Antibodies binding to human CSF1R extracellular domain 4 and their use

The present invention relates to antibodies directed against human CSF-1R (anti-CSF-1R antibodies), methods for their production, pharmaceutical compositions containing said antibodies, and their uses.

Background of the invention

Human CSF-1 receptor (CSF-1R; colony stimulating factor 1 receptor; synonyms: Μ-CSF receptor; macrophage colony stimulating factor receptor 1, proto-oncogene Fms, cfms, SEQ ID NO : 62) has been known since 1986 (Coussens, L., et al. Nature 320 (1986) 277-280). CSF-1R is a growth factor and is encoded by the proto-oncogene c-fms (developed in Roth, P., and Stanley, ER, Curr. Top. Microbiol. Immunol. 181 (1992) 141- 67).

ئ CSF-1R is the receptor for CSF-1 (colony stimulating factor 1, also called Μ-CSF, macrophage colony stimulating factor) and induces the biological effects of this cytokine (Sherr, CJ, et al .. Cell 41 (1985) 665-676).

The cloning of the colony stimulating factor 1 receptor (CSF-1R) (also called c-fms) was first described by Roussel, MF, et al., Ata 325 (1987) 549-552. In this publication, CSFIR was shown to have transformational potential dependent on changes in the c-terminal tail of the protein, including the loss of inhibitory tyrosine 969 phosphorylation which binds Cbl and thus regulates the downregulation of the protein. receptor (Le, PS, et al., £ 0 س 7. 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).

The cytokine CSF-1R (colony stimulating factor 1, also referred to as Μ-CSF) is found extracellularly as a disulfide-linked homodimer (Stanley, ER et al. Journal of Cellular Biochemistry 21 (1983). ) 151-159; Stanley, ER et al. Stem Cells 12 Suppl. 1 (1995) 15-24).

The main biological effects of CSF-1R signaling are the differentiation, proliferation, migration and survival of hematopoietic cells precursors of the macrophage lineage (including osteoclasts). Activation of CSF-1R is mediated by its ligands, CSF-1 (MCSF) and IL-34. Binding of CSF-1 (Μ-CSF) to CSF-1R induces the formation of homodimers and the activation of the kinase by phosphorylation of tyrosine (Li, w. Et al, EMBO Journal. 10 (1991) 277-288; Stanley, ER, et al. Mol. Reprod. Dev. 46 (1997) 4-10).

The biologically active CSF-1 homodimer binds to CSF-1R within the D1 to D3 subdomains of the extracellular domain of the CSF-1 receptor (CSF-

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1R-ECD). The CSF-IR-ECD comprises 5 immunoglobulin-like subdomains (called D1 to D5). The D4 to D5 subdomains of the extracellular domain (CSF-1RECD) are not involved in the binding of CSF-1. (Wang, Z., et al Molecular and Cellular Biology 13 (1993) 5348-5359). The D4 subdomain is involved in dimerization (Yeung, YG., Et al Molecular &amp; Cellular Proteomics 2 (2003) 11431155; Pixley, F. j., Et al .. Trends Cell Biol 14 (2004) 628-638) .

Another signaling is mediated by the ρ85 subunit of Ρ13Κ and Grb2 connecting respectively to the pathways of Ρ13Κ / ΑΚΤ and Ras / ΜΑΡΚ. These two important signaling pathways can regulate proliferation, survival and apoptosis. Other signaling molecules which bind to the intracellular phosphorus domain of CSF-1R include STAT1, STAT3, PLCy, and Cbl (Bourette, Rp and Rohrschneider, LR, Growth Factors 17 (2000) 155-166).

Signaling by CSF-1R has a physiological role in immune responses, in bone remodeling and in the reproductive system. The 15 knockout animals for CSF-1 (Pollard, JW, Mol. Reprod. Dev. 46 (1997) 54-61) or CSF-1R (Dai, Χ.Μ., et al .. Blood 99 (2002) 111-120) were found to have osteopetrosis, hematopoiesis, tissue macrophage, and reproductive phenotypes consistent with a role for CS F-1R in the respective cell types.

Sherr, CJ, et al. Blood 73 (1989) 1786-1793 relates to certain antibodies directed against CSF-1R which inhibit the activity of CSF-1 (see Sherr, CJ et al ..

Blood 73 (1989) 1786-1793). Ashmun, RA, et al. Blood 73 (1989) 827-837 relates to anti-CSF-1R antibodies. Lenda, D., et al. Journal of Immunology 170 (2003) 3254-3262 reports that a reduction in macrophage recruitment, proliferation and activation in CSF-1 deficient mice results in a decrease in tubular apoptosis during renal inflammation. Kitaura, H., et al .. Journal ofDental Research 87 (2008) 396-400 relates to an anti-CSF-1 antibody that balances orthodontic movement of teeth. The patent document

WO 2001/030381 mentions inhibitors of CSF-1 activity, comprising antisense nucleotides and antibodies, while describing only anti-sense nucleotides of CSF-1. Patent document WO 2004/045532 relates to the prevention of metastases and bone loss and the treatment of metastatic cancer by a CSF-1 antagonist, only describing antiCSF-1 antibodies as antagonists. Patent document wo 2005/046657 relates to the treatment of inflammatory bowel disease with anti-CSF-1 antibodies. The patent document US 2002/0141994 relates to inhibitors of colony stimulating factors.

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The patent document WO 2006/096489 relates to the treatment of rheumatoid arthritis with anti-CSF-1 antibodies. patent documents WO 2009/026303 and WO 2009/112245 relate to certain anti-CSF-1R antibodies binding to CSF-1R inside the first 3 subdomains (D1 to D3) of the extracellular domain (CSF1 ؤ R-ECD ).

Summary of the invention

The invention comprises an antibody binding to human CSF-1R, characterized in that the antibody binds to the delD4 fragment (SEQ ID NO: 65) of human CSFIR and to the extracellular domain (SEQ ID NO: 64) of CSF- Human 1R with a ratio of at most 1:50.

The invention further comprises an antibody according to the invention, characterized in 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 one of its humanized versions.

The invention further comprises an antibody according to the invention, characterized in 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;

or one of its humanized versions.

In one embodiment, the antibody according to the invention is characterized in 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

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d)

e) the heavy chain variable domain is SEQ ID NO: 47 and the light chain variable domain is SEQ ID NO: 48; or the heavy chain variable domain is SEQ ID NO: 55 and the light chain variable domain is SEQ ID NO: 56.

The invention further comprises an antibody according to the invention, characterized in that

a) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 1, a CDR2 region of SEQ ID NO: 2 and a CDR1 region 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 ED 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 ED NO: 11, and the light chain variable domain comprises a CDR3 region of

SEQ ED NO: 12, a CDR2 region of SEQ DD NO: 13 and a CDR1 region of

SEQ BD NO: 14, or

c) the heavy chain variable domain comprises a CDR3 region of SEQ DD NO: 17, a CDR2 region of SEQ ED 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 DD NO: 21 and a CDR1 region of

SEQ DD NO: 22, or

d) the heavy chain variable domain comprises a CDR3 region of SEQ DD 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 DD NO: 28, a CDR2 region of SEQ ID NO: 29 and a CDR1 region of

SEQ DD NO: 30, or

e) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 33, a CDR2 region of SEQ DD NO: 34 and a CDR1 region of SEQ ID NO: 35, and the light chain variable domain comprises a CDR3 region of

SEQ DD NO: 36, a CDR2 region of SEQ DD NO: 37 and a CDR1 region of

SEQ DD NO: 38, or

f) the heavy chain variable domain comprises a CDR3 region of SEQ D} NO: 41, a CDR2 region of SEQ ID NO: 42 and a CDR1 region of SEQ DD NO: 43, and the light chain variable domain comprises a region CDR3 of

SEQ DD NO: 44, a CDR2 region of SEQ ID NO: 45 and a CDR1 region of

SEQ DD NO: 46, or

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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 CDR3 region 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 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 ro NO: 77, a CDR2 region of SEQ ro NO: 78 and a CDR1 region of SEQ ro NO: 79, and the light chain variable domain comprises a CDR3 region of SEQ ro NO: 80, a CDR2 region of SEQ ro NO: 81 and a CDR1 region of SEQroNO: 82.

The antibody according to the invention is preferably from the human IgG1 subclass or from the human IgG4 subclass.

Another embodiment of the invention is a pharmaceutical composition comprising an antibody according to the invention.

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 induced by CSF-1R.

The invention further comprises the use of an antibody according to the invention for the manufacture of a medicament for the treatment of cancer.

3C

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.

The invention further comprises the use of an antibody according to the invention for the manufacture of a medicament for the treatment of metastases.

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.

The invention further comprises an antibody according to the invention for the treatment of a disease induced by CSF-1R.

The invention further comprises an antibody according to the invention for the treatment of cancer.

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The invention further comprises an antibody according to the invention intended for the treatment of bone loss.

The invention further comprises an antibody according to the invention intended for the treatment of metastases.

The invention further comprises an antibody according to the invention intended for the treatment of inflammatory diseases.

Another embodiment of the invention is a nucleic acid encoding an antibody according to the invention, characterized in that

a) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 1, a CDR2 region of SEQ ID NO: 2 and a CDR1 region 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 CDR3 region of SEQ ED NO: 12, a CDR2 region of SEQ DD NO: 13 and a CDR1 region of SEQ DD NO: 14, or

c) the heavy chain variable domain comprises a CDR3 region of SEQ DD NO: 17, a CDR2 region of SEQ ID NO: 18 and a CDR1 region of SEQ DD NO: 19, and the light chain variable domain comprises a CDR3 region of SEQ ED NO: 20, a CDR2 region of SEQ BD NO: 21 and a CDR1 region of SEQ BD 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 light chain variable domain comprises a CDR3 region of SEQ DD NO: 28, a CDR2 region of SEQ ED NO: 29 and a CDR1 region of SEQ BD NO: 30, or

e) the heavy chain variable domain comprises a CDR3 region of SEQ DD NO: 33, a CDR2 region of SEQ DD NO: 34 and a CDR1 region of SEQ DD NO: 35, and the light chain variable domain comprises a CDR3 region of SEQ DD NO: 36, a CDR2 region of SEQ ED NO: 37 and a CDR1 region of SEQ ID NO: 38, or

f) the heavy chain variable domain comprises a CDR3 region of SEQ BD NO: 41, a CDR2 region of SEQ ID NO: 42 and a CDR1 region of SEQ DD NO: 43, and the light chain variable domain comprises a CDR3 region of

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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 CDR3 region 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 DD

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 light chain variable domain comprises a CDR3 region of

SEQ ID NO: 80, a CDR2 region of SEQ ID NO: 81 and a CDR1 region of SEQ ID NO: 82.

Another embodiment of the invention is a nucleic acid encoding an antibody according to the invention characterized in that:

20 at)

(b

(vs ;

25

(d the heavy chain variable domain is SEQ ID NO: 7 and the light chain variable domain is SEQ ID NO: 8;

the heavy chain variable domain is SEQ ID NO: 15 and the light chain variable domain is SEQ ID NO: 16;

the heavy chain variable domain is SEQ ID NO: 75 and the light chain variable domain is SEQ ID NO: 76;

the heavy chain variable domain is SEQ ID NO: 83 and the light chain variable domain is SEQ ID NO: 84;

or one of its humanized versions.

Another embodiment of the invention is a nucleic acid encoding an antibody according to the invention characterized in 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

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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 ED NO: 56.

The invention further provides expression vectors containing a nucleic acid according to the invention, capable of expressing said nucleic acid in a prokaryotic or eukaryotic host cell, and host cells containing such vectors for the recombinant production of a antibody according to the invention.

The invention further comprises a prokaryotic or eukaryotic host cell comprising a vector according to the invention.

The invention further comprises a method for producing a recombinant human or humanized antibody according to the invention, characterized by the expression of a nucleic acid according to the invention in a prokaryotic or eukaryotic host cell and the recovery of said antibody to from said cell or from the cell culture supernatant. The invention further comprises the antibody obtained by such a recombinant technique.

the antibodies according to the invention have benefits for patients in need of therapy targeting CSF-1R. The antibodies according to the invention exhibit effective antiproliferative activity against ligand independent and ligand dependent proliferation and are therefore particularly useful in the treatment of cancer and metastases.

The invention further provides a method of treating a patient suffering from cancer, 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.

Another embodiment of the invention is a method of treating a patient suffering from cancer, characterized by administering to the patient an antibody according to the invention.

It was surprisingly found that, using a delD4 fragment of human CSFIR in which the D4 subdomain of CSFIR-ECD has been deleted (SEQ ID NO: 65), one could select the new anti-CSF-1R antibodies according to the 'invention. These antibodies exhibit interesting properties such as excellent inhibition of ligand dependent cell growth and at the same time inhibition of ligand independent cell growth of cells.

NIH 3Τ3, infected etrovirally with an expression vector for CSF-1R

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Wild-type whole 34780Β1 (SEQ ID NO: 62) or mutant CSF-1R L301S Y969F (SEQ ID NO: 63), whereby cells recombined with mutant CSF-1R are able to form sphero'1'des independently of the CSF-1 ligand. In addition, the antibodies according to the invention inhibit the differentiation of macrophages both in ؛؛ man and the cynomolgus monkey, since they unbalance the survival of human monocytes and the cynomolgus monkey.

Detailed description of the invention

The invention comprises an antibody binding to human CSF-1R, characterized in that the antibody binds to the delD4 fragment of human CSF-1R 1 ؛ (comprising the extracellular subdomains D1-D3 and D5) (SEQ ID NO: 65) and to the extracellular domain of human CSF-1R (CSEIR-ECD) (comprising the extracellular subdomains D1-D5) (SEQ ID NO: 64) with a ratio of at most 1:50.

The invention further comprises an antibody according to the invention, characterized in that it comprises as CDR3 region of the heavy chain variable domain a CDR3 region of 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.

The invention further comprises an antibody according to the invention, characterized in that

a) the heavy chain variable domain is SEQ ID NO: 7 and the variable domain is

2 ؛ of light chain 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:

or one of its humanized versions.

The invention further comprises an antibody according to the invention, characterized

! ؛ 2 in that

a) the heavy chain variable domain is SEQ ID NO: 7 and the light chain variable domain is SEQ JD 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 one of its humanized versions.

The invention further comprises an antibody according to the invention, characterized

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34780Β1 in that the heavy chain variable domain is SEQ ID NO: 7 and the light chain valid domain is SEQ ID NO: 8;

or one of its humanized versions.

In one embodiment, the antibody according to the invention is characterized in that:

a) the heavy chain valid domain is SEQ ID NO: 23 and the light chain valid domain is SEQ ID NO: 24; or the heavy chain variable domain is SEQ ID NO: 31 and the light chain variable domain is SEQ ID NO: 32; or the heavy chain variable domain is SEQ ID NO: 39 and the light chain variable domain is SEQ ID NO: 40; or the heavy chain variable domain is SEQ ID NO: 47 and the light chain variable domain is SEQ ID NO: 48; or the heavy chain variable domain is SEQ ID NO: 55 and the light chain variable domain is SEQ ID NO: 56.

In one embodiment, the antibody according to the invention is characterized in that:

at)

b)

vs)

d)

e) the heavy chain variable domain is SEQ ID NO: 23 and the light chain variable domain is SEQ ID NO: 24; or the heavy chain variable domain is SEQ ID NO: 31 and the light chain variable domain is SEQ ID NO: 32; or the heavy chain valid domain is SEQ ID NO: 39 and the light chain variable domain is SEQ ID NO: 40; or the heavy chain variable domain is SEQ ID NO: 47 and the light chain variable domain is SEQ ID NO: 48.

In one embodiment, the antibody according to the invention is characterized in that:

the heavy chain variable domain is SEQ ID NO: 23 and the light chain variable domain is SEQ ID NO: 24.

In one embodiment, the antibody according to the invention is characterized in that:

the heavy chain variable domain is SEQ ID NO: 31 and the light chain variable domain is SEQ ID NO: 32.

In one embodiment, the antibody according to the invention is characterized in that:

b)

vs)

d)

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34780Β1 the heavy chain variable domain is SEQ ID NO: 39 and the light chain variable domain is SEQ ID NO: 40.

In one embodiment, the antibody according to the invention is characterized in that:

the heavy chain variable domain is SEQ ID NO: 47 and the light chain variable domain is SEQ ID NO: 48.

The invention further comprises an antibody according to the invention, characterized in that:

the heavy chain variable domain is SEQ ID NO: 15 and the light chain variable domain is SEQ ID NO: 16, or a humanized version thereof.

The invention further comprises an antibody according to the invention, characterized in 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 thereof.

The invention further comprises an antibody according to the invention, characterized in that:

the heavy chain variable domain is SEQ ID NO: 83 and the light chain variable domain is SEQ DD NO: 84, or a humanized version thereof.

