CSF-1R. The anti-CCR5 m <CCR5> control antibody Pz03.1C5 also does not bind to the delD4 fragment of CSF-1R.
[0182] Figure 2f Biacore sensogram of the binding of different anti-CSF-1R antibodies to the extracellular domain of the immobilized human CSF-1R (CSF-1R-ECD) (comprising the extracellular subdomains D1-D5) (SEQ ID NO: 64) ( y-axis: binding signal in Response Units (RU), baseline = 0 RU, x-axis: time in seconds (s)): All anti-CSF-1R antibodies - 1.2.SM, CXIIG6, ab10676 and MAB3291 exhibited connection to CSF-1R-ECD. The anti-CCR5 m <CCR5> control antibody Pz03.1C5 does not bind to CSF-1R-ECD.
[0183] Figure 3a-d levels of CSF-1 in a cynomolgus monkey after the application of different dosages of anti-CSF-1R antibody according to the invention.
[0184] Figure 4 In vivo efficacy in inhibiting tumor growth of anti-CSF-1R antibodies according to the invention in BT20 breast cancer xenograft.
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Example 1
Generation Of A Hybridoma Cell Line That Produces Anti-CSF-1R Antibodies
NMRI Mouse Immunization Procedure [0185] NMRI mice were immunized with an expression vector pDisplay® (Invitrogen, USA), which encodes the extracellular domain of huCSF-1R, using electroporation. Each mouse was immunized 4 times with 100pg of DNA. When anti-huCSF-1R antibody titers in serum were found to be sufficient, the mice were additionally subjected to intensifying immunizations for once with 50 pg of a 1: 1 mixture of huCSF-1R ECD / huCSF-1R ECDhuFc in 200 pl of PBS intravenously (iv) 4 and 3 days before fusion.
Antigen-Specific ELISA [0186] Anti-CSF-1R titers in the serum of immunized mice were determined by antigen-specific ELISA.
[0187] 0.3 pg / ml of huCSF-1R-huFc chimera (soluble extracellular domain) was captured on a streptavidin plate (Maxisorb; MicroCoat, DE, Cat.No. 11974998 / MC1099) with 0.1 mg / ml of biotinylated anti Fcy (Jackson ImmunoResearch, Cat.No. 109-066-098) and mouse anti-IgG F (ab ') 2 conjugated to horseradish peroxidase (HRP) - (GE Healthcare, United Kingdom, Cat.No .NA9310V) diluted 1/800 in PBS / 0.05% Tween20 / and 0.5% BSA was added. All collected sera were diluted 1/40 in PBS / 0.05% Tween20 / 0.5% BSA and diluted in series until 1/1638400. The diluted sera were added to the wells. A pre-collected serum was used as a negative control. A serial dilution of Mab3291 mouse anti-human CSF-1R (R&D Systems, UK) from 500 ng / ml to 0.25 ng / ml was used as a positive control. All components were incubated together for 1.5 hours, the wells were
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71/91 washed 6 times with PBST (PBS / 0.2% Tween20) and the assays were developed ABTS® solution prepared at the time of use (1 mg / ml) (ABTS: 2,2'-azino bis (3 -ethylbenzothiazoline-6-sulfonic) for 10 minutes at room temperature, absorbance was measured at 405 nm.
Hybridoma Generation [0188] Mouse lymphocytes can be isolated and fused with a mouse myeloma cell line using standard PEG-based protocols to generate hybridomas. The resulting hybridomas are then screened for the production of antigen-specific antibodies. For example, suspensions of a single cell type of lymphocytes derived from the spleen from immunized mice are fused to P3X63Ag8.653 mouse myeloma cells (ATCC, CRL-1580) that do not secrete Ag8 with 50% PEG. The cells are plated at approximately 104 in a 96-well flat-bottomed microtiter plate, followed by incubation for about two weeks in selective media. The individual wells are then screened by ELISA for the presence of human IgG and IgM antiCSF-1R monoclonal antibodies. Once extensive hybridoma growth occurs, antibody-secreting hybridomas are re-plated, re-screened, and if still positive for human IgG anti-CSF-1R monoclonal antibodies, they can be subcloned by FACS. The stable subclones are then cultured in vitro to produce the antibody in tissue culture medium for characterization. The antibodies according to the invention can be selected using the determination of the binding of the anti-CSF-1R antibody to the human CSF-1R delD4 fragment and to the human CSF-1R Extracellular Domain (CSF-1R-ECD) as described in the Example 4, as well as determining the growth inhibition of NIH3T3 cells transfected with wild-type CSF-1R (ligand-dependent signaling) or L301S Y969F mutant (ligand-independent signaling) mutant under treatment
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72/91 with anti-CSF-1R monoclonal antibodies as described in Example 5.
Hybridoma Culture [0189] The generated muMAb hybridomas were grown in RPMI 1640 medium (PAN - catalog no. (Cat. No.) PO4-17,500) supplemented with 2 mM L-glutamine (GIBCO - Cat. No. 35050- 038), 1 mM Na-Pyruvate (GIBCO - Cat. No. 11360-039), 1x NEAA (GIBCO - Cat. No. 11140. -035), 10% SBF (PAA - Cat. No. A15 -649), 1x Pen Strep (Roche - Cat. No. 1074440), 1x Nutridoma CS (Roche Cat. No. 1363743), 50 mM Mercaptoethanol (GIBCO - Cat. No. 31350-010) and 50 U / ml of mouse IL 6 (Roche - Cat. No. 1 444 581), at 37 ° C and 5% CO2. Some of the resulting mouse antibodies have been humanized (for example, Mab 2F11) and have been expressed recombinantly.
Example 2
Inhibition of CSF-1 Binding to CSF-1R (ELISA) [0190] When establishing this assay, it was possible to first test the binding of anti-CSF-1R antibody to CSF-1R-ECD followed by the detection of ligands not bound to either receptor by antibodies that displace ligands and by anti-CSF-1R antibodies that inhibit dimerization. The test was performed on 384-well microtiter plates (MicroCoat, DE, Cat.N ° 464,718) at room temperature. After each incubation step, the plates were washed 3 times with PBST.
