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WO2011070024A1 - Antibodies binding preferentially human csf1r extracellular domain 4 and their use - Google Patents

Antibodies binding preferentially human csf1r extracellular domain 4 and their use Download PDF

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Publication number
WO2011070024A1
WO2011070024A1 PCT/EP2010/069090 EP2010069090W WO2011070024A1 WO 2011070024 A1 WO2011070024 A1 WO 2011070024A1 EP 2010069090 W EP2010069090 W EP 2010069090W WO 2011070024 A1 WO2011070024 A1 WO 2011070024A1
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WIPO (PCT)
Prior art keywords
seq
variable domain
chain variable
region
csf
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PCT/EP2010/069090
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French (fr)
Inventor
Nikolaos Dimoudis
Georg Fertig
Alexander Fidler
Klaus Kaluza
Marlene Pickl
Carola Ries
Stefan Seeber
Martin Lanzendoerfer
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F Hoffmann La Roche AG
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F Hoffmann La Roche AG
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Priority to UAA201208177A priority Critical patent/UA111322C2/en
Priority to KR1020127017818A priority patent/KR101434070B1/en
Priority to CN201080063488.2A priority patent/CN102791738B/en
Priority to JP2012542522A priority patent/JP5677451B2/en
Priority to DK10794936.4T priority patent/DK2510010T3/en
Priority to RU2012128651/10A priority patent/RU2565541C2/en
Priority to SI201031097T priority patent/SI2510010T1/en
Priority to AU2010329934A priority patent/AU2010329934B2/en
Priority to HRP20160131TT priority patent/HRP20160131T1/en
Priority to HK13103659.1A priority patent/HK1176361B/en
Priority to EP10794936.4A priority patent/EP2510010B1/en
Priority to CA2780692A priority patent/CA2780692C/en
Priority to RS20160074A priority patent/RS54596B1/en
Application filed by F Hoffmann La Roche AG filed Critical F Hoffmann La Roche AG
Priority to PH1/2012/500881A priority patent/PH12012500881A1/en
Priority to SG2012042313A priority patent/SG181589A1/en
Priority to MA34923A priority patent/MA34780B1/en
Priority to PL10794936T priority patent/PL2510010T3/en
Priority to PL15168902T priority patent/PL2949670T3/en
Priority to NZ599516A priority patent/NZ599516A/en
Priority to MX2012006553A priority patent/MX2012006553A/en
Priority to ES10794936.4T priority patent/ES2557454T3/en
Priority to EP15168902.3A priority patent/EP2949670B1/en
Priority to BR112012013717A priority patent/BR112012013717B1/en
Publication of WO2011070024A1 publication Critical patent/WO2011070024A1/en
Priority to IL219595A priority patent/IL219595A/en
Priority to ZA2012/03782A priority patent/ZA201203782B/en
Anticipated expiration legal-status Critical
Priority to AU2015213308A priority patent/AU2015213308B2/en
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to antibodies against human CSF-IR (anti-CSF-lR antibodies), methods for their production, pharmaceutical compositions containing said antibodies, and uses thereof.
  • CSF-IR colony stimulating factor 1 receptor
  • M-CSF receptor Macrophage colony-stimulating factor 1 receptor, Fms proto-oncogene, c-fms, SEQ ID NO: 62
  • CSF-IR is a growth factor and encoded by the c-fms proto-oncogene (reviewed e.g. in Roth, P., and Stanley, E.R., Curr. Top. Microbiol. Immunol. 181 (1992) 141-67).
  • CSF-IR is the receptor for CSF-1 (colony stimulating factor 1, also called M-CSF, macrophage colony- stimulating factor) and mediates the biological effects of this cytokine (Sherr, C.J., et al., Cell 41 (1985) 665-676).
  • CSF-IR colony stimulating factor- 1 receptor
  • CSF-IR had transforming potential dependent on changes in the C- terminal tail of the protein including the loss of the inhibitory tyrosine 969 phosphorylation which binds Cbl and thereby regulates receptor down regulation (Lee, P.S., et al., Embo J. 18 (1999) 3616-3628).
  • IL-34 interleukin-34
  • cytokine CSF-1 colony stimulating factor 1, also called M-CSF, macrophage
  • M-CSF colony stimulating factor 1
  • CSF-IR signaling The main biological effects of CSF-IR signaling are the differentiation, proliferation, migration, and survival of hematopoietic precursor cells to the macrophage lineage (including osteoclast). Activation of CSF-IR is mediated by its ligands, CSF-1 (M-CSF) and IL-34. Binding of CSF-1 (M-CSF) to CSF-IR induces the formation of homodimers and activation of the kinase by tyrosine phosphorylation (Li, W. et al, EMBO Journal.10 (1991) 277-288; Stanley, E.R., et al, Mol. Reprod. Dev. 46 (1997) 4-10).
  • the biologically active homodimer CSF-1 binds to the CSF-IR within the subdomains Dl to D3 of the extracellular domain of the CSF-1 receptor (CSF-1 R- ECD).
  • the CSF-1R-ECD comprises five immunoglobulin-like subdomains
  • the subdomains D4 to D5 of the extracellular domain are not involved in the CSF-1 binding. (Wang, Z., et al Molecular and Cellular Biology 13 (1993) 5348-5359).
  • the subdomain D4 is involved in dimerization (Yeung, Y-G., et al Molecular & Cellular Proteomics 2 (2003) 1143- 1155; Pixley, F. J., et al, Trends Cell Biol 14 (2004) 628-638).
  • PI3K and Grb2 Further signaling is mediated by the p85 subunit of PI3K and Grb2 connecting to the PI3K/AKT and Ras/MAPK pathways, respectively. These two important signaling pathways can regulate proliferation, survival and apoptosis.
  • Other signaling molecules that bind the phosphorylated intracellular domain of CSF-IR include STAT1 , STAT3, PLCy, and Cbl (Bourette, R.P. and Rohrschneider, L.R.,
  • CSF-I R signaling has a physiological role in immune responses, in bone remodeling and in the reproductive system.
  • the knockout animals for either CSF-1 (Pollard, J.W., Mol. Reprod. Dev. 46 (1997) 54-61) or CSF-IR (Dai, X.M., et al., Blood 99 (2002) 1 11-120) have been shown to have osteopetrotic, hematopoietic, tissue macrophage, and reproductive phenotypes consistent with a role for CSF-IR in the respective cell types.
  • Sherr, C.J., et al., Blood 73 (1989) 1786-1793 relates to some antibodies against CSF-IR that inhibit the CSF-1 activity (see Sherr, C.J. et al., Blood 73 (1989) 1786-1793).
  • Ashmun, R.A., et al., Blood 73 (1989) 827-837 relates to CSF-IR antibodies.
  • Lenda, D., et al, Journal of Immunology 170 (2003) 3254-3262 relates to reduced macrophage recruitment, proliferation, and activation in CSF-1 -deficient mice results in decreased tubular apoptosis during renal inflammation. Kitaura, H., et al., Journal of Dental Research 87 (2008) 396-400 refers to an anti-CSF-1 antibody which inhibits orthodontic tooth movement.
  • CSF-1 activity inhibitors including antisense nucleotides and antibodies while disclosing only CSF-1 antisense nucleotides.
  • WO 2004/045532 relates to metastases and bone loss prevention and treatment of metastatic cancer by a CSF-1 antagonist disclosing as antagonist anti-CSF-1 -antibodies only.
  • WO 2005/046657 relates to the treatment of inflammatory bowel disease by anti-CSF-1 -antibodies.
  • US 2002/0141994 relates to inhibitors of colony stimulating factors.
  • WO 2006/096489 relates to the treatment of rheumatoid arthritis by anti-CSF-1 - antibodies.
  • WO 2009/026303 and WO 2009/112245 relate to certain anti-CSF-lR antibodies binding to CSF-IR within the first three subdomains (Dl to D3) of the Extracellular Domain (CSF-1R-ECD).
  • the invention comprises an antibody binding to human CSF-IR, characterized in that the antibody binds to human CSF-IR fragment delD4 (SEQ ID NO: 65) and to human CSF-IR Extracellular Domain (SEQ ID NO: 64) with a ratio of 1 :50 or lower.
  • 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,
  • 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 a humanized version thereof.
  • 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,
  • 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 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
  • 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.
  • 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 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
  • 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:
  • 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:
  • 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 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
  • 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
  • the light chain variable domain comprises a CDR3 region of
  • 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
  • the light chain variable domain comprises a CDR3 region of
  • 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
  • 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
  • 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.
  • the antibody according to the invention is of human IgGl subclass or of human IgG4 subclass.
  • a further embodiment of the invention is a pharmaceutical composition comprising an antibody according to the invention.
  • the invention further comprises the use an of an antibody according to the invention for the manufacture of a medicament for treatment of a CSF-IR mediated disease.
  • the invention further comprises the use an of an antibody according to the invention for the manufacture of a medicament for treatment of cancer.
  • the invention further comprises the use an of an antibody according to the invention for the manufacture of a medicament for treatment of bone loss.
  • the invention further comprises the use an of an antibody according to the invention for the manufacture of a medicament for treatment of metastasis.
  • the invention further comprises the use an of an antibody according to the invention for the manufacture of a medicament for treatment of inflammatory diseases.
  • the invention further comprises an antibody according to the invention for treatment of a CSF-IR mediated disease.
  • the invention further comprises an antibody according to the invention for treatment of cancer.
  • the invention further comprises an antibody according to the invention for treatment of bone loss.
  • the invention further comprises an antibody according to the invention for treatment of metastasis.
  • the invention further comprises an antibody according to the invention for treatment of inflammatory diseases.
  • a further 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:
  • 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
  • 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
  • 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
  • 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
  • the light chain variable domain comprises a CDR3 region of
  • 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
  • the light chain variable domain comprises a CDR3 region of
  • 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
  • the light chain variable domain comprises a CDR3 region of SEQ ID NO:52, a CDR2 region of SEQ ID NO: 53, and a CDRl region of SEQ ID NO: 54
  • the heavy chain variable domain comprises a CDR3 region of SEQ ID NO:69, a CDR2 region of SEQ ID NO: 70, and a CDRl region of SEQ ID NO:71
  • the light chain variable domain comprises a CDR3 region of
  • the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 77, a CDR2 region of SEQ ID NO: 78, and a CDRl region of SEQ ID NO: 79
  • the light chain variable domain comprises a CDR3 region of
  • SEQ ID NO:80 a CDR2 region of SEQ ID NO: 81 , and a CDRl region of SEQ ID NO: 82.
  • a further 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: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 a humanized version thereof.
  • a further 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
  • 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 d) 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 provides expression vectors containing 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 an 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 the production of a recombinant human or humanized antibody according to the invention, characterized by expressing a nucleic acid according to the invention in a prokaryotic or eukaryotic host cell and recovering said antibody from said cell or the cell culture supernatant.
  • the invention further comprises the antibody obtained by such a recombinant method.
  • Antibodies according to the invention show benefits for patients in need of a CSF-1R targeting therapy.
  • the antibodies according to the invention show efficient antiproliferative activity against ligand-independent and ligand-dependant proliferation and are therefore especially useful in the treatment of cancer and metastasis.
  • the invention further provides a method for treating a patient suffering from cancer, comprising administering to a patient diagnosed as having such a disease (and therefore being in need of such a therapy) an effective amount of an antibody according to the invention.
  • the antibody is administered preferably in a pharmaceutical composition.
  • a further embodiment of the invention is a method for the treatment of a patient suffering from cancer characterized by administering to the patient an antibody according to the invention.
  • mutant CSF-IR recombinant cells are able to form spheroids independent of the CSF-1 ligand.
  • the antibodies according to the invention inhibit (both) human and cynomolgous macrophage differentiation, as they inhibit survival of human and cynomolgous monocytes.
  • the invention comprises an antibody binding to human CSF-IR, characterized in that the antibody binds to human CSF-IR fragment delD4 (comprising the extracellular subdomains Dl -D3 and D5) (SEQ ID NO: 65) and to human CSF-IR Extracellular Domain (CSF-1R-ECD) (comprising the extracellular subdomains Dl -D5) (SEQ ID NO: 64) with a ratio of 1 :50 or lower.
  • human CSF-IR fragment delD4 comprising the extracellular subdomains Dl -D3 and D5
  • CSF-1R-ECD human CSF-IR Extracellular Domain
  • the invention further comprises an antibody according to the invention characterized in comprising as heavy chain variable domain CDR3 region 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 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 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;
  • 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 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:7 and the light chain variable domain is SEQ ID NO: 8, or a humanized version thereof.
  • 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
  • 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.
  • 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
  • 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.
  • 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, or
  • 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.
  • the antibody according to the invention is characterized in that 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 ID 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: l , a CDR2 region of SEQ ID NO: 2, and a CDRl region of SEQ ID NO:
  • the light chain variable domain comprises a CDR3 region of SEQ ID NO: 4, a CDR2 region of SEQ ID NO:5, and a CDRl 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 CDRl region of SEQ ID NO:
  • the light chain variable domain comprises a CDR3 region of SEQ ID NO: 12, a CDR2 region of SEQ ID NO: 13, and a CDRl 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 CDRl region of SEQ ID NO:
  • the light chain variable domain comprises a CDR3 region of SEQ ID NO: 20, a CDR2 region of SEQ ID NO:21 , and a CDRl 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 CDRl region of SEQ ID NO:
  • the light chain variable domain comprises a CDR3 region of SEQ ID NO:28, a CDR2 region of SEQ ID NO: 29, and a CDRl region of SEQ ID NO: 30, or e) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 33, a CDR2 region of SEQ ID NO: 34, and a CDRl region of SEQ ID NO:
  • the light chain variable domain comprises a CDR3 region of SEQ ID NO:36, a CDR2 region of SEQ ID NO: 37, and a CDRl region of SEQ ID NO: 38, or f) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO:41 , a CDR2 region of SEQ ID NO: 42, and a CDRl region of SEQ ID NO:
  • the light chain variable domain comprises a CDR3 region of SEQ ID NO: 44, a CDR2 region of SEQ ID NO:45, and a CDRl 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 CDRl 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 CDRl region of SEQ ID NO: 54.
  • 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: l , a CDR2 region of SEQ ID NO: 2, and a CDRl 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 CDRl 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 CDRl region of SEQ ID NO: 1 1 , 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 CDRl 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
  • 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 SEQ ID NO:74, or b) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 77, a CDR2 region of SEQ ID NO: 78, and a CDR1 region of SEQ ID NO: 79, and the 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.
  • the antibody according to the invention is characterized in that a) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 17, a CDR2 region of SEQ ID NO: 18, and a CDR1 region of SEQ ID NO: 19, and the 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 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 S
  • the antibody according to the invention is characterized in that a) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 17, a CDR2 region of SEQ ID NO: 18, and a CDR1 region of SEQ ID NO: 19, and the 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 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 S
  • 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: 20, a CDR2 region of SEQ ID NO:21, and a CDR1 region of SEQ ID NO:22.
  • 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:28, a CDR2 region of SEQ ID NO: 29, and a CDR1 region of SEQ ID NO: 30.
  • the antibody according to the invention is characterized in that the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 33, a CDR2 region of SEQ ID NO: 34, and a CDR1 region of SEQ ID NO: 35, and the light chain variable domain comprises a CDR3 region of SEQ ID NO:36, a CDR2 region of SEQ ID NO: 37, and a CDR1 region of SEQ ID NO: 38.
  • the antibody according to the invention is characterized 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: 44, a CDR2 region of SEQ ID NO:45, and a CDR1 region of SEQ ID NO:46.
  • the antibody binding to human CSF-1R characterized in that the antibody binds to human CSF-1R fragment delD4 (SEQ ID NO: 65) and to human CSF-IR-ECD (SEQ ID NO: 64) with a ratio of 1 :50 or lower, is further characterized in not binding to human CSF-1R fragment D1-D3 (SEQ ID NO: 66).
  • antibody encompasses the various forms of antibodies including but not being limited to whole antibodies, antibody fragments, human antibodies, humanized antibodies, chimeric antibodies, T cell epitope depleted antibodies, and further genetically engineered antibodies as long as the characteristic properties according to the invention are retained.
  • Antibody fragments comprise a portion of a full length antibody, preferably the variable domain thereof, or at least the antigen binding site thereof. Examples of antibody fragments include diabodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. scFv antibodies are, e.g., described in Houston, J.S., Methods in Enzymol. 203 (1991) 46-88).
  • antibody fragments comprise single chain polypeptides having the characteristics of a V H domain binding to CSF-1R, namely being able to assemble together with a V L domain, or of a V L domain binding to CSF-IR, namely being able to assemble together with a V H domain to a functional antigen binding site and thereby providing the property.
  • monoclonal antibody or “monoclonal antibody composition” as used herein refer to a preparation of antibody molecules of a single amino acid composition.
  • chimeric antibody refers to a monoclonal antibody comprising a variable region, i.e., binding region, from mouse and at least a portion of a constant region derived from a different source or species, usually prepared by recombinant DNA techniques. Chimeric antibodies comprising a mouse variable region and a human constant region are especially preferred. Such rat/human chimeric antibodies 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" encompassed by 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 referred to as "class-switched antibodies.” Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques now well known in the art. See, e.g., Morrison, S.L., et al. , Proc. Natl. Acad Sci. USA 81 (1984) 6851-6855; US 5,202,238 and US 5,204,244.
  • humanized antibody refers to antibodies in which the framework or "complementarity determining regions” (CDR) have been modified to comprise the CDR of an immunoglobulin of different specificity as compared to that of the parent immunoglobulin.
  • CDR complementarity determining regions
  • a murine CDR is grafted into the framework region of a human antibody to prepare the "humanized antibody.”
  • the framework region can be modified by further mutations.
  • the CDRs can be modified by one or more mutations to generate antibodies according to the invention e.g. by mutagenesis based upon molecular modeling as described by Riechmann, L., et al, Nature 332 (1988) 323-
  • a "humanized version of an antibody according to the invention” refers to an antibody, which is based on the mouse antibody sequences in which the V H and V L are humanized by standard techniques (including CDR grafting and optionally subsequent mutagenesis of certain amino acids in the framework region and the CDRs ).
  • Preferably such humanized version is chimerized with a human constant region (see e.g. Sequences SEQ ID NO:57-61).
  • humanized antibodies encompassed by the present invention are those in which the constant region has been additionally modified or changed from that of the original antibody to generate the properties according to the invention, especially in regard to Clq binding and/or Fc receptor (FcR) binding.
  • FcR Fc receptor
  • Mab or “muMab” refer to murine monoclonal antibodies such as Mab 2F11 or Mab 2E10
  • hMab refers to humanized monoclonal versions of such murine antibodies such as hMab 2F11-cl 1, hMab 2F1 l-d8, hMab 2F1 l-e7, hMab 2F1 l-fl2, etc.
  • human antibody as used herein, is intended to include antibodies having variable and constant regions derived from human germ line immunoglobulin sequences.
  • Human antibodies are well-known in the state of the art (van Dijk, M.A., and van de Winkel, J.G., Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human antibodies can also be produced in transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire or a selection of human antibodies in the absence of endogenous immunoglobulin production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90 ( 1993) 255 1-2555; Jakobovits, A., et al, Nature 362 (1993) 255-258;
  • Human antibodies can also be produced in phage display libraries (Hoogenboom, H.R., and Winter, G.J. Mol. Biol. 227 (1992) 381-388; Marks, J.D., et al, J. Mol. Biol. 222 (1991) 581- 597).
  • the techniques of Cole, et al., and Boerner, et al. are also available for the preparation of human monoclonal antibodies (Cole, S.P.C., et al, Monoclonal
  • human antibody as used herein also comprises such antibodies which are modified in the constant region to generate the properties according to the invention, especially in regard to C lq binding and/or FcR binding, e.g. by "class switching” i.e. change or mutation of Fc parts (e.g. from IgGl to IgG4 and/or IgGl/IgG4 mutation).
  • recombinant human antibody is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from a host cell such as a NSO or CHO cell or from an animal (e.g. a mouse) that is transgenic for human immunoglobulin genes or antibodies expressed using a recombinant expression vector transfected into a host cell.
  • recombinant human antibodies have variable and constant regions in a rearranged form.
  • the recombinant human antibodies according to the invention have been subjected to in vivo somatic hypermutation.
  • the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germ line VH and VL sequences, may not naturally exist within the human antibody germ line repertoire in vivo.
  • the antibodies according to the invention include, in addition, such antibodies having "conservative sequence modifications", nucleotide and amino acid sequence modifications which do not affect or alter the above-mentioned characteristics of the antibody according to the invention. Modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR- mediated mutagenesis. Conservative amino acid substitutions include ones 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.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • a predicted nonessential amino acid residue in a human anti-CSF-lR antibody can be preferably replaced with another amino acid residue from the same side chain family.
  • Amino acid substitutions can be performed by mutagenesis based upon 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.
  • CSF-1R The human CSF-1R (CSF-1 receptor; synonyms: M-CSF receptor; Macrophage colony-stimulating factor 1 receptor, Fms proto-oncogene, c-fms, SEQ ID NO: 22)) is known since 1986 (Coussens, L., et al, Nature 320 (1986) 277-280).
  • CSF-1R is a growth factor and encoded by the c-fms proto-oncogene (reviewed e.g. in Roth, P. and Stanley, E.R., Curr. Top. Microbiol. Immunol. 181 (1992) 141-67).
  • CSF-1 R is the receptor for CSF-1 (macrophage colony stimulating factor, also called M-CSF) and IL-34 and mediates the biological effects of these cytokines
  • CSF-1 R is a single chain, transmembrane receptor tyrosine kinase (RTK) and a member of the family of immunoglobulin (Ig) motif containing RTKs characterized by 5 repeated Ig-like subdomains D1-D5 in the extracellular domain
  • CSF-1R Extracellular Domain (SEQ ID NO: 64) comprises all five extracellular Ig-like subdomains Dl -D5.
  • the human CSF-1 R fragment delD4 (SEQ ID NO: 65) comprises the extracellular Ig-like subdomains D1-D3 and D5, but is missing the D4 subdomain.
  • the human CSF-1 R fragment delD4 (SEQ ID NO: 65) comprises the extracellular Ig-like subdomains D1-D3 and D5, but is missing the D4 subdomain.
  • CSF-1 R fragment D1-D3 (SEQ ID NO: 66) comprises the respective subdomains D1-D3. The sequences are listed without the signal peptide MGSGPGVLLL LLVATAWHGQ G (SEQ ID NO: 67).
  • the intracellular protein tyrosine kinase domain is interrupted by a unique insert domain that is also present in the other related RTK class III family members that include the platelet derived growth factor receptors (PDGFR), stem cell growth factor receptor (c-Kit) and fins-like cytokine receptor (FLT3).