The invention further comprises an antibody according to the invention, characterized in that

a) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 1, a CDR2 region of SEQ ID NO: 2 and a CDRI region 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 CDRI 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 CDRI region of SEQ ID

NO: 11, and the light chain variable domain comprises a CDR3 region of

SEQ ID NO: 12, a CDR2 region of SEQ ID NO: 13 and a CDRI 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 CDRI 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 CDRI region of SEQ ro NO: 22, or

d) the heavy chain variable domain comprises a CDR3 region of SEQ JD mA

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<td></td><td></td><td>12 NO: 25, a CDR2 region of SEQ ID NO: 26 and a CDR1 region of SEQ ID</td>

<td></td><td></td><td>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</td>

<td> 5</td><td>e)</td><td>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: 35 ID NO: 36, a CDR2 region of SEQ ID NO: 37 and a CDR1 region of SEQ ID NO: 38, or</td>

<td> 10</td><td>f)</td><td>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: 42 ID NO: 44, a CDR2 region of SEQ ID NO: 45 and a CDR1 region of SEQ ID NO: 46, or</td>

<td> 15</td><td>g)</td><td>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 CDR3 region of SEQ ID NO: 52, a CDR2 region of SEQ ID NO: 53 and a CDR1 region of SEQ ID NO: 54.</td>

<td colspan="2"> 20</td><td>The invention further comprises an antibody according to the invention, characterized</td>

<td></td><td colspan="2">in that</td>

<td></td><td>at)</td><td>the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 1, a CDR2 region of SEQ ID NO: 2 and a CDR1 region of SEQ ID NO: 3, and the light chain variable domain comprises a CDR3 region of</td>

<td> ؛2</td><td></td><td>SEQ ID NO: 4, a CDR2 region of SEQ ID NO: 5 and a CDR1 region of SEQ ED NO: 6, or</td>

<td></td><td>b)</td><td>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 CDR3 region of</td>

<td colspan="2"> 30</td><td>SEQ ID NO: 12, a CDR2 region of SEQ ID NO: 13 and a CDR1 region of SEQ ID NO: 14, or</td>

<td>. إ . .</td><td>vs)</td><td>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</td>

<td> 35</td><td></td><td>SEQ ED NO: 20, a CDR2 region of SEQ ID NO: 21 and a CDR1 region of SEQ ID NO: 22, or</td>

<img file="MA34780B1_D0002.tif" />

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d)

e)

g)

أ 2 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, or 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, or the heavy chain variable domain comprises a region CDR3 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, or 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 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 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 the heavy chain variable domain comprises a region CDR3 of SEQ ID NO: 77, a CDR2 region of SEQ ID NO: 78 and a CDR1 region of SEQ ID NO: 79, and the light chain variable domain comprises a CDR3 region of SEQ ID NO: 80, a CDR2 region of SEQ ID NO: 81 and a CDR1 region of SEQ ID NO: 82.

In one embodiment, the antibody according to the invention is characterized in 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 CDR3 region of SEQ ID NO: 72, a CDR2 region of SEQ ID NO: 73 and a CDR1 region of

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SEQ ID NO: 74, or

b) the heavy chain variable domain comprises a CDR3 region of SEQ BD NO: 77, a CDR2 region of SEQ BD NO: 78 and a CDR1 region of SEQ ro NO: 79, and the light chain variable domain comprises a CDR3 region of SEQ ro NO: 80, a CDR2 region of SEQ ro NO: 81 and a CDR1 region of SEQroNO: 82.

In one embodiment, the antibody according to the invention is characterized in that:

at)

b)

vs)

d)

e) the heavy chain variable domain comprises a CDR3 region of SEQ ro NO: 17, a CDR2 region of SEQ ro NO: 18 and a CDR1 region of SEQ ro NO: 19, and the light chain variable domain comprises a CDR3 region of SEQ ro NO: 20, a CDR2 region of SEQ ro NO: 21 and a CDR1 region of SEQroNO: 22, or the heavy chain variable domain comprises a CDR3 region of SEQ ro NO: 25, a CDR2 region of SEQ ro NO : 26 and a CDR1 region of SEQ ro NO: 27, and the light chain variable domain comprises a CDR3 region of SEQ ro NO: 28, a CDR2 region of SEQ ro NO: 29 and a CDR1 region of SEQroNO: 30, or the heavy chain variable domain comprises a CDR3 region of SEQ ro NO: 33, a CDR2 region of SEQ ro NO: 34 and a CDR1 region of SEQ ro NO: 35, and the light chain variable domain comprises a CDR3 region of SEQ ro NO: 36, a CDR2 region of SEQ ro NO: 37 and a CDR1 region of SEQroNO: 38, or the heavy chain variable domain comprises a CDR3 region of SEQ ro NO: 41, a CDR2 region of SEQ ro NO: 42 and a CDR1 region of SEQ ro NO: 43, and the light chain variable domain comprises a region CDR3 of SEQ ro NO: 44, a CDR2 region of SEQ ro NO: 45 and a CDR1 region of SEQroNO: 46, or the heavy chain variable domain comprises a CDR3 region of SEQ ro NO: 49, a CDR2 region of SEQ ID NO: 50 and a CDR1 region of SEQ ro NO: 51, and the light chain variable domain comprises a CDR3 region of SEQroNO: 52, a CDR2 region of SEQroNO: 53 and a CDR1 region of SEQroNO: 54.

In one embodiment, the antibody according to the invention is characterized in that:

a) the heavy chain variable domain comprises a CDR3 region of SEQ ro

MY

34780Β1

b)

؟3

vs)

d)

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 ED NO: 20, a CDR2 region of SEQ ID NO: 21 and a CDR1 region of SEQ ID NO: 22, or 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, or 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 SEQIDNO: 38, or 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 ED NO: 45 and a CDR1 region of SEQ ro NO: 46.

In one embodiment, the antibody according to the invention is characterized in 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: 19 ID NO: 20, a CDR2 region of SEQ ID NO: 21 and a CDR1 region of SEQ ID NO: 22.

In one embodiment, the antibody according to the invention is characterized in 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: 27 ID NO: 28, a CDR2 region of SEQ ID NO: 29 and a CDR1 region of SEQ ID NO: 30.

In one embodiment, the antibody according to the invention is characterized in that:

the heavy chain variable domain comprises a CDR3 region of SEQ ID

MY

34780Β1

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.

In one embodiment, the antibody according to the invention is characterized

3 ¢ in 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: 42 ED NO: 44, a CDR2 region of SEQ DD NO: 45 and a CDR1 region of SEQ ID NO: 46.

In one embodiment, the antibody binding to human CSF-1R, characterized in that the antibody binds 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) with a ratio of at most 1:50, is further characterized in that it does not bind to the D1-D3 fragment of human CSF-1R (SEQ BD NO: 66).

The term antibody includes the various forms of antibodies including, but not limited to, whole antibodies, antibody fragments, human antibodies, humanized antibodies, chimeric antibodies, T-cell epitope-depleted antibodies, and other antibodies produced by genetic engineering, insofar as the characteristic properties according to the invention are retained. Antibody fragments comprise a portion of an entire antibody, preferably its variable domain, or at least its antigen binding site. Examples of antibody fragments include diabodies, single chain antibody molecules, and multispecific antibodies formed from antibody fragments. scFv antibodies are described, for example, by Houston, JS, Methods in Enzymol. 203 (1991) 4688). In addition, the antibody fragments comprise single-chain polypeptides having the characteristics of a Vh domain binding to CSF-1R, that is to say capable of assembling with a ٧L domain, or of a Vl domain. binding to CSF-1R, i.e. capable of assembling with a Vh domain, to form a functional antigen binding site and thus provide the property.

The terms monoclonal antibody or monoclonal antibody composition refer to a preparation of antibody molecules having a unique amino acid composition.

The expression chimeric antibody denotes a monoclonal antibody

MY

34780Β1 comprising a variable region, i.e., a binding region, of mouse, and at least a portion of a constant region 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 particularly preferred. Rat / human chimeric antibodies of this type are the product of expressed immunoglobulin genes comprising DNA segments encoding rat immunoglobulin variable regions and DNA segments encoding human immunoglobulin constant regions. Other forms of chimeric antibodies included in the present invention are those in which the class or subclass has been modified or changed from that of the original antibody. Such chimeric antibodies are also called class-switched antibodies. Methods of producing chimeric antibodies involve conventional techniques of DNA recombination and gene transfection now well known in the art. See, e.g., Morrison, SL, et al., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855; US Patents Nos. 5,202,238 and 5,204,244.

The expression humanized antibody denotes antibodies whose framework or regions determining complementarity (CDR) have been modified 2٩ to comprise a CDR of an immunoglobulin of different specificity compared to that of the mother immunoglobulin. In a preferred embodiment, a mouse CDR is grafted into the framework region of a human antibody to prepare the humanized antibody. See, e.g., Riechmann, L., et al .. Nature 332 (1988) 323-327; and Neuberger, M. s., et al. Nature 314 (1985) 2682270 و. The framework region can optionally be modified by other mutations. CDRs too can be modified by one or more mutations to produce antibodies according to the invention, for example by molecular modeling based mutagenesis as described by Riechmann, L., et al., Afatart? 332 (1988) 323-327 and Queen, v., Et al., Proc. Natl. Acad. Sci. USA 86 (1989) 1002930 10033, or others. Particularly preferred CDRs correspond to those which represent sequences recognizing the antigens indicated above for the chimeric antibodies. A humanized version of an antibody according to the invention (which is for example of murine origin) refers to an antibody based on the sequences of the mouse antibody in which the Vhs and Vls are humanized by standard techniques (including CDR grafting, optionally followed by mutagenesis of certain amino acids in the framework region and the CDRs). Such a version

<img file="MA34780B1_D0003.tif" />

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Humanized 34780Β1 is preferably chimerized with a human constant region (see for example the sequences SEQ ED NO: 57-61).

Other forms of humanized antibodies included in the present invention are those in which the constant region has been additionally modified or changed from that of the original antibody to give the properties according to the invention, in particular. with regard to the binding of Clq eU the binding of the Fc receptor (FcR).

In the examples below, the terms mAb or mAcM denote murine monoclonal antibodies such as mAb 2F11 or mAb 2Ε10, while the term hAcM denotes humanized monoclonal versions of these murine antibodies, such as hAcM 2Fll-cll١ hAcM 2F11-d8, hAcM 2Fll-e7, hAcM 2Fll-fl2, etc.

The term human antibody, as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies are well known in the art (van Dijk, Μ.Α., and van de Winkel, JG, Curr. Opin. Chem. Β ، '٠ /. 5 (2001) 368-374). Human antibodies can also be produced in transgenic animals (eg, mice) which are capable, upon immunization, of producing a whole repertoire or selection of human antibodies in the absence of production of endogenous immunoglobulins. Transfer of the human germline immunoglobulin gene set into such germline mutant mice will result in the production of human antibodies upon challenge with antigens (see, e.g., 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. Year Immunol. 7 (1993) ) 33-40). Human antibodies can also be produced in phage display libraries (Hoogenboom, HR, and Winter, GJ Mol. Biol. 227 (1992) 381-388; Marks, JD, et al., 7. Mol. Biol. 222. (1991) 581-597). There are also the techniques of Cole, et al., And Boemer, et al., For the preparation of human monoclonal antibodies (Cole, SPC, et al. Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, P. 77 (1985); and Boemer, P., et al., 7. Immunol. 147 (1991) 8695). As already indicated for chimeric and humanized antibodies according to the invention, the term human antibody, as used herein, includes antibodies of this type which are modified in the constant region to give the properties according to the invention. , especially with regard to Clq binding and / or FcR receptor binding, for example by class switching, i.e.

ΜΑ 34780Β1 change or mutation of parts of Fc (eg from IgG1 to IgG4 and / or IgGl /! GG4 mutation).

The term recombinant human antibody, as used herein, is intended to include all human antibodies which are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from a host cell such as an NSO cell or CHO or from an animal (eg, mouse) which is transgenic for human immunoglobulin genes, or antibodies expressed using a recombinant expression vector transfected into a host cell. These recombinant human antibodies have variable and constant regions in a rearranged form. The recombinant human antibodies according to the invention were subjected to somatic hypermutation in vivo. the amino acid sequences of the VH and VL regions of the recombinant antibodies are therefore sequences which, although originating from VH and VL sequences of human germ lines and related to these sequences, may not naturally exist in the repertoire of geninal lines of human antibodies in vivo.

The antibodies according to the invention further comprise antibodies having conservative sequence modifications, which are modifications of the nucleotide sequence and of the amino acid sequence which do not affect or alter the aforementioned characteristics of the substance. antibody according to the invention. The modifications can be introduced by standard techniques known to those skilled in the art, such as site-directed mutagenesis and PCR-induced mutagenesis. Conservative amino acid substitutions include those in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids having basic side chains (eg lysine, arginine, 1 histidine), acidic side chains (eg aspartic acid, glutamic acid), uncharged polar side chains (eg. eg glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (eg alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (eg threonine, valine, isoleucine) and aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a human anti-CSF-1R antibody can preferably be replaced by another amino acid residue from the same family of side chains.

<img file="MA34780B1_D0004.tif" />

MA 34780Β1 amino acid substitutions can be made 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.

Human CSF-1R (CSF-1 receptor; synonyms: MCSF receptor; macrophage colony stimulating factor receptor 1, protooncogene Fms, c-fms, SEQ ID NO: 22) has been known since 1986 (Coussens, L ., et al. Nature 320 (1986) 277-280). CSF-1R is a growth factor and is encoded by the proto-oncogene c-fms (developed for example in Roth, P., and Stanley, ER, Curr. Top. Microbiol. Immunol. 181 (1992) 141 -67).

ممل CSF-1R is the receptor for CSF-1 (macrophage colony stimulating factor 1, also called Μ-CSF) and IL-34 and induces the biological effects of these cytokines (Sherr, CJ, et al .. Cell 41 (1985) 665-676), Lin, H., et al. Science 320 (2008) 807-811). The cloning of the colony stimulating factor receptor 1) (also referred to as c-fms) was first described by Roussel, MF, et al. Nature 325 (1987) 549-552. In this publication, CSF-1R was shown to have transformation potential dependent on changes in the C-terminal tail of the protein, including the loss of inhibitory tyrosine 969 phosphorylation which binds Cbl and thus regulates regulation. receptor negative (Lee, PS, et al., Embo j. 18 (1999) 3616-3628).

ممل CSF-1R is a single-chain transmembrane tyrosine kinase (RTK) receptor and member of the RTK family containing an immunoglobulin (Ig) motif characterized by 5 Ig-like subdomains repeated in the D1-D5 domain. extracellular (ECD) receptor (Wang, z., et al Molecular and Cellular Biology 13 (1993) 5348-5359). ممل extracellular domain of human CSF-1R (CSF-IR-ECD) (SEQ ID NO: 64) comprises the 5 extracellular subdomains of type Ig D1-D5. The delD4 fragment of human CSF-1R (SEQ ID NO: 65) comprises the extracellular Ig-like subdomains D1-D3 and D5, but does not have the D4 subdomain. The D1-D3 fragment of human CSF-1R (SEQ ED NO: 66) comprises the respective subdomains D1-D3. The sequences are listed without the signal peptide MGSGPGVLLLLLVATAWHGQ G (SEQ ID NO: 67).

ممل intracellular domain of protein tyrosine kinase is interrupted by a single insert domain which is also present in other related members of the class III RTK family which includes platelet-derived growth factor (PGDFR) receptors, the stem cell growth factor receptor (c-Kit) and the fms-type cytokine receptor (FLT3). Despite

<img file="MA34780B1_D0005.tif" />

MK

34780Β1 structural homology in this family of growth factors, they have distinct tissue-specific functions.

ICSF-IR is expressed primarily on cells of the monocytic lineage and in the female reproductive tract and placenta. In addition, expression of CSF-1R has been reported in Langerhans cells, in the skin, a subset of smooth muscle cells (Inaba, T., et al., J. Biol. Chem. 267 (1992) 5693-5699), B lymphocytes (Baker, Α.Η., et al .. Oncogene 8 (1993) 371378) 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 independent of line stimulation.

As used herein, binding to human CSF-1R or specifically binding to human CSF-1R refers to an antibody that specifically binds to human CSF-IR antigen with a binding affinity having a value of KD. of at most 1.0 X 10'8 mol / 1 at 35٥c, in one embodiment a value of KD of at most 1.0 X 10 و mol / 1 at 35٥c. Binding affinity is determined using a standard 35٥c binding assay, such as the surface plasmon resonance technique (BIAcore®, GEHealthcare Uppsala, Sweden). A technique for determining the KD value of binding affinity is described in Example 9. Thus, human CSF-1R binding antibody, as used herein, refers to an antibody specifically binding to human CSF-1R antigen with a KD binding affinity of up to 1.0X. 10'8 mol / 1 (preferably 1.0 X 10'8 mol / 1 -l, 0x 10 '2 mol / 1) at 35٥c, preferably KD of at most 1.0 X 10'5 mol / 1 to 35٥c (preferably 1.0 X 10٠ mol / 1 -1.0 X 10٤2 mol / 1).

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), as used herein, is measured by plasmon resonance analysis of surface (Biacore) as described in Example 4. The delD4 fragment of human CSF-1R (SEQ ID NO: 65) or the extracellular domain of human CSF-1R (SEQ ID NO: 64) are respectively captured on the surface (each on a separate surface) and the antibodies are added. test (each in a separate measurement) and the respective link signals (Response Units (UR)) are determined. The reference signals are subtracted (white surface). When non-binding test antibody signals are slightly below 0, the values ​​are set to 0. Next, the ratio of the respective binding signals (binding signal (UR) to the delD4 fragment is determined. CSF-1R / binding signal (UR) to the extracellular domain of human CSF-1R (CSF-IRECD)). the antibodies according to the invention have a binding signal ratio

<img file="MA34780B1_D0006.tif" />

I

34780Β1 (UR (delD4 / UR (CSF-IR-ECD) at most 1:50, preferably at most 1: 100 (the included lower limit is 0 (for example, if UR is 0, the ratio is 0:50 OR 0: 100)).