[0191] At first, the plates were coated with 0.5 mg / ml of biotinylated goat anti-Fcy F (ab ') 2 (Jackson ImmunoResearch., Cat. No. 109006-170) for 1 hour (h).
[0192] Subsequently, the wells were blocked with PBS supplemented with 0.2% Tween-20® and 2% BSA (Roche Diagnostics GmbH, DE) for 0.5 h. 75 ng / ml of the huCSF-1R-huFc chimera (which constitutes the soluble extracellular dimeric domain of huCSF-1R) was immobilized on the plate for 1 h. Then, dilutions of purified antibodies in PBS / 0.05% Tween20 / 0.5%
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BSA were incubated for 1 h. After adding a mixture of 3 ng / ml CSF-1 (Biomol, DE, Cat.No.60530), 50 ng / ml biotinylated anti-CSF-1 clone BAF216 (R&D Systems, UK) and streptavidin- HRP diluted 1: 5000 (Roche Diagnostics GmbH, DE, Cat.No.11089153001) for 1 h, the plates were washed 6 times with PBST. The anti-CSF-1R SC 2-4a5 (Santa Cruz Biotechnology, USA), which inhibits ligand-receptor interaction, was used as a positive control. The plates were developed with substrate solution “BM Blue POD® 'prepared at the time of use (BM Blue®: 3,3'-5,5'-tetramethylbenzidine, Roche Diagnostics GmbH, DE, Cat.N ⁰ 11484281001) for 30 minutes at room temperature. Absorbance was measured at 370 nm. A decrease in absorbance is found if the anti-CSF-1R antibody causes the release of CSF-1 from the dimeric complex. All anti-CSF-1R antibodies exhibited significant inhibition of the interaction of CSF-1 with CSF-1R (see Table 1). The anti-CSF-1R SC 2-4a5 (Santa Cruz Biotechnology, USA, see also Sherr, CJ et al., Blood 73 (1989) 1786-1793), which inhibits ligand-receptor interaction, was used as a control of reference.
Table 1
IC50 Values Calculated For CSF-1 / CSF-1R Interaction Inhibition
CSF-1R Mab IC50 of Inhibition CSF-1 / CSF-1R [ng / ml] Mab 2F11 19.3 Mab 2E10 20, 6 Mab2H7 18.2 Mab1G10 11.8 SC-2-4A5 35.2
Example 3
Inhibition of CSF-1-Induced CSF-1R Phosphorylation In Recombinant NIH3T3-CSF-1R Cells [0193] 4.5 x 10 ³ NIH 3T3 cells, infected by retrovirus with a full-length CSF-1R expression vector were cultured in
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74/91 DMEM medium (Cat PAA. No. E15-011), 2 mM L-glutamine (Sigma, Cat No. G7513), 2 mM sodium pyruvate, 1x non-essential amino acids, 10% FKS (PAA, Cat No. A15-649) and 100 pg / ml PenStrep (Sigma, Cat No. P4333 [10mg / ml]) until they reach confluence. Then, the cells were washed with DMEM without serum (PAA Cat No. EE-0-011) supplemented with sodium selenite [5ng / ml] (Sigma, Cat No. S9133), transferrin [10pg / ml] (Sigma , Cat No. T8158), BSA [400pg / ml] (Roche Diagnostics GmbH, Cat No. 10,735,078), 4mM L-glutamine (Sigma, Cat No.G7513), 2mM sodium pyruvate ( Gibco, Cat No. 11,360), 1x non-essential amino acids (Gibco, Cat No. 11,140035), 2-mercaptoethanol [0.05 mm] (Merck, Cat No. M7522), 100pg / ml and PenStrep (Sigma, Cat No. P4333) and incubated in 30 pL of the same medium for 16 hours to allow positive regulation (up-regulation) of the recipient. 10 µl of diluted anti-CSR-1R antibodies were added to the cells for 1.5 h. Then, the cells were stimulated with 10 µl of 100 ng / ml hum-CSF-1 (Biomol Cat No. 60530) for 5 min. After incubation, the supernatant was removed, cells were washed twice with 80 µl of cold PBS and 50 µl of cold lysis buffer prepared at the time of use (150 mM NaCl / pH 7.5 Tris 20 mM / EDTA 1 mM / EGTA 1 mM / 1% Triton X-100/1 protease inhibitor tablet (Roche Diagnostics GmbH Cat No. 1836170) per 10 ml of buffer / 10 pl / ml of phosphatase inhibitor cocktail (Sigma Cat No. P -2850, 100x stock solution / 10pl / ml protease inhibitor 1 (Sigma Cat No. P-5726, 100x stock solution) / 10pl / ml 1M NaF) was added. After 30 minutes on ice, the plates were shaken vigorously on a plate shaker for 3 minutes and then centrifuged for 10 minutes at 2200 rpm (Heraeus Megafuge 10).
[0194] The presence of phosphorylated receptor and total CSF-1 in the cell lysate was analyzed by ELISA. For the detection of the phosphorylated receptor, the R&D Systems kit (Cat. No. DYC3268-2) was used according to the
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75/91 supplier instructions. For the detection of total CSF-1R, 10 μΙ of the lysate was immobilized on a plate by using a capture antibody contained in the kit. Thereafter, the biotinylated anti-CSF-1R antibody diluted 1: 750 BAF329 (R&D Systems) and the 1: 1000 diluted streptavidin-HRP conjugate were added. After 60 minutes, the plates were developed with a freshly prepared ABTS ^® solution and absorbance was detected. The data were calculated as% positive control without the antibody and the value of the phosphorus / total receptor ratio expressed. The negative control was defined without the addition of M-CSF-1. The anti-CSF-1R SC 2-4a5 (Santa Cruz Biotechnology, USA, see also Sherr, CJ et al., Blood 73 (1989) 1786-1793), which inhibits ligand-receptor interaction, was used as a control of reference.
