MX2008008948A - Compositions and methods of use for antibodies of dickkopf-1 and/or -4 - Google Patents

Abstract

Antibodies and fragments that bind to the protein target Dickkopf (DKK1) are provided, as are methods of use and kits, for treating a target cell, in particular, a cell associated with an osteolytic condition.

Description

COM POSITIONS AND METHODS OF USE FOR ANTICU ERPOS OF DICKKOPF-1 AND / OR -4 FIELD OF USE The present invention relates to compositions and methods of use for antibodies of Dickkopf-1 ("DKK1"), Dickkopf-4 ("DKK4"), or both, for the treatment of DKK-related abnormalities of bone density. , metabolism, diabetes, cancers, and the like. BACKGROUND OF THE INVENTION The Wnt signaling pathway is involved in the control of embryonic development and neoplastic processes. The extracellular Wnt proteins are responsible for the growth and differentiation of many cell types, including in particular the growth, differentiation, and regulation of osteoblasts, osteoclasts, and adipocytes.Many cancers are associated with bone tissues , and may result in osteoblastic lesions, such as those found in prostate cancer, or osteolytic lesions, such as those found in lung cancer, breast cancer, and multiple myeloma (eg, Tian et al., 2003). New England J. Med. 349: 2483-2494.) Members of the Wnt gene family encode secreted glycoproteins that are required for a variety of developmental processes (Fedi et al., 1999 9 J. Bio. Chem. 274: 1 9465-1 9472) A protein member of the family Wnt initiates a signaling pathway that is important for the growth and differentiation of osteoblasts, which cause bone deposition. In addition to bone formation, bone resorption is a continuous normal process driven by cells known as osteoclasts. In contrast, the nuclear factor-kappa B ligand receptor activator (RANKL) is the final mediator of osteoclastic bone resorption, where it has an important role in the pathogenesis of post-menopausal osteoporosis, as well as in bone loss. associated with rheumatoid arthritis, metastatic cancer, multiple myeloma, aromatase inhibitor therapy, and androgen deprivation therapy (see, for example, Lewiecki (2006) Expert Opin, Biol. Ther 6: 1 041 -50). Osteoprotegrin (OPG), which is expressed by osteoblasts, inhibits RAN KL, thus decreasing the activity and formation of osteoclasts. The balance between anabolic bone formation and analytical bone resorption regulates normal bone density, while an increase in one or the other leads to higher bone density or greater bone loss, respectively. Wnt binds and acts through other cell surface proteins, and Wnt signaling can contribute to the neoplastic process. Additionally, genetic alterations can affect a cellular protein complex known as adenomatous polyposis coli, which involves a β-catenin protein, and these complexes have been observed in the cells of patients with diseases such as human colon cancer, melanomas , Y hepatocellular carcinomas, indicating that the aberrations of the Wnt signaling pathways are relevant for the development of these and possibly other human cancers (Fedi et al., 1999 J. Bio, Chem. 274: 1 9465-1 9472). There are at least two protein families that inhibit Wnt signaling, that is, the family related to secreted frizzled, and the Dickkopf family (DKK). The DKK family currently contains four members of the family, namely, DKK1 (Human DNA, Accession No. M_012242; PRT, Accession number 094907), DKK2 (human, accession number NM_01 4421; PRT Accession Number N P_055236), DKK3 (human, number of Access NM_015881; PRT, Accession Number AAQ88744), and DKK4 (human, Accession Number N M_01 4420; PRT, Accession Number N P_055235). Dickkopf-1 (DKK1) is a secreted inhibitor of the Wnt / β-catenin signaling pathway. See, for example, U.S. Patent Application Number 2005-0079173 to Niehrs; U.S. Patent Application Number 2004-023451 5 to McCarthy. DKK1 possesses the ability to inhibit axis duplication induced by Wnt, and genetic analysis indicates that DKK1 acts upstream to inhibit Wnt signaling. DKK1 is also important in skeletal development, as demonstrated by the effects on the loss of bone structures in embryos of chickens and mice after exposure to high levels of DKK1 (Tian et al. 2003 New England J. Med. 349: 2483-2494). Elevated levels of serum DKK1 have been associated with prostate cancer, and elevated levels of DKK1 and RAN KL in bone marrow plasma and in peripheral blood of patients with multiple myeloma are associated with the presence of focal bone lesions. See, for example, Tian 2003, OMI M, Accession No. 6051 89. DKK1 also has a role in adipogenesis, chondrogenesis, gastrointestinal epithelial proliferation, bone loss associated with rheumatism, and initiation of plaque formation of hair follicles. . OMI M, Accession number 6051 89. Dickkopf-4 (DKK4) is less well characterized, but in the same way it is a secreted inhibitor of the Wnt pathway. It has been shown that DKK4 is plaque deposited in patients with Alzheimer's disease, and is expressed in the muscle. Wnt has no known role in muscle development, and therefore, it is postulated in the present that DKK4 has an inhibitory role on muscle development. There is a need for compositions and methods for treating cancers and bone density abnormalities, including agents that interfere with, or neutralize, the antagonism of Wnt signaling mediated by DKK1 and / or DKK4. BRIEF DESCRIPTION OF THE INVENTION One embodiment of the invention herein provides an antibody or antigen binding portion thereof that selectively binds to, and neutralizes, a DKK1 polypeptide and / or DKK4, or a fragment thereof. In a preferred embodiment, the antibody is a neutralizing antibody to DKK1 / 4. In different embodiments, the antigen binding portion of the DKK1 / 4 neutralizing antibody does not bind to DKK2 or to DKK3. In one embodiment, the antibody or an antigen binding portion thereof is configured within an immunoglobulin-type scaffolding, such as a structure selected, for example, from human scaffolding, humanized, humanered ("humanly engineered"), of shark, or camelid, and / or may additionally be a recombinant, chimeric, or CDR-grafted antibody. For example, the technology designed to minimize the response of the human anti-murine antibody (the humanering [humanodiseño] technology of Kalobios or the PDL humanization technology) is contemplated within the invention. In addition, the antigen binding portions specific for DKK1 or DKK4 may also be within a scaffolding that is not of the immunoglobulin type, including, for example, arranged within an adnectin, a fibrinogen, ankyrin-derived repeats, etc. , in the type of structure. In a particular embodiment, the Dkk1 antibody is characterized by having an antigen binding region that is specific for the target protein DKK1, and the antibody or functional fragment is linked to DKK1 or a fragment thereof. In a related embodiment, the DKK4 antibody is characterized as having an antigen binding region that is specific for the target protein DKK4, and the antibody or functional fragment is linked to DKK4 or a fragment thereof. In a related embodiment, the Dkk4 antibody is characterized by having an antigen binding region that is specific for the target protein DKK4, and the antibody or functional fragment is linked to DKK4 or a fragment thereof. In a preferred embodiment, the antibody or antigen binding portion thereof, binds to a DKK1 and DKK4 polypeptide, but not to a DKK2 or DKK3 polypeptide. In another embodiment, the antibody or an antigen binding portion thereof is monoclonal. In another embodiment, the antigen binding portion is polyclonal. In different embodiments, the DKK1 antibody, or an antigen binding portion thereof, is linked to a peptide consisting of 30 contiguous amino acids of a DKK1 or DKK4 polypeptide. In a related embodiment, the binding to DKK1 or DKK4 is determined at least by one of the following assays: inhibition of DKK1 or DKK4 antagonism of transcription signalized by Wnt; affinity determination of surface plasmon resonance, binding in immunosorbent assay bound with enzymes; link analysis based on electrochemiluminescence; FMAT, SET, SPR, ALP, TopFlash, concentration of biomarkers in blood serum, such as osteocalcin (OCN), propeptide nitrogenous pro-collagen type 1 (P1 NP), and osteoprotegrin (OPG), and the binding to the receptors of Cell surface such as Frizzled (Fz), LRP (LRP5 / 6) or Kremen (Krm). In certain embodiments, the Dkk1 antibody or the antigen binding portion possesses at least one of the following properties: selectivity for DKK1 which is at least 1 03 times, 1 04 times, or 1 05 times higher than for DKK2 or DKK3 humans; binds to DKK1 or DKK4 with a Kacti ada of less than 1 00 n M, 50 n M, 1 0 n M, 1 .0 n M, 500 pM, 1 00 pM, 50 pM or 1 0 pM; and has a deactivation velocity for DKK1 of less than 1 0"2 per second, 1 0'3 per second, 10" 4 per second, or 1 0.5 per second. In a related embodiment, an antibody of the invention competes with DKK1 and / or DKK4 for binding to LPR5 / 6. In a related embodiment, an antibody of the invention competes with DKK1 and / or DKK4 for binding to Krm. In still another embodiment, the invention provides an antigen binding region isolated from any of the foregoing antibodies or functional fragments of these antibodies, and the amino acid sequences thereof. Accordingly, in certain embodiments, the invention provides isolated amino acid sequences selected from the group of SEQ ID NOs: 2 to 20, and SEQ ID NOs: 40 to 72, and the conservative or humanered ("humanly engineered") variants of these sequences. In addition, in certain additional embodiments, the invention provides the amino acid sequences SEQ ID NOs: 2-20, 65-72, and the conservative or humaneered ("humanly engineered") variants of these sequences, which provide binding regions of isolated antigen, each of which is an H-CDR-3. In a related embodiment, the isolated antigen binding region is an H-CDR2 region having an amino acid sequence selected from SEQ ID NOs: 2-20, 57-64, and the humaneered conservative variants ("humanized"). ) from the same. In yet another related embodiment, the isolated antigen binding region is a consensus H-CDR2 region illustrated in the sequence selected from the group of SEQ ID NO: 40 having the amino acid sequence GISGSGSYTYYADSVKF, SEQ ID NO: 41 which has the amino acid sequence GISYYGGNTYYADSVKF, and SEQ ID NO: 42 which has the amino acid sequence GISYYGGSTYYADSVKF, and the conservative or humaneered variants ("humanodiseadas") of the amino acid residues of these sequences. In certain embodiments, the novel sequence provided is one of SEQ ID NOs: 2-5, 8-11, 20, 49-56, and the conservative or "humanered" variants thereof, which provide a region of isolated antigen binding, which is a H-CDR1 region. In a related embodiment, the isolated antigen binding region is a consensus H-CDR1 region having an amino acid sequence (using the one letter amino acid code) selected from the amino acid sequences GFTFSSYGMT (SEQ ID NO: 43), GFTFNSYGMT (SEQ ID NO: 44), GFTFSNYGMT (SEQ ID NO: 45), GFTFSSYWMT (SEQ ID NO: 46), GFTFSSYAMT (SEQ ID NO: 47), GFTFSSYGMS (SEQ ID NO: 48), and the conservative or humaneered variants ("humanodiseadas") of any of these sequences. In another embodiment, the invention provides amino acid sequences that are isolated antigen binding regions that have an L-CDR3 region, such as amino acid sequences selected from SEQ ID NO: 21 -39, 89-96, and Conservative or humanered variants ("human-designed") of these sequences. In still another related embodiment, the isolated antigen binding region is an L-CDR 1 region having an amino acid sequence selected from SEQ ID NOs: 21-39, 73-80, and the conservative or humaneered variants ( "humanized") of these sequences. In still another related embodiment, the isolated antigen binding region is an L-CDR2 region having an amino acid sequence selected from SEQ ID NOs: 21 -39, 81 -88, and the conservative or humaneered variants (" humanized ") of these sequences. In certain embodiments, the isolated antigen binding region is a variable light chain having an amino acid sequence selected from SEQ ID NOs: 21-39, and the humanered ("human-diseased") variants of these sequences. In another embodiment, the invention provides a nucleotide sequence selected from the group of SEQ I D NOs: 97- 1 03. In a related embodiment, each of these nucleotide sequences encodes an amino acid sequence, and the humaneered ("human-engineered") variants of these encoded amino acid sequences, which are also within the scope of the present invention. In another related embodiment, the nucleotide sequence of each of SEQ ID NOs: 97-100 encodes an antigen binding light chain. In a further related embodiment, the nucleotide sequence of each of SEQ ID NOs: 101 -1 03, encodes an antigen binding heavy chain. In yet another related embodiment, each of these nucleotide sequences is further optimized for expression in a cell. Preferred cells include, but are not limited to, for example, Chinese hamster ovary cells (e.g., CHO), baculovirus, yeast, bacteria, myeloma cells, and / or H. sapiens. Preferably, the sequences are optimized for expression and for production and for clinical use. Characteristics for optimization for clinical use include, but are not limited to, for example, half-life, pharmacokinetics (PK), antigenicity, effector function, FcRn elimination, and patient response, including antibody-dependent cellular cytotoxicity (ADCC). ), or complement dependent cytotoxicity (CDC) activities. In a further embodiment, the invention provides an isolated antigen binding region having a heavy chain encoded by a nucleotide sequence selected from from the group of SEQ ID NOs: 101-103. In a related embodiment, the invention provides an isolated antigen binding region having a light chain encoded by a nucleotide sequence selected from the group of SEQ ID NOs: 97-100. In still another related embodiment, the invention provides an isolated antigen binding region having a light chain encoded by a nucleotide sequence selected from the group of SEQ ID NOs: 97-100, and a heavy chain encoded by a sequence of nucleotides selected from the group of SEQ ID NOs: 101-103. In certain embodiments, the invention provides a nucleotide sequence selected from the group of SEQ ID NOs: 104-110. In a related embodiment, each of these nucleotide sequences encodes an amino acid sequence, and the humaneered ("human-engineered") variants of these amino acid sequences, which are also within the scope of the present invention. In another related embodiment, the nucleotide sequence of each of SEQ ID NOs: 104-107, encodes an antigen binding light chain. In a further related embodiment, the nucleotide sequence of each of SEQ ID NOs: 108-110, encodes an antigen binding heavy chain. In yet another related embodiment, each of these nucleotide sequences is optimized for expression and / or for clinical use. In a further embodiment, the invention provides a isolated antigen binding region having a heavy chain encoded by a nucleotide sequence selected from the group of SEQ ID NOs: 108-110. In another related embodiment, the invention provides an antigen binding region having a light chain encoded by a nucleotide sequence selected from the group of SEQ ID NOs: 104-107. In still another related embodiment, the invention provides an isolated antigen binding region having a light chain encoded by a nucleotide sequence selected from the group of SEQ ID NOs: 104-107, and a heavy chain encoded by a sequence of nucleotides selected from the group of SEQ ID NOs: 108-110. In other embodiments, the invention provides an amino acid sequence selected from the group of SEQ ID NOs: 111-117, and provides conservative variants of these sequences. In a related embodiment, each of SEQ ID NOs: 111-114 illustrates an antigen binding light chain. In another embodiment, each of SEQ ID NOs: 115-117 illustrates an antigen binding heavy chain. In yet another related embodiment, the amino acid sequence is optimized for expression and / or for clinical use. In a further embodiment, the invention provides an isolated antigen binding region having a heavy chain screened in an amino acid sequence selected from the group of SEQ ID NOs: 115-117, and provides variants conservative of these sequences. In a related embodiment, the invention provides an isolated antigen binding region having a light chain illustrated in an amino acid sequence selected from the group of SEQ ID NOs: 1 1 1 -1 14, and provides conservative variants of these sequences. In still another related embodiment, the invention provides an isolated antigen binding region having a heavy chain illustrated in an amino acid sequence selected from the group of SEQ ID NOs: 1 15-1 17, and provides conservative variants of these sequences, and a light chain illustrated in an amino acid sequence selected from the group of SEQ ID NOs: 1 1 1-14, and provides conservative variants of these sequences. In another embodiment, the invention provides a nucleotide sequence selected from the group of SEQ ID NOs: 120-1 21, and / or the isolated antigen binding region having a variable region of a light chain or a chain heavy coding by the respective nucleotide sequences. In a related embodiment, each of these nucleotide sequences encodes an amino acid sequence, and the humaneered ("human-engineered") variants of these amino acid sequences, which are also within the scope of the invention. In another related embodiment, the nucleotide sequence of SEQ I D NO: 120 encodes an antigen binding light chain. In an additional related embodiment, the nucleotide sequence of SEQ ID NO: 121 codes for an antigen binding heavy chain. In yet another related embodiment, each of these nucleotide sequences is further optimized for expression and / or for clinical use. In certain embodiments, the invention provides an amino acid sequence selected from the group of SEQ ID NOs: 1 1 8-1 19, and the conservative or "humanered" variants of these sequences. In a related embodiment, SEQ I D NO: 1 1 8 illustrates a variable antigen binding region of a light chain. In another related embodiment, SEQ ID NO: 1 1 9 illustrates a variable antigen binding region of a heavy chain. In yet another related embodiment, the amino acid sequence is optimized for expression and / or for clinical use. In other embodiments, the invention provides an amino acid sequence having at least 60, 70, 80, 90, 95, 96, 97, 98, or 99 percent identity with the CD R regions illustrated in SEQ ID NOs: 2-39. In a related embodiment, the invention provides an amino acid sequence having an identity of at least 60, 70, 80, 90, 95, 96, 97, 98, or 99 percent with a sequence illustrated in SEQ ID NOs: 1 1 1 -120. In yet another related embodiment, the invention provides a nucleotide sequence having at least 60, 70, 80, 90, 95, 96, 97, 98, or 99 percent identity with a sequence illustrated in SEQ ID NOs : 97-1 10 and 120-1 21.

In some embodiment, any of the above isolated antibodies is an IgG. In a related embodiment, any of the above isolated antibodies is a lgG1, a lgG2, a lgG3, or a lgG4. In another embodiment, the antibody is an IgE, IgM, IgD, or IgA. In a related embodiment, the invention is selected from a monoclonal or polyclonal antibody composition. In the additional modalities, the antibody is chimeric, humanized, humanered ("humanly engineered"), recombinant, etc. In yet another embodiment, the invention provides an isolated human or humanized antibody or a functional fragment thereof, which has an antigen binding region that is specific for an epitope of DKK1, and the antibody or functional fragment is linked to DKK1 or DKK4 , or otherwise blocks the binding of DKK1 or DKK4 with a cell surface receptor (e.g., receivers such as LRP5 / 6, Kremen, Frizzled). In ain embodiments, the antibody or fragment thereof prevents, treats, or improves the development of osteolytic lesions. In other embodiments, the anti-DKK composition of the invention prevents, treats, or ameliorates a canor disease associated with DKK1 or DKK4. In a related embodiment, the invention provides an isolated human or humanized antibody or functional fragment thereof, which has an antigen binding region that is specific for an epitope of target DKK1 or DKK4, and the epitope contains six or more amino acid residues. s from a fragment of polypeptide comprising the CYS 1 -linker-CYS2 domains of DKK1 and / or DKK4. In a related embodiment, the epitope is a conformational epitope. In a preferred embodiment, the epitope resides within the CYS2 domain. In a particular embodiment, the epitope comprises a modified amino acid residue. In a related embodiment, the epitope contains at least one glycosylated amino acid residue. Functional fragments include the Fv and Fab fragments (including the single-stranded versions, such as scFv), as well as other antigen binding regions of an antibody, including those that bind to a non-immunoglobulin scaffold, and heavy chain antibodies, such as camelid and shark antibodies, and nanobodies. In a related embodiment, the isolated antibody, as described above, is an IgG. In another related embodiment, the isolated antibody, as described above, is a IgG1, IgG2, IgG3, or IgG4. In one embodiment, the antibody is an IgG, an IgM, or an IgA. In a related embodiment, the invention is a polyclonal antibody composition. In another embodiment, the invention provides a pharmaceutical composition having at least one of the above antibodies or functional fragments or conservative variants, and a pharmaceutically acceptable carrier or excipient therefor.

In still another embodiment, the invention provides a transgenic animal that carries a gene encoding any of the previous antibodies or functional fragments thereof. In certain embodiments, the invention provides a method for the treatment of a disorder or condition associated with the expression of DKK1 or DKK4. As used herein, "diseases associated with DKK1" or "diseases associated with DKK4", include, but are not limited to, osteolytic lesions - especially osteolytic lesions associated with a myeloma, especially multiple myeloma, or with cancers of bone; breast, colon, melanocytes, hepatocytes, epithelium, esophagus, brain, lung, prostate, or pancreas, or metastasis thereof; Bone loss associated with transplant. Other diseases or disorders include, but are not limited to, for example, osteosarcoma, prostate cancer, hepatocellular carcinoma (HCC), myeloma, including multiple myeloma, diabetes, obesity, muscular waste, Alzheimer's disease, osteoporosis, osteopenia, rheumatism, colitis, and / or loss of hair ndeseada. The method involves administering to a subject in need, an effective amount of any of the above pharmaceutical compositions. In a related embodiment, the disorder or condition to be treated is an abnormality of bone density. In another related embodiment, the abnormality of the bone density to be treated is the presence of an osteolytic lesion in the subject. In another embodiment, the disorder or condition to be treated is an osteoporotic condition, such as an osteolytic lesion associated with a cancer. In a related modality, cancer which is to be treated is a myeloma, such as multiple myeloma, or a cancer of bone, breast, colon, melanocytes, hepatocytes, epithelium, esophagus, brain, lung, prostate, or pancreas, or metastasis thereof. In other modalities, the osteoporotic condition is osteoporosis or osteopenia or osteosarcoma. In certain embodiments, any of the above methods also involves administering a chemotherapeutic agent. In a related embodiment, the chemotherapeutic agent is an agent against cancer. In another related embodiment, the chemotherapeutic agent is an anti-osteoporotic agent. In still another embodiment, the invention provides a method for the treatment of a target cell, the method involving blocking the interaction of DKK1 or DKK4 with the cell, with any of the foregoing antibodies or functional fragments thereof. In general, the target cell carries a receptor that binds to DKK1 or DKK4. In one embodiment, the target cell is an osteoblast, wherein treatment with the neutralizing anti-DKK1 / 4 composition of the invention will improve proliferation and stimulate bone formation. In one embodiment, the target cell is a muscle cell, where the treatment will counteract the muscle waste. In a related embodiment, the method further involves the treatment of a patient with the target cell, with a chemotherapeutic agent or radiation. In a related mode, immediately after the administration or contact, any of the previous methods also involves observing the improvement or retardation of the development of an osteolytic lesion. In yet another embodiment, the invention provides a method for identifying DKK1 or DKK4 in serum. This method involves detecting DKK1 or DKK4 with any of the above antibodies or fragments of antibodies, which also have a detectable label. The brand is radioactive, fluorescent, magnetic, paramagnetic, or chemiluminescent. In another embodiment, any of the human or humanized antibodies, or fragments of antibodies, are synthetic. In another embodiment, the invention provides a pharmaceutical composition of any of the foregoing antibodies or functional fragments of these antibodies, and an additional therapeutic agent. The additional therapeutic agent can be selected from the group consisting of an anticancer agent; an anti-osteoporotic agent; an antibiotic; an anti-metabolic agent; an anti-diabetic agent; an anti-inflammatory agent; an anti-angiogenic agent; a growth factor; and a cytokine. The invention further relates to a method for preventing or treating proliferative diseases, or diseases such as cancer, in a mammal, in particular in a human, with a combination of pharmaceutical agents comprising: (a) a neutralizing agent of DKK1 / 4 of the invention; and (b) one or more pharmaceutically active agents; where at least one pharmaceutically active agent It is a therapeutic agent against cancer. The invention further relates to pharmaceutical compositions comprising: (a) a neutralizing agent of DKK1 Y4; (b) a pharmaceutically active agent; and (c) a pharmaceutically acceptable carrier; wherein at least one pharmaceutically active agent is a therapeutic agent against cancer. The present invention further relates to a commercial package or product comprising: (a) a pharmaceutical formulation of a neutralizing antibody of DKK1 / 4; and (b) a pharmaceutical formulation of a pharmaceutically active agent for simultaneous, concurrent, separate, or sequential use; wherein at least one pharmaceutically active agent is a therapeutic agent against cancer. In a certain embodiment, the invention provides an antibody having a first amino acid sequence that is a heavy chain selected from SEQ ID NOs: 2-20, and a sequence having a sequence identity of at least 60, 70 , 80, 90, 95, 96, 97, 98, or 99 percent in the CD R regions, with the CDR regions having the SEQ ID NOs: 2-20; and a second amino acid sequence that is a light chain selected from SEQ I D NOs: 21 -39, and a sequence that has a sequence identity of at least 60, 70, 80, 90, 95, 96, 97, 98, or 99 percent in the CDR regions, with the CDR regions shown in SEQ I D NO: 21 -39. In still another embodiment, the invention provides an immunoconjugate made of a first component that is an antibody or fragment as described above, and a second component having a second amino acid sequence. For example, the immunoconjugate is a cytotoxin, or the immunoconjugate is a binding protein or an antibody that has a binding specificity for an objective that is different from DKK1 or DKK4. For example, the aim of the binding specificity different from DKK1 or DKK4 is a tumor antigen or a tumor-associated protein on the surface of a cancer cell. In certain embodiments, the invention provides any of the foregoing antibodies as a bispecific antibody. In another embodiment, the invention provides a kit having any of the above antibodies or antibody fragments. In some embodiments, the kit further contains a pharmaceutically acceptable carrier or excipient thereof. In other related embodiments, any of the above antibodies in the kit is present in a unit dose. In still another modality, the kit is an ELISA diagnostic kit. In a related embodiment, the kit includes instructions for use in the administration of any of the antibodies prior to a subject. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph illustrating that there is no significant loss of His-Strep-tagged DKK1 from the Strep-Tactin-coated beads, when the unwashed beads are compared to the beads washed with different astringency from HuCAL®. Figure 2 is a bar graph illustrating the results of a Wnt activity assay with 1000 microliters of Bright-Glo luciferase reagent, as provided in Example 3. Figure 3 is a graph of a linked immunosorbent assay with enzymes (ELISA). Fluorescence is measured on a TECAN Spectrafluor plate reader, and link curves are shown.

Figure 4 is a graph illustrating the standard Wnt3a dependent TCF / LEF luciferase reporter assay, as provided in Example 6. Figure 5 is a graph illustrating an improved version of the TCF luciferase reporter assay / LEF, which shows the highly enhanced sensitivity to DKK1, mediated by the co-expression of the Kremen co-receptor protein. Figure 6A is a graphic illustration of the surface plasmon resonance measurement of an anti-DKK1 / 4 antibody that binds to DKK1. Figure 6B is a tabulation of the calculated link affinity and kinetic values. Figure 7A is a schematic representation of DKK1 of full length and truncated, to be used in epitope mapping. Figure 7B illustrates the binding of an antibody of the invention with the DKK1 proteins, and the fragments of Figure 7A.

Figure 8 is a graphic illustration of the antibody binding of DKK1 / 4 in a competition ELISA assay. Figure 9 is a graphic illustration of the reactivation associated with the DKK1 / 4 antibody of the gene transcription regulated by downstream Wnt. Figure 10 is a graphic illustration of reversed DKK1 of the DKK1 / 4 antibody inhibited from ALP secretion. Figure 11 is a graphic illustration of the effects of the DKK1 / 4 antibody on xenografts in vivo. Figure 12 is a graphic illustration of the elevation of bone density associated with the DKK1 / 4 antibody in vivo. Figure 13 is a graphic illustration comparing the anti-osteolytic efficacy of Zometa with a DKK1 / 4 antibody. Figure 14 is a graphic illustration of the dose-dependent efficacy of a DKK1 / 4 antibody on anabolic bone. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to isolated DKK1 / 4 antibodies, in particular human antibodies, which specifically bind to DKK1 or DKK4, and which inhibit the functional properties of DKK1 or DKK4. In a preferred embodiment, the DKK1 / 4 antibody does not specifically bind to DKK2 or DKK3. In certain embodiments, the antibodies of the invention are derived from particular heavy and light chain sequences, and / or comprise particular structural features, such as CDR regions that comprise particular amino acid sequences. The invention provides isolated antibodies, methods for making these antibodies, immunoconjugates, and bispecific molecules comprising these antibodies, and pharmaceutical compositions containing the antibodies, immunoconjugates, or bispecific molecules of the invention. The invention also relates to methods for using the antibodies in order to inhibit a disorder or condition associated with DKK1 or DKK4. The contemplated diseases and disorders include, but are not limited to, osteolytic lesions - especially osteolytic lesions associated with a myeloma, especially multiple myeloma, or with cancers of bone, breast, colon, melanocytes, hepatocytes, epithelium, esophagus, brain , lung, prostate, or pancreas, or metastasis thereof; Bone loss associated with transplant. Other diseases or disorders include, but are not limited to, for example, osteosarcoma, prostate cancer, hepatocellular carcinoma (HCC), myeloma, including multiple myeloma, diabetes, obesity, muscular waste, Alzheimer's disease, osteoporosis, osteopenia, rheumatism, colitis, and / or unwanted hair loss. In order that the present invention can be understood more easily, certain terms are defined first. They are stipulated additional definitions throughout the detailed description. The term "immune response" refers to the action of, for example, lymphocytes, antigen-presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or by the liver (including antibodies, cytokines , and supplements), which result in selective damage to, destruction of, or elimination of the human body from, invading pathogens, cells or tissues infected with pathogens, cancer cells, or in the cases of autoimmunity or pathological inflammation, the normal human cells or tissues. A "signal transduction path" refers to the biochemical relationship between a variety of signal transduction molecules that have a role in the transmission of a signal from one cell portion to another portion of a cell. As used herein, the phrase "cell surface receptor" includes, for example, molecules and complexes of molecules capable of receiving a signal, and capable of transmitting this signal through the plasma membrane of a cell. An example of a "cell surface receptor" of the present invention is a receptor with which the protein molecule of DKK1 or DKK4 is linked. These cell surface receptors include, but are not limited to, Frizzled (Fz), LRP (LRP5 and LRP6), and Kremen (Krm). As used herein, the term "antibody" refers to polyclonal antibodies, monoclonal antibodies, humanized antibodies, single chain antibodies, and fragments thereof, such as Fab, (ab ') 2, Fv, and other fragments that retain the antigen binding function of the parent antibody. As such, an antibody can refer to an immunoglobulin or glycoprotein, or a fragment or portion thereof, or to a construct comprising an antigen binding portion comprised within a modified immunoglobulin-like structure, or to an antigen binding portion. comprised within a construction comprising a structure or scaffolding that is not of the immunoglobulin type. As used herein, the term "monoclonal antibody" refers to an antibody composition having a homogeneous antibody population. The term is not limited to considering the species or source of the antibody, nor is it intended to be limited by the manner in which it is made. The term encompasses whole immunoglobulins, as well as fragments, such as Fa. (ab) 2. Fv, and others that retain the antigen binding function of the antibody. Monoclonal antibodies of any mammalian species can be used in this invention. However, in practice, the antibodies will typically be of rat or murine origin, due to the availability of rat or murine cell lines for use in the preparation of the required hybrid cell lines or hybridomas to produce the monoclonal antibodies. As used herein, the term "polyclonal antibody" refers to an antibody composition having a heterogeneous antibody population. Polyclonal antibodies are often derived from the secretory serum of immunized animals or selected humans. As used herein, the phrase "single chain antibodies" refers to antibodies prepared by determining the binding domains (both heavy and light chain) of a binding antibody; and the provision of a binding fraction that allows the preservation of the binding function. This forms, in essence, a radically abbreviated antibody, which has only the portion of the variable domain necessary to bind to the antigen. The determination and construction of single chain antibodies are described in United States Patent No. 4,946,778 to Ladner et al. A "naturally occurring antibody" is a glycoprotein comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH), and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH 1, CH 2 and CH 3. Each light chain is comprised of a light chain variable region (abbreviated herein as V), and a light chain constant region. The light chain constant region is comprised of a domain, CL. The VH and V regions can be further subdivide regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed as structure regions (FR). Each VH and V is composed of three complementarity determining regions and four structure regions configured from the amino terminus to the carboxyl terminus in the following order: FR1, CDR 1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of immunoglobulin to host tissues or factors, including different cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The term "antigen binding portion" of an antibody (or simply "antigen portion"), as used herein, refers to the sequence of the protein that binds to the target, eg, one or more regions determining complementarity. It includes, for example, full length antibodies, one or more fragments of an antibody, and / or complementarity determining regions on a scaffold unrelated to immunoglobulin that retains the ability to specifically bind to an antigen (eg, DKK1). The antigen binding function of an antibody can be carried out by fragments of a full-length antibody.

Examples of the link fragments encompassed within the term "antigen binding portion" of an antibody include a Fab fragment, a monovalent fragment consisting of the V, VH, C and CH 1 domains; an F (ab) 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the joint region; an Fd fragment consisting of the VH and CH 1 domains; an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a fragment of dAb (Ward et al., 1989 Nature 341: 544-546), which consists of a VH domain; and an isolated complementarity determining region (CDR). As used herein, an "antigen" or an "epitope" refers interchangeably to a polypeptide sequence on a target protein specifically recognized by an antigen binding portion of an antibody, antibody fragment, or its equivalents. An antigen or epitope comprises at least six amino acids, which may be contiguous within an objective sequence, or non-contiguous. A conformation epitope may comprise non-contiguous residues, and optionally may contain naturally occurring or synthetically modified amino acid residues. Modifications to the residues include, but are not limited to: phosphorylation, glycosylation, PEGylation, ubiquitination, furanilization, and the like. Additionally, although the two domains of the Fv, VL and VH fragment are encoded by separate genes, they can be joined, using recombinant methods, using a synthetic linker that makes it possible for them to be made as a single protein chain, where the V and VH regions pair to form monovalent molecules (known as single chain Fv (scFv), see, for example , Bird et al., 1 988 Science 242: 423-426; and H uston et al., 1 988 Proc. Nati, Acad. Sci. 85: 5879-5883). These single chain antibodies are also intended to be encompassed within the term "antigen binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same way as the antibodies are detected. As described herein, conservative variants include amino acid residues in any of the amino acid sequences identified, in particular conservative changes that are well known to an ordinary expert in the field of protein engineering. An "isolated antibody", as used herein, refers to an antibody that is substantially free of other antibodies having different antigenic specificities (for example, an isolated antibody that specifically binds to DKK1 is substantially free of antibodies that are specifically bind with antigens other than DKK1). An isolated antibody that binds specifically to DKK1, however, may have cross-reactivity with other antigens, such as DKK1 molecules of other species, or other members of the family, such as DKK4 or related paralogs. Moreover, an isolated antibody can be substantially free of other cellular material and / or chemicals. The term "human antibody", as used herein, is intended to include antibodies having variable regions wherein both the structure regions and the complementarity determining regions are derived from sequences of human origin. Additionally, if the antibody contains a constant region, the constant region is also derived from these human sequences, for example, human germline sequences, or mutated versions of human germline sequences. The human antibodies of the invention may include amino acid residues not encoded by human sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro, or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies wherein sequences of complementary determining regions derived from the germ line of other mammalian species, such as a mouse, have been grafted. , on human structure sequences. The term "human monoclonal antibody" refers to antibodies that exhibit a single binding specificity that have variable regions, wherein both the structure and the complementarity determining regions are derived from human sequences. In one embodiment, human monoclonal antibodies are produced by a hybridoma that includes a B-cell obtained from a transgenic non-human animal (e.g., a transgenic mouse, having a genome comprising a heavy chain transgene and a transgene of light chain fused to an immortalized cell As used herein, the term "humanized antibodies" means that at least a portion of the structure regions of an immunoglobulin is derived from human immunoglobulin sequences. ", such as antibodies with CDR sequences derived from the germline of other species, especially a mammalian species, for example a mouse, which have been grafted onto sequences of human structure. they include the PDL humanization technology.As used herein, the term "humanered antibodies (" humanly designed ")" means the antibodies that bind to the same epitope but that differ in sequence. Exemplary technologies include the humaneered ("human-designed") antibodies produced by the humaneering ("human-design") technology of Kalobios, wherein the sequence of the antigen binding region is derived, for example, from a mutation, instead of being due to conservative amino acid replacements.

The term "recombinant human antibody", as used herein, includes all human antibodies that are prepared, expressed, created, or isolated by recombinant means, such as antibodies isolated from an animal (e.g., a mouse). ) that is transgenic or transchromosomal to the human immunoglobulin genes, or a hybridoma prepared therefrom, the antibodies isolated from a host cell transformed to express the human antibody, for example, from a transfectome; antibodies isolated from a library of recombinant human combination antibodies; and antibodies prepared, expressed, created, or isolated by any other means involving the splicing of all or a portion of a human immunoglobulin gene; the sequences for other DNA sequences. These recombinant human antibodies have variable regions in which the structure regions and the complementarity determining regions are derived from the human germline immunoglobulin sequences. In certain modalities, however, these recombinant human antibodies can be subjected to in vitro mutagenesis (or, when a transgenic animal is used for human Ig sequences, somatic mutagenesis in vivo), and therefore, the amino acid sequences of the VH regions and V of the recombinant antibodies are sequences that, although they are derived from, and are related to, the VH and VL sequences of the germline human, may not exist naturally within the repertoire of the germline of the human antibody in vivo. As used in the present "isotype" it refers to an antibody class (IgA, IgD, IgM, IgE, IgG, such as lgG1, IgG2, IgG3 or IgG4), which is provided by the heavy chain constant region genes . The phrases "an antibody that recognizes an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody that specifically binds an antigen". As used herein, an antibody that "specifically binds to human DKK1" is intended to refer to an antibody that binds to human DKK1 with a KD of 5x10"9M or less, 2x10'9M or less, or 1x10 ' 10M or less: An antibody that "cross-reacts with a different antigen from human DKK1" is intended to refer to an antibody that binds to the antigen with a KD of 0.5x10"8M or less, 5x10'9M or less, or 2x10" 9M or less An antibody that "does not cross-react with a particular antigen" is intended to refer to an antibody that binds to that antigen, to a KD of 1.5x10"8M or more, or a KD of 5-10x10" 8M or 1x10 ' 7M or more In certain embodiments, these antibodies that do not cross-react with the antigen exhibit an essentially undetectable linkage to these proteins in conventional binding assays. As used herein, an antibody that "inhibits linkage of DKK1 with a "cell surface receptor", such as LRP, Fz, or Krm, refers to an antibody that inhibits the binding of DKK1 to the receptor with a KD of 1 nM or less, 0.75 nM or less, 0.5 n M or less, or 0.25 n M or less As used herein, "osteolysis" refers to a decrease in bone density, which may be due to different mechanisms of action, including, for example, a decrease in In osteoclasts activity, or an increase in osteoclast activity, osteolysis encompasses the mechanisms that generally affect bone density, as used herein, an antibody that "inhibits osteolytic activity "is intended to refer to an antibody that inhibits the loss of bone density, either by increasing bone formation or by blocking bone resorption.The term" Kas0c? ada "or" Ka ", as used herein, is intended to refer to to the rate of association of a particular antibody-antigen interaction, while the term "Kd, s" or "KD", as used herein, is intended to refer to the dissociation index of a particular antibody-antigen interaction. The term "KD", as used herein, is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka (that is, Kd / Ka), and is expressed as a molar concentration (M). The KD values for the antibodies can be determined using well-established methods in this field. A method for determining the KD of an antibody is through the use of shallow plasmon resonance, using FMAT, or using a biosensor system, such as a Biacore® system. As used herein, the term "affinity" refers to the strength of interaction between the antibody and the antigen at unique antigenic sites. Within each antigenic site, the variable region of the "arm" of the antibody interacts through weak non-covalent forces with the antigen at numerous sites; the more interactions there are, the stronger the affinity will be. As used herein, the term "avidity" refers to an information measure of the overall stability or strength of the antibody-antigen complex. It is controlled by three major factors: affinity of the antibody epitope; the valence of both the antigen and the antibody; and the structural configuration of the parts that interact. Finally, these factors define the specificity of the antibody, i.e., the likelihood that the particular antibody is binding to an accurate antigen epitope. In order to obtain a probe of higher avidity, a dimeric conjugate (two JWJ-1 molecules coupled with a FACS marker) can be constructed, thereby making the interactions of low affinity (such as with the antibody of the germ line) are more easily detected by FACS. In addition, another means of increasing the avidity of the antigen binding involves the generation of dimers or multimers of any of the fibronectin constructs described herein, of the antibodies of DKK1 or DKK4. These multimers can be generated through the covalent bond between the individual modules, for example, by mimicking the natural bond of the term C with N, or by mimicking the antibody dimers that are held together through their constant regions. The bonds designed at the Fc / Fc interface can be covalent or non-covalent. In addition, dimerization or multimerization components other than Fe can be used in the hybrids of DKK1 or DKK4 to create these higher order structures. As used herein, the term "cross-reactivity" refers to an antibody or a population of antibodies that bind epitopes on other antigens. This can be caused either by the low avidity or specificity of the antibody, or by multiple different antigens having identical or very similar epitopes. Cross-reactivity is sometimes desirable when general linkage with a related group of antigens is desired, or when cross-species marking is sought when the epitope sequence of the antigen is highly conserved in evolution.