  • PDGFR platelet derived growth factor receptors
  • c-Kit stem cell growth factor receptor
  • FLT3 fins-like cytokine receptor
  • CSF-IR is mainly expressed on cells of the monocytic lineage and in the female reproductive tract and placenta.
  • CSF-I R has been reported in Langerhans cells in skin, a subset of smooth muscle cells (Inaba, T., et al, J. Biol. Chem. 267 (1992) 5693-5699), B cells (Baker, A.H., et al, Oncogene 8 (1993) 371-378) and microglia (Sawada, M., et al., Brain Res. 509 (1990) 1 19- 124).
  • Cells with mutant human CSF-IR (SEQ ID NO: 23) are known to proliferate independently of ligand stimulation.
  • binding to human CSF-IR or "specifically binding to human
  • CSF-IR refers to an antibody specifically binding to the human CSF-IR antigen with a binding affinity of KD-value of 1.0 x 10 ⁇ 8 mol/1 or lower at 35°C, in one embodiment of a KD-value of 1 .0 xlO 9 mol/1 or lower at 35°C.
  • the binding affinity is determined with a standard binding assay at 35°C, such as surface plasmon resonance technique (BIAcore®, GE-Healthcare Uppsala, Sweden)
  • a method for determining the KD-value of the binding affinity is described in Example 9.
  • an "antibody binding to human CSF-IR” as used herein refers to an antibody specifically binding to the human CSF-IR antigen with a binding affinity of KD 1.0 x 10 ⁇ 8 mol/1 or lower (preferably 1.0 x 10 "8 mol/1 - 1.0 x 10 ⁇ 12 mol/1) at 35°C, preferably of a KD 1.0 xlO 9 mol/1 or lower at 35°C (preferably 1.0 x 10 "9 mol/1 - 1.0 x 10 "12 mol/1).
  • binding to human CSF-IR fragment delD4 (SEQ ID NO: 65) and to human CSF-IR Extracellular Domain (SEQ ID NO: 64)" as used herein is measured by a Surface Plasmon Resonance assay (Biacore assay) as described in Example 4.
  • the human CSF-IR fragment delD4 (SEQ ID NO: 65) or human CSF-IR Extracellular
  • the antibodies according to the invention have a ratio of the binding signals (RU(delD4) / RU(CSF-IR-ECD) of 1 :50 or lower, preferably of 1 : 100 or lower (the lower included end is 0 ( e.g. if the RU is 0, then the ratio is 0:50 or 0: 100) ).
  • anti-CSF-lR antibodies according to the invention do not bind to the human CSF-1R fragment delD4 (like the anti-CCR5 antibody m ⁇ CCR5>Pz03.1C5 (deposited as DSM ACC 2683 on 18.08.2004 at DSMZ) and have binding signals for binding to the human CSF-1R fragment delD4 in the range of the anti-CCR5 antibody m ⁇ CCR5>Pz03.1C5, which are below 20 RU (Response Units), preferably below 10 RU in a Surface Plasmon Resonance (BIAcore) assay as shown in Example 4.
  • BiAcore Surface Plasmon Resonance
  • binding to human CSF-1R fragment D1-D3 refers to a binding affinity determination by a Surface Plasmon Resonance assay (Biacore assay).
  • the test antibody is captured to the surface and the human CSF-1R fragment D1-D3 (SEQ ID NO: 66) was added and the respective binding affinities were determined.
  • the term "not binding to human CSF-1R fragment D 1-D3" denotes that in such an assay the detected signal was in the area of no more than 1.2 fold of background signal and therefore 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 selecting antibodies according to the invention comprising the following steps: a) determining the binding signal (Response Units (RU)) of anti-CSF-lR antibodies to human CSF-1R fragment delD4 (SEQ ID NO: 65) and to human CSF-1R Extracellular Domain (CSF-1R-ECD) (SEQ ID NO: 64) by a Surface Plasmon Resonance assay (Biacore assay).
  • RU Response Units
  • the determination is performed at 25 °C.
  • the screening method comprises as further steps the measuring of the binding of anti-CSF-lR antibodies to human CSF-1R fragment D1-D3 (SEQ ID NO: 66) (D1-D3) and the selecting of antibodies which show no binding to said fragment.
  • epitope denotes a protein determinant of human CSF-IR capable of specifically binding to an antibody.
  • Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually epitopes have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.
  • an antibody according to the invention binds specifically to native and to denatured CSF-IR.
  • variable domain denotes each of the pair of light and heavy chain domains which are involved directly in binding the antibody to the antigen.
  • the variable light and heavy chain domains have the same general structure and each domain comprises four framework (FR) regions whose sequences are widely conserved, connected by three "hypervariable regions” (or complementary determining regions, CDRs).
  • the framework regions adopt a ⁇ -sheet conformation and the CDRs may form loops connecting the ⁇ -sheet structure.
  • the CDRs in each chain are held in their three-dimensional structure by the framework regions and form together with the CDRs from the other chain the antigen binding site.
  • the antibody's heavy and light chain CDR3 regions play a particularly important role in the binding specificity/affinity of the antibodies according to the invention and therefore provide a further object of the invention.
  • antigen-binding portion of an antibody when used herein refer to the amino acid residues of an antibody which are responsible for antigen-binding.
  • the antigen-binding portion of an antibody comprises amino acid residues from the "complementary determining regions" or "CDRs".
  • CDRs complementary determining regions
  • FR Framework regions are those variable domain regions other than the hypervariable region residues as herein defined. Therefore, the light and heavy chain variable domains of an antibody comprise from N- to C-terminus the domains FR1 , CDR1 , FR2, CDR2,
  • CDR3 of the heavy chain is the region which contributes most to antigen binding and defines the antibody's properties.
  • CDR and FR regions are determined according to the standard definition of Kabat et al, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) and/or those residues from a "hypervariable loop".
  • nucleic acid or “nucleic acid molecule”, as used herein, are intended to include DNA molecules and RNA molecules.
  • a nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA.
  • amino acid denotes the group of naturally occurring carboxy a-amino acids comprising alanine (three letter code: ala, one 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), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).
  • the antibodies according to the invention inhibit CSF-1 binding to CSF-1R. In one embodiment with an IC50 of 200 ng/ml or lower, in one embodiment with an IC50 of 50 ng/ml or lower.
  • the IC50 of inhibition of CSF-1 binding to CSF-1R can be determined as shown in Example 2.
  • the antibodies according to the invention inhibit CSF-1 - induced CSF-1R phosphorylation (in NIH3T3-CSF-1R recombinant cells).
  • CSF-1 - induced CSF-1R phosphorylation in NIH3T3-CSF-1R recombinant cells.
  • IC50 of 800 ng/ml or lower in one embodiment with an IC50 of 600 ng/ml or lower, in one embodiment with an IC50 of 250 ng/ml or lower.
  • the IC50 of CSF-l-induced CSF-1R phosphorylation can be determined as shown in Example 3.
  • the antibodies according to the invention inhibit the growth of recombinant NIH3T3 cells expressing human CSF-1R (SEQ ID No: 62).
  • SEQ ID No: 62 human CSF-1R
  • an IC50 of 10 ⁇ g/ml or lower in one embodiment with an IC50 of 5 ⁇ g/ml or lower, in one embodiment with an IC50 of 2 ⁇ g/ml or lower.
  • an IC30 of 10 ⁇ g/ml or lower in one embodiment with an IC30 of 5 ⁇ g/ml or lower, in one embodiment with an IC30 of 2 ⁇ g/ml or lower.
  • the IC50 value, the IC30 value or the % growth inhibition is determined as shown in
  • the antibodies according to the invention inhibit the growth of recombinant NIH3T3 cells expressing human mutant CSF-1R L301S Y969F (SEQ ID No : 63). In one embodiment with an IC50 of 15 ⁇ g/ml or lower, in one embodiment with an IC50 of 10 ⁇ g/ml or lower. In one embodiment with an IC30 of 10 ⁇ g/ml or lower, in one embodiment with an IC50 of 5 ⁇ g/ml ng/ml or lower; in one embodiment with an IC50 of 2 ⁇ g/ml or lower. The IC50 value, the IC30 value or the % growth inhibition is determined as shown in Example 5.
  • the antibodies according to the invention inhibit the growth of BeWo tumor cells (ATCC CCL-98) by 65 % or more (at an antibody concentration of 10 ⁇ g/ml ; and as compared to the absence of antibody).
  • the % growth inhibition is determined as shown in Example 8.
  • E.g. Mab 2F11 shows a growth inhibition of BeWo tumor cells of 70 %.
  • the antibodies according to the invention inhibit (both) human and cynomolgous macrophage differentiation (which is indicated by the inhibition of the survival of human and cynomolgous monocytes as shown in Examples 7 and
  • the antibodies according to the invention inhibit the survival of human monocytes with an IC50 of 0.15 ⁇ g/ml or lower, in on embodiment with an IC50 of 0.10 ⁇ g/ml or lower.
  • the inhibition of the survival of human monocytes is determined as shown in Example 7.
  • the antibodies according to the invention inhibit the survival of cynomolgous monocytes by 80 % or more, in one embodiment by 90 % or more (at an antibody concentration of 5 ⁇ g/ml ;and as compared to the absence of antibody).
  • the inhibition of the survival of human monocytes is determined as shown in Example 8.
  • a further embodiment of the invention is a method for the production of an antibody against CSF-1R characterized in that the sequence of a nucleic acid encoding the heavy chain of a human IgGl class antibody binding to human CSF-1R according to the invention said modified nucleic acid and the nucleic acid encoding the light chain of said antibody are inserted into an expression vector, said vector is inserted in a eukaryotic host cell, 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. Therefore the antibody is preferably an isolated monoclonal antibody.
  • recombinant methods are widely known in the state of the art and comprise protein expression in prokaryotic and eukaryotic cells with subsequent isolation of the antibody polypeptide and usually purification to a pharmaceutically acceptable purity.
  • nucleic acids encoding light and heavy chains or fragments thereof are inserted into expression vectors by standard methods. Expression is performed in appropriate prokaryotic or eukaryotic host cells like
  • CHO cells NSO cells, SP2/0 cells, HEK293 cells, COS cells, yeast, or E.coli cells, and the antibody is recovered from the cells (supernatant or cells after lysis).
  • the antibodies may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. Purification is performed in order to eliminate other cellular components or other contaminants, e.g. other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others well known in the art. See Ausubel, F., et al, ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987). Expression in NSO cells is described by, e.g., Barnes, L.M., et al, Cytotechnology
  • HEK 293 A preferred transient expression system (HEK 293) is described by Schlaeger, E.-J., and Christensen, K., in Cytotechnology 30 (1999) 71-83 and by Schlaeger, E.-J., in J. Immunol. Methods 194 (1996) 191-199.
  • control sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site.
  • Eukaryotic cells are known to utilize promoters, enhancers and polyadenylation signals.
  • Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that 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.
  • "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
  • the monoclonal antibodies are suitably separated from the culture medium by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • DNA and RNA encoding the monoclonal antibodies are readily isolated and sequenced using conventional procedures.
  • the hybridoma cells can serve as a source of such DNA and RNA.
  • the DNA may be inserted into expression vectors, which are then transfected into host cells such as HEK 293 cells, CHO cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of recombinant monoclonal antibodies in the host cells.
  • the expressions "cell”, “cell line”, and “cell culture” are used interchangeably and all such designations include progeny.
  • the words “transformants” and “transformed cells” include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological activity as screened for in the originally transformed cell are included.
  • the "Fc part” of an antibody is not involved directly in binding of an antibody to an antigen, but exhibit various effector functions.
  • a “Fc part of an antibody” is a term well known to the skilled artisan and defined on the basis of papain cleavage of antibodies.
  • antibodies or immunoglobulins are divided in the classes: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g. IgGl, IgG2, IgG3, and IgG4, IgAl, and IgA2.
  • subclasses e.g. IgGl, IgG2, IgG3, and IgG4, IgAl, and IgA2.
  • the different classes of immunoglobulins are called a, ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the Fc part of an antibody is directly involved in ADCC
  • CDC complement-dependent cytotoxicity
  • complement activation is initiated by binding of complement factor Clq to the Fc part of most IgG antibody subclasses. While the influence of an antibody on the complement system is dependent on certain conditions, binding to C lq is caused by defined binding sites in the Fc part. Such binding sites are known in the state of the art and described e.g. by Boackle, R.J., et al., Nature 282 (1979) 742-743, Lukas, T.J., et al, J. Immunol.
  • binding sites are e.g. L234, L235, D270, N297, E318, K320, K322, P331 and P329 (numbering according to EU index of Kabat, E.A., see below).
  • Antibodies of subclass IgGl, IgG2 and IgG3 usually show complement activation and Clq and C3 binding, whereas IgG4 do not activate the complement system and do not bind Clq and C3.
  • the antibody according to the invention comprises a Fc part derived from human origin and preferably all other parts of the human constant regions.
  • Fc part derived from human origin denotes a Fc part which is either a Fc part of a human antibody of the subclass IgGl, IgG2, IgG3 or IgG4, preferably a Fc part from human IgGl subclass, a mutated Fc part from human IgGl subclass (preferably with a mutation on L234A + L235A), a Fc part from human IgG4 subclass or a mutated Fc part from human IgG4 subclass (preferably with a mutation on S228P).
  • the human heavy chain constant regions of SEQ ID NO: 58 (human IgGl subclass), SEQ ID NO: 59 (human IgGl subclass with mutations L234A and L235A) , SEQ ID NO: 60 human IgG4 subclass), or SEQ ID NO: 61 (human IgG4 subclass with mutation S228P).
  • the antibody according to the invention is of human IgGl subclass or of human IgG4 subclass.
  • the antibody according to the invention is of human IgGl subclass.
  • the antibody according to the invention is of human IgG4 subclass.
  • the antibody according to the invention is characterized in that the constant chains are of human origin.
  • a useful human heavy chain constant region comprises an amino acid sequence of SEQ ID NO: 58.
  • a useful human light chain constant region comprises an amino acid sequence of a kappa-light chain constant region of SEQ ID 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,
  • the heavy chain variable domain is SEQ ID NO: 15 and the light chain variable domain is SEQ ID NO: 16;
  • 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,
  • 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 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: 7 and the light chain variable domain is SEQ ID NO: 8, 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 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 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.
  • 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
  • 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.
  • 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 :23 and the light chain variable domain is SEQ ID NO:24, or
  • 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:31 and the light chain variable domain is SEQ ID 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 ID NO :39 and the light chain variable domain is SEQ ID NO:40.
  • 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 :47 and the light chain variable domain is SEQ ID 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 ID 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: l, 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 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
  • 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 ID NO: 18, and a CDR1 region of SEQ ID NO: 19, and the light chain variable domain comprises a CDR3 region of
  • 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
  • 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
  • the light chain variable domain comprises a CDR3 region of SEQ ID NO:36, a CDR2 region of SEQ ID NO: 37, and a CDRl region of SEQ ID NO: 38
  • the heavy chain variable domain comprises a CDR3 region of SEQ ID NO:41, a CDR2 region of SEQ ID NO: 42, and a CDRl region of SEQ ID NO:43
  • the light chain variable domain comprises a CDR3 region of SEQ ID NO: 44, a CDR2 region of SEQ ID NO:45, and a CDRl region of SEQ ID NO:46, or e) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 44, a CDR2 region of SEQ ID NO:45, and a CDRl region of SEQ ID NO:46, or
  • 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 ID NO : 18, and a CDRl 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 CDRl region of SEQ ID NO:22, or b) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 25, a CDR2 region of SEQ ID NO: 26, and a CDRl 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 CDRl 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:
  • 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 ID NO: 18, and a CDRl 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 CDRl region of SEQ ID 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 ID NO: 25, a CDR2 region of SEQ ID NO: 26, and a CDRl 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 CDRl region of SEQ ID 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 ID NO: 1
  • the light chain variable domain comprises a CDR3 region of SEQ ID NO:36, a CDR2 region of SEQ ID NO: 37, and a CDRl 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 ID NO: 42, and a CDRl 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.
  • the invention comprises a method for the treatment of 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 comprises the use of an antibody according to the invention for therapy.
  • CSF-1R antibodies of the present invention for use in the treatment of "CSF-1R mediated diseases" or the CSF-1R antibodies of the present invention for use for the manufacture of a medicament in the treatment of "CSF-1R mediated diseases", which can be described as follows:
  • CSF-1R signaling is likely involved in tumor growth and metastasis.
  • the first is that expression of CSF-ligand and receptor has been found in tumor cells originating in the female reproductive system (breast, ovarian, endometrium, cervical) (Scholl, S.M., et al., J. Natl. Cancer Inst. 86 (1994) 120-126; Kacinski, B.M., Mol. Reprod. Dev. 46 (1997) 71- 74; Ngan, H.Y., et al., Eur. J. Cancer 35 (1999) 1546-1550; Kirma, N., et al., Cancer Res 67 (2007) 1918-1926) and the expression has been associated with breast cancer xenograft growth as well as poor prognosis in breast cancer patients.
  • a landscape effect is proposed to be responsible for the resulting tumor mass that consists of monocytic cells attracted by cells that express M-CSF.
  • TGCTs are smaller tumors that can be relatively easily removed from fingers where they mostly occur.
  • PVNS is more aggressive as it can recur in large joints and is not as easily controlled surgically.
  • the second mechanism is based on blocking signaling through M-CSF/CSF-IR at metastatic sites in bone which induces osteoclastogenesis, bone resorption and osteolytic bone lesions.
  • Breast, multiple myeloma and lung cancers are examples of cancers that have been found to metastasize to the bone and cause osteolytic bone disease resulting in skeletal complications.
  • M-CSF released by tumor cells and stroma induces the differentiation of hematopoietic myeloid monocyte progenitors to mature osteoclasts in collaboration with the receptor activator of nuclear factor kappa-B ligand-RANKL.
  • M-CSF acts as a permissive factor by giving the survival signal to osteoclasts (Tanaka, S., et al., J. Clin. Invest. 91 (1993) 257-263).
  • Inhibition of CSF-1R activity during osteoclast differentiation and maturation with a anti-CSF-l R antibody is likely to prevent unbalanced activity of osteoclasts that cause osteolytic disease and the associated skeletal related events in metastatic disease.
  • breast, lung cancer and multiple myeloma typically result in osteolytic lesions
  • metastasis to the bone in prostate cancer initially has an osteoblastic appearance in which increased bone forming activity results in 'woven bone' which is different from typical lamellar structure of normal bone.
  • bone lesions display a significant osteolytic component as well as high serum levels of bone resorption and suggests that anti-resorptive therapy may be useful.
  • Bisphosphonates have been shown to inhibit the formation of osteolytic lesions and reduced the number of skeletal- related events only in men with hormone-refractory metastatic prostate cancer but at this point their effect on osteoblastic lesions is controversial and bisphosphonates have not been beneficial in preventing bone metastasis or hormone responsive prostate cancer to date .
  • the effect o f anti-resorptive agents in mixed osteolytic/osteoblastic prostate cancer is still being studied in the clinic (Choueiri, M.B., et al., Cancer Metastasis Rev.
  • TAM tumor associated macrophages
  • the macrophages can then contribute to tumor progression through the secretion of angiogenic factors, proteases and other growth factors and cytokines and may be blocked by inhibition of CSF-1R signaling. Recently it was shown by Zins et al (Zins, K., et al., Cancer Res. 67 (2007) 1038-1045) that expression of siRNA of Tumor necrosis factor alpha (TNF alpha), M-CSF or the combination of both would reduce tumor growth in a mouse xenograft model between 34% and 50% after intratumoral injection of the respective siRNA. SiRNA targeting the TNF alpha secreted by the human SW620 cells reduced mouse M-CSF levels and led to reduction of macrophages in the tumor.
  • TNF alpha Tumor necrosis factor alpha
  • TAMs are only one example of an emerging link between chronic inflammation and cancer.
  • inflammation and cancer There is additional evidence for a link between inflammation and cancer as many chronic diseases are associated with an increased risk of cancer, cancers arise at sites of chronic inflammation, chemical mediators of inflammation are found in many cancers; deletion of the cellular or chemical mediators of inflammation inhibits development of experimental cancers and long-term use of anti-inflammatory agents reduce the risk of some cancers.
  • Macrophages are key cells in chronic inflammation and respond differentially to their microenvironment.
  • M l macrophages are involved in Type 1 reactions. These reactions involve the activation by microbial products and consequent killing of pathogenic microorganisms that result in reactive oxygen intermediates.
  • M2 macrophages involved in Type 2 reactions that promote cell proliferation, tune inflammation and adaptive immunity and promote tissue remodeling, angiogenesis and repair
  • Chronic inflammation resulting in established neoplasia is usually associated with M2 macrophages.
  • a pivotal cytokine that mediates inflammatory reactions is TNF alpha that true to its name can stimulate anti-tumor immunity and hemorrhagic necrosis at high doses but has also recently been found to be expressed by tumor cells and acting as a tumor promoter (Zins, K., et al., Cancer Res.
  • cancer may be, for example, lung cancer, non small cell lung (NSCL) cancer, bronchioloalviolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colon cancer, breast cancer, uterine cancer, 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, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the
  • cancer is a breast cancer, ovarian cancer , cervical cancer, lung cancer or prostate cancer.
  • cancers are further characterized by CSF-1 or CSF-1 R expression or overexpression.
  • the invention are the CSF-1 R antibodies of the present invention for use in the simultaneous treatment of primary tumors and new metastases.
  • CSF-IR antibodies of the present invention for use in the treatment of periodontitis, histiocytosis X, osteoporosis, Paget's disease of bone (PDB), bone loss due to cancer therapy, periprosthetic osteolysis, glucocorticoid-induced osteoporosis, rheumatoid arthritis, psiratic arthritis, osteoarthritis, inflammatory arthridities, and inflammation.
  • Histiocytosis X also called Langerhans cell histiocytosis, LCH
  • Langerhans cells are derived from circulating monocytes.
  • M-CSF Increased levels of M-CSF that have been measured in sera and lesions where found to correlate with disease severity (da Costa, C.E., et al, J. Exp. Med. 201 (2005) 687-693).
  • the disease occurs primarily in a pediatric patient population and has to be treated with chemotherapy when the disease becomes systemic or is recurrent.
  • Paget's disease of bone 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, pathological fractures and deafness.
  • Mutations in four genes have been identified that regulate normal osteoclast function and predispose individuals to PDB and related disorders: insertion mutations in TNFRSF11 A, which encodes receptor activator of nuclear factor (NF) kappaB (RANK)-a critical regulator of osteoclast function, inactivating mutations of TNFRSF1 IB which encodes osteoprotegerin (a decoy receptor for
  • VCP valosin-containing protein
  • Targeted CSF-1R inhibitors provide an opportunity to block the deregulation of the RANKL signaling indirectly and add an additional treatment option to the currently used bisphosphonates.