Table 2
IC50 Values Calculated For Inhibition Of Receptor Phosphorylation
CSF-1.
CSF-1R Mab IC50 of CSF-1R phosphorylation [ng / ml] Mab2F11 219.4 Mab 2E10 752.0 Mab2H7 703.4 1G10 Mab 56.6 SC-2-4A5 1006.6
Example 4
Determination of Binding of Anti-CSF-1R Antibodies to the delD4 Fragment of Human CSF-1R and to the Extracellular Domain of Human CSF-1R (CSF-1R-ECD)
Preparation of the Human CSF-1R Extracellular Domain (CSF-1R-ECD) (Comprising the D1-D5, hCSF-1R-ECD Extracellular Subdomains) of SEQ ID NO: 64:
[0195] pCMV-preS-Fc-hCSF-1R-ECD (7836bp) encodes the complete EDC of human CSF-1R (SEQ ID NO: 64) fused C-terminally to a
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76/91 PreScission protease cleavage site, followed by aa 100-330 from human IgG1 and a 6xHis-7ag, under the control of the CMV promoter. The natural signal peptide was varied by inserting amino acids G and S after the first M, in order to create a BamHI restriction site.
Preparation of the DelD4 Fragment of Human CSF-1R (Comprising Extracellular Subdomains D1-D3 and D5, hCSF-1R-delD4) of SEQ ID NO: 65: 65:
[0196] hCSF1R-delD4-V1-PreSc-hFc-His was cloned from pCMV-preS-Fc-hCSF-1R-ECD using the “Stratagene QuikChange XL” mutagenesis protocol using delD4-for with the sequence CACCTCCATGTTCTTCCGGTACCCCCCAGAGGTAAG (SEQ ID NO: 68) as a forward primer and delD4-rev with the complementary reverse sequence as the reverse primer. A variation of the protocol published in Bio7echniques 26 (1999) 680 was used to extend both primers in separate reactions in three cycles that precede the standard Stratagene protocol:
[0197] Two separate 50 pL reaction mixtures were created according to the manufacturer's manual, each containing 10 ng of plasmid pCMV-preS-Fc-hCSF1R-ECD as the template and 10 pM of one of the primers, delD4- for or delD4-rev, and 0.5 μ Pfu of DNA polymerase as supplied with the kit. Three PCR cycles at 95 ° C - 30 sec / 55 ° C - 60 sec / 68 ° C 8 min were performed, then 25 μl each of both reaction mixtures were combined in a new tube and 0.5 μL Pfu of fresh DNA polymerase was added. The regular PCR protocol with 18 temperature cycles as specified by Stratagene in the kit manual was performed, followed by 2 hours of final digestion with the restriction enzyme Dpn1 provided with the kit. Clones that carry the deletion were detected by digestion with CelII and NotI and verified by sequencing.
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77/91 [0198] The protein was prepared by transient transfection in the HEK293 cell system in FreeStyle suspension (Invitrogen) according to the manufacturer's specifications. After 1 week 500 ml of the supernatant was filtered and loaded onto a 1ml HiTrap MabSelect Xtra protein GE column (GE Healthcare) (0.2 ml / min). The colomn was washed first with PBS, then with 50 mM Tris / 150 mM NaCl / 1 mM EDTA / pH 7.3. 75 pL of Protease PreScission (GE # 27-0843-01) diluted in 375 pL of the same buffer was loaded onto the column and the closed column was incubated overnight at 4 ° C. The column was mounted on top of a 1 ml GSTrap FF column (GE helthcare) and the desired protein was eluted (0.2 ml / min, 0.2 ml fractions). The pooled fractions were concentrated from 1.8 ml to 0.4 ml by ultrafiltration using a 3k Nanosep centrifuge and were then chromatographed on HR S200 SEC in PBS (0.5 ml / min).
[0199] The delD4 fragment of human CSF-1R was obtained in two fractions as a dimeric molecule (pool1, V = 1.5 ml; c = 0.30 mg / ml; apparent mass in the SDS-PAGE 83 kDa, 62 kDa reduced) and as a monomer (pool 2, V = 1.4 ml, C = 0.25 mg / ml apparent mass in the 62 kDa SDS-PAGE). The dimer shape was used for all experiments.
Determination of the Binding of Anti-CSF-1R Antibodies to the delD4 Fragment of Human CSF-1R And to the Extracellular Domain of Human CSF-1R (CSF-1R-ECD) (Binding Signals as Response Units (RU)):
Instrument: Biacore T100 (GE Healthcare)
Software:
T100 Control, Version 2.0.1 T100
Evaluation,, Version 2.0.2
Test format:
Chip: CM5
Temperature: 25 ° C
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78/91 [0200] The CSF-1R fragments were immobilized by amine coupling. To compare the binding of different antiCSF-1R antibodies according to the invention, a concentration of the test antibody was injected. Mab3291 anti-CSF-1R (R & D-Systems) and SC 2-4a5 (Santa Cruz Biotechnology, USA, see also: Sherr, CJ et al., Blood 73 (1989) 17861793), were used as reference controls, the anti -CCR5 m <CCR5> Pz03.1C5 (deposited as DSM ACC 2683 on 08/18/2004 in DSMZ) was used as a negative control, all under the same conditions as the anti-CSF-1R antibodies according to the invention.
Amine Coupling of CSF-1R Fragments:
[0201] Standard amine coupling according to manufacturer's instructions: Running buffer: PBS-T (Roche: 11 666 789 + 0.05% Tween-20: 11 332 465), activation by EDC / NHS, injection of the delD4 fragment of human CSF-1R (comprising the extracellular subdomains D1-D3 and D5) (SEQ ID NO: 65) and the Extracellular Domain of human CSF-1R (CSF-1R-ECD) (comprising the subdomains extracellular D1-D5) (SEQ ID NO: 64) for 600 seconds at a flow rate of 10pl / min; diluted in NaAc coupling buffer, pH 5.0, c = 10 µg / ml; finally the remaining activated carboxyl groups were blocked by the injection of 1 M ethanolamine.