This means that these anti-CSF-1R antibodies according to the invention do not bind to the delD4 fragment of human CSF-1R (like the anti-CCR5 m &lt;CCR5&gt; PzO3.1C5 antibody (deposited at the DSMZ on 08/18/2004 under DSM No. ACC 2683)) and have binding signals for binding to the delD4 fragment of human CSF-1R which are of the order of those of the anti-CCR5 m &lt;CCR5&gt; Pz03.1C5 antibody, au- below 20 UR (Response Units), preferably below 10 UR in a surface plasmon resonance (BIAcore) assay, as shown in Example 4.

The expression binding to the D1-D3 fragment of human CSF-1R refers to the determination of the binding affinity by surface plasmon resonance analysis (Biacore analysis). The test antibody is captured on the surface and the D1-D3 fragment of human CSF-1R (SEQ ID NO: 66) is added and the respective binding affinities are determined. Expression not binding to the D1-D3 fragment of human CSF-1R indicates that, in such an analysis, the detected signal was in the region of at most 1.2 times the background noise signal and therefore that No significant binding could be detected, and no binding affinity could be determined (see Example 10).

One embodiment of the invention is a screening method for the selection of antibodies according to the invention, comprising the following steps:

a) the binding signal (Response Units (UR)) of antiCSF-1R antibodies to a delD4 fragment of human CSF-1R (SEQ ID NO: 65) and to the extracellular domain of human CSF-1R (CSF -IR-ECD) (SEQ ID NO: 64) by surface plasmon resonance analysis (Biacore analysis).

b) antibodies showing a binding signal ratio (delD4 fragment of human CSF-1R / extracellular domain of human CSF-1R (CSF-IR-ECD) of at most 50: 1 are selected.

In one embodiment, the determination is made at 25٠c.

In one embodiment, the screening method comprises as further steps measuring the binding of anti-CSF-1R antibodies to the D1-D3 fragment of human CSF-1R (SEQ JD NO: 66) (D1-D3) and the selection of antibodies not exhibiting binding to said fragment.

The term epitope refers to a determinant of human CSF-1R protein capable of specifically binding to an antibody. Epitopes usually consist of chemically active surface groups of

MY

34780Β1 molecules like amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished by the fact that binding to the former, but not to the latter, is lost in the presence of denaturing solvents. An antibody according to the invention preferably binds specifically to native CSF-1R and denatures. The variable domain (variable domain of a light chain (Vl), variable domain of a heavy chain (Vh)), as used herein, refers to each member of the pair of heavy and light chain domains which is involved. directly into the binding of the antibody to the antigen. The domains of the variable human heavy and light chains have the same general structure and each domain comprises four framework regions (FR) whose sequences are largely conserved, connected by three hypervariable regions (or regions determining complementarity, the CDRs). The framework regions adopt a ول β-sheet conformation and the CDRs can form loops connecting the β-sheet structure. The CDRs of each chain are maintained in their three-dimensional structure by the framework regions and together with the CDRs of 1 other chain form the antigen binding site. The CDR3 regions of the light and heavy chains of the antibody play a particularly important role in the binding specificity / affinity of the antibodies according to the invention and therefore represent a further subject of the invention.

The term antigen-binding portion of an antibody used herein refers to the amino acid residues of an antibody which are responsible for binding to the antigen. The antigen-binding portion of an antibody comprises amino acid residues of the complementarity determining regions or CDRs. Framework regions or FRs are the regions of variable domains other than the residues of the hypervariable region as defined herein. Accordingly, the variable domains of the light and heavy chains of an antibody include, from the N-terminus to the C-terminus, the FRI, CDR1, FR2, CDR2, FR3, CDR3 and FR4 domains. In particular, the heavy chain CDR3 is the region that contributes the most to antigen binding and defines the properties of the antibody. The CDR and FR regions are determined according to the standard definition of Kabat et al., ئديسة of Proteins of Immunological Interest, 5th edition. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and / or the residues of a hypervariable loop.

The expressions nucleic acid or nucleic acid molecule, such as

MY

34780Β1 as used herein are believed to include DNA molecules and RNA molecules. A nucleic acid molecule can be single stranded or double stranded, but is preferably double stranded DNA.

The term amino acid, as used in this patent application, designates the group of natural α-amino carboxylic acids comprising alanine (3 letter code: ala, 1 letter code: A), arginine (arg, R ), asparagine (asn, N), aspartic acid (asp, D), cysteine ​​(cys, C), glutamine (gin, Q), glutamic acid (glu, E), glycine ( gly. G), lfristidine (his. H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, 10 P), serine (ser, s), threonine (thr, T), tryptophan (trp, w), tyrosine (tyr. Y) and lavaline (val) V).

In one embodiment, the antibodies according to the invention inhibit the binding of CSF-1 to CSF-1R. In one embodiment with an IC of at most 200 ng / ml, in one embodiment with an IC of at most 50 ng / ml٠ The IC of inhibition of CSF-1 binding to CSF-1R can be determined as described in Example 2.

In one embodiment, the antibodies according to the invention inhibit the phosphorylation of CSF-1R induced by CSF-1 (in recombinant NIH3T3-CSF-1R cells).

In one embodiment, with an IC50 of at most 800 ng / ml, in one embodiment with an IC of at most 600 ng / ml, in one embodiment with an IC of at most 250 ng / ml ml. The IC of Inhibition of CSF-1R-Induced Phosphorylation of CSF-1 can be determined as described in Example 3.

In one embodiment, the antibodies according to the invention inhibit the growth of recombinant ΝΙΗ3Τ3 cells expressing human CSF-1R (SEQ ID NO: 62). In one embodiment with an IC of at most 10 pg / ml, in one embodiment with an IC of at most 5 pg / ml, in one embodiment with an IC of at most 2 pg / ml . In one embodiment with an IC of at most 30 10 pg / ml, in one embodiment with an IC of at most 5 pg / ml, in one embodiment with an IC 30 of at most 2 pg / ml . The IC50 value, IC value or% inhibition of growth is determined as described in Example 5.

In one embodiment, the antibodies according to the invention inhibit the growth of recombinant ΝΙΗ3Τ3 cells expressing mutant human CSF-1R L301S Y969F (SEQ ID NO: 63). In one embodiment with an IC of at

MK

34780Β1 plus 15 pg / ml, in one embodiment with an IC of no more than 10 pg / ml. In one embodiment with an IC of at most 10 pg / ml, in one embodiment with an IC of at most 5 pg / ml, in one embodiment with an IC of at most 2 pg / ml . The IC value, the IC value or the% inhibition of growth was determined as described in Example 5.

In one embodiment, the antibodies according to the invention inhibit the growth of BeWo tumor cells (ATCC CCI 98) by at least 65% (at an antibody concentration of 10 pg / ml; and compared to the absence antibodies). The% growth inhibition is determined as described in Example 8.

2¢

For example, mAb 2FH exhibits an inhibition of the growth of BeWo tumor cells by 70%.

In one embodiment, the antibodies according to the invention inhibit the differentiation of both human and cynomolgus monkey macrophages (which is indicated by inhibiting the survival of human monocytes and cynomolgus monkeys as described in Examples 7 and 8). In one embodiment, the antibodies according to the invention inhibit the survival of human monocytes with an IC of at most 0.15 pg / ml, in one embodiment with an IC50 of at most 0.10 pg / ml . The inhibition of the survival of human monocytes is determined as described in Example 7. In one embodiment, the antibodies according to the invention inhibit the survival of monocytes of the cynomolgus monkey by at least 80%, in a embodiment of at least 90% (at an antibody concentration of 5 µg / ml; and compared to the absence of antibody). The inhibition of monocyte survival of the cynomolgus monkey is determined as described in Example 8.

Another embodiment of the invention is a method for producing an antibody directed against CSF-1R, characterized in that the sequence of a nucleic acid encoding the heavy chain of an antibody of the class of human IgG1 binding to human CSF-1R according to the invention and a nucleic acid encoding the light chain of said antibody in an expression vector, said vector is inserted into a eukaryotic host cell, and the encoded protein is expressed and recovered from the host cell or the supernatant.

The antibodies according to the invention are preferably produced by recombinant means. The antibody is therefore preferably an isolated monoclonal antibody. Recombinant techniques of this type are well known in the art and involve expression of proteins in prokaryotic and eukaryotic cells followed by isolation of the antibody polypeptide and usually purification.

V

MY

34780Β1 at a pharmaceutically acceptable purity. For expression of the protein, nucleic acids encoding the heavy and light chains or fragments thereof are inserted into expression vectors by standard techniques.

Expression takes place in appropriate prokaryotic or eukaryotic host cells such as CHO cells, NSO cells, SP2 / 0 cells, ΗΕΚ293 cells, COS cells, yeast, or £ l coh 'cells, and the antibody is recovered from the cells (supernatant or cells after lysis).

The production of antibodies by recombination is well known in the art and is described, for example, in the review articles by Makrides, sc. Protein Expr. Purif. 17 (1999) 183-202; Geisse, s., Et al. Protein Expr. Purif. 8 (1996) 271-282; Kaufman, R. L, Mol. Biotechnol. 16 (2000) 151-161; Wemer, RG, Drug Res. 48 (1998) 870-880.

The antibodies can be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. Purification to remove other cellular constituents or other impurities, eg, other nucleic acids or cellular proteins, is performed by standard techniques including treatment with base / SDS, banding on CsCl, column chromatography, agarose gel electrophoresis, and others well known to those skilled in the art. See Ausubel, F., et al., Eds.

Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

Expression in NSO cells is described, for example, by Barnes, LM, et al., Cytotechnology 32 (2000) 109-123; and Barnes, LM) et al. Biotech. Bioeng. 73 (2001) 261-270. Temporary expression is described, for example, by Durocher, Y., et al., Nucl. Acids. Res. 30 (2002) Ε9. Variable domain cloning is described by Orlandi, R., et al., Proc. Natl. Acad. Sci. USA 86 (1989) 38333837; Carter, P., et al., Proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289; and Norderhaug, L., et al., 7. 87-77 (1997) 204 تصسؤ .سربءد. A preferred temporary expression system (HEK 293) is described by Schlaeger, E.-J., and Christensen, K., Cytotechnology 30 (1999) 71-83 and by Schlaeger, E.-J., J. Immunol .

.191-199 (1996) 194 دجه

Control sequences which are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. It is known that eukaryotic cells use promoters, enhancers and polyadenylation signals.

A nucleic acid is operably linked when placed in

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34780Β1 a functional relationship with another nucleic acid sequence. For example, DNA encoding a presequence or a secretory leader sequence is operably linked to DNA encoding a polypeptide if it is expressed as a preprotein which participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, operably linked means that the linked DNA sequences are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, the amplifiers do not have to be contiguous.

Binding occurs by ligation at convenient restriction sites. when such sites do not exist, synthetic oligonucleotide adapters or linkers are used in accordance with current practice.

the monoclonal antibodies are suitably separated from the culture medium by conventional immunoglobulin purification techniques such as, for example, chromatography with protein A - Sepharose, hydroxyapatite, gel electrophoresis, dialysis, or affinity chromatography. DNA and RNA encoding monoclonal antibodies are readily isolated and sequenced by standard techniques. Hybridoma cells can serve as a source for these DNA and RNA. Once isolated, the DNA can be inserted into expression vectors, which are then transfected into host cells such as ΗΕΚ293 cells, CHO cells, or myeloma cells which otherwise do not produce the 293 protein. immunoglobulin, to achieve the synthesis of recombinant monoclonal antibodies in host cells.

As used herein, the terms cell, cell line and cell culture are used interchangeably and all such designations include progeny. Thus, the words transformed cells include the primary subject cell and the cultures derived therefrom, regardless of the number of transfers. It is also understood that all offspring may not be exactly identical in DNA content, due to deliberate or accidental mutations. Variant progeny which have the same function or biological activity as that screened in the original transformed cells are included.

The Fc part of an antibody is not directly involved in the binding of the antibody to the antigen, but exhibits several effector functions. An Fc portion of an antibody is an expression well known to those skilled in the art and is defined on the basis of the cleavage of antibodies by papain. According to the sequence

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34780Β1 of amino acids from the constant region of their heavy chains, antibodies or immunoglobulins are divided into the classes IgA, IgD, IgE, IgG and IgM, and several of them can be further divided into subclasses (isotypes) , for example IgG1, IgG2, IgG3 and IgG4, IgAl and IgA2. Depending on the constant regions of the heavy chains, the different classes of immunoglobulins are called respectively a, δ, ε, γ and μ. The Fc part of an antibody is directly involved in ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity) based on complement activation, C1q binding and binding to receiver of Fc. Complement activation (CDC) is initiated by binding of complement factor Clq to the Fc portion of most antibody subclasses.

IgG. While the influence of an antibody on the complement system depends on certain conditions, binding to Clq is due to binding sites defined in the Fc part. These binding sites are known in the art and described, for example, by BoackleRJ., Et al., Atowre 282 (1979) 742-743, Lukas, TJ., Et al., 7 سسد.

127 (1981) 2555-2560, Bmnhouse, R., and Cebra, JJ, Mol. Immunol. 16 (1979) 907917, Burton, DR, et al., Mztare 288 (1980) 338-344, Thommesen, JE, et al., Λ7٠1.

Immunol. 37 (2000) 995-1004, Idusogie, Ε.Ε., et al., 7. Immunol. 164 (2000) 41784184, Hezareh, M., et al., Virology 75 (2001) 12161-12168, Morgan, A., et al., TtfWiwrcology 86 (1995) 319-324, patent document EP 0307434. These Binding sites are for example L234, L235, D27O, Ν297, Ε318, Κ320, Κ322, Ρ331 and Ρ329 (numbering according to the EU index of Kabat, Ε.Α., see below). antibodies of the IgG1, IgG2 and IgG3 subclasses usually show activation of complement and binding to C1q and C3, while IgG4 does not activate the complement system and does not bind to C1q and C3.

In one embodiment, the antibody according to the invention comprises an Fc part of human origin and preferably all the other parts of the human constant regions. As used herein, the expression Fc part of human origin denotes an Fc part which is an Fc part of a human antibody of the IgG1, IgG2, IgG3 or IgG4 subclass, preferably an Fc part of the subclass. -class 30 of human IgGl, a mutated Fc part of the human IgGl subclass (preferably with an L234A + L235A mutation), an Fc part of the human IgG4 subclass or a mutated Fc part of the human IgG4 subclass (preferably with an S228P mutation). Preferred are the constant regions of human heavy chains of SEQ ID NO: 58 (human IgG1 subclass), SEQ 35 ID NO: 59 (human IgG1 subclass with L234A and L235A mutations),

SEQ ID NO: 60 (subclass of human IgG4) or SEQ ID NO: 61 (subclass

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34780Β1 of human IgG4 with the S228P mutation).

Preferably, the antibody according to the invention is from the subclass of human IgG1 or from the subclass of human IgG4. In one embodiment, the antibody according to the invention is from the subclass of human IgG1. In one embodiment, the antibody according to the invention is of the human IgG4 subclass.

In one embodiment, the antibody according to the invention is characterized in that the constant chains are of human origin. These constant chains are well known in the art and are described, for example, by Kabat, Ε.Α. (see eg Johnson, G. and Wu, Τ.Τ., Nucleic Acids Res. 28 (2000) 214-218). For example, a useful human heavy chain constant region comprises an amino acid sequence of SEQ DD NO: 58. For example, a useful human light chain constant region comprises an amino acid sequence of a kappa light chain constant region of SEQ DD NO: 57.

Another aspect of the invention is an antibody binding to human CSF-1R, characterized in 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 DD NO: 16;

or one of its humanized versions.

Another aspect of the invention is an antibody binding to human CSF-1R, characterized in that

a) the heavy chain variable domain is SEQ ID NO: 7 and the light chain variable domain is SEQ DD NO: 8;

b) the heavy chain variable domain is SEQ DD 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 DD 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 one of its humanized versions.

Another aspect of the invention is an antibody binding to human CSF-1R, characterized in that the heavy chain variable domain is SEQ DD NO: 7 and the light chain variable domain is SEQ DD NO: 8;

or one of its humanized versions.

ΜΑ 34780Β1

Another aspect of the invention is an antibody binding to human CSF-1R, characterized in 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 ro NO: 55 and the light chain variable domain is SEQ ro NO: 56.

Another aspect of the invention is an antibody binding to human CSF-1R, characterized in that:

a) the heavy chain variable domain is SEQ ro NO: 23 and the light chain variable domain is SEQ ro NO: 24; or

b) the heavy chain variable domain is SEQ ro NO: 31 and the light chain variable domain is SEQ ro NO: 32; or

c) the heavy chain variable domain is SEQ ro NO: 39 and the light chain variable domain is SEQ ro NO: 40; or

d) the heavy chain variable domain is SEQ ro NO: 47 and the light chain variable domain is SEQ ro NO: 48.