  • Cancer therapy induced bone loss especially in breast and prostate cancer patients is an additional indication where a targeted CSF-1R inhibitor could prevent bone loss (Lester, J.E., et al., Br. J. Cancer 94 (2006) 30-35).
  • the long-term consequences of the adjuvant therapies become more important as some of the therapies including chemotherapy, irradiation, aromatase inhibitors and ovary ablation affect bone metabolism by decreasing the bone mineral density, resulting in increased risk for osteoporosis and associated fractures (Lester, J.E., et al., Br. J. Cancer 94 (2006) 30-35).
  • Targeted inhibition of CSF-1R signaling is likely to be beneficial in other indications as well when targeted cell types include osteoclasts and macrophages e.g. treatment of specific complications in response to joint replacement as a consequence of rheumatoid arthritis.
  • Implant failure due to periprosthetic bone loss and consequent loosing of prostheses is a major complication of joint replacement and requires repeated surgery with high socioeconomic burdens for the individual patient and the health-care system.
  • there is no approved drug therapy to prevent or inhibit periprosthetic osteolysis (Drees, P., et al., Nat. Clin. Pract. Rheumatol. 3 (2007) 165-171).
  • Glucocorticoid- induced osteoporosis is another indication in which a CSF- 1R inhibitor could prevent bone loss after longterm glucocorticocosteroid use that is given as a result of various conditions among those chronic obstructive pulmonary disease, asthma and rheumatoid arthritis (Guzman-Clark, J.R., et al., Arthritis Rheum. 57 (2007) 140-146; Feldstein, A.C., et al., Osteoporos. Int. 16
  • Rheumatoid arthritis, psioratic arthritis and inflammatory arthridities are in itself potential indications for CSF-1R signaling inhibitors in that they consist of a macrophage component and to a varying degree bone destruction (Ritchlin, C.T., et al., J. Clin. Invest. I l l (2003) 821-831).
  • Osteoarthritis and rheumatoid arthritis are inflammatory autoimmune disease caused by the accumulation of macrophages in the connective tissue and infiltration of macrophages into the synovial fiuid, which is at least partially mediated by M-CSF. Campbell, I., K., et al., J. Leukoc. Biol.
  • M-CSF is produced by human-joint tissue cells (chondrocytes, synovial fibroblasts) in vitro and is found in synovial fluid of patients with rheumatoid arthritis, suggesting that it contributes to the synovial tissue proliferation and macrophage infiltration which is associated with the pathogenesis of the disease.
  • Inhibition of CSF-1R signaling is likely to control the number of macrophages in the joint and alleviate the pain from the associated bone destruction.
  • one method is to specifically inhibit CSF-1R without targeting a myriad other kinases, such as Raf kinase.
  • M-CSF influences the atherosclerotic process by aiding the formation of foam cells (macrophages with ingested oxidized LDL) that express CSF-1R and represent the initial plaque (Murayama, T., et al, Circulation 99 (1999) 1740-1746). Expression and signaling of M-CSF and CSF-1R is found in activated microglia.
  • Microglia which are resident macrophages of the central nervous system, can be activated by various insults, including infection and traumatic injury.
  • M-CSF is considered a key regulator of inflammatory responses in the brain and M-CSF levels increase in HIV-1, encephalitis, Alzheimer's disease (AD) and brain tumors.
  • Microgliosis as a consequence of autocrine signaling by M-CSF/CSF-1R results in induction of inflammatory cytokines and nitric oxides being released as demonstrated by e.g. using an experimental neuronal damage model (Hao, A.J., et al, Neuroscience 112 (2002) 889-900; Murphy, G.M., Jr., et al, J. Biol. Chem. 273
  • Microglia that have increased expression of CSF-IR are found to surround plaques in AD and in the amyloid precursor protein V717F transgenic mouse model of AD (Murphy, G.M., Jr., et al., Am. J. Pathol. 157 (2000) 895-904).
  • op/op mice with fewer microglia in the brain resulted in fibrilar deposition of A-beta and neuronal loss compared to normal control suggesting that microglia do have a neuroprotective function in the development of AD lacking in the op/op mice (Kaku, M., et al., Brain Res. Brain Res. Protoc. 12 (2003) 104-108).
  • inflammatory bowel disease refers to serious, chronic disorders of the intestinal tract characterized by chronic inflammation at various sites in the gastrointestinal tract, and specifically includes ulcerative colitis (UC) and Crohn's disease.
  • the invention comprises an antibody binding to human CSF-IR being characterized by the above mentioned epitope binding properties or alternatively by the above mentioned amino acid sequences and amino acid sequence fragments for the treatment of cancer.
  • the invention comprises an antibody binding to human CSF-IR being characterized by the above mentioned epitope binding properties or alternatively by the above mentioned amino acid sequences and amino acid sequence fragments for the treatment of bone loss.
  • the invention comprises an antibody binding to human CSF-IR being characterized by the above mentioned epitope binding properties or alternatively by the above mentioned amino acid sequences and amino acid sequence fragments for the prevention or treatment of metastasis.
  • the invention comprises an antibody binding to human CSF-IR being characterized by the above mentioned epitope binding properties or alternatively by the above mentioned amino acid sequences and amino acid sequence fragments for treatment of inflammatory diseases.
  • the invention comprises the use of an antibody characterized in comprising the antibody binding to human CSF-IR being characterized by the above mentioned epitope binding properties or alternatively by the above mentioned amino acid sequences and amino acid sequence fragments for the treatment of cancer or alternatively for the manufacture of a medicament for the treatment of cancer.
  • the invention comprises the use of an antibody characterized in comprising the antibody binding to human CSF-IR being characterized by the above mentioned epitope binding properties or alternatively by the above mentioned amino acid sequences and amino acid sequence fragments for the treatment of bone loss or alternatively for the manufacture of a medicament for the treatment of bone loss.
  • the invention comprises the use of an antibody characterized in comprising the antibody binding to human CSF-IR being characterized by the above mentioned epitope binding properties or alternatively by the above mentioned amino acid sequences and amino acid sequence fragments for the prevention or treatment of metastasis or alternatively for the manufacture of a medicament for the prevention or treatment of metastasis.
  • the invention comprises the use of an antibody characterized in comprising the antibody binding to human CSF-IR being characterized by the above mentioned epitope binding properties or alternatively by the above mentioned amino acid sequences and amino acid sequence fragments for treatment of inflammatory diseases or alternatively for the manufacture of a medicament for the treatment of inflammatory diseases.
  • a further embodiment of the invention is a method for the production of an antibody against CSF-IR characterized in that the sequence of a nucleic acid encoding the heavy chain of a human IgG l class antibody binding to human CSF-IR according to the invention said modified nucleic acid and the nucleic acid encoding the light chain of said antibody are inserted into an expression vector, said vector is inserted in a eukaryotic host cell, 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. Such methods are widely known in the state of the art and comprise protein expression in prokaryotic and eukaryotic cells with subsequent isolation of the antibody polypeptide and usually purification to a pharmaceutically acceptable purity.
  • nucleic acids encoding light and heavy chains or fragments thereof are inserted into expression vectors by standard methods. Expression is performed in appropriate prokaryotic or eukaryotic host cells, such as CHO cells, NS0 cells, SP2/0 cells, HEK293 cells, COS cells, yeast, or E. coli cells, and the antibody is recovered from the cells (from the supernatant or after cells lysis).
  • the antibodies may be present in whole cells, in a cell lysate, or in a partially purified, or substantially pure form. Purification is performed in order to eliminate other cellular components or other contaminants, e.g. other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others well known in the art. See Ausubel, F., et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987). Expression in NS0 cells is described by, e.g., Barnes, L.M., et al, Cytotechnology
  • HEK 293 A preferred transient expression system (HEK 293) is described by Schlaeger, E.-J. and Christensen, K., in Cytotechnology 30 (1999) 71-83, and by Schlaeger, E.-J., in J. Immunol. Methods 194 (1996) 191-199.
  • Nucleic acid molecules encoding amino acid sequence variants of anti-CSF-lR antibody are prepared by a variety of methods known in the art.
  • These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of humanized anti-CSF-lR antibody.
  • the heavy and light chain variable domains according to the invention are combined with sequences of promoter, translation initiation, constant region, 3' untranslated region, polyadenylation, and transcription termination to form expression vector constructs.
  • the heavy and light chain expression constructs can be combined into a single vector, co-transfected, serially transfected, or separately transfected into host cells which are then fused to form a single host cell expressing both chains.
  • the present invention provides a composition, e.g. a pharmaceutical composition, containing one or a combination of monoclonal antibodies, or the antigen-binding portion thereof, of the present invention, formulated together with a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption/resorption delaying agents, and the like that are physiologically compatible.
  • the carrier is suitable for injection or infusion.
  • composition of the present invention can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results.
  • Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the preparation of sterile injectable solutions or dispersion.
  • the use of such media and agents for pharmaceutically active substances is known in the art.
  • the carrier can be, for example, an isotonic buffered saline solution.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient (effective amount).
  • the selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • the invention comprises the use of the antibodies according to the invention for the treatment of a patient suffering from cancer, especially from colon, lung or pancreas cancer.
  • the invention comprises also a method for the treatment of a patient suffering from such disease.
  • the invention further provides a method for the manufacture of a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of an antibody according to the invention together with a pharmaceutically acceptable carrier and the use of the antibody according to the invention for such a method.
  • 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 together 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 effective amount for the manufacture of a pharmaceutical agent, preferably together with a pharmaceutically acceptable carrier, for the treatment of a patient suffering from cancer.
  • SEQ ID NO: 28 light chain CDR3, hMab 2F1 l-d8
  • SEQ ID NO: 48 light chain variable domain, hMab 2F1 l-fl2
  • SEQ ID NO: 58 human heavy chain constant region derived from IgGl
  • SEQ ID NO: 59 human heavy chain constant region mutated on L234A and L235A
  • X axis viability normalized mean relative light units (RLU) corresponding to the ATP-content of the cells (CellTiterGlo assay).
  • Y axis tested probes: Minimal Medium (0.5% FBS), mouse IgGl (mlgGl , ⁇ ⁇ ), mouse IgG2a (mIgG2a l( ⁇ g/ml), CSF-1 only, Mab 2F1 1 , Mab 2E10, Mab2H7, MablGlO and SC 2-4A5.
  • Figure 2 Biacore sensogram of binding of different anti-CSF-lR antibodies to immobilized human CSF-1 R fragment delD4 (comprising the extracellular subdomains Dl -D3 and D5) (SEQ ID NO: 1)
  • CSF-1 R-ECD immobilized human CSF-IR Extracellular Domain
  • All anti-CSF-lR antibodies show binding to CSF-1R-ECD.
  • the control anti-CCR5 antibody m ⁇ CCR5>Pz03.1C5 did not bind to the CSF-1R-ECD.
  • the control anti-CCR5 antibody m ⁇ CCR5>Pz03.1C5 did also not bind to the CSF-IR fragment delD4.
  • CSF-1R-ECD CSF-IR Extracellular Domain
  • the control anti-CCR5 antibody m ⁇ CCR5>Pz03.1C5 did not bind to the CSF-1R-ECD.
  • the control anti-CCR5 antibody m ⁇ CCR5>Pz03.1C5 did also not bind to the CSF-IR fragment delD4.
  • CSF-1R-ECD CSF-IR Extracellular Domain
  • All anti-CSF-lR antibodies 1.2.SM, CXIIG6, abl0676 and MAB3291 show binding to CSF-1R-ECD.
  • the control anti- CCR5 antibody m ⁇ CCR5>Pz03.1C5 did not bind to the CSF-1R- ECD.
  • mice were immunized with an expression vector pDisplayTM (Invitrogen, USA) encoding the extracellular domain of huCSF-lR by utilizing electroporation. Every mouse was 4 times immunized with 100 ⁇ g DNA. When serum titers of anti- huCSF-lR were found to be sufficient, mice were additionally boosted once with 50 ⁇ g of a 1 : 1 mixture huCSF-lR ECD/huCSF-lR ECDhuFc chimera in 200 ⁇ PBS intravenously (i.v.) 4 and 3 days before fusion.
  • pDisplayTM Invitrogen, USA
  • Antigen specific ELISA Anti-CSF-lR titers in sera of immunized mice were determined by antigen specific
  • huCSF-lR-huFc chimera soluble extracellular domain
  • streptavidin plate MaxiSorb; MicroCoat, DE, Cat.No. 11974998/MC1099
  • biotinylated anti Fey Jackson Immuno Research., Cat.No. 109-066-098
  • HRP horse radish peroxidase
  • the mouse lymphocytes can be isolated and fused with a mouse myeloma cell line using PEG based standard protocols to generate hybridomas.
  • the resulting hybridomas are then screened for the production of antigen-specific antibodies.
  • single cell suspensions of splenic derived lymphocytes from immunized mice are fused to Ag8 non-secreting mouse myeloma cells P3X63Ag8.653 (ATCC, CRL-1580) with 50% PEG.
  • Cells are plated at approximately 10 4 in flat bottom 96 well micro titer plate, followed by about two weeks incubation in selective medium. Individual wells are then screened by ELISA for human anti-CSF-lR monoclonal IgM and IgG antibodies.
  • the antibody secreting hybridomas are replated, screened again, and if still positive for human IgG, anti-CSF-lR monoclonal antibodies, can be subcloned by FACS. The stable subclones are then cultured in vitro to produce antibody in tissue culture medium for characterization.
  • Antibodies according to the invention could be selected using the determination of the binding of anti-CSF-lR antibodies to human CSF-1 R fragment delD4 and to human CSF-1R Extracellular Domain (CSF-1R-ECD) as described in Example 4, as well as the determination of growth inhibition of NIH3T3 cells transfected with wildtype CSF-1R (ligand dependent signalling) or mutant CSF-1R L301S Y969F (ligand independent signalling) under treatment with anti-CSF-lR monoclonal antibodies as described in Example 5.
  • CSF-1R-ECD human CSF-1R Extracellular Domain
  • 4.5xl0 3 NIH 3T3 cells retrovirally infected with an expression vector for full- length CSF-1R, were cultured in DMEM (PAA Cat. No.E 15-011), 2mM L- glutamine (Sigma, Cat.No.G7513, 2mM Sodium pyruvate , lx nonessential aminoacids, 10% FKS (PAA, Cat.No.Al 5-649) and 100 ⁇ g/ml PenStrep (Sigma,
  • the presence of phosphorylated and total CSF-1 receptor in the cell lysate was analyzed with Elisa.
  • the kit from R&D Systems Cat. No. DYC3268-2
  • For detection of total CSF-1 R 10 ⁇ of the lysate was immobilized on plate by use of the capture antibody contained in the kit. Thereafter 1 :750 diluted biotinylated anti CSF-IR antibody BAF329 (R&D Systems) and 1 : 1000 diluted streptavidin-HRP conjugate was added. After 60 minutes plates were developed with freshly prepared ABTS ® solution and the absorbance was detected.
  • pCMV-preS-Fc-hCSF-lR-ECD (7836bp) encodes the complete ECD of human
  • CSF-IR (SEQ ID NO: 64) C-terminally fused to a PreScission protease cleavage site, followed by aal 00-330 of human IgGl and a 6xHis-Tag, under the control of CMV promoter.
  • the natural signal peptide has been varied by insertion of amino acids G and S after the first M, in order to create a BamHI restriction site.
  • Two separate 50 ⁇ reaction mixtures were set up according to the manufacturer's manual, each containing 10 ng plasmid pCMV-preS-Fc-hCSFlR-ECD as the template and 10 pM of one of the primers delD4-for or delD4-rev, and 0,5 ⁇ Pfu DNA polymerase as provided with the kit.
  • Three PCR cycles 95 °C 30 sec / 55 °C 60 sec / 68 °C 8 min were run, then 25 ⁇ each of both reaction mixtures were combined in a new tube and 0,5 ⁇ fresh Pfu DNA polymerase were added.
  • the regular PCR protocol with 18 temperature cycles as specified by Stratagene in the kit manual was carried out, followed by 2 hrs final digestion with the Dpnl restriction enzyme provided with the kit. Clones bearing the deletion were detected by digestion with Cel II and Not I and verified by sequencing.
  • Protein was prepared by transient transfection in the Hek293 FreeStyle suspension cell system (Invitrogen) according to the manufacturer's specifications. After 1 week 500 ml supernatant was filtered and loaded onto a 1ml HiTrap MabSelect Xtra (GE healthcare) protein A column (0,2 ml /min). The colomn was washed first with PBS, then with 50 mM Tris/ 150 mM NaCl/ 1 mM EDTA/ pH 7,3. 75 ⁇ PreScission Protease (GE #27-0843-01) diluted in 375 ⁇ of the same buffer were loaded onto the column and the closed column was incubated over night at 4 °C with rolling.
  • the column was mounted on top of a 1 ml GSTrap FF column (GE helthcare) and the desired protein was eluted (0,2 ml/min, 0,2 ml fractions). Pooled fractions were concentrated from 1,8 ml to 0,4 ml by centrifugal ultrafiltration via a 3k Nanosep and chromatographed over an S200 HR SEC in PBS (0,5 ml/min).
  • the dimeric form was used for all experiments.
  • CSF-1R-ECD binding signals as Response Units (RU): Instrument: Biacore T 100 (GE Healthcare)
  • Mab 2H7 0 147 0/147 0 m ⁇ CCR5>Pz03.1C5 2 5 - Mab 2F1 1 and Mab 2E10 showed binding to the human CSF-IR Extracellular Domain (CSF-IR-ECD) (see Fig. 2b); however no binding was detected to CSF-IR fragment delD4. (see Fig. 2a).
  • CSF-IR-ECD human CSF-IR Extracellular Domain
  • Mab 1G10, Mab 2H7 and humanized hMab 2F1 l-e7 showed binding to the human CSF-IR Extracellular Domain (CSF-IR-ECD) (see Fig. 2d); however no binding was detected to CSF-IR fragment delD4. (see Fig. 2c).
  • CSF-IR-ECD human CSF-IR Extracellular Domain
  • anti-CSF-lR antibodies 1.2.SM ligand displacing CSF-IR antibody described in WO2009026303
  • CXIIG6 ligand displacing CSF-IR antibody described in WO 2009/112245
  • the goat polyclonal anti-CSF-lR antibody ab 10676 (abeam) were investigated.
  • Anti-CSF-lR antibody Mab3291 (R&D-Systems) was used as reference control.
  • NIH 3T3 cells retrovirally infected with either an expression vector for full-length wildtype CSF-1R (SEQ ID NO: 62) or mutant CSF-1R L301 S Y969F (SEQ ID NO: 63), were cultured in DMEM high glucose media (PAA, Pasching, Austria) supplemented with 2mM L-glutamine, 2mM sodium pyruvate and non-essential amino acids and 10% fetal bovine serum (Sigma, Taufkirchen, Germany) on poly- HEMA (poly(2-hydroxyethylmethacrylate)) (Polysciences, Warrington, PA, USA)) coated dishes to prevent adherence to the plastic surface. Cells are seeded in medium replacing serum with 5ng/ml sodium selenite, l Omg/ml transferrin,
  • Reference control Mab R&D-Systems 3291 did not show inhibition of mutant CSF-IR recombinant cell proliferation.
  • the anti-CSF-lR antibody according to the invention hMab
  • BeWo choriocarcinoma cells (ATCC CCL-98) were cultured in F12K media (Sigma, Steinheim, Germany) supplemented with 10% FBS (Sigma) and 2mM L-glutamine. 5xl0 4 cells/well were seeded in 96-well poly-HEMA (poly(2-hydroxyethylmethacrylate)) coated plates containing F12K medium supplemented with 0.5 % FBS and 5% BSA. Concomitantly, 200 ng/ml huCSF-1 and ⁇ of different anti-CSF-lR monoclonal antibodies were added and incubated for 6 days.
  • the CellTiterGlo assay was used to detect cell viability by measuring the ATP-content of the cells in relative light units (RLU).
  • RLU relative light units
  • BeWo spheroid cultures were treated with different anti-CSF-lR antibodies (10 ⁇ ) inhibition of CSF-1 induced growth was observed.
  • Mean RLU value of unstimulated BeWo cells was subtracted from all samples.
  • Mean RLU value of CSF-1 stimulated cells was set arbitrarily to 100%.
  • Mean RLU values of cells stimulated with CSF-1 and treated with anti-CSF-lR antibodies were calculated in % of CSF-1 stimulated RLUs.
  • the Table 6 shows the calculated data of growth inhibition of BeWo tumor cells in 3D culture under treatment with anti-CSF-lR monoclonal antibodies; Fig. la and b depicts normalized mean RLU values. Table 6:
  • Human monocytes were isolated from peripheral blood using the RosetteSepTM Human Monocyte Enrichment Cocktail (StemCell Tech. - Cat. No.15028). Enriched monocyte populations were seeded into 96 well microtiterplates (2.5x10 4 cells/well) in 100 ⁇ RPMI 1640 (Gibco - Cat. No.31870) supplemented with 10% FCS (GIBCO - Cat. No.01 1-090014M), 4 mM L-glutamine (GIBCO - Cat.
  • Cynomolgous monocytes were isolated from peripheral blood using the CD14 MicroBeads non- human primate kit (Miltenyi Biotec - Cat.No. 130-091-097) according to the manufacturers description. Enriched monocyte populations were seeded into 96 well microtiterplates (l-3xl0 4 cells/well) in 100 ⁇ RPMI 1640 (Gibco - Cat. No.31870) supplemented with 10% FCS (GIBCO - Cat. No.01 1- 090014M), 4 mM L-glutamine (GIBCO - Cat. No.25030) and lx PenStrep (Roche Cat. No.
  • Chip CM5 (Biacore BR- 1006-68)
  • Buffer PBS (Biacore BR- 1006-72), pH 7.4, 35°C
  • CSF-1R-ECD Human CSF-1R Extracellular Domain
  • SEQ ID NO: 64 R&D-Systems 329-MR or subcloned pCMV-presS-HisAvitag-hCSF-lR- ECD
  • Antibodies against CSF-IR were captured via amine coupled capture molecules. Using the single cycle kinetics five increasing concentrations of human CSF-IR fragment D1-D3 (SEQ ID NO: 66) were injected. Human CSF-IR fragment D1-D3 was subcloned into pCMV-presS-HisAvitag expression vector.
  • Anti CSF-IR SC 2-4A5 (Santa Cruz Biotechnology, US; Sherr, C.J. et al, Blood 73 (1989) 1786-1793) which inhibits the ligand-receptor interaction, and Mab 3291 (R&D-Systems) were used as reference controls.