Binding of <CSF-1 R> Mab 2F11, Mab 2E10, Mab 3291 and sc2-4a5 and other anti-CSF-1R antibodies to the delD4 fragment of human CSF-1R and extracellular domain of human CSF-1R (CSF-1R-ECD ) at 25 ° C:
[0202] Running buffer: PBS-T (Roche: 11 666 789 + 0.05% Tween 20: 11 332 465).
Analyte sample [0203] The binding was measured at a flow rate of 30 pL / min by an injection of the analyte with a concentration c = 10 nM. (for Mab 1G10,
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Mab 2H7 and hMab 2F11-e7 humanized in a second experiment). Each injection was 700 seconds long, followed by a 180 second dissociation phase. The final regeneration was carried out after each cycle, using 50 mM NaOH, contact time of 60 seconds, flow rate of 30 pL / min.
[0204] The signals were measured by a reference point 10 seconds after the end of the injection. The reference signals (signals from a blank reference cell flow (treated with EDC / NHS and ethanolamine, only) have been subtracted to give the binding signals (such as RU). switched on were slightly below 0 (Mab 2F11 = -3; Mab 2E10 = -2; Mab 1G10 = - 6, Mab 2H7 = -9; and hMab 2F11 -e7 humanized = -7) the values were set to 0.
Table 3A
Linking Mabs <CSF-1 R> To The DelD4 Fragment Of Human CSF-1R And
CSF-1R-ECD E Ratio at 25 ° C, Measured by SPR
Link to delD4 [UK] Connection to CSF-1Recd [RU] Relationship of the binding of anti-CSF1R antibodies to the delD4 fragment of CSF1R / CSF-1R-ECD Mab 3291 1015 627 1015/627 = 1.61 sc2-4A5 374 249 374/249 = 1.50 Mab 2F11 0 176 0/176 = 0 hFab 2F11-e7 0 237 0/237 = 0 Mab 2E10 0 120 0/120 = 0 1G10 Mab 0 2708 0/2708 = 0 Mab2H7 0 147 0/147 = 0 m <CCR5> Pz03,1C5 2 5 -
[0205] Mab 2F11 and Mab 2E10 exhibited connection to the Domain
Extracellular human CSF-1R (CSF-1R-ECD) (see Fig. 2b), but no binding was detected to the delD4 CSF-1R fragment. (see fig. 2a).
[0206] Sc2-4a5 and MAB3291 exhibited a connection to CSF-1R-ECD and delD4 (see figs. 2b and 2a).
[0207] Therefore, the binding relationship of anti-CSF1R antibodies
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Mab 2F11 and Mab 2E10: delD4 fragment from CSF1R / CSF-1R-ECD were clearly less than 1:50 (= 0.02), whereas the binding ratio of MAB3291 and SC2-4a5 antibodies was 1.61 and 1 , 50, respectively, and were well above 1:50 (= 0.02). The negative control antibody m <CCR5> Pz03.1C5 showed no binding (as expected).
[0208] Mab 1G10, Mab 2H7 and humanized hMab 2F11-e7 exhibited binding to the human CSF-1R Extracellular Domain (CSF-1R-ECD) (see Fig. 2d), but no binding was detected to the delD4 fragment of CSF1R. (see fig. 2c) Therefore, the binding ratio of anti-CSF1R, Mab 1G10, Mab 2H7 and humanized hMab 2F11-e7 antibodies: delD4 fragment of CSF1R / CSF-1R-ECD was clearly less than 1:50 (= 0 , 02).
[0209] In an additional experiment the anti-CSF-1 R 1,2.SM antibodies (CSF-1R antibody that causes the ligand to be displaced described in WO2009026303), CXIIG6 (CSF-1R antibody that causes the ligand to be displaced in WO 2009/112245) and the goat polyclonal antibody antiCSF-1R ab10676 (Abcam) were investigated. The anti-CSF-1R antibody Mab3291 (R&D Systems) was used as a reference control. The antiCCR5 m <CCR5> Pz03.1C5 (deposited as DSM ACC 2683 on 08/18/2004 at DSMZ) was used as a negative control.
Table 3B
Linking Mabs <CSF-1 R> To The DelD4 Fragment Of Human CSF-1R And
CSF-1R-ECD E Ratio at 25 ° C, Measured by SPR
Link to delD4 [UK] Connection to CSF1R-ECD [RU] Relationship of the binding of anti-CSF1R antibodies to the delD4 fragment of CSF1R / CSF-1R-ECD MAB3291 1790 1222 1790/1222 = 1.47 1.2, SM 469 704 469/704 = 0.67 CXIIG6 1983 1356 1983/1356 = 1.46 ab10676 787 547 787/547 = 1.44 m <CCR5> Pz03,1C5 0 0 -
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81/91 [0210] Antibodies 1.2.SM, CXIIG6, ab10676 and MAB3291 exhibited binding to CSF-1R-ECD and delD4 (see figs. 2f and 2e).
[0211] The binding ratio of the 1.2.SM, CXIIG6, ab10676 and MAB3291 antibodies was highly greater than 1:50 (= 0.02). The negative control antibody m <CCR5> Pz03.1C5 showed no binding (as expected).
Example 5
Growth Inhibition of Recombinant NIH3T3-CSF-1R Cells in 3D Culture Under Treatment with Anti-CSF-1R Monoclonal Antibodies (CellTiterGlo Assay) [0212] NIH 3T3 cells, infected using retrovirus with an expression vector for the wild type CSF-1R total length (SEQ ID NO: 62) or CSF-1R mutant L301S Y969F (SEQ ID NO: 63), were cultured in DMEM medium with high glucose concentration (PAA, Pasching, Austria) supplemented with 2 mM of L-glutamine , 2 mM sodium pyruvate and non-essential amino acids and 10% fetal bovine serum (Sigma, Taufkirchen, Germany) on poly-HEMA (poly (2 hydroxyethylmethacrylate)) coated plates (Polysciences, Warrington, PA, USA)) for avoid sticking to the plastic surface. The cells were seeded in medium with the substitution of serum with sodium selenite 5ng / ml, transferrin 10 mg / ml, BSA 400 pg / ml and 0.05 mM 2-mercaptoethanol. When treated with 100 ng / ml huCSF-1 (Biomol, Hamburg, Germany) cells that express wtCSF-1R form dense spheres that grow three-dimensionally, a property that is called anchorage independence. These spheroids closely resemble the three-dimensional architecture and organization of solid tumors in situ. Recombinant CSF-1R mutant cells are capable of forming spheroids independent of the CSF-1 ligand. Spheroid cultures were incubated for 3 days in the presence of different
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82/91 antibody concentrations in order to determine an IC50 (Concentration with 50 percent inhibition of cell viability). The CellTiterGlo assay was used to detect cell viability by measuring the ATP content of cells.