As used herein, the term "high affinity" or "high specificity" for an IgG antibody, refers to an antibody having a KD of 10"8M or less, 10.9M or less, or 10 However, the "high affinity" linkage may vary for other antibody isotypes, eg, the "high affinity" linkage for an IgM isotype refers to an antibody that has a KD of 10"7M or less, or 1 0'8M or less. As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, for example mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, etc. As used herein, the term "optimized" means that a nucleotide sequence has been altered to purify an amino acid sequence using codons that are preferred in the production cell or organism, and / or that the nucleotide sequence has been altered to remove splice donor sites or dormant splice acceptors. Codon optimized tables are well known in the art for a wide variety of species. Sequences for splice donor sites and splice acceptors are also known in this field, and latent splice sites can be identified, for example, by analysis of transcription or expression data. Production cells include, but are not limited to, a prokaryotic cell, such as, for example, a prokaryotic cell such as a baculovirus or bacteria (E. coli), or a eukaryotic cell, eg, yeast (e.g. , Pichia), a Chinese hamster ovary cell (CHO), a myeloma cell, or a human cell. The optimized nucleotide sequence is designed to completely retain or as much as possible the amino acid sequence and the number of residues originally encoded by the starting nucleotide sequence, which is also known as the "progenitor" sequence. The optimized sequences herein are designed to have codons that are preferred in production cells; however, optimized expression of these sequences in other eukaryotic and prokaryotic cells is also envisioned herein. The amino acid sequences encoded by the optimized nucleotide sequences are optionally referred to as optimized. In the related embodiments, the polypeptide sequences of the neutralizing anti-DKK1 / 4 compositions of the invention, and the nucleotides encoding them, are preferably optimized for production and clinical use. Features that can be optimized for clinical use include, but are not limited to, for example, half-life, pharmacokinetics (PK), antigenicity, effector function, elimination of FcRn, and patient response, including the activities of antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) ). As used herein, "diseases associated with DKK1" or "diseases associated with DKK4" include, but are not limited to, osteolytic lesions - especially osteolytic lesions associated with a myeloma, especially multiple myeloma, or with cancers of bone, breast, colon, melanocytes, hepatocytes, epithelium, esophagus, brain, lung, prostate, or pancreas, or metastasis thereof; Bone loss associated with transplant. Other diseases or disorders include, but are not limited to, for example, osteosarcoma, prostate cancer, hepatocellular carcinoma (HCC), myeloma, including multiple myeloma, diabetes, obesity, muscular waste, Alzheimer's disease, osteoporosis, osteopenia, rheumatism, colitis, and / or unwanted hair loss. As used herein, a "treatment" is an intervention performed with the intent to prevent the development or alter the pathology of a disorder. In accordance with the above, "treatment" refers to both therapeutic treatment and prophylactic or preventive measures. Those in need of treatment include those who already have the disorder, as well as those in whom the disorder is to be prevented. In the treatment of tumors (eg cancer), a therapeutic agent can directly decrease the pathology of the tumor cells, or make the tumor cells more susceptible to treatment by other therapeutic agents, for example radiation and / or chemotherapy. The "pathology" of cancer includes all phenomena that compromise the well-being of the patient. This includes, without limitation, abnormal or uncontrollable cell growth, metastasis, interference with normal functioning of neighboring cells, release of cytokines or other secretory products to abnormal levels, suppression or aggravation of the inflammatory or immunological response, etc. The treatment of patients suffering from clinical, biochemical, radiological, or subjective symptoms of the disease, such as osteolysis, may include alleviating some or all of these symptoms, or reducing the predisposition to the disease. In general, a neutralizing anti-DKK1 / 4 composition of the invention prevents, treats, or ameliorates the Wnt-related diseases associated with DKK1 or DKK4, or both, but not the diseases associated with DKK2 or DKK3, or with other modulators of the Wnt path. Different aspects of the invention are described in greater detail in the following subsections. The Wnt pathway is an important regulator of the differentiation of mesenchymal totipotent cells (MSCs) to osteoblasts. It is also an important survival factor for active osteoblasts. Dickkopf-1 (DKK1) is a Wnt pathway antagonist expressed predominantly in adult bone, and it increases in myeloma patients with osteolytic lesions. A neutralizing anti-DKK1 / 4 antibody is a truly anabolic agent, which acts through increased osteoblastic activity, while simultaneously decreasing osteoclastic activity. In contrast, current drugs, such as PTH, which are marketed as anabolic agents, in fact increase the markers associated with both osteoblasts and osteoclasts. In the invention, polyclonal and monoclonal antibodies selected to bind to DKK1 are provided. In some embodiments, the anti-DKK1 antibody cross-reacts with DKK4 (Kd -300 pM), but not with DKK2 (continuous affinity analysis). A preferred epitope for an anti-DKK1 or anti-DKK4 antibody is mapped to the Cys-2 domain (amino acids 189-263), which is known to be responsible for the binding of both LRP6 and Kremen. In one embodiment, the epitope includes at least six, and at most 30 amino acid residues from the Cys-2 domain of a DKK1 or DKK4 polypeptide. In a certain embodiment, the epitope includes a stretch of at least six contiguous amino acids. In other embodiments, the preferred binding site is non-linear, i.e., it includes non-contiguous amino acid residues. In some embodiments, the linkage depends on N-glycosylation. Only one N-glycosylation site is predicted at residue 256 of the Cys-2 domain. In the present invention, a neutralizing anti-DKK1 antibody blocks the interaction of DKK1 with LRP6 in both ELISA and cell surface binding assays. As expected, this effectively neutralizes the Wnt suppressor activity of DKK1 at EC50s below 1 nM in vitro. In an in vitro model of osteoblast differentiation, mouse 10T1 / 2 cells are treated with Wnt3A to stimulate the secretion of alkaline phosphatase (AP), a marker for the activity of osteoblasts. DKK1 blocks the production of AP in this model, and the antibodies of the invention completely reverse this inhibition.

In certain embodiments, an antibody of the invention exhibits a linear dose pharmacokinetics (AUC) in mice, with a dose-dependent terminal half-life of 35 to 96 hours in mice, over a dose of 20 to 200 micrograms / mouse . Using the intratibial model of osteolytic prostate tumor metastasis, the antibodies of the invention inhibit tumor-induced cortical bone damage. The effects on trabecular bone are confused in this model by the observation that both the tumor implants and the false implants cause mechanical damage to the bone, which results in an initial increase in the bone tissue that is later remodeled, causing this way a decrease in apparent osseous volume. In a certain embodiment, an antibody of the invention increases the production of the bone tissue in both tibias implanted with tumor and implanted falsely, and inhibits the decrease in bone volume that accompanies remodeling. In the related embodiments, changes in bone markers (osteocalcin, sRANKL, OPG, AP, TRAF) are used to demonstrate the clinical effects of the antibodies of the invention on bone-related diseases, and to predict clinical outcomes. of treatments with pharmaceutically effective levels of a neutralizing anti-DKK1 antibody, as provided herein. Krm binds to DKK in approximately the same region as LRP. Krm is needed for the inhibition of the Wnt signal by means of the interaction of DKK with LRP. Krm-DKK-LRP form complex in order to internalize for the deactivation of the Wnt path. A neutralizing anti-DKK1 / 4 composition of the invention preferably binds to DKK1 or DKK4, blocking its interaction with LRP and / or Krm, thereby neutralizing the effect of DKK1 or DKK4 on the Wnt signaling pathway. The reactivation of the Wnt path occurs only when DKK1 and / or DKK4 are limiting. Conventional assays to assess the binding capacity of antibodies to DKK1 of different species are known in the art, including, for example, ELISAs, Western blots, and RIAs. Suitable assays are described in detail in the Examples. The binding kinetics (e.g., binding affinity) of the antibodies can also be assessed by conventional assays known in the art, such as by Biacore analysis, the BioVeris SET assay, FMAT, chemiluminescence, and / or SPR assays. inhibition or signal release assays. Assays to evaluate the effects of antibodies on the functional properties of DKK1 are described in greater detail in the Examples. In accordance with the foregoing, it will be understood that an antibody that "inhibits" one or more of these functional properties of DKK1 (eg, biochemical, immunochemical, cellular, physiological, or other biological activities, or the like), determined in accordance with the known methodologies in this Field and described herein, is related to a statistically significant decrease in the particular activity relative to that seen in the absence of the antibody (for example, or when a control antibody of an irrelevant specificity is present). An antibody that inhibits the effects of DKK1 activity, such as a statistically significant decrease, by at least 10 percent of the parameter measured, by at least 50 percent, 80 percent, or 90 percent, and in certain embodiments, an antibody of the invention can inhibit more than 95 percent, 98 percent, or 99 percent of the functional activity of DKK1. Members of the Dickkopf Family The appropriate bone metabolism involves a complex network of regulatory pathways and interconnections between osteoblasts, osteoclasts, and the surrounding stroma. Imbalances in these regulatory mechanisms are involved in osteolytic conditions, such as osteoporosis, osteolytic lesions induced by tumor, bone injury induced by kidney and liver transplantation, and bone loss induced by anti-hormone chemotherapy. These conditions can have severe symptoms, including bone pain, fractures, vertebral compression, and reduced mobility. Most approved treatments or currently under clinical evaluation, act predominantly inhibiting the function of osteoclasts. For example, bisphosphonates, such as zoledronic acid (Zometa), are the current standard of care, and they act by inhibiting the function and survival of osteoclasts. Although bisphosphonates are generally effective, some patients do not respond robustly, or interfere with use due to renal toxicity or osteonecrosis [Markowitz 2003] [Ruggiero 2004]. In addition, there are multiple research drugs that target the development of osteoclasts, such as RAN KL and neutralizing antibodies to M-CSF, or the function of osteoclasts, such as Catepsin inhibitors. the activity of osteoblasts would provide a novel mechanism by which to treat osteolytic disease. The Wnt pathway has an important role in the differentiation and activity of osteoblasts, whereas the D ickkopf antagonist family (DKK) in particular has been identified as key regulators of this in vitro process (see [Krishnan 2006]. ] for a ew). In vitro, Wnt is a factor of survival and essential differentiation of osteoblasts. The classical validation for the role of the Wnt pathway is provided by mutations in the Wnt co-receptor, LRP5. Inactivating mutations in LR P5 result in the osteoporosis pseudo-gyneoma syndrome (OPPG) [Ai 2005], whereas activating mutations result in high bone mass [Boyden 2002]. These activating mutations are found in the extracellular domain, and have been shown to impair linkage with the Wnt Dickkopf family of antagonists (DKK). It has been reported that DKK 1 is overexpressed in the cells of Myeloma of patients with bone lesions, but is absent in normal plasma cells or in plasma cells of patients without bone lesions [Tian 2004] [Politou 2006]. This over-expression of DKK1 by myeloma cells, can alter the normal balance between osteoblasts and osteoclasts, by blocking the differentiation of osteoblasts, and therefore, bone resorption is promoted. Furthermore, it has been reported that some of the anti-tumor treatments used for myeloma, such as dexamethasone, increase DKK1 [Ohnaka 2004]. Accordingly, a neutralizing anti-DKK1 antibody should allow the reactivation of the Wnt pathway in osteolytic lesions, while not affecting Wnt signaling in other tissues where DKK1 levels are relatively low, or where other antagonists predominate . In adults, it has been reported that DKK1 is highly expressed only in bone [Li 2006], suggesting a continuous role for DKK1 in the regulation of bone metabolism in adults. Transgenic mice that overexpress Wnt1 in breast tissue develop mammary carcinomas [Tsukamoto 1 988], and approximately 90% of human colorectal cancers have mutations in either APC or β-catenin, two cytoplasmic components of the path of Wnt [Morin 1997] [Rowan 2000]. This type of evidence presents concerns that the activation of the Wnt pathway could be a risk for an increase in the initiation or progression of tumors. In addition, the DKKs decrease in colorectal tumors and in melanomas, suggesting a potential tumor suppressor role. A DKK polypeptide of the invention includes DKK1 (SEQ ID NO: 1) and DKK4 (SEQ ID NO: 124), as well as DKK2 (SEQ ID NO: 122), and DKK3 (SEQ ID NO: 123). Members of the DKK family have two CYS domains (CYS1 and CYS2), as shown in Table A - PileUp member of the DKK1 family. The DKK proteins contain an N-terminal acid signal peptide, two CYS domains containing clusters of cysteine residues separated by a divergent linker region, and a potential C-terminal N-glycosylation site. The CYS2 domain in DKK4 has a lipid binding function that can facilitate Wnt / DKK interactions in the plasma membrane. OMIM, Accession Number 605417. Table A: PileUp Member of the DKK1 Family hDKKl hDKK2 hDKK3 MQRLGAT LC L LAAAVPTA PAPAPTATSA PV PGPA SY PQEEATLNEM hDKK -; 51 100 hDKKl M MALGAAGATR VFVAMVAAAL GGHPL GVSA TLNSVLNSNA hDKK2 M AALMRSKDSS CCLLLLAAVL .... MVESSQ IGSSRAK NS hDKK3 FREVEE MED TQHK RSAVE EMEAEEAAAK ASSEVN ANL PPSYHNETNT hDKK 101 150 hDKKl IKN PPP GG AAGHPGSAVS AAPGILYPG. ..GNKYQTID NYQPY | PCAED] hDKK2 IKS ... SLGG ET ..PGQAAN RSAG.MYQGL AFGGSKKGKN LGQAY | PCSS51 hDKK3 DTKVGNNTIH VHREIHKITN NQTGQMVFSE TVITSVGDEE GRRSH C ?? Dl hDKK4 MVAA VLLGLSWLCS PLGALVLDFN NIRSSADLHG ARKGS ^ CLSD] 151 CYS1 200 hDKKl lEECGTDEYCA SPTRGGDAGV QICLACRKRR KRCMRHAMCC PGNYCKNGiq hDKK2 IKECEVGRYCH SPHQGSSA .. .. CMVCRRKK KRCHRDGMCC PSTRCNMGIC | hDKK3 lEDCGPSMYCQ FASFQ YTCQPCRGQR MLCTRDSECC GDQLCVWGHC | CY§1 hDKK4 frDCNTRKFCL QPRD .... EK PFCATCRGLR RRCQRDAMCC PGTLCVNDVCJ 201 250 hDKKl @ SSDQN..HF RGEI ... EET ITESFGNDH. STLD.GYSRR TTLSSKMYHT hDKK2 [? P] VTES .. IL TPHIPALDGT RHRDRNHGHY SNHDLG QNL GRPHTKMSHI hDKK3 ffKVlA T hD ?? 4 | TT | MEDATPIL ERQLDEQDGT .HAEGTTGH. .PVQENQPKR KPSIKKSQGR 251 CYS2 300 hDKKl KGQEGSVJCLR SSDCASGLCC A..RHF SKI CKPVLKEGQV CTKHRRK ... | hDKK2 KGHEGDP | CLR SSDCIEGFCC A..RHFWTKI CKPVLHQGEV CTKQRKK ... | hDKK3 RGSNGTI | CDN QRDCQPGLCC AFQRGLLFPV CTPLPVEGEL CHDPASRLLD | hDKK4 KGQEGES | CLR TFDCGPGLCC A..RHF TKI CKPVLLEGQV CSRRGHK ... | CYS2 301 350 hDKKl | ... G.SHGLE IFQRCYCGEG LSCRIQKDHH QASNSSRLHT J | QRH hDKK2 | ... G.SHGLE IFQRCDCAKG LSCKV KD.A TYSSKARLHV ^ C | QKI hDKK3 ILIT ELEPDG ALDRCPCASG LLC QPHSHSLVYV ^ KPTFVGSRD hDKK4 | ... DTAQAPE IFQRCDCGPG LLCRSQLTSN R .. QHARLRV ÜQKIEKL 351 400 hDKKl hDKK2 hDKK3 QDGEILLPRE VPDEYEVGSF MEEVRQELED LERSLTEEMA LGEPAAAAAA 401 hDKK2 (SEQ ID NO: 122) hDKK3 LLGGEEI (SEQ ID NO: 123) hDKK4 (SEQ ID NO: 124) Antibodies against D KK1 and DKK4 In a preferred embodiment, the antibody of the invention is specific for a human DKK protein. A neutralizing antibody to DKK1 or DKK4 is distinct from the modifications of the Wnt pathway that have been linked to tumor promotion. The Wnt pathway is regulated by a complex network of extracellular ligands, receptors, and antagonists, of which DKK1 is only one. Due to the restricted expression of DKK1 in adults, and to its functional redundancy with other Wnt antagonists, it is unlikely that a neutralizing DKK1 antibody will cause a widely extended activation of Wnt signaling, or consequently, tumorigenesis. This is further supported by two observations: first, the activation of LRP5 mutations (which inhibit DKK binding) induce a phenotype of high bone mass, but do not have an apparent increased cancer risk [Moon 2004], whereas mice heterozygous nulls of DKK1 or Doubleridge have decreased levels of DKK1, the phenotype of high bone mass, but no increased rate of tumor formation is reported [MacDonald 2004]. An anti-DKK1 antibody must positively impact osteolytic disease induced by myeloma, while not increasing the risk of de novo tumorigenesis. It is expected that this antibody would be used in combination with antitumor chemotherapies, and possibly with anti-bone resorption drugs that inhibit the function of osteoclasts. Polyclonal Antibodies The antibodies of the invention can be polyclonal antibodies, especially human polyclonal antibodies. Polyclonal antibodies are derived from the secreted serum of immunized animals or selected humans. Monoclonal Antibodies The antibodies of the invention are preferably human monoclonal antibodies, such as those isolated and structurally characterized, for example, in Examples 1 to 8. VH-specific amino acid sequences are shown. antibodies, for example, in SEQ ID NOs: 2-20. The antibody-specific VL amino acid sequences are shown, for example, in SEQ ID NOs: 21 -39. A VH amino acid sequence of the antibody can be optimized for expression in a mammalian cell, for example, such as the sequence shown in SEQ ID NO: 1 1 9. A VL amino acid sequence of the antibodies can be optimized for expression in a mammalian cell, for example, such as the sequence shown in SEQ ID NO: 1 18. In the same manner, the sequences can be optimized for expression in, for example, yeast, bacteria, hamster cells and other cells, depending on the system preferred expression for the feature that is being optimized. Other antibodies of the invention include amino acids that have mutated, and yet have at least 60, 70, 80, 90, 95, 96, 97, 98, or 99 percent identity in CDR regions, with the CDR regions illustrated in the sequences described above. In addition, the full-length light chain progenitor nucleotide sequences are shown in SEQ ID NOs: 97-100. The full-length heavy chain progenitor nucleotide sequences are shown in SEQ ID NOs: 101-103. The full length light chain nucleotide sequences optimized for expression in a mammalian cell are shown in SEQ ID NOs: 104-107. The full-length heavy chain nucleotide sequences for the expression in a mammalian cell in SEQ ID NOs: 108-1 1 0. The full length light chain amino acid sequences encoded by the light chain nucleotide sequences optimized in SEQ ID NOs are shown: 1 1 1 - 1 1 4. The full length heavy chain amino acid sequences encoded by the heavy chain nucleotide sequences optimized in SEQ ID NOs are shown: 1 1 5-1 1 7. Other antibodies of the invention include amino acids or nucleic acids that have mutated, and that nonetheless, have an identity of at least 60, 70, 80, 90, 95, 96, 97, 98, or 99 percent with the sequences described above. Because each of these antibodies can bind to DKK1, the full length light chain, VH, VL, and heavy chain sequences (nucleotide sequences and amino acid sequences) can be "mixed and matched" to create other anti-DKK1 binding molecules of the invention. The DKK1 binding of these "mixed and matched" antibodies can be tested using the binding assays described above, and in the Examples (eg, ELISAs). When these chains are mixed and matched, a VH sequence from a particular VH / V pair must be replaced with a structurally similar VH sequence. In the same way, a full-length heavy chain sequence from a particular pair of full-length heavy chain / full-length light chain, must be replaced with a sequence of full-length heavy chain structurally similar. In the same way, a V sequence from a particular VH / VL pair must be replaced with a structurally similar VL sequence. In the same manner, a full length light chain sequence from a particular full length heavy chain / full length light chain pair must be replaced with a structurally similar full length light chain sequence. The full-length light chain, VH, V chain and heavy chain sequences of the antibodies of the present invention are particularly susceptible to mixing and pairing, because these antibodies use the VH, VL sequences. , full length light chain, and full length heavy chain derived from the same germline sequences, and therefore, exhibit structural similarity. In accordance with the above, in one aspect, the invention provides an isolated monoclonal antibody or an antigen binding portion thereof, having: a VH region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-20 and 1 1 9; and a V region comprising an amino acid sequence selected from the group consisting of SEQ I D NOs: 21 -39 and 1 1 8; wherein the antibody specifically binds to DKK1. Examples of heavy and light chain combinations include: a VH region comprising the amino acid sequence of SEQ ID NO: 2, and a V region comprising the amino acid sequence of SEQ ID NO: 21; or a VH region comprising SEQ ID NO: 3, and a VL region comprising SEQ ID NO: 22; or a VH region comprising SEQ ID NO: 4 and a VL region comprising SEQ ID NO: 23; or a VH region comprising SEQ ID NO: 5 and a V region comprising SEQ ID NO: 24; or a VH region comprising SEQ ID NO: 6 and a VL region comprising SEQ ID NO: 25, or a VH region comprising SEQ ID NO: 7 and a VL region comprising SEQ ID NO: 28; or a VH region comprising SEQ ID NO: 8 and a V region comprising SEQ ID NO: 29; or a VH region comprising SEQ ID NO: 9 and a V region comprising SEQ ID NO: 30; or a VH region comprising SEQ ID NO: 10 and a V region comprising SEQ ID NO: 31; or a VH region comprising SEQ ID NO: 11 and a V region comprising SEQ ID NO: 32; or a VH region comprising SEQ ID NO: 12 and a Vu region comprising SEQ ID NO: 33; or a VH region comprising SEQ ID NO: 119 and a VL region comprising SEQ ID NO: 118. In another aspect, the invention provides an isolated monoclonal antibody or an antigen binding portion thereof having: a full-length heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 115- 117; and a full-length light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 111- 114. Accordingly, examples of the full length heavy chain and full length light chain combinations, respectively, include: SEQ ID NO: 115 with SEQ ID NO: 111; or SEQ ID NO: 116 with SEQ ID NO: 112; or SEQ ID NO: 117 with SEQ ID NO: 113; or SEQ ID NO: 117 with SEQ ID NO: 114. In another aspect, the invention provides an isolated monoclonal antibody, or an antigen binding portion thereof, comprising a full length heavy chain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 101 -103; and a full-length light chain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 97-100. Accordingly, examples of the nucleotides encoding the full length light and heavy chains, respectively, which may be combined, include: SEQ ID NO: 101 and 97; or SEQ ID NO: 102 and 98; or SEQ ID NO: 103 and 99; or SEQ ID NO: 104 and 100. In still another aspect, the invention provides an isolated monoclonal antibody, or an antigen binding portion thereof, which has been optimized for expression in the cell having: a length heavy chain complete comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 108-110; and a full-length light chain comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 104-107. In yet another aspect, the invention provides antibodies comprising the heavy chain and light chain CDR1s, CDR2s, and CDR3s of the antibodies, or combinations thereof. The amino acid sequences of the VH CDR1s of the antibodies are shown in SEQ ID NOs: 2-5, 8-11, 20, and 49-56. The amino acid sequences of the VH CDR2s of the antibodies are shown in SEQ ID NOs: 2-20 and 57-64. The amino acid sequences of the VH CDR3s of the antibodies are shown in SEQ ID NOs: 2-20 and 65-72. The amino acid sequences of the Vu CDR1s of the antibodies are shown in SEQ ID NOs: 21-39 and 73-80. The amino acid sequences of the V CDR2s of the antibodies are shown in SEQ ID NOs: 21-39 and 81-88. The amino acid sequences of the V CDR3s of the antibodies are shown in SEQ ID NOs: 21-39 and 89-96. The CDR regions are delineated using the Kabat system (Kabat, E. A. et al., 1991 Sequences of Proteins of Immunological Interest, Fifth Edition, United States Department of Health and Human Services, NIH Publication Number 91-3242). Provided that each of these antibodies can bind to DKK1, and that the antigen binding specificity is provided primarily by the CDRI, 2, and 3 regions, the VH CDR1, 2, and 3 sequences and the CDR1, 2 sequences. , and 3 of VL can be "mixed and matched" (ie, the CDRs of different antibodies can be mixed and matched, although each antibody must contain a VH CDR1, 2, and 3 and a VL CDR 1, 2, and 3 to create other anti-DKK1 binding molecules of the invention. The binding of DKK1 with these "mixed and matched" antibodies can be tested using the binding assays described above and in the examples (e.g., ELISAs). When the VH CDR sequences are mixed and matched, the sequence of CDR1, CDR2, and / or CDR3 from a particular VH sequence must be replaced with a structurally similar CDR sequence. In the same way, when the CDR sequences of V are mixed and matched, the sequence of CDR 1, CDR2, and / or CDR3 from a particular V sequence must be replaced with a structurally similar CDR sequence. Additionally, the sequence of CDR1, CDR2, and / or CDR3 from a particular VH or VL sequence can be mutated in a specific or random manner to create antibodies, which can be tested to determine their affinity or binding characteristics. It will be readily apparent to the ordinary skilled person that the novel VH and V sequences can be created by substituting one or more sequences of the VH and / or V CDR region with structurally similar sequences from the CDR sequences shown herein., to have the monoclonal antibodies of the present invention. An isolated monoclonal antibody, or an antigen binding portion thereof, has: a CDR1 region of VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-5, 8-11, 20 and 49-56; a CDR2 region of VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-20 and 57-64; a CDR3 region of VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-20 and 65-72; a CDR1 region of V comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 21-39 and 73-80; a CDR2 region of VL comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 21-39 and 81-88; and a VR CDR3 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 21-39 and 89-96; wherein the antibody specifically binds to DKK1. In a certain modality, the antibody consists of: a region VH CDR1 comprising SEQ ID NO: 2; a CDR2 region of VH comprising SEQ ID NO: 6; a CDR3 region of VH comprising SEQ ID NO: 7; a CDR1 region of V comprising SEQ ID NO: 21; a CDR2 region of VL comprising SEQ ID NO: 22; and a CDR3 region of VL comprising SEQ ID NO: 23. In another embodiment, the antibody consists of: a VH CDR1 region comprising SEQ ID NO: 3; a CDR2 region of VH comprising SEQ ID NO: 12; a CDR3 region of VH comprising SEQ ID NO: 13; a CDR1 region of VL comprising SEQ ID NO: 24; a CDR2 region of VL comprising SEQ ID NO: 25; Y a CDR3 region of VL comprising SEQ ID NO: 26. In still another embodiment, the antibody consists of: a VH CDR1 region comprising SEQ ID NO: 4; a CDR2 region of VH comprising SEQ ID NO: 14; a CDR3 region of VH comprising SEQ ID NO: 15; a CDR1 region of VL comprising SEQ ID NO: 27; a CDR2 region of V comprising SEQ ID NO: 28; and a CDR3 region of VL comprising SEQ ID NO: 29. In another embodiment, the antibody consists of: a CDR1 region of VH comprising SEQ ID NO: 5; a CDR2 region of VH comprising SEQ ID NO: 16; a CDR3 region of VH comprising SEQ ID NO: 17; a CDR1 region of V comprising SEQ ID NO: 30; a CDR2 region of V comprising SEQ ID NO: 31; and a CDR3 region of VL comprising SEQ ID NO: 32. In a certain modality, the antibody consists of: a region VH CDR1 comprising SEQ ID NO: 8; a CDR2 region of VH comprising SEQ ID NO: 18; a CDR3 region of VH comprising SEQ ID NO: 19; a CDR1 region of V comprising SEQ ID NO: 33; a CDR2 region of VL comprising SEQ ID NO: 34; and a CDR3 region of VL comprising SEQ ID NO: 35. In another embodiment, the antibody consists of: a VH CDR1 region comprising SEQ ID NO: 9; a CDR2 region of VH comprising SEQ ID NO: 10; a CDR3 region of VH comprising SEQ ID NO: 11; a CDR1 region of VL comprising SEQ ID NO: 36; a CDR2 region of VL comprising SEQ ID NO: 37; Y a CDR3 region of VL comprising SEQ ID NO: 38. As used herein, a human antibody comprises the heavy or VL regions, or the full-length heavy and light chains that are "the product of" or "derived from" a particular germline sequence, if the variable regions or the full length chains of the antibody are obtained from a system using the human germline immunoglobulin genes. These systems include immunizing a transgenic mouse carrying the human immunoglobulin genes with the antigen of interest, or screening a library of human immunoglobulin genes displayed on the phage with an antigen of interest. A human antibody that is "the product of" or "derived from" a human germline immunoglobulin sequence can be identified as such by comparing the amino acid sequence of the human antibody with the amino acid sequences of the line immunoglobulins. human germinal, and selecting the human germline immunoglobulin sequence that is closest in sequence (ie, has the highest percentage of identity) to the sequence of the human antibody. A human antibody that is "the product of" or "derived from" a particular human germline immunoglobulin sequence, may contain amino acid differences, compared to the germline sequence, due, for example, to somatic mutations that occur naturally, or to the intentional introduction of mutation directed to the site. Without However, a human antibody selected is typically at least 90 percent identical in amino acid sequence to an amino acid sequence encoded by a human germline immunoglobulin gene, and contains amino acid residues that identify the human antibody as a human , when compared to the germline immunoglobulin amino acid sequences of other species (eg, murine germ line sequences). In certain cases, a human antibody can be at least 60 percent, 70 percent, 80 percent, 90 percent, or at least 95 percent, or even at least 96 percent, 97 percent, 98 percent, or 99 percent identical in the amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, a human antibody derived from a particular human germline sequence will not display more than 10 amino acid differences of the amino acid sequence encoded by the human germline immunoglobulin gene. In certain cases, the human antibody can exhibit no more than five, or even no more than 4, 3.2, or 1 amino acid differences of the amino acid sequence encoded by the germline immunoglobulin gene. Homologous antibodies In yet another embodiment, an antibody of the invention has the heavy and light chain amino acid sequences of full length; the heavy and light chain full-length nucleotide sequences, the heavy and light chain nucleotide sequences of variable regions, or the heavy and light chain amino acid sequences of variable regions, which are homologous to the amino acid and nucleotides of the antibodies described herein, and wherein the antibodies retain the desired functional properties of the neutralizing anti-DKK1 / 4 composition of the invention. For example, the invention provides an isolated monoclonal antibody, or an antigen binding portion thereof, comprising a VH region and a V region, wherein: the VH region comprises an amino acid sequence that is at least 80 percent homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-20 and 1 19; the V region comprises an amino acid sequence that is at least 80 percent homologous to an amino acid sequence selected from the group consisting of SEQ I D NOs: 21 -39 and 1 1 8; the antibody binds specifically to DKK1 and / or DKK4, and the antibody exhibits at least one of the following functional properties: the antibody neutralizes the binding of a DKK1 protein with LPR6, Fz, and / or Krm, or the antibody neutralizes the binding of a DKK4 protein with LRP, Pz, and / or Krm. In a further example, the invention provides an isolated monoclonal antibody, or an antigen binding portion thereof, comprising a full length heavy chain and a full length light chain, wherein: the full length heavy chain comprises an amino acid sequence that is at least 80 percent homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 1 5 -1 17; the full length light chain comprises an amino acid sequence that is at least 80 percent homologous to an amino acid sequence selected from the group consisting of SEQ I D NOs: 1 1 1 -1 14; the antibody binds specifically to DKK1, and the antibody exhibits at least one of the following functional properties: the antibody inhibits the binding of the DKK1 protein to the DKK1 receptor, or the antibody inhibits the binding of the DKK1 receptor, preventing or improving osteolysis, or the antibody inhibits the binding of the DKK1 receptor by preventing or improving osteolytic lesions, or the antibody inhibits the binding of the DKK1 receptor by preventing or improving cancer. In another example, the invention provides an isolated monoclonal antibody, or an antigen binding portion thereof, which comprises a full length heavy chain and a full length light chain, wherein: the full length heavy chain comprises a nucleotide sequence which is at least 80 percent homologous to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 101 -103; the full-length light chain comprises a nucleotide sequence that is at least 80 percent homologous to a nucleotide sequence selected from the group consisting of SEQ I D NOs: 97-100; the antibody binds specifically to DKK1, and the antibody exhibits at least one of the following functional properties: the antibody inhibits the binding of the DKK1 protein to the DKK1 receptor, or the antibody inhibits the binding of the DKK1 receptor by preventing or improving the osteolysis, or the antibody inhibits the binding of the DKK1 receptor by preventing or ameliorating the osteolytic lesions, or the antibody inhibits the binding of the DKK1 receptor by preventing or ameliorating the cancer. In another example, the invention provides an isolated monoclonal antibody, or an antigen binding portion thereof, that has been optimized for expression in a cell, which comprises a full-length heavy chain and a full-length light chain, wherein: the full-length heavy chain comprises a nucleotide sequence that is at least 80 percent homologous to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 108-1 10; the full-length light chain comprises a nucleotide sequence that is at least 80 percent homologous to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 104-1 07; the antibody binds specifically to DKK1, and the antibody exhibits at least one of the following functional properties: the antibody inhibits the binding of the DKK1 protein to the DKK1 receptor, or the antibody inhibits the binding of the DKK1 receptor by preventing or ameliorating osteolysis, or the antibody inhibits the binding of the DKK1 receptor by preventing or ameliorating the osteolytic lesions, or the antibody inhibits the binding of the DKK1 receptor by preventing or ameliorating the cancer. In another example, the invention provides an isolated monoclonal antibody, or an antigen binding portion thereof, that has been optimized for expression in a cell, which comprises a VH region and a VL region, wherein: the heavy chain Full length comprises a nucleotide sequence that is at least 80 percent homologous to a nucleotide sequence selected from the group consisting of SEQ ID NO: 121; the full-length light chain comprises a nucleotide sequence that is at least 80 percent homologous to a nucleotide sequence selected from the group consisting of SEQ ID NO: 120; the antibody binds specifically to DKK1, and the antibody exhibits at least one of the following functional properties: the antibody inhibits the binding of the DKK1 protein to the DKK1 receptor, or the antibody inhibits the binding of the DKK1 receptor by preventing or improving the osteolysis, or the antibody inhibits the binding of the DKK1 receptor by preventing or ameliorating the osteolytic lesions, or the antibody inhibits the binding of the DKK1 receptor by preventing or ameliorating the cancer. In different modalities, the antibody can exhibit one or more, two or more, or three of the functional properties discussed above. The antibody can be, for example, a human antibody, a humanized antibody, or a chimeric antibody. As used herein, the percentage of homology between two amino acid sequences or two nucleotide sequences is equivalent to the percent identity between the two sequences. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (ie, percentage of homology = number of identical positions / total number of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be entered for optimal alignment of the two sequences. The comparison of the sequences and the determination of the percentage of identity between two sequences, can be carried out using a mathematical algorithm, as described in the non-limiting Examples that follow later. The percentage of identity between two amino acid sequences can be determined using the algorithm of Meyers and W. Miller (Comput. Appl. Biosci., 4: 1 1 7 7, 1 988), which has been incorporated into the ALIGN program (version 2.0), using a weight residue table PAM 120, a fine per gap length of 1 2, and a gap fine of 4. In addition, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48: 444-453, 1 970), which has been incorporated into the GAP program, in the package of GCG software (available at http://www.gcg.com), using either a Blosson 62 matrix, or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6. In an additional or alternative manner, the protein sequences of the present invention can be additionally used as a "requested sequence", to carry conduct a search against public databases, for example, to identify related sequences. These searches can be carried out using the XBLAST program (version 2.0) of Altschul et al., 1990 J. Mol. Biol. 215-403-10. Searches of BLAST protein can be carried out with the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to the antibody molecules of the invention. To obtain alignments with gaps for comparison purposes, Gapped BLAST can be used, as described in Altschul et al., 1997 Nucleic Acids Res. 25 (17): 3389-3402. When using the BLAST and Gapped BLAST programs, the default parameters of the respective programs can be used (for example, XBLAST and NBLAST). See http: www.ncbi.nhn.nih.gov. Antibodies with conservative modifications In certain embodiments, an antibody of the invention has a VH region consisting of the sequences of CDR1, CDR2, and CDR3, and a V region consisting of the sequences of CDR1, CDR2, and CDR3, wherein one or more of these CDR sequences have specified amino acid sequences based on the antibodies described herein, or conservative modifications thereof, and wherein the antibodies retain the desired functional properties of the neutralizing DKK1 / 4 composition. of the invention. In accordance with the above, the invention provides an isolated monoclonal antibody, or an antigen binding portion thereof, consisting of a VH region consisting of the sequences of CDR 1, CDR2, and CDR3, and a VL region consisting of in the sequences of CDR 1, CDR2, and CDR3, wherein: the VH regions of CDR 1 are the sequences consisting of the amino acid sequences selected from the group consisting of the amino acid sequences of SEQ ID NOs: 2-5, 8-1,15, 49-56, and the conservative modifications thereof; the VH region of the CDR2 sequences consisting of the amino acid sequences selected from the group consisting of the amino acid sequences of SEQ ID NOs: 2-20, 57-64, and the conservative modifications thereof; the VH region of CDR3 are the sequences consisting of the amino acid sequences selected from the group consisting of the amino acid sequences of SEQ I D NOs: 2-20, 65-72, and the conservative modifications thereof; the V regions of CDR1 are the sequences consisting of the amino acid sequences selected from the group consisting of the amino acid sequences of SEQ ID NOs: 21 - 39, 73-80, and the conservative modifications thereof; the V regions of the CDR2 are the sequences consisting of the amino acid sequences selected from the group consisting of the amino acid sequences of SEQ I D NOs: 21 -39, 81 -88, and the conservative modifications thereof; the V regions of the CDR3 are the sequences consisting of the amino acid sequences selected from the group consisting of the amino acid sequences of SEQ ID NOs: 21 -39, 89-96, and the conservative modifications thereof; the antibody specifically binds to DKK1; and the antibody exhibits at least one of the following functional properties: the antibody inhibits the binding of the DKK1 protein to the DKK1 receptor, or the antibody inhibits the binding of the DKK1 receptor by preventing or improving osteolysis, or the antibody inhibits the binding of the DKK1 receptor by preventing or improving osteolytic lesions, or the antibody inhibits the binding of the DKK1 receptor by preventing or improving cancer. In different embodiments, the antibody may exhibit one or more, two or more, or three or more of the listed functional properties discussed above. These antibodies can be, for example, human antibodies, humanized antibodies, or chimeric antibodies. In other embodiments, an antibody of the invention has a full length heavy chain sequence and a full length light chain sequence, wherein one or more of these sequences have the specified amino acid sequences, based on the antibodies described herein, or conservative modifications thereof, and wherein the antibodies retain the desired functional properties of the neutralizing anti-DKK1 / 4 composition of the invention. In accordance with the above, the invention provides an isolated monoclonal antibody, or an antigen binding portion thereof, consisting of a full-length heavy chain and a full-length light chain, wherein: the full-length heavy chain has the amino acid sequences selected from the group of SEQ ID NOs: 1 1 5-1 1 7, and the conservative modifications thereof; and the full-length light chain has the amino acid sequences selected from the group of SEQ I D NOs: 1 1 1 -1 1 4, and the conservative modifications thereof; the antibody specifically binds to DKK1; and the antibody exhibits at least one of the following functional properties: the antibody inhibits the binding of the DKK1 protein to the DKK1 receptor, or the antibody inhibits the binding of the DKK1 receptor by preventing or improving osteolysis, or the antibody inhibits the binding of the DKK1 receptor by preventing or ameliorating osteolytic lesions, or the antibody inhibits the binding of the DKK1 receptor by preventing or ameliorating the cancer. In different embodiments, the antibody may exhibit one or more, two or more, or three or more of the functional properties listed previously discussed. These antibodies can be, for example, human antibodies, humanized antibodies, or chimeric antibodies. In other embodiments, an antibody of the invention optimized for expression in a cell has a sequence of the VH region and a sequence of the VL region, wherein one or more of these sequences have the specified amino acid sequences, based on the antibodies described herein, or conservative modifications thereof, and wherein the antibodies retain the desired functional properties of the neutralizing anti-DKK1 / 4 composition of the invention. In accordance with the above, the invention provides an isolated monoclonal antibody, or an antigen binding portion thereof, which consists of a VH region and a V region, wherein: the VH region has the amino acid sequences selected from of the group of SEQ ID NO: 1 1 9, and the conservative modifications thereof; and the VL region has the amino acid sequence selected from the group of SEQ ID NO: 1 1 8, and the conservative modifications thereof; the antibody binds specifically to DKK1, and the antibody exhibits at least one of the following functional properties: the antibody inhibits the binding of the DKK1 protein to the DKK1 receptor, or the antibody inhibits the binding of the DKK1 receptor by preventing or improving the osteolysis, or the antibody inhibits the binding of the DKK1 receptor by preventing or ameliorating osteolytic lesions, or antibody inhibits the binding of the DKK1 receptor by preventing or improving cancer. In different embodiments, the antibody may exhibit one or more, two or more, or three or more of the listed functional properties discussed above. These antibodies can be, for example, human antibodies, humanized antibodies, or chimeric antibodies. As used herein, the term "conservative sequence modifications" is intended to refer to amino acid modifications that do not affect or significantly alter the binding characteristics of the antibody containing the amino acid sequence. These conservative modifications include substitutions, additions, and deletions of amino acids. Modifications can be introduced into an antibody of the invention by conventional techniques known in the art, such as site-directed mutagenesis., and mutagenesis mediated by polymerase chain reaction. Conservative amino acid substitutions are those in which the amino acid residue is replaced with an amino acid residue having a similar side chain. The families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (eg, lysine, arginine, histidine), acid side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (eg, alanine, valine, leucine, isoieucine, proline, phenylalanine, methionine), beta-branched side chains (eg, threonine , valine, isoieucine), and aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine). Accordingly, one or more amino acid residues within the CDR regions of an antibody of the invention can be replaced with other amino acid residues of the same side chain family, and the altered antibody can be tested to have a retained function using the functional assays described herein. Antibodies that bind to the same epitope as the neutralizing anti-DKK1 / 4 composition of the invention In another embodiment, the invention provides antibodies that bind to the same epitope as the different neutralizing anti-DKK1 / 4 compositions of the invention provided in the present. These additional antibodies can be identified based on their ability to cross compete (eg, to competitively inhibit the binding of, in a statistically significant manner) with other antibodies of the invention, in conventional DKK1 binding assays. The ability of a test antibody to inhibit binding of the antibodies of the present invention to human DKK1 demonstrates that the test antibody can compete with that antibody for binding to human DKK1; this antibody, according to the hypothesis limiting, it can be linked to the same or a related epitope (eg, a structurally similar or spatially proximal epitope) on human DKK1 than the antibody with which it competes. In a certain embodiment, the antibody that binds to the same epitope on human DKK1 as the antibodies of the present invention is a human monoclonal antibody. These human monoclonal antibodies can be prepared and isolated as described in the Examples. Camelid antibodies and other heavy chain antibodies Antibody proteins obtained from members of the camel and dromedary family (Camelus bactrianus and Calelus dromaderius), including members of the New World, such as llama species, have been characterized. (Lama páceos, Lama glama, and Lama vicugna), with respect to size, structural complexity, and antigenicity for human subjects. Certain IgG antibodies of this family of mammals, as found in nature, lack light chains, and therefore, are structurally distinct from the typical four-chain quaternary structure that has two heavy chains and two light chains, for antibodies from other animals. See TCP Publication No. PCT / EP93 / 0221 4 (International Publication Number WO 94/04678 published March 3, 1994). A region of the camelid antibody which is the small individual variable domain identified as VHH can be obtained. by genetic engineering, to produce a small protein having a high affinity for a target, resulting in a protein derived from low molecular weight antibody known as a "camelid nanobody". See U.S. Patent No. 5,759,808, issued June 2, 1,998; see also Stijlemans, B. et al., 2004 J. Biol. Chem. 279: 1256-1261; Dumoulin, M. et al., 2003 Nature 424: 783-788; Pleschberger, M. et al., 2003 Bioconjugate Chem. 1 4: 440-448; Cortez-Retamozo, V. et al., 2002 Int. J. Cancer 89: 456-62; and Lauwereys, M. et al., 1 998 EMBO J. 1 7: 351 2-3520. The designed libraries of antibodies and camelid antibody fragments are commercially available from, for example, Ablynx, Ghent, Belgium. As with other antibodies of non-human origin, an amino acid sequence of a camelid antibody can be altered in a recombinant manner to obtain a sequence that more closely resembles a human sequence, that is, the nanobody can be "humanized". Accordingly, the low natural antigenicity of the camelid antibodies for humans can be further reduced. The camelid nanobody has a molecular weight of about one-tenth that of a human IgG molecule, and the protein has a physical diameter of only a few nanometers. One consequence of the small size is the ability of the camelid nanobodies to link with the antigenic sites that are functionally invisible to the larger antibody proteins, i.e., the camelid nanobodies are useful as reagents for detecting antigens that are otherwise cryptic using classical immunological techniques, and as possible therapeutic agents. Therefore, still another consequence of the small size, is that a camelid nanobody can inhibit, as a result of binding to a specific site in a narrow slot or slit of a target protein, and therefore, can serve in a capacity that resembles more closely to the function of a classic low molecular weight drug than that of a classical antibody. The low molecular weight and compact size also result in camelid nanobodies that are extremely thermostable, stable at extreme pH and proteolytic digestion, and poorly antigenic. Another consequence is that the camelid nanobodies move easily from the circulatory system to the tissues, and even cross the blood-brain barrier, and can treat disorders that affect nervous tissue. Nanobodies can further facilitate the transport of drugs through the blood-brain barrier. See U.S. Patent Application No. 200401 61738, published August 1, 2004. These characteristics, combined with the low antigenicity for humans, indicate the great therapeutic potential. In addition, these molecules can be expressed completely in prokaryotic cells, such as E. coli, and are expressed as fusion proteins with bacteriophages and are functional. In accordance with the above, a feature of the present invention is an antibody or camelid nanobody that has a high affinity for DKK1. In certain embodiments herein, the camelid antibody or nanobody is naturally produced in the camelid animal, ie, it is produced by the camelid following immunization with DKK1 or with a peptide fragment thereof, employing the techniques described herein. for other antibodies. Alternatively, the neutralizing anti-DKK1 / 4 camelid nanobody is designed, i.e., produced by selection, e.g., from a library of phages displaying proteins from appropriately mutated camelid nanobodies using extension procedures with DKK1 and / or DKK4 as a target, as described in the Examples herein. The designed nanobodies can be further made to the extent by genetic engineering to have a half-life in a recipient subject from 45 minutes to two weeks. In addition to camelid antibodies, heavy chain antibodies occur naturally in other animals, including, but not limited to, for example, certain species of shark and pufferfish (see, for example, Publication of the TCP Number WO). 03/014161). Although variable domains derived from these heavy chain antibodies can be used In the invention, the use of heavy chain antibodies derived from camelid and / or the variable domain sequences thereof, is the preferred optimization, humanization, humaneering ("humanodiseño"), and the like, and / or for clinical use in humans. An antibody of the invention can be further prepared using an antibody having one or more of the VH and / or V sequences shown herein as a starting material for designing a modified antibody, which modified antibody can have altered antibody departure. An antibody can be designed by modifying one or more residues within one or both of the variable regions (i.e., VH and / or VL), for example, within one or more CDR regions, and / or within one or more structure regions. Additionally or in an alternative way, an antibody can be designed by modifying the residues within the constant regions, for example, to alter the effector functions of the antibody. One type of variable region design that can be carried out is the CDR graft. The antibodies interact with the target antigens predominantly through the amino acid residues that are located in the six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences within the CDRs are more diverse between the individual antibodies than the sequences outside the CDRs. Because the CDR sequences are responsible for In most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific antibodies that occur naturally, by constructing expression vectors that include CDR sequences from the specific antibody that is naturally occurring grafted onto the structure sequences of a different antibody with different properties (see, for example, Riechmann, L. et al, 1998 Nature 332: 323-327; Jones, P. et al., 1986 Nature 321: 522-525; C. et al., 1 989 Proc. Nati, Acad. Sci. USA 86: 1 0029-10033; United States of America Patent No. 5,225,539 to Winter, and Patents of the United States of North America Nos. 5,530, 101 5,585,089, 5,693,762 and 6,180,370 to Queen et al.). In accordance with the above, another embodiment of the invention pertains to an isolated monoclonal antibody, or an antigen binding portion thereof, comprising a VH region comprising the CDR 1 sequences having an amino acid sequence selected in part from from the group consisting of SEQ ID NOs: 2-5, 8-1 1, 20, 49-56; the CDR2 sequences having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-20 and 57-64; the CDR3 sequences having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-20 and 65-72, respectively; and a V region having the CDR 1 sequences having an amino acid sequence selected from the group consisting of SEQ ID NOs: 21-39 and 73-80; the CDR2 sequences having an amino acid sequence selected from the group consisting of SEQ ID NOs: 21-39 and 81-88; and CDR3 sequences consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 21-39 and 89-96, respectively. Accordingly, these antibodies contain the VH and V CDR sequences of the monoclonal antibodies, and nevertheless, may contain different structure sequences of these antibodies. These structure sequences can be obtained from public DNA databases, or from published references that include genetic sequences of germline antibodies. For example, the germline DNA sequences for the human heavy chain and VL region genes can be found in the human germline sequence database "VBase" (available on the Internet at www.mrccpe. cam.ac.uk/vbase), as well as in Kabat, EA, et al., 1991 Sequences of Proteins of Immunological Interest, Fifth Edition, United States Department of Health and Human Services, NIH Publication Number 91-3242; Tomlinson, I. M., et al., 1992 J. Mol. Biol. 227: 776-798; and Cox, J. P. L. et al., 1994 Eur. J. Immunol. 24: 827-836; the content of each of them being expressly incorporated herein by reference. An example of structure sequences to be used in the Antibodies of the invention are those that are structurally similar to the structure sequences used by the antibodies selected from the invention, for example, the sequences in consensus and / or the structure sequences used by the monoclonal antibodies of the invention. VH CDR 1, 2, and 3 sequences and VR CDR 1, 2, and 3 sequences can be grafted to regions of structure that have the sequence identical to that found in the germline immunoglobulin gene from which the structure sequence is derived, or the CDR sequences can be grafted onto the structure regions containing one or more mutations, compared to the germline sequences. For example, it has been found that, in certain cases, it is beneficial to mutate the residues within the structure regions to maintain or enhance the antigen binding capacity of the antibody (see, for example, United States of America Patents Numbers). 5,530, 101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.). Another type of modification of the variable region is to mutate the amino acid residues within the CDR 1, CDR2, and / or CDR3 regions of VH and / or V, to thereby improve one or more binding properties (e.g., affinity ) of the antibody of interest, known as "affinity maturation". Site-directed mutagenesis or mutagenesis mediated by polymerase chain reaction can be carried out to introduce the mutations, and the effect on the binding of the antibody or other functional property of interest in in vitro or in vivo assays, as described herein and as provided in the Examples. Conservative modifications can be introduced (as discussed in the above). Mutations can be substitutions, additions, or deletions of amino acids. Moreover, typically more than 1, 2, 3, 4, or 5 residues are altered within a CDR region. In accordance with the foregoing, in another embodiment, the invention provides an isolated neutralizing anti-DKK1 / 4 composition, or antigen binding portions thereof, which consists of a VH region having: a VH CDR1 region consisting of an amino acid sequence selected from the group having SEQ ID NOs: 2-5, 8-1,15, 49-56, or an amino acid sequence having one, two, three, four, or five substitutions, deletions, or additions of amino acids, compared to SEQ ID NOs: 2-5, 8-1 1, 20, 49-56; a CDR2 VH region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-20 and 57-64, or an amino acid sequence having one, two, three, four, or five substitutions, deletions, or additions of amino acids, compared to SEQ ID NOs: 2-20 and 57-64; a CDR3 VH region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-20 and 65-72, or an amino acid sequence having one, two, three, four, or five substitutions, deletions, or amino acid additions, compared to SEQ ID NOs: 2-20 and 65-72; a 1 V CDR region that has a amino acid sequence selected from the group consisting of SEQ ID NOs: 21 -39 and 73-80, or an amino acid sequence having one, two, three, four, or five substitutions, deletions, or amino acid additions, comparing with SEQ ID NOs: 21 -39 and 73-80; a CDR2 VL region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 21 -39 and 81 -88, or an amino acid sequence having one, two, three, four, or five substitutions, deletions, or amino acid additions, compared to SEQ ID NOs: 21 -39 and 81 -88; and a CDR3 VL region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 21 -39 and 89-96, or an amino acid sequence having one, two, three, four, or five substitutions , deletions, or additions of amino acids, comparing with SEQ ID NOs: 21 -39 and 89-96. The designed antibodies of the invention include those wherein modifications to the framework residues have been made within VH and / or VL, for example, to improve the properties of the antibody. Typically, these structural modifications are made to decrease the immunogenicity of the antibody. For example, one approach is to "retromue" one or more structure residues up to the sequence of the corresponding germ line. More specifically, an antibody that has undergone somatic mutation may contain structure residues that differ from the germline sequence from which it is derived. the antibody is derived. These can be identified by comparing the structure sequences of the antibody with the germline sequences from which the antibody is derived. To return to the region of structure sequences up to their germline configuration, somatic mutations can be "retromutated" up to the germline sequence, for example, by site-directed mutagenesis or by mutagenesis-mediated chain reaction mutagenesis. the polymerase These "retromutated" antibodies are also encompassed by the present invention. Another type of structure modification involves the mutation of one or more residues within the framework region, or even within one or more CDR regions, to remove the T cell epitopes, in order to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization", and is described in greater detail in U.S. Patent Publication No. 200301 53043, by Carr et al. In addition or in an alternative manner to modifications made within the framework or CDR regions, the antibodies of the invention can be designed to include modifications within the Fe region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, receptor binding, and / or antigen-dependent cellular cytotoxicity. Additionally, an antibody of the invention can be chemically modified (for example, one or more chemical fractions can be bound to the antibody), or it can be modified to alter its glycosylation, again in order to alter one or more functional properties of the antibody. Each of these modalities is described in more detail below. The numbering of the residues in the Fe region is that of the United States index of Kabat. In one embodiment, the joint region of CH1 is modified, such that the number of cysteine residues in the joint region is altered, for example, that is increased or decreased. This approach is further described in U.S. Patent No. 5,677,425 by Bodmer et al. The number of cysteine residues in the joint region of CH1 is altered, for example, to facilitate the assembly of the heavy and light chains, or to increase or decrease the stability of the antibody. In another embodiment, the Fe-articulation region of an antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the interface region of the CH2-CH3 domain of the Fe joint fragment, such that the antibody has impaired the binding of Staphylococcyl protein A (SpA) with the SpA linkage of the native Fe articulation domain. This approach is described in greater detail in U.S. Patent No. 6,165,745 by Ward et al.

