HK1176948B

HK1176948B - Antibodies against human csf-1r and uses thereof

Info

Publication number: HK1176948B
Application number: HK13104333.3A
Authority: HK
Inventors: Georg Fertig; Alexander Fidler; Klaus Kaluza; Marlene Thomas; Carola Ries; Stefan Seeber
Original assignee: 霍夫曼-拉罗奇有限公司
Priority date: 2010-03-05
Filing date: 2011-03-03
Publication date: 2017-06-30

Description

Anti-human CSF-1R antibodies and uses thereof

Technical Field

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

Background

The CSF-1 receptor (CSF-1R; synonym: M-CSF receptor; macrophage colony stimulating factor 1 receptor, EC2.7.10.1, Fms proto-oncogene, c-Fms, SwissProtP07333, CD 115) has been known since 1986 (Coissens, L., et al, Nature320 (1986) 277-. CSF-1R is a growth factor and is encoded by the c-fms proto-oncogene (reviewed, for example, in Roth, P., and Stanley, E.R., curr. Top. Microbiol. Immunol.181 (1992) 141-67).

CSF-1R is a receptor for M-CSF (macrophage colony stimulating factor, also known as CSF-1) and mediates the biological effects of this cytokine (Sherr, C.J., et al, Cell41(1985) 665-676). Cloning of the colony stimulating factor-1 receptor (also known as c-fms) was first described in Roussel, M.F., et al, Nature325 (1987) 549-one 552. In this publication, CSF-1R was shown to have a transforming potential dependent on changes in the C-terminal tail of the protein, including loss of phosphorylation of the inhibitory tyrosine 969, which binds to Cbl and thereby modulates receptor downregulation (Lee, P.S., et al, EmboJ.18 (1999) 3616-.

CSF-1R is a single-chain, transmembrane Receptor Tyrosine Kinase (RTK) and a member of the RTK family containing immunoglobulin (Ig) motifs, characterized by a repetitive Ig domain in the extracellular portion of the receptor. The intracellular protein tyrosine kinase domain is interrupted by a unique insertion domain that is also present in other related RTKIII family members, including platelet-derived growth factor receptor (PDGFR), stem cell growth factor receptor (c-Kit), and fins-like cytokine receptor (FLT 3). Despite structural homology in this family of growth factor receptors, they have distinct tissue-specific functions. CSF-1R is expressed predominantly on cells of the monocytic lineage and in the female reproductive tract and placenta. In addition, CSF-1R expression has been reported in Langerhans cells (a smooth muscle cell subset) in skin (Inaba, T., et al, J.biol.chem.267 (1992) 5693-.

The major biological effects of CSF-1R signaling are differentiation, proliferation, migration, and survival of hematopoietic precursor cells to macrophage lineages, including osteoclasts. Activation of CSF-1R is mediated by its ligand M-CSF. Binding of CSF-1R by M-CSF induces homodimer formation and activation of kinases by tyrosine phosphorylation (Stanley, e.r., et al, mol.reprod.dev.46 (1997) 4-10). Additional signaling is mediated by the p85 subunit and Grb2 of PI3K, which are linked to the PI3K/AKT and Ras/MAPK pathways, respectively. These two important signaling pathways regulate proliferation, survival and apoptosis. Other signaling molecules that bind to the intracellular domain of phosphorylated CSF-1R include STAT1, STAT3, PLCy, and Cb1 (Bourette, R.P., Rohrschneider, L.R., GrowthFactors17 (2000) 155-.

CSF-1R signaling has physiological roles in immune responses, bone remodeling, and in the reproductive system. It has been shown that either M-CSF (Pollad, J.W., mol. reprod.Dev.46 (1997) 54-61) or CSF-1R (Dai, X.M., et al, Blood99 (2002) 111-120) knockout animals have osteopetrotic, hematopoietic, tissue macrophage, and reproductive phenotypes consistent with the role of CSF-1R in various cell types.

Sherr, C.J., et al, Blood73 (1989) 1786-1793 relates to antibodies against CSF-1R which inhibit CSF-1 activity (see Sherr, C.J., et al, Blood73 (1989) 1786-1793). Ashmun, R.A., et al, Blood73 (1989) 827-837 relates to CSF-1R antibodies. Lenda, d.et al, journal of immunology170 (2003) 3254-3262, involved reduced macrophage recruitment, proliferation, and activation in CSF-1 deficient mice, leading to reduced tube apoptosis during renal inflammation. Kitaura, H.et al, journal of denttalresearch 87 (2008) 396-. WO2001/030381 mentions inhibitors of CSF-1 activity, including antisense nucleotides and antibodies, although only CSF-1 antisense nucleotides are disclosed. WO2004/045532, which relates to the prevention of cancer metastasis and bone loss and the treatment of metastatic cancer by M-CSF antagonists, discloses only antagonistic anti-CSF-1 antibodies. WO2005/046657 relates to the treatment of inflammatory bowel disease by anti-CSF-1 antibodies. US2002/0141994 relates to inhibitors of colony stimulating factors. WO2006/096489 relates to the treatment of rheumatoid arthritis by anti-CSF-1 antibodies. WO2009/026303 and WO2009/112245 relate to anti-CSF-1R antibodies.

Summary of The Invention

The invention encompasses an antibody that binds human CSF-1R characterized by binding to the same epitope as the deposited antibody DSMACC 2922.

In one embodiment, the antibody is characterized by comprising the CDR3 region of seq id No.1, seq id No. 9, or seq id No. 17 as the heavy chain variable domain CDR3 region.

In one embodiment, the antibody is characterized by:

a) the heavy chain variable domain comprises the CDR3 region SEQ ID NO:1, the CDR2 region SEQ ID NO:2, and the CDR1 region SEQ ID NO:3, and the light chain variable domain comprises the CDR3 region SEQ ID NO:4, the CDR2 region SEQ ID NO:5, and the CDR1 region SEQ ID NO:6, or

b) The heavy chain variable domain comprises the CDR3 region SEQ ID NO 9, the CDR2 region SEQ ID NO 10, and the CDR1 region SEQ ID NO 11, and the light chain variable domain comprises the CDR3 region SEQ ID NO 12, the CDR2 region SEQ ID NO 13, and the CDR1 region SEQ ID NO 14, or

c) The heavy chain variable domain comprises the CDR3 region SEQ ID NO:17, the CDR2 region SEQ ID NO:18, and the CDR1 region SEQ ID NO:19, and the light chain variable domain comprises the CDR3 region SEQ ID NO:20, the CDR2 region SEQ ID NO:21, and the CDR1 region SEQ ID NO:22, or

d) A CDR-grafted antibody variant, a humanized antibody variant, or a T cell epitope-depleted antibody variant of antibody a), b), or c).

In one embodiment, the antibody is characterized by comprising:

a) the amino acid sequence of the heavy chain variable domain is SEQ ID NO:7 and the amino acid sequence of the light chain variable domain is SEQ ID NO:8, or

b) The amino acid sequence of the heavy chain variable domain is SEQ ID NO:15 and the amino acid sequence of the light chain variable domain is SEQ ID NO:16, or

c) The amino acid sequence of the heavy chain variable domain is SEQ ID NO:23 and the amino acid sequence of the light chain variable domain is SEQ ID NO:24, or

In one embodiment, the antibody that binds human CSF-1R and is characterized by the amino acid sequences and amino acid sequence fragments described above belongs to the subclass human IgG1 or to the subclass human IgG 4.

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

The invention further comprises a pharmaceutical composition characterized in that it comprises an antibody that binds human CSF-1R, characterized by the epitope binding properties described above or the amino acid sequences and amino acid sequence fragments described above.

The invention further comprises the use of an antibody characterized in that it comprises an antibody that binds to human CSF-1R, characterized by the epitope binding properties described above or the amino acid sequences and fragments of the amino acid sequences described above for the manufacture of a pharmaceutical composition.

The invention further includes the use of an antibody characterized by comprising an antibody that binds human CSF-1R, characterized by the epitope binding properties described above or the amino acid sequences and amino acid sequence fragments described above, for the manufacture of a medicament for the treatment of CSF-1R mediated diseases.

The invention further includes the use of antibodies characterized by binding to human CSF-1R, antibodies characterized by the epitope binding properties described above or fragments of the amino acid sequences and amino acid sequences described above for the treatment of cancer.

The invention further includes the use of antibodies characterized by binding to human CSF-1R, antibodies characterized by the epitope binding properties described above or fragments of the amino acid sequences and amino acid sequences described above for the treatment of bone loss.

The invention further includes the use of antibodies characterized by binding to human CSF-1R, antibodies characterized by the epitope binding properties described above or fragments of the amino acid sequences and amino acid sequences described above for the prevention or treatment of metastasis.

The invention further includes the use of antibodies characterized by binding to human CSF-1R, antibodies characterized by the epitope binding properties described above or fragments of the amino acid sequences and amino acid sequences described above for the treatment of inflammatory diseases.

One aspect of the present invention is an antibody that binds to human CSF-1R, characterized by comprising the CDR3 region of SEQ ID NO.1, SEQ ID NO. 9, or SEQ ID NO. 17 as the heavy chain variable domain CDR3 region.

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

In one embodiment, the antibody is characterized by comprising:

In one aspect of the invention, an antibody according to the invention is administered in an amount of at least 10^-8mol/l to 10^-12The affinity of mol/l binds to human CSF-1R.

In one aspect of the invention, the antibody according to the invention is a humanized antibody.

Yet another embodiment of the invention is a nucleic acid encoding the heavy chain variable domain and/or the light chain variable domain of an antibody according to the invention. Preferably, the nucleic acid encodes the heavy chain of an antibody that binds human CSF-1R, which antibody is characterized by comprising the CDR3 region of SEQ ID NO:1, SEQ ID NO:9, or SEQ ID NO:17 as the heavy chain variable domain CDR3 region.

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

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

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 in that a nucleic acid according to the invention is expressed in a prokaryotic or eukaryotic host cell and the antibody is recovered from the cell or cell culture supernatant. The invention further comprises antibodies obtainable by such recombinant methods.

The antibodies according to the invention show benefits to patients in need of CSF-1R targeted therapy. The antibodies according to the invention have the novel and inventive property of giving benefit to patients suffering from a neoplastic disease, in particular from cancer.

The invention further provides a method for treating a patient suffering from cancer comprising administering to a patient diagnosed with such a disease (and therefore in need of such therapy) an effective amount of an antibody according to the invention that binds human CSF-1R. The antibody is preferably administered in a pharmaceutical composition.

Yet another embodiment of the invention is a method for treating a patient suffering from cancer, characterized in that an antibody according to the invention is administered to the patient.

The invention further comprises the use of an antibody according to the invention for the treatment of a patient suffering from cancer and for the manufacture of a pharmaceutical composition according to the invention. In addition, the present invention comprises a method for manufacturing a pharmaceutical composition according to the present invention.