Table 5A
CSF-1R Mab wtCSF-1R IC50 [g / ml] IC50 mutant CSF-1R [g / ml] Mab2F11 1.1 8.0 Mab 2E10 0.49 4.9 Mab2H7 0.31 5.3 1G10 Mab 0.29 14.2 SC 2-4A5 10.0 10.0
[0213] The reference control Mab 3291 R&D Systems showed no inhibition of the cell proliferation of the recombinant mutant CSF-1R.
[0214] In an additional experiment the anti-CSF-1R antibody according to the invention the hMab 2F11-e7 and anti-CSF-1R 1.2.SM antibodies (CSF-1R antibody that causes the displacement of the ligand described in WO2009026303), CXIIG6 (CSF-1R antibody that causes displacement of the ligand described in WO 2009/112245), polyclonal goat antiCSF-1 R ab10676 (Abcam) and SC 2-4a5 (Santa Cruz Biotechnology, USA see also, Sherr, CJ et al., Blood 73 (1989) 1786-1793) were investigated.
[0215] Spheroid cultures were incubated for 3 days in the presence of different concentrations of antibody in order to determine an IC30 (Concentration with 30 percent inhibition of cell viability). The maximum concentration was 20 pg / ml The CellTiterGlo assay was used to detect cell viability by measuring the ATP content of cells.
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Table 5B
CSF-1R Mab wtCSF-1R IC50 [g / ml] IC30 mutant CSF-1R [g / ml] hFab 2F11-e7 4.91 0.54 1.2.SM 1.19 > 20 pg / ml (-19% inhibition at 20 pg / ml = 19% stimulation) CXIIG6 > 20 pg / ml (21% inhibition at 20 pg / ml) > 20 pg / ml (-36% inhibition at 20 pg / ml = 36% stimulation) ab10676 14.15 > 20 pg / ml (0% inhibition at 20 pg / ml) SC 2-4A5 16.62 2.56
Example 6 Inhibition of BeWo Tumor Cell Growth in 3D Culture Under Treatment of Anti-CSF-1R Monoclonal Antibodies (CellTiterGlo Assay) [0216] BeWo Choriocarcinoma cells (ATCC CCL-98) were cultured in F12K (Sigma, Steinheim, Germany) supplemented with 10% FBS (Sigma) and 2 mM L-glutamine. 5 x 104 cells / well were seeded in 96-well plates coated with poly-HEMA (poly (2hydroxyethylmethacrylate)) containing F12K medium supplemented with 0.5% FBS and 5% BSA. Concomitantly, 200 ng / ml of huCSF-1 and 10 pg / ml of different anti-CSF-1R monoclonal antibodies were added and incubated for 6 days. The CellTiterGlo assay was used to detect cell viability by measuring the ATP content of cells in relative units of light (RLU). When the BeWo spheroid cultures were treated with different anti-CSF-1R antibodies (10 pg / ml) the inhibition of CSF1-induced growth was observed. To calculate antibody-mediated inhibition, the mean RLU value of unstimulated BeWo cells was subtracted from all samples. The mean RLU value of cells stimulated with CSF-1 was arbitrarily set at 100%. The average RLU values of cells stimulated with CSF-1 and treated with anti-CSF-1R antibodies were calculated in%
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CSF-1 stimulated RLUs. Table 6 shows the calculated data for inhibiting the growth of BeWo tumor cells in 3D culture under treatment with anti-CSF-1R monoclonal antibodies; Figs.1a and b represent the normalized mean RLU values.
Table 6
CSF-1R Mab % inhibition with 10pg / ml antibody concentration OnlyCSF-1 0 Mab 2F11 70 Mab 2E10 102 Mab2H7 103 Mab1G10 99 SC 2-4A5 39
Example 7
Inhibition of Differentiation of Human Macrophages Under Treatment with Monoclonal Anti-CSF-1R Antibodies (CellTiterGlo Assay) [0217] Human monocytes were isolated from peripheral blood using the cocktail “RosetteSep® Human Monocyte Enrichment Cocktail” (StemCell Tech - Cat No. 15028 ). The enriched monocyte populations were seeded in 96-well microtiter plates (2.5 x 10 ⁴ cells / well) in 100 pL of RPMI 1640 medium (Gibco -. Cat No. 31870) supplemented with 10% SBF (GIBCO -. Cat No. 011-090014M), 4 mM Lglutamine (GIBCO - Cat No. 25030) and 1x PenStrep (Roche - Cat No. 1074440), at 37 ° C and 5% CO2 in a humid atmosphere. When 150 ng / ml of huCSF-1 was added to the medium, a clear differentiation in adherent macrophages could
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85/91 be observed. This differentiation can be inhibited by the addition of anti-CSF-IR antibodies. In addition, monocyte survival is affected and can be analyzed by CellTiterGlo (CTG) analysis. From the concentration-dependent inhibition of monocyte survival by treatment with the antibody, an IC50 was calculated (see Table 7).