In another embodiment, the antibody is modified to increase its biological half-life. Different approaches are possible. For example, one or more of the following mutations may be introduced: T252L, T254S, T256F, as described in U.S. Patent No. 6,277,375 to Ward. Alternatively, in order to increase the biological half-life, the antibody can be altered within the CH1 or CL region, to contain a salvage receptor binding epitope taken from two cycles of a CH2 domain of a Fe region of an IgG, as described in the Patents of the United States of North America Nos. 5,869,046 and 6,121,022 by Presta et al. In still other embodiments, the Fe region is altered by replacing at least one amino acid residue with a different amino acid residue in order to alter the effector functions of the antibody. For example, one or more amino acids may be replaced with a different amino acid residue, such that the antibody has an altered affinity for an effector ligand, but retains the antigen binding capacity of the parent antibody. The effector ligand with which the affinity is altered can be, for example, a Fe receptor, or the C1 component of the complement. This approach is described in greater detail in the Patents of the United States of North America Numbers, 624,821 and 5,648,260, both by Winter et al. In another embodiment, one or more amino acids selected at from the amino acid residues can be replaced with a different amino acid residue, such that the antibody has the altered C1 q bond, and / or the reduced or abolished complement-dependent cytotoxicity (CDC). This approach is described in greater detail in U.S. Patent No. 6,141,551 by Idusogie et al. In another embodiment, one or more amino acid residues are altered to thereby alter the ability of the antibody to bind the complement. This approach is further described in PCT Publication Number WO 94/29351 by Bodmer et al. In yet another embodiment, the Fe region is modified to increase the ability of the antibody to measure antibody dependent cellular cytotoxicity (ADCC), and / or to increase the affinity of the antibody for an Fcy receptor., by modifying one or more amino acids. This approach is further described in the Publication of TCP Number WO 00/42072 by Presta. Moreover, the binding sites on human IgG 1 for FcγRI, FcγRI, FcγRIII, and FcRn have been mapped, and variants with a better binding have been described (see Shields, R. L. et al. 2001, J. Biol. Chem. 276: 6591-6604). In still another embodiment, the glycosylation of an antibody is modified. For example, an aglycosylated antibody can be made (ie, the antibody lacks glycosylation). Glycosylation can be altered, for example, to increase the affinity of the antibody for an "antigen." These carbohydrate modifications can be carried out, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the removal of one or more glycosylation sites of variable region structure, to thereby eliminate glycosylation at that site. This aglycosylation can increase the affinity of the antibody for the antigen. This approach is described in greater detail in the Patents of the United States of North America Nos. 5,714,350 and 6,350,861 by Co et al. In an additional or alternative way, an antibody having an altered type of glycosylation, such as a hypophosphorylated antibody having reduced amounts of fucosyl, or an antibody having more GIcNac bisecting structures can be made. It has been shown that these altered glycosylation patterns increase the ADCC capacity of the antibodies. These carbohydrate modifications can be carried out, for example, by expression of the antibody in a host cell with an altered glycosylation machinery. Cells with the altered glycosylation machinery have been described in the art, and can be used as host cells in which the recombinant antibodies of the invention can be expressed, in order to thereby produce an antibody with altered glycosylation. For example, European Patent Number EP 1, 176, 195 by Hang and collaborators, describes a cell line with a functionally altered FUT8 gene, which encodes a fucosyltransferase, such that antibodies expressed in this cell line exhibit hypophosphorylation. PCT Publication Number WO 03/035835 by Presta, describes a variant CHO cell line, Lecl3 cells, with a reduced ability to bind fucose to Asn linked carbohydrates (297), which also result in hypophosphorylation of the antibodies expressed in that host cell (see also Shields RL et al., 2002 J. Biol. Chem. 277: 26733-26740). TCP Publication Number WO 99/54342 by Umana et al. Describes cell lines designed to express glycoprotein-modifying glycosyl transferases (eg, beta- (1,4-N-acetyl-glucosaminyl-transferase III (Gn TI N )), such that the antibodies expressed in the designed cell lines exhibit more bisection GIcNac structures, which results in increased ADCC activity of the antibodies (see also Umana et al., 1999 Nat. Biotech. : 176-1 80) Another modification of the antibodies of the present which is contemplated by the invention is pegylation: An antibody can be pegylated, for example, to increase the biological (eg, serum) half-life of the antibody. To pegylate an antibody, the antibody, or fragment thereof, is typically reacted with polyethylene glycol (PEG), such as a reactive ester or a PEG aldehyde derivative, under conditions wherein one or more PEG groups come to bind with the antibody or with the antibody fragment. PEGylation can be carried out by an acylation reaction or by an alkylation reaction with a reactive PEG molecule (or an analogous reactive water soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any of the PEG forms that have been used to derive other proteins, such as mono (C1-C1 O) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for protein pegylation are known in the art, and can be applied to the antibodies of the invention. See, for example, European Patent Number EP 0, 1 54.31 6 by Nishimura et al., and European Patent Number EP 0,401, 384 by Ishikawa et al. Non-immunoglobulin scaffolds A wide variety of antibody or immunoglobulin structures or scaffolds may be employed, provided that the resulting polypeptide includes at least one binding region that is specific for the target protein. These structures or scaffolds include the five major idiotypes of human immunoglobulins, or fragments thereof (such as are disclosed elsewhere herein), and include the immunoglobulins of other animal species, preferably having humanized aspects. Antibodies from a single heavy chain, such as those identified in camelids and / or in the shark, are of particular interest in this regard. Experts in this field continue to discover and develop novel structures, scaffolds, and fragments. In one aspect, the invention pertains to the generation of non-immunoglobulin-based antibodies, using non-immunoglobulin scaffolds, on which the complementarity determining regions of the invention can be grafted. Non-known or future immunoglobulin structures or scaffolds may be employed, provided they comprise a specific binding region for the target protein of SEQ ID NO: 1 or SEQ ID NO: 122. These compounds are known herein as "polypeptides comprising a target-specific binding region". Non-immunoglobulin structures or scaffolds known include, but are not limited to, adnectins (fibronectin) (Compound Therapeutics, Inc., Waltham, MA), ankyrin (Molecular Partners AG, Zurich, Switzerland), domain antibodies (Domantis , Ltd., Cambridge, MA) and Ablynx nv (Zwijnaarde, Belgium), lipocalin (Anticalin) (Pieris Proteolab AG, Freising, Germany), small modular immuno-pharmaceutical products (Trubion Pharmaceuticals Inc., Seattle, WA), mammalian ( Avidia, Inc. (Mountain View, CA)), Protein A (Affibody AG, Sweden), and afilin (gamma-crystalline or ubiquitin) (Scil Proteins GmbH, Halle, Germany). (i) Adnectins - Compound Therapeutics Adnectin scaffolds are based on the domain of fibronectin type III (for example, the tenth module of fibronectin type III (domain 10 Fn3). The domain of fibronectin type III has 7 or 8 beta chains that are distributed between two beta sheets, which are packaged against each other to form the nucleus of the protein, and that also contain cycles (analogous to the complementarity determining regions), that connect the beta chains with each other, and are exposed to the solvent.There are at least three of these cycles in each edge of the sandwich of leaves beta, where the border is the limit of the protein perpendicular to the direction of the beta chains (U.S. Patent Number US 6,818,418) .These scaffolds based on fibronectin are not an immunoglobulin, although the overall fold is tightly related to that of the smallest functional antibody fragment, the variable region of the heavy chain, comprising the entire antigen recognition unit in the to camel and llama IgG. Because of this structure, the non-immunoglobulin antibody mimics the antigen binding properties that are similar in nature and affinity to those of the antibodies. These scaffolds can be used in a cyclic strategy of random selection and in vitro mixing that is similar to the affinity maturation process of antibodies in vivo. These molecules based on fibronectin can be used as scaffolds, where the cycle regions of the molecule can be replaced with the complementarity determining regions of the molecule. invention, using conventional cloning techniques. (ii) Ankyrin - Molecular Partners The technology is based on the use of proteins with repetitive modules derived from ankyrin as scaffolding to support the variable regions that can be used to link with different targets. The ankyrin repeat module is a 33 amino acid polypeptide consisting of two antiparallel-a and one-b-turn helices. The linkage of the variable regions is optimized for the most part using the ribosome display. (iii) Virus / Avimer - Avidia Avimer is derived from the protein containing the natural A-domain, such as LRP-1. These domains are used by nature for protein-protein interactions, and in humans, more than 250 proteins are structurally based on A-domains. Avimeres consist of a number of different "A-domain" monomers (2-10) linked via amino acid linkers. Avimeres that can bind to the target antigen can be created, using the methodology described, for example, in Publications Numbers 20040175756; 20050053973; 20050048512; and 20060008844. (iv) Protein A - Affibodv Affibody® affinity ligands are small single proteins composed of a three-helix bundle based on the scaffolding of one of the binding domains of Protein A. Protein A is a surface protein from the bacteria Staphylococcus aureus. This scaffolding domain consists of 58 amino acids, thirteen of which are randomly selected to generate Affibody libraries with a large number of ligand variants (see, for example, U.S. Patent Number US 5,831,012). The Affibody® molecules mimic the antibodies, they have a molecular weight of 6 kDa, compared to the molecular weight of the antibodies, which is 150 kDa. Despite its small size, the binding site of Affibody® molecules is similar to that of an antibody. (v) Anticalinas - Pieris Las Anticalinas® are products developed by the company Pieris ProteoLab AG. They are derived from lipocalins, a widely extended group of small, robust proteins that are usually involved in the physiological transport or storage of chemically sensitive or insoluble compounds. Several natural lipocalins occur in human tissues or in body fluids. The architecture of the protein resembles that of immunoglobulins, with hypervariable cycles on the upper part of a rigid structure. However, in contrast to antibodies or their recombinant fragments, lipocalins are composed of a single polypeptide chain with 160 to 180 amino acid residues, which is just marginally larger than a single immunoglobulin domain. The set of four cycles, which forms the link pocket, shows a pronounced structural plasticity, and tolerates a variety of side chains. Therefore, the binding site can be reconfigured in a registered process, in order to recognize the target molecules prescribed in different ways with high affinity and specificity. A protein of the lipocalin family, the biline binding protein (BBP) of Pieris Brassicae, has been used to develop the anticalines, mutating the set of the four cycles. An example of a patent application describing the "anticalines" is the PCT Publication PCT Number WO 199916873. (vi) Afiline - Scil Proteins The molecules of Afilina ™ are small non-immunoglobulin proteins, which are designed to have specific affinities towards proteins and small molecules. The new inaMR Afil molecules can be selected very rapidly from two libraries, each of which is based on different scaffold proteins derived from humans. The Afilin ™ molecules do not show any structural homology with the immunoglobulin proteins. Scil Proteins employs two scaffolds of Afilina ™, one of which is gamma-crystalline, a lens protein of the human structural eye, and the other is of the proteins of the "ubiquitin" superfamily. Both human scaffolds are very small, show a high temperature stability, and are almost resistant to changes in pH and denaturing agents. This high stability is mainly due to the expanded beta sheet structure of the proteins. Examples of gamma-crystalline derived proteins are described in International Publication Number WO2001 041 44, and examples of "ubiquitin-like" proteins are described in International Publication Number WO2004106368. Methods for designing antibodies As described above, anti-DKK1 antibodies having the VH and VL sequences or the full length heavy and light chain sequences shown herein, can be used to create new anti-DKK1 / 4 antibodies, by modifying the sequences of the heavy chain and / or the full length light chain, the VH and / or VL sequences, or the constant regions attached thereto. Accordingly, in another aspect of the invention, the structural features of an anti-DKK1 antibody of the invention are used to create structurally related anti-DKK1 / 4 antibodies that retain at least a functional property of the antibodies of the invention, such as binding to human DKK1 or DKK4, or both, and also inhibiting one or more functional properties of DKK1 or DKK4, or both. For example, one or more complementary determining regions of the antibodies of the invention, or mutations thereof, can be combined in a recombinant manner with the known structure regions and / or other regions. complementarity determinants to create additional recombinantly designed anti-DKK1 antibodies of the invention, as described above. Other types of modifications include those described in the previous section. The starting material for the design method is one or more of the VH and / or V sequences provided herein, or one or more complementarity determining regions thereof. In order to create the engineered antibody, it is not necessary to actually prepare (ie, express as a protein) an antibody having one or more of the VH and / or V sequences provided herein, or one or more of the regions that determine their complementarity. Rather, the information contained in the sequences is used as the starting material to create "second generation" sequences derived from the original sequences, and then the "second generation" sequences are prepared and expressed as a protein. In accordance with the foregoing, in another embodiment, the invention provides a method for the preparation of an anti-DKK1 antibody, which consists of: an antibody sequence of the VH region having a CDR 1 sequence selected from the group which consists of SEQ ID NOs: 2-5, 8-1 1, 20, 49-56; a sequence of CDR2 selected from the group consisting of SEQ ID NOs: 2-20 and 57-64; and / or a CDR3 sequence selected from the group consisting of SEQ ID NOs: 2-20 and 65-72; and an antibody sequence of the V region having a sequence of CDR 1 selected from the group consisting of SEQ ID NOs: 21 -39 and 73-80; a sequence of CDR2 selected from the group consisting of SEQ I D NOs: 21 -39 and 81 -88; and / or a sequence of CDR3 selected from the group consisting of SEQ ID NOs: 21 -39 and 89-96; altering at least one amino acid residue within the VH region antibody sequence and / or within the V region antibody sequence to create at least one altered antibody sequence; and expressing the altered antibody sequence as a protein. In accordance with the foregoing, in another embodiment, the invention provides a method for the preparation of an anti-DKK1 antibody, which consists of: a full length heavy chain antibody sequence having a sequence selected from the group of SEQ ID NOs: 1 1 5-1 17; and a full-length light chain antibody sequence having a sequence selected from the group of SEQ ID NOs: 1 1 1 -1 14; altering at least one amino acid residue within the full length heavy chain antibody sequence and / or within the full length light chain antibody sequence, to create at least one altered antibody sequence; and expressing the altered antibody sequence as a protein. In another embodiment, the invention provides a method for the preparation of a neutralizing anti-DKK1 / 4 composition optimized for expression in mammals, consisting of: antibody sequence of the VL region having a sequence selected from the group of SEQ ID NO: 119; and an antibody sequence of the V region having a sequence selected from the group of SEQ ID NO: 118; altering at least one amino acid residue within the VH region antibody sequence and / or within the V region antibody sequence, to create at least one altered antibody sequence; and expressing the altered antibody sequence as a protein. Conventional molecular biology techniques can be employed to prepare and express the altered antibody sequence. The antibody encoded by the altered antibody sequences, is one that retains one, some, or all of the functional properties of the neutralizing anti-DKK1 / 4 compositions described herein, whose functional properties include, but are not limited to, specifically binding with the human DKK1; and the antibody exhibits at least one of the following functional properties: the antibody inhibits the binding of the DKK1 protein to the DKK1 receptor, or the antibody inhibits the binding of the DKK1 receptor by preventing or improving osteolysis, or the antibody inhibits the binding of the DKK1 receptor thereby preventing or enhancing the osteolytic lesions, or the antibody inhibits the binding of the DKK1 receptor by preventing or ameliorating the cancer. The altered antibody may exhibit one or more, two or more, or three or more of the functional properties discussed above.

The functional properties of the altered antibodies can be evaluated using conventional assays available in the art and / or described herein, such as those stipulated in the Examples (eg, ELISAs). In certain embodiments of the methods for designing the antibodies of the invention, mutations may be introduced in a random or selective manner throughout all or part of an anti-DKK1 antibody coding sequence, and the resulting modified anti-DKK1 antibodies. they can be traced to determine their binding activity and / or other functional properties, as described herein. Methods of mutation have been described in the art. For example, PCT Publication Number WO 02/092780 by Short discloses methods for creating and tracking antibody mutations using saturation mutagenesis, synthetic linkage assembly, or a combination thereof. Alternatively, the PCT Publication Number WO 03/074679 by Lazar et al. Describes methods for using computer tracking methods to optimize the physicochemical properties of the antibodies. The Fe constant region of an antibody is critical for determining serum half-life and effector functions, i.e., antibody-dependent cellular cytotoxicity (ADCC), or complement-dependent cytotoxicity (CDC) activities. Specific mutants of the Fe fragment can be designed for altering effector function and / or serum half-life (see, for example, Xencor Technology, see also, for example, International Publication Number WO2004029207). A method for altering the effector function and the serum half-life of an antibody is to graft the variable region of an antibody fragment with an Fe fragment having the appropriate effector function. Isotypes of lgG 1 or lgG4 can be selected for the activity of cell annihilation, while the lgG2 isotype can be used for silent or neutralizing antibodies (without cell killing activity). Silent antibodies with a long serum half-life can be obtained by making the chimeric fusion of the variable regions of an antibody with a whey protein, such as the HSA binding protein. Effector functions can also be altered by modulating the glycosylation pattern of the antibody. Glycart (e.g., U.S. Patent Number US6,602,684), Biowa (e.g., U.S. Patent Number US6,946,292) and Genentech (e.g., International Publication Number WO03 / 035835), have designed mammalian cell lines to produce antibodies with increased or decreased effector function. In particular, non-fucosylated antibodies will have better ADCC activities. Glycofi has also developed yeast cell lines capable of producing specific glycoforms of antibodies.