The invention further comprises a pharmaceutical composition comprising an antibody according to the invention, optionally together with buffers and/or adjuvants useful for formulating the antibody for pharmaceutical purposes.

The invention further provides a pharmaceutical composition comprising an antibody according to the invention in a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition may be included in an article of manufacture or a kit.

Drawings

FIG. 110. mu.g/ml concentration of different anti-CSF-1R monoclonal antibodies on the growth inhibition of BeWo tumor cells in 3D cultures.

An X axis: relative Light Units (RLU) mean viability corresponding to ATP content of cells (CellTiterGlo assay).

Y-axis: and (3) testing a probe: minimal medium (0.5% FBS), mouse IgG1 (mIgG 1, 10 μ g/ml), mouse IgG2a (mIgG 2a10 μ g/ml), CSF-1 only, < CSF-1R >7H5.2G10, < CSF-1R >10A4.1G11, and SC-02, clone 2-4a 5.

The highest inhibition of CSF-1 induced growth was observed with anti-CSF-1R antibodies according to the invention.

Detailed description of embodiments of the invention

I. Definition of

The term "antibody" encompasses various forms of antibodies, including but not limited to whole antibodies, antibody fragments, humanized antibodies, chimeric antibodies, T cell epitope-depleting antibodies, and other genetically engineered antibodies, so long as the characteristic properties according to the present invention are retained.

An "antibody fragment" comprises a portion of a full-length antibody, preferably its variable domain, or at least its antigen-binding site. Examples of antibody fragments include diabodies, single chain antibody molecules, and multispecific antibodies formed from antibody fragments. scFv antibodies are described, for example, in Houston, J.S., MethodsinEnzymol.203 (1991) 46-88. In addition, antibody fragments comprise single chain polypeptides having a V that binds CSF-1R_HDomain (i.e. capable of associating with V)_LDomains assembled together) or V binding CSF-1R_LDomain (i.e. capable of associating with V)_HDomains assembled together) assemble into a functional antigen binding site and thereby provide a feature of this property.

As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibody molecules of a single amino acid composition.

The term "chimeric antibody" refers to a monoclonal antibody comprising at least a portion of the mouse-derived variable, i.e., binding, region and constant regions derived from different sources or species, typically prepared by recombinant DNA techniques. Chimeric antibodies comprising mouse variable regions and human constant regions are particularly preferred. Such rat/human chimeric antibodies are the product of an expressed immunoglobulin gene comprising a DNA segment encoding a rat immunoglobulin variable region and a DNA segment encoding a human immunoglobulin constant region. Other forms of "chimeric antibodies" encompassed by the invention are those in which the class or subclass has been modified or altered relative to the original antibody. Such "chimeric" antibodies are also referred to as "switch-like antibodies". Methods for generating 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.Acadsi.USA 81 (1984) 6851-6855; US5,202,238 and US5,204,244.

As used herein, the term "CDR-grafted variant" refers to an antibody variable domain comprising complementarity determining regions (CDRs or hypervariable regions) from one source or species and Framework Regions (FRs) from a different source or species, typically prepared by recombinant DNA techniques. CDR grafted variants comprising the murine CDRs and the variable domains of human FRs are preferred.

As used herein, the term "T cell epitope-depleting variant" refers to an antibody variable domain that has been modified to eliminate or reduce immunogenicity by removing human T cell epitopes (peptide sequences within the variable domain that have the ability to bind mhc class ii molecules). By this method, the interaction between the variable domain amino acid side chains and specific binding pockets within the MHC class II binding groove is identified. The identified immunogenic region is mutated to eliminate immunogenicity. Such processes are generally described in, for example, WO 98/52976.

As used herein, the term "humanized variant" refers to an antibody variable domain that has been reconstituted from Complementarity Determining Regions (CDRs) of non-human origin (e.g., from a non-human species) and from Framework Regions (FRs) of human origin, and further modified to also reconstitute or improve the binding affinity and specificity of the original non-human variable domain. Such humanized variants are typically prepared by recombinant DNA techniques. Reconstitution of the affinity and specificity of the parent non-human variable domains is a crucial step for which different approaches are currently used. In one approach, it is determined whether it is beneficial to introduce mutations in the non-human CDRs as well as in the human FRs (so-called back mutations). Suitable positions for such back-mutations can be identified, for example, by sequence or homology analysis, by selecting human frameworks (fixed framework approach; homology matching or best fit), by using consensus sequences, by selecting FRs from several different human mabs, or by replacing non-human residues on the three-dimensional surface with the most common residues found in human mabs ("resurfacing" or "mosaicing").

In addition, antibodies according to the invention include antibodies having "conservative sequence modifications," nucleotide and amino acid sequence modifications that do not affect or alter the characteristics of the antibodies according to the invention described above. 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 the replacement of an amino acid residue with one 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 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, preferably, a predicted nonessential amino acid residue in a human anti-CSF-1R antibody may be replaced with another amino acid residue from the same side chain family.

Amino acid substitutions may be made by mutagenesis based on molecular modeling, as described in Riechmann, L., et al, Nature332 (1988) 323-.

As used herein, the term "CSF-1R" refers to human CSF-1R (SEQ ID NO: 31). Human CSF-1R (synonyms: CSF-1 receptor; M-CSF receptor; macrophage colony stimulating factor 1 receptor, EC2.7.10.1, Fms proto-oncogene, c-Fms, SwissProtP07333, CD 115) has been known since 1986 (Coussens, L., et al, Nature320 (1986) 277-280). CSF-1R is a growth factor and is encoded by the c-fms proto-oncogene (reviewed, for example, in Roth, P. and Stanley, E.R., curr. Top. Microbiol. Immunol.181 (1992) 141-67).

CSF-1R is a member of the single-chain, transmembrane Receptor Tyrosine Kinase (RTK) and RTK families that contain immunoglobulin (Ig) motifs, characterized by repetitive Ig domains in the extracellular domain of the receptor. The intracellular protein tyrosine kinase domain is interrupted by a unique insertion domain that is also present in other related RTKIII family members, including platelet-derived growth factor receptor (PDGFR), stem cell growth factor receptor (c-Kit), and fins-like cytokine receptor (FLT 3). Despite structural homology in this family of growth factor receptors, they have distinct tissue-specific functions. CSF-1R is expressed predominantly on cells of the monocytic lineage and in the female reproductive tract and placenta. In addition, CSF-1R expression has been reported in Langerhans cells (a smooth muscle cell subset) in skin (Inaba, T., et al, J.biol.chem.267 (1992) 5693-.

As used herein, the terms "bind to human CSF-1R" or "anti-CSF-1R" are used interchangeably and refer to an antibody that specifically binds to human CSF-1R antigen. Binding affinity is K at 35 ℃_DValue 1.0x10^-8K at mol/l or less, preferably 35 ℃_DValue 1.0x10^-9mol/l or less. Binding affinity is determined at 35 ℃ using standard binding assays, such as surface plasmon resonance (BIAcore) technology) (see example 4).

The term "epitope" refers to a protein determinant capable of specifically binding to an antibody. Epitopes are usually composed of chemically active surface groupings of molecules such as amino acids or sugar side chains, and epitopes usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that binding to the former, but not the latter, is lost in the presence of denaturing solvents. Preferably, the antibodies according to the invention specifically bind native CSF-1R rather than denatured CSF-1R.

As used herein, the term "binds to the same epitope as deposited antibody DSMACC 2922" refers to an anti-CSF-1R antibody of the invention that binds to the same epitope on CSF-1R to which antibody < CSF-1R >7H5.2G10 (deposited under accession number DSMACC 2922) binds. The epitope binding properties of the anti-CSF-1R antibodies of the invention can be determined using techniques known in the art. CSF-1R antibodies were measured in an in vitro competitive binding inhibition assay by Surface Plasmon Resonance (SPR) at 25 ℃ to determine the ability of the test antibody to inhibit the binding of antibody < CSF-1R >7H5.2G10 (accession number DSMACC 2922) to CSF-1R. This can be investigated by BIAcore assay (pharmacia biosensorab, Uppsala, Sweden), for example as in example 5. In example 5, the expected percent (%) of binding response of the CSF-1R antibodies of the invention competing for binding to antibody < CSF-1R >7H5.2G10 (accession number DSMACC 2922) was calculated by "100 relative response (general _ stability _ early)/rmax", where rmax was calculated by "relative response (general _ stability _ late) antibody molecular weight/antigen molecular weight", as described in the BIAcore assay epitope mapping instructions. The minimum binding response was also calculated from the pair of identified antibodies 1 and 2 (see example 5). Thus, the maximum value of +50% obtained was set as the threshold for significant competition and thus significant binding to the same epitope (see example 5, for antibody < CSF-1R >7H5.2G10, the calculated threshold was 7+3.5= 10.5). Thus, an antibody binding to human CSF-1R characterized as "binding to the same epitope as < CSF-1R >7H5.2G10 (accession number DSMACC 2922)" has a percent (%) expected binding response (%) (% expected binding response < 10.5) below 10.5.

In one aspect, an antibody according to the invention competes with the deposited antibody DSMACC2922 for binding to human CSF-1R. Such binding competition can be determined using techniques known in the art. CSF-1R antibodies were measured in an in vitro competitive binding inhibition assay by Surface Plasmon Resonance (SPR) at 25 ℃ to determine the ability of the test antibody to inhibit the binding of antibody < CSF-1R >7H5.2G10 (accession number DSMACC 2922) to human CSF-1R. This can be investigated by BIAcore assay (pharmacia biosensorab, Uppsala, Sweden), for example as in example 5.

As used herein, "variable domain" (variable domain of light chain (V)_L) Variable domain of heavy chain (V)_H) Variable light and heavy chain domains have the same overall structure, and each domain comprises four Framework (FR) regions, the sequences of which are widely conserved, connected by three "hypervariable regions" (or complementarity determining regions, CDRs). the framework regions adopt an β -sheet conformation, and the CDRs can form loops connecting a β -sheet structure.

The term "antigen-binding portion of an antibody" as used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The antigen-binding portion of an antibody comprises amino acid residues from a "complementarity determining region" or "CDR". The "framework" or "FR" regions are those regions of the variable domain which differ from the hypervariable region residues defined herein. Thus, the light and heavy chain variable domains of the antibody comprise, from N to C terminus, the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR 4. In particular, the CDR3 of the heavy chain is the region that contributes most to antigen binding and defines the properties of the antibody. The CDR and FR regions are determined according to the standard definition of Kabat, E.A., et al, sequences of proteins of immunologicalcalest, 5 th edition, public health service, national institutes of health, Bethesda, MD (1991) and/or those residues from "hypervariable loops".

As used herein, the term "nucleic acid" or "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA.

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

An "immunoconjugate" refers to an antibody conjugated to one or more heterologous molecules, including but not limited to cytotoxic agents.

An "individual" or "subject" refers to a mammal. Mammals include, but are not limited to, livestock (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, an individual or subject refers to a human.