  • Capture molecules Anti mouse Fey antibodies (from goat, Jackson Immuno Reasearch JIR115-005-071) for antibodies according to the invention and the R&D-Systems control Mab 3291 and Anti rat Fey antibodies (from goat, Jackson Immuno Reasearch JIR112-005-071) for the reference control anti CSF-IR SC 2- 4A5.
  • Running buffer PBS (Biacore BR- 1006-72) Capturing of Mabs ⁇ CSF-1R> on flow cells 2 to 4: Flow 20 ⁇ / ⁇ , contact time
  • the antibodies Mab 2F11, Mab 2E10 and Mab 1G10 showed no binding to human
  • the reference control Mab R&D-Systems 3291 showed binding to the human CSF- 1R fragment D1-D3.
  • Serum CSF-1 levels provide a pharmacodynamic marker of CSF-1 R neutralizing activity of anti-human CSF-1R dimerization inhibitor hMab 2Fl l-e7.
  • One male and one female cynomolgus monkey per dosage group (1 and 10 mg/kg) were intravenously administered anti-CSFIR antibody hMab 2Fl l-e7.
  • Blood samples for analysis of CSF-1 levels were collected 1 week before treatment (pre-dose), 2, 24, 48, 72, 96, 168 hours post-dose and weekly for two additional weeks.
  • CSF-1 levels were determined using a commercially available ELISA kit (Quantikine® human M-CSF) according to the manufacturer's instructions (R&D Systems, UK ).
  • Monkey CSF-1 level were determined by comparison with CSF-1 standard curve samples provided in the kit.
  • Administration of hMab 2F1 l-e7 induced a dramatic increase in CSF-1 by ⁇ 1000- fold, which depending on the dose administered lasted for 48 hr (lmg/kg) or 15 days (lOmg/kg).
  • a dimerization inhibitor for CSF-1 R offers the advantage to not directly compete with the dramatically upregulated ligand for binding to the receptor in contrast to a ligand displacing antibody.
  • the human breast cancer cell line BT-20 expresses human CSF-1R but lacks CSF-1 expression (Sapi, E. et al Cancer Res 59 (1999) 5578-5585). Since the mouse derived CSF-1 fails to activate human CSF-1R on the tumor cells recombinant human CSF-1 (Biomol, Hamburg, Germany) was supplemented via osmotic minipumps (ALZET, Cupertino, CA) providing a continuous CSF-1 infusion rate of 2 ⁇ g/day (Martin, T.A., Carcinogenesis 24 (2003) 1317-1323). To directly compare the efficacy of an antibody interfering with dimerization of
  • CSF-1R with a ligand displacing CSF-1R antibody we tested the chimeric anti- CSF-1R Mab 2F11 (antibody interfering with dimerization of CSF-1 R) and 1.2.SM (ligand displacing CSF-1R antibody described in WO2009026303) in the BT-20 xenograft model.
  • SCID beige mice (Charles River, Sulzfeld, Germany) were subcutaneously coinjected with lx 107 cells BT-20 cells (ATCC HTB-19) and ⁇ of Matrigel . Treatment of animals started at day of randomization at a mean tumor volume of 100 mm3. Mice are treated once weekly i.p.

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Abstract

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

Description

ANTIBODIES BINDING PREFERENTIALLY HUMAN CSF1 R
EXTRACELLULAR DOMAIN 4 AND THEIR USE
The present invention relates to antibodies against human CSF-IR (anti-CSF-lR antibodies), methods for their production, pharmaceutical compositions containing said antibodies, and uses thereof.
Background of the Invention The human CSF-1 receptor (CSF-IR; colony stimulating factor 1 receptor; synonyms: M-CSF receptor; Macrophage colony-stimulating factor 1 receptor, Fms proto-oncogene, c-fms, SEQ ID NO: 62) is known since 1986 (Coussens, L., et al, Nature 320 (1986) 277-280). CSF-IR is a growth factor and encoded by the c-fms proto-oncogene (reviewed e.g. in Roth, P., and Stanley, E.R., Curr. Top. Microbiol. Immunol. 181 (1992) 141-67).
CSF-IR is the receptor for CSF-1 (colony stimulating factor 1, also called M-CSF, macrophage colony- stimulating factor) and mediates the biological effects of this cytokine (Sherr, C.J., et al., Cell 41 (1985) 665-676). The cloning of the colony stimulating factor- 1 receptor (CSF-IR) (also called c-fms) was described for the first time in Roussel, M.F., et al., Nature 325 (1987) 549-552. In that publication, it was shown that CSF-IR had transforming potential dependent on changes in the C- terminal tail of the protein including the loss of the inhibitory tyrosine 969 phosphorylation which binds Cbl and thereby regulates receptor down regulation (Lee, P.S., et al., Embo J. 18 (1999) 3616-3628). Recently a second ligand for CSF-IR termed interleukin-34 (IL-34) was identified (Lin, FL, et al, Science 320
(2008) 807-811).
The cytokine CSF-1 (colony stimulating factor 1, also called M-CSF, macrophage) is found extracellularly as a disulfide-linked homodimer (Stanley, E.R. et al, Journal of Cellular Biochemistry 21 (1983) 151-159; Stanley, E.R. et al, Stem Cells 12 Suppl. 1 (1995) 15-24).
The main biological effects of CSF-IR signaling are the differentiation, proliferation, migration, and survival of hematopoietic precursor cells to the macrophage lineage (including osteoclast). Activation of CSF-IR is mediated by its ligands, CSF-1 (M-CSF) and IL-34. Binding of CSF-1 (M-CSF) to CSF-IR induces the formation of homodimers and activation of the kinase by tyrosine phosphorylation (Li, W. et al, EMBO Journal.10 (1991) 277-288; Stanley, E.R., et al, Mol. Reprod. Dev. 46 (1997) 4-10).
The biologically active homodimer CSF-1 binds to the CSF-IR within the subdomains Dl to D3 of the extracellular domain of the CSF-1 receptor (CSF-1 R- ECD). The CSF-1R-ECD comprises five immunoglobulin-like subdomains
(designated Dl to D5). The subdomains D4 to D5 of the extracellular domain (CSF- lR-ECD)are not involved in the CSF-1 binding. (Wang, Z., et al Molecular and Cellular Biology 13 (1993) 5348-5359). The subdomain D4 is involved in dimerization (Yeung, Y-G., et al Molecular & Cellular Proteomics 2 (2003) 1143- 1155; Pixley, F. J., et al, Trends Cell Biol 14 (2004) 628-638).
Further signaling is mediated by the p85 subunit of PI3K and Grb2 connecting to the PI3K/AKT and Ras/MAPK pathways, respectively. These two important signaling pathways can regulate proliferation, survival and apoptosis. Other signaling molecules that bind the phosphorylated intracellular domain of CSF-IR include STAT1 , STAT3, PLCy, and Cbl (Bourette, R.P. and Rohrschneider, L.R.,
Growth Factors 17 (2000) 155-166).
CSF-I R signaling has a physiological role in immune responses, in bone remodeling and in the reproductive system. The knockout animals for either CSF-1 (Pollard, J.W., Mol. Reprod. Dev. 46 (1997) 54-61) or CSF-IR (Dai, X.M., et al., Blood 99 (2002) 1 11-120) have been shown to have osteopetrotic, hematopoietic, tissue macrophage, and reproductive phenotypes consistent with a role for CSF-IR in the respective cell types.
Sherr, C.J., et al., Blood 73 (1989) 1786-1793 relates to some antibodies against CSF-IR that inhibit the CSF-1 activity (see Sherr, C.J. et al., Blood 73 (1989) 1786-1793). Ashmun, R.A., et al., Blood 73 (1989) 827-837 relates to CSF-IR antibodies. Lenda, D., et al, Journal of Immunology 170 (2003) 3254-3262 relates to reduced macrophage recruitment, proliferation, and activation in CSF-1 -deficient mice results in decreased tubular apoptosis during renal inflammation. Kitaura, H., et al., Journal of Dental Research 87 (2008) 396-400 refers to an anti-CSF-1 antibody which inhibits orthodontic tooth movement. WO 2001/030381 mentions
CSF-1 activity inhibitors including antisense nucleotides and antibodies while disclosing only CSF-1 antisense nucleotides. WO 2004/045532 relates to metastases and bone loss prevention and treatment of metastatic cancer by a CSF-1 antagonist disclosing as antagonist anti-CSF-1 -antibodies only. WO 2005/046657 relates to the treatment of inflammatory bowel disease by anti-CSF-1 -antibodies. US 2002/0141994 relates to inhibitors of colony stimulating factors. WO 2006/096489 relates to the treatment of rheumatoid arthritis by anti-CSF-1 - antibodies. WO 2009/026303 and WO 2009/112245 relate to certain anti-CSF-lR antibodies binding to CSF-IR within the first three subdomains (Dl to D3) of the Extracellular Domain (CSF-1R-ECD).
Summary of the Invention
The invention comprises an antibody binding to human CSF-IR, characterized in that the antibody binds to human CSF-IR fragment delD4 (SEQ ID NO: 65) and to human CSF-IR Extracellular Domain (SEQ ID NO: 64) with a ratio of 1 :50 or lower.
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 a humanized version thereof.
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 a humanized version thereof. 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
d) the heavy chain variable domain is SEQ ID NO:47 and the light chain variable domain is SEQ ID NO:48, or
e) the heavy chain variable domain is SEQ ID NO:55 and the light chain variable domain is SEQ ID NO:56.
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 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 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 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 ID NO: 38, or f) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO:41, a CDR2 region of SEQ ID NO: 42, and a CDR1 region of SEQ ID NO:43, and the light chain variable domain comprises a 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 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 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.
Preferably the antibody according to the invention is of human IgGl subclass or of human IgG4 subclass. A further embodiment of the invention is a pharmaceutical composition comprising an antibody according to the invention. The invention further comprises the use an of an antibody according to the invention for the manufacture of a medicament for treatment of a CSF-IR mediated disease.
The invention further comprises the use an of an antibody according to the invention for the manufacture of a medicament for treatment of cancer.
The invention further comprises the use an of an antibody according to the invention for the manufacture of a medicament for treatment of bone loss.
The invention further comprises the use an of an antibody according to the invention for the manufacture of a medicament for treatment of metastasis. The invention further comprises the use an of an antibody according to the invention for the manufacture of a medicament for treatment of inflammatory diseases.
The invention further comprises an antibody according to the invention for treatment of a CSF-IR mediated disease. The invention further comprises an antibody according to the invention for treatment of cancer.
The invention further comprises an antibody according to the invention for treatment of bone loss.
The invention further comprises an antibody according to the invention for treatment of metastasis.
The invention further comprises an antibody according to the invention for treatment of inflammatory diseases.
A further 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 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 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 ID NO: 38, or f) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO:41, a CDR2 region of SEQ ID NO: 42, and a CDR1 region of SEQ ID NO:43, and the light chain variable domain comprises a 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 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 CDRl 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 CDRl 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 CDRl 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 CDRl 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 CDRl region of SEQ ID NO: 82.
A further 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:7 and the light chain variable domain is SEQ ID NO: 8,
b) the heavy chain variable domain is SEQ ID NO: 15 and the light chain variable domain is SEQ ID NO: 16;
c) the heavy chain variable domain is SEQ ID NO: 75 and the light chain variable domain is SEQ ID NO:76;
d) the heavy chain variable domain is SEQ ID NO: 83 and the light chain variable domain is SEQ ID NO: 84; or a humanized version thereof.
A further 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 d) the heavy chain variable domain is SEQ ID NO:47 and the light chain variable domain is SEQ ID NO:48, or
e) the heavy chain variable domain is SEQ ID NO:55 and the light chain variable domain is SEQ ID NO:56. The invention further provides expression vectors containing 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 an 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 the production of a recombinant human or humanized antibody according to the invention, characterized by expressing a nucleic acid according to the invention in a prokaryotic or eukaryotic host cell and recovering said antibody from said cell or the cell culture supernatant. The invention further comprises the antibody obtained by such a recombinant method.
Antibodies according to the invention show benefits for patients in need of a CSF-1R targeting therapy. The antibodies according to the invention show efficient antiproliferative activity against ligand-independent and ligand-dependant proliferation and are therefore especially useful in the treatment of cancer and metastasis.
The invention further provides a method for treating a patient suffering from cancer, comprising administering to a patient diagnosed as having such a disease (and therefore being in need of such a therapy) an effective amount of an antibody according to the invention. The antibody is administered preferably in a pharmaceutical composition.
A further embodiment of the invention is a method for the treatment of a patient suffering from cancer characterized by administering to the patient an antibody according to the invention. Surprisingly it has been found that, using a human CSF-1R fragment delD4 in which the D4 subdomain of human CSF-1R-ECD was deleted (SEQ ID NO:65), the new anti-CSF-lR antibodies according to the invention could be selected. These antibodies show valuable properties like excellent ligand-dependant cell growth inhibition and at the same time ligand independent cell growth inhibition of NIH 3T3 cell, retrovirally infected with either an expression vector for full-length wildtype CSF-IR (SEQ ID NO:62) or mutant CSF-IR L301 S Y969F (SEQ ID
NO:63) whereby mutant CSF-IR recombinant cells are able to form spheroids independent of the CSF-1 ligand. Furthermore the antibodies according to the invention inhibit (both) human and cynomolgous macrophage differentiation, as they inhibit survival of human and cynomolgous monocytes. Detailed Description of the Invention
The invention comprises an antibody binding to human CSF-IR, characterized in that the antibody binds to human CSF-IR fragment delD4 (comprising the extracellular subdomains Dl -D3 and D5) (SEQ ID NO: 65) and to human CSF-IR Extracellular Domain (CSF-1R-ECD) (comprising the extracellular subdomains Dl -D5) (SEQ ID NO: 64) with a ratio of 1 :50 or lower.
The invention further comprises an antibody according to the invention characterized in comprising as heavy chain variable domain CDR3 region 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 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 a humanized version thereof.
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 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:7 and the light chain variable domain is SEQ ID NO: 8, or a humanized version thereof.
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
d) the heavy chain variable domain is SEQ ID NO:47 and the light chain variable domain is SEQ ID NO :48, or
e) the heavy chain variable domain is SEQ ID NO:55 and the light chain variable domain is SEQ ID NO:56.
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
d) 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, or
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 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 ID 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: l , a CDR2 region of SEQ ID NO: 2, and a CDRl 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 CDRl 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 CDRl region of SEQ ID
NO: 1 1 , 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 CDRl 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 CDRl 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 CDRl 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 CDRl 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 CDRl region of SEQ ID NO: 30, or e) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 33, a CDR2 region of SEQ ID NO: 34, and a CDRl 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 CDRl region of SEQ ID NO: 38, or f) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO:41 , a CDR2 region of SEQ ID NO: 42, and a CDRl 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 CDRl 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 CDRl 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 CDRl region of SEQ ID NO: 54.
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: l , a CDR2 region of SEQ ID NO: 2, and a CDRl 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 CDRl 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 CDRl region of SEQ ID NO: 1 1 , 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 CDRl 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 CDRl 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 CDRl 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 CDRl 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 CDRl region of SEQ ID NO: 30, or e) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 33, a CDR2 region of SEQ ID NO: 34, and a CDRl 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 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, 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; 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 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. 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 SEQ ID NO:74, or b) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 77, a CDR2 region of SEQ ID NO: 78, and a CDR1 region of SEQ ID NO: 79, and the 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. 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: 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 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 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 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.
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: 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 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 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 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. 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: 20, a CDR2 region of SEQ ID NO:21, and a CDR1 region of SEQ ID NO:22. 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: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 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 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: 44, a CDR2 region of SEQ ID 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 human CSF-1R fragment delD4 (SEQ ID NO: 65) and to human CSF-IR-ECD (SEQ ID NO: 64) with a ratio of 1 :50 or lower, is further characterized in not binding to human CSF-1R fragment D1-D3 (SEQ ID NO: 66).
The term "antibody" encompasses the various forms of antibodies including but not being limited to whole antibodies, antibody fragments, human antibodies, humanized antibodies, chimeric antibodies, T cell epitope depleted antibodies, and further genetically engineered antibodies as long as the characteristic properties according to the invention are retained. "Antibody fragments" comprise a portion of a full length antibody, preferably the variable domain thereof, or at least the antigen binding site thereof. Examples of antibody fragments include diabodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. scFv antibodies are, e.g., described in Houston, J.S., Methods in Enzymol. 203 (1991) 46-88). In addition, antibody fragments comprise single chain polypeptides having the characteristics of a VH domain binding to CSF-1R, namely being able to assemble together with a VL domain, or of a VL domain binding to CSF-IR, namely being able to assemble together with a VH domain to a functional antigen binding site and thereby providing the property.
The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of a single amino acid composition.
The term "chimeric antibody" refers to a monoclonal antibody comprising a variable region, i.e., binding region, from mouse and at least a portion of a constant region derived from a different source or species, usually prepared by recombinant DNA techniques. Chimeric antibodies comprising a mouse variable region and a human constant region are especially preferred. Such rat/human chimeric antibodies 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" encompassed by 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 referred to as "class-switched antibodies." Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques now well known in the art. See, e.g., Morrison, S.L., et al. , Proc. Natl. Acad Sci. USA 81 (1984) 6851-6855; US 5,202,238 and US 5,204,244.
The term "humanized antibody" refers to antibodies in which the framework or "complementarity determining regions" (CDR) have been modified to comprise the CDR of an immunoglobulin of different specificity as compared to that of the parent immunoglobulin. In a preferred embodiment, a murine 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) 268-270. Optionally the framework region can be modified by further mutations. Also the CDRs can be modified by one or more mutations to generate antibodies according to the invention e.g. by mutagenesis based upon 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, or others. Particularly preferred CDRs correspond to those representing sequences recognizing the antigens noted above for chimeric antibodies. A "humanized version of an antibody according to the invention" (which is e.g. of mouse origin) refers to an antibody, which is based on the mouse antibody sequences in which the VH and VL are humanized by standard techniques (including CDR grafting and optionally subsequent mutagenesis of certain amino acids in the framework region and the CDRs ). Preferably such humanized version is chimerized with a human constant region (see e.g. Sequences SEQ ID NO:57-61).
Other forms of "humanized antibodies" encompassed by the present invention are those in which the constant region has been additionally modified or changed from that of the original antibody to generate the properties according to the invention, especially in regard to Clq binding and/or Fc receptor (FcR) binding.
In the following examples the terms "Mab" or "muMab" refer to murine monoclonal antibodies such as Mab 2F11 or Mab 2E10, whereas the term "hMab" refers to humanized monoclonal versions of such murine antibodies such as hMab 2F11-cl 1, hMab 2F1 l-d8, hMab 2F1 l-e7, hMab 2F1 l-fl2, etc.. The term "human antibody", as used herein, is intended to include antibodies having variable and constant regions derived from human germ line immunoglobulin sequences. Human antibodies are well-known in the state of the art (van Dijk, M.A., and van de Winkel, J.G., Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human antibodies can also be produced in transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire or a selection of human antibodies in the absence of endogenous immunoglobulin production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90 ( 1993) 255 1-2555; 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, H.R., and Winter, G.J. Mol. Biol. 227 (1992) 381-388; Marks, J.D., et al, J. Mol. Biol. 222 (1991) 581- 597). The techniques of Cole, et al., and Boerner, et al., are also available for the preparation of human monoclonal antibodies (Cole, S.P.C., et al, Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); and Boerner, P., et al, J. Immunol. 147 (1991) 86-95). As already mentioned for chimeric and humanized antibodies according to the invention the term "human antibody" as used herein also comprises such antibodies which are modified in the constant region to generate the properties according to the invention, especially in regard to C lq binding and/or FcR binding, e.g. by "class switching" i.e. change or mutation of Fc parts (e.g. from IgGl to IgG4 and/or IgGl/IgG4 mutation). The term "recombinant human antibody", as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from a host cell such as a NSO or CHO cell or from an animal (e.g. a mouse) that is transgenic for human immunoglobulin genes or antibodies expressed using a recombinant expression vector transfected into a host cell. Such recombinant human antibodies have variable and constant regions in a rearranged form. The recombinant human antibodies according to the invention have been subjected to in vivo somatic hypermutation. Thus, the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germ line VH and VL sequences, may not naturally exist within the human antibody germ line repertoire in vivo.
The antibodies according to the invention include, in addition, such antibodies having "conservative sequence modifications", nucleotide and amino acid sequence modifications which do not affect or alter the above-mentioned characteristics of the antibody according to the invention. Modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR- mediated mutagenesis. Conservative amino acid substitutions include ones 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 with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a human anti-CSF-lR antibody can be preferably replaced with another amino acid residue from the same side chain family. Amino acid substitutions can be performed by mutagenesis based upon 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.
The human CSF-1R (CSF-1 receptor; synonyms: M-CSF receptor; Macrophage colony-stimulating factor 1 receptor, Fms proto-oncogene, c-fms, SEQ ID NO: 22)) is known since 1986 (Coussens, L., et al, Nature 320 (1986) 277-280). CSF-1R is a growth factor and encoded by the c-fms proto-oncogene (reviewed e.g. in Roth, P. and Stanley, E.R., Curr. Top. Microbiol. Immunol. 181 (1992) 141-67).
CSF-1 R is the receptor for CSF-1 (macrophage colony stimulating factor, also called M-CSF) and IL-34 and mediates the biological effects of these cytokines
(Sherr, C.J., et al, Cell 41 (1985) 665-676 (Lin, H., et al, Science 320 (2008) 807- 81 1). The cloning of the colony stimulating factor-1 receptor (also called c-fms) was described for the first time in Roussel, M.F., et al, Nature 325 (1987) 549-552. In that publication, it was shown that CSF-1R had transforming potential dependent on changes in the C-terminal tail of the protein including the loss of the inhibitory tyrosine 969 phosphorylation which binds Cbl and thereby regulates receptor down regulation (Lee, P.S., et al, Embo J. 18 (1999) 3616-3628).
CSF-1 R is a single chain, transmembrane receptor tyrosine kinase (RTK) and a member of the family of immunoglobulin (Ig) motif containing RTKs characterized by 5 repeated Ig-like subdomains D1-D5 in the extracellular domain
(ECD) of the receptor (Wang, Z., et al Molecular and Cellular Biology 13 (1993) 5348-5359). The human CSF-1R Extracellular Domain (CSF-1R-ECD) (SEQ ID NO: 64) comprises all five extracellular Ig-like subdomains Dl -D5. The human CSF-1 R fragment delD4 (SEQ ID NO: 65) comprises the extracellular Ig-like subdomains D1-D3 and D5, but is missing the D4 subdomain. The human
CSF-1 R fragment D1-D3 (SEQ ID NO: 66) comprises the respective subdomains D1-D3. The sequences are listed without the signal peptide MGSGPGVLLL LLVATAWHGQ G (SEQ ID NO: 67).