Nucleic acid molecules encoding the antibodies of the invention Another aspect of the invention pertains to nucleic acid molecules encoding the antibodies of the invention. Examples of the full-length light chain progenitor nucleotide sequences are shown in SEQ I D NOs: 97-100. Examples of the full length heavy chain progenitor nucleotide sequences are shown in SEQ ID NOs: 1 01 -1 03. Examples of full length light chain nucleotide sequences optimized for expression in a cell are shown in SEQ ID NOs: 104-1 07. Examples of the full-length heavy chain nucleotide sequences optimized for expression in a cell are shown in SEQ ID NOs: 108-1 10. Nucleic acids may be present in whole cells, in a cell lysate, or they may be nucleic acids in a partially purified or substantially pure form. A nucleic acid is "isolated" or "made substantially pure", when it is purified from other cellular components or from other contaminants, for example, other nucleic acids or cellular proteins, by conventional techniques, including alkaline / SDS treatment, CsCl band, column chromatography, agarose gel electrophoresis, and other well known in this field. See F. Ausubel et al., Editors, 1 987 Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. A The nucleic acid of the invention, for example, may be A DN or RNA, and may or may not contain intronic sequences. In one modality, the nucleic acid is a cDNA molecule. The nucleic acid may be present in a vector, such as in a phage display vector, or in a recombinant plasmid vector. The nucleic acids of the invention can be obtained using conventional molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice that will carry out immunoglobulin genes, as will be described further below), the cDNAs encoding the light and heavy chains of the antibody made by hybridoma can be obtained by amplification with conventional polymerase chain reaction, or by cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (for example, using phage display techniques), the n-nucleic acid encoding the antibody can be recovered from different clones of phages that are members of the library. Once the DNA fragments encoding the VH and VL segments are obtained, these DNA fragments can be further manipulated by conventional recombinant DNA techniques, for example, to convert the variable region genes to the antibody chain genes. full length, up to Fab genes, or up to a scFv gene. In these manipulations, a fragment of DNA that encode V or VH is operably linked to another DNA molecule, or to a fragment encoding another protein, such as an antibody constant region, or a flexible linker. The term "operably linked", as used in this context, is intended to mean that the two DNA fragments are linked in a functional manner, for example, such that the amino acid sequences encoded by the two DNA fragments remain within the frame, or in such a way that the protein is expressed under the control of a desired promoter. The isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operably linking the VH-encoding DNA with another DNA molecule that encodes heavy chain constant regions (CH1, CH2, and CH3) . The sequences of the human heavy chain constant region genes are known in this field (see, for example, Kabat, EA et al., 1 991 Sequences of Proteins of Immunological Interest, Fifth Edition, Department of Health and Human Services of the United States. United, NIH Publication Number 91 -3242), and DNA fragments spanning these regions can be obtained by conventional polymerase chain amplification. The heavy chain constant region can be a constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD. For a heavy chain gene of a Fab fragment, the DNA encoding VH can be operably linked to another DNA molecule that encodes only the CH 1 constant region heavy chain. The isolated DNA encoding the V region can be converted to the full-length light chain gene (as well as to a Fab light chain gene), by operatively linking the VL-encoding DNA to another DNA molecule encoding the constant region of light chain, CL. The sequences of the human light chain constant region genes are known in the art (see, for example, Kabat, EA et al., 1991 Sequences of Proteins of Immunological Interest, Fifth Edition, United States Department of Health and Human Services. , NIH Publication Number 91-3242), and DNA fragments spanning these regions can be obtained by conventional polymerase chain reaction amplification. The light chain constant region may be a kappa or lambda constant region. In order to create a scFv gene, DNA fragments encoding VH and V are operably linked to another fragment encoding a flexible linker, for example, encoding the amino acid sequence (Gly4 -Ser) 3, such that the VH and V sequences can be expressed. as a contiguous single chain protein, with the V and VH regions linked by the flexible linker (see, eg, Bird et al., 1988 Science 242: 423-426; Huston et al., 1988 Proc. Natl. Acad. Sci. USA 85: 5879-5883; McCafferty et al., 1990 Nature 348: 552-554).

Production of monoclonal antibodies of the invention Monoclonal antibodies (mAbs) can be produced by a variety of techniques, including conventional monoclonal antibody methodology, for example, the conventional somatic cell hybridization technique of Kohler and Milstein, 1975 Nature , 256: 495. Many techniques can be used to produce monoclonal antibodies, for example the viral or oncogenic transformation of B lymphocytes. An animal system for preparing hybridomas is the murine system. The production of hybridomas in the mouse is a well-established procedure. Immunization protocols and techniques for the isolation of splenocytes immunized for fusion are known in the art. Fusion components (eg, murine myeloma cells) and fusion procedures are also known. The chimeric or humanized antibodies of the present invention can be prepared based on the sequence of a murine monoclonal antibody prepared as described above. The DNA encoding the heavy and light chain immunoglobulins can be obtained from the hybridoma of interest, and can be designed to contain immunoglobulin sequences other than murine (e.g., human), employing conventional molecular biology techniques. For example, in order to create a chimeric antibody, murine variable regions can be linked to human constant regions, using methods known in the art (see, for example, U.S. Patent No. 4,816,567 to Cabilly et al.). In order to create a humanized antibody, the murine CDR regions can be inserted into a human structure, using methods known in the art. See, for example, U.S. Patent No. 5,225,539 to Winter, and U.S. Patent Nos. 5,530, 101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al. In a certain embodiment, the antibodies of the invention are human monoclonal antibodies. These human monoclonal antibodies directed against DKK1 can be generated using transgenic or transchromosomal mice carrying parts of the human immune system, instead of the mouse system. These transgenic and transchromosomal mice include the mice referred to herein as HuMAb mice and KM mice, respectively, and are collectively referred to herein as "human Ig mouse". The HuMAb® mouse (Medarex, Inc.) contains mini- / oc of the human immunoglobulin gene encoding non-reconfigured heavy chain immunoglobulin (μ and y) and human K sequences, together with targeted mutations that inactivate the μ and α-chain loci Endogenous K (see, for example, Lonberg et al., 1994 Nature 368 (6474): 856-859). In accordance with the above, the mice exhibit a reduced expression of IgM or K of mouse, and in response to immunization, introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high affinity monoclonal human IgG K (Lonberg, N. et al., 1994 supra; reviewed in Lonberg, N., 1994 Handbook of Experimental Pharmacology 113: 49-101; Lonberg, N. and Huszar, D., 1995 Intern. Rev. Immunol. 13: 65-93, and Harding, F. and Lonberg, N ., 1995 Ann. N. Y. Acad. Sci. 764: 536-546). The preparation and use of HuMAb mice, and the genomic modifications carried by these mice, are further described in Taylor, L. et al., 1992 Nucleic Acids Research 20: 6287-6295; Chen, J. et al., 1993 International Immunology 5: 647-656; Tuaillon et al., 1993 Proc. Nati Acad. Sci. USA 94: 3720-3724; Choi et al., 1993 Nature Genetics 4: 117-123; Chen, J. et al., 1993 EMBO J. 12: 821-830; Tuaillon et al., 1994 J. Immunol. 152: 2912-2920; Taylor, L. et al., 1994 International Immunology 579-591; and Fishwild, D. and collaborators, 1996 Nature Biotechnology 14: 845-851, the content of all of which is specifically incorporated herein by reference in its entirety. See also, Patents of the United States of North America Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429; all to Lonberg and Kay; U.S. Patent Number 5,545,807 to Surani et al .; The Publications of the TCP Numbers WO 92103918, WO 93/12227, WO 94/25585, WO 971 1 3852, WO 98/24884 and WO 99/45962, all to Lonberg and Kay; and Publication of TCP Number WO 01/1 4424 to Korman et al. In another embodiment, the human antibodies of the invention can be reproduced using a mouse carrying human immunoglobulin sequences on transgenes and transchromosomes, such as a mouse carrying a human heavy chain transgene and a human light chain transchromosome. These mice, referred to herein as "KM mice," are described in detail in PCT Publication Number WO 02/43478 to Ishida et al. Still further, there are alternative transgenic animal systems that express the human immunoglobulin genes available in the art, and can be used to reproduce the anti-DKK1 antibodies of the invention. For example, an alternative transgenic system referred to as the Xenomouse® (Abgenix, Inc.) may be used. These mice are described, for example, in U.S. Patent Nos. 5,939,598; 6,075, 181; 6,1, 14,598; 6, 150,584, and 6, 162,963 to Kucherlapati et al. Furthermore, there are alternative transchromosomal animal systems that express human immunoglobulin genes available in the art, and can be used to reproduce the anti-DKK1 antibodies of the invention. For example, mice carrying both a transchromosomal chain can be used heavy human as a human light chain transchromosome, referred to as "TC mice"; these mice are described in Tomizuka et al., 2000 Proc. Nati Acad. Sci. USA 97: 722-727. Additionally, cows carrying human heavy and light chain transchromosomes have been described in the art (Kuroiwa et al., 2002 Nature Biotechnology 20: 889-894), and can be used to reproduce the anti-DKK1 antibodies of the invention. The human monoclonal antibodies of the invention can also be prepared using phage display methods to screen libraries of human immunoglobulin genes. These methods of phage display to isolate human antibodies are established in the art. See for example: Patents of the United States of North America Nos. 5,223,409; 5,403,484; and 5,571, 698 to Ladner et al; Patents of the United States of North America Numbers 5,427,908 and 5,580,717 to Dower et al .; Patents of the United States of North America Nos. 5,969, 1 08 and 6, 1 72, 1 97 to McCafferty et al .; and Patents of the United States of North America Nos. 5,885,793; 6,521, 404; 6,544,731; 6,555.31 3; 6,582.91 5 and 6,593,081 to Griffiths et al. The human monoclonal antibodies of the invention can also be prepared using SCI D mice, in which human immune cells have been reconstituted, such that a response to human antibody can be generated after immunization. These mice are described, for example, in Patents of the United States of North America Numbers. 5,476,996 and 5,698,767 to Wilson et al. Traces of human antibody libraries can be used to identify an antibody of the invention. The choice of tracking technologies include, but are not limited to, phage display (Morphosys), type of libraries (eg, Morphosys HuCal library), affinity maturation technology, and other optimization sequences. codons. Generation of human monoclonal antibodies against DKK1 Purified recombinant human DKK1 derived from E. coli, baculovirus, or HEK-EBNA cells, or purified recombinant human DKK1 conjugated with orifice limpet hemocyanin (KLH), is used as the aen. Fully human monoclonal aodies to DKK1 are prepared using the HCo7, HCo12, and HCo17 strains of transgenic HuMAb mice, and the KM strain of transgenic transchromosomal mice, each of which expresses the human aody genes. In each of these mouse races, the endogenous mouse kappa light chain gene can be homozygously altered, as described in Chen et al., 1 993 EMBO J. 12:81 1 -820, and the endogenous mouse heavy chain gene can be altered homozygously as described in Example 1 of the Publication of TCP Number WO01 1 091 87. Each of these mouse races carries a chain transgene. light human kappa, KCo5, as described in Fishwild et al, 13 1996 Nature Biotechnology 14: 845-851. The HCo7 race carries the HCo7 human heavy chain transgene, as described in U.S. Patent Nos. 5,545,806; 5,625,825; and 5,545,807. The HCo12 race carries the human heavy chain transgene HCo12, as described in Example 2 of the PCT Publication Number WO 01/09187. Race HC017 carries the human heavy chain transgene HCo17. The KNM race contains the transchromosome SC20, as described in the Publication of the TCP Number WO 02/43478. To generate the fully human monoclonal aodies to DKK1, HuMAb mice and KM mice are immunized with the purified recombinant DKK1 derived from E. coli, or the conjugate of DKK1-KLH as the aen. The general immunization schemes for HuMAb mice are described in Lonberg, N. et al., 1994, Nature 368 (6474): 856-859; Fishwild, D. et al., 1996 Nature Biotechnology 14: 845-851, and in the Publication of the TCP Number WO 98/24884. The mice are 6 to 16 weeks of age after the first aen infusion. A purified recombinant preparation (5 to 50 micrograms) of the DKK1 aen (eg, purified from transfected E. coli cells expressing DKK1) is used to immunize HuMAb mice and KM mice intraperitoneally, subcutaneously ( Se), or by injection into the sole of the leg. The transgenic mice are immunized twice with the aen in complete Freund's adjuvant or in Ribi's adjuvant, either intraperitoneally (IP), subcutaneously (Se), or by injection in the paw (FP), followed by 3 to 21 days of intraperitoneal, subcutaneous immunization, or by injection into the paw (up to a total of 11 immunizations) with the aen in incomplete Freund or Ribi adjuvant. The immune response is monitored by retro-orbital bleeds. Plasma is screened by ELISA, and mice are used with sufficient titers of human aDKK1 immunoglobulin. The mice are boosted intravenously with aen 3 and 2 days before sacrifice and removal of the spleen. Typically, 1 to 35 fusions are carried out for each aen. Several dozen mice are immunized for each aen. A total of 82 mice from the races of HCo7, HCo1 2, HCo1 7, and KM mice are immunized with DKK1. In order to select HuMAb or KM mice that produce aodies that bind to DKK1, sera from mice immunized by ELISA can be tested, as described by Fishwild, D. et al., 1996. Briefly stated. , the microtitre plates are coated with purified recombinant E. coli DKK1 at 1-2 micrograms / milliliter in phosphate-buffered serum, at 50 micrograms / well, incubated at 4 ° C overnight, and then blocked with 200 microliters / 5 percent chicken serum well in phosphate / Tween regulated serum (0.05 percent). Plasma dilutions are added from mice immunized with DKK1 to each well, and incubated for 1 hour. fifteen at 2 hours at room temperature. The plates are washed with phosphate-regulated serum / Tween, and then incubated with polyclonal antibody goat anti-human IgG Fe-conjugated with red radicle peroxidase (HRP) for 1 hour at room temperature. After washing, the plates are developed with ABTS substrate (Sigma, A-1 888, 0.22 milligrams / milliliter), and analyzed by the spectrophotometer at an OD of 41 5-495. The mice that developed the highest titers of the anti-DKK1 antibodies are those that are used for the fusions. The fusions are carried out, and the hybridoma supernatants are tested for anti-DKK1 activity by ELISA. Mouse splenocytes, isolated from HuMAb mice and KM mice, are fused with PEG to a mouse myeloma cell line, based on conventional protocols. The resulting hybridomas are then screened to determine the production of antigen-specific antibodies. The individual cell suspensions of the splenic lymphocytes of the immunized mice are fused to a quarter of the number of SP2 / 0 non-secretory mouse myeloma cells (ATCC, CRL 1 581) with 50 percent PEG (Sigma). Cells are applied at approximately 1 x 10 5 / well in flat bottom microtiter plates, followed by approximately two weeks of incubation in a selective medium containing 1 0 percent fetal bovine serum, P388D 1 to 1 0 percent conditioned medium (ATCC , CRL TIB-63), Origin® at 3-5 percent (IGEN) in DMEM (Mediatech, CRL 1 001 3, 16 with high glucose, L-glutamine, and sodium pyruvate), plus 5 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 milligrams / milliliter of gentamicin, and 1 x HAT (Sigma, CRL P-71 85). After one to two weeks, the cells are cultured in the medium where the HAT is replaced with HT. Individual wells are then screened by ELISA to determine human anti-DKK1 monoclonal IgG antibodies. Once extensive hybridoma growth occurs, the medium is monitored usually after 10 to 14 days. The antibody-secreting hybridomas are reapplied on the plate, screened again, and if they are still positive for human IgG, anti-DKK1 monoclonal antibodies are subcloned at least twice by limiting dilution. The stable subclones are then cultured in vitro to generate small amounts of antibody in a tissue culture medium for further characterization. Immunization of mice with human Ig When mice with human Ig are used to reproduce the human antibodies of the invention, these mice can be immunized with a purified or enriched preparation of the DKK1 antigen and / or the recombinant DKK1, or a fusion protein. of DKK1, as described by Lonberg, N. et al., 1 994 Nature 368 (6474): 856-859; Fishwald, D. et al., 1 996 Nature Biotechnology 14: 845-851; and in the Publications of the TCP Numomers WO 98124884 and WO 01/1 4424. The mice can be 6 to 16 weeks of age after the first infusion. For example, a purified or recombinant preparation (5 to 50 micrograms) of DKK1 antigen can be used to immunize human Ig mice intraperitoneally. The detailed procedures for generating fully human monoclonal antibodies to DKK1 are described above. Cumulative experience with different antigens has shown that transgenic mice respond when initially immunized intraperitoneally (IP) with the antigen in complete Freund's adjuvant, followed by intraperitoneal immunizations every third week (up to a total of 6) with the antigen in incomplete adjuvant of Freund. However, it was also found that other adjuvants other than Freund's are effective. In addition, it is found that whole cells in the absence of adjuvant are highly immunogenic. The immune response can be monitored during the course of the immunization protocol with plasma samples obtained by retro-orbital bleeds. Plasma can be screened by ELISA, and mice with sufficient titers of human anti-DKK1 immunoglobulin for infusions can be used. Mice can be boosted intravenously with antigen three days before slaughter, and the spleen is removed. It is expected that two to three mergers may be required for each immunization. Typically, between 6 and 24 mice are immunized for each antigen. Usually, both strains HCo-7 and HCo-12 are used. In addition, both transgenes of HCo-7 and HCo-12 can be reproduced together in a single mouse that has two different human heavy chain transgenes (HCo7 / HCo12). Generation of Human Monoclonal Antibodies Producing Hybridomas With the object of hybridomas that produce human monoclonal antibodies of the invention, splenocytes and / or lymph node cells of immunized mice can be isolated and can be fused with an appropriate immortalized cell line , such as a mouse myeloma cell line. The resulting hybridomas can be screened for the production of antigen-specific antibodies. For example, single cell suspensions of splenic lymphocytes from immunized mice can be fused with one sixth of the number of non-secretory mouse myeloma cells P3X63-Ag8.653 (ATCC, CRL 1580) with 50 percent PEG. Cells are applied at approximately 2 x 145 in flat bottom microtiter plates, followed by a two week incubation in a selective medium containing 20 percent fetal clone serum, conditioned medium "653" at 1 8 percent, ® at 5 percent (IGEN), 4 mM L-glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 0.055 m 2-mercaptoethanol, 50 units / milliliter of penicillin, 50 milligrams / milliliter streptomycin, 50 milligrams / milliliter of gentamicin, and 1 X HAT (Sigma, the HAT is added 24 hours after the fusion). After approximately two weeks, the cells can be cultured in the medium where the HAT is 19 replaced with HT. Then individual wells can be screened by ELISA to determine human monoclonal IgM and IgG antibodies. Once extensive hybridoma growth occurs, the medium can usually be observed after 10 to 14 days. Antibody-secreting hybridomas can be reapplied on plates, screened again, and if they are still positive for human IgG, monoclonal antibodies can be subcloned at least twice by limiting dilution. Then the stable subclones can be cultured in vitro to generate small amounts of antibody in a tissue culture medium for characterization. In order to purify the human monoclonal antibodies, the selected hybridomas can be cultured in two liter centrifuge flasks for the purification of the monoclonal antibody. The supernatants can be filtered and concentrated before affinity chromatography with protein A-Sepharose (Pharmacia, Piscataway, N. J.). The eluted IgG can be verified by gel electrophoresis and by chromatography of high-performance liquids, to ensure purity. The regulatory solution can be exchanged in phosphate-regulated serum, and the concentration can be determined by OD2β. using an extinction coefficient of 1.43. The monoclonal antibodies can be aliquoted and stored at -80 ° C.

Generation of Transfectoma Producing Monoclonal Antibodies The antibodies of the invention can also be produced in a host cell transfectoma using, for example, a combination of recombinant DNA techniques and genetic transfection methods, as is well known in the art. example, Morrison, S. (1 985) Science 229: 1202). For example, to express the antibodies, or antibody fragments thereof, DNAs encoding partial or full-length heavy and light chains can be obtained by conventional molecular biology techniques (e.g., amplification with chain reaction of the polymerase or cloning of cDNA using a hybridoma expressing the antibody of interest), and the DNAs can be inserted into expression vectors, such that the genes are operably linked to the transcriptional and translational control sequences. In this context, the term "operably linked" is intended to mean that an antibody gene is ligated into a vector, such that the transcriptional and translational control sequences within the vector serve their intended function of regulating transcription and transcription. translation of the antibody gene. The expression vector and the expression control sequences are selected to be compatible with the expression host cell used. The light chain gene of the antibody and the heavy chain gene of the antibody can be inserted into a vector separately, or more typically, both genes are inserted into the same expression vector. The antibody genes are inserted into the expression vector by conventional methods (eg, ligation of the complementary restriction sites on the antibody gene fragment and the vector, or blunt-ended ligation if no restriction sites are present) . The light and VH regions of the antibodies described herein can be used to create full-length antibody genes of any antibody isotype, by inserting them into expression vectors that already code the heavy chain constant and light chain constant regions. of the desired isotype, such that the VH segment is operatively linked to the CH segments within the vector, and the V segment is operatively linked to the CL segment within the vector. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates the secretion of the antibody chain from a host cell. The gene of the antibody chain can be cloned into the vector, such that the signal peptide is linked within the framework with the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a protein other than immunoglobulin). In addition to the antibody chain genes, the recombinant expression vectors of the invention carry sequences regulators that control the expression of the antibody chain genes in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. These regulatory sequences are described, for example, in Goeddel (Gene Expression Technology, Methods in Enzymology 1 85, Academic Press, San Diego, CA 1 990). Those skilled in the art will appreciate that the design of the expression vector, including the selection of regulatory sequences, may depend on factors such as the choice of the host cell to be transformed, the level of protein expression desired, etc. Regulatory sequences for the expression of mammalian host cells include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV), simian virus 40 (SV40). ), adenovirus (e.g., adenovirus major late promoter (AdMLP)), and polyoma. Alternatively, non-viral regulatory sequences can be used, such as the ubiquitin promoter or the p-globin promoter. Still further, regulatory elements composed of sequences from different sources, such as the S Ra promoter system, which contains sequences from the SV40 early promoter, and the long terminal repeat of the human T-cell leukemia virus type 1 (Takebe, Y. and collaborators, 1 988 Mol. Cell. Biol. 8: 466-472). In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention can carry additional sequences, such as sequences that regulate the replication of the vector in the host cells (e.g., replication origins), and selectable marker genes. The selectable marker gene facilitates the selection of the host cells into which the vector has been introduced (see, for example, U.S. Patent Nos. 4,399,216; 4,634,665; and 5,179.01 7, all of Axel et al. ). For example, typically the selectable marker gene confers resistance to drugs, such as G41 8, hygromycin, or methotrexate, in a host cell into which the vector has been introduced. Selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with selection / amplification of methotrexate), and the neo gene (for the selection of G41 8). For the expression of the light and heavy chains, the expression vectors encoding the heavy and light chains are transfected to a host cell by conventional techniques. The different forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, for example electroporation, calcium phosphate precipitation, transfection of DEAE-dextran, and the like. Theoretically, it is It is possible to express the antibodies of the invention in prokaryotic or eukaryotic host cells. The expression of antibodies in eukaryotic cells, in particular in mammalian host cells, is discussed because these eukaryotic cells, and in particular mammalian cells, are more likely than prokaryotic cells to assemble and secrete an appropriately folded antibody. and antigen binding. The prokaryotic expression of the antibody genes has been reported as ineffective for the production of high yields of active antibodies (Boss, M. A. and Wood, C.R., 1 985 Immunology Today 6: 12-1 3). Mammalian host cells for expressing the recombinant antibodies of the invention include Chinese hamster ovary cells (CHO cells) (including CHO-DHFR cells, described in U rlaub and Chasin, 1 980 Proc. Nati. Acad. Sci. USA 77: 4216-4220, used with a selectable marker of DHFR, for example as described in RJ Kaufman and PA Sharp, 1982 Mol. Biol. 59: 601-621, NSO myeloma cells, COS cells, and SP2 cells In particular, for use with NSO myeloma cells, another expression system is the expression system of the GS gene shown in Patent Numbers WO 87/04462, WO 89/01036, and EP 338,841. Recombinant expression encoding antibody genes in mammalian host cells, the antibodies are produced by culturing the host cells for a sufficient period of time to to allow the expression of the antibody in the host cells, or the secretion of the antibody in the culture medium where the host cells are cultured. The antibodies can be recovered from the culture medium using conventional protein purification methods. Pharmaceutical compositions In another aspect, the present invention provides a composition, for example, a pharmaceutical composition, containing one or a combination of a neutralizing anti-DKK1 / 4 composition, or antigen binding portions thereof, of the present invention , formulated together with a pharmaceutically acceptable vehicle. These compositions may include one or a combination of (eg, two or more different) antibodies, or immunoconjugates, or bispecific molecules of the invention. For example, a pharmaceutical composition of the invention may comprise a combination of antibodies (or immunoconjugates or bispecifics) that bind to different epitopes on the target antigen, or that have complementary activities. The pharmaceutical compositions of the invention can also be administered in a combination therapy, i.e., combined with other agents. For example, the combination therapy may include an anti-DKK1 antibody of the present invention, combined with at least one other anti-inflammatory or anti-osteoporotic agent. The Examples of the therapeutic agents that can be used in the combination therapy are described in greater detail later in the section on uses of the agents of the invention. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption retarding agents, and the like, that are physiologically compatible. The vehicle should be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., the antibody, immunoconjugate, or bispecific molecule, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound. The pharmaceutical compounds of the invention may include one or more pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound, and does not impart any undesired toxicological effect (see, for example, Berge, S. M. et al., 1 977 J. Pharm, Sci. 66: 1-9). Examples of these salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic acid, phosphorous, and the like, as well as from non-toxic organic acids, such as aliphatic mono- and dicarboxylic acids, phenyl substituted alkanoic acids, alkanoic hydroxy acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like . The base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium, and the like, as well as from non-toxic organic amines, such as N, N'-dibenzyl-ethylene diamine. , N-methyl-glucamine, chloro-procaine, choline, diethanolamine, ethylene diamine, procaine, and the like. A pharmaceutical composition of the invention may also include a pharmaceutically acceptable antioxidant. Examples of the pharmaceutically acceptable antioxidants include: water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; oil soluble antioxidants, such as ascorbyl palmitate, butylated hydroxy-anisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and metal chelating agents, such as citric acid, ethylene diamine tetra-acetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. Examples of suitable aqueous and non-aqueous vehicles that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and mixtures thereof. suitable therefrom, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents, and dispersing agents. The prevention of the presence of microorganisms can be ensured both by sterilization procedures, supra, and by the inclusion of different antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be advisable to include isotonic agents, such as sugars, sodium chloride, and the like, in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be caused by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of this medium and agents for pharmaceutically active substances is known in the art. Except where conventional means or agents are incompatible with the active compound, the use thereof is contemplated in the pharmaceutical compositions of the invention. Supplementary active compounds can also be incorporated into the compositions. Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for a high concentration of drug. The carrier may be a solvent or a dispersion medium containing, for example, water, ethanol, polyol (eg, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion., or by the use of surfactants. In many cases, isotonic agents may be included, for example sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be caused by the inclusion in the composition of an agent that retards absorption, for example, salts of monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent, with one or a combination of the ingredients listed above, as required, followed by sterilization by microfiltration. In general terms, the dispersions are prepared by incorporating the active compound in a sterile vehicle containing a basic dispersion medium and the other required ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and freeze drying (lyophilization), which provides a powder of the active ingredient plus any additional desired ingredients from a pre-solution Sterile filtered from it. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the subject being treated, and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form in general will be the amount of the composition that produces a therapeutic effect. In general, one hundred percent, this amount will be in the range of about 0.01 percent to about 99 percent active ingredient, from about 0.1 percent to about 70 percent, or about 1 percent to about 30 percent active ingredient, in combination with a pharmaceutically acceptable carrier. Dosage regimens are adjusted to provide the desired optimal response (e.g., a therapeutic response).

For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be reduced or increased proportionally, as indicated by the exigencies of the therapeutic situation. It is especially convenient to formulate the parenteral compositions in a unit dosage form for ease of administration and uniformity of dosage. The unit dosage form, as used herein, refers to physically separate units suitable as unitary dosages for the subjects to be treated; each unit contains a predetermined amount of the active compound, calculated to produce the desired therapeutic effect, in association with the required pharmaceutical carrier. The specification for dosage unit forms of the invention is dictated by, and directly depends on, the unique characteristics of the active compound, and the particular therapeutic effect to be achieved, and the limitations inherent in the composition technique of This active compound for the treatment of sensitivity in individuals. For administration of the antibody, the dosage is in the range of about 0.0001 to 200 milligrams / kilogram, and more usually 0.01 to 50 milligrams / kilogram of the host's body weight. For example, dosages may be 0.3 milligrams / kilogram of body weight, 1 milligram / kilogram of body weight, 3 milligrams / kilogram of body weight, 5 milligrams / kilogram of body weight, or 10 milligrams / kilogram of body weight, or within the range of 1 to 20 milligrams / kilogram. An example treatment regimen involves administration once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, or once every 3 to 6 weeks. months Dosage regimens for an anti-DKK1 antibody of the invention include 1 milligram / kilogram of body weight or 3 milligrams / kilogram of body weight by intravenous administration, the antibody being given using one of the following dosing schedules: every four weeks for six dosages, then every three months; every three weeks; 3 milligrams / kilogram of body weight once, followed by 1 milligram / kilogram of body weight every three weeks. In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case, the dosage of each antibody administered falls within the indicated ranges. The antibody is usually administered multiple times. The intervals between the individual dosages can be, for example, weekly, monthly, every three months, or annually. The intervals may also be irregular, as indicated by measuring the antibody levels in the patient's blood for the target antigen or a biomarker, such as OCN, OPG, or P1 NP. In some methods, the dosage is adjusted to achieve a plasma antibody concentration of about 1 to 1,000 micrograms / miller, and in some methods of about 25 to 300 micrograms / miller. Alternatively, the antibody can be administered as a sustained release formulation, in which case, less frequent administration is required. Dosage and frequency vary depending on the antibody's half-life in the patient. In general, human antibodies show the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. The dosage and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, relatively high dosing is sometimes required at relatively short intervals, until the progression of the disease is reduced or stopped, or until the patient shows a partial or complete decrease in the symptoms of the disease. Subsequently, a prophylactic regimen can be administered to the patient. The actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a patient, composition, and particular mode of administration, without being toxic to the patient. The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the particular compositions of the present invention employed, or the ester, salt, or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compositions employed, age, sex, weight, condition, general health, and medical history prior to the patient being treated, and similar factors well known in the medical art. A "therapeutically effective dosage" of an anti-DKK1 antibody of the invention may result in a decrease in the severity of disease symptoms, an increase in the frequency and duration of periods without disease symptoms, or a prevention of impairment or disaby due to the affliction of the disease. A composition of the present invention can be administered by one or more routes of administration, using one or more of a variety of methods known in the art. As will be appreciated by the skilled person, the route and / or mode of administration will vary depending on the desired results. The administration routes for the antibodies of the invention include administration intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration", as used herein, means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal injection and infusion. , intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal. Alternatively, an antibody of the invention can be administered by a non-parenteral route, such as a topical, epidermal, or mucosal route of administration, for example intranasally, orally, vaginally, rectally, sublingually, or topically. The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biocompatible and biodegradable polymers can be used, such as ethylene-vinyl acetate, polyanhydrides, poly-glycolic acid, collagen, poly-ortho esters, and poly-lactic acid. Many methods for the preparation of these formulations are patented or are generally known to those skilled in the art. See, for example, Sustained and Controlled Relay Drug Delivery Systems, J. R. Robinson, ed. , Marcel Dekker, Inc., New York, 1978. Therapeutic compositions can be administered with medical devices known in the art. For example, in one embodiment, a therapeutic composition of the invention can be administered with a needleless hypodermic injection device, such as the devices shown in U.S. Patent Nos. 5,399,163; 5,383,851; 5.31, 2.335; 5,064.41 3; 4,941, 880; 4,790,824 or 4,596,556. Examples of the well-known implants and modules useful in the present invention include: U.S. Patent No. 4,487,603, which shows a implantable micro-infusion pump for dosing medication at a controlled rate; U.S. Patent No. 4,486,194, which shows a therapeutic device for administering drugs through the skin; U.S. Patent No. 4,447,233, which shows a pump for drug infusion to deliver medication at a precise infusion rate; U.S. Patent No. 4,447,224, which shows an implantable variable flow infusion apparatus for continuous delivery of the drug; U.S. Patent Number 4,439, 1 96, which shows an osmotic drug delivery system having multi-chamber compartments; and U.S. Patent No. 4,475, 196, which shows an osmotic drug delivery system. These patents are they are incorporated herein by reference. Many other of these implants, delivery systems, and modules are known to those skilled in the art. In certain embodiments, the human monoclonal antibodies of the invention can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophobic compounds. In order to ensure that the therapeutic compounds of the invention cross the blood-brain barrier (if desired), they can be formulated, for example, in liposomes. For liposome manufacturing methods, see, for example, US Patents Nos. 4,522.81 1; 5,374,548; and 5,399,331. The liposomes may comprise one or more fractions, which are selectively transported to specific cells or organs, and thus improve the delivery of the targeted drug (see, for example, VV Ranade, 1989 J. Cline Pharmacol. 29: 685 ). Exemplary address fractions include folate or biotin (see, for example, U.S. Patent No. 5,41 6,01 6 to Low et al.); mannosides (Umezawa et al., 1988 Biochem Biophys., Res. Commun. 1 53: 1038); antibodies (P. G Bloeman et al., 1 995 FEBS Lett 357: 1 40; M. Owais et al., 1 995 Antimicrob Agents Chemother., 39: 1 80); surfactant protein A receptor (Briscoe et al., 1 995 Am. J. Physiol., 1 233: 134); page 120 (Schreier et al., 1 994 J. Biol. Chem. 269: 9090); see also K.