An "isolated" antibody is one that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis), or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity see, e.g., Flatman et al, J.Chromatogr.B848:79-87 (2007).

An "isolated" nucleic acid is a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

"isolated nucleic acid encoding an anti-CSF-1R antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of an antibody, including such nucleic acid molecules in a single vector or in separate vectors, and such nucleic acid molecules present at one or more locations in a host cell.

"Natural antibody" refers to a naturally occurring immunoglobulin molecule having a different structure. For example, a native IgG antibody is a heterotetrameric glycoprotein of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains that are disulfide-bonded. From N-terminus to C-terminus, each heavy chain has one variable region (VH) (also known as variable or heavy chain variable domain) followed by three constant domains (CH 1, CH2, and CH 3). Similarly, from N-terminus to C-terminus, each light chain has a variable region (VL) (also known as a variable light domain or light chain variable domain) followed by a Constant Light (CL) domain. The "light chains" of antibodies can be classified into one of two types, called kappa (κ) and lambda (λ), depending on the amino acid sequence of their constant domains.

The term "package insert" is used to refer to instructions typically included in commercial packaging for therapeutic products that contain information regarding indications, usage, dosage, administration, combination therapy, contraindications, and/or warnings relating to the use of such therapeutic products.

"percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Comparison for the purpose of determining percent amino acid sequence identity can be performed in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or megalign (dnastar) software. One skilled in the art can determine suitable parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared. However, for the purposes of the present invention,% amino acid sequence identity values are obtained using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech corporation and the source code has been submitted to the us copyright office (USCopyrightOffice, washington d.c.,20559) along with the user document and registered with us copyright registration number TXU 510087. The ALIGN-2 program is publicly available from Genentech corporation (South SanFrancisco, Calif.) or may be compiled from source code. The ALIGN2 program should be compiled for use on UNIX operating systems, including digital UNIXV4.0D. All sequence comparison parameters were set by the ALIGN-2 program and were not changed.

In the case of employing ALIGN-2 to compare amino acid sequences, the% amino acid sequence identity of a given amino acid sequence a relative to (to), with (with), or against (against) a given amino acid sequence B (or may be stated as having or comprising a given amino acid sequence a with respect to, with, or against a given amino acid sequence B) is calculated as follows:

fractional X/Y times 100

Wherein X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in the A and B alignments of this program, and wherein Y is the total number of amino acid residues in B. It will be appreciated that if the length of amino acid sequence a is not equal to the length of amino acid sequence B, then the% amino acid sequence identity of a relative to B will not be equal to the% amino acid sequence identity of B relative to a. Unless specifically stated otherwise, all% amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

Compositions and methods

In one aspect, the invention is based, in part, on the same epitope as the deposited antibody DSMACC 2922. The antibodies of the invention are useful, for example, in the diagnosis or treatment of cancer, inflammatory diseases, or bone loss; or preventing or treating metastasis.

Exemplary anti-CSF-1R antibodies

In one aspect, the invention provides antibodies that bind to human CSF-1R. In certain embodiments, the anti-CSF-1R antibody is characterized by binding to the same epitope as the deposited antibody DSMACC 2922.

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

d) A CDR-grafted antibody variant, a humanized antibody variant or a T-cell epitope-depleted antibody variant of antibody a), b) or c), and

having one or more of the following characteristics (determined in the assays described in examples 2,3, 4, 6, 7 and 8):

-the anti-CSF-1R antibody inhibits CSF-1 binding to CSF-1R with an IC50 of 75ng/ml or less, in one embodiment with an IC50 of 50ng/ml or less;

-the anti-CSF-1R antibody inhibits CSF-1-induced CSF-1R phosphorylation (in NIH3T3-CSF-1R recombinant cells) with an IC50 of 100ng/ml or less, in one embodiment with an IC50 of 50ng/ml or less;

-the anti-CSF-1R antibody inhibits the growth of recombinant NIH3T3 cells expressing human CSF-1R (SEQ ID NO:15) by 80% or more (compared to the antibody deletion), preferably by 90% or more;

-the anti-CSF-1R antibody inhibits the growth of BeWo tumor cells (ATCCCL-98) by 70% or more (at a concentration of 10. mu.g/ml antibody; and compared to the absence of antibody), preferably by 80% or more;

-the anti-CSF-1R antibody inhibits macrophage differentiation (in one embodiment, the anti-CSF-1R antibody inhibits monocyte survival with an IC50 of 1.5nM or less, preferably with an IC50 of 1.0nM or less); or

-the anti-CSF-1R antibody at 35 ℃ with KD =2.0x10^-9Binding affinity of mol/l or less binds to human CSF-1R.

In another aspect, the anti-CSF-1R antibody according to the invention comprises in the heavy chain variable domain (VH) sequence a) CDR1H having an amino acid sequence identical to seq id no:3, seq id no:11 or seq id no:19 or comprising 1, 2 or 3 amino acid residue substitutions relative to seq id no:3, seq id no:11 or seq id no:19, b) CDR2H having an amino acid sequence identical to seq id no:2, seq id no:10 or seq id no:18 or comprising 1, 2 or 3 amino acid residue substitutions relative to seq id no:2, seq id no:10 or seq id no:18, and c) CDR3H having an amino acid sequence identical to seq id no:1, seq id no:9 or seq id no:17 or comprising 1, 2 or 3 amino acid residue substitutions relative to seq id no:1, seq id no:9 or seq id no: 17.

In certain embodiments, the heavy chain variable domain (VH) sequence comprising CDR1H having an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue substitutions relative to seq id No.3, seq id No.11, or seq id No. 19, b) a CDR2H having an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue substitutions relative to seq id No.2, seq id No.10, or seq id No.18, and c) a heavy chain variable domain (VH) sequence comprising CDR3H having an amino acid sequence comprising 1, 2, or 3 amino acid residue substitutions relative to seq id No.1, seq id No. 9, or seq id No. 17, or comprising 1, 2, or 3 amino acid residue substitutions relative to seq id No.1, seq id No. 9, or seq id No. 17, contains substitutions (e.g., insertions, or deletions) relative to the reference sequence of the anti-CSF antibody that retains the ability to bind to R1.

In another aspect, an anti-CSF-1R antibody according to the invention comprises in the light chain variable domain (VL) sequence a) CDR1L having an amino acid sequence identical to seq id No.6, seq id No.14 or seq id No. 22 or comprising 1, 2 or 3 amino acid residue substitutions relative to seq id No.6, seq id No.14 or seq id No. 22, b) CDR2L having an amino acid sequence identical to seq id No.5, seq id No.13 or seq id No. 21 or comprising 1, 2 or 3 amino acid residue substitutions relative to seq id No.5, seq id No.13 or seq id No. 21, and c) CDR3L having an amino acid sequence identical to seq id No.4, seq id No. 12 or seq id No. 20 or comprising 1, 2 or 3 amino acid residue substitutions relative to seq id No.4, seq id No. 12 or seq id No. 20.

In certain embodiments, the anti-CSF-1-binding antibody comprising a) CDR1L having an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue substitutions relative to seq id No.6, seq id No.14, or seq id No. 22, b) CDR2L having an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue substitutions relative to seq id No.5, seq id No.13, or seq id No. 21, and c) CDR2 3L having an amino acid sequence comprising 1, 2, or 3 amino acid residue substitutions relative to seq id No.4, seq id No. 12, or seq id No. 20, or comprising 1, 2, or 3 amino acid residue substitutions relative to seq id No.4, seq id No. 12, or seq id No. 20, comprises a light chain variable domain (VL) sequence comprising a substitution (e.g., a substitution), insertion, or a conservative deletion of the anti-CSF-1-R sequence.

In another aspect, anti-CSF-1R antibodies according to the invention

-in the heavy chain variable domain (VH) sequence comprises a) a CDR1H having an amino acid sequence identical to seq id No.3 or comprising 1, 2 or 3 amino acid residue substitutions relative to seq id No.3, b) a CDR2H having an amino acid sequence identical to seq id No.2 or comprising 1, 2 or 3 amino acid residue substitutions relative to seq id No.2, and c) a CDR3H having an amino acid sequence identical to seq id No.1 or comprising 1, 2 or 3 amino acid residue substitutions relative to seq id No.1, and comprising in the light chain variable domain (VL) sequence d) a CDR1L having an amino acid sequence identical to seq id No.6 or comprising 1, 2 or 3 amino acid residue substitutions relative to seq id No.6, e) a CDR2L having an amino acid sequence identical to seq id No.5 or comprising 1, 2 or 3 amino acid residue substitutions relative to seq id No.5, and f) a CDR2L having an amino acid sequence identical to seq id No.4 or comprising 1 amino acid residue substitutions relative to seq id No.4, 2. Or 3 amino acid residue substitutions, or CDR 3L; or

-in the heavy chain variable domain (VH) sequence comprises a) a CDR1H having an amino acid sequence identical to seq id No.11 or comprising 1, 2 or 3 amino acid residue substitutions relative to seq id No.11, b) a CDR2H having an amino acid sequence identical to seq id No.10 or comprising 1, 2 or 3 amino acid residue substitutions relative to seq id No.10, and c) a CDR3H having an amino acid sequence identical to seq id No. 9 or comprising 1, 2 or 3 amino acid residue substitutions relative to seq id No. 9, and comprising in the light chain variable domain (VL) sequence d) a CDR1L having an amino acid sequence identical to seq id No.14 or comprising 1, 2 or 3 amino acid residue substitutions relative to seq id No.14, e) a CDR2L having an amino acid sequence identical to seq id No.13 or comprising 1, 2 or 3 amino acid residue substitutions relative to seq id No.13, and f) a CDR2L having an amino acid sequence identical to seq id No. 12 or comprising 1 amino acid residue substitutions relative to seq id No. 12, 2. Or 3 amino acid residue substitutions, or CDR 3L; or

-comprising in the heavy chain variable domain (VH) sequence a) a CDR1H having an amino acid sequence identical to seq id No. 19 or comprising 1, 2 or 3 amino acid residue substitutions relative to seq id No. 19, b) a CDR2H having an amino acid sequence identical to seq id No.18 or comprising 1, 2 or 3 amino acid residue substitutions relative to seq id No.18, and c) a CDR3H having an amino acid sequence identical to seq id No. 17 or comprising 1, 2 or 3 amino acid residue substitutions relative to seq id No. 17, and comprising in the light chain variable domain (VL) sequence d) a CDR1L having an amino acid sequence identical to seq id No. 22 or comprising 1, 2 or 3 amino acid residue substitutions relative to seq id No. 22, e) a CDR2L having an amino acid sequence identical to seq id No. 21 or comprising 1, 2 or 3 amino acid residue substitutions relative to seq id No. 21, and f) a CDR2L having an amino acid sequence identical to seq id No. 20 or comprising amino acid residue substitutions relative to seq id No. 20, 2. Or 3 amino acid residue substitutions of CDR 3L.