Table 7
CSF-1R Mab IC50 [g / ml] Mab2F11 0.08 Mab2E10 0.06 Mab2H7 0.03 Mab1G10 0.06 SC 2-4A5 0.36
[0218] In a separate test the humanized series versions of Mab 2 F11, for example, hMab 2F11-c11, hMab 2F11 -d8, hMab 2F11 -e7, hMab 2F11-f12, showed IC50 values of 0.07 pg / ml (hMab 2F11-c11),
0.07 pg / ml (hMab 2F11 -d8), 0.04 pg / ml (hMab 2F11 -e7) and 0.09 pg / ml (hMab
2F11-F12).
Example 8
Inhibition of Differentiation of Cinomolgos Macrophages Under Treatment With Anti-CSF-1R Monoclonal Antibodies (CellTiterGlo Assay) [0219] Cinomolgos monocytes were isolated from peripheral blood using the non-human primate CD14 microsphere kit (Miltenyi Biotec - Cat.N ⁰ 130-091-097) according to the manufacturers description. The enriched monocyte populations were seeded in 96-well microtiter plates (1-3 x 10 ⁴ cells / well) in 100 pL of RPMI 1640 medium (Gibco -. Cat No. 31870) supplemented with 10% SBF (GIBCO -. Cat No. 011-090014M), 4 mM L-glutamine (GIBCO - Cat No. 25030) and 1x PenStrep (Roche - Cat No. 1074440), at 37 ° C and 5% CO2 in an atmosphere
Petition 870190118206, of 11/14/2019, p. 93/105
86/91 wet. When 150 ng / ml of huCSF-1 was added to the medium, a clear differentiation in adherent macrophages could be observed. This differentiation can be inhibited by the addition of anti-CSF-1R antibodies. In addition, monocyte survival is affected and can be analyzed by CellTiterGlo (CTG) analysis. Viability was analyzed at a concentration of 5 pg / ml of antibody treatment (see Table 8).
Table 8
CSF-1R Mab % of survival % inhibition (of survival) = (100% -% survival) Mab 2F11 4 * 96 Mab 2E10 17 ** 83 Mab2H7 8 92 1G10 Mab 2 98 SC 2-4A5 31 69
* Average of four experiments (3 experiments using the murine mAb, 1 experiment using the chimeric mAb).
** Average of two experiments using only the murine mAb.
Example 9
Determination of the binding affinity of anti-CSF-1R antibodies to human CSF-1R
- Instrument: BIACORE® A100
- Chip: CM5 (Biacore BR-1006-68)
- Coupling: amine coupling
- Buffer: PBS (Biacore BR-1006-72), pH 7.4, 35 ° C [0220] For affinity measurements 36 pg / ml anti-mouse antibodies (goat, Jackson Immuno Reasearch JIR115-005-071 ) were coupled to the chip surface to capture antibodies against CSF-1 R. The CSF-1R Human Extracellular Domain (CSF-1R-ECD) (comprising the extracellular subdomains D1-D5) (SEQ ID: 64) (R & D- Systems 329-MR or
Petition 870190118206, of 11/14/2019, p. 94/105
87/91 subcloned pCMV-presS-HisAvitag-hCSF-1R-ECD) was added in various concentrations in the solution. The association was measured by an injection of CSF-1R for 1.5 minutes at 35 ° C; dissociation was measured by washing the chip surface with buffer for 10 minutes at 35 ° C. For the calculation of kinetic parameters the Langmuir model 1: 1 was used.
Table 9
Affinity Data Measured by SPR
CSF-1R Mab Kd (nM) ka (1 / Ms) kd (1 / s) t1 / 2 (min) Mab 2F11 0.29 1.77E ⁺⁰⁵ 5.18E ^-05 223 Mab 2E10 0.2 1.52E ⁺⁰⁵ 2.97E ^-05 389 Mab2H7 0.21 1.47E ⁺⁰⁵ 3.12E ^-05 370 1G10 Mab 0.36 1.75E ⁺⁰⁵ 6.28E ^-05 184
[0221] In a separate BIAcore binding assay using the CSF1R ECD (data not shown) some competition from the Mab 2F11 and Mab 2E10 antibodies with the Ab SC-2-4a5 antibody was displayed. However, Mab 2F11 / Mab 2E10 did not bind to the delD4 fragment of human CSF-1R, while Ab SC-2-4a5 did bind to this delD4 fragment (see Example 4 and Fig. 2a). Thus, the Mab 2F11 / Mab 2E10 binding region is clearly distinct from the Ab SC-2-4a5 binding region, but is probably located in a nearby area. In the competition trial both antibodies, Mab 2F11 and Mab 2E10, did not compete with R&D Systems' Mab3291 (data not shown).
Example 10
Determination of the binding affinity of anti-CSF-1R antibodies to the human CSF-1R fragment D1-D3
- Instrument: Biacore T100 (GE Healthcare)
- Software: T100 Control, Version 1.1.11
B3000 Evaluation, Version 4.01
Scrubber, Version 2.0a
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88/91
- Assay format: Chip: CM5-Chip [0222] The antibodies against CSF-1R were captured using coupled amine capture molecules. Using single-cycle kinetics, five increasing concentrations of the human CSF-1R fragment D1-D3 (SEQ ID NO: 66) were injected. The D1-D3 fragment of human CSF-1R was subcloned into the pCMV-presS-HisAvitag expression vector.
[0223] The anti-CSF-1R SC 2-4A5 (Santa Cruz Biotechnology, USA, see also Sherr, CJ et al., Blood 73 (1989) 1786-1793), which inhibits ligand-receptor interaction, and the Mab 3291 (R & D-Systems) were used as a reference control.
[0224] Capture molecules: mouse Fcy antibodies (goat, Jackson Reasearch Immuno JIR115-005-071) for the antibodies according to the invention and the R & D-Systems Mab 3291 controls and mouse anti-Fcy antibodies (from goat, Jackson Reasearch Immuno JIR112- 005071) for the anti-CSF-1R SC 2-4a5 reference control.
Capture Molecule Amine Coupling [0225] The standard amine coupling was made according to the manufacturer's instructions: Running buffer: HBS-N buffer, activation by EDC / NHS mixture, target for 2000 RU binder density; the captured Acs were diluted in NaAc coupling buffer, pH 4.5, c = 10 pg / ml; finally, the remaining activated carboxyl groups were blocked by the injection of 1M ethanolamine.