The intracellular protein tyrosine kinase domain is interrupted by a unique insert domain that is also present in the other related RTK class III family members that include the platelet derived growth factor receptors (PDGFR), stem cell growth factor receptor (c-Kit) and fins-like cytokine receptor (FLT3). In spite of the structural homology among this family of growth factor receptors, they have distinct tissue-specific functions.
CSF-IR is mainly expressed on cells of the monocytic lineage and in the female reproductive tract and placenta. In addition expression of CSF-I R has been reported in Langerhans cells in skin, a subset of smooth muscle cells (Inaba, T., et al, J. Biol. Chem. 267 (1992) 5693-5699), B cells (Baker, A.H., et al, Oncogene 8 (1993) 371-378) and microglia (Sawada, M., et al., Brain Res. 509 (1990) 1 19- 124). Cells with mutant human CSF-IR ((SEQ ID NO: 23) are known to proliferate independently of ligand stimulation. As used herein, "binding to human CSF-IR" or "specifically binding to human
CSF-IR" refers to an antibody specifically binding to the human CSF-IR antigen with a binding affinity of KD-value of 1.0 x 10~8 mol/1 or lower at 35°C, in one embodiment of a KD-value of 1 .0 xlO 9 mol/1 or lower at 35°C. The binding affinity is determined with a standard binding assay at 35°C, such as surface plasmon resonance technique (BIAcore®, GE-Healthcare Uppsala, Sweden) A method for determining the KD-value of the binding affinity is described in Example 9. Thus an "antibody binding to human CSF-IR" as used herein refers to an antibody specifically binding to the human CSF-IR antigen with a binding affinity of KD 1.0 x 10~8 mol/1 or lower (preferably 1.0 x 10"8 mol/1 - 1.0 x 10~12 mol/1) at 35°C, preferably of a KD 1.0 xlO 9 mol/1 or lower at 35°C (preferably 1.0 x 10"9 mol/1 - 1.0 x 10"12 mol/1).
The "binding to human CSF-IR fragment delD4 (SEQ ID NO: 65) and to human CSF-IR Extracellular Domain (SEQ ID NO: 64)" as used herein is measured by a Surface Plasmon Resonance assay (Biacore assay) as described in Example 4. The human CSF-IR fragment delD4 (SEQ ID NO: 65) or human CSF-IR Extracellular
Domain (SEQ ID NO: 64), respectively, are captured to the surface (each to a separate surface) and the test antibodies were added (each in a separate measurement) and the respective binding signals (Response Units (RU)) were determined. Reference signals (blank surface) were subtracted. If signals of nonbinding test antibodies were slightly below 0 the values were set as 0. Then the ratio of the respective binding signals (binding signal (RU) to human CSF-IR fragment delD4 /binding signal (RU) to human CSF-IR Extracellular Domain (CSF-1R-ECD)) is determined. The antibodies according to the invention have a ratio of the binding signals (RU(delD4) / RU(CSF-IR-ECD) of 1 :50 or lower, preferably of 1 : 100 or lower (the lower included end is 0 ( e.g. if the RU is 0, then the ratio is 0:50 or 0: 100) ).
This means that such anti-CSF-lR antibodies according to the invention do not bind to the human CSF-1R fragment delD4 (like the anti-CCR5 antibody m<CCR5>Pz03.1C5 (deposited as DSM ACC 2683 on 18.08.2004 at DSMZ) and have binding signals for binding to the human CSF-1R fragment delD4 in the range of the anti-CCR5 antibody m<CCR5>Pz03.1C5, which are below 20 RU (Response Units), preferably below 10 RU in a Surface Plasmon Resonance (BIAcore) assay as shown in Example 4.
The term "binding to human CSF-1R fragment D1-D3" refers to a binding affinity determination by a Surface Plasmon Resonance assay (Biacore assay). The test antibody is captured to the surface and the human CSF-1R fragment D1-D3 (SEQ ID NO: 66) was added and the respective binding affinities were determined. The term "not binding to human CSF-1R fragment D 1-D3" denotes that in such an assay the detected signal was in the area of no more than 1.2 fold of background signal and therefore 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 selecting antibodies according to the invention comprising the following steps: a) determining the binding signal (Response Units (RU)) of anti-CSF-lR antibodies to human CSF-1R fragment delD4 (SEQ ID NO: 65) and to human CSF-1R Extracellular Domain (CSF-1R-ECD) (SEQ ID NO: 64) by a Surface Plasmon Resonance assay (Biacore assay).
b) selecting antibodies with ratio of the binding signals (human CSF-1R fragment delD4/ human CSF-1R Extracellular Domain (CSF-1R-ECD)) of 50: 1 or lower.
In one embodiment the determination is performed at 25 °C.
In one embodiment the screening method comprises as further steps the measuring of the binding of anti-CSF-lR antibodies to human CSF-1R fragment D1-D3 (SEQ ID NO: 66) (D1-D3) and the selecting of antibodies which show no binding to said fragment.
The term "epitope" denotes a protein determinant of human CSF-IR capable of specifically binding to an antibody. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually epitopes have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. Preferably an antibody according to the invention binds specifically to native and to denatured CSF-IR.
The "variable domain" (variable domain of a light chain (VL), variable domain of a heavy chain (VH)) as used herein denotes each of the pair of light and heavy chain domains which are involved directly in binding the antibody to the antigen. The variable light and heavy chain domains have the same general structure and each domain comprises four framework (FR) regions whose sequences are widely conserved, connected by three "hypervariable regions" (or complementary determining regions, CDRs). The framework regions adopt a β-sheet conformation and the CDRs may form loops connecting the β-sheet structure. The CDRs in each chain are held in their three-dimensional structure by the framework regions and form together with the CDRs from the other chain the antigen binding site. The antibody's heavy and light chain CDR3 regions play a particularly important role in the binding specificity/affinity of the antibodies according to the invention and therefore provide a further object of the invention.
The term "antigen-binding portion of an antibody" when used herein refer to the amino acid residues of an antibody which are responsible for antigen-binding. The antigen-binding portion of an antibody comprises amino acid residues from the "complementary determining regions" or "CDRs". "Framework" or "FR" regions are those variable domain regions other than the hypervariable region residues as herein defined. Therefore, the light and heavy chain variable domains of an antibody comprise from N- to C-terminus the domains FR1 , CDR1 , FR2, CDR2,
FR3, CDR3, and FR4. Especially, CDR3 of the heavy chain is the region which contributes most to antigen binding and defines the antibody's properties. CDR and FR regions are determined according to the standard definition of Kabat et al, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) and/or those residues from a "hypervariable loop".
The terms "nucleic acid" or "nucleic acid molecule", as used herein, are intended to include DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA.
The term "amino acid" as used within this application denotes the group of naturally occurring carboxy a-amino acids comprising alanine (three letter code: ala, one 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), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).
In one embodiment the antibodies according to the invention inhibit CSF-1 binding to CSF-1R. In one embodiment with an IC50 of 200 ng/ml or lower, in one embodiment with an IC50 of 50 ng/ml or lower. The IC50 of inhibition of CSF-1 binding to CSF-1R can be determined as shown in Example 2.
In one embodiment the antibodies according to the invention inhibit CSF-1 - induced CSF-1R phosphorylation (in NIH3T3-CSF-1R recombinant cells). In one embodiment with an IC50 of 800 ng/ml or lower, in one embodiment with an IC50 of 600 ng/ml or lower, in one embodiment with an IC50 of 250 ng/ml or lower. The IC50 of CSF-l-induced CSF-1R phosphorylation can be determined as shown in Example 3.
In one embodiment the antibodies according to the invention inhibit the growth of recombinant NIH3T3 cells expressing human CSF-1R (SEQ ID No: 62). In one embodiment with an IC50 of 10 μg/ml or lower, in one embodiment with an IC50 of 5 μg/ml or lower, in one embodiment with an IC50 of 2 μg/ml or lower. In one embodiment with an IC30 of 10 μg/ml or lower, in one embodiment with an IC30 of 5 μg/ml or lower, in one embodiment with an IC30 of 2 μg/ml or lower. The IC50 value, the IC30 value or the % growth inhibition is determined as shown in
Example 5. In one embodiment the antibodies according to the invention inhibit the growth of recombinant NIH3T3 cells expressing human mutant CSF-1R L301S Y969F (SEQ ID No : 63). In one embodiment with an IC50 of 15 μg/ml or lower, in one embodiment with an IC50 of 10 μg/ml or lower. In one embodiment with an IC30 of 10 μg/ml or lower, in one embodiment with an IC50 of 5 μg/ml ng/ml or lower; in one embodiment with an IC50 of 2 μg/ml or lower. The IC50 value, the IC30 value or the % growth inhibition is determined as shown in Example 5.
In one embodiment the antibodies according to the invention inhibit the growth of BeWo tumor cells (ATCC CCL-98) by 65 % or more (at an antibody concentration of 10μg/ml ; and as compared to the absence of antibody). The % growth inhibition is determined as shown in Example 8. E.g. Mab 2F11 shows a growth inhibition of BeWo tumor cells of 70 %.
In one embodiment the antibodies according to the invention inhibit (both) human and cynomolgous macrophage differentiation ( which is indicated by the inhibition of the survival of human and cynomolgous monocytes as shown in Examples 7 and
8). In one embodiment the antibodies according to the invention inhibit the survival of human monocytes with an IC50 of 0.15 μg/ml or lower, in on embodiment with an IC50 of 0.10 μg/ml or lower. The inhibition of the survival of human monocytes is determined as shown in Example 7. In one embodiment the antibodies according to the invention inhibit the survival of cynomolgous monocytes by 80 % or more, in one embodiment by 90 % or more (at an antibody concentration of 5 μg/ml ;and as compared to the absence of antibody). The inhibition of the survival of human monocytes is determined as shown in Example 8.
A further embodiment of the invention is a method for the production of an antibody against CSF-1R characterized in that the sequence of a nucleic acid encoding the heavy chain of a human IgGl class antibody binding to human CSF-1R according to the invention said modified nucleic acid and the nucleic acid encoding the light chain of said antibody are inserted into an expression vector, said vector is inserted in a eukaryotic host cell, 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. Therefore the antibody is preferably an isolated monoclonal antibody. Such recombinant methods are widely known in the state of the art and comprise protein expression in prokaryotic and eukaryotic cells with subsequent isolation of the antibody polypeptide and usually purification to a pharmaceutically acceptable purity. For the protein expression, nucleic acids encoding light and heavy chains or fragments thereof are inserted into expression vectors by standard methods. Expression is performed in appropriate prokaryotic or eukaryotic host cells like
CHO cells, NSO cells, SP2/0 cells, HEK293 cells, COS cells, yeast, or E.coli cells, and the antibody is recovered from the cells (supernatant or cells after lysis).
Recombinant production of antibodies is well-known in the state of the art and described, for example, in the review articles of Makrides, S.C., Protein Expr. Purif. 17 (1999) 183-202; Geisse, S., et al., Protein Expr. Purif. 8 (1996) 271-282;
Kaufman, R.J., Mol. Biotechnol. 16 (2000) 151-161; Werner, R.G., Drug Res. 48 (1998) 870-880.
The antibodies may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. Purification is performed in order to eliminate other cellular components or other contaminants, e.g. other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others well known in the art. See Ausubel, F., et al, ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987). Expression in NSO cells is described by, e.g., Barnes, L.M., et al, Cytotechnology
32 (2000) 109-123; and Barnes, L.M., et al, Biotech. Bioeng. 73 (2001) 261-270. Transient expression is described by, e.g., Durocher, Y., et al, Nucl. Acids. Res. 30 (2002) E9. Cloning of variable domains is described by Orlandi, R., et al, Proc. Natl. Acad. Sci. USA 86 (1989) 3833-3837; Carter, P., et al, Proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289; and Norderhaug, L., et al., J. Immunol. Methods 204
(1997) 77-87. A preferred transient expression system (HEK 293) is described by Schlaeger, E.-J., and Christensen, K., in Cytotechnology 30 (1999) 71-83 and by Schlaeger, E.-J., in J. Immunol. Methods 194 (1996) 191-199.
The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, enhancers and polyadenylation signals. Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that 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 DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
The monoclonal antibodies are suitably separated from the culture medium by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. DNA and RNA encoding the monoclonal antibodies are readily isolated and sequenced using conventional procedures. The hybridoma cells can serve as a source of such DNA and RNA. Once isolated, the DNA may be inserted into expression vectors, which are then transfected into host cells such as HEK 293 cells, CHO cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of recombinant monoclonal antibodies in the host cells.
As used herein, the expressions "cell", "cell line", and "cell culture" are used interchangeably and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. The "Fc part" of an antibody is not involved directly in binding of an antibody to an antigen, but exhibit various effector functions. A "Fc part of an antibody" is a term well known to the skilled artisan and defined on the basis of papain cleavage of antibodies. Depending on the amino acid sequence of the constant region of their heavy chains, antibodies or immunoglobulins are divided in the classes: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g. IgGl, IgG2, IgG3, and IgG4, IgAl, and IgA2. According to the heavy chain constant regions the different classes of immunoglobulins are called a, δ, ε, γ, and μ, respectively. 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, C l q binding and Fc receptor binding. Complement activation (CDC) is initiated by binding of complement factor Clq to the Fc part of most IgG antibody subclasses. While the influence of an antibody on the complement system is dependent on certain conditions, binding to C lq is caused by defined binding sites in the Fc part. Such binding sites are known in the state of the art and described e.g. by Boackle, R.J., et al., Nature 282 (1979) 742-743, Lukas, T.J., et al, J. Immunol. 127 (1981) 2555-2560, Brunhouse, R., and Cebra, J.J., Mol. Immunol. 16 (1979) 907-917, Burton, D.R., et al, Nature 288 (1980) 338-344, Thommesen, J.E., et al, Mol. Immunol. 37 (2000) 995-1004,
Idusogie, E.E., et al., J. Immunol.164 (2000) 4178-4184, Hezareh, M., et al., J. Virology 75 (2001) 12161-12168, Morgan, A., et al, Immunology 86 (1995) 319- 324, EP 0307434. Such binding sites are e.g. L234, L235, D270, N297, E318, K320, K322, P331 and P329 (numbering according to EU index of Kabat, E.A., see below). Antibodies of subclass IgGl, IgG2 and IgG3 usually show complement activation and Clq and C3 binding, whereas IgG4 do not activate the complement system and do not bind Clq and C3.
In one embodiment the antibody according to the invention comprises a Fc part derived from human origin and preferably all other parts of the human constant regions. As used herein the term "Fc part derived from human origin" denotes a Fc part which is either a Fc part of a human antibody of the subclass IgGl, IgG2, IgG3 or IgG4, preferably a Fc part from human IgGl subclass, a mutated Fc part from human IgGl subclass (preferably with a mutation on L234A + L235A), a Fc part from human IgG4 subclass or a mutated Fc part from human IgG4 subclass (preferably with a mutation on S228P). Mostly preferred are the human heavy chain constant regions of SEQ ID NO: 58 (human IgGl subclass), SEQ ID NO: 59 (human IgGl subclass with mutations L234A and L235A) , SEQ ID NO: 60 human IgG4 subclass), or SEQ ID NO: 61 (human IgG4 subclass with mutation S228P). Preferably the antibody according to the invention is of human IgGl subclass or of human IgG4 subclass. In one embodiment the antibody according to the invention is of human IgGl subclass. In one embodiment the antibody according to the invention is of human IgG4 subclass. In one embodiment the antibody according to the invention is characterized in that the constant chains are of human origin. Such constant chains are well known in the state of the art and e.g. described by Kabat, E.A., (see e.g. Johnson, G. and Wu, T.T., Nucleic Acids Res. 28 (2000) 214-218). For example, a useful human heavy chain constant region comprises an amino acid sequence of SEQ ID NO: 58. For example, a useful human light chain constant region comprises an amino acid sequence of a kappa-light chain constant region of SEQ ID 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 ID NO: 16;
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 is SEQ ID NO: 7 and the light chain variable domain is SEQ ID NO: 8,
b) the heavy chain variable domain is SEQ ID NO: 15 and the light chain variable domain is SEQ ID NO: 16;
c) the heavy chain variable domain is SEQ ID NO:75 and the light chain variable domain is SEQ ID NO:76;
d) the heavy chain variable domain is SEQ ID NO: 83 and the light chain variable domain is SEQ ID NO: 84; or a humanized version 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: 7 and the light chain variable domain is SEQ ID NO: 8, 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 is SEQ ID NO: 23 and the light chain variable domain is SEQ ID NO:24, or
b) the heavy chain variable domain is SEQ ID NO:31 and the light chain variable domain is SEQ ID NO:32, or
c) the heavy chain variable domain is SEQ ID NO: 39 and the light chain variable domain is SEQ ID NO:40, or
d) the heavy chain variable domain is SEQ ID NO:47 and the light chain variable domain is SEQ ID NO:48, or
e) the heavy chain variable domain is SEQ ID NO:55 and the light chain variable domain is SEQ ID NO:56. 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. 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 :23 and the light chain variable domain is SEQ ID NO:24, or
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:31 and the light chain variable domain is SEQ ID 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 ID NO :39 and the light chain variable domain is SEQ ID NO:40.
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 :47 and the light chain variable domain is SEQ ID 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 ID 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: l, 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 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 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 ID NO: 38, or f) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO:41, a CDR2 region of SEQ ID NO: 42, and a CDR1 region of SEQ ID NO:43, and the light chain variable domain comprises a CDR3 region of SEQ ID NO: 44, a CDR2 region of SEQ ID NO:45, and a CDR1 region of SEQ ID NO:46, g) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 49, a CDR2 region of SEQ ID NO: 50, and a CDR1 region of SEQ ID NO: 51, and the light chain variable domain comprises a CDR3 region of SEQ ID NO:52, a CDR2 region of SEQ ID NO: 53, and a CDR1 region of SEQ ID NO: 54; h) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO:69, a CDR2 region of SEQ ID NO: 70, and a CDR1 region of SEQ ID NO:71, and the 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 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 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 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 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 CDRl 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 CDRl 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 CDRl 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 CDRl 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 CDRl 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 ID NO: 17, a CDR2 region of SEQ ID NO : 18, and a CDRl 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 CDRl region of SEQ ID NO:22, or b) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO: 25, a CDR2 region of SEQ ID NO: 26, and a CDRl 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 CDRl 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 CDRl 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 CDRl 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 CDRl 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 CDRl region of SEQ ID 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 ID NO: 18, and a CDRl 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 CDRl region of SEQ ID 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 ID NO: 25, a CDR2 region of SEQ ID NO: 26, and a CDRl 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 CDRl region of SEQ ID 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 ID
NO: 33, a CDR2 region of SEQ ID NO: 34, and a CDRl 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 CDRl 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 ID NO: 42, and a CDRl 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.
The invention comprises a method for the treatment of 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 comprises the use of an antibody according to the invention for therapy.
One preferred embodiment of the invention are the CSF-1R antibodies of the present invention for use in the treatment of "CSF-1R mediated diseases" or the CSF-1R antibodies of the present invention for use for the manufacture of a medicament in the treatment of "CSF-1R mediated diseases", which can be described as follows:
There are 3 distinct mechanisms by which CSF-1R signaling is likely involved in tumor growth and metastasis. The first is that expression of CSF-ligand and receptor has been found in tumor cells originating in the female reproductive system (breast, ovarian, endometrium, cervical) (Scholl, S.M., et al., J. Natl. Cancer Inst. 86 (1994) 120-126; Kacinski, B.M., Mol. Reprod. Dev. 46 (1997) 71- 74; Ngan, H.Y., et al., Eur. J. Cancer 35 (1999) 1546-1550; Kirma, N., et al., Cancer Res 67 (2007) 1918-1926) and the expression has been associated with breast cancer xenograft growth as well as poor prognosis in breast cancer patients.
Two point mutations were seen in CSF-1R in about 10-20% of acute myelocytic leukemia, chronic myelocytic leukemia and myelodysplasia patients tested in one study, and one of the mutations was found to disrupt receptor turnover (Ridge, S .A., et al. , Proc. Natl. Acad. Sci USA 87 ( 1990) 1377-1380). However the incidence of the mutations could not be confirmed in later studies (Abu-Duhier,
F.M., et al, Br. J. Haematol. 120 (2003) 464-470). Mutations were also found in some cases of hepatocellular cancer (Yang, D.H., et al., Hepatobiliary Pancreat. Dis. Int. 3 (2004) 86-89) and idiopathic myelofibrosis (Abu-Duhier, F.M., et al., Br. J. Haematol. 120 (2003) 464-470). Recently, in the GDM-1 cell line derived from a patient with myelomonoblastic leukemia the Y571D mutation in CSF-1R was identified (Chase, A., et al, Leukemia 23 (2009) 358-364). Pigmented villonodular synovitis (PVNS) and Tenosynovial Giant cell tumors (TGCT) can occur as a result of a translocation that fuses the M-CSF gene to a collagen gene COL6A3 and results in overexpression of M-CSF (West, R.B., et al, Proc. Natl. Acad. Sci. USA 103 (2006) 690-695). A landscape effect is proposed to be responsible for the resulting tumor mass that consists of monocytic cells attracted by cells that express M-CSF. TGCTs are smaller tumors that can be relatively easily removed from fingers where they mostly occur. PVNS is more aggressive as it can recur in large joints and is not as easily controlled surgically.