In another aspect, anti-CSF-1R antibodies according to the invention

-comprising in the heavy chain variable domain (VH) sequence a) a CDR1H having an amino acid sequence identical to seq id No. 19 or comprising 1, 2 or 3 amino acid residue substitutions relative to seq id No. 19, b) a CDR2H having an amino acid sequence identical to seq id No.18 or comprising 1, 2 or 3 amino acid residue substitutions relative to seq id No.18, and c) a CDR3H having an amino acid sequence identical to seq id No. 17 or comprising 1, 2 or 3 amino acid residue substitutions relative to seq id No. 17, and comprising in the light chain variable domain (VL) sequence d) a CDR1L having an amino acid sequence identical to seq id No. 22 or comprising 1, 2 or 3 amino acid residue substitutions relative to seq id No. 22, e) a CDR2L having an amino acid sequence identical to seq id No. 21 or comprising 1, 2 or 3 amino acid residue substitutions relative to seq id No. 21, and f) a CDR2L having an amino acid sequence identical to seq id No. 20 or comprising amino acid residue substitutions relative to seq id No. 20, 2. Or 3 amino acid residues substituted at3 CDR3L, and

the anti-CSF-1R antibody has one or more of the following properties (determined in the assays described in examples 2,3, 4, 6, 7 and 8):

Recombinant methods and compositions

Preferably, the antibodies according to the invention are generated by recombinant means. Such methods are widely known in the art and include protein expression in prokaryotic and eukaryotic cells and subsequent isolation of the antibody polypeptide and purification, usually to a pharmaceutically acceptable purity. For protein expression, nucleic acids encoding the light and heavy chains or fragments thereof are inserted into the expression vector by standard methods. Expression is carried out in suitable 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 (either from the supernatant or after cell lysis).

Recombinant production of antibodies is well known in the art and is described, for example, in reviewed article 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-.

The antibody may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. Purification is carried out by standard techniques, including alkali/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques known in the art to eliminate other cellular components or other contaminants, such as other cellular nucleic acids or proteins. See Ausubel, F., et al, eds, Current protocols in molecular biology, Greene publishing and Wiley Interscience, New York (1987).

Expression in NS0 cells is described, for example, in Barnes, L.M., et al, Cytotechnology32 (2000) 109-123; barnes, L.M., et al, Biotech.Bioeng.73 (2001) 261-. Transient expression is described, for example, in Durocher, Y., et al, Nucl. Cloning of the variable domains is described in Orlandi, R., et al, Proc.Natl.Acad.Sci.USA86 (1989) 3833-; carter, p., et al, proc.natl.acad.sci.usa89 (1992) 4285-; norderhaug, l., et al, j.immunol.methods204 (1997) 77-87. A preferred transient expression system (HEK 293) is described in Schlaeger, E.J., Christensen, K., Cytotechnology30 (1999) 71-83 and Schlaeger, E.J., J.Immunol.Methods194 (1996) 191-199.

Suitable control sequences for prokaryotes include, for example, promoters, optional operator sequences, and ribosome binding sites. Eukaryotic cells are known to utilize promoters, enhancers, and polyadenylation signals.

A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, 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 operably linked to a coding sequence if the ribosome binding site is positioned so as to facilitate translation. In general, "operably linked" means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers need not be contiguous. Ligation is achieved by ligation at convenient restriction sites. If such sites are not present, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

The monoclonal antibodies are suitably separated from the culture broth by conventional immunoglobulin purification procedures, such as, for example, protein a-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. DNA and RNA encoding monoclonal antibodies are readily isolated and sequenced using conventional procedures. Hybridoma cells may serve as a source of such DNA and RNA. Once isolated, the DNA may be inserted into an expression vector, which is then transfected into a host cell (such as a HEK293 cell, CHO cell, or myeloma cell) that does not otherwise produce immunoglobulin protein, to obtain synthesis of recombinant monoclonal antibodies in the host cell.

Nucleic acid molecules encoding amino acid sequence variants of anti-CSF-1R antibodies are prepared by a variety of techniques 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 a previously prepared variant or non-variant form of the humanized anti-CSF-1R antibody.

The heavy and light chain variable domains according to the invention are combined with sequences for promoters, translation initiation, constant regions, 3' untranslated regions, polyadenylation, and translation termination to form expression vector constructs. The heavy and light chain expression constructs may be combined into a single vector, co-transfected, sequentially transfected, or separately transfected into a host cell and then fused to form a single host cell expressing the double chain.

As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the words "transformant" and "transformed cell" include the primary test cell and cultures derived therefrom, regardless of the number of transfers. It is also understood that the DNA content of all progeny may not be exactly the same 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 portion" of an antibody is not directly involved in binding of the antibody to an antigen, but exhibits multiple effector functions. The "Fc portion of an antibody" is a term well known to the skilled artisan and is defined based on the cleavage of the antibody by papain. Antibodies or immunoglobulins are classified according to the amino acid sequence of their heavy chain constant region: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, and IgG4, IgA1, and IgA 2. Depending on the heavy chain constant region, the different classes of immunoglobulins are called α, γ, and μ, respectively. The Fc portion of an antibody is directly involved in ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity) based on complement activation, C1q binding and Fc receptor binding. Complement activation (CDC) is initiated by the binding of complement factor C1q to the Fc portion of most IgG antibody subclasses. Although the effect of antibodies on the complement system depends on certain conditions, binding to C1q is caused by a defined binding site in the Fc portion. Such binding sites are known in the art and are described, for example, in Boackle, R.J., et al, Nature282 (1979) 742-743, Lukas, T.J., et al, J.Immunol.127 (1981) 2555-2560, Brunhouse, R.R., Cebra, J.J., mol.Immunol.16 (1979) 907-917, Burton, D.R., et al, Nature288 (1980) 338-344, Thommesen, J.E., et al, mol.Immunol.37 (2000) 995-1004, Idusogene, E.e., et al, J.164 (2000) 4178-4184, Hezareh, M.et al, J.Vioglol (61) 2001-319, EP-12134, EP 03031, EP 12134, et al. Such binding sites are for example L234, L235, D270, N297, E318, K320, K322, P331 and P329 (numbering according to the EU index of Kabat, e.a., see below). Antibodies of subclasses IgG1, IgG2 and IgG3 generally show complement activation and binding to C1q and C3, whereas IgG4 does not activate the complement system and does not bind to C1q and C3.

In one embodiment, the antibody according to the invention comprises an Fc part derived from human origin and preferably all other parts of the human constant region. As used herein, the term "Fc portion derived from human origin" denotes an Fc portion of a human antibody belonging to subclass IgG1, IgG2, IgG3 or IgG4, preferably an Fc portion from subclass human IgG1, a mutant Fc portion from subclass human IgG1 (preferably having a mutation on L234A + L235A), an Fc portion from subclass human IgG4 or a mutant Fc portion from subclass human IgG4 (preferably having a mutation on S228P). Most preferred is the human heavy chain constant region of SEQ ID NO:27 (subclass human IgG 1), SEQ ID NO:28 (subclass human IgG1 with mutations L234A and L235A), SEQ ID NO:29 (subclass human IgG 4), or SEQ ID NO:30 (subclass human IgG4 with mutation S228P).

In one embodiment, the antibody according to the invention is characterized in that the constant chains are of human origin. Such constant strands are well known in the art and are described, for example, in Kabat, E.A. (see, e.g., Johnson, G., Wu, T.T., nucleic acids Res.28 (2000) 214-218). For example, one useful human heavy chain constant region comprises the amino acid sequence of SEQ ID NO. 25. For example, one useful human light chain constant region comprises the kappa light chain constant region amino acid sequence of SEQ ID NO: 26. It is further preferred that the antibody is of mouse origin and comprises the antibody variable sequence framework of a mouse antibody according to Kabat.

Immunoconjugates

The invention also provides immunoconjugates comprising an anti-CSF-1R antibody herein conjugated to one or more cytotoxic agents such as a chemotherapeutic agent or drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or a radioisotope.

In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC), wherein the antibody is conjugated to one or more drugs, including but not limited to maytansinoids (maytansinoids) (see U.S. Pat. nos. 5,208,020, 5,416,064, and european patent EP0425235B 1); auristatins such as monomethyl auristatin drug modules DE and DF (MMAE and MMAF) (see U.S. Pat. nos. 5,635,483 and 5,780,588, and No.7,498,298); dolastatin (dolastatin); calicheamicin (calicheamicin) or a derivative thereof (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al, cancer Res.53:3336-3342 (1993); and Lode et al, cancer Res.58:2925-2928 (1998)); anthracyclines such as daunomycin (daunomycin) or doxorubicin (doxorubicin) (see Kratz et al, Current Med. chem.13: 477-; methotrexate; vindesine (vindesine); taxanes such as docetaxel (docetaxel), paclitaxel (paclitaxel), larotaxel, tesetaxel, and ortataxel; trichothecenes (trichothecenes); and CC 1065.

In another embodiment, the immunoconjugate comprises an antibody described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria toxin A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin (sarcin), Aleuties fordii (Aleuties fordii) toxic protein, Dianthus caryophyllus (dianthin) toxic protein, Phytolacca americana (Phytolacca) toxic protein (PAPI, PAPII, and PAP-S), Momordica charantia (Mordicacharantia) inhibitor, curculin (curcin), crotin (crotin), Saponaria officinalis (saparonia officinalis) inhibitor, gelonin (gelonin), mitogelonin (mitogelonin), restrictocin (chrysin), trichothecin (strictoxin), trichothecin (trichothecin), and enomycin (trichothecin).

In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for use in generating radioconjugates. Examples include At²¹¹、I¹³¹、I¹²⁵、Y⁹⁰、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、Bi²¹²、P³²、Pb²¹²And radioactive isotopes of Lu. Where the radioconjugate is used for detection, it may contain a radioactive atom for scintigraphic studies, e.g. Tc^99mOr I¹²³Or spin labels for Nuclear Magnetic Resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

Conjugates of the antibody and cytotoxic agent may be prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), Iminothiolane (IT), imidoesters (such as dimethyl adipimidate hcl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis (p-diazoniumbenzoyl) ethylenediamine), diisocyanates (such as toluene 2, 6-diisocyanate), and bis-active fluorine compounds (such as 1, 5-difluoro-2, 4-dinitrobenzene) is used. For example, a ricin immunotoxin may be prepared as described in Vitettaetaet, Science238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelator for conjugating radionucleotides to antibodies. See WO 94/11026. The linker may be a "cleavable linker" that facilitates release of the cytotoxic drug in the cell. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers can be used (Charieral., cancer research52: 127-.

Immunoconjugates or ADCs herein expressly encompass, but are not limited to, such conjugates prepared with the following crosslinking agents, including, but not limited to: commercial BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl- (4-vinylsulfone) benzoate), e.g., from Pierce Biotechnology Inc., Rockford, IL, U.S.A..