Kinetic Characterization of the Connection of D1-D3 Fragments of Human CSF-1R to MAbs <CSF-1 R> At 37 ° C [0226] Running buffer: PBS (Biacore BR-1006-72) [0227] Mabs capture <CSF -1 R> in 2-4 flow cells: 20 pL / min flow, 90 second contact time, c (Abs <CSF-1 R>) = 50 nM, diluted with running buffer + 1 mg / ml of BSA.
Petition 870190118206, of 11/14/2019, p. 96/105
89/91
Analyte sample [0228] Single cycle kinetics was measured at a flow rate of 30 pL / min for five consecutive injections of analyte with concentrations, c = 7.8, 31.25, 125 500 and 2000 nM, without regeneration . Each injection was 30 seconds long and followed by a dissociation phase of 120 seconds for the first four injections and, finally, 1200 seconds, for the highest concentration (= last injection).
[0229] Final regeneration was carried out after each cycle, using 10 mM Glycine pH 1.5 (Biacore BR-1003-54), contact time of 60 seconds, flow rate of 30 pL / min.
[0230] The kinetic parameters were calculated using the usual double reference (control reference: binding of the analyte to the capture molecule; flow cell: subdomain of CSF-1R in concentration “0” as white) and calculation with the model “kinetic titration 1: 1 connection with the project ”.
Table 10
Affinity Data for CSF-1R Fragment D1 D3 Binding
Human Measured By SPR
CSF-1R Mab Sub domain Kd (nM) ka (1 / Ms) kd (1 / s) t1 / 2 (min) Mab 2F11 D1-D3 no connection Mab 2E10 D1-D3 no connection Mab2H7 D1-D3 Not determined 1G10 Mab D1-D3 no connection SC-2-4A5 D1-D3 no connection R & D-Systems 3291 D1-D3 5.4 2.2E ⁺⁵ 1.2E ^-3 9.6
[0231] The antibodies Mab 2F11, Mab 2E10 and Mab 1G10 showed no binding to the human CSF-1R fragment D1-D3.
[0232] The Ab SC-2-4a5 reference control also did not bind to the human CSF-1 R fragment D1-D3.
Petition 870190118206, of 11/14/2019, p. 97/105
90/91 [0233] The 3291 R & D-Systems Mab reference control exhibited binding to the human CSF-1R fragment D1-D3.
Example 11 CSF-1 Level Increases During Inhibition Of CSF-1R In Cinomolgo Monkey [0234] Serum CSF-1 levels provide a pharmacodynamic marker of the neutralizing activity of CSF-1R of the human anti-CSF-1R dimerization inhibitor hMab 2F11-E7. One male and one female cynomolgus monkey per dosage group (1 and 10 mg / kg) were administered intravenously with the anti-CSF1R hMab 2F11-e7 antibody. Blood samples for analysis of CSF-1 levels were collected 1 week before treatment (pre-dose), 2, 24, 48, 72, 96, 168 hours after administration, and weekly for two additional weeks. CSF-1 levels were determined using a commercially available ELISA kit (Quantikine ^® M-CSF human), according to the manufacturer's instructions (R&D Systems, UK). CSF-1 levels were determined by comparing samples from a standard CSF-1 curve provided in the kit.
[0235] Administration of hMab 2F11-e7 induced a dramatic increase in CSF-1 by ~ 1000 times, which, depending on the dose administered, lasted 48 hr (1mg / kg) or 15 days (10mg / kg). Therefore, a dimerization inhibitor for CSF-1R offers the advantage of not competing directly with the ligand that has been dramatically regulated by binding to the receptor, in contrast to an antibody that causes ligand displacement.
Example 12
In Vivo Efficacy - Tumor Growth Inhibition of AntiCSF-1R Antibodies in BT20 Xenograft Tumor Cell Breast Cancer In SCID Beige Mice [0236] The BT-20 Human Breast Cancer Cell Line
Petition 870190118206, of 11/14/2019, p. 98/105
91/91 expresses CSF-1R, but lacks the expression of CSF-1 (Sapi, E. et al, Cancer Res. 59 (1999) 5578-5585). Since mouse-derived CSF-1 fails to activate human CSF-1R in tumor cells, recombinant human CSF-1 (Biomol, Hamburg, Germany) was supplemented by means of osmotic minipumps (ALZET, Cupertino, CA) that provide a rate of continuous infusion of CSF-1 of 2pg / day (Martin, TA, Carcinogenesis 24 (2003) 1317-1323).
[0237] To directly compare the effectiveness of an antibody that interferes with CSF-1R dimerization and an antibody that causes displacement of the CSF-1R ligand, the inventors tested the chimeric anti-CSF1R Mab 2F11 (antibody that interferes with dimerization CSF-1R) and 1.2.SM (antibody that causes CSF-1R ligand displacement described in WO2009026303) in the BT-20 xenograft model.
[0238] SCID-beige mice (Charles River, Sulzfeld, Germany) were co-injected subcutaneously with 1 x 10 ⁷ BT-20 cells (ATCC HTB-19) and 100 pL of Matrigel. The treatment of the animals started on the day of randomization until an average tumor volume of 100 mm ³ . The mice are treated once a week via ip with the respective antibodies (see figure 4) in buffer with 20 mM histidine, 140 mM NaCl, pH 6.0. Tumor dimensions are measured using a caliper starting on the day of staging and then twice a week for the duration of the treatment. The tumor volume is calculated according to the NCI protocol (tumor weight 1 / 2ab2, where "a" and "b" are the long and short diameters of the tumor, respectively).
[0239] Analysis of tumor growth is shown in Figure 4. Inhibition of human CSF-1R in tumor cells with the chimeric antiCSF-1R 2F11 Mab was statistically more effective in mediating inhibition of tumor growth than anti-CSF antibody -1R 1,2.SM (CSF1R antibody described in WO2009026303).