The second mechanism is based on blocking signaling through M-CSF/CSF-IR at metastatic sites in bone which induces osteoclastogenesis, bone resorption and osteolytic bone lesions. Breast, multiple myeloma and lung cancers are examples of cancers that have been found to metastasize to the bone and cause osteolytic bone disease resulting in skeletal complications. M-CSF released by tumor cells and stroma induces the differentiation of hematopoietic myeloid monocyte progenitors to mature osteoclasts in collaboration with the receptor activator of nuclear factor kappa-B ligand-RANKL. During this process, M-CSF acts as a permissive factor by giving the survival signal to osteoclasts (Tanaka, S., et al., J. Clin. Invest. 91 (1993) 257-263). Inhibition of CSF-1R activity during osteoclast differentiation and maturation with a anti-CSF-l R antibody is likely to prevent unbalanced activity of osteoclasts that cause osteolytic disease and the associated skeletal related events in metastatic disease. Whereas breast, lung cancer and multiple myeloma typically result in osteolytic lesions, metastasis to the bone in prostate cancer initially has an osteoblastic appearance in which increased bone forming activity results in 'woven bone' which is different from typical lamellar structure of normal bone. During disease progression bone lesions display a significant osteolytic component as well as high serum levels of bone resorption and suggests that anti-resorptive therapy may be useful. Bisphosphonates have been shown to inhibit the formation of osteolytic lesions and reduced the number of skeletal- related events only in men with hormone-refractory metastatic prostate cancer but at this point their effect on osteoblastic lesions is controversial and bisphosphonates have not been beneficial in preventing bone metastasis or hormone responsive prostate cancer to date . The effect o f anti-resorptive agents in mixed osteolytic/osteoblastic prostate cancer is still being studied in the clinic (Choueiri, M.B., et al., Cancer Metastasis Rev. 25 (2006) 601-609; Vessella, R.L. and Corey, E., Clin. Cancer Res. 12 (20 Pt 2) (2006) 6285s-6290s). The third mechanism is based on the recent observation that tumor associated macrophages (TAM) found in solid tumors of the breast, prostate, ovarian and cervical cancers correlated with poor prognosis (Bingle, L., et al., J. Pathol. 196 (2002) 254-265; Pollard, J.W., Nat. Rev. Cancer 4 (2004) 71-78). Macrophages are recruited to the tumor by M-CSF and other chemokines. The macrophages can then contribute to tumor progression through the secretion of angiogenic factors, proteases and other growth factors and cytokines and may be blocked by inhibition of CSF-1R signaling. Recently it was shown by Zins et al (Zins, K., et al., Cancer Res. 67 (2007) 1038-1045) that expression of siRNA of Tumor necrosis factor alpha (TNF alpha), M-CSF or the combination of both would reduce tumor growth in a mouse xenograft model between 34% and 50% after intratumoral injection of the respective siRNA. SiRNA targeting the TNF alpha secreted by the human SW620 cells reduced mouse M-CSF levels and led to reduction of macrophages in the tumor. In addition treatment of MCF7 tumor xenografts with an antigen binding fragment directed against M-CSF did result in 40% tumor growth inhibition, reversed the resistance to chemotherapeutics and improved survival of the mice when given in combination with chemotherapeutics (Paulus, P., et al., Cancer Res. 66 (2006) 4349-4356).
TAMs are only one example of an emerging link between chronic inflammation and cancer. There is additional evidence for a link between inflammation and cancer as many chronic diseases are associated with an increased risk of cancer, cancers arise at sites of chronic inflammation, chemical mediators of inflammation are found in many cancers; deletion of the cellular or chemical mediators of inflammation inhibits development of experimental cancers and long-term use of anti-inflammatory agents reduce the risk of some cancers. A link to cancer exists for a number of inflammatory conditions among- those H.pylori induced gastritis for gastric cancer, Schistosomiasis for bladder cancer, HHVX for Kaposi's sarcoma, endometriosis for ovarian cancer and prostatitis for prostate cancer (Balkwill, F., et al., Cancer Cell 7 (2005) 21 1-217). Macrophages are key cells in chronic inflammation and respond differentially to their microenvironment. There are two types of macrophages that are considered extremes in a continuum of functional states: M l macrophages are involved in Type 1 reactions. These reactions involve the activation by microbial products and consequent killing of pathogenic microorganisms that result in reactive oxygen intermediates. On the other end of the extreme are M2 macrophages involved in Type 2 reactions that promote cell proliferation, tune inflammation and adaptive immunity and promote tissue remodeling, angiogenesis and repair (Mantovani, A., et al, Trends Immunol. 25 (2004) 677-686). Chronic inflammation resulting in established neoplasia is usually associated with M2 macrophages. A pivotal cytokine that mediates inflammatory reactions is TNF alpha that true to its name can stimulate anti-tumor immunity and hemorrhagic necrosis at high doses but has also recently been found to be expressed by tumor cells and acting as a tumor promoter (Zins, K., et al., Cancer Res. 67 (2007) 1038-1045; Balkwill, F., Cancer Metastasis Rev. 25 (2006) 409-416). The specific role of macrophages with respect to the tumor still needs to be better understood including the potential spatial and temporal dependence on their function and the relevance to specific tumor types.
Thus one embodiment of the invention are the CSF-1 R antibodies of the present invention for use in the treatment of cancer. The term "cancer" as used herein may be, for example, lung cancer, non small cell lung (NSCL) cancer, bronchioloalviolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colon cancer, breast cancer, uterine cancer, 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, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, mesothelioma, hepatocellular cancer, biliary cancer, neoplasms of the central nervous system (CNS), spinal axis tumors, brain stem glioma, glioblastoma multiforme, astrocytomas, schwanomas, ependymonas, medulloblastomas, meningiomas, squamous cell carcinomas, pituitary adenoma, lymphoma, lymphocytic leukemia, including refractory versions of any of the above cancers, or a combination of one or more of the above cancers. Preferably such cancer is a breast cancer, ovarian cancer , cervical cancer, lung cancer or prostate cancer. Preferably such cancers are further characterized by CSF-1 or CSF-1 R expression or overexpression. One further embodiment the invention are the CSF-1 R antibodies of the present invention for use in the simultaneous treatment of primary tumors and new metastases. Thus another embodiment of the invention are the CSF-IR antibodies of the present invention for use in the treatment of periodontitis, histiocytosis X, osteoporosis, Paget's disease of bone (PDB), bone loss due to cancer therapy, periprosthetic osteolysis, glucocorticoid-induced osteoporosis, rheumatoid arthritis, psiratic arthritis, osteoarthritis, inflammatory arthridities, and inflammation.
Rabello, D., et al., Biochem. Biophys. Res. Commun. 347 (2006) 791-796 has demonstrated that SNPs in the CSF1 gene exhibited a positive association with aggressive periodontitis: an inflammatory disease of the periodontal tissues that causes tooth loss due to resorption of the alveolar bone. Histiocytosis X (also called Langerhans cell histiocytosis, LCH) is a proliferative disease of Langerhans dendritic cells that appear to differentiate into osteoclasts in bone and extra osseous LCH lesions. Langerhans cells are derived from circulating monocytes. Increased levels of M-CSF that have been measured in sera and lesions where found to correlate with disease severity (da Costa, C.E., et al, J. Exp. Med. 201 (2005) 687-693). The disease occurs primarily in a pediatric patient population and has to be treated with chemotherapy when the disease becomes systemic or is recurrent.
The pathophysiology of osteoporosis is mediated by loss of bone forming osteoblasts and increased osteoclast dependent bone resorption. Supporting data has been described by Cenci et al showing that an anti-M-CSF antibody injection preserves bone density and inhibits bone resorption in ovariectomized mice (Cenci, S., et al., J. Clin. Invest. 105 (2000) 1279-1287). Recently a potential link between postmenopausal bone loss due to estrogen deficiency was identified and found that the presence of TNF alpha producing T-cell affected bone metabolism (Roggia, C, et al., Minerva Med. 95 (2004) 125-132). A possible mechanism could be the induction of M-CSF by TNF alpha in vivo. An important role for M-CSF in TNF- alpha-induced osteoclastogenesis was confirmed by the effect of an antibody directed against M-CSF that blocked the TNF alpha induced osteolysis in mice and thereby making inhibitors of CSF-IR signaling potential targets 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, pathological fractures and deafness. Mutations in four genes have been identified that regulate normal osteoclast function and predispose individuals to PDB and related disorders: insertion mutations in TNFRSF11 A, which encodes receptor activator of nuclear factor (NF) kappaB (RANK)-a critical regulator of osteoclast function, inactivating mutations of TNFRSF1 IB which encodes osteoprotegerin (a decoy receptor for
RANK ligand), mutations of the sequestosome 1 gene (SQSTM1), which encodes an important scaffold protein in the NFkappaB pathway and mutations in the valosin-containing protein (VCP) gene. This gene encodes VCP, which has a role in targeting the inhibitor of NFkappaB for degradation by the proteasome (Daroszewska, A. and Ralston, S.H., Nat. Clin. Pract. Rheumatol. 2 (2006) 270-
277). Targeted CSF-1R inhibitors provide an opportunity to block the deregulation of the RANKL signaling indirectly and add an additional treatment option to the currently used bisphosphonates.
Cancer therapy induced bone loss especially in breast and prostate cancer patients is an additional indication where a targeted CSF-1R inhibitor could prevent bone loss (Lester, J.E., et al., Br. J. Cancer 94 (2006) 30-35). With the improved prognosis for early breast cancer the long-term consequences of the adjuvant therapies become more important as some of the therapies including chemotherapy, irradiation, aromatase inhibitors and ovary ablation affect bone metabolism by decreasing the bone mineral density, resulting in increased risk for osteoporosis and associated fractures (Lester, J.E., et al., Br. J. Cancer 94 (2006) 30-35). The equivalent to adjuvant aromatase inhibitor therapy in breast cancer is androgen ablation therapy in prostate cancer which leads to loss of bone mineral density and significantly increases the risk of osteoporosis-related fractures (Stoch, S.A., et al, J. Clin. Endocrinol. Metab. 86 (2001) 2787-2791).
Targeted inhibition of CSF-1R signaling is likely to be beneficial in other indications as well when targeted cell types include osteoclasts and macrophages e.g. treatment of specific complications in response to joint replacement as a consequence of rheumatoid arthritis. Implant failure due to periprosthetic bone loss and consequent loosing of prostheses is a major complication of joint replacement and requires repeated surgery with high socioeconomic burdens for the individual patient and the health-care system. To date, there is no approved drug therapy to prevent or inhibit periprosthetic osteolysis (Drees, P., et al., Nat. Clin. Pract. Rheumatol. 3 (2007) 165-171). Glucocorticoid- induced osteoporosis (GIOP) is another indication in which a CSF- 1R inhibitor could prevent bone loss after longterm glucocorticocosteroid use that is given as a result of various conditions among those chronic obstructive pulmonary disease, asthma and rheumatoid arthritis (Guzman-Clark, J.R., et al., Arthritis Rheum. 57 (2007) 140-146; Feldstein, A.C., et al., Osteoporos. Int. 16
(2005) 2168-2174).
Rheumatoid arthritis, psioratic arthritis and inflammatory arthridities are in itself potential indications for CSF-1R signaling inhibitors in that they consist of a macrophage component and to a varying degree bone destruction (Ritchlin, C.T., et al., J. Clin. Invest. I l l (2003) 821-831). Osteoarthritis and rheumatoid arthritis are inflammatory autoimmune disease caused by the accumulation of macrophages in the connective tissue and infiltration of macrophages into the synovial fiuid, which is at least partially mediated by M-CSF. Campbell, I., K., et al., J. Leukoc. Biol. 68 (2000) 144-150, demonstrated that M-CSF is produced by human-joint tissue cells (chondrocytes, synovial fibroblasts) in vitro and is found in synovial fluid of patients with rheumatoid arthritis, suggesting that it contributes to the synovial tissue proliferation and macrophage infiltration which is associated with the pathogenesis of the disease. Inhibition of CSF-1R signaling is likely to control the number of macrophages in the joint and alleviate the pain from the associated bone destruction. In order to minimize adverse affects and to further understand the impact of the CSF-1R signaling in these indications, one method is to specifically inhibit CSF-1R without targeting a myriad other kinases, such as Raf kinase.
Recent literature reports correlate increased circulating M-CSF with poor prognosis and atherosclerotic progression in chronic coronary artery disease (Saitoh, T., et al., J. Am. Coll. Cardiol. 35 (2000) 655-665; Ikonomidis, I., et al, Eur. Heart. J. 26
(2005) p. 1618-1624); M-CSF influences the atherosclerotic process by aiding the formation of foam cells (macrophages with ingested oxidized LDL) that express CSF-1R and represent the initial plaque (Murayama, T., et al, Circulation 99 (1999) 1740-1746). Expression and signaling of M-CSF and CSF-1R is found in activated microglia.
Microglia, which are resident macrophages of the central nervous system, can be activated by various insults, including infection and traumatic injury. M-CSF is considered a key regulator of inflammatory responses in the brain and M-CSF levels increase in HIV-1, encephalitis, Alzheimer's disease (AD) and brain tumors. Microgliosis as a consequence of autocrine signaling by M-CSF/CSF-1R results in induction of inflammatory cytokines and nitric oxides being released as demonstrated by e.g. using an experimental neuronal damage model (Hao, A.J., et al, Neuroscience 112 (2002) 889-900; Murphy, G.M., Jr., et al, J. Biol. Chem. 273
(1998) 20967-20971). Microglia that have increased expression of CSF-IR are found to surround plaques in AD and in the amyloid precursor protein V717F transgenic mouse model of AD (Murphy, G.M., Jr., et al., Am. J. Pathol. 157 (2000) 895-904). On the other hand op/op mice with fewer microglia in the brain resulted in fibrilar deposition of A-beta and neuronal loss compared to normal control suggesting that microglia do have a neuroprotective function in the development of AD lacking in the op/op mice (Kaku, M., et al., Brain Res. Brain Res. Protoc. 12 (2003) 104-108).
Expression and signaling of M-CSF and CSF-IR is associated with inflammatory bowel disease (IBD) (WO 2005/046657). The term "inflammatory bowel disease" refers to serious, chronic disorders of the intestinal tract characterized by chronic inflammation at various sites in the gastrointestinal tract, and specifically includes ulcerative colitis (UC) and Crohn's disease.
The invention comprises an antibody binding to human CSF-IR being characterized by the above mentioned epitope binding properties or alternatively by the above mentioned amino acid sequences and amino acid sequence fragments for the treatment of cancer.
The invention comprises an antibody binding to human CSF-IR being characterized by the above mentioned epitope binding properties or alternatively by the above mentioned amino acid sequences and amino acid sequence fragments for the treatment of bone loss.
The invention comprises an antibody binding to human CSF-IR being characterized by the above mentioned epitope binding properties or alternatively by the above mentioned amino acid sequences and amino acid sequence fragments for the prevention or treatment of metastasis.
The invention comprises an antibody binding to human CSF-IR being characterized by the above mentioned epitope binding properties or alternatively by the above mentioned amino acid sequences and amino acid sequence fragments for treatment of inflammatory diseases.
The invention comprises the use of an antibody characterized in comprising the antibody binding to human CSF-IR being characterized by the above mentioned epitope binding properties or alternatively by the above mentioned amino acid sequences and amino acid sequence fragments for the treatment of cancer or alternatively for the manufacture of a medicament for the treatment of cancer.
The invention comprises the use of an antibody characterized in comprising the antibody binding to human CSF-IR being characterized by the above mentioned epitope binding properties or alternatively by the above mentioned amino acid sequences and amino acid sequence fragments for the treatment of bone loss or alternatively for the manufacture of a medicament for the treatment of bone loss.
The invention comprises the use of an antibody characterized in comprising the antibody binding to human CSF-IR being characterized by the above mentioned epitope binding properties or alternatively by the above mentioned amino acid sequences and amino acid sequence fragments for the prevention or treatment of metastasis or alternatively for the manufacture of a medicament for the prevention or treatment of metastasis.
The invention comprises the use of an antibody characterized in comprising the antibody binding to human CSF-IR being characterized by the above mentioned epitope binding properties or alternatively by the above mentioned amino acid sequences and amino acid sequence fragments for treatment of inflammatory diseases or alternatively for the manufacture of a medicament for the treatment of inflammatory diseases.
A further embodiment of the invention is a method for the production of an antibody against CSF-IR characterized in that the sequence of a nucleic acid encoding the heavy chain of a human IgG l class antibody binding to human CSF-IR according to the invention said modified nucleic acid and the nucleic acid encoding the light chain of said antibody are inserted into an expression vector, said vector is inserted in a eukaryotic host cell, 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. Such methods are widely known in the state of the art and comprise protein expression in prokaryotic and eukaryotic cells with subsequent isolation of the antibody polypeptide and usually purification to a pharmaceutically acceptable purity. For the protein expression nucleic acids encoding light and heavy chains or fragments thereof are inserted into expression vectors by standard methods. Expression is performed in appropriate prokaryotic or eukaryotic host cells, such as CHO cells, NS0 cells, SP2/0 cells, HEK293 cells, COS cells, yeast, or E. coli cells, and the antibody is recovered from the cells (from the supernatant or after cells lysis).
Recombinant production of antibodies is well-known in the state of the art and described, for example, in the review articles of Makrides, S.C., Protein Expr. Purif. 17 (1999) 183-202; Geisse, S., et al., Protein Expr. Purif. 8 (1996) 271-282;
Kaufman, R.J., Mol. Biotechnol. 16 (2000) 151-161 ; Werner, R.G., Drug Res. 48 (1998) 870-880.
The antibodies may be present in whole cells, in a cell lysate, or in a partially purified, or substantially pure form. Purification is performed in order to eliminate other cellular components or other contaminants, e.g. other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others well known in the art. See Ausubel, F., et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987). Expression in NS0 cells is described by, e.g., Barnes, L.M., et al, Cytotechnology
32 (2000) 109-123; Barnes, L.M., et al., Biotech. Bioeng. 73 (2001) 261-270. Transient expression is described by, e.g., Durocher, Y., et al, Nucl. Acids. Res. 30 (2002) E9. Cloning of variable domains is described by Orlandi, R., et al., Proc. Natl. Acad. Sci. USA 86 (1989) 3833-3837; Carter, P., et al, Proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289; Norderhaug, L., et al., J. Immunol. Methods 204
(1997) 77-87. A preferred transient expression system (HEK 293) is described by Schlaeger, E.-J. and Christensen, K., in Cytotechnology 30 (1999) 71-83, and by Schlaeger, E.-J., in J. Immunol. Methods 194 (1996) 191-199. Nucleic acid molecules encoding amino acid sequence variants of anti-CSF-lR antibody are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of humanized anti-CSF-lR antibody.
The heavy and light chain variable domains according to the invention are combined with sequences of promoter, translation initiation, constant region, 3' untranslated region, polyadenylation, and transcription termination to form expression vector constructs. The heavy and light chain expression constructs can be combined into a single vector, co-transfected, serially 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, e.g. a pharmaceutical composition, containing one or a combination of monoclonal antibodies, or the antigen-binding portion thereof, of the present invention, formulated together with a pharmaceutically acceptable carrier.
As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption/resorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for injection or infusion.
A composition of the present invention can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results.
Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the preparation of sterile injectable solutions or dispersion. 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, an isotonic buffered saline solution. Regardless of the route of administration selected, the compounds of 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 conventional methods known to those of skill in the art.
Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient (effective amount). The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
The invention comprises the use of the antibodies according to the invention for the treatment of a patient suffering from cancer, especially from colon, lung or pancreas cancer.
The invention comprises also a method for the treatment of a patient suffering from such disease.
The invention further provides a method for the manufacture of a pharmaceutical composition comprising an effective amount of an antibody according to the invention together with a pharmaceutically acceptable carrier and the use of the antibody according to the invention for such a method.
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 together 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 effective amount for the manufacture of a pharmaceutical agent, preferably together with a pharmaceutically acceptable carrier, for the treatment of a patient suffering from cancer. The following examples, sequence listing and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.
Description of the Sequences
SEQ ID NO: 1 heavy chain CDR3, Mab 2F11
SEQ ID NO: 2 heavy chain CDR2, Mab 2F11
SEQ ID NO: 3 heavy chain CDR1, Mab 2F11
SEQ ID NO: 4 light chain CDR3, Mab 2F11
SEQ ID NO: 5 light chain CDR2, Mab 2F11
SEQ ID NO: 6 light chain CDR1, Mab 2F11
SEQ ID NO: 7 heavy chain variable domain, Mab 2F11
SEQ ID NO: 8 light chain variable domain, Mab 2F11
SEQ ID NO: 9 heavy chain CDR3, Mab 2E10
SEQ ID NO: 10 heavy chain CDR2, Mab 2E10
SEQ ID NO: 11 heavy chain CDR1, Mab 2E10
SEQ ID NO: 12 light chain CDR3, Mab 2E10
SEQ ID NO: 13 light chain CDR2, Mab 2E10
SEQ ID NO: 14 light chain CDR1, Mab 2E10
SEQ ID NO: 15 heavy chain variable domain, Mab 2E10
SEQ ID NO: 16 light chain variable domain, Mab 2E10
SEQ ID NO: 17 heavy chain CDR3, hMab 2F11-cl 1
SEQ ID NO: 18 heavy chain CDR2, hMab 2F11-cl 1
SEQ ID NO: 19 heavy chain CDR1, hMab 2F11-cl 1
SEQ ID NO: 20 light chain CDR3, hMab 2F11-cl 1
SEQ ID NO: 21 light chain CDR2, hMab 2F11-cl 1
SEQ ID NO: 22 light chain CDR1, hMab 2F11-cl 1
SEQ ID NO: 23 heavy chain variable domain, hMab 2F11-cl 1
SEQ ID NO: 24 light chain variable domain, hMab 2F11-cl 1 SEQ ID NO: 25 heavy chain CDR3, hMab 2F1 l-d8
SEQ ID NO: 26 heavy chain CDR2, hMab 2F1 l-d8
SEQ ID NO: 27 heavy chain CDR1, hMab 2F1 l-d8
SEQ ID NO: 28 light chain CDR3, hMab 2F1 l-d8
SEQ ID NO: 29 light chain CDR2, hMab 2F1 l-d8
SEQ ID NO: 30 light chain CDR1, hMab 2F1 l-d8
SEQ ID NO: 31 heavy chain variable domain, hMab 2F1 l-d8
SEQ ID NO: 32 light chain variable domain, hMab 2F1 l-d8
SEQ ID NO: 33 heavy chain CDR3, hMab 2F1 l-e7
SEQ ID NO: 34 heavy chain CDR2, hMab 2F1 l-e7
SEQ ID NO: 35 heavy chain CDR1, hMab 2F1 l-e7
SEQ ID NO: 36 light chain CDR3, hMab 2F1 l-e7
SEQ ID NO: 37 light chain CDR2, hMab 2F1 l-e7
SEQ ID NO: 38 light chain CDR1, hMab 2F1 l-e7
SEQ ID NO: 39 heavy chain variable domain, hMab 2F1 l-e7
SEQ ID NO: 40 light chain variable domain, hMab 2F1 l-e7
SEQ ID NO: 41 heavy chain CDR3, hMab 2F1 l-fl2
SEQ ID NO: 42 heavy chain CDR2, hMab 2F1 l-fl2
SEQ ID NO: 43 heavy chain CDR1, hMab 2F1 l-fl2
SEQ ID NO: 44 light chain CDR3, hMab 2F1 l-fl2
SEQ ID NO: 45 light chain CDR2, hMab 2F11-Ω2
SEQ ID NO: 46 light chain CDR1, hMab 2F11-Ω2
SEQ ID NO: 47 heavy chain variable domain, hMab 2F1 l-fl2
SEQ ID NO: 48 light chain variable domain, hMab 2F1 l-fl2
SEQ ID NO: 49 heavy chain CDR3, hMab 2F11-gl
SEQ ID NO: 50 heavy chain CDR2, hMab 2F11-gl
SEQ ID NO: 51 heavy chain CDR1, hMab 2F11-gl
SEQ ID NO: 52 light chain CDR3, hMab 2F11-gl
SEQ ID NO: 53 light chain CDR2, hMab 2F11-gl
SEQ ID NO: 54 light chain CDR1, hMab 2F11-gl
SEQ ID NO: 55 heavy chain variable domain, hMab 2F11-gl
SEQ ID NO: 56 light chain variable domain, hMab 2F11-gl
SEQ ID NO: 57 human kappa light chain constant region
SEQ ID NO: 58 human heavy chain constant region derived from IgGl SEQ ID NO: 59 human heavy chain constant region mutated on L234A and L235A
SEQ ID NO: 60 human heavy chain constant region deriv
SEQ ID NO: 61 human heavy chain constant region
mutated on S228P
SEQ ID NO: 62 human wildtype CSF-1R (wt CSF-1R)
SEQ ID NO: 63 human mutant CSF-1R L301S Y969F
SEQ ID NO: 64 human CSF-1R Extracellular Domain
SEQ ID NO: 65 human CSF-1R fragment delD4
SEQ ID NO: 66 human CSF-1R fragment D1-D3
SEQ ID NO: 67 signal peptide
SEQ ID NO: 68 Primer
SEQ ID NO: 69 heavy chain CDR3, Mab 1G10
SEQ ID NO: 70 heavy chain CDR2, Mab 1G10
SEQ ID NO: 71 heavy chain CDR1, Mab 1G10
SEQ ID NO: 72 light chain CDR3, Mab 1G10
SEQ ID NO: 73 light chain CDR2, Mab 1G10
SEQ ID NO: 74 light chain CDR1, Mab 1G10
SEQ ID NO: 75 heavy chain variable domain, Mab 1G10
SEQ ID NO: 76 light chain variable domain, Mab 1G10
SEQ ID NO: 77 heavy chain CDR3, Mab 2H7
SEQ ID NO: 78 heavy chain CDR2, Mab 2H7
SEQ ID NO: 79 heavy chain CDR1, Mab 2H7
SEQ ID NO: 80 light chain CDR3, Mab 2H7
SEQ ID NO: 81 light chain CDR2, Mab 2H7
SEQ ID NO: 82 light chain CDR1, Mab 2H7
SEQ ID NO: 83 heavy chain variable domain, Mab 2H7
SEQ ID NO: 84 light chain variable domain, Mab 2H7
The following examples, sequence listing and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention. Description of the Figures
Figure 1 Growth inhibition of BeWo tumor cells in 3D culture under
treatment with different anti-CSF-lR monoclonal antibodies at a concentration of
Figure imgf000054_0001
X axis: viability normalized mean relative light units (RLU) corresponding to the ATP-content of the cells (CellTiterGlo assay).