Keinanen; M. L. Laukkanen, 1994 FEBS Lett. 346: 123; J. J. Killion; I. J. Fidler, 1 994 Immunomethods 4: 273. The combinations The invention further relates to a method for preventing or treating proliferative diseases or disorders, such as cancer, in a mammal, in particular in a human, with a combination of pharmaceutical agents comprising: (a) an anti-inflammatory composition; Neutralizing DKK1 / 4; and (b) one or more pharmaceutically active agents. The invention further relates to pharmaceutical compositions comprising: (a) a neutralizing anti-DKK1 / 4 composition; (b) a pharmaceutically active agent; and (c) a pharmaceutically acceptable carrier. The present invention further relates to a commercial package or a product comprising: (a) a pharmaceutical formulation of a neutralizing anti-DKK1 / 4 composition; and (b) a pharmaceutical formulation of a pharmaceutically active agent for simultaneous, concurrent, separate, or sequential use. Pharmaceutically active agents The term "pharmaceutically active agents" is broad and covers many pharmaceutically active agents that have different mechanisms of action. The combinations of some of these with antibodies / neutralizing compositions of DKK1 / 4 can result in improvements in cancer therapy. In general terms, pharmaceutically active agents are classified according to the mechanism of action. Many of the available agents are anti-metabolites of the development pathways of different tumors, or react with the DNA of the tumor cells. There are also agents that inhibit enzymes, such as topoisomerase I and topoisomerase II, or that are anti-mitotic agents. The term "pharmaceutically active agent" means in particular any pharmaceutically active agent other than a neutralizing anti-DKK1 / 4 composition, or a derivative thereof. It includes, but is not limited to: i. an aromatase inhibitor; ii. an anti-estrogen, an anti-androgen, or a gonadorelin agonist; iii. a topoisomerase I inhibitor or a topoisomerase II inhibitor; iv. an active agent in microtubules, an alkylating agent, an anti-neoplastic anti-metabolite, or a platinum compound; v. a compound that directs / reduces the activity of a protein or lipid kinase, or the activity of a protein or lipid phosphatase, an additional anti-angiogenic compound, or a compound that induces cell differentiation processes; saw. monoclonal antibodies; vii. a cyclo-oxygenase inhibitor, a bisphosphonate, a heparanase inhibitor, a biological response modifier; viii. an inhibitor of ras oncogenic isoforms; and ix. a telomerase inhibitor; x. a protease inhibitor, a matrix metalloproteinase inhibitor, a methionine aminopeptidase inhibitor, or a proteasome inhibitor; xi. agents used in the treatment of hematological malignancies, or compounds that direct, reduce, or inhibit the activity of Flt-3; xii. an inhibitor of HSP90; xiii. anti-proliferative antibodies; xiv. a histone deacetylase inhibitor (HDAC); xv. a compound that directs, reduces, or inhibits the activity / function of mTOR serine / threonine kinase; xvi. a somatostatin receptor antagonist; xvii. an anti-leukemic compound; xviii. approaches that damage tumor cells; xix. an EDG linker; xx. a ribonucleotide reductase inhibitor; xxi. an S-adenosyl-methionine decarboxylase inhibitor; xxii. a monoclonal antibody of VEGF or VEGFR; xxiii. photodynamic therapy; xxiv. an angiostatic steroid; xxv. an implant that contains corticosteroids; xxvi. an AT1 receptor antagonist; and xxvii. an ACE inhibitor. The term "aromatase inhibitor", as used herein, refers to a compound that inhibits the production of estrogen, ie, the conversion of the substrates androstenedione and testosterone to estrone and estradiol, respectively. The term includes, but is not limited to, steroids, especially atamestane, exemestane, and formestane; and in particular, non-steroids, in particular aminoglutethimide, rogletimide, pyridoglutethimide, trilostane, testolactone, ketoconazole, vorozole, fadrozole, anastrozole, and letrozole. Exemestane is traded as AROMASIN; the formestane as LENTARON; fadrozole as AFEMA; anastrozole as ARIMIDEX; letrozole as FEMARA or FEMAR; and aminoglutethimide as ORIMETEN. A combination of the invention comprising a pharmaceutically active agent that is an aromatase inhibitor is particularly useful for the treatment of hormone receptor-positive tumors, for example breast tumors. The term "anti-estrogen," as used herein, refers to a compound that antagonizes the effect of estrogen at the level of the estrogen receptor. The term includes, but is not limited to, tamoxifen, fulvestrant, raloxifene, and raloxifene hydrochloride. Tamoxifen can be administered in the form as it is traded, for example NOLVADEX; and raloxifene hydrochloride is traded as EVISTA. The fulvestrant can be formulated as disclosed in the Patent of the United States of North America Number 4,659,516, and is traded as FASLODEX. A combination of the invention comprising a pharmaceutically active agent that is an anti-estrogen, is particularly useful for the treatment of tumors positive for the estrogen receptor, for example breast tumors. The term "anti-androgen", as used herein, refers to any substance that is capable of inhibiting the biological effects of androgenic hormones, and includes, but is not limited to, bicalutamide (CASODEX), which can be formulate, for example, as disclosed in United States Patent Number 4,636,505. The term "gonadorelin agonist" as used herein, includes, but is not limited to, abarelix, goserel, and goserelin acetate. Goserelin is disclosed in U.S. Patent Number 4,100,274, and is marketed as ZOLADEX. Abarelix can be formulated, for example, as disclosed in U.S. Patent No. 5,843,901. The term "topoisomerase I inhibitor", as used herein, includes, but is not limited to, topotecan, gimatecan, irinotecan, camptothecin and its analogues, 9-nitro-camptothecin, and the macromolecular camptothecin conjugate PNU-166148 (compound A1 of International Publication Number WO 99/17804). The irinotecan can be administered, for example, in the manner trade, for example under the trademark registered CAMPTOSAR. The topotecan can be administered, for example, in the form as it is traded, for example under the registered trademark HYCAMTIN. The term "topoisomerase M inhibitor" as used herein, includes, but is not limited to, anthracyclines, such as doxorubicin, including the liposomal formulation, for example CAELYX, daunorubicin, including the liposomal formulation, eg, DAUNOSOME, epirubicin, idarubicin, and nemorubicin; the anthraquinones mitoxantrone and losoxantrone; and the podophyllotoxins etoposide and teniposide. The etoposide is traded as ETOPOPHOS; teniposide as VM 26-BRISTOL; doxorubicin as ADRIBLASTIN or ADRIAMYC N; epirubicin as FARMORUBICIN; idarubicin as ZAVEDOS; and mitoxantrone as NOVANTRON. The term "microtubule active agent", as used herein, refers to microtubule stabilizing agents, microtubule destabilizing agents, and inhibitors of microtubulin polymerization, including, but not limited to, taxanes, e.g., paclitaxel. and docetaxel; vinca alkaloids, for example vinblastine, especially vinblastine sulphate, vincristine, especially vincristine sulfate, and vinorelbine; discodermolidas; colchicine and epothilone and derivatives thereof, for example epothilone B or a derivative thereof. Paclitaxel is marketed as TAXOL; docetaxel as TAXOTERE; sulfate vinblastine as VI N BLASTI N RP; and vincristine sulfate as FARMISTI N. Also included are the generic forms of paclitaxel, as well as different dosage forms of paclitaxel. Generic forms of paclitaxel include, but are not limited to, betaxolol hydrochloride. Different dosage forms of paclitaxel include, but are not limited to, paclitaxel in albumin nanoparticles traded as ABRAXANE; ONXOL, CYTOTAX. The discodermolide can be obtained, for example, as disclosed in United States Patent Number 5,01,099. Also included are epothilone derivatives, which are disclosed in U.S. Patent No. 6,141,981, and in International Publications Num. WO 98/101 21, WO 98/25929, WO 98/08849, WO 99/43653, WO 98/22461 and WO 00/31 247. Epothilone A and / or B are especially preferred. The term "alkylating agent", as used herein, includes, but is not limited to, is limited to, cyclophosphamide, ifosfamide, melphalan, or nitrosourea (BCN U or Gliadel), or temozolamide (TEMODAR). The cyclophosphamide can be administered, for example, in the form as it is traded, for example under the registered trademark CYCLOSTIN; and ifosfamide as HOLOXAN. The term "anti-neoplastic anti-metabolite" includes, but is not limited to, 5-fluoro-uracil (5-FU); capecitabine; gemcitabine; DNA demethylating agents, such as 5-azacytidine and decitabine; methotrexate; edatrexate; and folic acid antagonists, such as, but not limited to, pemetrexed. Capecitabine can be administering, for example, in the way it is traded, for example under the registered trademark XELODA; and the gemcitabine as GEMZAR. The term "platinum compound", as used herein, includes, but is not limited to, carboplatin, cisplatin, cisplatin, oxaliplatin, Satraplatin, and platinum agents, such as ZD0473. Carboplatin can be administered, for example, in the form as it is traded, for example CARBOPLAT; and oxaliplatin as ELOXATIN. The term "compounds that direct / reduce the activity of protein or lipid kinase, or a protein or lipid phosphatase activity, or other anti-angiogenic compounds", as used herein, includes, but is not limited to, a, protein tyrosine kinase inhibitors and / or serine and / or threonine kinase inhibitors, or lipid kinase inhibitors, for example: i) compounds that direct, reduce, or inhibit the activity of endothelial growth factor receptors vascular (VEGF), such as compounds that direct, reduce, or inhibit VEGF activity, especially compounds that inhibit the VEGF receptor, such as, but not limited to, 7H-pyrrolo- [2,3 -d] -pyrimidine (AEE788); BAY 43-9006; isolcholine compounds disclosed in International Publication Number WO 00/09495, such as (4-tert-butyl-phenyl) -94-yl-methyl-isoquinolin-1-yl) -amine (AAL881); and ii) compounds that direct, reduce, or inhibit activity of platelet-derived growth factor receptor (PDGFR) receptors, such as the compounds that direct, reduce, or inhibit the activity of PDGFR, especially compounds that inhibit the PDGF receptor, for example an N-phenyl derivative. 2-pyrimidine-amine, for example imatinib, SU101, SU6668, and GFB-111; iii) compounds that direct, reduce, or inhibit the activity of fibroblast growth factor receptors (FGFR); iv) compounds that direct, reduce, or inhibit the activity of insulin-like growth factor-1 receptor (IGF-1R), such as compounds that direct, reduce, or inhibit IGF-1R activity, especially compounds that inhibit the IGF-1R receptor. The compounds include, but are not limited to, the compounds disclosed in International Publication Number WO 02/092599, and the derivatives thereof of 4-amino-5-phenyl. -7-cyclobutyl-pyrrolo- [2,3-d] -pyrimidine (AEW541); v) compounds that direct, reduce, or inhibit the activity of the Trk receptor tyrosine kinase family; vi) compounds that direct, reduce, or inhibit the activity of the Axl receptor tyrosine kinase family; vii) compounds that direct, reduce, or inhibit the activity of the c-Met receptor; viii) compounds that direct, reduce, or inhibit the activity of the receptor tyrosine kinase Ret; ix) compounds that direct, reduce, or inhibit receptor tyrosine kinase activity Kit / SCFR; x) compounds that direct, reduce, or inhibit the activity of receptor tyrosine kinases C-kit (part of the PDGFR family), such as the compounds that direct, reduce, or inhibit the activity of the receptor tyrosine kinase family C-kit, especially compounds that inhibit the C-kit receptor, for example imatinib; xi) compounds that direct, reduce, or inhibit the activity of members of the c-Abl family, and their gene fusion products, for example BCR-Abl kinase, such as compounds that direct, reduce, or inhibit activity of members of the c-Abl family and their gene fusion products, for example, a derivative of N-phenyl-2-pyrimidine-amine, for example imatinib, PD180970, AG957, NSC 680410 or PD173955 of ParkeDavis; BMS354825; xii) compounds that direct, reduce, or inhibit the activity of protein kinase C (PKC) members and the Raf family of serine / threonine kinases, members of the MEK family, SRC, JAK, FAK, PDK and Ras / MAPK, either the kinase family Pl (3), or the kinase family related to the PI (3) kinase, and / or the members of the cyclin dependent kinase family (CDK), and are especially staurosporine derivatives disclosed in U.S. Patent No. 5,093,330, for example, midostaurin; examples of other compounds include, for example, UCN-01; safingol; BAY 43-9006; Bryostatin 1; Periphosin; Ilmofosin; RO 318220 and RO 320432; GO 6976; Isis 3521; LY333531 / LY3791 96; Isoquinoline compounds, such as those disclosed in International Publication Number WO 00/09495; FTIs; PD 1 84352 or QAN697, to a P 1 3K inhibitor; xiii) compounds that direct, reduce, or inhibit the activity of tyrosine protein kinase, such as imatinib mesylate (GLEEVEC); ti rfostin, or pyridyl-amino-benzamide and its derivatives (AMN 107). A tyrphostin is preferably a low molecular weight compound (Mr < 1 500), or a pharmaceutically acceptable salt thereof, especially a compound selected from the benzylidene malonitrile class, or the S class of compounds. -aryl-benzene-malonitrile or bis-substrate quinoline, more especially any compound selected from the group consisting of Tyrphostin A23 / RG-50810, AG 99, Tyrphostin AG 213, Tyrphostin AG 1 748, Tyrphostin AG 490, Tyrphostin B44, enantiomer ( +) of Tirfostin B44, Tirfostin AG 555, AG 494, Tirfostin AG 556; AG957 and adafostin (4- [{(2,5-dihydroxy-phenyl) -methyl] -aminoj-benzoic acid adamantyl ester, NSC 680410, adaphostin); xiv) compounds that direct, reduce, or inhibit the activity of the tyrosine kinase family of epidermal growth factor receptors (EGFR, ErbB2, ErbB3, ErbB4 as homo- or hetero-dimers), such as the compounds they direct, reduce , or inhibit the activity of the epidermal growth factor receptor family, which are in particular compounds, proteins, or antibodies that inhibit members of the EGF receptor tyrosine kinase family, for example the EGF receptor, ErbB2, ErbB3, and ErbB4, or that bind to EGF or to EGF-related ligands, and are in particular the compounds, proteins, or generic monoclonal antibodies and specifically disclosed in International Publication Number WO 97/02266, by example the compound of Example 39, or in Patent Numbers EP 0 564 409, WO 99/03854, EP 0520722, EP 0 566 226, EP 0 787 722, EP 0 837 063, Patent of the United States of America Number 5,747,498, WO 98/1 0767, WO 97/30034, WO 97/49688, WO 97/38983, and especially in International Publication No. WO 96/30347, for example the compound known as CP 358774, in International Publication WO Number 96/33980, for example the compound ZD 1 839; and in International Publication No. WO 95/03283, for example the compound ZM 1051 80, for example trastuzumab (H ERCEPTI N®), cetuximab, Isa ssa, OS I-774, Cl 1 033, EKB-569, GW- 201 6, E1 .1, E2.4, E2.5, E6.2, E6.4, E2.1 1, E6.3, or E7.6.3, and 7H-pyrrolo- [2,3-d] -pyrimidine derivatives, which are disclosed in International Publication Number WO 03/01 3541, erlotinib and gefitinib. Erlotinib can be administered in the form as it is traded, for example TARCEVA, and gefitinib as I RESSA, and human monoclonal antibodies against the epidermal growth factor receptor, including ABX-EGFR; and xv) compounds that direct, reduce, or inhibit the activity / function of mTOR serine / threonine kinase, which are especially compounds, proteins, or antibodies that direct / inhibit members of the mTOR kinase family, eg RAD, RAD001, CC I-779, ABT578, SAR543, rapamycin and its derivatives / analogs, AP23573 and AP23841 from Ariad, everolimus (CERTICAN) and sirolimus. CERTICAN (everolimus, RAD), a novel proliferation signal inhibitor that prevents proliferation of T cells and vascular smooth muscle cells. When referring to an antibody, intact monoclonal antibodies, nanobodies, polyclonal antibodies, multispecific antibodies formed from at least two intact antibodies, and antibody fragments should be included, provided they exhibit the desired biological activity. The phrase "compound that directs, reduces, or inhibits the activity of a protein or lipid phosphatase", as used herein, includes, but is not limited to, phosphatase 1, phosphatase 2A, PTEN or CDC25 inhibitors. , for example okadaic acid or a derivative thereof. The term "monoclonal antibodies," as used herein, includes, but is not limited to, bevacizumab, cetuximab, trastuzumab, Ibritumomab-tiuxetan, denosumab, anti-CD40, anti-GM-CSF, and tositumomab, and iodine I 131 Bevacizumab can be administered in the way it is sold, for example AVASTI N; cetuximab as ERBITUX; trastuzumab as HERCEPTI N; Rituximab as MABTHERA; Ibritumomab-tiuxetane as ZEVULI N; anti-RANKL as denosumab (AMG 1 62), anti-CD40 as HCD122 (U.S. Patent Application Number 2002-0106371), and tositumomab and iodine I 131 as BEXXAR. The phrase "additional anti-angiogenic compounds" includes, but is not limited to, compounds having another mechanism for their activity, for example, unrelated to the inhibition of the protein or lipid kinase, for example thalidomide (THALOMID), and TNP-470. The phrase "compounds that induce cell differentiation processes", as used herein, includes, but is not limited to, retinoic acid, a-, and-, or d-tocopherol, or a- , and-, or d-tocotrienol. The term "cyclooxygenase inhibitor", as used herein, includes, but is not limited to, for example, COX-2 inhibitors, 2-aryl-amino-phenyl-acetic acid substituted by 5-alkyl and its derivatives, such as celecoxib (CELEBREX), rofecoxib (VIOXX), etoricoxib, valdecoxib, or a 5-alkyl-2-aryl-amino-phenyl-acetic acid, for example 5-methyl-2- (2'-chloro) -6'-fluoro-anilino) -phenyl-acetic, lumiracoxib. The term "bisphosphonates", as used herein, includes, but is not limited to, etridonic, clodronic, tiludronic, pamidronic, alendronic, ibandronic, risedronic, and zoledronic acid. The "etridonic acid" can be administered, for example, in the form as it is traded, for example DIDRONEL; the "clodronic acid" as BONEFOS; "tiludronic acid" as SKELID; the "acid pamidronic acid "as AREDIA," alendronate "as FOSAMAX," ibandronic acid "as BONDRANAT," risedronic acid "as ACTONEL, and" zoledronic acid "as ZOMETA, the term" heparanase inhibitor ", as used in present, refers to compounds that direct, reduce, or inhibit the degradation of heparin sulfate The term includes, but is not limited to, PI88.The term "biological response modifier", as used herein , includes, but is not limited to, lymphosine or interferons, for example interferon and I. The term "inhibitor of the Ras oncogenic isoforms", as used herein, includes, but is not limited to, H-Ras, K- Ras, or N-Ras, as used herein, and refers to compounds that direct, reduce, or inhibit the oncogenic activity of Ras, for example a farnesyl transferase inhibitor (FTI), for example L-744832 , DK8G557 or R115777 (ZARNESTRA) The term "telomerase inhibitor", as used herein, includes, but is not limited to, compounds that direct, reduce, or inhibit telomerase activity. The compounds that direct, reduce, or inhibit telomerase activity are in particular compounds that inhibit the telomerase receptor, for example telomestatin. The term "matrix metalloproteinase inhibitor" or (MMP inhibitor), as used herein, includes, but is not limited to, peptidomimetic and non-peptidomimetic inhibitors of collagen; tetracycline derivatives, for example the peptidomimetic hydroxamate inhibitor, batimastat; and its orally bioavailable analog, marimastate (BB-2516), prinomastat (AG3340), metastate (NSC 683551) BMS 279251, BAY 12-9566, TAA211, MMI270B or AAJ996. The term "methionine amino-peptidase inhibitor", as used herein, includes, but is not limited to, compounds that direct, reduce, or inhibit the activity of the methionine aminopeptidase. The compounds that direct, reduce, or inhibit the activity of the methionine aminopeptidase are, for example, bengamide or a derivative thereof. The term "proteasome inhibitors", as used herein, includes those compounds that direct, reduce, or inhibit proteasome activity. Compounds that direct, reduce, or inhibit proteasome activity include, but are not limited to, PS-341; MLN 341. Bortezomib or Velcade. The phrase "agent used in the treatment of hematological malignancies", as used herein, includes, but is not limited to, tyrosine kinase inhibitors type FMS, for example compounds that direct, reduce, or inhibit the activity of tyrosine kinase receptors type FMS (3R); interferon, 1-b-D-arabino-furanosyl-cytosine (ara-c), and bisulfan; and ALK inhibitors, for example the compounds that direct, reduce, or inhibit the anaplastic lymphoma kinase. The phrase "compounds that direct, reduce, or inhibit Flt-3"activity, as used herein, includes, but is not limited to, compounds, proteins, or antibodies that inhibit Flt-3, for example N-benzoyl-staurosporine, midostaurin, a staurosporine derivative, SU11248 and MLN518 The term "HSP90 inhibitors", as used herein, includes, but is not limited to, compounds that direct, reduce, or inhibit the intrinsic activity of the HSP90 ATPase; reduce or inhibit HSP90 client proteins by means of the proteasome pathway of ubiquitin Compounds that direct, reduce, or inhibit the intrinsic activity of HSP90 ATPase are especially compounds, proteins, or antibodies that inhibit the activity of ATPase HSP90, for example, 17-allyl-amino-17-demethoxy-geldanamycin (17AAG), a derivative of geldanamycin, other compounds related to geldanamycin, radicicol, and HDAC inhibitors.The term "an anti-proliferative antibody", how is it used in the present, includes, but is not limited to, trastuzumab (HERCEPTIN), trastuzumab-DM1, erlotinib (TARCEVA), bevacizumab (AVASTIN), rituximab (RITUXAN), PR064553 (anti-CD40), and 2C4 antibody. Antibodies means, for example, intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies formed from at least two intact antibodies, and antibody fragments, as long as they exhibit the desired biological activity. The term "HDAC inhibitor", as used herein, refers to compounds that inhibit histone deacetylase, and which possess anti-proliferative activity. This includes, but is not limited to, the compounds disclosed in International Publication Number WO 02/22577, especially N-hydroxy-3- [4 - [[(2-hydroxy-ethyl) - [2- (1H-indol-3-yl) -ethyl] -amino] -methyl] -phenyl] -2-E-2-propenamide, and N-hydroxy-3- [4 - [[[2- (2-methyl-1 H -indol-3-yl) -ethyl] -amino] -methyl] -phenyl] -2E-2-propenamide, and the pharmaceutically acceptable salts thereof (LBH589). It also includes, in particular, suberoyl anilide hydroxamic acid (SAHA); pyridin-3-yl-methyl ester of [4- (2-amino-phenyl-carbamoyl) -benzyl] -carbamic acid and its derivatives; butyric acid; piroxamide, trichostatin A, Oxamflatine, apicidin, depsipeptide; depudecin, and trapoxin. The phrase "compound that directs, reduces, or inhibits the activity / function of the serine / threonine mTOR kinase", as used herein, includes, but is not limited to, the compounds, proteins, or antibodies that direct / inhibit members of the mTOR kinase family, eg RAD, RAD001, CCI-779, ABT578, SAR543, rapamycin and its derivatives / analogs, AP23573 and AP23841 from Ariad, everolimus (CERTICAN), and sirolimus (RAPAMUNE), CCI -779 and ABT578. CERTICAN (everolimus, RAD), a novel proliferation signal inhibitor that prevents the proliferation of T-cells and vascular smooth muscle cells. The term "somatostatin receptor antagonist", as used herein, includes, but is not limited to, agents that direct, treat, or inhibit the somatostatin receptor, such as octreoride and SOM230. The term "anti-leukemic compound", as used herein, includes, but is not limited to, Ara-C, a pyrimidine analog, which is the 2'-a-hydroxy-ribose (arabinoside) derivative of the deoxycytidine. Also included is the purine analog of hypoxanthine, 6-mercapto-purine (6-MP), and fludarabine phosphate. The phrase "approaches that damage tumor cells" refers to approaches, such as ionizing radiation. The term "ionizing radiation", referred to above and hereinafter, means ionizing radiation that occurs either as electromagnetic rays, such as X-rays and gamma rays; or particles, such as alpha, beta, and gamma particles. Ionizing radiation is provided in, but is not limited to, radiation therapy, and is known in the art. See Hellman, Cancer, 4th Edition, Volume 1, Devita et al., Editors, pages 248-275 (1 993). The term "EDG linker", as used herein, includes, but is not limited to, a class of immunosuppressants that modulates the recirculation of lymphocytes, such as FTY720. The term "ribonucleotide reductase inhibitor", as used herein, includes, but is not limited to, pyrimidine or purine nucleoside analogues, including, but not limited to, fludaravin and / or ara-C; 6-thioguanine; 5-FU; cladribine; 6-mercapto-purine, especially in combination with ara-C against leukemia acute lymphocytic; and / or pentostatin. Inhibitors of ribonucleotide reductase are in particular hydroxy urea or derivatives of 2-hydroxy-1H-isoindol-1,3-dione, such as PL-1, PL-2, PL-3, PL-4, PL-5 , PL-6, PL-7, or PL 8. See Nandy et al., Acta Oncológica, Volume 33, Number 8, pages 953-961 (1994). The term "S-adenosyl-methionine decarboxylase", as used herein, includes, but is not limited to, the compounds disclosed in U.S. Patent No. 5,461,076. The phrase "VEGF or VEGFR monoclonal antibodies", as used herein, includes, but is not limited to, the compounds disclosed in International Publication Number WO 98/35958, eg, 1- (4) -chloro-anilino) -4- (4-pyridyl-methyl) -phthalazine or a pharmaceutically acceptable salt thereof, for example succinate, or in Patent Numbers WO 00/09495, WO 00/27820, WO 00/59509 , WO 98/11223, WO 00/27819 and EP 0769947; those described by Prewett et al., Cancer Res, Volume 59, pages 5209-5218 (1999); Yuan et al., Proc. Nati Acad. Sci. USA, Volume 93, pages 14765-14770 (1996); Zhu et al., Cancer Res, Volume 58, pages 3209-3214 (1998); and Mordenti et al., Toxicol Pathol, Volume 27, Number 1, pages 14-21 (1999), in International Publications Nos. 00/37502 and WO 94/10202; ANGIOSTATIN, described by O'Reilly et al., Cell, Volume 79, pages 315-328 (1994); ENDOSTATINE, described by O'Reilly et al., Cell, Volume 88, pages 277-285 (1 997); ammonium acid amides; ZD41 90; ZD6474; SU5416; SU6668; bevacizumab; or anti-VEGF antibodies or anti-VEGF receptor antibodies, for example rhuMAb and RH UFab; VEGF aptamer, for example Macugon; FLT-4 inhibitors; FLT-3 inhibitors; lgG 1 antibody of VEGFR-2; Angiozyme (RPI 4610); and Avastan. The term "photodynamic therapy", as used herein, refers to therapy that uses certain chemicals known as photosensitizing agents to treat or prevent cancers. Examples of photodynamic therapy include, but are not limited to, treatment with agents, such as VISUDYNE, and porfimer-sodium. The term "angiostatic steroid", as used herein, includes, but is not limited to, agents that block or inhibit angiogenesis, such as, for example, anecortave, triamcinolone, hydrocortisone, 1 1-a-epihydrocortisol, cortexolone , 1 7a-hydroxyprogesterone, corticosterone, deoxy-corticosterone, testosterone, estrone, and dexamethasone. The phrase "corticosteroid-containing implant", as used herein, includes, but is not limited to agents such as, for example, fluocinolone and dexamethasone. The term "AT1 receptor antagonist", as used herein, includes, but is not limited to, agents such as DIOVAN. The term "ACE inhibitor", as used herein, includes, but is not limited to, CIBACEN, benazepril, enazepril (LOTENSI N), captopril, enalapril, fosinopril, lisinopril, moexipril, quinapril, ramipril, perindopril, and trandolapril. Other pharmaceutically active agents include, but are not limited to, plant alkaloids, hormonal agents and antagonists, modifications of the biological response, preferably lymphokines or interferons, antisense oligonucleotides or oligonucleotide derivatives; or various agents, or agents with other unknown mechanisms of action. In each case where citations of patent applications or scientific publications are given, in particular with respect to the claims of the respective compound and to the final products of the examples of processing thereof, the subject matter of the final products, the preparations Pharmaceuticals, and the claims, are incorporated in the present application as a reference to these publications. In the same way, the corresponding stereoisomers are included, as well as the corresponding crystal modifications, for example solvates and polymorphs, which are disclosed therein. The compounds used as active ingredients in the combinations disclosed herein can be prepared and administered as described in the cited documents, respectively. The structure of the active agents identified by code numbers, generic or commercial names, can be taken from the current edition of the standard compendium "The Merck Index", or from the databases, for example Patents International, for example IMS World Publications, or of the publications mentioned above and later. The corresponding content thereof is incorporated herein by reference. It will be understood that references to components (a) and (b) are intended to also include pharmaceutically acceptable salts of any of the active substances. If the active substances comprised by the components (a) and / or (b) have, for example, at least one basic center, they can form acid addition salts. The corresponding acid addition salts may also be formed having, if desired, a basic core additionally present. The active substances having an acid group, for example COOH, can form salts with bases. Active substances comprised in components (a) and / or (b), or a pharmaceutically acceptable salt thereof, may also be used in the form of a hydrate, or may include other solvents used for crystallization. Accordingly, in a first aspect, the present invention relates to a method for the prevention or treatment of proliferative diseases, or diseases that are triggered by persistent angiogenesis in a mammal, preferably in a human patient, which comprises treating the patient in a concurrent or sequential manner with pharmaceutically effective amounts of a combination of: (a) a neutralizing anti-DKK1 / 4 composition; and (b) a pharmaceutically active agent.

In a preferred embodiment, the present invention provides a pharmaceutical preparation comprising: (a) a neutralizing anti-DKK1 / 4 composition; and (b) one or more pharmaceutically active agents selected from the group consisting of an aromatase inhibitor; an anti-estrogen; an anti-androgen; a gonadorelin agonist; a topoisomerase I inhibitor; an inhibitor of topoisomerase I I; an active agent in microtubules; an alkylating agent; an anti-neoplastic anti-metabolite; a platinum compound; a compound that directs / reduces a protein or lipid kinase activity, or a protein or lipid phosphatase activity; an anti-angiogenic compound; a compound that induces cellular differentiation processes; monoclonal antibodies; a cyclo-oxygenase inhibitor; a bisphosphonate; a heparanase inhibitor; a biological response modifier; an inhibitor of the Ras oncogenic isoforms; a telomerase inhibitor; a protease inhibitor; a matrix metalloproteinase inhibitor; an inhibitor of methionine amino peptidase; a proteasome inhibitor; agents that direct, reduce, or inhibit the activity of Flt-3; an inhibitor of HSP90; anti-proliferative antibodies; an HDAC inhibitor; a compound that directs, reduces, or inhibits the activity / function of mTOR serine / threonine kinase; a somatostatin receptor antagonist; an antileukemic compound; approaches that damage tumor cells; an EDG linker: a ribonucleotide reductase inhibitor; a S-adenosyl-methionine decarboxylase inhibitor; a monoclonal antibody of VEGF or VEGFR; photodynamic therapy; an angiostatic ster an implant that contains corticoster; an AT1 receptor antagonist; and an ACE inhibitor. Any of the combination of components (a) and (b), the method for the treatment of a warm-blooded animal comprising administering these two components, a pharmaceutical composition comprising these two components for simultaneous, separate use, or sequence, the use of the combination for the delay of progress or treatment of a proliferative disease or for the manufacture of a pharmaceutical preparation for these purposes, or a commercial product comprising this combination of components (a) and (b), all as mentioned or defined above, will also be referred to subsequently as the combination of the invention. In such a way that this term refers to each one of these modalities (that therefore, can replace this term where appropriate). For example, the simultaneous administration may take place in the form of a fixed combination with two or more active ingredients, or by the simultaneous administration of two or more active ingredients that are formulated in an independent manner. The use in sequence (administration) preferably means the administration of one (or more) components of a combination at a point of time, other components at a different point of time, i.e. in a chronically staggered manner, preferably such so that the combination shows more efficiency than the individual compounds administered independently (especially, showing synergism). The preferred use (administration) means the administration of the components of the combination independently of one another at different points of time, preferably meaning that components (a) and (b) are administered in such a way that there is no overlap present measurable blood levels of both compounds in an overlapping manner (at the same time). Combinations of two or more administrations in sequence, separately, and simultaneously, are also possible, preferably in such a way that the component drugs of the combination show a joint therapeutic effect that exceeds the effect that is found when the component drugs of the combination are use independently at such large time intervals that no mutual effect on their therapeutic efficiency can be found, with a synergistic effect being especially preferred.

The term "progress delay", as used herein, means the administration of the combination to patients who are in a previous stage or in a first phase, of the first manifestation or a recurrence of the disease that is going to treat, in whose patients, for example, a pre-form of the corresponding disease is diagnosed, or whose patients are in a condition, for example, during a medical treatment, or a condition resulting from an accident, under which it is probable that a corresponding disease develops. "Jointly therapeutically active" or "joint therapeutic effect" means that the compounds can be given separately (in a chronically staggered manner, especially in a specific manner in sequence), at such time intervals that preferably, in the animal of warm blood, especially in the human being, to be treated, still show an interaction (preferably synergistic) (joint therapeutic effect). The fact that this is the case can be determined, among other things, by following blood levels, showing that both compounds are present in the blood of the human being to be treated at least during certain time intervals. "Pharmaceutically effective" preferably refers to an amount that is therapeutically, or in a broader sense, also prophylactically effective against the progress of a proliferative disease. The term "a commercial package" or "a product", as used herein, especially defines a "kit of parts", in the sense that the components (a) and (b), as defined above, they can be dosed in an independent way, or by using different fixed combinations with distinguished quantities of components (a) and (b), that is, in a simultaneous manner or at different points of time. Moreover, these terms comprise a commercial package comprising (especially combining), as active ingredients, the components (a) and (b), together with instructions for simultaneous use, in sequence (chronically staggered, in a specific sequence in time, preferentially), or (less preferably) separated from them, in the delay of progress or treatment of a proliferative disease. The parts of the kit of parts, for example, can then be administered in a simultaneous or chronologically staggered manner, that is, at different points of time and with equal or different time intervals for any part of the kit of parts. Most preferably, the time intervals are selected such that the effect on the disease treated in the combined use of the parts is greater than the effect that would be obtained by using only any of the combination components ( a) and (b) (as may be determined in accordance with conventional methods). The ratio of the total amounts of the combination component (a) to the combination component (b) to be administered in the combined preparation, can be varied, for example, in order to deal with the needs of a sub-population of patients to be treated, or with the needs of the individual patient, whose different needs may be due to the particular disease, age , sex, body weight, etc. , from the patients. Preferably, there is at least one beneficial effect, for example a mutual improvement of the effect of the combination components (a) and (b), in particular an effect rather than additive, which, therefore, could be achieved with doses plus lowers of each of the combined drugs, respectively, of what is tolerable in the case of treatment with the individual drugs only without combination, producing additional convenient effects, for example fewer side effects, or a combined therapeutic effect in an ineffective dosage of one or both of the combination components (components (a) and (b)), and most preferably, a strong synergism of the combination components (a) and (b). Both in the case of the use of the combination of components (a) and (b), and of the commercial package, any combination of simultaneous, sequential, and separate use is also possible, meaning that components (a) and (b) ) can be administered at a point of time in a simultaneous manner, followed by the administration of only one component with a lower toxicity to the host, either chronically, for example more than 3 to 4 weeks of daily dosing, at one point of the subsequent time, and subsequently the other component or the combination of both components at a later time point (in the following courses of treatment of the drug combination for an optimal anti-tumor effect), or the like. The COMBINATION OF THE INVENTION may also be applied in combination with other treatments, for example surgical intervention, hyperthermia, and / or irradiation therapy. The pharmaceutical compositions according to the present invention can be prepared by conventional means, and are those suitable for enteral, such as oral or rectal, and parenteral administration to mammals, including man, comprising a therapeutically effective amount of a VEGF inhibitor, and at least one pharmaceutically active agent alone or in combination with one or more pharmaceutically acceptable carriers , especially those suitable for enteral or parenteral application. The pharmaceutical compositions comprise from about 0.00002 to about 100 percent, especially, for example, in the case of infusion dilutions that are ready to be used, from 0.0001 to 0.02 percent, or, for example, in the case of concentrates for injection or infusion, or especially in parenteral formulations, from about 0.1 percent to about 95 percent, preferably from about 1 percent to about 90 percent, more preferably about 20 percent a approximately 60 percent active ingredient (weight by weight, in each case). The pharmaceutical compositions according to the invention, for example, can be in a unit dosage form, such as in the form of ampoules, flasks, dragees, tablets, infusion bags, or capsules. The effective dosage of each of the combination components employed in a formulation of the present invention may vary depending on the particular compound or pharmaceutical composition employed, the mode of administration, the condition that is being treated, and the severity of the condition being treated. A physician, clinician, or veterinarian of ordinary experience, can easily determine the effective amount of each of the active ingredients, necessary to prevent, treat, or inhibit the progress of the condition. Tyrphostins, especially Adafostin, are preferably administered to a warm-blooded animal, especially a human, at a dosage in the range of about 1 to 6,000 milligrams / day, more preferably 25 to 5,000 milligrams / day. , and in a very preferable way from 50 to 4,000 milligrams / day. Unless otherwise reported herein, the compound is preferably administered from 1 to 5, especially from 1 to 4 times per day. Pharmaceutical preparations for combination therapy for enteral or parenteral administration, for example, are those that are in unit dosage forms, such as sugar-coated tablets, capsules, or suppositories, and also ampoules. If not stated otherwise, these formulations are prepared by conventional means, for example by means of conventional mixing, granulating, sugar coating, dissolving, or lyophilizing processes. It will be appreciated that the unit content of a combination component contained in an individual dose of each dosage form does not need to constitute an effective amount by itself, because the effective amount necessary can be achieved by the administration of a plurality of dosage units. A person skilled in the art has the ability to determine the appropriate pharmaceutically effective amounts of the components of the combination. Preferably, the compounds or pharmaceutically acceptable salts thereof are administered as an oral pharmaceutical formulation in the form of a tablet, capsule, or syrup; or as parenteral injections, if appropriate. In preparing the compositions for oral administration, any pharmaceutically acceptable medium, such as water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, may be employed. Pharmaceutically acceptable carriers include starches, sugars, microcrystalline celluloses, diluents, granulating agents, lubricants, binders, disintegrating agents. The solutions of the active ingredient, and also the suspensions, and especially the isotonic aqueous solutions or suspensions, are useful for the parenteral administration of the active ingredient, being possible, for example, in the case of the lyophilized compositions, which comprise the active ingredient alone or together with a pharmaceutically acceptable carrier, for example mannitol, for these solutions or suspensions to be produced before use. The pharmaceutical compositions can be sterilized and / or can comprise excipients, for example preservatives, stabilizers, wetting agents and / or emulsifiers, solubilizers, salts for regulating the osmotic pressure and / or pH regulators, and are prepared in a manner known per se, for example by means of conventional dissolution or lyophilization processes. The solutions or suspensions may comprise viscosity-increasing substances, such as sodium carboxy-methyl-cellulose, carboxy-methyl-cellulose, dextran, polyvinyl-pyrrolidone, or gelatin. Suspensions in oil comprise, as the oil component, the vegetable, synthetic, or semi-synthetic oils customary for injection purposes. The isotonic agent can be selected from any of those known in the art, for example mannitol, dextrose, glucose, and sodium chloride. The formulation for infusion can be diluted with the aqueous medium. The amount of the aqueous medium used as diluent is selected according to the desired concentration of the active ingredient in the solution for infusion. Infusion solutions may contain other excipients commonly employed in the formulations to be administered intravenously, such as antioxidants. The present invention further relates to "a combined preparation", which, as used herein, especially defines a "kit of parts", in the sense that the combination components (a) and (b), as are defined above, can be dosed independently, or by using different fixed combinations with quantities distinguished from the combination components (a) and (b), that is, in a simultaneous manner or at different points of time. The parts of the kit of parts can then be administered, for example, in a simultaneous or chronologically staggered manner, ie in different points of time and with equal or different time intervals for any part of the kit of parts. The ratio of the total amounts of the combination component (a) to the combination component (b) to be administered in the combined preparation can be varied, for example, in order to deal with the needs of a sub-population of patients to be treated, or with the needs of the individual patient, based on the severity of any of the side effects that the patient experiences. USES AND METHODS OF THE INVENTION The antibodies (and immunoconjugates and bispecific molecules) of the present invention have diagnostic and therapeutic utilities in vitro and in vivo. For example, these molecules can be administered to cells in culture, for example in vitro or in vivo, or to a subject, e.g., in vivo, to treat, prevent, or diagnose a variety of disorders. The term "subject", as used herein, is intended to include human and non-human animals. Non-human animals include all vertebrates, for example mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cattle, horses, chickens, amphibians, and reptiles. The methods are particularly suitable for the treatment of human patients who have a disorder associated with the expression of DKK1. When the antibodies to DKK1 are administered together with another agent, the two can be administered in any order or in a simultaneous manner. In one embodiment, the antibodies (and immunoconjugates and bispecific molecules) of the invention can be used to detect levels of DKK1, or levels of cells containing DKK1. This can be achieved, for example, by contacting a sample (such as an in vitro sample), and a control sample, with the anti-DKK1 antibody, under conditions that allow the formation of a complex between the antibody and DKK1. Any complexes formed between the antibody and DKK1, are detected and compared in the sample and in the control. As a non-limiting example, conventional detection methods that are well known in the art can be carried out, such as, for example, ELISA, MALDI, and flow cytometric assays, using the compositions of the invention. Accordingly, in one aspect, the invention further provides methods for detecting the presence of DKK1 (e.g., human DKK1 antigen) in a sample, or for measuring the amount of DKK1, which comprises contacting the sample. , and a control sample, with an antibody of the invention, or an antigen binding portion thereof, that specifically binds to DKK1, under conditions that allow the formation of a complex between the antibody or a portion of the antibody. same and DKK1. The formation of a complex is then detected, where a difference in the formation of a complex between the sample compared to the control sample indicates the presence of DKK1 in the sample. Also, within the scope of the invention, are kits consisting of the compositions (e.g., antibodies, human antibodies, immunoconjugates, and bispecific molecules) of the invention, and instructions for their use. The kit may further contain at least one additional reagent, or one or more additional antibodies of the invention (for example, an antibody having a complementary activity that binds to an epitope on the target antigen other than the first antibody). The kits typically include a mark indicating the intended use of the kit contents. The term "trademark" includes any written, or registered material supplied on or with the kit, or which otherwise accompanies the kit. The invention having been fully described, it is further illustrated by the following examples and claims, which are illustrative and are not intended to be additionally limiting. Those skilled in the art will recognize or may assert, employing no more than routine experimentation, numerous equivalents to the specific procedures described herein. These equivalents are within the scope of the present invention and the claims. The content of all references, including patents issued and applications for published patents, cited throughout this application, are incorporated herein by reference. EXAMPLES Example 1: Generation of specific antibodies of human DKK1 from the HuCAL GOLD® library. Therapeutic antibodies against the human DKK1 protein are generated by the selection of clones having high binding affinities, using the source of the variant antibody proteins of a commercially available phage display library, the MorphoSys library HuCAL GOLD®. HuCAL GOLD® is a Fab library (Knappik et al., 2000, J. Mol. Biol. 296: 57-86, Krebs et al., 2001 J. Immunol. Methods 254: 67-84; Rauchenberger et al., 2003 J. Biol. Mol. Chem. 278 (40): 38194-38205), where the six complementarity determining regions are diversified by appropriate mutation, and which employs CysDisplay® technology to link the Fab fragments with the phage surface (Publication International Number WO 01/05950, Lóhning 2001). General Procedures: Rescue of faqémidos. phage amplification, and purification The HuCAL GOLD® library is amplified in a standard rich bacterial medium (2xYT) containing 34 micrograms / milliliter of chloramphenicol, and 1 percent glucose (2xYT-CG). After infection of the cells at an OD60onm of 0.5 with auxiliary phages VSCM13 (incubating the mixture of phages and cells for 30 minutes at 37 ° C without shaking, followed by 30 minutes at 37 ° C with shaking at 250 revolutions per minute), cells are centrifuged (4.120 g, 5 minutes, 4 ° C), resuspended in 2xYT / 34 micrograms / milliliter of chloramphenicol / 50 micrograms / milliliter of kanamycin / I PTG 0.25 mM, and grown overnight at 22 ° C. At the end of this period, the cells are removed by centrifugation, and the phages are precipitated in PEG twice from the supernatant, resuspended in 20% phosphate / glycerol-regulated serum, and stored at -80 °. C. The amplification of phages between two rounds of panning is conducted as follows: the cells of the TG1 strain of E. coli in the mid-log phase are infected with phages and eluted following selection with the DKK1 protein, and are applied on LB-agar supplemented with 1 percent glucose and 34 micrograms / milliliter of chloramphenicol (LB-CG plates). After overnight incubation of the plates at 30 ° C, the bacterial colonies are scraped from the agar surface, and used to inoculate the 2xYT-CG broth to obtain an OD6oonm of 0.5, and then add the phage helper. VSCM1 3 to obtain a productive infection as described above. Previous Experiments for Solution Panning Using Strep-Tacti Magnetic Pearls It has been reported that Strep-tag II has a low affinity for the Strep-Tactin matrix (KD of approximately 1 μM according to Voss and Skerra, 1 997, Protein Eng. 10: 975-982), and therefore, A previous experiment is carried out to evaluate the suitability of using Strep-Tactin-coated MagStrep beads for antigen capture during antibody selections, and to avoid antigen loss during panning. For this purpose, 8 milligrams of pearls are incubated MagStrep with 46 micrograms of DKK1 labeled with His-Strep for 1 hour at room temperature, and the sample is divided into four previously blocked Eppendorf tubes. One tube served as the positive control (without washing), and the other three samples are washed with different astringencies according to the panning section of the HuCAL GOLD® manual. The detection of the linkage of DKK1 labeled with His-Strep to the MagStrep beads (magnetic beads coated with Strep-Tactin obtained from IBA, Góttingen, Germany), is carried out in BioVeris, using a goat anti-DKK1 antibody, and a anti-goat detection antibody marked with rubidium. As shown in Figure 1 herein, no significant loss of His-Strep-labeled DKK1 can be detected from Strep-Tactin-coated beads when comparing unwashed beads with beads washed with different astringencies. of H uCAL®. Therefore, it appears that DKK1 labeled with His-Strep is suitable for use in the pans in solution with magnetic beads coated with Strep-Tactin (MagStrep beads).