Another aspect of the invention is an antibody binding to human CSF-1R, characterized in that:

the heavy chain variable domain is SEQ ro NO: 23 and the light chain variable domain is SEQ ro NO: 24.

Another aspect of the invention is an antibody binding to human CSF-1R, characterized in that:

the heavy chain variable domain is SEQ ro NO: 31 and the light chain variable domain is SEQ ro NO: 32.

Another aspect of the invention is an antibody binding to human CSF-1R, characterized in that:

the heavy chain variable domain is SEQ ro NO: 39 and the light chain variable domain is SEQ ro NO: 40.

Another aspect of the invention is an antibody binding to human CSF-1R, characterized in that:

<img file="MA34780B1_D0008.tif" />

34780Β1 the heavy chain variable domain is SEQ ID NO: 47 and the light chain variable domain is SEQ ED NO: 48.

Another aspect of the invention is an antibody binding to human CSF-1R, characterized in that:

the heavy chain variable domain is SEQ ID NO: 15 and the light chain variable domain is SEQ ID NO: 16, or a humanized version thereof.

Another aspect of the invention is an antibody binding to human CSF-1R, characterized in that:

the heavy chain variable domain is SEQ ID NO: 75 and the light chain variable domain is SEQ ED NO: 76, or a humanized version thereof.

Another aspect of the invention is an antibody binding to human CSF-1R, characterized in 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 thereof.

Another aspect of the invention is an antibody binding to human CSF-1R, characterized in that

a) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 1, a CDR2 region of SEQ ID NO: 2 and a CDR1 region 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 ro 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 CDR3 region 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 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, 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 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

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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 CDR3 region of SEQ ID NO: 36, a CDR2 region of SEQ ID NO: 37 and a CDR1 region of

SEQ ED 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 CDR3 region 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 BD NO: 51, and the light chain variable domain comprises a CDR3 region of SEQ DD NO: 52, a CDR2 region of SEQ ED NO: 53 and a CDR1 region of

SEQ DD NO: 54,

h) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 69, a CDR2 region of SEQ ED NO: 70 and a CDR1 region of SEQ BD NO: 71, and the light chain variable domain comprises a CDR3 region of SEQ DD NO: 72, a CDR2 region of SEQ DD NO: 73 and a CDR1 region of

SEQ DD NO: 74, or

i) the heavy chain variable domain comprises a CDR3 region of SEQ ED NO: 77, a CDR2 region of SEQ DD NO: 78 and a CDR1 region of SEQ DD NO: 79, and the light chain variable domain comprises a CDR3 region of SEQ DD NO: 80, a CDR2 region of SEQ DD NO: 81 and a CDR1 region of

SEQ DD NO: 82.

Another aspect of the invention is an antibody binding to human CSF-1R, characterized in that:

a) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 17, a CDR2 region of SEQ ED NO: 18 and a CDR1 region of SEQ DD

NO: 19, and the light chain variable domain comprises a CDR3 region of

SEQ ED NO: 20, a CDR2 region of SEQ DD NO: 21 and a CDR1 region of SEQ DD 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 BD

NO: 27, and the light chain variable domain comprises a CDR3 region of

SEQ DD NO: 28, a CDR2 region of SEQ DD NO: 29 and a CDR1 region of

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SEQ ID NO: 30, OR

c) the heavy chain variable domain comprises a CDR3 region of SEQ BD 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 ED 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 CDR3 region 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 light chain variable domain comprises a CDR3 region of SEQ ID NO: 52, a CDR2 region of SEQ ID NO: 53 and a CDR1 region of SEQ ID NO: 54.

Another aspect of the invention is an antibody binding to human CSF-1R, characterized in that:

a) the heavy chain variable domain comprises a CDR3 region of SEQ ED NO: 17, a CDR2 region of SEQ ID NO: 18 and a CDR1 region of SEQ ED 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 ro 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 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 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 BD NO: 35, and the light chain variable domain comprises a CDR3 region of SEQ DD NO: 36, a CDR2 region of SEQ DD 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 DD NO: 43, and the light chain variable domain comprises a CDR3 region of

<img file="MA34780B1_D0009.tif" />

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34780Β1

SEQ ID NO: 44, a CDR2 region of SEQ ID NO: 45 and a CDR1 region of SEQ BD NO: 46.

Another aspect of the invention is an antibody binding to human CSF-1R, characterized in that:

the heavy chain variable domain comprises a CDR3 region of SEQ ID

NO: 17, a CDR2 region of SEQ DD NO: 18 and a CDR1 region of SEQ DD NO: 19, and the light chain variable domain comprises a CDR3 region of SEQ DD NO: 20, a CDR2 region of SEQ DD NO: 21 and a CDR1 region of SEQ DD NO: 22.

Another aspect of the invention is an antibody binding to human CSF-1R, characterized in that:

the heavy chain variable domain comprises a CDR3 region of SEQ BD NO: 25, a CDR2 region of SEQ DD NO: 26 and a CDR1 region of SEQ ID NO: 27, and the light chain variable domain comprises a CDR3 region of 15 SEQ ID NO: 28, a CDR2 region of SEQ ID NO: 29 and a CDR1 region of

SEQ BD NO: 30.

Another aspect of the invention is an antibody binding to human CSF-1R, characterized in that:

the heavy chain variable domain comprises a CDR3 region of SEQ DD 20 NO: 33, a CDR2 region of SEQ JD NO: 34 and a CDR1 region of SEQ BD

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.

Another aspect of the invention is an antibody binding to human CSF-1R, characterized in that:

the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 41, a CDR2 region of SEQ BD NO: 42 and a CDR1 region of SEQ ID NO: 43, and the light chain variable domain comprises a CDR3 region of SEQ DD NO: 44, a CDR2 region of SEQ BD NO: 45 and a CDR1 region of SEQ ID NO: 46.

The invention comprises a method of treating a patient in need of therapy, characterized by administering to the patient a therapeutically effective amount of an antibody according to the invention.

The invention includes the use of an antibody according to the invention for therapy.

A preferred embodiment of the invention is the

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34780Β1 anti-CSF-1R antibody of the present invention, for use in the treatment of diseases mediated by CSF-1R or anti-CSF-1R antibodies of the present invention for use in the manufacture of a medicament for the treatment of diseases mediated by CSF-1R, which can be described as follows:

5 ا There are 3 distinct mechanisms by which CSF-1R signaling is probably involved in tumor growth and metastasis. ئ first is that the expression of the CSF receptor and ligand has been observed in tumor cells originating from the female reproductive system (breast, ovary, endometrium, cervix) (Scholl, SM, et al.,]. Natl. Cancer Inst. 86 (1994) 120-126; Kacinski, Β.Μ., Mol.

Reprod. Dev. 46 (1997) 71-74; Ngan, Η.Υ., et al., Eur. ]. Cancer 35 (1999) 15461550; Kirma, N., et al .. Cancer Res 67 (2007) 1918-1926) and expression has been associated with breast cancer xenograft growth as well as poor prognosis in cancer patients. breast. Two points of mutation in CSF-IR were observed in approximately 10-20% of patients with acute myelocytic leukemia, chronic myelocytic leukemia and myelodysplasia tested in one study, and one of the mutations was shown to disrupt the cycle of reconstitution of the receptor (Ridge, SA, et al., Proc. Natl. Acad. Sci USA 87 (1990) 1377-1380). However, the incidence of the mutations could not be confirmed in subsequent studies (Abu-Duhier, FM, et al., Br. J. Haematol. 120 (2003) 464-470). Mutations have also been found in some cases of hepatocellular cancer (Yang, DH, et al., Hepatobiliary Pancreat. Dis. Int. 3 (2004) 86-89) and idiopathic myelofibrosis (Abu-Duhier, FM, et al. ., Br. J. Haematol. 120 (2003) 464-470). The Y571D mutation in CSF-1R was recently identified in the GDM-1 cell line from a patient suffering from myelomonoblastic leukemia (Chase, A., et al., 23 (2009) 358-364).

Pigmented villonodular synovitis (SVNP) and tenosynovial giant cell tumors (TTCG) can occur as a result of a translocation that fuses the Μ-CSF gene to a COL6A3 collagen gene and results in overexpression of Μ-CSF (West , RB, et al., Proc. Natl. Acad. Sci. USA 30103 (2006) 690-695). A landscape effect is proposed as being responsible for the resulting tumor mass, which is made up of monocytic cells attracted to cells which express Μ-CSF. ITTCG are small tumors that are easily removed from the fingers where they appear most often. PVNS is more aggressive because it can recur in large joints and is not as easily treated with surgery.

The second mechanism is based on blocking signaling by

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ΜΑ 34780Β1 Intermediate Μ-CSF / CSF-IR at metastatic sites in bone, which induces osteoclastogenesis, bone resorption and osteolytic bone lesions. Breast cancers, multiple myelomas, and lung cancer are examples of cancers that have been shown to metastasize into bone and lead to osteolytic bone disease leading to skeletal complications. MCSF released by tumor cells and stroma induces the differentiation of progenitors of hematopoietic myeloid monocytes into mature osteoclasts in conjunction with the nuclear factor activator receptor ligand kappa-B (RANKL). During this process, Μ-CSF acts as a permissive factor by giving the signal for osteoclast survival (Tanaka, s., Et al., J. Clin. Invest. 91 (1993) 257-263). Inhibition of CSF-1R activity during osteoclast differentiation and maturation with anti-CSF-1R antibody is expected to prevent the imbalanced osteoclast activity that results in osteolytic disease and the skeletal-specific phenomena associated with the disease. metastatic. While breast cancer, lung cancer, and multiple myeloma typically result in osteolytic lesions, bone metastases in prostate cancer initially have an osteoblastic appearance where increased bone-forming activity. results in a woven bone that is different from the typical lamellar structure of normal bone. During the course of the disease, bone lesions show a strong osteolytic component, as well as elevated serum levels of bone resorption, and this suggests that anti-resorption therapy may be useful. Bisphosphonates have been shown to inhibit the formation of osteolytic lesions and reduce the number of skeletal phenomena only in men with metastatic hormone refractory prostate cancer, but at this point their effect on osteoblastic lesions is controversial. and bisphosphonates have heretofore not been beneficial in preventing bone metastasis or hormone-responsive prostate cancer. The effect of anti-resorption agents in mixed osteolytic osteoblastic cancer of the prostate is still under clinical investigation (Choueiri, Μ.Β., et al .. Cancer Metastasis Rev. 25 (2006) 601-609; Vessella, RL and Corey, E., Clin. Cancer Res. 12 (20 Pt 2) (2006) 6285s-6290s).

The third mechanism is based on the recent observation that tumor associated macroplations (TATs) found in solid tumors of breast, prostate, ovarian and cervical cancers correlate with a poor prognosis ( Bingle, L., et al., J. Pathol. 796 (2002) 254-265; Pollard, JW, Nat. Rev. Cancer 4 (2004) 71-78). Macrophages are recruited to the tumor by

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34780Β1 Μ-CSF and other chemokines. Macrophages can then contribute to tumor progression through the secretion of angiogenic factors, proteases and other growth factors and cytokines and can be blocked by inhibiting CSF-1R signaling. Zins et al. (Zins, K., et al., Cancer Res. 67 (2007) 1038-1045) recently showed that the expression of the siRNA of tumor necrosis factor alpha (TNF-a), Μ-CSF or a combination of the two would reduce, in a mouse xenograft model, tumor growth of between 34% and 50% after intratumoral injection of the corresponding siRNA. SiRNA targeting TNFα secreted by human SW620 cells reduced Μ-CSF levels in mice and resulted in reduction of macrophages in the tumor. In addition, treatment of MCF7 tumor xenografts with an antigen-binding fragment directed against MCSF effectively resulted in 40% inhibition of tumor growth, reversed resistance to chemotherapy, and improved survival in mice when was administered in combination with chemotherapeutics (Paulus, P., et al. Cancer Res.

66(2006)4349-4356).

MAT is just one example of an emerging link between chronic inflammation and cancer. There is further evidence for a link between inflammation and cancer, as many chronic diseases are associated with an increased risk of cancer, cancers occur at sites of chronic inflammation, there is evidence for this. chemical mediators of inflammation in many cancers; deletion of cellular or chemical mediators of inflammation inhibits the development of experimental cancers, and prolonged use of anti-inflammatory agents reduces the risk of certain cancers. A link to cancer exists for a number of inflammatory disorders, among which gastritis induced by H. pylori 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) 211217). Macrophages are key cells in chronic inflammation and respond differentially to their microenvironment. There are two types of macrophages which are considered to be extreme in a continuum of functional states: M1 macrophages are involved in type 1 reactions. These reactions involve activation by microbial products, and the resulting destruction of pathogenic microorganisms which give reactive oxygenated intermediates. At the other end of the spectrum, there are Μ2 macrophages involved in type 2 reactions which promote cell proliferation, good inflammation and

<img file="MA34780B1_D0011.tif" />

MA 34780Β1 adaptive immunity and promote tissue remodeling, angiogenesis and repair (Mantovani, A., et al. Trends Immunol. 25 (2004) 677-686). Chronic inflammation leading to established neoplasia is usually associated with Μ2 macrophages. A pivotal cytokine that induces inflammatory reactions is TNF-α which, as its name suggests, can stimulate anti-tumor immunity and high dose hemorrhagic necrosis, but which has also recently been shown to be expressed by tumor cells and act as a tumor promoter (Zins, K., et al. Cancer Res. 67 (2007) 1038-1045; Balkwill, F., Cancer Metastasis Rev. 25 (2006) 409-416). There is still a need to better understand the specific role of macrophages in relation to tumor, including the potential spatial and temporal dependence of their function and the relevance for specific tumor types.

Thus, one embodiment of the invention is the anti-CSF-1R antibodies of the present invention for use in the treatment of cancer. The term cancer, as used herein, can include, for example, lung cancer, non-small cell lung cancer, bronchio-alveolar lung cancer, bone cancer, pancreatic cancer, cancer of the breast. skin, head or neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, cancer of the stomach, gastric cancer, colon cancer, breast cancer, cancer of the uterus, carcinoma of the fallopian tubes, carcinoma of the endometrium; carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, 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 of the urethra, cancer of the penis, cancer of the prostate, cancer of the bladder, cancer of the kidney or urethra, renal cell carcinoma, renal pelvis carcinoma, mesothelioma, hepatocellular cancer, biliary cancer, central nervous system (CNS) neoplasms, spinal cord tumors, brainstem glioma, glioblastoma multiforme, astrocytomas, schwannomas, ependymomas, medulloblastomas, meningiomas, squamous cell carcinomas, pituitary adenoma, lymphoma, lymphocytic leukemia, including the refractory versions of the aforementioned cancers, or a combination of one or more of the above cancers. This cancer is preferably breast cancer, ovarian cancer, cervical cancer, lung cancer or prostate cancer. These cancers are preferably further characterized by the expression or

<img file="MA34780B1_D0012.tif" />

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34780Β1 overexpression of CSF-1 or CSF-1R. Another embodiment of the invention is the anti-CSF-1R antibodies of the present invention, for use in the simultaneous treatment of primary tumors and new metastases.

Thus, another embodiment of the invention is constituted by the anti-CSF-1R antibodies of the present invention, for use in the treatment of periodontitis, histiocosis X, osteoporosis, bone disease of Paget (MOP), bone loss due to cancer therapy, periprosthetic osteolysis, glucocorticoid-induced osteoporosis, rheumatoid arthritis, psoriatic arthritis, osteoarthritis, inflammatory arthritis and inflammation.

Rabello, D., et al., Biochem. Biophys. Res. Common. 347 (2006) 791-796, demonstrated that SNPs of the CSF-1 gene exhibited a positive association with aggressive periodontitis: an inflammatory disease of periodontic tissue that results in tooth loss due to resorption of alveolar bone.

Histiocytosis X (also called Langerhansian histiocytosis) is a proliferative disease of Langerhans dendritic cells that is seen to differentiate into osteoclasts in bone and extraosseous lesions of Langerhans histiocytosis. Langerhans cells originate from circulating monocytes. Increased levels of Μ-CSF measured in serum and lesions have been shown to correlate with disease severity (da Costa, CE, et al., J. Exp. Med. 201 (2005) 687-693). The disease occurs primarily in a pediatric patient population and should be treated with chemotherapy when it becomes systemic or recurs.

The pathophysiology of osteoporosis is induced by loss of bone forming osteoblasts and increased osteoclast dependent bone resorption. Supporting data has been described by Cenci et al., Showing that injection of anti-M-CSF antibodies conserves bone density and inhibits bone resorption in ovariectomized mice (Cenci, s., Et al. ,]. Clin. Invest. 105 (2000) 1279-1287). A potential link with postmenopausal bone loss due to estrogen deficiency has recently been identified and the presence of TNF-α-producing T cells has been observed to affect bone metabolism (Roggia, v., Et al., Minerva Med. 95 (2004) 125-132). One possible mechanism could be the induction of Μ-CSF by TNF-α n w'vo. The important role of MCSF in TNF-α-induced osteoclastogenesis was confirmed by the effect of an antibody directed against Μ-CSF, which blocked TNF-α-induced osteolysis in mice, causing thus inhibitors of CSF-1R signaling of targets

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34780Β1 potential for inflammatory arthritis (Kitaura, H., et al., J. Clin. Invest. 115 (2005) 3418-3427).