Therapeutic methods and compositions

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

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

A preferred embodiment of the invention is a CSF-1R antibody of the invention for use in the treatment of a "CSF-1R mediated disease", or a CSF-1R antibody of the invention for use in the preparation of a medicament for the treatment of a "CSF-1R mediated disease", as may be described by:

CSF-1R signaling is likely to be involved in tumor growth and metastasis through 3 unique mechanisms. The first is to find expression of CSF ligands and receptors in tumor cells derived from the female reproductive system (breast/breast, ovary, endometrium, cervix) (Scholl, S.M., et al, J.Natl.cancer Inst.86 (1994) 120-126; Kacinski, B.M., mol.Reprod.Dev.46 (1997) 71-74; Ngan, H.Y., et al, Eur.J.cancer35 (1999) 1546-1550; Kirma, N.et al, cancer Res67 (2007) 1918-1926) and this expression is associated with growth of breast cancer xenografts and poor prognosis in breast cancer patients. Two point mutations in CSF-1R were seen in about 10-20% of patients with acute myelogenous leukemia, chronic myelogenous leukemia, and myelodysplasia tested in one study, and one of the mutations was found to disrupt receptor turnover (Ridge, S.A., et al, Proc. Natl. Acad. SciUSA87 (1990) 1377-1380). However, the incidence of mutations could not be confirmed in later studies (Abu-Duhier, F.M., et al, Br.J.Haematol.120 (2003) 464-470). Mutations have also been found in some hepatocellular carcinoma (Yang, D.H., et al, hepatobiliaryPancreat. Dis. Int.3 (2004) 86-89) and idiopathic myelofibrosis (Abu-Duhier, F., M., et al, Br. J. Haematol.120 (2003) 464-470) cases.

Pigmented villonodular synovitis (PVNS) and tenosynostomegalomatosis (TGCT) can occur due to translocations that fuse the M-CSF gene to the collagen gene COL6A3 and result in overexpression of M-CSF (West, R.B., et al, Proc. Natl. Acad. Sci. USA103 (2006) 690-. It has been proposed that the landscape effect (landscapeffect) is associated with causing a tumor mass consisting of monocytes attracted by M-CSF expressing cells. TGCT is a small tumor that can be excised relatively easily from the finger (where it occurs predominantly). PVNS is more aggressive because it can replicate in large joints and is not easily controlled by surgery.

The second mechanism is based on blocking signaling through M-CSF/CSF-1R at metastatic sites in bone, which induces osteoclastogenesis, bone resorption and osteolytic bone damage. Breast, multiple myeloma and lung cancers are examples of cancers that have been found to metastasize to bone and cause osteolytic bone disease leading to skeletal complications. M-CSF released by tumor cells and stroma in cooperation with receptor activator nuclear factor kappa-B ligand-RANKL induces differentiation of hematopoietic myeloid monocyte progenitors into mature osteoclasts. In this process, M-CSF functions as a permissive factor by giving osteoclasts a survival signal (Tanaka, S., et al, J.Clin.invest.91 (1993) 257-263). Inhibition of CSF-1R activity during osteoclast differentiation and maturation using an anti-CSF-1R antibody is likely to prevent osteoclast activity imbalance in metastatic disease leading to osteolytic disease and related skeletal related events. Although breast, lung and multiple myeloma cancers often result in osteolytic lesions, in prostate cancer, metastases to the bone initially have an osteoblastic appearance, with increased bone forming activity resulting in "braided bone" as opposed to the typical layered structure of normal bone. During disease progression, bone lesions exhibit significant osteolytic components and high serum levels of bone resorption and suggest that anti-resorptive therapy may be useful. Bisphosphonates have been shown to inhibit the formation of osteolytic lesions and to reduce the number of skeletal-related events (only in men with hormone-refractory metastatic prostate cancer), but in this regard their effect on osteoblast damage is controversial and bisphosphonates have not been shown to be beneficial in the prevention of bone metastasis or hormone-responsive prostate cancer to date. The efficacy of antiresorptive agents in mixed osteolytic/osteoblastic prostate cancer is still under clinical investigation (Choueri, M.B., et al, cancer MetastasisRev.25 (2006) 601-609; Vessella, R.L. and Corey, E., Clin. cancer Res.12 (20 Pt 2) (2006) 6285s-6290 s).

The third mechanism is based on the observation that tumor-associated macrophages (TAMs), which have been recently found in solid tumors of breast, prostate, ovarian and cervical cancers, are associated with a poor prognosis (big, L., et al, J.Pathol.196 (2002) 254-. Macrophages are recruited to the tumor by M-CSF and other chemokines. Macrophages can then promote tumor progression via secretion of angiogenic factors, proteases, and other growth factors and cytokines, and can be blocked by inhibiting CSF-1R signaling. Recently, Zins, k.et al, cancer res.67 (2007) 1038-. TNF α -targeting sirnas secreted by human SW620 cells reduced mouse M-CSF levels and resulted in a reduction of macrophages in tumors. In addition, treatment of MCF7 tumor xenografts with antigen-binding fragments against M-CSF did result in 40% tumor growth inhibition when administered in combination with chemotherapeutic agents, reversing resistance to chemotherapeutic agents and improving mouse survival (Paulus, p., et al, cancer res.66 (2006) 4349-.

TAMs are the only example of a link that occurs 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, cancer occurs at the site of chronic inflammation, and chemical mediators of inflammation are found in many cancers; cellular or chemical mediators that eliminate inflammation inhibit the development of experimental cancers, and chronic use of anti-inflammatory agents reduces the risk of some cancers. There are many inflammatory conditions linked to cancer, among which is h.pylori (h.pyri) -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 cell7 (2005) 211-. Macrophages are key cells in chronic inflammation and respond differentially to their microenvironment. There are two classes of macrophages considered to be the extremes of successive functional states: m1 macrophages are involved in type 1 responses. These reactions involve activation by microbial products and thus killing of pathogenic microorganisms, producing reactive oxygen mediators. At the other extreme, M2 macrophages, which are involved in type 2 responses, promote cell proliferation, regulate inflammation and adaptive immunity, and promote tissue remodeling, angiogenesis and repair (Mantovani, a., et al, trends immunol.25 (2004) 677-. Chronic inflammation leading to the establishment of neoplasia is often associated with M2 macrophages. One key cytokine mediating the inflammatory response is TNF α, which is notoriously able to stimulate anti-tumor immunity and hemorrhagic necrosis at high doses, but has recently been found to be expressed by tumor cells and to act as a tumor promoter (Zins, k., et al, cancer res.67 (2007) 1038-. There remains a need to better understand the specific role of macrophages with respect to tumors, including the potential spatial and temporal dependence on their function and association with specific tumor types.

Thus, one embodiment of the invention is a CSF-1R antibody of the invention for use in the treatment of cancer. As used herein, the term "cancer" may be, for example, lung cancer, non-small cell lung (NSCL) cancer, bronchoalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, cancer of the stomach, colon cancer, breast cancer, cancer of the uterus, cancer of the fallopian tubes, cancer of the endometrium, cancer of the cervix, cancer of the vagina, cancer 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, cancer of the renal cells, carcinoma of the renal pelvis, mesothelioma, hepatocellular carcinoma, cancer of the gall bladder, Central Nervous System (CNS) neoplasms, spinal axis tumors, brain stem glioma, glioblastoma multiforme, Schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, lymphoma, lymphocytic leukemia, including refractory forms of any of the foregoing cancers, or a combination of one or more of the foregoing cancers. Preferably, such cancer is breast cancer, ovarian cancer, cervical cancer, lung cancer or prostate cancer. Preferably, such cancers are further characterized by CSF-1 or CSF-1R expression or overexpression. Yet another embodiment of the invention is a CSF-1R antibody of the invention for use in the simultaneous treatment of primary tumors and new metastases.

Thus, another aspect of the invention is a CSF-1R antibody of the invention for use in the treatment of periodontitis, histiocytosis X, osteoporosis, Paget's disease of the bone (PDB), bone loss due to cancer therapy, periprosthetic osteolysis, glucocorticoid-induced osteoporosis, rheumatoid arthritis, psoriatic arthritis, osteoarthritis, inflammatory joint disease (inflammatorthritis), and inflammation.

Rabello, d.et al, biochem. biophysis. res. commun.347 (2006) 791-796 demonstrated that SNP in the CSF1 gene exhibited a positive association with aggressive periodontitis, a periodontal inflammatory disease in which tooth loss is caused by resorption of alveolar bone.

Histiocytosis disease X (also known as Langerhans' histiocytosis, LCH) is a proliferative disease of Langerhans dendritic cells that appear to differentiate into osteoclasts in bone and additional bone LCH damage. Langerhans cells are derived from monocytes in circulation. Elevated M-CSF levels measured in serum and lesions were found to correlate with disease severity (daCosta, c.e., et al, j.exp. med.201 (2005) 687-693). The disease occurs primarily in pediatric patient populations and has to be treated with chemotherapy when the disease becomes systemic or relapses.

The pathophysiology of osteoporosis is mediated by osteoblast loss and increased osteoclast-dependent bone resorption to form bone. Supportive data is described in Cenci et al, showing that injection of anti-M-CSF antibody maintains bone density and inhibits bone resorption in ovariectomized mice (Cenci, s., et al, j.clin.invest.105 (2000) 1279-. Recently, a potential link between postmenopausal bone loss due to estrogen deficiency was identified and the presence of T cells producing TNF α was found to affect bone metabolism (Roggia, C., et al, Minervamed.95 (2004) 125-132). One possible mechanism is the induction of M-CSF by TNF α in vivo. The important role of M-CSF in TNF- α -induced osteoclastogenesis is demonstrated by the effect that antibodies directed against M-CSF block TNF α -induced osteolysis in mice, thereby making inhibitors of CSF-1R signaling a potential target for inflammatory arthritis (Kitaura, H., et al, J.Clin.invest.115 (2005) 3418-3427).

Paget's disease of the bone (PDB) is the second most common disorder of bone metabolism after osteoporosis, where focal abnormalities of increased bone turnover lead to complications such as bone pain, deformity, pathological fracture and deafness. Mutations in four genes have been identified that modulate normal osteoclast function and predispose individuals to PDB and related disorders: insertional mutations in TNFRSF11A encoding receptor activator of Nuclear Factor (NF) kb (RANK), a key regulator of osteoclast function, inactivating mutations in TNFRSF11B encoding osteoprotegerin (decoy receptor for RANK ligand), mutations in the spacer (sequestosome) 1 gene (SQSTM 1) encoding an important scaffold protein in the NF κ B pathway, and mutations in the Valosin Containing Protein (VCP) gene. This gene encodes VCP, which has a role in targeting NF κ B inhibitors to proteasome-induced degradation (Daroszewska, a., Ralston, s.h., nat. clin. pract. rheumato.2 (2006) 270-277). Targeted CSF-1R inhibitors offer the opportunity to indirectly block dysregulation of RANKL signaling and add additional treatment options to currently used bisphosphonates.