Selection by panning of DKK1-specific antibodies from the library For the selection of antibodies that recognize human DKK1, two panning strategies are applied. In summary, HuCAL GOLD® phage-antibodies are divided into four groups comprising different combinations of VH master genes (group 1 contains VH1 / 5 AK, group 2 contains VH3, group 3 contains VH2 / 4/6 AK, and group 4 contains VH1 -6 AK). These groups are subjected individually to two rounds of panning in DKK1 solution labeled with His-Strep captured on magnetic Strep-Tactin beads (Mega Strep beads, IBA), and for the third round of selection only, either on DKK1 labeled with His -Strep captured on Strep-Tactin magnetic beads, or on APP-labeled human DKK1 protein captured by streptavidin beads (Dynabeads® M-280, Streptavidin, Dynal), with a biotinylated anti-APP antibody. In detail, for panning in solution using DKK1 labeled with His-Strep coupled with Strep-Tactin magnetic beads, the following protocol is applied: pre-blocked tubes (1.5 milliliter Eppendorf tubes) are prepared by treatment with 1.5 milliliters of ChemiBLOCKER 2x diluted 1: 1 with phosphate-buffered serum overnight at 4 ° C. Pre-blocked beads are prepared by treatment as follows: 580 microliters (28 milligrams of beads) of Strep-Tactin magnetic beads are washed once with 580 microliters of regulated serum with phosphate, and re-suspended in 580 microliters of ChemiBLOCKER (diluted in a volume of serum regulated with phosphate 1 x). The blocking of the beads is carried out in the tubes previously blocked overnight at 4 ° C. Phage particles diluted in phosphate buffered saline to a final volume of 500 microliters for each panning condition are mixed with 500 microliters of ChemiBLOCKER / 0.1 percent Tween, and held for 1 hour at room temperature on a rotating wheel. The pre-adsorption of the phage particles to remove the Strep-Tactin or the phages bound with the beads is carried out twice: 160 microliters of blocked Strep-Tactin magnetic beads (4 milligrams) are added to the blocked phage particles. , and incubate for 30 minutes at room temperature on a rotating wheel. After separation of the beads by a magnetic device (Dynal MPC-E), the phage supernatant (approximately 1.1 milliliters) is transferred to a fresh blocked reaction tube, and the pre-absorption is repeated using 1 60 microliters. of pearls blocked for 30 minutes. Then, DKK1 labeled with His-Strep, either at 400 μM or 1 00 mM, is added to the blocked phage particles, in a fresh blocked 1.5 ml reaction tube, and the mixture is incubated for 60 minutes. minutes at room temperature on a rotating wheel. The phage-antigen complexes are cred using 320 microliters or 1 60 microliters of Strep-Tactin magnetic beads blocked aggregates to the phage panning groups at 400 nM or 1 00 nM, respectively, which is then incubated for 20 minutes at room temperature on a rotating wheel. The phage particles bound to the Strep-Tactin magnetic beads are collected again with the magnetic particle separator. The beads are then washed seven times with phosphate-buffered saline / 0.05 percent Tween (PBST), followed by washing three more times with phosphate-buffered serum only. Elution of the phage particles from the Strep-Tactin magnetic beads is carried out by adding 200 microliters of 20 mM DTT in 1 0 mM Tris-HCl, pH 8.0 to each tube for 10 minutes. The eluate is collected, and the beads are washed once with 200 microliters of phosphate-buffered serum, and the phosphate-regulated serum eluate is added to the DTT eluate. This eluted sample is used to infect 14 milliliters of a TG-1 culture of E. coli, which had been grown to OD60onm from 0.6 to 0.8. After infection and the next centrifugation for 1 0 minutes at 5,000 revolutions per minute, each bacterial granule is resuspended in 500 microliters of the 2xYT medium, applied to 2xYT-CG agar plates, and incubated overnight at 30 ° C. The next morning, the resulting colonies are scraped from the plates, and the phage is prepared by rescue and amplification as described above. The second round of panes in solution on DKK1 labeled with His-Strep is carried out according to the protocol of the first round, except that decreasing amounts of antigen (50 nM, and 10 nM) are used, and the astringency of the washing procedure is appropriately altered. Two different panning strategies are applied for the third round of selection: the amplified phage production of the second round of panning is divided and subjected to two different panning conditions. The first half of phage production is used for the standard panning strategy on DKK1 labeled with human His-Strep cred on Strep-Tactin beads, as described above (the amounts of antigen are 10 nM or 1 nM, respectively) . The second panning variation for the third round of selection is carried out on DKK1 labeled with human APP. Protein DKK1 labeled with APP at a final concentration of 50 nM or 1 0 nM, it is mixed with 1 milliliter of phage particles from the second round previously rinsed, and the mixture is incubated at room temperature for 1 hour on a rotating wheel. In parallel, 8 milligrams of Dynabeads M-280 Streptavidin previously blocked (Dynal) were incubated with 40 micrograms of biotinylated mouse anti-APP antibody for 30 minutes at room temperature on a rotating wheel, followed by two washing steps with PBST. Pre-formed complexes consisting of phage-antibodies bound to APP-labeled DKK1 are captured by M-280 streptavidin magnetic beads coated with anti-APP for 30 minutes at room temperature. ambient. The elution and amplification of phages are carried out as described in the above. Subcloning and expression of ubiquitous Fab sun fragments The inserts that encode Fab of the selected HuCAL GOLD® phagemids are subcloned into the expression vector pMORPH®X9_Fab_FH, in order to facilitate the rapid and efficient expression of soluble Fabs. For this purpose, the plasmid DNA of the selected clones is digested with the restriction enzyme endonucleases Xba \ and EcoRI, thereby separating the insert encoding Fab (ompA-VLCL and phoA-Fd). This insert is then cloned into the expression vector pMORPH®X9_Fab_FH digested by Xoal and EcoRI. Fab proteins are expressed from this vector, and as a result, they carry two C-terminal labels (FLAGMR and 6xHis, respectively), both for detection and for purification. Expression of HuCAL GOLD® Fab antibodies in E. coli To obtain sufficient amounts of protein encoded by each of the clones obtained above, individual bacterial colonies resistant to chloramphenicol are selected, after subcloning of the selected Fabs, into the vector of expression pMORPH®X9_Fab_FH. Each of these colonies is then used to inoculate the wells of a sterile 96-well microtiter plate, each well containing 100 microliters of the 2xYT-CG medium per well, and the bacteria are grown overnight at 37 ° C. One sample (5 microliters) of each culture of TG-1 from E. coli is transferred to a fresh 96-well sterile microtiter plate previously filled with 100 microliters of 2xYT medium supplemented with 34 micrograms / milliliter of chloramphenicol, and 0.1 percent glucose per well. The microtiter plates are incubated at 30 ° C with shaking at 400 revolutions per minute on a microplate shaker, until the cultures are slightly turbid (approximately 2 to 4 hours), with an OD6oon of approximately 0.5. For expression in the format of these plates, 20 microliters of the 2xYT medium supplemented with 34 micrograms / milliliter of 3 mM IPTG chloramphenicol (isopropyl-β-D-thiogalactopyranoside) per well (final concentration of 0.5 mM IPTG), the plates are added. Microtiter cells are sealed with a gas-permeable tape, and incubated overnight at 30 ° C, shaking at 400 revolutions per minute. Generation of whole cell lysates (BEL extracts) To each well of the expression plates, we add 40 microliters of BEL regulator (2xBBS / EDTA: 24.7 grams / liter of boric acid, 18.7 grams of NaCl / liter, 1.49 grams of EDTA / liter, pH 8.0), containing 2.5 milligrams / milliliter of lysozyme, and the plates are incubated for 1 hour at 22 ° C on a microtiter plate shaker (400 revolutions per minute). The BEL extracts are used for binding analysis by FMAT (see Example 2).

Expression of microgram quantities of Fab HuCAL GOLD® antibodies in E. coli and purification Expression of Fab fragments purified with pMORPH®X9_Fab_FH in E. coli TG1 F cells is carried out in plastic tubes of 50 milliliters. For this purpose, the pre-cultures inoculated with individual clones are grown in a 2xYT-CG medium overnight at 30 ° C. The next morning, 50 microliters of each pre-culture is used to inoculate 25 milliliters of the 2xYT medium supplemented with 34 micrograms / milliliter of chloramphenicol, 1 mM IPTG, and 0.1 percent glucose, in 50-milliliter sterile plastic tubes, and they are incubated overnight at 30 ° C. The cells of E. coli are harvested, the cellular granules are frozen, and finally they are altered with the Bug Buster (Novagen). The Fab fragments are isolated using Ni-NTA Agarose (Qiagen, Hilden, Germany). Expression of the milligram amounts of Fab HuCAL GOLD® antibodies in E. coli and purification Expression of Fab fragments purified by pMORPH®X9_Fab_FH in TG1 F cells is carried out in shake flask cultures using 750 milliliters of 2xYT medium supplemented with 34 micrograms / milliliter of chloramphenicol. The cultures are shaken at 30 ° C until the OD6oonm reaches 0.5. Expression is induced by the addition of 0.75 mM IPTG, followed by incubation for 20 hours at 30 ° C. The cells are disrupted using lysozyme, and the Fab fragments are isolated by chromatography with Ni-NTA (Qiagen, Hilden, Germany). Protein concentrations are determined by ultraviolet spectrophotometry (Krebs et al., 2001). Example 2: identification of H uCAL® antibodies specific for DKK1. The BEL extracts of the individual E. coli clones selected by the aforementioned panning strategies are analyzed by the Fluorometric Microvolume Assay Technology (FMATM R, 8200 Cellular Detection System analyzer, Applied Biosystems, Foster City, Calif.), to identify the clones that encode the specific Fabs of DKK1. The FMATM R 8100 HTS system is a high-throughput, macro-confocal, fluorescence screening instrument that automates the detection of non-radioactive assays of mixing and reading with living cells or beads (Miraglia, J. Biomol. Screening (1 999) ), 4 (4) 1 93-204). Link analysis based on fluorometric microvolume assay technology (FMAT), for the detection of Fabs that bind to DKK1. from bacterial lysates For the detection of Fab antibodies that bind to DKK1 from the lysates of E. coli (BEL extracts), the binding with the FMAT cell detection system 8200 (Applied Biosystems) is analyzed. To attach the His-Strep-labeled DKK1 onto the M-450 Epoxy beads (Dynal), a sample of 300 microliters of M-450 Epoxy beads (1 .2x1 08 beads) is transferred to a reaction tube, and captured with a magnetic particle separator. He The supernatant is removed, and the beads are washed four times in 1 milliliter of 1 00 mM sodium phosphate buffer, pH 7.4. For antigen coating, 60 micrograms of His-Strep-labeled DKK1 is added to the bead suspension in 1 50 microliters of 1 00 mM sodium phosphate buffer, pH 7.4. The antigen-bead suspension is incubated for 1 6 hours at room temperature on a rotating wheel. The coated beads are then washed three times with phosphate-buffered serum, and resuspended in a final volume of 250 microliters of phosphate-buffered serum. For each 384-well plate, a 20-milliliter mixture of phosphate-buffered serum containing 3 percent bovine serum albumin, 0.005 percent Tween 20, 4 microliters of DKK1-coated beads (1.9 x 106 beads) is prepared, and 4 microliters of Cy5M R detection antibody. A 45 microliter sample of this solution is dosed per well in a 384 well black / clear FMAT bottom plate (Applied Biosystems). The BEL extract containing the Fab (5 microliters) is added to each well. The FMAT plates are incubated at room temperature overnight. The next morning, the plates are analyzed in the cell detection system 8200 (Applied Biosystems). The positive clones are obtained, and the heavy and light chain sequences of the clones that produce specific positive signals in the FMAT are analyzed. It is observed that 57 unique anti-DKK1 clones (non-redundant) are identified, which show a link strong enough with human DKK1. These clones are expressed, purified, and tested to determine their affinity, and in functional assays. Determination of nanomolar affinities using surface plasmon resonance Using these clones, the kinetic analysis of SPR on a CM5 chip (Biacore, Sweden), which had been coated with a density of approximately 400 RU of either recombinant human DKK1, was carried out. , Mouse DKK1 (R &D System), or cynomolgus monkey DKK1, in 10 mM sodium acetate, pH 4.5, using the standard amine coupling chemistry of EDC-NHS. A comparable amount of human serum albumin (HSA) is immobilized on the reference flow cell. Phosphate-regulated serum (136 mM NaCl, 2.7 mM KCl, 10 mM Na2HP04, 1.76 mM KH2P04, pH 7.4) is used as the execution regulator. The Fab preparations are applied in series of concentrations of 16 to 500 nM, at a flow rate of 20 microliters / minute. The association phase is set at 60 seconds, and the dissociation phase at 120 seconds. A summary of the affinities in nM for each of the human, mouse, and cynomolgus monkey DKK1, determined by this method is shown in Table 1 herein.

Table 1 Affinities of the Fabs selected for each of human DKK1, mouse, and cynomolgus monkey * A single measurement. N.d. = Not determined. Example 3: Identification of human anti-DKK1 candidate Fabs that inhibit Wnt antagonist activity of DKK1.

The different resulting DKK1-specific antibodies selected from the HuCAL GOLD® library are used to obtain the purified antibody, which is then tested for its potency to inhibit the Wnt antagonist activity of human DKK1. Of these, 17 candidate antibodies are functionally active, as shown in Figures 2 and 4.

The functional activity of each of the HuCAL® Fabs is verified using a luciferase reporter gene assay. Twelve TCF / Lef binding sites upstream of the luciferase reporter gene are cloned, causing the luciferase gene to respond to TCF / Lef. The canonical Wnt proteins lead to a stabilization of beta-catenin, thus activating the transcription of TCF / Lef, and producing the luciferase protein. The addition of the DKK1 protein blocks the activity of Wnt, and consequently, also the transcription of the luciferase gene. Accordingly, the luciferase levels produced by the respective cells are expressed to correlate with the potency of the Fabs selected to block the action of DKK1. Reporter cell line HEK293T / 17-12xSTF that responds to TCF / stable Lef Bioassays are carried out using the stable human embryonic kidney cell reporter cell line HEK293T / 17-12xSTF. The cells are cultured in high glucose DMEM medium (Invitrogen), containing 10 percent fetal calf serum (PAN or BioWhittaker), and 1 microgram / milliliter of puromycin (BD Biosciences), until a confluence of 90 is reached. percent. The cells are then trypsinized, counted, and diluted in the culture medium without puromycin, to a concentration of 4 × 10 5 cells per milliliter. Subsequently, the cells are seeded on a 96-well flat-bottom white plate (Corning, 100 microliters of cell suspension per well), and incubated at 37 ° C and with C02 at 5 percent overnight. The next day, the test medium is prepared: 500 nanograms / milliliter of DKK1 / APP are added to the medium conditioned with Wnt3a (CM). The HuCAL® anti-DKK1 Fabs (final concentration of 20 micrograms / milliliter), and the goat anti-human DKK1 antibody (R & D Systems) used as a positive control (final concentration of 1.5 micrograms / milliliter), are diluted in the conditioned medium with Wnt3a. A volume of 60 microliters of medium is removed from each well of the assay plate without altering the adherent cells, and replaced by 60 microliters of the test or control antibody, diluted in the conditioned medium in Wnt3a. The cells are incubated for a further 24 hours, and 100 microliters of Bright-Glo luciferase reagent is added to each well. After a 5 minute incubation time, the luminescence is read in a luminometer (GenioPro, Tecan). The results obtained with 20 of the 57 Fabs are shown in Figure 2 in the present. The extent of expressed luciferase is a measure of the extent of antibody present. Example 4: Quantitative analysis of binding affinities: Determination of human anti-DKK1 candidate Fabs that inhibit Wnt antagonist activity of DKK1. Affinity Determination In order to further characterize the anti-DKK1 antibodies, the affinity is determined with human DKK1, cynomolgus monkey, and mouse. The recombinant DKK1 protein is immobilized on a CM5 Biacore chip, and the Fabs are applied in the mobile phase in different concentrations. For a reliable determination of monovalent affinities, only Fab batches are used for Biacore measurements that show a monomer fraction > .90 percent in a chromatography by size exclusion. The summarized affinity data on human DKK1, of mouse, and of cynomolgus monkey, are shown in Table 2. It is found that the 17 tested Fabs have affinity with human DKK1 below 100 nM. In addition, nine of the clones produced antibodies with affinities less than 10 nM. In all the tested cases, the affinities for DKK1 of cynomolgus and mouse monkey are almost identical to those for human DKK1. Table 2 Affinity data of the selected Fabs on human DKK1, mouse, and cynomolgus monkey N. d. = Not determined. Determination of the ECsn The data showing the effective concentration for 50 percent inhibition for the clones of the antibodies that have the highest affinity for DKK1, are shown in Table 3 of this. The data show that the EC50 of the effective concentrations is in the range of 39 to 95 nM, with an average value between 58 and 83 nM. Table 3 Effective concentration for 50 percent inhibition of the selected Fabs Example 5: Affinity maturation of the selected anti-DKK1 Fabs by parallel exchange of the LCDR3 and HCDR2 cassettes. To optimize the affinities of the antibodies described herein for DKK1 for a group of Fab progenitor fragments, the LCDR3, structure 4, and the constant region of the light chains (405 base pairs) of each parent Fab, using Bp / I and Sph \, and are replaced by a repertoire of diversified LCDR3s, along with structure 4 and the constant domain. A 0.5 microgram sample of the linker vector is ligated with a three-fold molar excess of the insert fragment carrying the diversified LCDR3s. In a similar approach, the HCDR2 is diversified using the X GÍOI and BssH I l sites, and the connection structure regions remain constant. In order to increase the efficiency of the cloning, the progenitor HCDR2 is replaced by a embedded sequence of 590 base pairs before insertion of the diversified HCD R2 cassette. Linkage mixtures from 11 different libraries are electroporated into 4 milliliters of TOP10 F 'cells from E. coli (Invitrogen, Carlsbad, CA, USA), yielding from 2 × 10 7 to 2 × 1 08 independent colonies. The amplification of the libraries is carried out as described above (Rauchenberger et al., 2003 J. Biol. Chem. 278 (40): 381 94-38205). For quality control, several clones are randomly collected per library, and sequenced (SequiServe, Vaterstetten, Germany), using the primers CFR84 (VL) and OCAL_Seq_Hp (VH). Selection of candidates for affinity maturation Six selected maturing candidates ("Fabs progenitors") are selected, having been characterized as having the following properties: affinities with human DKK1 less than nM, with significant cross-reactivity with cynomolgus monkey and mouse DKK1, an EC50 less than 100 nM, and good to moderate Fab expression levels in E. coli, and lack accumulation after Fab purification . During the course of affinity measurements, it became apparent that MOR04480 is highly unstable at high dilutions. For this reason, MOR04480 is omitted from the list of maturation candidates, despite having the highest affinity (1 nM) and the best EC50 (7 nM) of all the Fabs tested. MOR04483 had a high affinity of 5.5 nM for human DKK1, but it is shown to cross-react with mouse DKK1, and MOR04453 contained a high proportion of Fab aggregates after purification. Therefore, these two antibodies are also excluded from maturation. After a careful evaluation of all available data, six maturation candidates are selected (MOR04454, MOR04455, MOR04456, MOR04461, MOR04470, and MOR04516). The properties of these candidates are listed in Table 4 herein.

Table 4 Properties of the selected Fabs * A single measurement. N. d. = Not determined. # Monomeric portion > 90 percent. Generation of Fab libraries selected for affinity maturation In order to obtain clones having a higher affinity and inhibitory activity of the anti-DKK1 antibodies, the selected Fab clones MOR04454, MOR04455, MOR04456, MOR04461, MOR04470, and MOR4516 shown in the previous example, they undergo additional rounds of diversification and selection, a process known as maturity by affinity. For this purpose, the complementarity determining regions are diversified using the corresponding LCDR3 and HCDR2 maturation cassettes previously constructed by trinucleotide mutagenesis (Virnekás et al., 1 994 Nucleic Acids Res. 22: 5600-5607; Nagy et al., 2002 Nature Medicine 8: 801-807). Table 5 of the present shows the LCDR3 sequences for the progenitor clones MOR04454, MOR04455, MOR04456, MOR061, MOR04470 and MOR451 6. Table 5 LCDR3 sequences for the selected Fabs Table 6 of the present shows the sequences of HCDR2 for the parent clones MOR04454, MOR04455, MOR04456, MOR061, MOR04470 and MOR4516. Table 6 HCDR2 sequences for the selected Fabs The Fab fragments of the expression vector pMORPH®X9_Fab_FH are subcloned into the phagemid vector pMORPH®25 (see U.S. Pat. No. 6,753, 136). This vector provides the plll phage protein N-terminally fused to a cysteine residue, as well as a C-terminal cysteine for the Fd antibody chain, and therefore, permits disulfide-linked display of the respective Fab fragments on the phage surface. Two different strategies are applied in parallel to optimize both the affinity and effectiveness of the parent Fabs.

Five libraries of Fab phage antibodies are generated, where the LCDR3 of five of the six parent clones is replaced by a repertoire of individual light chain CDR3 sequences. Maturation with LCDR3 of MOR04454 is not carried out, because this clone has an additional Bp / 'l restriction site in one of the complementarity determining regions, and the Bp /' l restriction enzyme is used for the procedure of cloning the library. In parallel, the HCDR2 region of each parent clone is replaced by a repertoire of individual heavy chain CDR2 sequences. Each parent Fab is separated and replaced by a sausage of 590 base pairs. This DNA sausage facilitates the separation of individually digested and double digested vector bands, and reduces the background of high affinity progenitor Fabs during maturation panning. In a subsequent step, the sausage is separated from the plasmids encoding the Fab of each parent clone, and is replaced by the highly diversified HCDR2 maturing cassette. Large high affinity maturation libraries of more than 2x107 members are generated by conventional cloning procedures, and the diversified clones are transformed into electro-competent E. coli TOP10F 'cells (Invitrogen). Fab-presenting phages are prepared as described in Example 1 above.

Four maturing groups are constructed in order to facilitate the following selection process: group 1 a consisted of the LCDR3 libraries MOR04470 and MOR04516; group 1 b consisted of the libraries of HCDR2 MOR04470 and MOR0451 6; group 2a consisted of the LCDR3 libraries MOR04454, MOR04455, MOR04456, and MOR04461; and group 2b consisted of the libraries of HCDR2 MOR04454, MOR04455, MOR04456, and MOR04461. For each group, the panning is carried out in solution, using decreasing amounts of DKK1 labeled with His-Strep and phage-antigen captured by Strep-Tactin beads. In parallel, each group is applied in the panes using decreasing amounts of biotinylated DKK1, which is captured on plates coated with neutravidin. In order to increase the astringency of the panning, and to select better deactivation rates, competition is carried out with the purified parent Fabs, as well as with the unlabeled antigen, during the prolonged incubation periods. Immediately after the panning, the enriched phagemid groups are subcloned into the expression vector pMORPH®X9_FH. Approximately 2,300 individual clones are collected, and the Fabs are induced with I PTG. Panning strategies of maturation The panning procedures using the four antibody groups are carried out with the DKK1 labeled with His-Strep, and with DKK1 labeled with His-Strep biotinylated in solution for two or three rounds, respectively. For each wave of panning strategies, competition is used with purified progenitor Fab proteins or with DKK1 labeled with unlabeled APP, as well as low concentrations of antigen and extensive washing, to increase the astringency. The panning in solution on the DKK1 labeled with unlabeled His-Strep is carried out in two rounds of selection, mainly in accordance with the conventional protocol described in Example 1. Exceptions to these procedures are the application of reduced amounts of antigen (decreasing from 5 nM down to 1 nM), the high stringency of the washing procedure, either with or without competitor, and prolonged incubation periods of phage-antibodies together with the antigen. For the first round of selection using the biotinylated DKK1, the wells of a Neutravidin plate are washed twice with 300 microliters of phosphate-buffered serum. The wells are blocked with ChemiBLOCKER (Chemicon, Temecula, CA) diluted 1: 1 in phosphate-regulated serum (Blocking Regulator). Prior to selections, HuCAL GOLD® phages are also blocked with a volume of Blocking Regulator containing 0.1 percent Tween 20 for 30 minutes at room temperature. The blocked phage preparations are transferred in 1 00 microlitre to the wells of a plate coated with Neutravidin for 30 minutes at room temperature. This step of pre absorption is repeated once. The blocked and previously cleared phage preparations are incubated with 5 nM biotinylated DKK1 for 2 hours at 22 ° C on a rotating wheel. The parent Fab, APP-DKK1, or no competitor is added, and the samples are incubated overnight at 4 ° C on a rotating wheel. The antigen-phage complexes are captured in the wells of a neutravidin plate for 30 minutes at room temperature. After extensive washing steps, the bound phage particles are eluted by the addition of 200 microliters of 20 mM DTT in 10 mM Tris, pH 8.0, per well, for 10 minutes at room temperature. The eluate is removed and added to 1 4 milliliters of E. coli TG 1 cells grown to an OD60on of 0.6 to 0.8. The wells are rinsed once with 200 microliters of phosphate buffered serum, and this solution is also added to the TG 1 cells of E. coli. Infection with E. coli phage is allowed for 45 minutes at 37 ° C without agitation. After centrifugation for 10 minutes at 5,000 revolutions per minute, the bacterial granules are resuspended each in 500 microliters of the 2xYT medium, applied to the agar plates with 2xYT-CG, and incubated overnight at 30 minutes. ° C. The colonies are harvested by scraping from the surface of the plates, and the phage particles are rescued and amplified as described above.

The second and third rounds of the selection are carried out as described above for the first round of selection, except that the washing conditions are more astringent, and the Antigen concentrations are 1 and 0.1 nM, respectively. Bond analysis based on electroauimiluminescence (BioVeris) of Fabs that bind to DKK1 For the detection of affinity-enhanced DKK1-specific antibody fragments in E. coli lysates (BEL extracts), a BioVeris workstation is used M-384 SERIES® (BioVeris Europe, Witney, Oxfordshire, United Kingdom). The assay is carried out in 96 well polypropylene microtiter plates, and phosphate buffered serum supplemented with 0.5 percent bovine serum albumin and 0.02 percent Tween 20, as the assay regulator. Biotinylated human DKK1 is immobilized on paramagnetic beads of streptavidin M-280 (Dynal) according to the supplier's instructions. A 1:25 dilution of the pearl supply solution is added per well. 100 microliter samples of diluted BEL extract and beads are incubated overnight at room temperature on a shaker. For detection, the anti-human (Fab) '2 (Dianova) labeled with BV-tag ™ is used according to the supplier's instructions (BioVeris Europe, Witney, Oxfordshire, United Kingdom). A set of approximately 2,300 randomly picked clones is analyzed by the method described above. A subset of 160 clones is selected that give the highest values for the additional analysis in the equilibrium titration in solution.

Determination of picomolar affinities using the Equilibrium Titration in Solution (SET) For the determination of the KD, fractions of monomers (at least 90 percent monomer content, analyzed by analytical SEC, Superdex75, Amersham Pharmacia) are used. Fab. The determination of the affinity based on electrochemiluminescence (ECL) in solution, and the evaluation of the data, are carried out basically as described by Haenel et al., 2005. A constant amount of Fab is equilibrated with different concentrations (3n dilutions in series) of human DKK1 (initial concentration of 4 nM) in solution. The biotinylated human DKK1 coupled with the paramagnetic beads (M-280 Streptavidin, Dynal), and the anti-human (Fab) '2 labeled with BV-tag ™ (BioVeris Europe, Witney, Oxfordshire, United Kingdom) (Dianova), is added. and the mixture is incubated for 30 minutes. Subsequently, the unbound Fab concentration is quantified by ECL detection using the M-SERIES® 384 analyzer (BioVeris Europe). For this purpose, 160 individual clones are selected, and purified by Ni-NTA Agarose on the microgram scale. Preliminary affinities are determined by titration in equilibrium in four point solution (SET) in BioVeris. From these data, 20 clones are selected that show affinities. These Fabs are purified on the milligram scale. MOR04950 is excluded from affinity determination, and its evaluation additional, due to the partial accumulation of Fab that is detected in the chromatography by size exclusion. The final affinities are determined from two independent batches of each Fab clone using a SET measurement of 8 points, and human, mouse, and cynomolgus monkey DKK1. The affinity determination for mouse DKK1 and cynomolgus monkey is essentially done as described above, using mouse DKK1 (R &D Systems) and cynomolgus monkey DKK1 as the analyte in solution, instead of human DKK1 . For the detection of free Fab, biotinylated human DKK1 coupled with paramagnetic beads is used. The affinities are calculated according to Haenel et al., 2005 Anal. Biochem. 339.1: 182-184. Using the assay conditions described above, the affinities for affinity-optimized anti-DKK1 Fabs are determined in solution. The affinities are determined for MOR04910 and MOR04946, with KDs below 30 pM for human DKK1, and between 36 and 42 pM for mouse DKK1 and cynomolgus monkey. Seven other antibodies showed affinities below 100 pM for all three antigens. The clone MOR04950 showed no linkage with the biotinylated DKK1. The affinities are summarized in Table 7 of this.

Table 7 Fab Affinities *: At least two independent measurements are carried out from two different batches of Fab. #: A single measurement. Example 6: Characterization of human anti-DKK1 Fabs opti fi ed by affinity. Techniques of essay inm unasorbente linked with enzymes (ELISA) The binding specificity of matured Fabs is determined in the presence of 50 percent human serum (HS). The Serial dilutions of the recombinant human biotinylated DKK1 in TBS are coated onto the neutravidin microtiter plates for 2 hours at room temperature, from 8 nanograms of DKK1 per well to a concentration of 125 nanograms of DKK1 per well. After antigen coating, the wells are blocked with PBS / 0.05 percent Tween (TBS-T) supplemented with 1 percent bovine serum albumin for 1 hour at room temperature. The purified Fabs described above are diluted either in TBS / 4 percent bovine serum albumin, or in TBS / 50 percent human serum, in a final concentration of 1 microgram / milliliter, added to the coated wells and blocked, and the plates are incubated for 1 hour at room temperature. For detection, an antibody conjugated with alkaline phosphatase (AP) anti-FLAG (dilution of 1: 5000 in TBST), and the fluorogenic substrate AttoPhos (Roche) are used. After each incubation, the wells of the microtiter plates are washed with TBST five times, except after the final incubation step with the labeled secondary antibody, when the wells are washed three times. Fluorescence is measured in a TECAN plate reader Spectrafluor. The example link curves are shown in Figure 3 hereof. Table 8 summarizes the binding activity of anti-DKK1 Fabs optimized in the presence of 50 percent human serum, compared to binding activity in 4 percent bovine serum albumin, which is in the range of 83%. one hundred to one hundred percent. It is found that the mean value is 93 percent, and therefore, it is found that the anti-DKK1 Fabs are completely bound to the target in the presence of human serum. Table 8 Fabs link activity * A single measurement. Assay of luciferase reporter cells in the presence of human serum, using the U2QS cell line For further determination of the binding specificity of the optimized anti-DKK1 Fabs, the luciferase reporter cell assay is repeated in the presence of human serum at 15 percent, using the U20S osteosarcoma cell line. U20S cells (ATCC No. HTB-96) are cultured according to the supplier's protocol (ATCC, Manassas, VA, USA). The cells are trypsinized, counted, and diluted in the culture medium (McCoy's 5a / 10% FCS), to a concentration of 2x105 cells / milliliter. For each 2x104 cells, a solution is prepared which is a mixture of 0.075 micrograms of pTA-LUC-12xSuperTopFlash, and 0.004 micrograms of phRL-SV40. These are mixed in a final volume of 9.8 microliters of OPTI-MEM. Then 0.2 microliters of Fugene 6 Transfection Reagent (Roche, Mannheim, Germany) is added. This transfection mixture is incubated briefly, and then mixed with the previously prepared cells. Subsequently, the cells are seeded in 100 microliters per well of a 96-well flat-bottomed white cell culture plate, and incubated at 37 ° C and 5% C02 overnight. The next day, 75 microliters of medium is removed from each well of the assay plate, and replaced by 10 microliters of FaC HuCAL® antibody dilutions from (10 to 0.01 micrograms / milliliter diluted in the culture medium without serum ), and 15 microliters of either 70% FCS or human serum, and 50 microliters of the Wnt3a conditioned medium, containing 600 nanograms / milliliter of DKK1-ATP are added to each well. For the negative control, the serum-free medium is added in place of the antibody dilutions. In order to obtain a maximal luciferase signal, controls containing 10 microliters of serum-free medium are added in place of antibody dilutions, and 50 microliters of Wnt3a-conditioned medium without DKK1-APP. After a 24-hour incubation at 37 ° C, with 5 percent CO 2, the luminescence is measured with the Dual-Glo Luciferase Assay System (Promega, Madison, Wl, USA) according to the manufacturer's instructions. Table 9 shows the inhibitory activity of anti-DKK1 Fabs optimized in the presence of 15 percent human serum (compared to 15 percent inhibitory activity in FCS). The data show that the activity obtained in the presence of serum is in the range of 26 percent to 90 percent, with a twenty-one average value of 70 to 74 percent. These data show that clones of anti-DKK1 Fabs are obtained that function in the presence of human serum. Table 9 Inhibitor activity of the Fabs Determination of the EC ^ n of affinity-optimized anti-DKK1 Fabs by assay of luciferase reporter cells The affinity-enhanced Fabs test in the standard Wnt3a-dependent TCF assay / luciferase reporter LEF, used 10 nM DKK1 to obtain the inhibition of luciferase expression. It is seen that EC50 values could not be generated by this method, because the sensitivity of the assay is too low. This is indicated by very pronounced inhibition curves and similar EC50 values for all tested Fabs, as seen in Figure 4 hereof. An improved version of the TCF / luciferase reporter LEF is developed. DKK1 binds to the Kremen-1 and -2 transmembrane proteins, and this interaction leads to strong synergistic inhibition of Wnt signaling (Mao et al., 2002 Nature 417: 664-67). Accordingly, the Kremen cDNA is co-transfected with the TCF / luciferase reporter LEF assay. The resulting Wnt3a dependent reporter assay showed a highly improved sensitivity to DKK1, mediated by the co-expression of the Kremen co-receptor protein. In In this assay, DKK1 0.33 nM is sufficient to induce a complete inhibition of Wnt signaling. The titrations of Fabs (in 10 concentrations) are repeated using DKK1 0.33 nM, and produce sigmoidal inhibition curves (Figure 5 of this), from which the EC50 values can be calculated. In this way the anti-DKK1 Fabs optimized by affinity are analyzed with respect to the EC50, as described above. As shown in Table 10 hereof, the EC50 values obtained by this method were in the range of 0.2 nM to 5.6 nM. Table 10 EC50 of the Fabs Sequence analysis of affinity-optimized Fabs The nucleotide sequences of the heavy and V regions (VH and VL) of the twenty Fabs are determined. The amino acid sequences of the complementarity determining regions (CDRs) are listed in Table 11 A and Table 11B herein.