Paget's disease of bone (PDB) is the second most common bone metabolism disorder after osteoporosis, in which focal abnormalities of increased bone turnover lead to complications such as bone pain, deformity, bone pain, bone strain, bone loss, bone loss and bone formation. pathological fractures and deafness. Mutations have been identified in 4 genes that regulate the normal function of osteoclasts and predispose individuals to POM and related disorders: insertional mutations in TNFRSFllA, which encodes a nuclear factor 10 activating receptor (NF) kappa B (RANK), a critical regulator of osteoclast function, inactivating mutations in TNFRSFllB which encodes osteoprotegerin (decoy receptor for the RANK ligand), mutations in the sequestosome 1 gene (SQSTMl), which encodes an important scaffold protein in the NFkappaB pathway, and mutations in the gene for valosin-containing protein (VTP). This gene encodes PCV, which has a role in targeting the NFkappaB inhibitor for proteasome degradation (Daroszewska, A. and Ralston, SH, Nat. Clin. Pract. Rheumatol. 2 (2006) 270- 277). Targeted antiCSF-1R inhibitors provide an opportunity to indirectly block the deregulation of RANKL signaling and add an additional treatment option to the bisphosphonates in use today.

Bone loss induced by cancer therapy, particularly in patients with breast or prostate cancer, is a further indication in which a target CSF-1R inhibitor could prevent bone loss (Lester, JE, et al. ., Br. J. Cancer 94 (2006) 30-35). With the improved prognosis for early breast cancer, the long-term consequences of adjuvant therapies become more important since some of the therapies, including chemotherapy, irradiation, aromatase inhibitors, and ovarian ablation. , alter bone metabolism by lowering bone mineral density, leading to an increased risk of osteoporosis and associated fractures (Lester, JE, et al., 30 Br. j. Cancer 94 (2006) 30-35). The equivalent of adjuvant therapy with aromatase inhibitors in breast cancer is androgen ablation therapy in prostate cancer, which results in loss of bone mineral density and significantly increases the risk of associated fractures. to osteoporosis (Stoch, SA, et al., j. Clin. Endocrinol. Metab. 86 (2001) 2787-2791).

Targeted inhibition of CSF-1R signaling is also expected to be beneficial in other indications where the cell types targeted include

<img file="MA34780B1_D0014.tif" />

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34780Β1 osteoclasts and macrophages, for example in the treatment of specific complications in response to joint replacement following rheumatoid arthritis. Failure of implantation due to periprosthetic bone loss and the resulting looseness of the prosthesis is a major complication of joint replacement and requires repeated chemotherapy operations, with high socioeconomic burdens for the individual patient and patient. the health system. There is currently no authorized drug therapy to prevent or inhibit periprosthetic osteolysis (Drees, p., Et al., Nat. Clin. Pract.

. (165-171 (2007) 3 لحسد

Glucocorticoid-induced osteoporosis is another indication in which a CSF-1R inhibitor may prevent bone loss after prolonged use of glucocorticosteroids which are administered as a result of various pathologies, including chronic obstructive pulmonary disease, asthma. and rheumatoid arthritis (Guzman-Clark, JR, et al., Arthritis Rheum. 57 (2007) 140-146; Feldstein, AC, et al., Osteoporos. Int. 16 (2005) 2168-2174).

Rheumatoid arthritis, psoriatic arthritis and inflammatory arthritis are in themselves potential indications for inhibitors of CSF-1R signaling, as they consist of a macrophage component and to a varying degree of. bone destruction (Ritchlin, CT, et al., Clin. Invest. Ill (2003) 821-831). Osteoarthritis and rheumatoid arthritis are autoimmune inflammatory diseases caused by the build-up of macrophages in connective tissue and the infiltration of macrophages into synovial fluid, which are at least partially induced by Μ-CSF. Campbell, I., K., et al. 7. Leukoc. 25 Biol. 68 (2000) 144-150, have shown that Μ-CSF is produced by cells of human joint tissues (chondrocytes, synovial fibroblasts) in vitro and that it is found in the synovial fluid of patients suffering from rheumatoid arthritis , suggesting that it contributes to the proliferation of synovial tissue and the infiltration of macrophages which is associated with the pathogenesis of the disease. 30 Inhibition of CSF-1R signaling is expected to lower the number of macrophages in the joint and relieve pain due to associated bone destruction. To minimize the adverse effects and further understand the impact of CSF-1R signaling in these indications, one technique is to inhibit CSF-1R specifically without targeting a multitude of other kinases such as Raf kinase.

Recent literature reports a correlation between the increase in

MA 34780Β1

Circulating Μ-CSF with poor prognosis and atherosclerotic progression in chronic coronary insufficiency (Saitoh, T., et al., J. Am. Coll. Cardiol. 35 (2000) 655-665; Ikonomidis, I., et al. al., Eur. Heart. 2005) 26 .ل) p. 1618-1624); MCSF influences the atherosclerotic process by promoting the formation of foam cells (macrophages with ingested oxidized LDL) which express CSF-1R and represent the initial plaque (Murayama, T., et al .. Circulation 99 (1999) 17401746).

Expression and signaling of Μ-CSF and CSF-1R are seen in activated microglia. Microglia, which are resident macrophages of the central nervous system, can be activated by a variety of insults, including infection and traumatic injury. Μ-CSF is believed to be a key regulator of inflammatory responses in the brain, and levels of Μ-CSF are increased in HIV-1, encephalitis, Alzheimer's disease (AD) and brain tumors. Microgliosis, as a consequence of autocrine signaling by MCSF / CSF-IR, induces the release of inflammatory cytokines and nitric oxides, as demonstrated for example by an experimental neuronal lesion model ( Hao, AJ, et al. Neuroscience 112 (2002) 889-900; Murphy, GM, Jr., et al., 7. Biol. Chem. 273 (1998) 20967-20971). Microglia which have accme expression of CSF-1R appear to surround plaques in AD and in the mouse model of AD transgenic with the amyloid precursor protein V717F (Murphy, GM, Jr., et al., Am. ] Pathol 157 (2000) 895904). On the other hand, ορ / ορ mice with less microglia in the brain gave fibril deposition of ill-β and neuronal loss compared to the normal control, suggesting that the microglia did indeed have a neuroprotective function in the brain. development of AD, which does not exist in ορ / ορ mice (Kaku, M., et al. Brain Res. Brain Res. Protoc. 12 (2003) 104-108).

The expression and signaling of Μ-CSF and CSF-1R are associated with inflammatory bowel disease (Mil) (patent document wo 2005/046657). The term inflammatory bowel disease refers to severe chronic disorders of the intestinal tract characterized by chronic inflammation at various sites of the gastrointestinal tract, and specifically includes ulcerative colitis and Crohn's disease.

The invention comprises an antibody binding to human CSF-1R, characterized by the aforementioned epitope binding properties or alternatively by the aforementioned amino acid sequences and fragments of amino acid sequences, for the treatment of cancer.

MK

34780Β1

The invention comprises an antibody binding to human CSF-1R, characterized by the aforementioned epitope-binding properties or alternatively by the aforementioned amino acid sequences and fragments of amino acid sequences, for the treatment of bone loss. .

The invention comprises an antibody which binds to human CSF-1R, characterized by the aforementioned epitope-binding properties or alternatively by the aforementioned amino acid sequences and fragments of amino acid sequences, for the prevention or treatment of metastases.

The invention comprises an antibody binding to human CSF-1R, characterized by the aforementioned epitope binding properties or alternatively by the aforementioned amino acid sequences and fragments of amino acid sequences, for the treatment of inflammatory diseases.

The invention comprises the use of an antibody binding to human CSF-1R, characterized by the aforementioned epitope binding properties or alternatively by the aforementioned amino acid sequences and fragments of amino acid sequences, for for the treatment of cancer or for the manufacture of a medicament for the treatment of cancer.

The invention comprises the use of an antibody binding to human CSF-1R, characterized by the aforementioned epitope binding properties or alternatively by the aforementioned amino acid sequences and fragments of amino acid sequences, for for the treatment of bone loss or for the manufacture of a medicament for the treatment of bone loss.

The invention comprises the use of an antibody binding to human CSF-1R, characterized by the aforementioned epitope binding properties or alternatively by the aforementioned amino acid sequences and fragments of amino acid sequences, for for the prevention or treatment of metastases or for the manufacture of a medicament for the prevention or treatment of metastases.

The invention comprises the use of an antibody binding to human CSF-1R, characterized by the aforementioned epitope binding properties or alternatively by the aforementioned amino acid sequences and fragments of amino acid sequences, for for the treatment of inflammatory diseases or for the manufacture of a medicament for the treatment of inflammatory diseases.

Another embodiment of the invention is a method of producing an antibody directed against CSF-1R, characterized by inserting the sequence of a nucleic acid encoding the heavy chain of an antibody. of the human IgG class binding to human CSF-1R and a nucleic acid encoding the chain

<img file="MA34780B1_D0015.tif" />

ΜΑ 34780Β1 of said antibody into an expression vector, said vector is inserted into a eukaryotic host cell, and the encoded protein is expressed and recovered from the host cell or the supernatant.

The antibodies according to the invention are preferably produced by recombinant means. Methods of this type are well known in the art and involve expression of proteins in prokaryotic and eukaryotic cells, followed by isolation of the antibody polypeptide and usually purification to a pharmaceutically acceptable purity. For protein expression, nucleic acids encoding the heavy and light chains or fragments thereof are inserted into expression vectors by standard techniques. Expression takes place in appropriate prokaryotic or eukaryotic host cells such as CHO cells, NSO cells, SP2 / 0 cells, ΗΕΚ293 cells, cos cells, yeast, or dΈ cells. coli, and the antibody is recovered from the cells (supernatant or after cell lysis).

The production of antibodies by recombination is well known in the state of the art and is described, for example, in the review articles by Makrides, sc. Protein Expr. Purif. 17 (1999) 183-202; Geisse, s., Et al. Protein Expr. Purif. 8 (1996) 271-282; Kaufuaan, R. L, Mol. Biotechnol. 16 (2000) 151-161; Wemer, R. G., Drug Res. 48 (1998) 870-880.

the antibodies can be present in whole cells, in a cell lysate, or in a partially purified or essentially pure form. Purification to remove other cellular constituents or other impurities, eg, other nucleic acids or cellular proteins, is performed by standard techniques, including treatment with base / SDS, banding on CsCl, column chromatography, agarose gel electrophoresis, and others well known to those skilled in the art. See Ausubel, F., et al., Eds. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

Expression in NSO cells is described, for example, by Barnes, LM, et al., Cytotechnology 32 (2000) 109-123; and Banes, LM, et al. Biotech. Bioeng. 73 (2001) 261-270. Temporary expression is described, for example, by Durocher, Y., et al., Nucl. Acids. Res. 30 (2002) Ε9. ممل variable domain cloning is described by Orlandi, R., et al., Proc. Natl. Acad. Sci. USA 86 (1989) 38333837; Carter, p., Et al., Proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289; and Norderhaug, L., et al. 7. Immunol. Methods 204 (1997) 77-87. A preferred temporary expression system (HEK 293) is described by Schlaeger, E.-L, and

<img file="MA34780B1_D0016.tif" />

I

34780Β1

Christensen, K., Cytotechflo / ogy 30 (1999) 71-83 and by Schlaeger, E.-J., 7.! ™ moi

.191-199 (1996) 194 ده

Nucleic acid molecules encoding variants of the amino acid sequence of the anti-CSF-1R antibody are prepared by various methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring variants of the amino acid sequence), or preparation by mutagenesis via oligonucleotides (or site-directed), by PCR mutagenesis, and by mutagenesis with a cassette of a previously prepared variant or a non-variant version of the humanized anti-CSF-1R antibody.

The heavy and light chain variable regions are combined with promoter, translation initiation, constant region, 3 'untranslated region, polyadenylation and transcription termination sequences to form vector constructs. expression. The heavy and light chain expression constructs can be combined into a single vector, co-transfected, successively transfected, or separately transfected into host cells which are then fused to form a single host cell expressing both chains.

In another aspect, the present invention provides a composition, for example a pharmaceutical composition, containing a monoclonal antibody or a combination of monoclonal antibodies, or their antigen-binding portion, of the present invention, formulated with a carrier. pharmaceutically acceptable.

As used herein, a pharmaceutically acceptable carrier includes all solvents, dispersing media, coatings, antibacterial and antifungal agents, isotonic agents and absorption / resorption delaying agents, and the like, which are physiologically compatible. The carrier is preferably suitable for injection or infusion.

A composition of the present invention can be administered by various methods known in the art. As one skilled in the art will recognize, the route and / or mode of administration will vary depending on the results desired.

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

Regardless of the route of administration chosen, the compounds of

/٦

The present invention, which may be used in a suitable hydrated form, and / or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by standard techniques known to those skilled in the art.

The effective dose levels of the active ingredients in the pharmaceutical compositions of the present invention can be varied to obtain an amount of active ingredient which is effective to provide the desired therapeutic response for a particular patient, composition and mode of administration. , without being toxic to the patient (effective amount). ماً dose level chosen will depend on various pharmacokinetic factors including the activity of the particular compositions of the present invention used, or their esters, salts or amides, the route of administration, the time of administration, the speed excretion of the particular compound used, other drugs, compounds and / or products used in combination with the particular compositions used, age, sex, weight, disease, the general health and previous medical history of the patient being treated, and like factors well known in the medical art.

The invention comprises the use of the antibodies according to the invention for the treatment of a patient suffering from cancer, in particular cancer of the colon, lung or pancreas.

The invention also includes a method of treating a patient suffering from such a disease.

The invention further provides a method of making a pharmaceutical composition comprising an effective amount of an antibody according to the invention with a pharmaceutically acceptable carrier and the use of the antibody according to the invention in such a method.

The invention further provides the use of an antibody according to the invention in an effective amount for the manufacture of a pharmaceutical agent, preferably with a pharmaceutically acceptable carrier, for the treatment of a patient suffering from cancer.

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 with a pharmaceutically acceptable carrier, for the treatment of a patient suffering from cancer.

The examples, sequence listing and figures below are

ΜΑ 34780Β1

1:

provided to facilitate understanding of the present invention, the true scope of which is set out in the appended claims. It is understood that modifications can be made to the techniques presented without departing from the spirit of the invention.

Description of sequences

Heavy chain CDR3, mAb 2FH heavy chain CDR2, mAb 2FH heavy chain CDR1, mAb 2FU light chain CDR3, mAb 2FH light chain CDR2, mAb 2FH light chain CDR1, mAb 2F11 heavy chain variable domain, mAb 2FH light chain variable domain, mAb 2F11 heavy chain CDR3, mAb 2Ε10 heavy chain CDR2, mAb 2Ε10 heavy chain CDR1, mAb 2Ε10 light chain CDR3, mAb 2Ε10 light chain CDR2, mAb 2Ε10 light chain CDR1, mAb 2Ε10 heavy chain variable domain, MAb 2Ε10 variable domain light chain, mAb 2Ε10 heavy chain CDR3, hAcM 2FH-dl CDR2 heavy chain, hAcM 2F11-C11 heavy chain CDR1, hAcM 2F11-C11 CDR3 light chain, hAcM 2FU-C11 CDR2 light chain , hAcM 2F11-C11 light chain CDR1, hAcM 2F11-C11 heavy chain variable domain, hAcM 2FH-C11 light chain variable domain, hAcM 2FH-C11 heavy chain CDR3, hAcM 2F11-d8 heavy chain CDR2, hAcM 2Fll -d8 heavy chain CDR1, hAcM 2F11-d8 light chain CDR3, hAcM 2F11-d8 light chain CDR2, hAcM 2Fll-d8 light chain CDR1, hAcM 2F11-d8 heavy chain variable domain, hAcM 2F11-d8

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 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 SEQDDNO SEQ ID NO SEQ ID NO SEQDDNO SEQ ID NO SEQ ED NO SEQDDNO SEQ DD NO SEQDDNO SEQDDNO

MK

34780Β1

<td></td><td>SEQ ID NO: 32</td><td>light chain variable domain, hAcM 2F11-d8</td>

<td> .</td><td>SEQ ID NO: 33</td><td>Heavy chain CDR3, hAcM 2Fll-e7</td>

<td> &gt; .</td><td>SEQ ID NO: 34</td><td>Heavy chain CDR2, hAcM 2Fll-e7</td>

<td></td><td>SEQ ID NO: 35</td><td>Heavy chain CDR1, hAcM 2Fll-e7</td>

<td> 5</td><td>SEQ ID NO: 36</td><td>Light chain CDR3, hAcM 2Fll-e7</td>

<td></td><td>SEQ ID NO: 37</td><td>Light chain CDR2, hAcM 2Fll-e7</td>

<td> .</td><td>SEQ ID NO: 38</td><td>Light chain CDRl, hAcM 2Fll-e7</td>

<td> . . .</td><td>SEQ ID NO: 39</td><td>heavy chain variable domain, hAcM 2F11-e7</td>

<td></td><td>SEQ ID NO: 40</td><td>light chain variable domain, hAcM 2Fll-e7</td>