Y axis: tested probes: Minimal Medium (0.5% FBS), mouse IgGl (mlgGl , ΙΟμ ηύ), mouse IgG2a (mIgG2a l(^g/ml), CSF-1 only, Mab 2F1 1 , Mab 2E10, Mab2H7, MablGlO and SC 2-4A5.
Highest inhibition of CSF-1 induced growth was observed with the anti-CSF-lR antibodies according to the invention.
Figure 2a Biacore sensogram of binding of different anti-CSF-lR antibodies to immobilized human CSF-1 R fragment delD4 (comprising the extracellular subdomains Dl -D3 and D5) (SEQ
ID NO: 65) (y-axis: binding signal in Response Units (RU), baseline = 0 RU, x-axis: time in seconds (s)): While the antibodies Mab 3291 and sc 2-4 A5 clearly show binding to this delD4 fragment, the antibodies according to the invention e.g. Mab 2F1 1 , and Mab 2E10, did not bind to the CSF-1 R fragment delD4. The control anti-CCR5 antibody m<CCR5>Pz03.1C5 did also not bind to the CSF-IR fragment delD4.
Figure 2b Biacore sensogram of binding of different anti-CSF-lR
antibodies to immobilized human CSF-IR Extracellular Domain (CSF-1 R-ECD) (comprising the extracellular subdomains Dl -
D5) (SEQ ID NO: 64) (y-axis: binding signal in Response Units (RU), baseline = 0 RU, x-axis: time in seconds (s)): :
All anti-CSF-lR antibodies show binding to CSF-1R-ECD. The control anti-CCR5 antibody m<CCR5>Pz03.1C5 did not bind to the CSF-1R-ECD.
Figure 2c Biacore sensogram of binding of different anti-CSF-lR antibodies to immobilized human CSF-IR fragment delD4 (comprising the extracellular subdomains Dl -D3 and D5) (SEQ ID NO: 65) (y-axis: binding signal in Response Units (RU), baseline = 0 RU, x-axis: time in seconds (s)): Mab 1G10, Mab 2H7 and humanized hMab 2Fl l-e7 did not bind to the CSF-IR fragment delD4. The control anti-CCR5 antibody m<CCR5>Pz03.1C5 did also not bind to the CSF-IR fragment delD4.
Biacore sensogram of binding of different anti-CSF-lR antibodies to immobilized human CSF-IR Extracellular Domain (CSF-1R-ECD) (comprising the extracellular subdomains Dl - D5) (SEQ ID NO: 64) (y-axis: binding signal in Response Units (RU), baseline = 0 RU, x-axis: time in seconds (s)): All anti-CSF- 1R antibodies Mab 1G10, Mab 2H7 and humanized hMab 2F11- e7 showed binding to CSF-1R-ECD. The control anti-CCR5 antibody m<CCR5>Pz03.1C5 did not bind to the CSF-1R-ECD. Biacore sensogram of binding of different anti-CSF-lR antibodies to immobilized human CSF-IR fragment delD4 (comprising the extracellular subdomains Dl -D3 and D5) (SEQ ID NO: 65) (y-axis: binding signal in Response Units (RU), baseline = 0 RU, x-axis: time in seconds (s)): All anti-CSF-lR antibodies 1.2.SM, CXIIG6, abl0676 and MAB3291 show binding to to the CSF-IR fragment delD4. The control anti-CCR5 antibody m<CCR5>Pz03.1C5 did also not bind to the CSF-IR fragment delD4.
Biacore sensogram of binding of different anti-CSF-lR antibodies to immobilized human CSF-IR Extracellular Domain (CSF-1R-ECD) (comprising the extracellular subdomains Dl - D5) (SEQ ID NO: 64) (y-axis: binding signal in Response Units (RU), baseline = 0 RU, x-axis: time in seconds (s))::
All anti-CSF-lR antibodies 1.2.SM, CXIIG6, abl0676 and MAB3291 show binding to CSF-1R-ECD. The control anti- CCR5 antibody m<CCR5>Pz03.1C5 did not bind to the CSF-1R- ECD.
CSF-1 levels in Cynomolgus monkey after application of different dosages of anti-CSF-lR antibody according to the invention Figure 4 In vivo efficacy - tumor growth inhibition of anti-CSF-lR antibodies according to th invention in breast cancer BT20 xenograft Example 1
Generation of a hybridoma cell line producing anti-CSF-lR antibodies
Immunization procedure of NMRI mice
NMRI mice were immunized with an expression vector pDisplay™ (Invitrogen, USA) encoding the extracellular domain of huCSF-lR by utilizing electroporation. Every mouse was 4 times immunized with 100μg DNA. When serum titers of anti- huCSF-lR were found to be sufficient, mice were additionally boosted once with 50μg of a 1 : 1 mixture huCSF-lR ECD/huCSF-lR ECDhuFc chimera in 200 μΐ PBS intravenously (i.v.) 4 and 3 days before fusion.
Antigen specific ELISA Anti-CSF-lR titers in sera of immunized mice were determined by antigen specific
ELISA.
0.3 μg/ml huCSF-lR-huFc chimera (soluble extracellular domain) was captured on a streptavidin plate (MaxiSorb; MicroCoat, DE, Cat.No. 11974998/MC1099) with 0.1 mg/ml biotinylated anti Fey (Jackson Immuno Research., Cat.No. 109-066-098) and horse radish peroxidase (HRP)-conjugated F(ab')2 anti mouse IgG (GE
Healthcare, UK, Cat.No .NA9310 V) diluted 1/800 in PBS/0.05% Tween20/0.5% BSA was added. Sera from all taps were diluted 1/40 in PBS/0.05% Tween20/0.5% BSA and serially diluted up to 1/1638400. Diluted sera were added to the wells. Pre-tap serum was used as negative control. A dilution series of mouse anti-human CSF-1R Mab3291 (R&D Systems, UK) from 500 ng/ml to 0,25 ng/ml was used as positive control. All components were incubated together for 1,5 hours, Wells were washed 6 times with PBST (PBS/0.2% Tween20) and assays were developed with freshly prepared ABTS® solution (1 mg/ml) (ABTS: 2,2'-azino bis (3-ethylbenzthiazoline-6-sulfonic acid) for 10 minutes at RT. Absorbance was measured at 405 nm. Hybridoma generation
The mouse lymphocytes can be isolated and fused with a mouse myeloma cell line using PEG based standard protocols to generate hybridomas. The resulting hybridomas are then screened for the production of antigen-specific antibodies. For example, single cell suspensions of splenic derived lymphocytes from immunized mice are fused to Ag8 non-secreting mouse myeloma cells P3X63Ag8.653 (ATCC, CRL-1580) with 50% PEG. Cells are plated at approximately 104 in flat bottom 96 well micro titer plate, followed by about two weeks incubation in selective medium. Individual wells are then screened by ELISA for human anti-CSF-lR monoclonal IgM and IgG antibodies. Once extensive hybridoma growth occurs, the antibody secreting hybridomas are replated, screened again, and if still positive for human IgG, anti-CSF-lR monoclonal antibodies, can be subcloned by FACS. The stable subclones are then cultured in vitro to produce antibody in tissue culture medium for characterization. Antibodies according to the invention could be selected using the determination of the binding of anti-CSF-lR antibodies to human CSF-1 R fragment delD4 and to human CSF-1R Extracellular Domain (CSF-1R-ECD) as described in Example 4, as well as the determination of growth inhibition of NIH3T3 cells transfected with wildtype CSF-1R (ligand dependent signalling) or mutant CSF-1R L301S Y969F (ligand independent signalling) under treatment with anti-CSF-lR monoclonal antibodies as described in Example 5.
Culture of hybridomas
Generated muMAb hybridomas were cultured in RPMI 1640 (PAN - Catalogue No. (Cat. No.) PO4-17500) supplemented with 2 mM L-glutamine (GIBCO - Cat. No.35050-038), 1 mM Na-Pyruvat (GIBCO - Cat. No.11360-039), lx NEAA (GIBCO - Cat. No.1 1140-035), 10% FCS (PAA - Cat. No.A15-649), lx Pen Strep
(Roche - Cat. No.1074440), lx Nutridoma CS (Roche - Cat. No.1363743), 50 μΜ Mercaptoethanol (GIBCO - Cat. No.31350-010) and 50 U/ml IL 6 mouse (Roche - Cat. No. l 444 581) at 37°C and 5% C02. Some of the resulting mouse antibodies have been humanized (e.g. Mab 2F11) and been expressed recombinantly. Example 2
Inhibition of CSF-1 binding to CSF-IR (ELISA)
By setting-up this assay to first allow for anti-CSF-lR antibody binding to the CSF-IR -ECD followed by detection of ligand not bound to the receptor both- ligand displacing antibodies and dimerization inhibitor anti-CSF-lR antibodies - can be tested. The test was performed on 384 well microtiter plates (MicroCoat, DE, Cat.No. 464718) at RT. After each incubation step plates were washed 3 times with PBST.
At the beginning, plates were coated with 0.5 mg/ml goat F(ab')2 biotinylated anti Fey (Jackson Immuno Research., Cat.No.109-006-170) for 1 hour (h).
Thereafter the wells were blocked with PBS supplemented with 0.2% Tween®-20 and 2% BSA (Roche Diagnostics GmbH, DE) for 0.5 h. 75 ng/ml of huCSF-lR- huFc chimera (which forms the dimeric soluble extracellular domain of huCSF-lR) was immobilized to plate for 1 h. Then dilutions of purified antibodies in PBS/0.05% Tween20/0.5% BSA were incubated for 1 h. After adding a mixture of
3 ng/ml CSF-1 (Biomol, DE, Cat.No.60530), 50ng/ml biotinylated anti CSF-1 clone BAF216 (R&D Systems,UK) and 1 :5000 diluted streptavidin HRP (Roche Diagnostics GmbH, DE, Cat.No.11089153001) for 1 h the plates were washed 6 times with PBST. Anti CSF-IR SC 2-4A5 (Santa Cruz Biotechnology, US), which inhibits the ligand- receptor interaction, was used as positive control. Plates were developed with freshly prepared BM blue® POD substrate solution (BM blue®: 3,3 '-5,5 '-Tetramethylbenzidine, Roche Diagnostics GmbH, DE, Cat.No . 11484281001) for 30 minutes at RT. Absorbance was measured at 370 nm.A decrease of absorbance is found, if the anti-CSF-lR antibody causes a release of CSF-1 from the dimeric complex. All anti-CSF-lR antibodies showed significant inhibition of the CSF-1 interaction with CSF-IR (see Table 1). Anti CSF-IR SC 2- 4A5 (Santa Cruz Biotechnology, US see also Sherr, C.J. et al, Blood 73 (1989) 1786-1793), which inhibits the ligand- receptor interaction, was used as reference control. Table 1:
Calculated IC50 values for the inhibition of the CSF-l/CSF-lR interaction
Figure imgf000059_0001
Example 3
Inhibition of CSF-l-induced CSF-1R phosphorylation in NIH3T3-CSF-1R recombinant cells
4.5xl03 NIH 3T3 cells, retrovirally infected with an expression vector for full- length CSF-1R, were cultured in DMEM (PAA Cat. No.E 15-011), 2mM L- glutamine (Sigma, Cat.No.G7513, 2mM Sodium pyruvate , lx nonessential aminoacids, 10% FKS (PAA, Cat.No.Al 5-649) and 100μg/ml PenStrep (Sigma,
Cat.No. P4333 [lOmg/ml]) until they reached confluency. Thereafter cells were washed with serum-free DMEM media (PAA Cat.No. E15-011) supplemented with sodium selenite [5ng/ml] (Sigma, Cat.No. S9133), transferrin [10μg/ml] (Sigma, Cat.No. T8158), BSA [400μg/ml] (Roche Diagnostics GmbH, Cat.No. 10735078), 4mM L-glutamine (Sigma, Cat.No. G7513), 2mM sodium pyruvate (Gibco, Cat.No.
11360), lx nonessential aminoacids (Gibco, Cat: 1 1 140-035), 2-mercaptoethanol [0,05mM] (Merck, Cat.No. M7522), 100μg/ml and PenStrep (Sigma, Cat. No. P4333) and incubated in 30 μΐ of the same medium for 16 hours to allow for receptor up-regulation. 10 μΐ of diluted anti-CSR-lR antibodies were added to the cells for 1.5 h. Then cells were stimulated with 10 μΐ of 100 ng/ml huM-CSF-1
(Biomol Cat.No.60530) for 5 min. After the incubation, supernatant was removed, cells were washed twice with 80 μΐ of ice-cold PBS and 50 μΐ of freshly prepared ice-cold lysis buffer (150mM NaCl/ 20mM Tris pH 7.5 / ImM EDTA/ ImM EGTA/ 1% Triton X-100 l\ protease inhibitor tablet (Roche Diagnostics GmbH Cat.No.1 836 170) per 10 ml buffer/ ΙΟμΙ/ml phosphatase inhibitor cocktail 1
(Sigma Cat.No. P-2850, lOOx Stock)/ ΙΟμΙ/ml protease inhibitor 1 (Sigma Cat.No.P-5726, lOOx Stock) /10μ1/ιη1 1 M NaF ) was added. After 30 minutes on ice the plates were shaken vigourously on a plateshaker for 3 minutes and then centrifuged 10 minutes at 2200 rpm (Heraeus Megafuge 10).
The presence of phosphorylated and total CSF-1 receptor in the cell lysate was analyzed with Elisa. For detection of the phosphorylated receptor the kit from R&D Systems (Cat. No. DYC3268-2) was used according to the instructions of the supplier. For detection of total CSF-1 R 10 μΐ of the lysate was immobilized on plate by use of the capture antibody contained in the kit. Thereafter 1 :750 diluted biotinylated anti CSF-IR antibody BAF329 (R&D Systems) and 1 : 1000 diluted streptavidin-HRP conjugate was added. After 60 minutes plates were developed with freshly prepared ABTS® solution and the absorbance was detected. Data were calculated as % of positive control without antibody and the ratio value phospho/total receptor expressed. The negative control was defined without addition of M-CSF-1. Anti CSF-IR SC 2-4A5 (Santa Cruz Biotechnology, US, see also Sherr, C.J. et al, Blood 73 (1989) 1786-1793), which inhibits the ligand- receptor interaction, was used as reference control.
Table 2:
Calculated IC50 values for the inhibition of CSF-1 receptor phosphorylation.
Figure imgf000060_0001
Example 4
Determination of the binding of anti-CSF-lR antibodies to human CSF-IR fragment delD4 and to human CSF-IR Extracellular Domain (CSF-1R-ECD)
Preparation of human CSF-IR Extracellular Domain (CSF-1R-ECD)
(comprising the extracellular subdomains Dl -D5, hCSF-lR-ECD) of SEQ ID NO: 64: pCMV-preS-Fc-hCSF-lR-ECD (7836bp) encodes the complete ECD of human
CSF-IR (SEQ ID NO: 64) C-terminally fused to a PreScission protease cleavage site, followed by aal 00-330 of human IgGl and a 6xHis-Tag, under the control of CMV promoter. The natural signal peptide has been varied by insertion of amino acids G and S after the first M, in order to create a BamHI restriction site.
Preparation of human CSF-1R fragment delD4 (comprising the extracellular subdomains Dl -D3 and D5, hCSF-lR-delD4) of SEQ ID NO: 65: hCSFlR-delD4-Vl-PreSc-hFc-His was cloned from pCMV-preS-Fc-hCSF-lR- ECD by means of the Stratagene QuikChange XL site-directed mutagenesis protocol, using delD4-for with sequence CACCTCCATGTTCTTCCGGTACCCCCCAGAGGTAAG (SEQ ID NO: 68) as the forward primer and delD4-rev with the reverse complement sequence as the reverse primer. A protocol variation published in BioTechniques 26 (1999) 680 was used to extend both primers in separate reactions in three cycles preceeding the regular Stratagene protocol:
Two separate 50 μΐ reaction mixtures were set up according to the manufacturer's manual, each containing 10 ng plasmid pCMV-preS-Fc-hCSFlR-ECD as the template and 10 pM of one of the primers delD4-for or delD4-rev, and 0,5 μΐ Pfu DNA polymerase as provided with the kit. Three PCR cycles 95 °C 30 sec / 55 °C 60 sec / 68 °C 8 min were run, then 25 μΐ each of both reaction mixtures were combined in a new tube and 0,5 μΐ fresh Pfu DNA polymerase were added. The regular PCR protocol with 18 temperature cycles as specified by Stratagene in the kit manual was carried out, followed by 2 hrs final digestion with the Dpnl restriction enzyme provided with the kit. Clones bearing the deletion were detected by digestion with Cel II and Not I and verified by sequencing.
Protein was prepared by transient transfection in the Hek293 FreeStyle suspension cell system (Invitrogen) according to the manufacturer's specifications. After 1 week 500 ml supernatant was filtered and loaded onto a 1ml HiTrap MabSelect Xtra (GE healthcare) protein A column (0,2 ml /min). The colomn was washed first with PBS, then with 50 mM Tris/ 150 mM NaCl/ 1 mM EDTA/ pH 7,3. 75 μΐ PreScission Protease (GE #27-0843-01) diluted in 375 μΐ of the same buffer were loaded onto the column and the closed column was incubated over night at 4 °C with rolling. The column was mounted on top of a 1 ml GSTrap FF column (GE helthcare) and the desired protein was eluted (0,2 ml/min, 0,2 ml fractions). Pooled fractions were concentrated from 1,8 ml to 0,4 ml by centrifugal ultrafiltration via a 3k Nanosep and chromatographed over an S200 HR SEC in PBS (0,5 ml/min).
Human CSF-1 R fragment delD4 was obtained in two fractions as a dimeric molecule (pooll , V=l ,5 ml; c= 0,30 mg/ml; apparent mass on SDS page 83 kDa, reduced 62 kDa) and as the monomer (pool 2, V=l,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-lR antibodies to human CSF-1R fragment delD4 and to human CSF-1R Extracellular Domain (CSF-1R-ECD) (binding signals as Response Units (RU): Instrument: Biacore T 100 (GE Healthcare)
Software: T 100 Control, Version 2.0.1
T100 Evaluation, Version 2.0.2
Assayformat Chip: CM5
Temperature: 25°C CSF-1R fragments were immobilized via amine coupling. To compare the binding of different anti-CSF-lR antibodies according to the invention one concentration of the test antibody was injected. Anti CSF-1R Mab3291 (R&D-Systems) and SC 2- 4A5 (Santa Cruz Biotechnology, US- see also Sherr, C.J. et al, Blood 73 (1989) 1786-1793), was used as reference control, anti-CCR5 m<CCR5>Pz03.1C5 (deposited as DSM ACC 2683 on 18.08.2004 at DSMZ) as negative control, all under the same conditions as the anti-CSF-lR antibodies according to the invention.
Amine coupling of CSF-1R fragments
Standard amine coupling according to the manufacturer's instructions: running buffer: PBS-T (Roche: 11 666 789 + 0.05% Tween20: 11 332 465), activation by mixture of EDC/NHS, injection of human CSF-1R fragment delD4 (comprising the extracellular subdomains Dl -D3 and D5) (SEQ ID NO: 65) and human CSF-1R Extracellular Domain (CSF-1R-ECD) (comprising the extracellular subdomains Dl -D5) (SEQ ID NO: 64) for 600 seconds at flow rate ΙΟμΙ/min; diluted in coupling buffer NaAc, pH 5.0, c = 10 μg/mL; finally remaining activated carboxyl groups were blocked by injection of 1 M Ethanolamin. Binding of <CSF-1R> Mab 2F11, Mab 2E10, Mab 3291 and sc2-4A5 and other anti-CSF-lR antibodies to human CSF-IR fragment delD4 and human CSF- IR Extracellular Domain (CSF-1R-ECD) at 25°C
Running buffer: PBS-T (Roche: 11 666 789 + 0.05% Tween20: 11 332 465) Analyte sample:
Binding was measured at a flow rate of 30 μί/ηιίη by one injection of the analyte with concentration c = 10 nM. (for Mab 1G10, Mab 2H7 and humanized hMab 2F1 l-e7 in second experiment) Each injection was 700 seconds long, followed by a dissociation phase of 180 seconds. Final regeneration was performed after each cycle using 50 mM NaOH, contact time 60 seconds, flow rate 30 μΕ/ηιίη.