Cancer treatment-induced bone loss, particularly in breast and prostate cancer patients, is another indication that targeted CSF-1R inhibitors can prevent bone loss (Lester, j.e., et al, br.j. cancer94 (2006) 30-35). The long-term consequences of adjuvant therapy become more important as the prognosis of early breast cancer improves, as some therapies (including chemotherapy, irradiation, aromatase inhibitors and ovariectomy) affect bone metabolism by reducing bone mineral density, leading to increased risk of osteoporosis and associated fractures (Lester, j.e., et al, br.j. cancer94 (2006) 30-35). Equivalent to adjuvant aromatase inhibitor therapy in breast cancer is androgen ablation therapy in prostate cancer, which results in a significant increase in loss of bone mineral density and 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 may be beneficial in other indications as well as when targeting cell types including osteoclasts and macrophages (e.g., to treat specific complications of joint replacement surgery in response to rheumatoid arthritis). Periprosthetic bone loss and hence implant failure due to prosthetic loosening is a major complication of joint replacement surgery and requires repeated surgery, placing a high socio-economic burden on the patient's individual and 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 where CSF-1R inhibitors can prevent bone loss following chronic administration of glucocorticosteroids due to various conditions such as chronic obstructive pulmonary disease, asthma, and rheumatoid arthritis (Guzman-Clark, J.R., et al, Arthritis Rheum.57 (2007) 140-2174; Feldstein, A.C., et al, Osteporos. int.16 (2005) 2168-2174).

Rheumatoid arthritis, psoriatic arthritis and inflammatory arthropathies are themselves potential indications for inhibitors of CSF-1R signaling, as they are composed of macrophage components and varying degrees of bone destruction (ritclin, c.t., et al, j.clin.invest.111 (2003) 821-. Osteoarthritis and rheumatoid arthritis are inflammatory autoimmune diseases caused by macrophage accumulation in connective tissue and macrophage infiltration into synovial fluid (which is mediated at least in part by M-CSF). Campbell, I.K., et al, J.Leukoc.biol.68 (2000) 144-. Inhibition of CSF-1R signaling has the potential to control macrophage numbers in joints and to reduce pain from associated bone destruction. To minimize adverse effects and to further understand the effects of CSF-1R signaling in these indications, one approach is to specifically inhibit CSF-1R without targeting a myriad of other kinases, such as Raf kinase.

Recent literature reports correlate elevated circulating M-CSF with atherosclerotic progression and poor prognosis in chronic coronary artery disease (Saitoh, T., et al, J.Am.Coll.Cardiol.35 (2000) 655-; M-CSF influences the atherosclerotic process by helping to form foam cells (macrophages with ingested oxidized LDL) that express CSF-1R and represent the initial plaque (Murayama, T., et al, Circulation99 (1999) 1740-1746).

Expression and signaling of M-CSF and CSF-1R are found in activated microglia. Microglia (i.e., resident macrophages of the central nervous system) can be activated by a variety of insults, including infection and trauma. M-CSF is considered to be a key regulator of inflammatory responses in the brain, and M-CSF levels are elevated in HIV-1, encephalitis, Alzheimer's Disease (AD) and brain tumors. Microgliosis (as a consequence of autocrine signaling through M-CSF/CSF-1R) results in the induction of inflammatory cytokine and nitric oxide release as demonstrated, for example, by using an experimental neuronal damage model (Hao, A.J., et al, Neuroscience112 (2002) 889-. Microglia with increased CSF-1R expression were 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 less microglia in the brain cause fibril deposition and neuronal loss of A-. beta.compared to normal controls, suggesting that microglia indeed have neuroprotective function in the development of AD depletion in op/op mice (Kaku, M., et al, BrainRes. Protoc.12 (2003) 104- "108).

Expression and signaling of M-CSF and CSF-1R are associated with Inflammatory Bowel Disease (IBD) (WO 2005/046657). The term "inflammatory bowel disease" refers to a severe, chronic intestinal disorder characterized by chronic inflammation at multiple sites in the gastrointestinal tract, and specifically includes Ulcerative Colitis (UC) and Crohn's disease.

The antibodies of the invention are characterized by including antibodies that bind to human CSF-1R, characterized by the epitope binding properties described above or by the amino acid sequences and amino acid sequence fragments described above, for use in the treatment of cancer.

The antibodies of the invention are characterized by comprising antibodies that bind to human CSF-1R, characterized by the epitope binding properties described above or by the amino acid sequences and amino acid sequence fragments described above, for use in the treatment of bone loss.

The antibodies of the invention are characterized by including antibodies that bind to human CSF-1R, characterized by the epitope binding properties described above or by the amino acid sequences and amino acid sequence fragments described above, for use in the prevention or treatment of metastasis.

The antibodies of the invention are characterized by including antibodies that bind to human CSF-1R, characterized by the epitope binding properties described above or by the amino acid sequences and amino acid sequence fragments described above, for use in the treatment of inflammatory diseases.

The antibodies of the invention are characterized by comprising the use of an antibody (characterized by comprising an antibody that binds human CSF-1R, characterized by the epitope binding properties described above or by the amino acid sequences and amino acid sequence fragments described above) for the treatment of cancer or for the manufacture of a medicament for the treatment of cancer.

The invention includes the use of an antibody (characterised in that it includes an antibody that binds human CSF-1R, characterised in that it is characterized by the epitope binding properties described above or in that it is an amino acid sequence and amino acid sequence fragment described above) for the treatment of bone loss or for the manufacture of a medicament for the treatment of bone loss.

The invention includes the use of an antibody (characterised in that it includes an antibody that binds human CSF-1R, characterised by the epitope binding properties described above or by the amino acid sequences and amino acid sequence fragments described above) for the prevention or treatment of metastasis or for the manufacture of a medicament for the prevention or treatment of metastasis.

The invention includes the use of an antibody (characterised in that it includes an antibody that binds human CSF-1R, characterised in that it is characterized by the epitope binding properties described above or in that it is an amino acid sequence and amino acid sequence fragment described above) for the treatment of an inflammatory disease or for the manufacture of a medicament for the treatment of an inflammatory disease. In one embodiment, an antibody according to the invention inhibits CSF-1 binding to CSF-1R with an IC50 of 75ng/ml or less, and in one embodiment, CSF-1 binding to CSF-1R with an IC50 of 50ng/ml or less. IC50 inhibiting 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 with an IC50 of 150ng/ml or less, in one embodiment CSF-1 induced CSF-1R phosphorylation with an IC50 of 100ng/ml or less, in one embodiment CSF-1 induced CSF-1R phosphorylation with an IC50 of 50ng/ml or less, and in one embodiment CSF-1 induced CSF-1R phosphorylation with an IC50 of 25ng/ml or less (in NIH3T3-CSF-1R recombinant cells). IC50 inhibiting CSF-1 induced CSF-1R phosphorylation can be determined as shown in example 3.

In one embodiment, the antibody according to the invention inhibits the growth of recombinant NIH3T3 cells expressing human CSF-1R (SEQ ID NO:15) by 80% or more (compared to the antibody deletion), preferably by 90% or more. % growth inhibition was determined as shown in example 6, where% survival was measured. The% growth inhibition was calculated from% survival as follows: % growth inhibition =100-% survival. For example, < CSF-1R >7G5.3B6 showed 100-2=98% inhibition of growth of NIH3T3 cells expressing wild-type human CSF-1R.

In one embodiment, the antibody according to the invention stimulates the growth of recombinant NIH3T3 expressing human mutant CSF-1RL301SY969F (SEQ ID NO:16) by 5% or more (compared to in the absence of antibody), in one embodiment by 20% or more. % growth stimulation was determined as shown in example 6, where% survival was measured. The% growth stimulation was calculated from% survival as follows: % growth stimulation = - (100-% survival). For example, < CSF-1R >7G5.3B6 shows growth stimulation of NIH3T3 cells expressing mutant human CSF-1R by- (100-0) = - (100-.

In one embodiment, an antibody according to the invention inhibits the growth of BeWo tumor cells (ATCCCL-98) by 70% or more (at a concentration of 10. mu.g/ml antibody; and compared to the absence of antibody), preferably by 80% or more. % growth inhibition was determined as shown in example 7. For example, < CSF-1R >7G5.3B6 showed 89% inhibition of growth of BeWo tumor cells.

In one embodiment, the antibodies according to the invention inhibit macrophage differentiation. In one embodiment, the antibody according to the invention inhibits the survival of monocytes with an IC50 of 1.5nM or lower than 1.5nM, preferably with an IC50 of 1.0nM or lower than 1.0 nM. Inhibition of monocyte survival was determined as shown in example 8.

Yet another embodiment of the invention is a method for the production of antibodies against CSF-1R, characterized in that the nucleic acid encoding the heavy chain of a human IgG 1-type antibody binding to human CSF-1R according to the invention (the modified nucleic acid) and the nucleic acid encoding the light chain of said antibody are inserted into an expression vector, said vector is inserted into a eukaryotic host cell, the encoded protein is expressed and recovered from the host cell or supernatant.

Pharmaceutical formulations

The term "pharmaceutical formulation" refers to a formulation in a form that allows the biological activity of the active ingredients included therein to be effective, and that does not contain other ingredients that have unacceptable toxicity to the subject to which the formulation is to be administered.

In another aspect, the invention provides compositions, e.g., pharmaceutical compositions, comprising one or a panel of monoclonal antibodies of the invention, or antigen-binding portions thereof, formulated with a pharmaceutically acceptable carrier.

"pharmaceutically acceptable carrier" refers to a component of a pharmaceutical formulation other than the active ingredient that is not toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives. As used herein, "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption/resorption delaying agents, and the like. Preferably, the carrier is suitable for injection or infusion.

The compositions of the present invention may be administered by a variety of methods known in the art. As the skilled artisan will appreciate, the route and/or pattern of administration will vary with the desired result.

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

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

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

The invention includes the use of an antibody according to the invention for the treatment of a patient suffering from cancer, in particular colon, lung or pancreatic cancer.

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

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

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

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

Article of manufacture

In another aspect of the invention, there is provided an article of manufacture comprising a material as described above useful for the treatment, prevention and/or diagnosis of a condition. The article comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, vials, cartridges, syringes, IV solution bags, and the like. The container may be made of a variety of materials, such as glass or plastic. The container contains a composition effective, alone or in combination with another composition, in the treatment, prevention and/or diagnosis of the condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an antibody of the invention. The label or package insert indicates that the composition is for use in treating the selected condition. In addition, an article of manufacture can comprise (a) a first container having a composition therein, wherein the composition comprises an antibody of the invention; and (b) a second container having a composition therein, wherein the composition includes an additional cytotoxic agent or other therapeutic agent. The article of manufacture in this embodiment of the invention may also include a package insert indicating that the composition is useful for treating a particular condition. Alternatively or additionally, the article of manufacture may further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. It may further include other materials as needed from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

It will be appreciated that any of the above preparations may include an immunoconjugate of the invention in place of or in addition to the anti-CSF-1R antibody.