<td> 10</td><td>SEQ © NO: 41</td><td>Heavy chain CDR3, hAcM 2F11-fl2</td>

<td> .</td><td>SEQ ID NO: 42</td><td>Heavy chain CDR2, hAcM 2F11-fl2</td>

<td> .</td><td>SEQ ID NO: 43</td><td>Heavy chain CDR1, hAcM 2Fll-fl2</td>

<td></td><td>SEQ ID NO: 44</td><td>Light chain CDR3, hAcM 2Fll-fl2</td>

<td> .</td><td>SEQ ID NO: 45</td><td>Light chain CDR2, hAcM 2Fll-fl2</td>

<td> 15</td><td>SEQ ID NO: 46</td><td>Light chain CDRl, hAcM 2Fll-fl2</td>

<td></td><td>SEQ ID NO: 47</td><td>heavy chain variable domain, hAcM 2F11-fl2</td>

<td> . . . .</td><td>SEQ ID NO: 48</td><td>light chain variable domain, hAcM 2Fll-fl2</td>

<td></td><td>SEQ © NO: 49</td><td>Heavy chain CDR3, hAcM 2FH-gl</td>

<td> .</td><td>SEQ © NO: 50</td><td>Heavy chain CDR2, hAcM 2FH-gl</td>

<td> 20</td><td>SEQ © NO: 51</td><td>Heavy chain CDR1, hAcM 2FH-gl</td>

<td></td><td>SEQ © NO: 52</td><td>Light chain CDR3, hAcM 2FH-gl</td>

<td></td><td>SEQ © NO: 53</td><td>Light chain CDR2, hAcM 2FH-gl</td>

<td></td><td>SEQ © NO: 54</td><td>Light chain CDRl, hAcM 2FH-gl</td>

<td></td><td>SEQ © NO: 55</td><td>heavy chain variable domain, hAcM 2FH-gl</td>

<td> 25</td><td>SEQ © NO: 56</td><td>light chain variable domain, hAcM 2FH-gl</td>

<td></td><td>SEQ © NO: 57</td><td>human kappa light chain constant region</td>

<td></td><td>SEQ © NO: 58</td><td>human heavy chain constant region derived from IgG1</td>

<td></td><td>SEQ © NO: 59</td><td>human heavy chain constant region derived from IgG1</td>

<td colspan="2"> .</td><td>mutated to L234A and L235A</td>

<td> 30</td><td>SEQ © NO: 60</td><td>human heavy chain constant region derived from IgG4</td>

<td></td><td>SEQ © NO: 61</td><td>human heavy chain constant region derived from IgG4</td>

<td colspan="2"></td><td>transferred to S228P</td>

<td></td><td>SEQ © NO: 62</td><td>Wild type CSF-1R (wt CSF-1R)</td>

<td></td><td>SEQ © NO: 63</td><td>CSF-1R L301S Y969F mutant human</td>

<td> 35</td><td>SEQ © NO: 64</td><td>extracellular domain of human CSF-1R</td>

<td></td><td>SEQ © NO: 65</td><td>human CSF-1R © 4 fragment</td>

<img file="MA34780B1_D0017.tif" />

MA 34780Β1

SEQ ID NO: 66

SEQ ID NO: 67

SEQDDNO: 68

SEQroNO: 69

SEQroNO: 70

SEQroNO: 71

SEQroNO: 72

SEQroNO: 73 SEQ ro NO: 74

SEQroNO: 75

SEQroNO: 76 SEQ ro NO: 77

SEQroNO: 78

SEQroNO: 79

SEQroNO: 80

SEQroNO: 81

SEQroNO: 82

SEQroNO: 83

SEQroNO: 84 fragment D1-D3 of human CSF-1R peptide signal primer

Heavy chain CDR3, mAb 1G1O Heavy chain CDR2, mAb 1G1O Heavy chain CDR1, mAb 1G1O light chain CDR3, mAb 1G1O light chain CDR2, mAb 1G1O light chain CDR1, mAb 1G1O heavy chain variable domain, mAb 1G1O light chain variable domain, mAb 1G1O heavy chain CDR3, mAb 2Η7 heavy chain CDR2, mAb 2Η7 heavy chain CDR1, mAb 2Η7 light chain CDR3, mAb 2Η7 light chain CDR2, mAb 2Η7 light chain CDR1, mAb 2Η7 heavy chain variable domain, MAb 2Η7 light chain variable domain, mAb 2Η7 examples, sequence listing and figures below are provided to facilitate understanding of the present invention, the true scope of which is set out in the appended claims. It is understood that modifications can be made to the techniques presented without departing from the spirit of the invention.

Description of figures

Figure 1 Inhibition of the growth of BeWo tumor cells in a 3D culture under treatment with different anti-CSF-1R monoclonal antibodies at a concentration of 10 µg / ml.

X-axis: viability in normalized mean relative light units (RHUs), corresponding to the ATP content of cells (CellTiterGlo assay)

Y axis: samples tested: minimal medium (0.5% FBS), mouse IgGl (mlgGl, 10 pg / ml), mouse IgG2a (mIgG2a, 10 pg / ml), CSF-1 alone, mAb 2FU, Mab 2Ε10, Mab 2Η7, Mab 1G1O and SC 24Α5.

The highest inhibition of growth induced by the

ΜΑ 34780Β1

Figure 2a

Figure 2b

Figure 2c

Figure 2d

Figure 2e

CSF-1 with the anti-CSF-1R antibodies according to the invention.

Biacore sensogram of the binding of different anti-CSFIR antibodies to the delD4 fragment (comprising the extracellular subdomains D1-D3 and D5) (SEQ ID NO: 65) of immobilized human CSF-1R (y-axis: binding signal in Units Response time (UR), baseline = 0 UR, x axis: time in seconds (s)): while the mAb 2391 and sc 2-4Α5 antibodies clearly show a binding to this delD4 fragment, the antibodies according to the invention, for example mAb 2FU and mAb 2Ε10, do not bind to the delD4 fragment of CSF-1R. The anti-COR5 m &lt;CCR5&gt; PzO3.1C5 control antibody also did not bind to the delD4 fragment of CSF-1R.

Biacore sensogram of the binding of different anti-CSFIR antibodies to the extracellular domain (CSF-IR-ECD) (including extracellular subdomains D1-D5) (SEQ ID NO: 64) of immobilized human CSF-1R (y-axis: signal from Binding in Response Units (UR), baseline = 0RU, x-axis: time in seconds (s)): all anti-CSF-1R antibodies show binding to CSF-IR-ECD. The anti-CCR5 m &lt;CCR5&gt; PzO3.1C5 control antibody does not bind to CSF-IR-ECD.

Biacore sensogram of the binding of different anti-CSFIR antibodies to the delD4 fragment (comprising the extracellular subdomains D1-D3 and D5) (SEQ ID NO: 65) of immobilized human CSF-1R (y-axis: binding signal in Units Response (UR), baseline = OUR, x-axis: time in seconds (s): mAb 1G1O, mAb 2Η7 and humanized mAb 2F11-e7 do not bind to the delD4 fragment of CSFIR . The anti-CCR5 m &lt;CCR5&gt; PzO3.1C5 control antibody also did not bind to the delD4 fragment of CSF-1R.

Biacore sensogram of the binding of different anti-CSFIR antibodies to the extracellular domain (CSF-IR-ECD) (including extracellular subdomains D1-D5) (SEQ ID NO: 64) of immobilized human CSF-1R (y-axis: signal from Binding in Response Units (UR), baseline = 0RU, x axis: time in seconds (s)): all anti-CSF-1R antibodies mAb 1G1O, mAb 2Η7 and hAcM 2F11-e7 humanized show binding at the CSF-IR-ECD. The anti-CCR5 m &lt;CCR5&gt; PzO3.1C5 control antibody does not bind to CSF-IR-ECD.

Biacore sensogram of the binding of different anti-CSF- antibodies

<img file="MA34780B1_D0018.tif" />

MA 34780Β1

Jl

IR to the delD4 fragment (comprising the extracellular subdomains D1-D3 and D5) (SEQ ID NO: 65) of immobilized human CSF-1R (y axis: Unit Response (UR) binding signal, baseline = OUR, X-axis: time in seconds (s)): all anti-CSFIR 1.2.SM, CXIIG6, Ab 10676 and mAb 3291 antibodies show binding to the delD4 fragment of CSF-1R. The anti-CCR5 m &lt;CCR5&gt; PzO3.1C5 control antibody also did not bind to the delD4 fragment of CSF-1R.

Figure 2f Biacore sensogram of the binding of different anti-CSFIR antibodies to the extracellular domain (CSF-IR-ECD) (including extracellular subdomains D1-D5) (SEQ ID NO: 64) of immobilized human CSF-1R (y-axis: binding signal in Response Units (UR), baseline = 0 UR, x-axis: time in seconds (s)): all anti-CSF-lR 1.2.SM, CXIIG6, Ac 10676 and mAb 3291 antibodies present a link to the CSF-IR-ECD. The anti-CCR5 m &lt;CCR5&gt; Pz03.1C5 control antibody does not bind to CSF-IR-ECD.

Figure s 3a-d Levels of CSF-1 in the cynomolgus monkey after application of different doses of anti-CSF-1R antibodies according to the invention.

Figure 4 Efficacy U vivo - inhibition of tumor growth with anti-CSF-1R antibodies according to the invention in a ΒΤ20 breast cancer xenograft.

Example 1

Production of a hybridoma cell line producing antiCSF-1R antibodies

NMRI mouse immunization technique

NMRI mice were immunized with a pDisplay® expression vector (Invitrogen, USA) encoding the extracellular domain of huCSF-1R using electroporation. Each mouse was immunized 4 times with 100 µg of DNA. When the serum titers of antihuCSF-1R were found to be sufficient, the mice were additionally boosted with 50 µg of a 1: 1 mixture of huCSFlR ECD / huCSF-1R-huFc ECD chimer in 200 µl of PBS intravenously (iv) 4 and 3 days before the fusion.

Antigen specific ELISA

The titers of anti-CSF-1R in the serum of immunized mice were determined by antigen-specific ELISA analysis.

0.3 µg / ml of huCSF-1R-huFc chimera (domain

<img file="MA34780B1_D0019.tif" />

MK

Soluble extracellular 34780Β1) on a streptavidin plate (MaxiSorb; MicroCoat, DE, η٥ cat. 11974998 / MC1099) with 0.1 mg / ml of biotinyl anti-Fcy (Jackson ImmunoResearch., Η٠ cat. 109-066-098) and on a adds an anti-mouse IgG F (ab ') 2 conjugated to horseradish peroxidase (HRP) (GE Healthcare, UK, cat.

NA9310V) diluted at 1/800 in PBS / 0.05% Tween20 / 0.5% BSA. The sera from all punctures were diluted 1/40 in PBS / 0.05% Tween20 / 0.5% BSA and successive dilutions were made to 1/1638400. The diluted sera were added to the wells. Pre-puncture serum was used as a negative control. A dilution series of 10 mouse anti-human CSF-1R mAb 3291 (RfcD Systems, UK) of 500 ng / ml to 0.25 ng / ml was used as a positive control. All of the components were incubated together for 1.5 hours. The wells were washed 6 times with PBST (PBS / 0.2% Tween2O) and the assays were developed with a freshly prepared ABTS® solution (1 mg / ml) (ABTS: 2,2'-azinobis acid ( 3ethylbenzothiazolin-6-sulfonic acid) for 10 minutes at room temperature.

The absorbance was measured at 405 nm.

Hybridoma production

Mouse lymphocytes can be isolated and fused with a mouse myeloma cell line using standard PEG-based protocols to produce hybridomas. The resulting hybridomas are then screened for the production of antibodies specific for the antigen. For example, suspensions of cells isolated from lymphocytes from the spleen of mice immunized with Ag8 non-secreting mouse myeloma cells P3X63Ag8.653 (ATCC CRL-1580) are fused with 50% PEG. Cells are plated to about 0 in a 96-well flat bottom microtiter plate, then incubated for about 2 weeks in selective medium. Individual wells are then screened by ELISA analysis to select for anti-CSF-1R human IgM and IgG monoclonal antibodies. Once extensive growth of the hybridomas occurs, the antibody-secreting hybridomas are subcultured, re-screened and, if they are still positive for human IgG anti-CSF-1R monoclonal antibodies, one can. subclone them by FACS. The stable n vtro subclones are then grown 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 anti-CSF-1R antibodies to the delD4 fragment of human CSF-1R and to the extracellular domain of human CSF-1R (CSF-IR-ECD) of the human CSF-1R. As described in Example 4, as well as the determination of the inhibition of the growth of ΝΙΗ3Τ3 cells transfected with wild type CSF-1R

<img file="MA34780B1_D0020.tif" />

ΜΑ 34780Β1 (ligand dependent signaling) or mutant CSF-1R L301S Y929F (ligand independent signaling) under treatment conditions with anti-CSF-1R monoclonal antibodies as described in Example 5.

Hybridoma culture

MuAcM hybridomas produced in RPMI1640 (PAN - catalog no. (Cat. No.) Ρ04-17500) supplemented with 2 mM L-glutamine (GIBCO - cat. 35050-038), Na 1 pyruvate were grown. mM (GIBCO - cat. no. 11360039), NEAA X l (GIBCO - cat. no. 11140-035), 10% FCS (PAA - cat. no. Α15649), Pen Strep X 1 (Roche - η44 cat. 1074440 ), Nutridoma CS X 1 (Roche - cat. no. 1363743), mercaptoethanol 50 μΜ (GIBCO - η٠ cat. 31350-010) and 50 υ / ml d'n. 6 mouse (Roche - cat. 1 444,581) at 37٥c and 5% CO2. Some of the resulting mouse antibodies were humanized (eg mAb 2FH and expressed recombinantly.

Example 2

Inhibition of CSF-1 binding to CSF ​​-IR (ELdSA)

By setting up this assay to first allow binding of the anti-CSF-1R antibody to CSFIR-ECD, followed by detection of the ligand not bound to the receptor, both the ligand displacing antibodies and the ligand-displacing antibodies can be tested. anti-CSF-1R dimerization inhibitor antibodies. The assay was performed on 384 well microtiter plates (MicroCoat, DE, cat. No. 464718) at room temperature. After each incubation step, the plates were washed 3 times with PBST.

Initially, the plates were coated with 0.5 mg / ml of anti-Fcy biotinyl goat F (ab ') 2 (Jackson ImmunoResearch., Η٥ cat. 109-006-170) for 1 hour (h).

The wells were then blocked with PBS supplemented with 0.2% Tween®-20 and 2 μ BSA (Roche Diagnostics GmbH, DE) for 0.5 hour. 75 ng / ml of huCSF-1R-huFc chimera (which forms the soluble, dimeric extracellular domain of huCSF-1R) was immobilized on the plate for 1 hour. Dilutions of purified antibodies in PBS / 0.05% Tween20 / 0.5% BSA were then incubated for 1 hour. After adding a mixture of 3 ng / ml of CSF-1 (Biomol, DE, cat. 60530), 50ng / ml of biotinylated anti-CSF-1 clone BAF216 (R&amp;D Systems, UK) and Streptavidin-HRP diluted 1: 5000 (Roche Diagnostics GmbH, DE, cat. No. 11089153001) for 1 hour , the plates were washed 6 times with PBST. Anti-CSF-1R SC 2-4Α5 (Santa Cruz Biotechnology, US), which inhibits ligand-receptor interaction, was used as a positive control. Plates were developed with a solution of POD BM blue® substrate (BM blue®: 3.3'-5.5'-

<img file="MA34780B1_D0021.tif" />

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34780Β1 tetramethylbenzidine, Roche Diagnostics GmbH, DE, cat. 11484281001) for 30 minutes at room temperature. Absorbance was measured at 370 nm. A decrease in absorbance is observed when the anti-CSF-1R antibody causes release of CSF-1 from the dimer complex. All anti-CSF-1R antibodies showed significant inhibition of the interaction of CSF-1 with CSF-1R (see Table 1). Anti-CSF-1R SC 2-4Α5 (Santa Cruz Biotechnology, us, see also Sherr, CJ et al. Blood 73 (1989) 1786-1793), which inhibits ligand-receptor interaction, was used as a control. reference.