Table 1 1 A Amino acid sequences of the heavy chain complementarity determining regions Table 11B Amino acid sequences of the light chain complementarity determining regions Sequence analysis showed that five of the six parent Fabs (P) produced improved affinity successors. MOR04461 and MOR04470 could be optimized in HCDR2, as well as in LCDR3. Optimized successors of MOR04454 are not obtained. In addition, a high homology between different progenitor antibodies appears, as shown in the sequences in consensus 1 and in consensus 2 for the different regions determining complementarity in Table 1 1 B. Similar consensus sequences can be provided for the progenitor sequences shown in Table 10A employing methods well known to one skilled in the art. In addition, it is determined that MOR04920 has a mutation in the HCDR2 region (a Ser residue up to a Gly at position 73 according to the numbering scheme published by Honegger and Pluckthum, 2001 J. Mol. Biol. 309.3: 657-670 ) deviating from the HuCAL® design in this way. It is shown that MOR04913 has a point mutation in structure 4 of the kappa light chain (exchange of Lys to Asn in position 148). Because this position is not expected to have an effect on the binding properties of the antibody, the mutation is reverted back to the germline / HuCAL® composition during the conversion of the IgG, producing the antibody MOR051 45. MOR04947 it has a potential glycosylation site in LCDR2. This site is not removed, because MOR04947 is select only as one of the backup candidates. Example 7: Production of HuCAL® immunoglobulins. Conversion to the IqG format In order to express the full-length immunoglobulin (Ig), variable domain fragments of the heavy (VH) and light (VL) chains are subcloned from the Fab expression vectors pMORPH®X9_FH , either in the vector series pMORPH®_h_lg or pMORPH®2_h_lg for human IgG1 and human IgG4. Alternative vectors can be used for human IgG2. Restriction enzymes EcoRI, M / el, and B / pl are used for subcloning the VH domain fragment into pMORPH®_h_lgG1 and pMORPH®_h_lgG4. Restriction enzymes Mfe \ and B / pl are used for subcloning the VH domain fragment into pMORPH®2_h_lgG1f and pMORPH®2_h_lgG4. Is the subcloning of the V domain fragment carried out in pMORPH®_h_lg? and pMORPH®2_h_lg ?, using the EcoRV and BsWI sites, while subcloning in pMORPH®_h_lg? and pMORPH®2_h_lg? 2 using EcoRV and Hpal. Transient Expression and Purification of Human IqG HEK293 cells are transfected with an equimolar amount of IgG heavy and light chain expression vectors. On days 4 or 5 after transfection, the supernatant of the cell culture is harvested. After adjusting the pH of the supernatant to 8.0, and sterile filtration, the solution is subjected to standard protein A column chromatography (Poros 20A, Biosystems). Conversion of the parent Fabs to the IqG formats Parallel to the start of affinity maturation, MOR04454, MOR04456, and MOR04470 are cloned into the expression vectors pMORPH®_h_lgG 1 and pMORPH®_h_lgG4. Alternative constructs can be used for the creation of IgG2 expression vectors. Small scale expression is carried out by transient transfection of the HEK293 cells, and the full length immunoglobulins are purified from the supernatant of the cell culture. The data shows, by size exclusion chromatography, that the antibodies are in a monomeric form. The Wnt3a dependent reporter assay test proved that the proteins are functional. Example 8: Amino acid sequences and nucleotide sequences of the genes optimized for expression. In order to increase mammalian expression, changes are introduced in the heavy and light chains of the Fabs of the present, to optimize the use of codons for their expression in a cell. It is known that several negatively cis action motifs decrease expression in mammals. The optimization process of the present one removes the negative action sites-c / 's (such as the splice sites or the poly (A) signals, which have a negative influence on the expression.) The optimization process of the present further enriches the content of GC, to prolong the half-life of the mRNA. The variable light and heavy chain regions are optimized using a Fab clone, MOR04945 (the full-lenlight chain progenitor nucleotide sequence is SEQ ID NO: 98, and the full-lenheavy chain progenitor nucleotide sequence. is SEQ ID NO: 102), isolated herein by selection with phage display. Then, each of the nucleotide sequences encoding each of the entire light and heavy chains of this and other clones is optimized using these methods. Optimization process for the VH and VI chains of MOR04945 To optimize the nucleotide sequence and the amino acid sequence of each of the VL and VH chains for expression in mammalian cells, the codon usage is adapted to the inclination of the codons of mammalian genes. In addition, where possible, regions of very high (> 80 percent) or very low (< 30 percent) GC content are reduced or eliminated. In an alternative way, optimization for expression in bacteria, yeast, or baculovirus would involve adapting the use of inclined codons for their respective genes. During the optimization process for mammalian expression, the following cis-action sequence motifs are eliminated: the internal TATA frames, the chi-sites, and the ribosomal entry sites, the stretches of AT rich or rich sequences. in GC, the sequence elements of the instability motive of RNA (ARE), RNA sequence inhibitor elements (INS), sequence elements that respond to cAMP (CRS), repeat sequences and secondary RNA structures, donor and acceptor sites of splices, including cryptic sites, and branch points. Except as indicated, the introduction of the Mlu l and Hindl l l sites is avoided in the process of optimizing the nucleotide sequence of the VL chain. Except as indicated, the introduction of the Mlyl and BstEI I sites is avoided in the process of optimizing the nucleotide sequence of the VH chain. Amino acid sequences of the VH VV chains of MOR04945 optimized for expression The use of codons is adapted to that of mammals to enable higher and more stable expression rates in a mammalian cell, for the resulting optimized amino acid sequences for the VH and V chains of clone MOR04945 described above. See Example 5. Table 12A below shows the sense and anti-sense nucleotide (AS) sequences of the variable light chain (SEQ ID NO: 120), and the resulting variable light chain amino acid sequence (designated AA ) (SEQ ID NO: 1 18), as it is optimized for the expression.

Table 12A Sense and anti-sense nucleotide sequences, and amino acid sequences of the VH chain optimized for expression 5 Mlul EcoRV BstNI ACGCGTTGCGATATCGCCCTGACCCAGCCCGCCAGCGTGTCCGGCAGCCCTGGCCAGAGC (meaning) TGCGCAACGCTATAGCGGGACTGGGTCGGGCGGTCGCACAGGCCGTCGGGACCGGTCTCG (AS) - | or T_R_C_D_L_A_L_T_Q_P_A_S_V_S_G_S_P_G_Q_S_ (AA) Pvull BstNI BstNI ATCACCATCAGCTGTACCGGCACCAGCAGCGACCTGGGCGGCTACAACTACGTGTCCTGG (meaning) TAGTGGTAGTCGACATGGCCGTGGTCGTCGCTGGACCCGCCGATGTTGATGCACAGGACC (AS) l_T_l_S_C__T_G_T _S_S_D_L_G_G_Y_N_Y_V_S_W_ (AA) 15 TATCAGCAGCACCCCGGCAAGGCCCCCAAGCTGATGATCTACGACGTGAACAACAGACCT (in sense) 121 + + + + + + ATAGTCGTCGTGGGGCCGTTCCGGGGGTTCGACTACTAGATGCTGCACTTGTTGTCTGGA (AS) Y_Q_a_H_P_G_K_A_P_K_L_M_l_Y_D_V_N_N_R_P_ (AA) Hmfl AGCGGCGTGTCCAACAGATTCAGCGGCAGCAAGAGCGGCAACACCGCCAGCCTGACCATC (meaning) 181 + + + + + + TCGCCGCACAGGTTGTCTAAGTCGCCGTCGTTCTCGCCGTTGTGGCGGTCGGACTGGTAG (AS) S_G_V_S_N_R_F_S_G_S_K_S_G_N_T_A_S_L__T_I_ (AA) 25 Pstl TCTGGCCTGCAGGCTGAGGACGAGGCCGACTACTACTGCCAGACCTACGACCAGATCAAG (in sense) 241 + + + + + + AGACCGGACGTCCGACTCCTGCTCCGGCTGATGATGACGGTCTGGATGCTGGTCTAGTTC (AS) S_G_L_Q_A_E_D_E_A_D_Y_Y_C_a_T_Y_D_Q_l_K_ (AA) Hindlll CTGTCCGCCGTGTTTGGCGGCGGAACAAAGCTT (sense) (SEQ ID NO: 120) 301 + + + - - - GACAGGCGGCACAAACCGCCGCCTTGTTTCGAA (AS) L_S_A_V_F_G_G_G_T_K_L_ (AA) (SEQ ID N0: 118) Table 12B shows the sense and anti-sense variable heavy chain nucleotide sequences (SEQ ID NO: 121), and the resulting variable heavy chain amino acid sequence (designated as AA) (SEQ ID NO: 119), as it is optimized for expression. Table 12B Sense and anti-sense nucleotide sequences and amino acid sequences of the VH chain optimized for expression Mlyl Hinfl BstNI Pvull GAGTCCATTGGGAGTGCAGGCCCAGGTGCAGCTGGTGGAGAGCGGCGGAGGACTGGTGCA (meaning) CTCAGGTAACCCTCACGTCCGGGTCCACGTCGACCACCTCTCGCCGCCTCCTGACCACGT (AS) G_V_Q_A_Q_V_Q_L_V_E_S_G_G_G_L_V_Q_ (AA) BstNI GCCTGGCGGCAGCCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAGCTACTG (in sense) + + + + + + CGGACCGCCGTCGGACTCTGACTCGACACGGCGGTCGCCGAAGTGGAAGTCGTCGATGAC (AS) _P_G_G_S_L_R_L_S_C_A_A_S_G_F_T_F_S_S_Y_W_ (AA) BstNI BstNI Bell GATGAGCTGGGTGAGGCAGGCCCCTGGCAAGGGCCTGGAGTGGGTGTCCGTGATCAGCAG (in sense) + + + + + + CTACTCGACCCACTCCGTCCGGGGACCGTTCCCGGACCTCACCCACAGGCACTAGTCGTC (AS) _M_S_W_V_R_Q_A_P_G_K_G_L_E_W_V_S_V_I_S_S_ (AA) CGATAGCAGCAGCACCTACTACGCCGATAGCGTGAAGGGCCGGTTCACCATCAGCCGGGA (meaning) GCTATCGTCGTCGTGGATGATGCGGCTATCGCACTTCCCGGCCAAGTGGTAGTCGGCCCT (AS) _D_S_S_S_T_Y_Y_A_D_S_V_K_G_R_F__T__I_S_R_D_ (AA) Pstl BspMI CAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGT (in sense) + + + + + + GTTGTCGTTCTTGTGGGACATGGACGTCTACTTGTCGGACTCTCGGCTCCTGTGGCGGCA (AS) _N_S_K_N_T__L_Y__L_Q_M_N_S_L_R_A_E_D_T_A_V_ (AA) BstNI BstNI BstNI GTACTACTGTGCCAGGCACGGCATCGACTTCGACCACTGGGGCCAGGGCACCCTGGTCAC BstEII (in sense) + + + + + + CATGATGACACGGTCCGTGCCGTAGCTGAAGCTGGTGACCCCGGTCCCGTGGGACCAGTG (AS) _Y_Y__C_A_R_H_G_I_D_F_D_H_W_G_Q_G_T_L_V_T_ C (in sense) (SEQ ID NO: 121) 361 G (AS) _ (AA) (SEQ ID NO: 119) The pre- and post-optimization diagrams can provide the percentages of codons of sequences for each of the progenitor sequences and optimized genes, respectively, and analyze the quality class of the respective nucleotide sequences encoding the VH and VL chains. The quality value, as used herein, means that the most frequent use of codons for a given amino acid in the desired expression system is set to 100, and the remaining codons are scaled according to the frequency of use. (Sharp, P. M., Li, W. H., Nucleic Acids Res. 1 5 (3), 1987). In addition, the codon adaptation index (CAI) is a number that describes how well the codons of the nucleotide sequence are paired with the preference of codon usage of the target organism. The maximum value of CAI is set at 1.0, and therefore, a CAI of > 0.9 makes high expression possible. The CAI for the VL chain before the optimization is found to be 0.73, and after the optimization, the CAI is determined to be 0.95. In a similar way, the CAI for the VH chain before the optimization is found to be 0.74, and after the optimization, it is determined that it is 0.98 in the optimized constructs, and the GC content in the VL chain is increased from 51 percent for the progenitor sequence of MOR04945 to 62 percent for the optimized sequence derived from MOR04945. As shown in Figures 9A and 9B, the GC content in the VH chain is increased from 54 percent for the progenitor sequence of MOR04945 to 64 percent for the optimized derivative of MOR04945. Optimization for the expression of light chains and the full-length heavy chains of MOR04910. MOR04945, MOR04946. V MOR05145 The optimization process is applied to each of the progenitor full length nucleotide sequences of the light chains of MOR04910 (SEQ ID NO: 97), MOR04945 (SEQ ID NO: 98), MOR04946 (SEQ ID NO: 99) ), and MOR05145 (SEQ ID NO: 100), and the progenitor full length nucleotide sequences of the heavy chains of MOR04910 (SEQ ID NO: 101), MOR04945 (SEQ ID NO: 102), MOR04946 (SEQ ID NO: 103), and MOR05145 (SEQ ID NO: 103). The optimization process is used to construct each of the following light chain nucleotide sequences associated with the numbers of progenitor clones: for clone MOR04910, the optimized nucleotide sequence is SEQ ID NO: 104; for clone MOR04945, the optimized nucleotide sequence is SEQ ID NO: 105; for clone MOR04946, the optimized nucleotide sequence is SEQ ID NO: 106; and for him clone MOR05145, the optimized nucleotide sequence is SEQ ID NO: 107. In addition, the optimization process is used to construct each of the following heavy chain nucleotide sequences associated with the numbers of progenitor clones: for the MOR04910 clone, the optimized nucleotide sequence is SEQ ID NO: 108; for clone MOR04945, the optimized nucleotide sequence is SEQ ID NO: 109; for clone MOR04946, the optimized nucleotide sequence is SEQ ID NO: 110; and for clone MOR05145, the optimized nucleotide sequence is SEQ ID NO: 110. The optimized light chain nucleotide sequences are associated with the following optimized light chain amino acid sequences: for clone MOR04910, the optimized amino acid sequence is SEQ ID NO: 111; for clone MOR04945, the optimized amino acid sequence is SEQ ID NO: 112; for clone MOR04946, the optimized amino acid sequence is SEQ ID NO: 113; and for clone MOR05145, the optimized amino acid sequence is SEQ ID NO: 114. The optimized heavy chain nucleotide sequences are associated with the following optimized heavy chain amino acid sequences: for clone MOR04910, the optimized amino acid sequence is SEQ ID NO: 115; for clone MOR04945, the optimized amino acid sequence is SEQ ID NO: 116; for clone MOR04946, the optimized amino acid sequence is SEQ ID NO: 117; and for clone MOR05145, the optimized amino acid sequence is SEQ ID NO: 1 1 7. In Table 1 3A and in Table 1 3B, a ligation of the nucleotide and polypeptide sequences of the full length light and heavy l chain sequences is provided. contemplated. Table 1 3A provides the optimized nucleotide sequences and the polypeptides encoded by the same. These n-nucleotide sequences are optimized to remove the latent splice sites that are recognized in mammalian expression systems. Table 1 3B provides the sequences of n progenitor nucleotides for the clones listed in Table 1 3A.

Tabl a 1 3A Light chain (LC) and heavy chain (HC) sequences Opti m ized LC (opt) 4910 nucleotides SEQ ID NO: 97 GATATCGCACTGACCCAGCCAGCTTCAGTGAGCGGCTCACCAGGTCAGAGC ATTACCATCTCGTGTACGGGTACTAGCAGCGATGTTGGTGGTTTTAATTATGT GTCTTGGTACCAGCAGCATCCCGGGAAGGCGCCGAAACTTATGATTCATGAT GGTTCTAATCGTCCCTCAGGCGTGAGCAACCGTTTTAGCGGATCCAAAAGCG GCAACACCGCGAGCCTGACCATTAGCGGCCTGCAAGCGGAAGACGAAGCGG ATTATTATTGCCAGTCTTGGGATGTTTCTCCTATTACTGCTGTGTTTGGCGGC GGCACGAAGCTTACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACT CTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGT GTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAG ATAGCAGCCCCGTCAAGGCGGGAGTGGAGACAACCACACCCCCCCAACAAA GCAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGT GGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCG TGGAAAAGACAGTGGCCCCTACAGAATGTTCATAG LC4910 (BHQ880) polypeptide SEQ ID NO: 111 DIALTQPASVSGSPGQSITISCTGTSSDVGGFNYVSWYQQHPGKAPKLMIHDGS NRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCQSWDVSPITAVFGGGTKL TVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAG VETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS LC (opt) 4945 nucleotides SEQ ID NO: 98 GATATCGCACTGACCCAGCCAGCTTCAGTGAGCGGCTCACCAGGTCAGAGC ATTACCATCTCGTGTACGGGTACTAGCAGCGATCTTGGTGGTTATAATTATGT GTCTTGGTACCAGCAGCATCCCGGGAAGGCGCCGAAACTTATGATTTATGAT GTTAATAATCGTCCCTCAGGCGTGAGCAACCGTTTTAGCGGATCCAAAAGCG GCAACACCGCGAGCCTGACCATTAGCGGCCTGCAAGCGGAAGACGAAGCGG ATTATTATTGCCAGACTTATGATCAGATTAAGTTGTCTGCTGTGTTTGGCGGC GGCACGAAGCTTACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACT CTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGT GTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAG ATAGCAGCCCCGTCAAGGCGGGAGTGGAGACAACCACACCCTCCAAACAAA GCAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGT GGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCG TGGAAAAGACAGTGGCCCCTACAGAATGTTCATAG LC4945 (BHQ892) polypeptide SEQ ID N0: 112 DIALTQPASVSGSPGQSITISCTGTSSDLGGYNYVSWYQQHPGKAPKLMIYDVNN RPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCQTYDQIKLSAVFGGGTKLTV LGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVE TTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS LC (opt) 4946 nucleotides SEQ ID NO: 99 GATATCGTGCTGACCCAGAGCCCGGCGACCCTGAGCCTGTCTCCGGGCGAA CGTGCGACCCTGAGCTGCAGAGCGAGCCAGAATCTTTTTTCTCCTTATCTGG CTTGGTACCAGCAGAAACCAGGTCAAGCACCGCGTCTATTAATTTATGGTGCT TCTAATCGTGCAACTGGGGTCCCGGCGCGTTTTAGCGGCTCTGGATCCGGC ACGGATTTTACCCTGACCATTAGCAGCCTGGAACCTGAAGACTTTGCGGTGT ATTATTGCCAGCAGTATCTTACTCTTCCTCTTACCTTTGGCCAGGGTACGAAA GTCGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCAT CTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAAC TTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAAT CGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCT ACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAA AGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAA GAGCTTCAACAGGGGAGAGTGTTAG LC4946 (BHQ898) polypeptide SEQ ID NO: 113 DIVLTQSPATLSLSPGERATLSCRASQNLFSPYLAWYQQKPGQAPRLLIYGASNR ATGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYLTLPLTFGQGTKVEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC LC (opt) 5145 nucleotides SEQ ID NO: 100 GATATCGTGCTGACCCAGAGCCCGGCGACCCTGAGCCTGTCTCCGGGCGAA CGTGCGACCCTGAGCTGCAGAGCGAGCCAGAATCTTTTTTCTCCTTATCTGG CTTGGTACCAGCAGAAACCAGGTCAAGCACCGCGTCTATTAATTTATGGTGCT TCTAATCGTGCAACTGGGGTCCCGGCGCGTTTTAGCGGCTCTGGATCCGGC ACGGATTTTACCCTGACCATTAGCAGCCTGGAACCTGAAGACTTTGCGGTGT ATTATTGCCAGCAGTATATGACTCTTCCTCTTACCTTTGGCCAGGGTACGAAA GTCGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCAT CTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAAC TTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAAT CGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCT ACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAA AGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAA GAGCTTCAACAGGGGAGAGTGTTAG LC5145 (BHQ901) polypeptide SEQ ID NO: 114 DIVLTQSPATLSLSPGERATLSCRASQNLFSPYLAWYQQKPGQAPRLLIYGASNR ATGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYMTLPLTFGQGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC HC (opt) 4910 nucleotides SEQ ID NO: 101 CAGGCACAGGTGCAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGG CGGCAGCCTGCGTCTGAGCTGCGCGGCCTCCGGATTTACCTTTTCTTCTTAT TGGATGTCTTGGGTGCGCCAAGCCCCTGGGAAGGGTCTCGAGTGGGTGAGC GGTATCTCTTATTCTGGTAGCAATACCCATTATGCGGATAGCGTGAAAGGCC GTTTTTACCATTTCACGTGATAATTCGAAAAACACCCTGTATCTGCAAATGAACA GCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCGCGCGTATGGGTATTGA TCTTGATTATTGGGGCCAAGGCACCCTGGTCACCGTCTCCTCAGCCTCCACC AAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGG GGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTG ACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCG GCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTCGTGACCGTGC CCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCC CAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACT CACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTC TTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTG AGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGT TCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGC GGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCC TGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAA AGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCC CCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAA GAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATC GCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACG CCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCG TGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGC ATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGG GTAAATGA HC4910 (BHQ880) SEQ ID O-.115 polypeptide QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVSGIS YSGSNTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARMGIDLDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVE PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK HC (opt) 4945 nucleotides SEQ ID NO: 102 CAGGCACAGGTGCAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGG CGGCAGCCTGCGTCTGAGCTGCGCGGCCTCCGGATTTACCTTTTCTTCTTAT TGGATGTCTTGGGTGCGCCAAGCCCCTGGGAAGGGTCTCGAGTGGGTGAGC GTTATCTCTTCTGATTCTAGCTCTACCTATTATGCGGATAGCGTGAAAGGCCG TTTTACCATTTCACGTGATAATTCGAAAAACACCCTGTATCTGCAAATGAACAG CCTGCGTGCGGAAGATACGGCCGTGTATTATTGCGCGCGTCATGGTATTGAT TTTGATCATTGGGGCCAAGGCACCCTGGTCACCGTCTCCTCAGCCTCCACCA AGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGG GCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGA CGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGG CTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTCGTGACCGTGCC CTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCC AGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAACTC ACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCT TCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGA GGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTT CAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCG GGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCT GCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAA GCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCC CGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAG AACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCG CCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGC CTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGT GGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCA TGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGT AAATGA HC4945 (BHQ892) polypeptide SEQ ID NO: 116 QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVSVIS SDSSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHGIDFDHW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVE PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK HC (opt) 4946 = 5145 nucleotides SEQ ID NO: 103 CAGGTGCAGCTGGTGGAGAGCGGCGGAGGACTGGTGCAGCCTGGCGGCAG CCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAACAACTACGGCATG ACCTGGGTGAGGCAGGCCCCTGGCAAGGGCCTGGAGTGGGTGTCCGGCAT CAGCGGCAGCGGCAGCTACACCTACTACGCCGACAGCGTGAAGGGCAGGTT CACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGC CTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCCGGACCATCTACATG GACTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGCCTCCACCAAG GGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGC ACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACG GTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCT GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTCGTGACCGTGCCCT CCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAG CAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCAC ACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTC CTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGG TCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCA ACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGG AGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGC ACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGC CCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCG AGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAA CCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCC GTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCT CCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGG ACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATG AGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAA ATGA HC4946 = 5145 (BHQ898 / 901) polypeptide SEQ ID NO: 117 QVQLVESGGGLVQPGGSLRLSCAASGFTFNNYGMTWVRQAPGKGLEWVSGIS GSGSYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTIYMDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK Table 1 3B Light chain (LC) and heavy chain (HC) sequences Progenitors LC (progenitor) 4910 nucleotides SEQ ID NO: 104 GATATCGCCCTGACCCAGCCCGCCAGCGTGTCCGGCAGCCCTGGCCAGAG CATCACCATCAGCTGTACCGGCACCAGCAGCGATGTGGGCGGCTTCAACT ACGTGTCCTGGTATCAGCAGCACCCCGGCAAGGCCCCCAAGCTGATGATC CACGACGGCAGCAATAGACCCAGCGGCGTGTCCAATAGATTCAGCGGCAG CAAGAGCGGCAACACCGCCAGCCTGACCATCAGCGGCCTGCAGGCTGAG GACGAGGCCGACTACTACTGCCAGAGCTGGGATGTGAGCCCCATCACCGC CGTGTTTGGCGGCGGAACAAAGCTTACCGTCCTAGGTCAGCCCAAGGCTG CCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACA AGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAG TGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACAAC CACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTGAG CCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCA CGCATGAAGGGAGCACCGTGGAAAAGACAGTGGCCCCTACAGAATGTTCA TAG LC (progenitor) 4945 SEQ ID NO: 105 GATATCGCCCTGACCCAGCCCGCCAGCGTGTCCGGCAGCCCTGGCCAGAG CATCACCATCAGCTGTACCGGCACCAGCAGCGACCTGGGCGGCTACAACT ACGTGTCCTGGTATCAGCAGCACCCCGGCAAGGCCCCCAAGCTGATGATC TACGACGTGAACAACAGACCTAGCGGCGTGTCCAACAGATTCAGCGGCAG CAAGAGCGGCAACACCGCCAGCCTGACCATCTCTGGCCTGCAGGCTGAGG ACGAGGCCGACTACTACTGCCAGACCTACGACCAGATCAAGCTGTCCGCC GTGTTTGGCGGCGGAACAAAGCTTACCGTCCTAGGTCAGCCCAAGGCTGC CCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAA GGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGT GGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACAACC ACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTGAGC CTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCAC GCATGAAGGGAGCACCGTGGAAAAGACAGTGGCCCCTACAGAATGTTCAT AG LC (progenitor) 4946 nucleotides SEQ ID NO: 106 GACATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCTGGCGA GAGAGCCACCCTGTCTTGTAGGGCCAGCCAGAACCTGTTCAGCCCTTACCT GGCCTGGTATCAGCAGAAGCCCGGCCAGGCCCCCAGACTGCTGATCTACG GCGCCAGCAACAGAGCCACCGGCGTGCCCGCCAGATTCAGCGGCAGCGG CTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCTGAGGATTT CGCCGTGTACTACTGCCAGCAGTACCTGACCCTGCCCCTGACCTTCGGCC AGGGCACCAAGGTCGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCA TCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGT GCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGG ATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGAC AGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGC AGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCT GAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG LC (progenitor) 5145 nucleotides SEQ ID NO: 107 GACATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCTGGCGA GAGAGCCACCCTGTCTTGTAGGGCCAGCCAGAACCTGTTCAGCCCTTACCT GGCCTGGTATCAGCAGAAGCCCGGCCAGGCCCCCAGACTGCTGATCTACG GCGCCAGCAACAGAGCCACCGGCGTGCCCGCCAGATTCAGCGGCAGCGG CTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCTGAGGATTT CGCCGTGTACTACTGCCAGCAGTACATGACCCTGCCTCTGACCTTCGGCCA GGGCACCAAGGTCGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCAT CTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTG CCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGA TAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACA GCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCA GACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCT GAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG HC (progenitor) 4910 nucleotides SEQ ID NO: 108 CAGGCCCAGGTGCAGCTGGTGGAGAGCGGCGGAGGACTGGTGCAGCCTG GCGGCAGCCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAGC TACTGGATGAGCTGGGTGAGGCAGGCCCCTGGCAAGGGCCTGGAGTGGG TGTCCGGCATCAGCTACAGCGGCAGCAATACCCACTACGCCGACAGCGTG AAGGGCAGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCT GCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCC GGATGGGCATCGACCTGGATTACTGGGGCCAGGGCACCCTGGTCACCGTC TCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCC AAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTA CTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCG GCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCA GCAGCGTCGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATC TGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGA GCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGA ACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACAC CCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGA GCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAG GTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTA CCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCA AGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAG AAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACAC CCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCT GCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGC AATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTC CGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTG GCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAA CCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA HC (progenitor) 4945 nucleotides SEQ ID NO: 109 CAGGCCCAGGTGCAGCTGGTGGAGAGCGGCGGAGGACTGGTGCAGCCTG GCGGCAGCCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAGC TACTGGATGAGCTGGGTGAGGCAGGCCCCTGGCAAGGGCCTGGAGTGGG TGTCCGTGATCAGCAGCGATAGCAGCAGCACCTACTACGCCGATAGCGTG AAGGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCT GCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCA GGCACGGCATCGACTTCGACCACTGGGGCCAGGGCACCCTGGTCACCGTC TCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCC AAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTA CTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCG GCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCA GCAGCGTCGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATC TGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGA GCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGA ACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACAC CCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGA GCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAG GTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTA CCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCA AGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAG AAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACAC CCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCT GCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGC AATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTC CGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTG GCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAA CCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA HC (progenitor) 4946 = 5145 nucleotides SEQ ID NO: 110 CAGGTGCAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGGCA GCCTGCGTCTGAGCTGCGCGGCCTCCGGATTTACCTTTAATAATTATGGTA TGACTTGGGTGCGCCAAGCCCCTGGGAAGGGTCTCGAGTGGGTGAGCGGT ATCTCTGGTTCTGGTAGCTATACCTATTATGCGGATAGCGTGAAAGGCCGTT TTACCATTTCACGTGATAATTCGAAAAACACCCTGTATCTGCAAATGAACAG CCTGCGTGCGGAAGATACGGCCGTGTATTATTGCGCGCGTACTATTTATAT GGATTATTGGGGCCAAGGCACCCTGGTCACCGTCTCCTCAGCCTCCACCAA GGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGG GCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTG ACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCC GGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTCGTGACCGT GCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAA GCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAA AACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGT CAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAG GTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGAC AAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCC TCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAG GTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCC AAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGA GGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTA TCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAAC AACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC TATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTT CTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAG CCTCTCCCTGTCCCCGGGTAAATGA Example 9: Bioactivity tests. The biological activity of a neutralizing anti-DKK1 / 4 antibody is measured in a reporter gene assay, using the genetically modified cell line HEK293 T / 1 7 STF_70IRES_Krm_ (17), named as SuperTopflash Krm17. This cell line is derived from the human embryonic kidney cell line HEK293, and is stably transfected with: i) a reporter construct where the TCF promoter is fused upstream of the firefly luciferase gene, and ii) a construct which leads to the over-expression of Krm on the surface of this cell. In this cell line, exposure to Wnt protein stimulates luciferase expression in a dose-dependent manner. The addition of graded amounts of anti-DKK1 / 4 antibody to a fixed sub-maximal dose of DKK1 in the presence of Wnt, causes an increase in luciferase expression during an incubation period of 16 hours. At the end of this period, the amount of luciferase is quantified based on its enzymatic activity in the cell lysate. Luciferase catalyzes the conversion of the luciferin substrate to oxy luciferin, a chemiluminescent product. The resultant brightness chemiluminescence is then determined with an appropriate luminometer. The biological potency of a test sample of neutralizing anti-DKK1 / 4 antibody is determined by comparing its ability to increase the expression of luciferase, with that of a reference standard. The samples and the standard are normalized based on the protein content. Then the relative power is calculated using a parallel line test according to the European Pharmaeia. The final result is expressed as the relative power (in percent) of a sample compared to the reference standard. Example 10: In vitro activity on relevant biological targets. The leading Fabs are selected that have affinities in the low nanomolar interval, and a powerful activity in the cellular assay. The physiological linkage components for DKK1 are LRP5 / 6 (KD of approximately 340 pM), and Kremen 1 and 2 (KD of approximately 280 pM) [Mao 2001] [Mao 2002]. Given these high affinity interactions, it is desirable to further improve the affinity, in order to better compete with the physiological interactions of DKK1. In order to increase the affinity and biological activity of the selected Fabs, the CDR-L3 and CDR-H2 regions are optimized in parallel by cassette mutagenesis, using trinucleotide-directed mutagenesis [Virnekas 1 994] [Knappik 2000] [ Nagy 2002]. Following affinity maturation, a Fab having a low picomolar affinity is selected, which reactivates Wnt signaling inhibited by DKK1 with an EC50 below 1 nM, and which cross-reacts with mouse cynomolgus monkey DKK1, and rat. The variable regions of FAb are then designed in two different human IgG1 structures. The anti-DKK1 / 4 antibody has a high affinity for human DKK1 (2 pM), with a typical binding kinetics for an antibody of this affinity. See Figure 6. Figure 6. METHODS: The binding affinity and kinetics of the leader candidates and of rhDKKI (recombinant human DKK1) (Lot BTP7757), using surface plasmon resonance, are measured with a Biacore T100 instrument (Biacore , Uppsala, Sweden), which contains a CM5 (S) sensor chip (Cat. # BR-1006-68). The anti-human IgG 1 Faith (Jackson I mmuno Research, Cat. # 1 09-006-098) was immobilized on each cell flow, followed by the capture of a lead candidate to an expected capture of approximately 1 00 RU. Finally, six concentrations of DKK1 (range from 0.1 95 to 6.25 nM) are executed, with a repetition concentration on the chip. The flow cells are activated for the DKK1 link for 240 seconds, and the dissociation continues for 30 minutes. The normalized data (the background is subtracted) are adjusted to a 1: 1 link with the mass transport model, using the kinetics analysis in the BIA evaluation 1 .0 software. This experiment is carried out in triplicate, and the data are presented as the average of these three experiments with the standard deviation. Example 1 1: Mapping of epitopes. Mature DKK1 is a 266 amino acid protein with two regions rich in cysteine (Cys-1 and Cys-2). The Cys-2 domain is responsible for the binding of both LRPs and Kremen proteins, and is necessary and sufficient for the inhibition of Wnt signaling [Li 2002] [Brott 2002]: The immunoprecipitation experiments (Figures 7A and 7B) show that the anti-DKK1 / 4 antibody binds specifically to the Cys-2 domain, but not with the Cys-1 domain. The anti-DKK1 / 4 antibody is only weakly active in the Western blot with the denatured DKK1, and in a peptide mapping experiment it is not found to bind specifically with any of the overlapping 15 amino acid peptides covering the length of the protein (JTP), suggesting that the anti-DKK1 / 4 antibody probably recognizes a non-linear epitope within Cys-2.

Figure 7 (A) shows a schematic representation of the full-length and truncated DKK1. The full length (FL, which contains residues 1 -266), the truncated carboxy-terminal (? C, which contains residues 1 -1 85), and the truncated amino-terminal (? N, which contains residues 1 - 60 plus residues 157-266), are fused to an HA epitope in their C-termini, and cloned into a mammalian expression vector under the control of the cytomegalovirus (CMV) promoter. Figure 7 (B) illustrates the binding of a neutralizing anti-DKK1 / 4 antibody and the DKK1 proteins. The conditioned medium from the transiently transfected HEK293 cells expressing the full-length, amino-truncated and truncated carboxy-terminal DKK1 proteins is incubated with the anti-lysozyme IgG control or with the anti-DKK1 / 4 antibodies for 2 hours. hours at room temperature, and the immunocomplexes are harvested on G protein beads, resolved by SDS-PAGE, transferred, and stained with an anti-HA antibody. 1/1 0 of the total input is loaded as control. Example 11: Mapping of epitopes - N-glycosylation. A number of proteins within the Wnt signaling pathway are covalently modified by post-translational enzymes, which regulate their cellular activity. Members of the DKK family, including DKK1, are modified by N-glycosylation [Krupnik 1 999]. DKK1 has a theoretical N-linked glycosylation site at amino acid 256 within the Cys-2 domain. Given the highly conserved nature of the Cys-2 domain, and the site of Potential binding of both DKK1 for LRP6 and the anti-DKK1 / 4 antibody for DKK1, we sought to determine whether the anti-DKK1 / 4 antibody recognized the N-glycosylated form of DKK1. An ELISA demonstrates that the anti-DKK1 / 4 antibody recognizes the N-glycosylated form of rhDKKI much better than the specifically deglycosylated N-linked form of rhDKKL Table 14A. Although the same proteins are recognized equally well with a second antibody (anti-HIS), directed towards the fused epitope tag region of the recombinant protein. This difference in affinity is quantified using surface plasmon resonance, and it is found that the anti-DKK1 / 4 antibody has a 100-fold higher KD for the glycosylated rhDKKI than for the deglycosylated protein; see Table 14B.

Table 14A Link percentage - Dependence on the glycosylation of the anti-DKK1 / 4 antibody binding with DKK1 e Table 14B Resonance of surface plasmen The binding of the anti-DKK1 / 4 antibody to the wild-type (WT) rhDKKI (Lot # BTP7757 marked with the HEK HIS epitope), and the N-linked deglycosylated rhDKKI (N-DESG LI), deglycosylated N-linked to the PNGase F enzyme (Sigma, Cat. # E-DEGLY)), is measured by ELISA. Briefly stated, a high-binding ELISA plate (Nunc # 442404) is coated with 1 microgram / milliliter of wild-type or N-deglycosylated DKK1. The ratio of the binding of both the anti-DKK1 / 4 antibody and the anti-HIS antibody to the wild-type DKK1 is shown, compared to its respective N-deglycosylated link. This experiment is carried out with three different concentrations (data for a representative concentration are shown), and all concentrations had similar results. B. The binding affinity and the kinetics of the anti-DKK1 / 4 antibody are measured for both wild type and N-deglycosylated DKK1 (HEK293, Lot # BTP7757), using a Biacore T1 00. Anti-DKK1 / 4 antibody consistently had a 1 00 fold lower affinity for N-deglycosylated DKK1 than for wild type DKK1.

Example 12: Percentage of identity of the members of the DKK family. The human Dickkopf family consists of four paralogs (see Table 15), three of which (DKK1, 2, and 4) bind to the LRP6 and Kremen proteins, induce the internalization of LRP5 / 6, and inhibit Wnt signaling canonical [Mao 2001] [Mao 2003]. DKK2 also has synergism with the over-expression of LRP6 to improve Wnt signaling, but the co-expression of LRP6 and Kremen 2 restores the inhibition of the pathway by DKK2 [Mao 2003]. Accordingly, DKK2 can act as an agonist as well as an antagonist, depending on the cellular context. DKK3 is the least conserved member of the family, including within the Cys-2 domain responsible for the interactions of LRP5 / 6 and Kremen, and is distinct from the other members of the DKK family, because it does not link to the LRPs or Kremens, and does not block Wnt signaling [Mao 2001] [Mao 2003].

Table 15 Percentage of identity of the members of the DKK family through the whole protein and within the Cys-2 domains The homology between the members of the DKK family is evaluated (Vector NTI Advanced 9.1 .0) using the AlignX algorithm for alignments of sequences by pairs, comparing the proportions of amino acid identities. The opening and hollow extension fines of 1 0 and 0.1 are applied, respectively. This evaluation includes whole protein comparisons, as well as comparisons of the Cys-2 domains only. As indicated in the above table, DKKs 1, 2, and 4 share amino acid sequence homology of 30 to 40 percent through the entire protein. Comparison of Cys-2 domains only shows that DKKs 1 and 2 share a homology of 69 percent within this region, while DKK4 shares approximately 57 percent with the same domain of DKKs 1 and 2. DKK3 shows the lowest level of homology with the other members of the family. Among all the members, homology is greater within the Cys-2 domain.