The following examples and sequence listing are provided to aid in the understanding of the present invention, the following claims being set forth to illustrate the true scope of the invention. Those skilled in the art will appreciate that changes may be made in the protocols set forth without departing from the spirit of the invention.

Antibody preservation

Cell lines	Accession number	Date of storage
			<CSF-1R>7H5.2G10	DSM ACC2922	10.06.2008

Description of sequence listing

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. Those skilled in the art will appreciate that changes may be made in the protocols set forth without departing from the spirit of the invention.

Example III

Example 1

Generation of hybridoma cell lines producing anti-CSF-1R antibodies

Immunization protocol for NMRI mice

Expression vector pDisplay encoding the extracellular domain of huCSF-1R for electroporation^TM(Invitrogen, USA) NMRI mice were immunized. Each mouse was immunized 4 times with 100. mu.g of DNA. When serum titers against huCSF-1R were found to be sufficient, mice were boosted intravenously (i.v.) once with an additional 50 μ g of huCSF-1RECD/huCSF-1RECDhuFc chimera 1: 1 mixture in 200 μ l PBS 4 and 3 days prior to fusion.

Antigen-specific ELISA

anti-CSF-1R titers in sera of immunized mice were determined by antigen-specific ELISA.

Mu.g/ml of the huCSF1R-huFc chimera (soluble extracellular domain) were captured on streptavidin plates (MaxiSorb; MicroCoat, DE, Cat. No.11974998/MC 1099) with 0.1mg/ml of biotinylated anti-Fc γ (Jackson ImmunoResearch., Cat. No. 109-066-Asca 098), followed by addition of 1/800 horseradish peroxidase (HRP) -conjugated F (ab')₂Anti-mouse IgG (GEHealthcare, UK, Cat. No. NA9310V). Serum 1/40 from all blood draws (tap) was diluted in PBS/0.05% Tween20/0.5% BSA and serially diluted to 1/1638400. Diluted serum was added to the wells. Pre-bleed (pre-tap) sera were used as negative controls. From 500ng/ml to 0.25ng/ml mouse anti-human CSF-1RMab3291 (R)&DSystems, UK) serial dilutions were used as positive controls. All components were incubated together for 1.5 hours. The wells were washed 6 times with PBST (PBS/0.2% Tween 20) and then with freshly prepared ABTSSolution (1 mg/ml) (ABTS: 2, 2' -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) the assay was developed for 10 minutes at room temperature. Absorbance at 405nm was measured.

Hybridoma production

Mouse lymphocytes can be isolated and fused with a mouse myeloma cell line with PEG based on standard protocols for hybridoma production. The resulting hybridomas are then screened for the production of antigen-specific antibodies. For example, a single cell suspension of spleen-derived lymphocytes from immunized mice was fused with 50% PEG to Ag8 non-secreting mouse myeloma cells P3X63Ag8.653 (ATCC, CRL-1580). The cells were expanded to about 10⁴Seeded into flat-bottomed 96-well microtiter plates and then incubated on selective media for about two weeks. Individual wells were then screened by ELISA for human anti-CSF-1R monoclonal IgM and IgG antibodies. Once extensive hybridoma growth has occurred, antibody-secreting hybridomas are re-inoculated, screened again, and then subcloned by FACS if the anti-CSF-1R monoclonal antibody remains positive for human IgG. The stable subclones were then cultured in vitro to produce antibodies in tissue culture medium for characterization.

Culture of hybridomas

The resulting muMAb hybridomas were treated in RPMI1640 (PAN-Cat.No. PO4-17500) supplemented with 2 mML-glutamine (GIBCO-Cat.No. 35050-038), 1mM sodium pyruvate (GIBCO-Cat.No. 11360-039), 1xNEAA (GIBCO-Cat.No. 11140-035), 10% FCS (PAA-Cat.No. A15-649), 1 xPhenStrep (Roche-Cat.No. 1074440), 1 xNeutridomaCS (Roche-Cat.No. 1363743), 50 μ M mercaptoethanol (GIBCO-Cat.No. 31350-010) and 50U/ml mouse IL-6 (Roche-Cat.No. 1444581) at 37 ℃ and 5% CO₂The culture is carried out.

Example 2

Inhibition of CSF-1 binding to CSF-1R (ELISA)

The tests were carried out in 384 well microtiter plates (Microcoat, DE, Cat. No. 464718) at room temperature. The plates were washed 3 times with PBST after each incubation step.

Initially, with 0.5mg/ml goat F (ab')₂Biotinylated anti-Fc γ (jacksonimmunoresearch, inc.,Cat.No. 109-006-.

The wells were then supplemented with 0.2% TweenPBS blocking of-20 and 2% BSA (Roche diagnostics GmbH, DE) for 0.5 h. 75ng/ml of the huCSF-1R-huFc chimera (soluble extracellular domain) was immobilized on the plate for 1 hour. Purified antibody diluted in PBS/0.05% Tween20/0.5% BSA was then incubated for 1 hour. BAF216 (R) was cloned after addition of 3ng/ml CSF-1 (Biomol, DE, Cat. No. 60530), 50ng/ml biotinylated anti-CSF-1&DSystems, UK) and 1:5000 dilution of streptavidin HRP (Roche diagnostics GmbH, DE, Cat. No. 11089153001) for 1 hour, the plates were washed 6 times with PBST. An anti-CSF-1 RSC-02, clone 2-4A5 (Santa Cruz Biotechnology, US), which inhibits ligand-receptor interaction, was used as a positive control. Plates were plated with freshly prepared BMbluePOD substrate solution (BMblue)3,3 '-5, 5' -tetramethylbenzidine, Roche diagnostics GmbH, DE, Cat. No. 11484281001) at room temperature for 30 minutes. The absorbance at 370nm was measured. All anti-CSF-1R antibodies showed significant inhibition of CSF-1 binding to CSF-1R (see Table 1). anti-CSF-1 RSC-02, clone 2-4A5 (Santa Cruz Biotechnology, US), which inhibits ligand-receptor interaction, was used as a reference control.

Table 1: IC for inhibition of CSF-1/CSF-1R interaction₅₀Calculated value

Antibodies	IC50CSF-1/CSF-1R inhibition [ ng/ml]
		<CSF-1R>7H5.2G10	26.9
<CSF-1R>10A4.1G11	63.4
		<CSF-1R>6G4.1C8	21.2
SC-02, clone 2-4A5	30.9

Example 3

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

4.5x10³Retroviral infection of NIH3T3 cells with the expression vector for full-length CSF-1R was performed in DMEM (PAACat. No. E15-011), 2 mML-glutamine (Sigma, Cat. No. G7513), 2mM sodium pyruvate, 1x non-essential amino acids, 10% FKS (PAA, Cat. No. A15-649) and 100. mu.g/ml PenStrep (Sigma, Cat. No. P4333[10 mg/ml)]) Until they reach confluence. Then supplemented with sodium selenite [5ng/ml ]](Sigma, Cat. No. S9133), transferrin [ 10. mu.g/ml]（Sigma，Cat.No.T8158）、BSA[400μg/ml](Roche diagnostics GmbH, Cat. No. 10735078), 4 mML-glutamine (Sigma, Cat. No. G7513), 2mM sodium pyruvate (Gibco, Cat. No. 11360), 1X non-essential amino acids (Gibco, Cat:11140-]Serum-free DMEM medium (PAACat. No. E15-011) (Merck, Cat. No. M7522), 100. mu.g/ml and PenStrep (Sigma, Cat. No. P4333) cells were washed and incubated in 30. mu.l of the same medium for 16 hours to incubateUp-regulating the receptor. Mu.l of the diluted anti-CSR-1R antibody was added to the cells for 1.5 hours. Cells were then stimulated with 10. mu.l of 100ng/ml hum-CSF-1 (BiomolCat. No. 60530) for 5 minutes. After incubation, the supernatant was removed and the cells were rinsed 2 times with 80. mu.l of ice cold PBS and 50. mu.l of freshly prepared ice cold lysis buffer (150 mM NaCl/20mM Tris pH7.5/1mM EDTA/1mM EGTA/1% Triton X-100/1 protease inhibitor tablets (Roche diagnostics GmbH, Cat. No. 1836170) per 10ml buffer/10. mu.l/ml phosphatase inhibitor cocktail 1 (Sigma, Cat. No. P-2850, 100 Xstock)/10. mu.l/ml protease inhibitor 1 (Sigma, Cat. No. P-5726, 100 Xstock)/10. mu.l/ml 1M Na F) was added. After 30 minutes of ice-deposition, the plates were shaken vigorously on a plate shaker for 3 minutes and then centrifuged at 2200rpm for 10 minutes (Heraeus Megafuge 10).

Cell lysates were analyzed by ELISA for the presence of phosphorylated and total CSF-1 receptors. Using a catalyst derived from R&The DSystems (Cat. No. DYC 3268-2) kit detects phosphorylated receptors according to the supplier's instructions. To detect total CSF-1R, 10. mu.l of lysate was immobilized on the plate using the capture antibody included in the kit. Followed by the addition of a 1:750 dilution of the biotinylated anti-CSF-1R antibody BAF329 (R)&DSystems) and a 1: 1000 dilution of streptavidin-HRP conjugate. After 60 minutes the plates were washed with freshly prepared ABTSThe solution was developed and the absorbance was measured. Data were calculated as the percentage of positive control in the absence of antibody and the expressed phosphorylation/total receptor ratio. The negative control was defined as no M-CSF-1 added. anti-CSF-1 RSC-02, clone 2-4A5 (Santa Cruz Biotechnology, US, also see Sherr, C.J., et al, Cell41(1985) 665-.

Table 2: IC for inhibition of CSF-1 receptor phosphorylation₅₀The values are calculated.

Antibodies	IC50 CSF-1R phosphorylation [ ng/ml]
		<CSF-1R>7H5.2G10	49.0
<CSF-1R>10A4.1G11	15.4
		<CSF-1R>6G4.1C8	82.6
SC-02,clone 2-4A5	412.0

Example 4

Determination of the affinity of an anti-CSF-1R antibody for CSF-1R

The instrument comprises the following steps: BIACOREA100

Chip: CM5 (Biacore BR-1006-68)

Coupling: amine coupling

Buffer solution: PBS (Biacore BR-1006-72), pH7.4, 35 deg.C

For affinity measurements, 36. mu.g/ml anti-mouse Fc γ antibody (from goat, Jackson ImmunoReasearChJIR 115-005-071) was coupled to the chip surface to capture antibody against CSF-1R. Human CSF-1RECD (R & D-Systems329-MR or internally subcloned pCMV-presS-HisAvitag-hCSF-1R-ECD) was added at various concentrations in the solution. Binding was measured by CSF-1R injection at 35 ℃ for 1.5 min; dissociation was measured by washing the chip surface with buffer for 10 min at 35 ℃. anti-CSF-IRSC-02, clone 2-4A5 (Santa Cruz Biotechnology, US; see also Sherr, C.J., et al, Cell41(1985) 665-676), which inhibits ligand-receptor interaction, was used as a reference control.