<img file="MA34780B1_D0022.tif" />

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Table 1

IC values ​​calculated for inhibition of the CSF-1 / CSF-lR interaction

<td>Anti-CSF-lR mAb</td><td>"50 for inhibition of CSF-1 / CSF-1R [ng / ml]</td>

<td>MAb 2FH</td><td> 19,3</td>

<td>MAb 2Ε10</td><td> 20,6</td>

<td>MAb 2Η7</td><td> 18,2</td>

<td>MAb 1G1O</td><td> 11,8</td>

<td>SC-2-4A5</td><td> 35,2</td>

Example 3

Inhibition of CSF-1 Induced CSF-1R Phosphorylation in Recombinant NIH3T3-CSF-1R Cells

4.5X0 NIH 3Τ3 cells, retrovirally infected with full length CSF-1R expression vector, were cultured in DMEM (PAA cat. # 1510 OR), 2 mM Lglutamine (Sigma, cat. No. . G7513, 2 mM sodium pyruvate, non-essential amino acids X 1, 10% FKS (PAA, cat. No. Α15-649) and 100 pg / ml PenStrep (Sigma, cat. No. Ρ4333 [10 mg / ml ]) until they reached confluence, the cells were then washed with serum-free DMEM medium (PAA cat. Ε15-011) supplemented with sodium selenite [5 ng / ml] (Sigma, cat. No. 15 S9133), transferrin [10 pg / ml] (Sigma, η٥ cat. Τ8158), BSA [400 pg / ml] (Roche Diagnostics GmbH, η٠ cat. 10735078), 4 mM L-glutamine (Sigma, cat. no. G7513), 2 mM sodium pyruvate (Gibco, cat. 11360), non-essential amino acids X 1 (Gibco, cat. No. 11140-035), 2-mercaptoethanol [0.05 mM] (Merck, cat. No. Μ7522), 100pg / ml and PenStrep (Sigma, cat. Ρ4333) and incubated in 30 µl of the same medium for 16 hours to allow upregulation of the receptor. 10 µl of diluted anti-CSF-1R antibodies were added to cells over 1.5 hours. Cells were then stimulated with 10 µl of 100 ng / ml huMCSF-1 (Biomol cat. No. 60530) for 5 minutes. After incubation, the supernatant was separated, the cells were washed twice with 80 µl ice-cold PBS and 50 µl of freshly prepared ice lysis buffer (150 mM NaCl / 20 mM Tris pH 7.5 was added. / 1 mM EDTA / 1 mM EGTA / 1% Triton Χ-100/1 tablet of protease inhibitor (Roche Diagnostics GmbH η٥ cat. 1,836 170) for 10 ml of buffer / 10 μl / ml of a cocktail of phosphatase 1 inhibitors (Sigma cat. Ρ-2850, stock solution X 100) / 10 ρΐ / ml protease inhibitor 1 (Sigma cat. Ρ-5726, l

٦1٩ هلآ MA stock solution X 100) / ΙΟμΙ / rnl of NaF 1 M). After 30 minutes on ice, the plates were stirred vigorously on a plate shaker for 30 minutes, then centrifuged for 10 minutes at 2200 rpm (Heraeus Megafuge 10).

The presence of phosphorylated and total CSF-1 receptor in the cell lysate was analyzed by ELISA. For the detection of the phosphorylated receptor, the kit from R&amp;D Systems (cat. No. DYC3268-2) was used according to the supplier's instructions. For detection of total CSF-1R, 10 µl of the lysate was immobilized on a plate using the capture antibody contained in the kit. Biotinylated anti-CSF-1R antibody BAF329 (R&amp;D Systems) diluted 1: 750 and Streptavidin-HRP conjugate diluted 1: 1000 were then added. After 60 minutes, the plates were developed with freshly prepared ABTS® solution and the absorbance detected. Results were calculated as% positive control without antibody and express the value of the ratio of phosphorylated receptor / total. The negative control was defined without addition of M-CSF-1. Anti CSF-1R SC 2-4 15 (Santa Cruz Biotechnology, us, see also Sherr, CJ et al. Blood 73 (1989) 1786-1793), which inhibits ligandreceptor interaction, was used as a reference control.

Table 2

IC50 values ​​calculated for inhibition of phosphorylation of the

CSF-1

<td>Anti-CSF-lR mAb</td><td>IC for CSF-1R phosphorylation [ng / ml]</td>

<td>AcM 2FU</td><td> 219,4</td>

<td>MAb 2Ε10</td><td> 752,0</td>

<td>MAb 2Η7</td><td> 703,4</td>

<td>AcMIGIO</td><td> 56,6</td>

<td>SC-2-4A5</td><td> 1006,6</td>

Example 4

Determination of the binding of anti-CSF-1R antibodies to the delD4 fragment of human CSFIR and to the extracellular domain of human CSF-1R (CSF-LR-ECD)

Preparation of the extracellular domain of human CSFIR (CSF-IRECD) (comprising the extracellular subdomains Ρ1-Ρ5. HCSFlR-ECD) of SEO ID

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NO: 64 عل ρΟΜν-preS-Fc-hCSF-lR-ECD (7836 bp) codes for the complete ECD of human Χ34 (SEQ ID NO: 64) fused at the c-terminal position with a cut site by the PreScission protease , followed by amino acids 100-330 of human IgG1 and a 6xHis tag, under the control of the CMV promoter. The natural signal peptide was modified by inserting the amino acids G and S after the first M, to create a BamHI restriction site.

Preparation of the! D4 fragment of human CSF-1R (comprising the extracellular subdomains D1-D3 and Ρ5. HCSF-1R-delD4) of SEO ID NO: 65

HCSF1R-delD4-Vl-PreSc-hFc-His was cloned from pCMV-preSFchCSF-lR-ECD using Stratagene's QuickChange XL site-directed mutagenesis protocol, using delD4-for having the sequence CACCTCCATGTTCTTCCG GTACCCCCCAGAGGTAAG ( SEQ ID NO: 68) as a sense primer and of! D4-rev having the reverse complementary sequence as an antisense primer. A variant of the protocol published in BioTechniques 26 (1999) 680 was used for extension of the two primers in separate reactions in 3 cycles preceding the normal Stratagene protocol:

According to the manufacturer's manual, two separate reaction mixtures of 50 μΐ were produced, each containing 10 ng of plasmid pCMV-preS-Fc-hCSFIR-ECD 20 as a template and 10 μM of one of the primers of Dfor or of D-rev, 0.5 µl of Pfu DNA polymerase as supplied in the kit. Three cycles of PCR of 30 s at 95٥C / 60 s at 55٠C / 8 min at 68٥c were carried out, then 25 µl of each of the two reaction mixtures were combined in a new tube and 0.5 µl of DNApolymerase was added. of fresh Pfu. The normal PCR protocol was performed with 18 temperature cycles as specified by Stratagene in the kit manual, followed by a final 2 hour digestion with the restriction enzyme Dpnl supplied with the kit. Clones carrying the deletion were detected by digestion with CelII and NotI and verified by sequencing.

The protein was prepared by temporary transfection in the Hek293 FreeStyle Suspension Cell System (Invitrogen) according to the manufacturer's specifications. After 1 week, 500 ml of supernatant was filtered and loaded onto a 1 ml (0.2 ml / min) HiTrap MabSelect Xtra Protein A column (GE Healthcare). The column was washed first with PBS, then with 50 mM Tris /

150 mM NaCl / 1 mM EDTA / pH 7.3. 75 µl of PreScission protease (GE # 27-0843-01) diluted in 375 µl of the same buffer was loaded onto the column and the capped column was incubated overnight at 4 ° C by rolling. We went up

MA 34780Β1 column on top of a 1 ml GSTrap FF (GE Healthcare) column and the desired protein was eluted (0.2 ml / min, 0.2 ml fractions). The pooled fractions from 1.8 ml to 0.4 ml were concentrated by centrifugal ultrafiltration through a Nanosep 3k membrane and chromatographed on an S200 HR SEC gel in PBS (0.5 ml / min).

The delD4 fragment of human CSF-1R was obtained in two fractions in the form of a dimeric molecule (pool 1, V = 1.5 ml; c = 0.30 mg / ml; apparent mass on SDS-PAGE 83 kDa, reduced 62 kDa) and in the form of the monomer (pool 2, V = 1.4 ml; c = 0.25 mg / ml; apparent mass on SDS-PAGE 62 kDa). The dimeric form was used for all experiments.

Determination of the binding of anti-CSF-1R antibodies to the! D4 fragment of human CSF-1R and to the extracellular domain of human CSF-1R (CSF-IR-ECD) (binding signals in the form of Response Units ( UR):

Instrument: BiacoreTlOO (GE Healthcare)

Software: Τ100 Control, version 2.0.1

Τ100 Evaluation, version 2.0.2

Test format: chip: CM5 Temperature: 25٠c

The fragments of CSF-1R were immobilized by amine coupling. To compare the binding of different anti-CSF-1R antibodies according to the invention, a concentration of the test antibody was injected. Anti-CSF-1R mAb 3291 (R &amp; D-Systems) and SC 2-4Α5 (Santa Cruz Biotechnology, US - see also Sherr, CJ et al ..) antibodies were used as reference controls. Blood 73 (1989) 1786-1793), and as negative control the anti-CCR5 m &lt;CCR5&gt; PzO3.1C5 (deposited on 08/18/2004 with the DSMZ under the reference DSM ACC 2683), all under the same conditions as the anti-CSF-1R antibody according to the invention.

Amine coupling of CSFIR fragments

Conventional amine coupling according to manufacturer's instructions: flow buffer: PBS-T (Roche: 11 666 789 +٠ 0.05% Tween2O: 11 332 465), activation with a mixture of EDC / NHS, injection of the fragment delD4 of human CSF-1R (comprising the extracellular subdomains D1-D3 and D5) (SEQ © NO: 65) and the extracellular domain of human CSF-1R (CSF-IR-ECD) (comprising the extracellular D1 subdomains -D5) (SEQ © NO: 64) for 600 seconds at a flow rate of 10 μΐ / min; diluted in NaAc coupling buffer, pH 5.0, c = 10 µg / ml; the remaining active carboxyl groups were finally blocked by injection of 1M ethanolamine.

<img file="MA34780B1_D0023.tif" />

ΜΑ 34780Β1 اً jaison ties anticoms anti-CSFIR mAb 2F11- mAb 2Ε10. MAb 3291 and SC2-4A5 and other anti-ÇSF-1R antibodies to the! D4 fragment of human CSF-1R and the extracellular domain of human CSF-1R (CSF-tR-ECD) at 25 ° ç

Flow buffer: PBS-T (Roche: 11 666 789 + 0.05% Tween2O: 11 332 465)

Analyte sample:

Binding was measured at a flow rate of 30 μΐ / min by injecting the analyte with a concentration c = 10 nM (for mAb 1G10, mAb 2Η7 and humanized mAb 2Fll-e7 in a second experiment. ). Each injection lasted 700 seconds, and was followed by a dissociation phase of 180 seconds. A final regeneration was performed after each cycle using 50 mM NaOH, with a contact time of 60 seconds, and a flow rate of 30 μΐ / min.

The signals were measured by a report point 10 seconds after the end of the injection. Reference signals (signals from a blank reference flow cell (treated with EDC / NHS and Ethanolamine only) were subtracted to obtain binding signals (as UR). When the nonbinding antibody binding signals were slightly below 0 (mAb 2FU = -3; mAb 2Ε10 = -2; mAb 1G1O = -6; mAb 2Η7 = -9; and humanized hAcM 2F11-e7 = - 7), the values ​​were set to 0.

Table 3a:

Binding, measured by RPS, of mAb &lt;CSF-1R&gt; to the delD4 fragment of human CSF-1R and to CSF-IR-ECD and ratio at 25 ° C

<td></td><td>Link to delD4 [UR]</td><td>Link to CSF-1R- ECD [UR]</td><td>Ratio of anti-CSF-1R antibody binding to CSF1R delD4 fragment / CSF-IR-ECD</td>

<td>AcM 3291</td><td> 1015</td><td> 627</td><td> 1015/627= 1,61</td>

<td>SC2-4A5</td><td> 374</td><td> 249</td><td> 374/249= 1,50</td>

<td>AcM 2FU</td><td> 0</td><td> 176</td><td> 0/176 = 0</td>

<td>hAcM 2Fll-e7</td><td> 0</td><td> 237</td><td> 0/237=0</td>

<td>MAb 2Ε10</td><td> 0</td><td> 120</td><td> 0/120 = 0</td>

<td>AcMIGIO</td><td> 0</td><td> 2708</td><td> 0/2708 = 0</td>

<td>MAb 2Η7</td><td> 0</td><td> 147</td><td> 0/147 = 0</td>

<td>m &lt;CCR5&gt; Pz03.1C5</td><td> 2</td><td> 5</td><td> -</td>

<img file="MA34780B1_D0024.tif" />

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MAb 2F11 and mAb 2Ε10 exhibited binding to the extracellular domain of human CSF-1R (CSF-IR-ECD) (see Figure 2b); however, no binding to the delD4 fragment of CSF-1R was detected (see Figure 2a).

SC2-4A5 and mAb 3291 exhibited binding to CSF-IR-ECD and delD4 (see Figures 2b and 2a).

Thus, the binding ratio of anti-CSF-1R antibodies mAb 2FH and mAb 2Ε10 to the delD4 fragment of CSF-1R / to CSF-IR-ECD was clearly less than 1:50 (= 0.02), while the ratio binding of mAb 3291 and SC2-4A5 was 1.61 and 1.50, respectively, and was much greater than 1:50 (= 0.02). The negative control antibody m &lt;CCR5&gt; Pz03.1C5 showed no binding (as expected).

Humanized mAb 1G10,1'AbM 2Η7 and humanized hAcM 2F11-e7 exhibited binding to the extracellular domain of human CSF-1R (CSF-IR-ECD) (see Figure 2d); however, no binding to the delD4 fragment of CSFIR was detected (see Figure 2c). Thus, the binding ratio of anti-CSF-1R antibodies mAb 1G10, mAb 2Η7 and hAcM 2F11-e7 humanized to the delD4 fragment of CSF-1R / to CSF-IR-ECD was clearly less than 1:50 (= 0, 02).

In another experiment, we studied the anti-CSF-1R 1.2.SM antibodies (anti-CSF-1R antibody displacing the ligand described in patent document WO2009026303), CXIIG6 (anti-CSF-1R antibody displacing the ligand described in patent document WO 2009/112245), goat anti-CSF-1R polyclonal antibody acl0676 (abcam). The anti-CSF-1R antibody mAb 3291 (R &amp; D-Systems) was used as a reference control. The anü-CCR5 m &lt;CCR5&gt; Pz03.1C5 (deposited on 08/18/2004 with the DSMZ under the reference DSM ACC2683) was used as negative control.

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Table 3b

Binding, measured by RPS, of mAb &lt;CSF-1R&gt; to the delD4 fragment of human CSF-1R and to CSFIR-ECD and ratio at 25٠c

<td></td><td>Link to delD4 [UR]</td><td>Link to CSF1R-ECD [UR]</td><td>Ratio of the binding of anti-CSF-1R antibodies to the delD4 fragment of CSF-1R / to CSF-IRECD</td>

<td>AcM3291</td><td> 1790</td><td> 1222</td><td> 1790/1222=1,47</td>

<td>1.2.SM</td><td> 469</td><td> 704</td><td> 469/704 = 0,67</td>

<td>CXIIG6</td><td> 1983</td><td> 1356</td><td> 1983/1356= 1,46</td>

<td>acl0676</td><td> 787</td><td> 547</td><td> 787/547= 1,44</td>

<td>m &lt;CCR5&gt; PzO3.1C5</td><td> 0</td><td> 0</td><td> -</td>

1.2.SM, CXIIG6, ac10676 and mAb 3291 exhibited binding to CSF-IRECD and delD4 (see Figures 2f and 2e).

The binding ratio of 1.2.SM, CXIIG6, acl0676 and mAb 3291 was well above 1:50 (= 0.02). The negative control antibody m &lt;CCR5&gt; PzO3.1C5 showed no binding (as expected).

Examples

Inhibition of growth of recombinant N1H3T3-CSF-1R cells in 3D culture under treatment with anti-CSF-1R monoclonal antibodies (CellTiterGlo analysis)

ΝΙΗ3Τ3 cells, infected etrovirally with an expression vector of either whole wild-type CSF-1R (SEQ ID NO: 62) or mutant CSF-1R L301S Y969F (SEQ ID NO: 62) were cultured. 63) in high glucose DMEM medium (PAA, Pasching, Austria) supplemented with 2 mM L-glutamine, 2 mM sodium pyrtivate and non-essential amino acids and 10% fetal bovine serum (Sigma, Taufkirchen , Germany) on boxes coated with poly-HEMA (poly (2-hydroxyethyl methacrylate)) (Polysciences, Wartington, PA, USA) to prevent adhesion to the plastic surface. The cells of the medium replacing the serum were inoculated with 5 ng / ml of sodium selenite, 10 mg / ml of transfenin, 400 pg / ml of BSA and 0.05 mM 2-mercaptoethanol. When treated with 100 ng / ml huCSF-1 (Biomol, Hamburg, Germany), cells expressing wtCSF-1R form dense spheroids which develop in

<img file="MA34780B1_D0025.tif" />

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34780Β1 three-dimensional property called anchor independence. These spheroids closely resemble the three-dimensional architecture and organization of solid tumors in situ. Cells recombined with mutant CSF-1R are capable of forming spheroids independent of CSF-1 ligand. The 5 spheroid cultures were incubated for 3 days in the presence of different concentrations of antibody to determine an IC (concentration resulting in inhibition of cell viability by 50%). CellTiterGlo assay was used to detect cell viability by measuring the ATP content of cells.

Table 5a

<td>AcManti-CSF٠lR</td><td>wtCSF-lR CI [pg / ml]</td><td>CSF-1R mutant IC50 [gg / ml]</td>

<td>AcM2FU</td><td> 1,1</td><td> 8,0</td>

<td>MAb 2Ε10</td><td> 0,49</td><td> 4,9</td>

<td>MAb 2Η7</td><td> 0,31</td><td> 5,3</td>

<td>MAb 1G1O</td><td> 0,29</td><td> 14,2</td>

<td>SC 2-4Α5</td><td> 10,0</td><td> 10,0</td>

R &amp; D-Systems Reference Control mAb 3291 did not exhibit inhibition of the proliferation of recombinant cells with mutant CSF-1R.