Signals were measured by a report point 10 seconds after end of injection. Reference signals (signals from a blank reference flow cell (treated with EDC/NHS and ethanolamine, only) were subtracted to give the binding signals (as RU). If binding signals of nonbinding antibodies were slightly below 0 (Mab 2F11 =-3; Mab 2E10 = -2; Mab 1G10 = - 6, Mab 2H7 =-9; and humanized hMab 2F1 l-e7 = - 7) the values were set as 0.
Table 3a:
Binding of <CSF-1R> MAbs to human CSF-IR fragment delD4 and CSF-1R- ECD and ratio at 25°C, measured by SPR
Binding to Binding Ratio of binding of anti-CSFIR delD4 to_CSF- antibodies
[RU1 1R-ECD to CSF1R fragment delD4 /
[RU1 to CSF-1R-ECD
Mab 3291 1015 627 1015/627= 1.61 sc2-4A5 374 249 374/249= 1.50
Mab 2Fl l 0 176 0/176 = 0 hMab 2Fl l-e7 0 237 0/237= 0
Mab 2E10 0 120 0/120 = 0
Mab 1G10 0 2708 0/2708 = 0
Mab 2H7 0 147 0/147 = 0 m<CCR5>Pz03.1C5 2 5 - Mab 2F1 1 and Mab 2E10 showed binding to the human CSF-IR Extracellular Domain (CSF-IR-ECD) (see Fig. 2b); however no binding was detected to CSF-IR fragment delD4. (see Fig. 2a).
Sc2-4A5 and MAB3291 showed binding to CSF-IR-ECD and to del D4 (see Fig. 2b and 2a).
Thus the ratio of binding of anti-CSFIR antibodies Mab 2F11 and Mab 2E10 to CSF1R fragment delD4 / to CSF-IR-ECD was clearly below 1 :50 (= 0.02), while the binding ratio of MAB3291 and Sc2-4A5 were 1.61 and 1.50, respectively and were highly above 1 :50 (= 0.02). Negative control antibody m<CCR5>Pz03.1C5 did not show any binding (as expected).
Mab 1G10, Mab 2H7 and humanized hMab 2F1 l-e7 showed binding to the human CSF-IR Extracellular Domain (CSF-IR-ECD) (see Fig. 2d); however no binding was detected to CSF-IR fragment delD4. (see Fig. 2c). Thus the ratio of binding of anti-CSFIR antibodies Mab 1G10, Mab 2H7 and humanized hMab 2Fl l-e7 to CSF1R fragment delD4 / to CSF-IR-ECD was clearly below 1 :50 (= 0.02).
In a further experiment anti-CSF-lR antibodies 1.2.SM (ligand displacing CSF-IR antibody described in WO2009026303), CXIIG6 (ligand displacing CSF-IR antibody described in WO 2009/112245), the goat polyclonal anti-CSF-lR antibody ab 10676 (abeam) were investigated. Anti-CSF-lR antibody Mab3291 (R&D-Systems) was used as reference control. Anti-CCR5 m<CCR5>Pz03.1C5
(deposited as DSM ACC 2683 on 18.08.2004 at DSMZ) was used as negative control.
Table 3b:
Binding of <CSF-1R> MAbs to human CSF-1R fragment delD4 and CSF-1R- ECD and ratio at 25°C, measured by SPR
Figure imgf000065_0001
1.2.SM, CXIIG6, abl0676 and MAB3291 showed binding to CSF-1R-ECD and to del D4 (see Fig. 2f and 2e).
The binding ratio of 1.2.SM, CXIIG6, abl0676 and MAB3291 was highly above 1 :50 (= 0.02). Negative control antibody m<CCR5>Pz03.1C5 did not show any binding (as expected). Example 5
Growth inhibition of NIH3T3-CSF-1R recombinant cells in 3D culture under treatment with anti-CSF-lR monoclonal antibodies (CellTiterGlo-assay)
NIH 3T3 cells, retrovirally infected with either an expression vector for full-length wildtype CSF-1R (SEQ ID NO: 62) or mutant CSF-1R L301 S Y969F (SEQ ID NO: 63), were cultured in DMEM high glucose media (PAA, Pasching, Austria) supplemented with 2mM L-glutamine, 2mM sodium pyruvate and non-essential amino acids and 10% fetal bovine serum (Sigma, Taufkirchen, Germany) on poly- HEMA (poly(2-hydroxyethylmethacrylate)) (Polysciences, Warrington, PA, USA)) coated dishes to prevent adherence to the plastic surface. Cells are seeded in medium replacing serum with 5ng/ml sodium selenite, l Omg/ml transferrin,
400μg/ml BSA and 0.05 mM 2-mercaptoethanol. When treated with lOOng/ml huCSF-1 (Biomol, Hamburg, Germany) wtCSF-lR ( expressing cells form dense spheroids that grow three dimensionally, a property that is called anchorage independence. These spheroids resemble closely the three dimensional architecture and organization of solid tumors in situ. Mutant CSF-IR recombinant cells are able to form spheroids independent of the CSF-1 ligand. Spheroid cultures were incubated for 3 days in the presence of different concentrations of antibody in order to determine an IC50 (concentration with 50 percent inhibition of cell viability). The CellTiterGlo assay was used to detect cell viability by measuring the ATP- content of the cells.
Table 5a:
Figure imgf000066_0001
Reference control Mab R&D-Systems 3291 did not show inhibition of mutant CSF-IR recombinant cell proliferation. In a further experiment the anti-CSF-lR antibody according to the invention hMab
2Fl l-e7 and the anti-CSF-lR antibodies 1.2.SM (ligand displacing CSF-IR antibody described in WO2009026303), CXIIG6 (ligand displacing CSF-IR antibody described in WO 2009/112245), the goat polyclonal anti-CSF-lR antibody abl0676 (abeam), and SC 2-4A5 (Santa Cruz Biotechnology, US- see also Sherr, C.J. et al, Blood 73 (1989) 1786-1793) were investigated.
Spheroid cultures were incubated for 3 days in the presence of different concentrations of antibody in order to determine an IC30 (concentration with 30 percent inhibition of cell viability). Maximum concentration was 20 μg/ml The CellTiterGlo assay was used to detect cell viability by measuring the ATP-content of the cells. Table 5b:
Figure imgf000067_0001
Example 6
Growth inhibition of BeWo tumor cells in 3D culture under treatment with anti-CSF-lR monoclonal antibodies (CellTiterGlo-assay)
BeWo choriocarcinoma cells (ATCC CCL-98) were cultured in F12K media (Sigma, Steinheim, Germany) supplemented with 10% FBS (Sigma) and 2mM L-glutamine. 5xl04 cells/well were seeded in 96-well poly-HEMA (poly(2-hydroxyethylmethacrylate)) coated plates containing F12K medium supplemented with 0.5 % FBS and 5% BSA. Concomitantly, 200 ng/ml huCSF-1 and ΙΟμ^πιΙ of different anti-CSF-lR monoclonal antibodies were added and incubated for 6 days. The CellTiterGlo assay was used to detect cell viability by measuring the ATP-content of the cells in relative light units (RLU). When BeWo spheroid cultures were treated with different anti-CSF-lR antibodies (10 μ^ιηΐ) inhibition of CSF-1 induced growth was observed. To calculate antibody-mediated inhibition the mean RLU value of unstimulated BeWo cells was subtracted from all samples. Mean RLU value of CSF-1 stimulated cells was set arbitrarily to 100%. Mean RLU values of cells stimulated with CSF-1 and treated with anti-CSF-lR antibodies were calculated in % of CSF-1 stimulated RLUs. The Table 6 shows the calculated data of growth inhibition of BeWo tumor cells in 3D culture under treatment with anti-CSF-lR monoclonal antibodies; Fig. la and b depicts normalized mean RLU values. Table 6:
Figure imgf000068_0001
Example 7
Inhibition of human macrophage differentiation under treatment with anti- CSF-1R monoclonal antibodies (CellTiterGlo-assay)
Human monocytes were isolated from peripheral blood using the RosetteSep™ Human Monocyte Enrichment Cocktail (StemCell Tech. - Cat. No.15028). Enriched monocyte populations were seeded into 96 well microtiterplates (2.5x104 cells/well) in 100 μΐ RPMI 1640 (Gibco - Cat. No.31870) supplemented with 10% FCS (GIBCO - Cat. No.01 1-090014M), 4 mM L-glutamine (GIBCO - Cat.
No.25030) and lx PenStrep (Roche Cat. No. l 074 440) at 37°C and 5% C02 in a humidified atmosphere. When 150 ng/ml huCSF-1 was added to the medium, a clear differentiation into adherent macrophages could be observed. This differentiation could be inhibited by addition of anti-CSF-lR antibodies. Furthermore, the monocyte survival is affected and could be analyzed by
CellTiterGlo (CTG) analysis. From the concentration dependent inhibition of the survival of monocytes by antibody treatment, an IC50 was calculated (see Table 7). Table 7:
Figure imgf000069_0001
In a separate test series humanized versions of Mab 2 Fl l, e.g. hMab 2F11-cl 1, hMab 2Fl l-d8, hMab 2Fl l-e7, hMab 2F11-A2, showed IC50 values of 0.07 μg/ml (hMab 2F11-cl 1), 0.07 μg/ml (hMab 2F1 l-d8), 0.04 μ^ιηΐ (hMab 2F1 l-e7) and 0.09 μg/ml (hMab 2F1 l-fl2).
Example 8
Inhibition of cynomolgous macrophage differentiation under treatment with anti-CSF-lR monoclonal antibodies (CellTiterGlo-assay)
Cynomolgous monocytes were isolated from peripheral blood using the CD14 MicroBeads non- human primate kit (Miltenyi Biotec - Cat.No. 130-091-097) according to the manufacturers description. Enriched monocyte populations were seeded into 96 well microtiterplates (l-3xl04 cells/well) in 100 μΐ RPMI 1640 (Gibco - Cat. No.31870) supplemented with 10% FCS (GIBCO - Cat. No.01 1- 090014M), 4 mM L-glutamine (GIBCO - Cat. No.25030) and lx PenStrep (Roche Cat. No. l 074 440) at 37°C and 5% C02 in a humidified atmosphere. When 150 ng/ml huCSF-1 was added to the medium, a clear differentiation into adherent macrophages could be observed. This differentiation could be inhibited by addition of anti-CSF-lR antibodies. Furthermore, the monocyte survival is affected and could be analyzed by CellTiterGlo (CTG) analysis. The viability was analyzed at a concentration of 5 μg/ml antibody treatment (see Table 8). Table 8:
Figure imgf000070_0001
* mean of four experiments (3 expts. using the murine, 1 expt. using the chimeric mAb)
** mean of two experiments using the murine mAb only
Example 9
Determination of the binding affinity of anti-CSF-lR antibodies to human CSF-1R
Instrument: BIACORE® A100
Chip: CM5 (Biacore BR- 1006-68)
Coupling: amine coupling
Buffer: PBS (Biacore BR- 1006-72), pH 7.4, 35°C
For affinity measurements 36 μg/ml anti mouse Fey antibodies (from goat, Jackson Immuno Reasearch JIRl 15-005-071) have been coupled to the chip surface for capturing the antibodies against CSF-1R. Human CSF-1R Extracellular Domain (CSF-1R-ECD) (comprising the extracellular subdomains Dl -D5) (SEQ ID NO: 64) (R&D-Systems 329-MR or subcloned pCMV-presS-HisAvitag-hCSF-lR- ECD) was added in various concentrations in solution. Association was measured by an CSF-1R- injection of 1.5 minutes at 35 °C; dissociation was measured by washing the chip surface with buffer for 10 minutes at 35 °C. For calculation of kinetic parameters the Langmuir 1 : 1 model was used. Table 9:
Affinity data measured by SPR
Figure imgf000071_0001
In a separate biacore binding assay using the CSF-IR ECD (data not shown) some competition of the antibodies Mab 2F11 and Mab 2E10 with the antibody Ab SC- 2-4A5 was shown. However Mab 2F11/Mab 2E10 do not bind to the human CSF- IR fragment delD4, whereas Ab SC-2-4A5 binds to this delD4 fragment (see Example 4 and Fig 2a). Thus the binding region of Mab 2F11/Mab 2E10 is clearly distinct from the binding region of Ab SC-2-4A5, but probably located in a vicinity area. In such competition assay both antibodies Mab 2F1 1 and Mab 2E10 did not compete with Mab3291 from R&D-Systems (data not shown).
Example 10
Determination of the binding of anti-CSF-lR antibodies to human CSF-IR fragment D1-D3
Instrument: Biacore T100 (GE Healthcare)
Software: T 100 Control, Version 1.1.11
B3000 Evaluation, Version 4.01
Scrubber, Version 2.0a
Assayformat Chip :CM5 -Chip
Antibodies against CSF-IR were captured via amine coupled capture molecules. Using the single cycle kinetics five increasing concentrations of human CSF-IR fragment D1-D3 (SEQ ID NO: 66) were injected. Human CSF-IR fragment D1-D3 was subcloned into pCMV-presS-HisAvitag expression vector.
Anti CSF-IR SC 2-4A5 (Santa Cruz Biotechnology, US; Sherr, C.J. et al, Blood 73 (1989) 1786-1793) which inhibits the ligand-receptor interaction, and Mab 3291 (R&D-Systems) were used as reference controls. Capture molecules: Anti mouse Fey antibodies (from goat, Jackson Immuno Reasearch JIR115-005-071) for antibodies according to the invention and the R&D-Systems control Mab 3291 and Anti rat Fey antibodies (from goat, Jackson Immuno Reasearch JIR112-005-071) for the reference control anti CSF-IR SC 2- 4A5.
Amine coupling of capture molecules
Standard amine coupling according to the manufacturer's instructions: running buffer: HBS-N buffer, activation by mixture of EDC/NHS, aim for ligand density of 2000 RU; the capture- Abs were diluted in coupling buffer NaAc, pH 4.5, c = 10 μg/mL; finally remaining activated carboxyl groups were blocked by injection of
1 M Ethanolamin.
Kinetic characterization of human CSF-IR fragments D1-D3 binding to MAbs <CSF-1R> at 37°C
Running buffer: PBS (Biacore BR- 1006-72) Capturing of Mabs <CSF-1R> on flow cells 2 to 4: Flow 20 μί/ηιίη, contact time
90 seconds, c(Abs<CSF-lR>) = 50 nM, diluted with running buffer + 1 mg/mL BSA;
Analyte sample:
Single Cycle Kinetics was measured at a flow rate of 30 μΐ,/ηιίη by five consecutive injections of the analyte with concentrations, c = 7.8 , 31.25, 125 500 and 2000 nM, without regeneration. Each injection was 30 seconds long and followed by a dissociation phase of 120 Seconds for the first four injections, and finally 1200 seconds for the highest concentration (=last injection).
Final regeneration was performed after each cycle using 10 mM Glycin pH 1.5 (Biacore BR- 1003-54), contact time 60 seconds, flow rate 30 μΕ/ηιίη.
Kinetic parameters were calculated by using the usual double referencing (control reference: binding of analyte to capture molecule; Flow Cell: subdomain CSF-IR concentration "0" as Blank) and calculation with model 'titration kinetics 1 : 1 binding with draft'. Table 10:
Affinity data for binding of human CSF-1R fragment D1-D3 measured by
SPR
Figure imgf000073_0001
The antibodies Mab 2F11, Mab 2E10 and Mab 1G10 showed no binding to human
CSF-1 R fragment D1-D3
Also reference contra 1-Ab SC-2-4A5 did not bind to human CSF-1 R fragment Dl- D3.
The reference control Mab R&D-Systems 3291 showed binding to the human CSF- 1R fragment D1-D3.
Example 11
CSF-1 level increase during CSF-1R inhibition in Cynomolgus monkey
Serum CSF-1 levels provide a pharmacodynamic marker of CSF-1 R neutralizing activity of anti-human CSF-1R dimerization inhibitor hMab 2Fl l-e7. One male and one female cynomolgus monkey per dosage group (1 and 10 mg/kg) were intravenously administered anti-CSFIR antibody hMab 2Fl l-e7. Blood samples for analysis of CSF-1 levels were collected 1 week before treatment (pre-dose), 2, 24, 48, 72, 96, 168 hours post-dose and weekly for two additional weeks. CSF-1 levels were determined using a commercially available ELISA kit (Quantikine® human M-CSF) according to the manufacturer's instructions (R&D Systems, UK ).
Monkey CSF-1 level were determined by comparison with CSF-1 standard curve samples provided in the kit. Administration of hMab 2F1 l-e7 induced a dramatic increase in CSF-1 by ~ 1000- fold, which depending on the dose administered lasted for 48 hr (lmg/kg) or 15 days (lOmg/kg). Hence, a dimerization inhibitor for CSF-1 R offers the advantage to not directly compete with the dramatically upregulated ligand for binding to the receptor in contrast to a ligand displacing antibody.
Example 12
In vivo efficacy - tumor growth inhibition of anti-CSF-lR antibodies in breast cancer BT20 xenograft tumor cells in SCID beige mice
The human breast cancer cell line BT-20 expresses human CSF-1R but lacks CSF-1 expression (Sapi, E. et al Cancer Res 59 (1999) 5578-5585). Since the mouse derived CSF-1 fails to activate human CSF-1R on the tumor cells recombinant human CSF-1 (Biomol, Hamburg, Germany) was supplemented via osmotic minipumps (ALZET, Cupertino, CA) providing a continuous CSF-1 infusion rate of 2μg/day (Martin, T.A., Carcinogenesis 24 (2003) 1317-1323). To directly compare the efficacy of an antibody interfering with dimerization of
CSF-1R with a ligand displacing CSF-1R antibody we tested the chimeric anti- CSF-1R Mab 2F11 (antibody interfering with dimerization of CSF-1 R) and 1.2.SM (ligand displacing CSF-1R antibody described in WO2009026303) in the BT-20 xenograft model. SCID beige mice (Charles River, Sulzfeld, Germany) were subcutaneously coinjected with lx 107 cells BT-20 cells (ATCC HTB-19) and ΙΟΟμΙ of Matrigel . Treatment of animals started at day of randomization at a mean tumor volume of 100 mm3. Mice are treated once weekly i.p. with the respective antibodies (see figure 4) in 20mM Histidine, 140 mM NaCl pH 6.0 buffer. The tumor dimensions are measured by caliper beginning on the staging day and subsequently 2 times per week during the whole treatment period. Tumor volume is calculated according to NCI protocol (Tumor weight = l/2ab2, where "a" and "b" are the long and the short diameters of the tumor, respectively).
Tumor growth analysis is shown in Figure 4. Inhibition of human CSF-1R on tumor cells with the chimeric anti-CSF-lR Mab 2F11 was statistically more efficacious in mediating tumor growth inhibition than anti-CSF-lR antibody 1.2.SM (CSF-1R antibody described in WO2009026303).

Claims

Patent Claims
An antibody binding to human CSF-IR, characterized in that the antibody binds to human CSF-IR fragment delD4 (SEQ ID NO: 65) and to human CSF-IR Extracellular Domain (SEQ ID NO : 64) with a ratio of 1 :50 or lower.
The antibody according to claim 1, 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 a humanized version thereof.
The antibody according to claim 1, 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 ID NO:55 and the light chain variable domain is SEQ ID NO:56. The antibody according to claims 1, 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 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 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 ID NO: 38, or f) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO:41, a CDR2 region of SEQ ID NO: 42, and a CDR1 region of SEQ ID NO:43, and the light chain variable domain comprises a 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 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 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.
The antibody according to any one of claims 1 to 4, characterized in that said antibody is of human IgGl subclass or is of human IgG4 subclass.
A pharmaceutical composition characterized in comprising an antibody according to claims 1 to 5.
The antibody according to claims 1 to 5 for the treatment of cancer.
The antibody according to claims 1 to 5 for the treatment of bone loss.
The antibody according to claims 1 to 5 for the prevention or treatment of metastasis.
The antibody according to claims 1 to 5 for the treatment of inflammatory diseases.
11. A nucleic acid encoding a heavy chain of an antibody binding to CSF-1R, characterized in that said antibody comprises a variable domain according to claim 2 or 3.
12. An expression vector characterized in comprising a nucleic acid according to claim 11 for the expression of the antibody according to claims 1 to 5 in a prokaryotic or eukaryotic host cell.
13. A prokaryotic or eukaryotic host cell comprising a vector according to claim 12.
14. A method for the production of a recombinant antibody according to claims 1 to 5, characterized by expressing a nucleic acid according to claim 11 in a prokaryotic or eukaryotic host cell and recovering said antibody from said cell or the cell culture supernatant.
15. Use of the antibody according to claims 1 to 5 for the manufacture of a medicament for the treatment of cancer.
16. Use of the antibody according to claims 1 to 5 for the manufacture of a medicament for the treatment of bone loss.
17. Use of the antibody according to claims 1 to 5 for the manufacture of a medicament for the treatment of metastasis.
18. Use of the antibody according to claims 1 to 5 for the manufacture of a medicament for the treatment of inflammatory diseases.
19. A method for the treatment of a patient suffering from cancer characterized by administering to the patient an antibody according to claims 1 to 5.
20. A method for the treatment of a patient suffering from bone loss characterized by administering to the patient an antibody according to claims 1 to 5.
21. A method for the treatment of a patient suffering from metastasis characterized by administering to the patient an antibody according to claims 1 to 5.
22. A method for the treatment of a patient suffering from inflammatory diseases characterized by administering to the patient an antibody according to claims 1 to 5.
23. 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 ID NO: 16; c) the heavy chain variable domain is SEQ ID NO: 75 and the light chain variable domain is SEQ ID NO:76; d) the heavy chain variable domain is SEQ ID NO: 83 and the light chain variable domain is SEQ ID NO: 84; or a humanized version thereof.
24. 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 ID NO:55 and the light chain variable domain is SEQ ID NO:56.
25. 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 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 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 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 ID NO: 38, or f) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO:41, a CDR2 region of SEQ ID NO: 42, and a CDR1 region of SEQ ID NO:43, and the light chain variable domain comprises a 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 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 - SO - li) the heavy chain variable domain comprises a CDR3 region of SEQ ID NO:69, a CDR2 region of SEQ ID NO: 70, and a CDRl 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 CDRl 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 CDRl 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 CDRl region of SEQ ID NO: 82.
26. The antibody according to any one of claims 23 to 25, characterized in that said antibody is of human IgGl subclass.
27. A pharmaceutical composition characterized in comprising an antibody according to claims 23 to 26.
PCT/EP2010/069090 2009-12-10 2010-12-07 Antibodies binding preferentially human csf1r extracellular domain 4 and their use Ceased WO2011070024A1 (en)

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