For the calculation of kinetic parameters, the Langmuir 1: 1 model was used.

Table 3: by SPR (BIACORE)A100) Affinity data measured at 35 ℃

Antibodies	K_D(nM)	k_a(1/Ms)	k_d(1/s)	t_1/2(min)
					<CSF-1R>7H5.2G10	0.54	7.0E+05	3.8E-04	30.40
<CSF-1R>10A4.1G11	1.77	7.4E+05	1.3E-03	8.89
					<CSF-1R>6G4.1C8	0.52	5.7E+05	2.9E-04	39.43
SC-02, clone 2-4A5	2.73	5.09E+05	1.39E-03	8.31

Example 5

Epitope mapping of anti-CSF-1R monoclonal antibodies based on cross-competition using SPR

The instrument comprises the following steps: BIACOREA100

Chip: CM5 (Biacore BR-1006-68)

Coupling: amine coupling

Buffer solution: PBS (Biacore BR-1006-72), pH7.4, 25 deg.C

For epitope mapping assay via cross-competition, 36. mu.g/ml anti-mouse Fc γ antibody or anti-rat Fc γ antibody (from goat, Jackson ImmunoReasearChJIR115-005-Antibodies to CSF-1R. After capture from 5. mu.g/ml of anti-CSF-1R monoclonal antibody, the free binding capacity of the capture antibody was blocked with 250. mu.g/ml mouse or rat immunoglobulins (PierceCat. No.31202 and PierceCat. No. 31233) followed by injection of 12.5. mu.g/ml CSF-1R (R)&D-SystemsCat. No. 329-MR) for 2 minutes. The binding of the second anti-CSF-1R antibody was analyzed by injection for 2 minutes and dissociation was measured by washing with buffer for 5 minutes. The determination and measurement were carried out at 25 ℃. Specific binding of the second anti-CSF-1R antibody was referenced to a point set up against the same chip but not injected with CSF-1R alone. Cross-competition data was calculated as percent (%) expected binding response of the second anti-CSF-1R antibody. The term "percent (%) expected binding response" for binding of the second antibody is calculated by "100 relative response (general _ stability _ early)/rmax", where rmax is calculated by "relative response (general _ stability _ late) antibody molecular weight/antigen molecular weight", as specified for Biacore epitope mapping (for Biacore epitope mapping)A100 apparatus).

The minimum binding response was also calculated from the pair of identified antibodies 1 and 2. Thus, the maximum value of +50% obtained was set as the threshold for significant binding competition (see table X, e.g. for antibody < CSF-1R >7H5.2G10, the calculated threshold was 7+3.5= 10.5). Thus, an "anti-CSF-1R antibody binds to the same epitope as < CSF-1R >7 H5.2G10" has an expected percent binding response (%) of > 10.5.

anti-CSF-IRSC-02, clone 2-4A5 (Santa Cruz Biotechnology, US; see also Sherr, C.J., et al, Cell41(1985) 665-676), which inhibits ligand-receptor interaction, was used as a reference control.

Table 4: epitope mapping of anti-CSF-1R antibodies via cross-competition data

The results indicate that antibodies < CSF-1R >7H5.2G10, < CSF-1R >10A4.1G11 all bound the same epitope, whereas, for example, SC-2-4a5 bound another epitope and did not cross-react (compete for binding) with the antibody according to the invention.

Example 6

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

NIH3T3 cells infected with retrovirus using expression vectors of full-length wild-type CSF-1R (SEQ ID NO: 31) or mutant CSF-1RL301SY969F (SEQ ID NO: 32) were cultured on poly-HEMA (poly (2-hydroxymethacrylate))) (Polysciences, Warrington, PA, USA)) coated (to prevent adhesion to plastic surfaces) on DMEM high glucose medium (PAA, Pasching, Austria) supplemented with 2 mML-glutamine, 2mM sodium pyruvate and non-essential amino acids and 10% fetal bovine serum (Sigma, Taufkirchen, Germany). Cells were seeded in medium with serum replaced with 5ng/ml sodium selenite, 10mg/ml transferrin, 400. mu.g/ml BSA and 0.05mM 2-mercaptoethanol. Cells expressing wtCSF-1R form three-dimensionally growing dense ellipsoids when treated with 100ng/ml huCSF-1 (Biomol, Hamburg, Germany), a property known as anchorage independence (anchorage independence). These ellipsoids approximate the three-dimensional structure and organization of solid tumors in situ. Mutant CSF-1R recombinant cells are capable of forming ellipsoids independent of CSF-1 ligand. The ellipsoid cultures were incubated in the presence of 10. mu.g/ml antibody for 3 days. Cell viability was measured by measuring the ATP content of the cells using the CellTiterGlo assay.

Table 5:

^**the average of 15 different experiments was found to be,

^***average of 6 different experiments.

Example 7

Growth inhibition of BeWo tumor cells in 3D culture under treatment with anti-CSF-1R monoclonal antibody (CellTiterGlo-assay)

BeWo choriocarcinoma cells (ATCCCL-98) were cultured in F12K medium (Sigma, Steinheim, Germany) supplemented with 10% FBS (Sigma) and 2 mML-glutamine. Mix 5x10⁴Individual cells/well were seeded in 96-well poly-HEMA (poly-2-hydroxyethyl methacrylate) coated plates containing F12K medium supplemented with 0.5% FBS and 5% BSA. 200ng/ml huCSF-1 and 10. mu.g/ml of a different anti-CSF-1R monoclonal antibody were concomitantly added and incubated for 6 days. Cell viability was measured by measuring the ATP content of the cells in Relative Light Units (RLU) using the CellTiterGlo assay. Inhibition of CSF-1-induced growth was observed when BeWo ellipsoid cultures were treated with different anti-CSF-1R antibodies (10. mu.g/ml). To calculate antibody-mediated inhibition, RLU means of unstimulated BeWo cells were subtracted from all samples. The mean RLU values for CSF-1-stimulated cells were arbitrarily set at 100%. The mean RLU values for cells stimulated with CSF-1 and treated with anti-CSF-1R antibody were calculated as the percentage of RLU stimulated with CSF-1. Table 6 shows the calculated data; fig. 1 depicts RLU means. Each mean was obtained from triplicate experiments.

Table 6:

example 8

Inhibition of macrophage differentiation/monocyte survival under treatment with anti-CSF-1R monoclonal antibody (CellTiterGlo assay)

Using RosetteSep^TMHuman monocyte-enriched cocktail (stemcelltech. -cat. No. 15028) mononuclear cells were isolated from peripheral blood. Enriching the monocyte population at 37 ℃ and 5% CO₂Seeded in 96-well microtiter plates (2.5X 10)⁴Individual cells/well) were supplemented with 10% FCS (GIBCO-cat.no. 011-090014M), 4 ml-glutamine (GIBCO-cat.no. 25030) and 1 xpensstrep (rocheccat.no. 1074440) in 100 μ l rpmi1640 (GIBCO-cat.no. 31870). When 150ng/ml huCSF-1 was added to the medium, clear differentiation into adherent macrophages was observed. This differentiation can be inhibited by the addition of an anti-CSF-1R antibody. Furthermore, monocyte survival was affected and could be analyzed by the celltiterglo (ctg) assay. IC was calculated from the concentration-dependent inhibition of monocyte survival by antibody treatment₅₀(see Table 7).

Table 7:

antibodies	IC₅₀[nM]
		<CSF-1R>7H5.2G10	1.0
<CSF-1R>10A4.1G11	0.4
		SC-02, clone 2-4A5	2.4

PCT printout (original electronic document form)

(the page is not part of the international application and is not counted as the number of pages of the international application)

Claims

1. An antibody or variable domain-containing antibody fragment thereof that binds human CSF-1R, characterized in that:

c) The heavy chain variable domain comprises the CDR3 region SEQ ID NO:17, the CDR2 region SEQ ID NO:18, and the CDR1 region SEQ ID NO:19, and the light chain variable domain comprises the CDR3 region SEQ ID NO:20, the CDR2 region SEQ ID NO:21, and the CDR1 region SEQ ID NO: 22.

2. The antibody or antibody fragment according to claim 1, characterized in that:

the heavy chain variable domain comprises the CDR3 region SEQ ID NO:1, the CDR2 region SEQ ID NO:2, and the CDR1 region SEQ ID NO:3, and the light chain variable domain comprises the CDR3 region SEQ ID NO:4, the CDR2 region SEQ ID NO:5, and the CDR1 region SEQ ID NO: 6.

3. The antibody or antibody fragment according to claim 1, characterized in that:

the heavy chain variable domain comprises the CDR3 region SEQ ID NO. 9, the CDR2 region SEQ ID NO.10, and the CDR1 region SEQ ID NO.11, and the light chain variable domain comprises the CDR3 region SEQ ID NO. 12, the CDR2 region SEQ ID NO.13, and the CDR1 region SEQ ID NO. 14.

4. The antibody or antibody fragment according to claim 1, characterized in that:

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

5. The antibody or antibody fragment according to claim 2, characterized in that:

the amino acid sequence of the heavy chain variable domain is SEQ ID NO.7, and the amino acid sequence of the light chain variable domain is SEQ ID NO. 8.

6. The antibody or antibody fragment according to claim 3, characterized in that:

the amino acid sequence of the heavy chain variable domain is SEQ ID NO.15 and the amino acid sequence of the light chain variable domain is SEQ ID NO. 16.

7. The antibody or antibody fragment according to claim 4, characterized in that:

the amino acid sequence of the heavy chain variable domain is SEQ ID NO:23 and the amino acid sequence of the light chain variable domain is SEQ ID NO: 24.

8. Antibody or antibody fragment according to any one of claims 1 to 7, characterized in that the antibody or antibody fragment belongs to the subclass human IgG4 or to the subclass human IgG 1.

9. A pharmaceutical composition characterized by comprising an antibody or antibody fragment according to any one of claims 1 to 8.

10. Use of an antibody or antibody fragment according to any one of claims 2,3, 5 and 6 for the manufacture of a medicament for the treatment of choriocarcinoma.

11. Use of an antibody or antibody fragment according to any one of claims 2,3, 5 and 6 for the preparation of a medicament for the prevention or treatment of choriocarcinoma metastasis.

12. A nucleic acid encoding an antibody or antibody fragment according to any one of claims 1 to 8.

13. An expression vector, characterized in that it comprises a nucleic acid according to claim 12.

14. A prokaryotic or eukaryotic host cell comprising a vector according to claim 13.

15. A method for the production of an antibody or antibody fragment according to any one of claims 1 to 8, characterized in that a nucleic acid according to claim 12 is expressed in a prokaryotic or eukaryotic host cell and the antibody or antibody fragment is recovered from the cell or cell culture supernatant.