In another experiment, the anti-CSFTR antibody according to the invention hAcM 2F1 l-e7 and the anti-CSF-lR 1.2.SM antibodies (anti-CSFIR antibody displacing the ligand described in patent document WO2009026303 were studied. ), CXIIG6 (anti-CSF-lR antibody displacing the ligand described in patent document WO 2009/112245), the goat anti-CSF-lR polyclonal antibody acl0676 (abcam), and SC 2-4Α5 (Santa Cruz Biotechnology, us - see also Sherr, CJ et al., B / ood 20 73 (1989) 1786-1793).

The spheroid cultures were incubated for 3 days in the presence of different concentrations of antibody to determine an io (concentration resulting in inhibition of cell viability by 30%). CellTiterGlo assay was used to detect cell viability by measuring the ATP content of the cells.

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Table 5b

<td>Anti-CSF-lR mAb</td><td>wtCSF-lR Cg / ml]</td><td>CSF٠1R mutant Clpg / ml]</td>

<td>hAcM 2Fll-e7</td><td> 4,91</td><td> 0,54</td>

<td>1.2.SM</td><td> 1,19</td><td>&gt; 20 pg / ml (-19% inhibition at 20 pg / ml = 19% stimulation)</td>

<td>CXIIG6</td><td>&gt; 20 pg / ml (21% inhibition at 20 pg / ml)</td><td>&gt; 20 pg / ml (-36% inhibition at 20 pg / ml = 36% stimulation)</td>

<td>abl0676</td><td> 14,15</td><td>&gt; 20 pg / ml (0% inhibition at 20 pg / ml)</td>

<td>SC 2-4Α5</td><td> 16,62</td><td> 2,56</td>

Example 6

Inhibition of the growth of BeWo tumor cells in a 3D culture under treatment with anti-CSF-1R monoclonal antibodies (CellTiterGlo analysis)

BeWo choriocarcinoma cells (ATCC CCL-98) were cultured in F12K medium (Sigma, Steinheim, Germany) supplemented with 10% FBS (Sigma) and 2 mM Lglutamine. Poly-ΗΕΜΑ (poly (2-hydroxyethyl methacrylate)) ١ coated 96-well plates containing F12K medium supplemented with 0.5% FBS and 5% BSA were seeded with 5 X 10.⁴cells / well. At the same time, 200 ng / ml huCSF-1 and 10 µg / ml different anti-CSF-1R monoclonal antibodies were added and incubated for 6 days. CellTiterGlo assay was used to detect cell viability by measuring the ATP content of cells in relative light units (ULR). When the cultures of BeWo spheroids were tr eated with different anti-CSF-1R antibodies (10 µg ml), inhibition of growth induced by CSF-1 was observed. To calculate antibody-mediated inhibition, the mean ULR value of unstimulated BeWo cells was subtracted from all samples. We have arbitrarily established at

100 % the mean ULR value of cells stimulated with CSF-1. The mean ULR values ​​were calculated for cells stimulated with CSF-1 and treated with

<img file="MA34780B1_D0026.tif" />

ΜΑ 34780Β1 of anti-CSF-1R antibodies as% of RLUs of cells stimulated with CSF-1. Table 6 shows the results calculated for the inhibition of the growth of BeWo tumor cells in a 3D culture under treatment with anti-CSF-1R monoclonal antibodies; Figures la and lb show the normalized mean ULR values.

Table 6

<td>AcMCSF-lR</td><td>% inhibition antibody concentration 10pg / ml</td>

<td>CSF-1 alone</td><td> ٥</td>

<td>ACM2F11</td><td> 70</td>

<td>MAb 2Ε10</td><td> 102</td>

<td>MAb 2Η7</td><td> 103</td>

<td>MAb 1G1O</td><td> 99</td>

<td>SC 2-4Α5</td><td> 39</td>

Example 7

Inhibition of the differentiation of human macrophages under treatment with anti-CSF-1R monoclonal antibodies (CellTiterGlo assay)

Human monocytes were isolated from peripheral blood using RosetteSep® Human Monocyte Enrichment Cocktail (StemCell Tech. Η٥ cat. 15028). 96-well microtiter plates were seeded with the enriched monocyte populations (2.5 X 0 cells / well) in 100 µl of RPMI 1640 (Gibco - cat. No. 31870) supplemented with 10% FCS (GIBCO - no. cat. or090014Μ), 4 mM L-glutamine (GIBCO - cat. no. 25030) from PenStrep X 1 (Roche - cat. 1 074 440) at 37٥c and 5% O in a humidified atmosphere. When 150 ng / ml of huCSF-1 were added to the medium, a clear differentiation into adherent macrophages was observed. This differentiation could be inhibited by the addition of anti-CSF-1R antibodies. In addition, there is an effect on the survival of monocytes which could be analyzed by CellTiterGlo (CTG) analysis. An IC was calculated from the concentration-dependent inhibition of monocyte survival by treatment with the antibodies (see Table 7).

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Table 7

<td>Anti-CSF-lR mAb</td><td>CI [pg / ml]</td>

<td>MAb 2FH</td><td> 0,08</td>

<td>ACM2E10</td><td> 0,06</td>

<td>MAb 2Η7</td><td> 0,03</td>

<td>MAb 1G1O</td><td> 0,06</td>

<td>SC 2-4Α5</td><td> 0,36</td>

In a separate series of tests, humanized versions of mAb 2Fl, e.g., hAcM 2Fll-cll١ 1'hAm 2Fll-d8.1'hAcM 2Fll-e7, hAcM 2FHT12, exhibited IC50 values. 0.07 pg / ml (hAcM 2Fll-cll) ١ 0.07 pg / ml (hAcM 2Fll-d8), 0.04 pg / ml (hAcM 2Fll-e7) and 0.09 pg / ml (hAcM 2Fll- fl2).

Example 8

Inhibition of differentiation of macrophages in the monkey no mogs under treatment with anti-CSF-1R monoclonal antibodies (CellTiterGlo assay)

Cynomolgus monkey monocytes were isolated from peripheral blood using the CD14 MicroBeads Non-Human Primate Kit (Miltenyi Biotec - Cat. No. 130-091-097) according to the manufacturer's description. 96-well microtiter plates were seeded with the enriched monocyte populations (1-3 X 10 cells / well) in 100 µl of RPMI1640 (Gibco - cat. No. 31870) supplemented with 10% FCS (GIBCO - cat. 011-090014Μ), 4 mM Lglutamine (GEBCO - cat. 25030) and PenStrep X 1 (Roche - Cat. No. 1,074,440) at 37 ° C and 5% O in a humidified atmosphere. When 150 ng / ml of huCSF-1 were added to the medium, a clear differentiation into adherent macrophages could be observed. This differentiation could be inhibited by the addition of anti-CSF-1R antibodies. In addition, there is an effect on the survival of monocytes which could be analyzed by CellTiterGlo (CTG) analysis. Viability was analyzed at a concentration of 5 µg / ml of treatment antibody (see Table 8)

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Table 8

<td>Anti-CSF-lR mAb</td><td>% survival</td><td>% inhibition (of survival) = (100% -% of survival)</td>

<td>AcM 2FU</td><td> 4*</td><td> 96</td>

<td>AcM2E10</td><td> 17 **</td><td> 83</td>

<td>MAb 2Η7</td><td> 8</td><td> 92</td>

<td>AcMIGIO</td><td> ٦</td><td> 98</td>

<td>SC 2-4Α5</td><td> 31</td><td> 69</td>

* average of 4 experiments (3 experiments using mouse mAb, 1 experiment using chimeric mAb) ** average of 2 experiments using only mouse mAb

Example 9

Determination of the binding affinity of anti٠CSF٠lR antibodies to human CSF-1R

Instrument: BIACORE® Α100

Chip: CM5 (Biacore BR-1OO6-68)

Coupling: amine coupling

Buffer: PBS (Biacore BR-1OO6-72), pH 7.4, 35٥c

For the affinity measurements, 36 µg / ml of anti-mouse Fcy antibody (from goat, Jackson Immuno Research JIRI 15-005-071) were coupled to the surface of the chip to capture the antibodies directed against CSF -1R. Human CSF-1R extracellular domain (CSF-IR-ECD) (including extracellular subdomains D1 - D5) (SEQ ID NO: 64) (R &amp; D-Systems 329-MR or pCMVpresS-HisAvitag-hCSFlR) was added. -ECD subclone) at different concentrations in 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 the kinetic parameters, the 1: 1 model of Langmuir was used.

<img file="MA34780B1_D0027.tif" />

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Affinity results measured by RPS

<td>AcManti-CSF-lR</td><td>K٠ (nM)</td><td>ka (l / Ms)</td><td>kd (Is)</td><td>(1/2 (min ؛</td>

<td>MAb 2FH</td><td> 0,29</td><td>l, 77E⁺⁰⁵</td><td>5.18Ε٥5</td><td> 223</td>

<td>MAb 2Ε10</td><td> 0,2</td><td>1.52Ε⁺٥5</td><td>2.97Ε٥5</td><td> 389</td>

<td>MAb 2Η7</td><td> 0,21</td><td>1.47Ε * ٥5</td><td>3.12Ε'٥5</td><td> 370</td>

<td>AcMIGIO</td><td> 0,36</td><td>1.75Ε + ٥5</td><td>6.28Ε٥5</td><td> 184</td>

In a separate Biacore binding assay with CSF-IR-ECD (results not shown), some competition of the mAb 2F11 and mAb 2Ε10 antibodies was observed with the Ab SC-24A5 antibody. However, the 2F11 mAbs / 2Ε10 mAbs do not bind to the delD4 fragment of human CSF-1R, whereas the SC-2-4A5 antibody binds to this delD4 fragment (see example 4 and FIG. 2a). The 2F11AM 2Ε10 mAb binding region is therefore clearly distinct from the SC-2-4A5 antibody binding region, but probably located in a neighboring area. In this competition analysis, the mAb 2FH and mAb 2Ε10 antibodies did not compete with the mAb 3291 from R &amp; D-Systems (results not shown).

Example 10 ا؟ ا Determination of the binding of anCSF1R antibody to the D1-D3 fragment of human CSFIR

Instmment: Biacore T100 (GE Healthcare) software: Τ100 Control, version 1.1.11

Β3000 Evaluation, version 4.01

Scmbber, version 2.0a

Test format: chip: CM5 chip

Antibodies directed against CSF-1R were captured via capture molecules by amine coupling. Using single cycle kinetics, 5 increasing concentrations of 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.

The anti-CSF-1R SC 2-4Α5 (Santa Cruz Biotechnology, US; Sherr, CJ et al .. Blood 73 (1989) 1786-1793) which inhibits the ligand-receptor interaction was used as reference controls, and MAb 3291 (R &amp; D-Systems).

Capture molecules: anti-Fcy mouse antibody (from goat, Jackson

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Immuno Research JIR115-OO5-O71) for the antibodies according to the invention and the mAb 3291 control from R &amp; D-Systems, and anti-rat Fcy antibodies (goat, Jackson Immuno Research JIR112-OO5-O71) for the control of anti-CSFIR SC 2-4Α5 reference.

Amine coupling of capture molecules

Conventional amine coupling according to the manufacturer's instructions: flow buffer: HBS-Ν buffer, activation by a mixture of EDC / NHS, target ligand density: 2000 UR; capture antibodies were diluted in NaAc coupling buffer, pH = 4.5, c = 10 µg / ml; the remaining active carboxyl groups were finally blocked by injection of 1M ethanolamine.

Kinetic characterization of the binding of Ρ1-Ρ3 fragments of human CSF-1R to mAbs &lt;CSF-lR&gt; at 37 ° C

Flow buffer: PBS (Biacore BR-1OO6-72)

Capture of mAbs &lt;CSF-1R&gt; on flow cells 2 to 4: flow rate 20 μΐ / min, contact time of 90 seconds, c (mAb &lt;CSF-1R&gt;) = 50 nM, diluted with buffer flow + 1 mg / ml BSA.

Analyte sample:

The kinetics of a single cycle were measured at a flow rate of 30 μΐ / min by 5 consecutive injections of the analyte at concentrations c = 7.8, 31.25, 125, 500 and 20 2000 nM, without regeneration. Each injection lasted 30 seconds and was followed by a dissociation phase of 120 seconds for the first four injections, and finally 1200 seconds for the highest concentration (= last injection).

Final regeneration was performed after each cycle with mM glycine, pH 1.5 (Biacore BR-1003-54), contact time 60 seconds, flow rate.

30pl / min.

Kinetic parameters were calculated using the usual double referencing (control reference: binding of the analyte to the capture molecule; flow cell: concentration of the CSF-1R 0 subdomain as a blank test) and the calculation was carried out with the kinetic model of titration binding 1: 1

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Table 10

Results of binding affinity of the D1-D3 fragment of human CSF-1R measured by

RPS

<td>Anti- mAbs CSF-1R</td><td>Subdomain</td><td>Kd (nM)</td><td>(1 / Ms)</td><td>kd (l / s)</td><td>TV₂(mn)</td>

<td>MAb 2FH</td><td>D1-D3</td><td>no connection</td><td></td><td></td><td></td>

<td>AcM2E10</td><td>D1-D3</td><td>no connection</td><td></td><td></td><td></td>

<td>MAb 2Η7</td><td>D1-D3</td><td>not determined</td><td></td><td></td><td></td>

<td>MAb 1G1O</td><td>D1-D3</td><td>no connection</td><td></td><td></td><td></td>

<td>SC-2-4A5</td><td>D1-D3</td><td>no connection لآ</td><td></td><td></td><td></td>

<td>R &amp; D- 3291 Systems</td><td>D1-D3</td><td> 5,4</td><td>2.2Ε * 5</td><td>l, 2E'3</td><td> 9,6</td>

The antibodies mAb 2F11, mAb 2Ε10 and mAb 1G1O did not show binding to the D1-D3 fragment of human CSF-1R.

The SC-2-4A5 reference control antibody also did not bind to the D1-D3 fragment of human CSF-1R.

R &amp; D-Systems Reference mAb 3291 exhibited binding to the D1-D3 fragment of human CSF-1R.

Example 11

Increased level of CSF-1 during inhibition of CSF-1R in, cynomolgus monkey

Serum CSF-1 levels provide a pharmacodynamic marker for the CSF-1R neutralizing activity of the hAcM 2F11-e7 anti-human CSE1R dimerization inhibitor. Anti-CSF-1R hAcM 2F11-e7 antibody was administered intravenously to one male cynomolgus monkey and one female cynomolgus monkey per dose group (1 and 10 mg / kg). Blood samples were taken for analysis of CSF-1 levels one week before treatment (pre-administration), 2, 24, 48, 72, 96, 168 hours after administration and once a week for 2 more weeks. CSF-1 levels were determined using a commercial ELISA kit (Quantikine® Μ-human CSF) according to the manufacturer's instructions (R &amp; D-Systems, UK). The level of CSF-1 in monkeys was determined by comparison with samples of a CSF-1 calibration curve provided in the kit.

Administration of hAcM 2Fll-e7 induced an increase

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ΜΑ 34780Β1 spectacular CSF-1, of the order of 1000 times, which, depending on the dose administered, lasted 48 hours (1 mg / kg) or 15 days (10 mg / kg). A CSF-1R dimerization inhibitor therefore has the advantage of not directly competing with the ligand exhibiting spectacular positive regulation for binding to the receptor, unlike an antibody displacing the ligand.

Example 12

Efficacy in vivo - inhibition of tumor growth by anti-CSFIR antibodies in tumor cells of a breast cancer xenograft ΒΤ20 in a beige SCID mouse

The ΒΤ-20 human breast cancer cell line expresses human CSF-1R, but does not express CSF-1 (Sapi, E. et al Cancer Res 59 (1999) 55785585). As CSF-1 from mice does not activate human CSF-1R on tumor cells, recombinant human CSF-1 ((Biomol, Hamburg, Germany) was added via mini osmotic pumps (ALZET, Cupertino, CA). ) giving a continuous infusion rate of CSF-1 of 2 pg / day (Martin, Τ.Α., Carcinogenesis 24 (2003) 1317-1323).

To directly compare the effectiveness of an antibody disturbing the dimerization of CSF-1R with an anti-CSF-1R antibody displacing the ligand, the chimeric anti-CSF-1R mAb 2FU (antibody disturbing the dimerization of CSFIR) was tested. and 1.2.SM (anti-CSF-1R ligand displacing antibody described in patent document WO2009026303) in the ΒΤ-20 xenograft model.

Beige SCID mice (Charles River, Sulzfeld, Germany) were COinjected subcutaneously 1 X 10? ΒΤ-20 cells (ATCC ΗΤΒ-19) and 100 μΐ of Matrigel. ئ treatment of animals started on the day of randomization at an average tumor volume of 100 mm '. The mice were treated once a week intraperitoneally with the respective antibodies (see Figure 4) in 20 mM histidine buffer, 140 mM NaCl, pH 6.0. The tumor dimensions are measured with a caliper starting on the day of staging, then twice a week throughout the treatment period. The tumor volume is calculated according to the NCI protocol (tumor mass = l / 2ab2, where a and b are the large and small diameter respectively).

The analysis of tumor growth is shown in Fig. 4. Inhibition of human CSF-1R on tumor cells with the chimeric anti-CSF-1R mAb 2FU was statistically more effective in mediating inhibition of anti-CSF-1R. tumor growth than! anti-CSF-1R 1.2.SM antibody (anti-CSF-1R antibody described in patent document WO2009026303).