权利要求:
Claims (14)
[1]
Claims
1. HUMAN CSF-1R BINDING ANTIBODY, characterized by the heavy chain variable domain being SEQ ID NO: 7 and the light chain variable domain being SEQ ID NO: 8.
[2]
2. HUMAN CSF-1R BINDING ANTIBODY, characterized by:
a) the heavy chain variable domain is SEQ ID NO: 23 and the light chain variable domain is SEQ ID NO: 24, or
b) the variable domain of the heavy chain is SEQ ID NO: 31 and the variable domain of the light chain is SEQ ID NO: 32, or
c) the variable domain of the heavy chain is SEQ ID NO: 39 and the variable domain of the light chain is SEQ ID NO: 40, or
d) the variable domain of the heavy chain is SEQ ID NO: 47 and the variable domain of the light chain is SEQ ID NO: 48, or
e) the heavy chain variable domain is SEQ ID NO: 55 and the light chain variable domain is SEQ ID NO: 56.
[3]
ANTIBODY according to claim 2, characterized in that the heavy chain variable domain is SEQ ID NO: 31 and the light chain variable domain is SEQ ID NO: 32.
[4]
ANTIBODY according to claim 2, characterized in that the variable domain of the heavy chain is SEQ ID NO: 39 and the variable domain of the light chain is SEQ ID NO: 40.
[5]
5. HUMAN CSF-1R BINDING ANTIBODY, characterized by:
a) the variable domain of the heavy chain comprises a region
CDR3 of SEQ ID NO: 1, a CDR2 region of SEQ ID NO: 2, and a region
CDR1 of SEQ ID NO: 3, and the light chain variable domain comprises a CDR3 region of SEQ ID NO: 4, a CDR2 region of SEQ ID NO: 5, and a
Petition 870190118206, of 11/14/2019, p. 100/105
2/4 CDR1 region of SEQ ID NO: 6, or
b) the variable domain of the heavy chain comprises a CDR3 region of SEQ ID NO: 17, a CDR2 region of SEQ ID NO: 18, and a CDR1 region of SEQ ID NO: 19, and the variable domain of the light chain comprises a region CDR3 of SEQ ID NO: 20, a CDR2 region of SEQ ID NO: 21, and a CDR1 region of SEQ ID NO: 22, or
c) the variable domain of the heavy chain comprises a CDR3 region of SEQ ID NO: 25, a CDR2 region of SEQ ID NO: 26, and a CDR1 region of SEQ ID NO: 27, and the variable domain of the light chain comprises a region CDR3 of SEQ ID NO: 28, a CDR2 region of SEQ ID NO: 29, and a CDR1 region of SEQ ID NO: 30, or
d) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 33, a CDR2 region of SEQ ID NO: 34, and a CDR1 region of SEQ ID NO: 35, and the light chain variable domain comprises a region CDR3 of SEQ ID NO: 36, a CDR2 region of SEQ ID NO: 37, and a CDR1 region of SEQ ID NO: 38, or
e) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 41, a CDR2 region of SEQ ID NO: 42, and a CDR1 region of SEQ ID NO: 43, and the light chain variable domain comprises a region CDR3 of SEQ ID NO: 44, a CDR2 region of SEQ ID NO: 45, and a CDR1 region of SEQ ID NO: 46, or
f) the variable domain of the heavy chain comprises a CDR3 region of SEQ ID NO: 49, a CDR2 region of SEQ ID NO: 50, and a CDR1 region of SEQ ID NO: 51, and the variable domain of the light chain comprises a region CDR3 of SEQ ID NO: 52, a CDR2 region of SEQ ID NO: 53, and a CDR1 region of SEQ ID NO: 54.
[6]
ANTIBODY according to claim 5, characterized in that the variable domain of the heavy chain comprises a region
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3/4
CDR3 of SEQ ID NO: 25, a CDR2 region of SEQ ID NO: 26, and a CDR1 region of SEQ ID NO: 27, and the light chain variable domain comprises a CDR3 region of SEQ ID NO: 28, a CDR2 region of SEQ ID NO: 29, and a CDR1 region of SEQ ID NO: 30.
[7]
ANTIBODY according to claim 5, characterized in that the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 33, a CDR2 region of SEQ ID NO: 34, and a CDR1 region of SEQ ID NO: 35, and the light chain variable domain comprises a CDR3 region of SEQ ID NO: 36, a CDR2 region of SEQ ID NO: 37, and a CDR1 region of SEQ ID NO: 38.
[8]
ANTIBODY according to any one of claims 1 to 7, characterized in that said antibody is of the human IgG1 subclass or the human IgG4 subclass.
[9]
ANTIBODY according to any one of claims 1 to 7, characterized in that said antibody is of the human IgG1 subclass.
[10]
10. PHARMACEUTICAL COMPOSITION, characterized in that it comprises an antibody as defined in any one of claims 1 to 9.
[11]
11. USE OF THE ANTIBODY, as defined in any one of claims 1 to 9, characterized in that it is for the manufacture of a medicament for the treatment of cancer.
[12]
12. USE OF THE ANTIBODY, as defined in any one of claims 1 to 9, characterized in that it is for the manufacture of a medicament for the treatment of bone loss.
[13]
13. USE OF THE ANTIBODY, as defined in any one of claims 1 to 9, characterized in that it is for the manufacture of a medicine for the treatment of metastases.
Petition 870190118206, of 11/14/2019, p. 102/105
4/4
[14]
14. USE OF THE ANTIBODY, as defined in any one of claims 1 to 9, characterized in that it is for the manufacture of a medicament for the treatment of inflammatory diseases.

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法律状态:
2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-05-28| B07E| Notice of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-06-25| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

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2019-09-03| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application [chapter 6.1 patent gazette]|

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2019-12-31| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-01-28| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 07/12/2010, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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EP09015310|2009-12-10|

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EP10173407|2010-08-19|

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PCT/EP2010/069090|WO2011070024A1|2009-12-10|2010-12-07|Antibodies binding preferentially human csf1r extracellular domain 4 and their use|

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