Example 13: Affinity of anti-DKK1 / 4 antibody for members of the human DKK family. In addition to the DKK1 linkage, the anti-DKK1 / 4 antibody also binds to DKK4; see Table 16. Although the affinity for DKK4 is approximately 100 times lower than for DKK1, it is still subnanomolar, and therefore, is probably biologically and chemically relevant. It should be noted that neither DKK2 nor DKK4 retain the asparagine residue that is predicted to be targeted for glycosylation in the Cys-2 domain of DKK1. Preliminary immunoprecipitation experiments suggest that the anti-DKK1 / 4 antibody does not specifically bind to DKK2. The binding affinity of the anti-DKK1 / 4 antibody to bind to DKK2 will be determined immediately after the successful purification of DKK2. In a manner consistent with the different functional and binding properties of DKK3, the anti-DKK1 / 4 antibody does not bind to DKK3. Table 16 Affinity of anti-DKK1 / 4 antibody for members of the human DKK family The binding affinity and kinetics of the anti-DKK1 / 4 antibody for other members of the human DKK protein family are measured using a Biacore T100. As before, the experiments are carried out in triplicate for proteins with a significant binding to the anti-DKK1 / 4 antibody, and are reported as the average of three experiments with the standard deviation. DKK3, which is the least homologous family member, had no link that was detectable above the fund levels, and in this way, it is considered NSB (non-significant link). In the same way, recent data suggest that an anti-DKK1 / 4 antibody of the invention also has no significant binding to DKK2. Example 14: Anti-DKK1 / 4 antibody blocks the binding of DKK1 with the LRP6. DKK1 mediates its Wnt antagonist activity through interactions with LRP5 / 6 and Kremen, inducing internalization, and blocking the Wnt-induced interaction of LRP5 / 6 with Frizzled receptors. The anti-DKK1 / 4 antibody competitively inhibits the binding of DKK1 with LRP6 in a competition ELISA assay in Figure 8. HEK293T cells do not express sufficient levels of endogenous LRP5 or 6 to allow visualization of the DKK1 linkage. Without However, after co-transfection of LRP6 with a surface traffic chaperone protein, MESD, GFP-labeled DKK1 can be detected on the cell surface, illustrating the specific nature of the interaction of DKK1 / LRP6. MOR0491 0, which shares the same variable regions as the anti-DKK1 / 4 antibody, specifically blocks this interaction. The ability of the anti-DKK1 / 4 antibody to inhibit the binding of DKK1 directly with LRP6 is measured by an ELISA. Briefly, the untreated plates (Fisher, Cat. # 1 2565501) are coated with 1 microgram / milliliter of recombinant LRP6 (R & amp; amp;; D Systems, Cat. # 1 505-LR), then with 500 nanograms / milliliter of rhDKKI, and a concentration curve of either anti-DKK1 / 4 or h lgG 1 antibody (anti MOR3207) is pre-incubated on ice. -lisozyme, ACE 1091 5) for 30 minutes, after which they are placed on plates coated with LRP6 for 2 hours. The plates are washed, and the level of DKK1 binding is detected with the anti-DKK1 antibody (R & D Systems AFI096). The raw OD values are displayed (subtracting the background). Increasing concentrations of the anti-DKK1 / 4 antibody inhibit the binding of DKK1 directly with LRP6 in a dose-dependent manner, whereas increasing concentrations of hlgG1 do not block the binding of DKK1 with LRP6. The ability of MOR04910 to inhibit the binding of DKK1 / LRP6 on the cell surface is measured by the fluorescence microscope. HEK293T cells are transfected simulated, and transiently transfected, with plasmids encoding LRP6 and MESD. The cells are incubated with a medium conditioned with DKK1 -GFP together with anti-lysozyme Fab or Fab MOR04910 anti-DKK1 for 1 hour at 37 ° C, and examined by fluorescence microscopy. The fluorescence of GFP reflects the binding of DKK1 / -GFP with the LRP6 over-expressed on the plasma membrane. The anti-DKK1 / 4 antibody blocks the interactions of DKK1 with LRP6 on cell surfaces. Example 14: Reporter assays - Reactivation of TCF / LEF gene transcription inhibited by DKK1. The canonical Wnt signaling culminates in the translocation of beta-catenin to the nucleus, where it is associated with the transcription factors of the TCF / LEF family, resulting in a better transcription of genes that respond to Wnt. A reporter trial is established using a promoter that responds to TCF / LEF that drives transcription of the luciferase gene, facilitating the detection of Wnt path modulation. DKK1 effectively blocks the luciferase activity induced by the Wnt3a (CM) conditioned medium in this assay. The anti-DKK1 / 4 antibody reactivates Wnt signaling suppressed by DKK1 with an apparent EC5o of 0.16 nM in Figure 9. Because the assay requires approximately 1 nM of DKK1 for complete deletion, and the affinity of the antibody is 2 pM, it is likely that this EC50 reflects the sensitivity of the assay and the relative amounts of each protein, rather than an absolute limit of competition of the anti-DKK1 / 4 antibody. 293T cells stably transfected with the reporter SuperTopflash and Kremen, are treated with 10 nanograms / milliliter of rhDKKI, 50 percent Wnt3a conditioned medium, and different amounts of the anti-DKK1 / 4 antibody. Eighteen hours later, the luciferase activity is measured by the Bright-Glo luciferase assay kit (Promega). Example 15: Reporter trials - Reversal of alkaline phosphatase secretion inhibited by DKK1 in pre-osteoblast type cells. In order to determine whether the anti-DKK1 / 4 antibody blocks the functions of DKK1 in a more physiologically relevant setting, an in vitro assay is established to measure Wnt-mediated osteoblast differentiation of the urotropic pl C3H mouse cell line 10T1 / 2 (1 0T1 / 2); see Figure 1 0. After differentiation of the osteoblasts, the 1 0T1 / 2 cells secrete alkaline phosphatase (AP), a phenomenon that can be inhibited by DKK1. The DKK1 / 4 antibody, but not the IgG control, blocks the suppression by DKK1 of 10T1 / 2 differentiation in the presence of the Wnt3a conditioned medium. It has been reported that Wnt induces proliferation and inhibits apoptosis in a number of cellular contexts, and activation of the Wnt pathway, as indicated by beta-catenin stabilization or nuclear stabilization, is often associated with progress of the tumor. Additionally, the decrease of DKK1 in some cancers (eg, colon carcinoma and melanoma) [Gonzalez-Sancho 2005] [Kuphal 2006], has led some researchers to suggest that DKK1 may be a tumor suppressor for some cancers. In order to test whether DKK1 has an effect on tumor proliferation or survival, the tumor cell lines are treated with the anti-DKK1 / 4 antibody, and analyzed for changes in growth. It is not found that any tumor cell line tested is significantly affected by the addition of the anti-DKK1 / 4 antibody. The effect of the anti-DKK1 / 4 antibody on the survival and proliferation of several cancer cell lines is evaluated in vitro. In this assay, the anti-DKK1 / 4 antibody (100 micrograms / milliliter) is incubated with a tumor cell line; after 3 days, the number of cells is evaluated by quantification of ATP (Promega, Cell Titer Glo Assay®), as a measure of metabolically active cells, with a linear relationship with the number of cells. This test is carried out in three different serum concentrations (without serum, minimal growth, and complete growth). No significant changes were found, compared with cells not treated and treated with hlgG1. Supernatants of the cell line are analyzed for the expression of DKK1 by ELISA. Example 16: Cross reactivity between species and neutralization of DKK1. A neutralizing anti-DKK1 / 4 antibody is selected, not by its high affinity against human DKK1 and neutralizing capacity, but also based on its cross-reactivity with other species that could be used for efficacy and safety studies. The anti-DKK1 / 4 antibody cross-reacts with the DKK1 of mouse, rat, and cynomolgus monkey (cynomolgus, Macaca fascicularis), with an affinity similar to that of human DKK1; see Table 17. Moreover, the anti-DKK1 / 4 antibody neutralizes the Wnt suppressor activity mediated by DKK1 of the four species (Table 17), suggesting that these species should be relevant for both safety and efficacy models. Table 17 Cross reactivity between species and neutralization of DKK1 The affinity determination is assayed for human DKK1, cynomolgus monkey, mouse, and rat, by titration in equilibrium in solution (SET), using the M-384 SERIES® analyzer (BioVeris, Europe). For the determination of the KD by titration in equilibrium in solution (SET), monomer fractions (at least 90 percent of monomer content, analyzed by analytical SEC, Superdex75, Amersham Pharmacia) of the IgG proteins are used. The determination of the affinity based on the electrochemiluminescence (ECL) in solution, and the evaluation of the data are basically carried out as described by [Haenel et al., 2005], the modified linkage adjustment model is applied in accordance with [Piehler et al., 1997]. A constant amount of MOR04910 IgG is equilibrated with different concentrations (3n serial dilutions) of human DKK1 (initial concentration of 4 nM) in solution. Biotinylated human DKK1 coupled with paramagnetic beads (M-280 Streptavidin, Dynal), and polyclonal antibody (Fab) '2 goat anti-human labeled BV-tag ™ (BioVeris Europe, Witney, Oxfordshire, UK) are added and incubated for 30 minutes. Subsequently, the concentration of unbound IgG is quantified by means of ECL detection, using the M-384 SERIES® analyzer (BioVeris, Europe). The affinity determination for rat, mouse, and cynomolgus monkey DKK1 is carried out essentially as described above, using mouse, rat, and cynomolgus monkey DKK1 as the analyte in solution, instead of the Human DKK1. For the detection of free IgG molecules, biotinylated human DKK1 coupled with paramagnetic beads is used. MOR04910 and the anti-DKK1 / 4 antibody neutralize human DKK1 (Novartis) with an equivalent EC50; the anti-DKK1 / 4 antibody also neutralizes DKK1 from monkey (Novartis), mouse (R &D Systems 1765-DK-01 0) and rat (Novartis). The report of the TOPFLASH reporter for human, rat, mouse, and cynomolgus monkey DKK1 is carried out essentially as described in the previous (Figure 9), using the recombinant DKK1 of each species as the inhibitor of the conditioned medium of Wnt, instead of the human DKK1. Rat recombinant DKK1 required higher concentrations of protein to achieve significant inhibition of the TOPFLASH assay. Example 17: Effect of anti-DKK1 / 4 antibody on the intratibial growth of PC3M2AC6 xenografts. Your prostate moral metastases are unique among bone metastases, because they are overwhelmingly osteoblastic, rather than osteolytic [Keller 2001]. However, even predominantly osteoblastic bone metastases have underlying regions of osteolysis, and often have low bone mass densities (BMD), especially when patients are on androgen ablation therapy [Saad 2006]. Recently, it was demonstrated that DKK1 can act as a switch, whereby the expression of DKK1 improves the osteolytic properties of a mixed osteoblastic / osteolytic prostate tumor cell line (C4-2B). In addition, suppression of DKK1 by shRNA inhibited the osteolytic activity of a predominantly osteolytic prostate tumor cell line (PC3) [Hall 2005] [Hall2006]. The Genetic elimination of DKK1 also inhibited the intratibial growth of the tumor xenograft, leading the authors to speculate that osteolytic activity may be important for the establishment of a metastatic niche, but after the loss of DKK1 in the prosthetic metastases, it converts the tumor up to an osteoblastic phenotype. An osteolytic prostate tumor model is adapted from a method of [Kim 2003]. A variant of the osteolytic prostatic tumor cell line (PC3M) stably expressing luciferase (PCM2AC6) is injected into the tibia of the mice. The growth of the tumor is monitored by luciferase, while the changes in the bone are monitored by micro-computerized tomography (micro-CT) and histology. Instead of improving tumor growth, anti-DKK1 / 4 antibody tended toward inhibition of tumor growth. Although inhibition is not significant in any study, it has consistently occurred in 5/5 studies conducted to date, with a representative study showing the effects of three doses of anti-DKK1 / 4 antibody on tumor growth, as shown in Figure eleven . A similar non-significant tendency towards inhibition was presented with the mice treated with the anti-DKK1 / 4 antibody, with subcutaneous PC3M2AC6 xenografts. The treatments are started on day 5 after implantation (0.2 million cells / animal). The anti-DKK1 / 4 antibody is administered intravenously, in doses of 20, 60, and 200 micrograms / mouse / day, every day, three times a week for two weeks. The control IgG is administered intravenously at 200 micrograms / mouse / day, every day, three times a week for two weeks. The vehicle control (serum phosphate buffered) is administered intravenously every day, three times a week for three weeks. After treatment, the final efficacy data and the change in body weight are calculated. Using this model, we found that an anti-DKK1 / 4 antibody inhibits tumor-induced cortical bone damage. The effects on trabecular bone are confused in this model by the observation that both tumor implants and false implants cause mechanical damage to the bone, which results in an initial increase in the bone tissue, which is later remodeled, causing a decrease in apparent osseous volume. The relative effects of newly formed tissue bone and trabeculae on the overall proportions of bone volume / trabecular volume (BV / TV) are therefore obscured. However, it is clear that anti-DKK1 / 4 antibody increases bone production in both implanted and falsely implanted tibias, and inhibits or delays the decrease in bone volume that accompanies remodeling. Using the same tumor-induced osteolytic model, the anti-DKK1 / 4 antibody demonstrates equivalent anti-osteolytic activity as Zometa; see Figure 1 2. The bone metabolic effects of the anti-DKK1 / 4 antibody respond to the dose in the range of 20 to 200 micrograms / mouse, with a minimally effective dose of between 20 and 60 micrograms / mouse; see Figure 1 3. Together, these data suggest that the anti-DKK1 / 4 antibody should have an impact on tumor-induced osteolytic disease, but may also be effective in non-tumorous bone diseases, such as osteoporosis, or improve the repair of bone fractures. Example 18: The anti-DKK1 / 4 antibody maintains a high bone density in implanted tibias both with tumor and falsely. In an effort to evaluate the pharmacodynamic markers of efficacy in mice, three serum markers of bone metabolism are analyzed: osteocalcin (OC), osteoprotegerin (OPG), and receptor activator of nuclear factor ligand KB (sRANKL). These osteoblast markers are used in place of the most typical osteoclast markers, due to the expected mechanism of action of the antibody. However, no consistent changes are detected in animals with tumors against clean animals. No correlation of bone loss, measured by micro-CT or IHC, with any of these markers is consistently observed. Figure 12A shows representative examples of the micro-CT reconstructions of the tibias of the treated mice. Cortical damage is scored from 0 = no damage to 3 = severe damage. Figure 1 2B. Cortical damage in the tibias implanted with Tumor is scored manually by micro-CT analysis that are blind with respect to the groups under study. No cortical damage is seen in any of the legs implanted falsely. Methods: Female 12-week-old hairless mice are implanted intratibially with 2x105 PC-3M2AC6 cells in the left tibia, and are injected falsely into the right tibia. The treatments are started on day 5 after implantation. The NVP-anti-DKK1 / 4-NX antibody (anti-DKK1 / 4 antibody) and the IgG control are administered intravenously at doses of 200 micrograms / mouse / day each day, three times a week for two weeks. The vehicle control (serum phosphate buffered) is also administered intravenously every day, three times a week for two weeks. The animals are screened on days 7,14, and 18 after tumor implantation using the μ-CT explorer VivaCT40 (SCANCO, Switzerland). Trabecular bone density (BV / TV) is analyzed as described in the methods. An asterisk (+) indicates the statistically significant difference of the vehicle and IgG controls (n = 12) at the same point in time at a p < 0.05. In Figure 13, to determine the bone mass, images of the secondary spongiosa of the tibia are taken with the Zeiss Imager Z.1 and the Axiovision software based on Giemsa. The reading is based on the percentage of calcified bone throughout the field. Each column represents the mean and standard deviation of the number of animals mentioned. In the groups treated with PBS, IgG, and anti-DKK1 / 4 antibody, only the animals with tumor are analyzed. The right legs had no false injections, and the left legs had a tumor. Statistics: Dunnett multiple comparisons test, one way ANOVA. The left legs or the right legs were compared with the respective leg in the PBS group p < 0.05 *, p < 0.01 * J p > 0.05 n .s. Figure 14 shows the anabolic bone efficacy of a dose-dependent anti-DKK1 / 4 antibody with a minimum efficacy dose of between 20 and 60 micrograms / mouse three times per week. Female 12-week-old hairless mice are implanted intratibially with 2x105 PC-3M2AC6 cells in the left tibia, and a false injection is placed in the right tibia. The treatments begin on day 6 after implantation. NVP-anti-DKK1 / 4-NX antibody (anti-DKK1 / 4 antibody) is administered intravenously, in doses of 20, 60, and 200 micrograms / mouse / day, every day, three times a week for two weeks. The control IgG is administered intravenously at 200 micrograms / mouse / day each day, three times a week for two weeks. The vehicle control (PBS) is administered intravenously every day, three times a week for two weeks. The animals are screened on days 7 and 20 after tumor implantation, using the μ-CT explorer VivaCT40 (SCANCO, Switzerland). Trabecular bone density (BV / TV) is analyzed as described in the methods. The asterisk (*) indicates the difference Statistical significance of all controls, including vehicle, IgG, drilling only, and clean animals at the same point of time at a p < 0.05. Example 18A: Status of the biomarker. Biomarkers of DKK1 The pattern of RNA expression of DKK1 has been described. Krupnik (1999) showed the expression in placenta by Northern blot analysis, without expression detected in heart, brain, lung, liver, skeletal muscle, or pancreas. Wirths (2003) showed a lack of RNA expression in liver, kidney, and breast, although RNA expression is seen in a subset of hepatoblastomas and Wilms tumors. Workers who examined expression in the gastrointestinal tract of DKK1 by in situ RNA hybridization did not show expression in the stomach and colon, either normal or malignant (Byun 2006). The analysis of RNA expression in mice revealed high levels of DKK1 expression in bone, a mean expression in fetus and placenta, and a weak expression in brown adipose tissue, in the thymus, and in the duodenum [Li 2006] . The expression of the DKK1 protein in myeloma samples is evaluated using the same goat antibody used in the current study (Tian, 2003). In this document, the expression in myeloma cells of patients with a low grade morphology is seen; no expression of the DKK1 protein is detected in bone marrow biopsy samples from five control subjects.

Tissue distribution and cross-reactivity between species of anti-DKK1 / 4 therapeutic antibody are studied by screening against a series of normal human and monkey tissues. Both whole tissue sections and tissue microarrays are evaluated. Positive controls included a commercial antibody to DKK1 that is evaluated in the same tissue. DDK1 _1 5 (anti-DKK1 / 4 antibody conjugated to FITC) and DKK1 _8 (goat anti-DKK1 conjugated to FITC, R & D Systems, # AF1096, batches GBL013101 and GBL141 1 1). Other biomarkers Because little is known about the pathophysiological role of DKK1, a significant amount of effort has been focused on the accumulation of the knowledge base about the in vivo effects of the anti-DKK1 / 4 antibody through biomarker studies, and how this could be exploited for further development. Key areas of focus have included: 1) Understanding the effect of anti-DKK1 / 4 antibody on normal and metastatic bone metabolism by measuring circulating markers of osteoclastic and osteoblastic activity. 2) The comparative expression levels of DKK1 in multiple myeloma and other tumors to confirm and expand the target indications. 3) Effects on the level of gene expression in key tissues such as the colon, bone marrow, lung, skin, and breast, for evaluate the activation of beta-catenin. Preliminary molecular epidemiological studies have confi rmed the higher serum levels of DKK1 in patients with multiple myeloma, and support a POC in this indication. Based on existing knowledge, Table 1 8 provides the proposed potential biomarkers for an anti-DKK1 / 4 antibody. Table 1 8 Biomarkers for the objectives of DK K1 and DKK4 Tissue Categories Tumor Subrogated blood Pharmacodynamics (PD) N / A DKK1 levels - Free and bound target with free antibody and anti-DKK1 / 4 Downstream Activation of beta-Adipose / catenin skin Mechanism of NTx, CTx, PINP, Osteocalcin action, RANKL, OPG, PTH, Vitamin D3, Tissue Categories Tumor Subrogated blood calcitonin Efficacy Protein M in NTx, CTx, PINP, serum, protein M Osteocalcin, total in urine, RANKL, OPG, microglobin b2, PTH, calcitonin LDH bone - ALP, CICP, CTx, NTx, etc.

Markers DKK-1, CTx, PINP, Predictors Osteocalcin, RANKL Stratification, OPG, PTH, Vitamin D3, calcitonin Pre-selection Levels of DKK1 Expression of DKK1 in serum Safety Immunogenicity Pharmacokinetics Antibody anti-DKK1 / 4 Example 19: Amino acid sequences of the heavy and light chain variable regions of the anti-DKK1 antibodies. The amino acid sequences of the light and heavy chain variable regions of the anti-DKK1 antibodies, whose complementarity determining regions are shown in Tables 5a, 6, 11A, and 11B, are given in full in Table 19.

TABLE 19 Amino acid sequences of the heavy and light chain variable regions of anti-DKK1 antibodies (SEQ ID NOs: 2-39) MOR04454 VH: (SEQ ID NO: 2) QVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGLHWVRQAPGKGLEWVSSIS YYGSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGSHMD KPPGYVFAFWGQGTLVTVSS MOR04454 VL: (SEQ ID NO: 21) DIQMTQSPSSLSASVGDRVTITCRASQGIKNYLNWYQQKPGKAPKLLIGAASSL QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYYGMPPTFGQGTKVEIK RT MOR04455 VH: (SEQ ID NO: 3) QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLEWVSGI SGSGSYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHYMDH WGQGTLVTVSS MOR04455 VL: (SEQ ID NO: 22) DIQMTQSPSSLSASVGDRVTITCRASQDISNYLHWYQQKPGKAPKLLIYGASNL QSGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQYDSIPMTFGQGTKVEIK RT MOR04456 VH: (SEQ ID NO: 4) QVQLVESGGGLVQPGGSLRLSCAASGFTFNNYGMTWVRQAPGKGLEWVSGI SGSGSYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTIYMDY WGQGTLVTVSS MOR04456 VL: (SEQ ID NO: 23) DIVLTQSPATLSLSPGERATLSCRASQNLFSPYLAWYQQKPGQAPRLLIYGASN RATGVPARFSGSGSGTDFTLTISSLEPEDFATYYCQQYGDEPITFGQGTKVEIK RT MOR04461 VH: (SEQ ID NO: 5) QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVSGI SYSGSNTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARMGIDLD YWGQGTLVTVSS MOR04461 VL: (SEQ ID NO: 24) DIALTQPASVSGSPGQSITISCTGTSSDVGGFNYVSWYQQHPGKAPKLMIHDG SNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSTWDMTVDFVFGGGT KLTVLGQ MOR04470 VH: (SEQ ID NO: 6) QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVSVI SSDSSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHGIDFD HWGQGTLVTVSS MOR04470 VL: (SEQ ID NO: 25) DIALTQPASVSGSPGQSITISCTGTSSDLGGYNYVSWYQQHPGKAPKLMIYDV NNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCQSYASGNTKVVFGGG TKLTVLGQ MOR04516 VH: (SEQ ID NO: 7) QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYIGWVRQMPGKGLEWMGIIYP TDSYTNYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGIPFRMRG FDYWGQGTLVTVSS MOR04516 VL: (SEQ ID NO: 26) DIVLTQPPSVSGAPGQRVTISCSGSSSNIGSSFVNWYQQLPGTAPKLLIGNNSN RPSGVPDRFSGSKSGTSASLAITGLQSEDEADYYCASFDMGSPNVVFGGGTK LTVLGQ MOR04907 VH: (SEQ ID NO: 8) QVQLVESGGGLVQPGGSLRLSCAASGFTFNNYGMTWVRQAPGKGLEWVSGI SGSGSYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTIYMDY WGQGTLVTVSS MOR04907 VL: (SEQ ID NO: 27) DIVLTQSPATLSLSPGERATLSCRASQNLFSPYLAWYQQKPGQAPRLLIYGASN RATGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYLSLPTTFGQGTKVEIK RT MOR04913 VH: (SEQ ID NO: 9) QVQLVESGGGLVQPGGSLRLSCAASGFTFNNYGMTWVRQAPGKGLEWVSGI SGSGSYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTIYMDY WGQGTLVTVSS MOR04913 VL: (SEQ ID NO: 28) DIVLTQSPATLSLSPGERATLSCRASQNLFSPYLAWYQQKPGQAPRLLIYGASN RATGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYMTLPLTFGQGTKVEIN RT MOR04946 VH: (SEQ ID NO: 10) QVQLVESGGGLVQPGGSLRLSCAASGFTFNNYGMTWVRQAPGKGLEWVSGI SGSGSYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTIYMDY WGQGTLVTVSS MOR04946 VL: (SEQ ID NO: 29) DIVLTQSPATLSLSPGERATLSCRASQNLFSPYLAWYQQKPGQAPRLLIYGASN RATGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYLTLPLTFGQGTKVEIK RT MOR04910 VH: (SEQ ID NO: 11) QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVSGI SYSGSNTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARMGIDLD YWGQGTLVTVSS MOR04910 VL: (SEQ ID NO: 30) DIALTQPASVSGSPGQSITISCTGTSSDVGGFNYVSWYQQHPGKAPKLMIHDG SNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCQSWDVSPITAVFGGGT KLTVLGQ MOR04921 VH: (SEQ ID NO: 12) QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVSGI SYSGSNTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARMGIDLD YWGQGTLVTVSS MOR04921 VL: (SEQ ID NO: 31) DIALTQPASVSGSPGQSITISCTGTSSDVGGFNYVSWYQQHPGKAPKLMIHDG SNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCQTWATSPLSSVFGGG TKLTVLGQ MOR04948 VH: (SEQ ID NO: 13) QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVSGI SYSGSNTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARMGIDLD YWGQGTLVTVSS MOR04948 VL: (SEQ ID NO: 32) DIALTQPASVSGSPGQSITISCTGTSSDVGGFNYVSWYQQHPGKAPKLMIHDG SNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCQTWDSLSFFVFGGGT KLTVLGQ MOR04914 VH: (SEQ ID NO: 14) QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVSVI SSDSSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHGIDFD HWGQGTLVTVSS MOR04914 VL: (SEQ ID NO: 33) DIALTQPASVSGSPGQSITISCTGTSSDLGGYNYVSWYQQHPGKAPKLMIYDV NNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCQSYTYTPISPVFGGGT KLTVLGQ MOR04920 VH: (SEQ ID NO: 15) QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVSSI EHKDAGYTTWYAAGVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHGID FDHWGQGTLVTVSS MOR04920 VL: (SEQ ID NO: 34) DIALTQPASVSGSPGQSITISCTGTSSDLGGYNYVSWYQQHPGKAPKLMIYDV NNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCQSYASGNTKVVFGGG TKLTVLGQ MOR04945 VH: (SEQ ID NO: 16) QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVSVI SSDSSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHGIDFD HWGQGTLVTVSS MOR04945 VL: (SEQ ID NO: 35) DIALTQPASVSGSPGQSITISCTGTSSDLGGYNYVSWYQQHPGKAPKLMIYDV NNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCQTYDQIKLSAVFGGGT KLTVLGQ MOR04952 VH: (SEQ ID NO: 17) QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVSVI SSDSSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHGIDFD HWGQGTLVTVSS MOR04952 VL: (SEQ ID NO: 36) DIALTQPASVSGSPGQSITISCTGTSSDLGGYNYVSWYQQHPGKAPKLMIYDV NNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCQSYDSPTDSVVFGGG TKLTVLGQ MOR04954 VH: (SEQ ID NO: 18) QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVSVI EHKDKGGTTYYAASVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHGID FDHWGQGTLVTVSS MOR04954 VL: (SEQ ID NO: 37) DIALTQPASVSGSPGQSITISCTGTSSDLGGYNYVSWYQQHPGKAPKLMIYDV NNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADSYASGNTKVVFGGG TKLTVLGQ MOR04947 VH: (SEQ ID NO: 19) QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYIGWVRQMPGKGLEWMGIIVP GTSYTIYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMRGIPFRMRGF DYWGQGTLVTVSS MOR04947 VL: (SEQ ID NO: 38) DIVLTQPPSVSGAPGQRVTISCSGSSSNIGSSFVNWYQQLPGTAPKLLIGNNSN RPSGVPDRFSGSKSGTSASLAITGLQSEDEADSFDMGSPNVVFGGGTK LTVLGQ MOR05145 VH: (SEQ ID NO: 20) QVQLVESGGGLVQPGGSLRLSCAASGFTFNNYGMTWVRQAPGKGLEWVSGI SGSGSYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVRTIYMDY WGQGTLVTVSS MOR05145 VL: (SEQ ID NO: 39) DIVLTQSPATLSLSPGERATLSCRASQNLFSPYLAWYQQKPGQAPRLLIYGASN RATGVPARFSGSGSGTDFTLTISSLEPEDFAVQYMTLPLTFGQGTKVEIK RT The CDR and FR sections of the variable regions of Table 19 are aligned in Table 20A for the heavy chains (SEQ ID NOS: 2-20, VH3 is SEQ ID NO: 125, VH5 is SEQ ID NO: 126 ), in Table 20B for the kappa light chains (SEQ ID NOS: 21, 22, 23, 27, 28 and 29, VK1 is SEQ ID NO: 127 and VK3 is SEQ ID NO: 128), and in the Table 20C for lambda light chains (SEQ ID NOS: 24, 25, 30, 31, 32, 33, 34, 35, 36 and 37; VL2 is SEQ ID NO: 129 and VL1 is SEQ ID NO: 130) .

Table 20A Alignment of the amino acid sequences of the heavy chain variable regions of anti-DKK1 antibodies (SEQ ID NOs: 2-20, 125-126) VH Table 20B Alignment of the amino acid sequences of the kappa light chain variable regions of anti-DKK1 antibodies (SEQ ID N Os: 24-25, 30-37, 129-130) Linkers of DKKl VL of the kappa VL sequences 1 2 3 4 5 6 7 8 9 10 5 6 7 8 9 oo Table 20C Alignment of the amino acid sequences of the lambda light chain variable regions of the anti-DKK1 antibodies (SEQ ID NOs :) Linkers of DKK 1 VL of the lambda sequences VL C0R1 C0R2 Structure 3 CDR3 1 2 3 4 5 6 7 8 9 10 p 12345678901234567890 1234567890 a b c d e 1123456789012345678 901234567890123456789012345678901234567890123458t678901234567 8 9 OO The consensus CDR sequences are provided at least in SEQ ID NOs: 40-48 and in Table 1 1 B. One skilled in the art can determine additional consensus CDR sequences from the alignments of Tables 20A- 20C, using conventional methods, and the methods provided herein. EQUIVALENTS From the above detailed description of the specific embodiments of the invention, it should be evident that novel antibodies and immunological fragments thereof have been described. Although particular embodiments have been disclosed in detail herein, this has been done by way of example for purposes of illustration only. In particular, the inventor contemplates that different substitutions, alterations, and modifications to the invention can be made without departing from the spirit and scope of the invention.

Claims (53)

1 . A composition comprising an antigen binding region that specifically binds to an epitope on a DKK1 polypeptide (SEQ ID NO: 1) and / or on a DKK4 polypeptide (SEQ ID NO: 122), wherein the antibody or the functional fragment thereof binds to at least one epitope on DKK1 or DKK4, or both. The antibody according to claim 1, wherein the binding to DKK1 or DKK4 is determined in at least one assay selected from: antagonism of transcription signalized by Wnt; link analysis based on electrochemiluminescence; linkage of immunosorbent assay linked with enzymes; FMAC; SET; SPR; concentration of osteocalcin (OCN) in serum; concentration of osteoprotegrin (OPG) in serum; concentration of propeptide nitrogenous procollagen type 1 (P 1 N P) in serum; ALP production; TopFlash, or improvement of osteolysis.

3. An isolated amino acid sequence selected from the group of SEQ ID NOs: 2-20 and SEQ I D NOs: 40-72, and the conservative or humaneered variants (humanized) thereof.

4. The isolated amino acid sequences according to claim 3, wherein the amino acid sequences are selected from:

a) the group of SEQ ID NOs: 2-20, 65-72, each comprising an antigen binding region comprising an H-CDR3 region; b) the group of SEQ ID NOs: 2-20, 57-64, each comprising an H-CDR2 region; c) the group of SEQ ID NO: 40 comprising the sequence GISGSGSYTYYADSVKF, the group of SEQ ID NO: 41 comprising the sequence GISYYGGNTYYADSVKF, and SEQ ID NO: 42 comprising the sequence GISYYGGSTYYADSVKF, each comprising the H region -CDR2 in consensus; d) the group of SEQ ID NOs: 2-5, 8-11, 20, 49-56, each comprising an H-CDR1 region; and e) the group of SEQ ID NO: 43 having the amino acid sequence GFTFSSYGMT, SEQ ID NO: 44 having the amino acid sequence GFTFNSYGMT, SEQ ID NO: 45 having the amino acid sequence GFTFSNYGMT, SEQ ID NO: 46 having the amino acid sequence GFTFSSYWMT, SEQ ID NO: 47 having the amino acid sequence GFTFSSYAMTF, and SEQ ID NO: 48 having the amino acid sequence GFTFSSYGMS, each comprising an H-CDR1 region in consensus; or f) SEQ ID NOs: 21-39 and 73-88; and the conservative or humaneered variants (human-designed) thereof.

5. The isolated antigen binding region according to claim 4, wherein the amino acid sequences

selected from the group of SEQ ID NOs: 21-39 each comprises an L-CDR3 region, the amino acid sequences selected from the group of SEQ ID NOs: 21-39, 73-80 each comprising a region L-CDR1, the amino acid sequences selected from the group of SEQ ID NOs: 21-39, 81-88 each comprise an L-CDR2 region, the amino acid sequences selected from the group of SEQ ID NOs: 21-39 each comprises a variable region of a light chain.

6. An isolated nucleotide sequence selected from the group of SEQ ID NOs: 97-103.

7. An isolated amino acid sequence encoded by a nucleotide sequence according to claim 6, and the humaneered (human-engineered) variants of the amino acid sequence, wherein: a) each of SEQ ID NOs: 97- 100 encodes a light chain of antigen binding; and b) each of SEQ ID NOs: 101-103 encodes an antigen binding heavy chain.

8. An isolated nucleotide sequence according to claim 6, wherein the nucleotide sequence is further optimized for its expression and / or for clinical use.

9. An isolated nucleotide sequence selected from the group of SEQ ID NOs: 104-110.

10. An isolated amino acid sequence encoded by a

nucleotide sequence according to claim 9, and the humaneered (human-engineered) variants of the amino acid sequence, wherein: a) each of SEQ ID NOs: 104-107 encodes an antigen binding light chain; and b) each of SEQ ID NOs: 108-110 encodes an antigen binding heavy chain.

11. An isolated nucleotide sequence according to claim 9, wherein the nucleotide sequence is optimized for its expression and / or for clinical use.

12. An isolated amino acid sequence selected from the group of SEQ ID NOs: 111-117, and the conservative or humaneered variants thereof, wherein: a) each of the SEQ ID NOs: 111- 114 encodes an antigen binding light chain; and b) each of SEQ ID NOs: 115-117 encodes an antigen binding heavy chain.

13. The isolated amino acid sequence according to claim 12, wherein the amino acid sequence is optimized for its expression and / or for clinical use.

14. An isolated nucleotide sequence selected from the group of SEQ ID NOs: 120-121.

15. An isolated amino acid sequence encoded by a nucleotide sequence according to claim 14, and

conservative or humaneered variants of the amino acid sequence, wherein: a) SEQ ID NO: 120 codes for an antigen binding variable region of a light chain, and b) SEQ ID NO: 121 codes for a variable region of antigen binding of a heavy chain.

16. An isolated nucleotide sequence according to claim 14, wherein the nucleotide sequence is optimized for its expression and / or for clinical use.

17. An isolated antigen binding region comprising a variable region of a light chain encoded by a nucleotide sequence selected from the SEQ group

ID NO: 120, and a variable region of a heavy chain encoded by a nucleotide sequence selected from the group of SEQ ID NO: 121.

18. An isolated amino acid sequence selected from the group of SEQ ID NOs: 118-119, and the conservative or humaneered variants thereof.

19. The isolated amino acid sequence according to claim 18, wherein SEQ ID NO: 118 illustrates an antigen binding variable region of a light chain.

20. The isolated amino acid sequence according to claim 18, wherein SEQ ID NO: 119 illustrates a variable antigen binding region of a heavy chain.

21. The isolated amino acid sequence according to

claim 18, wherein the amino acid sequence is optimized for its expression and / or for its clinical use.

22. An isolated amino acid sequence having an identity of at least 95 percent with at least one complementarity determining region selected from the regions illustrated in SEQ I D NOs: 2-121.

23. An isolated amino acid sequence having a consensus sequence of the complementarity determining regions, as shown in Tables 1 1 A and 1 1 B.

24. The isolated antibody according to claim 1, which comprises a scaffold selected from an IgM and an IgG, wherein the IgG is selected from a IgG1, IgG2, IgG3, or IgG4.

25. The isolated antibody according to claim 24, wherein the IgM or the IgG is selected from polyclonal or monoclonal.

26. An isolated antibody or a functional fragment thereof, which comprises an antigen binding region that is specific for an epitope of DKK1, wherein the antibody or functional fragment binds to DKK1 or DKK4, and prevents or ameliorates the development of a disease associated with DKK1 or a disease associated with DKK4.

27. An antigen binding region isolated from an antibody or functional fragment thereof according to claim 26.

28. An isolated antibody or a functional fragment thereof, which comprises an antigen binding region that is specific for an epitope of the target DKK1 or DKK4, or both, wherein the epitope comprises at least six or more amino acid residues of a Cys-2 domain of the target DKK1 or DKK4.

29. The isolated antibody or functional fragment according to any one of claims 1 to 28 above, which is selected from a whole immunoglobulin or a Fab fragment or a scFv antibody fragment thereof, a heavy chain antibody, and an antigen binding region thereof, on a non-immunoglobulin scaffold.

30. The isolated antibody or functional fragment thereof according to claim 29, wherein the epitope is a conformational epitope.

31 A pharmaceutical composition, which comprises at least one antibody or functional fragment according to claim 29, and a pharmaceutically acceptable carrier or excipient therefor.

32. A transgenic animal carrying a gene encoding an antibody or functional fragment thereof, according to claim 29.

33. A method for the treatment of a disorder or condition associated with the presence of DKK1 or DKK4, which comprises administering to a subject in need thereof, an effective amount of the pharmaceutical composition according to claim 31.

34. The method according to claim 33, wherein the disorder or condition is selected from osteolytic lesions - especially osteolytic lesions associated with a myeloma, especially a multiple myeloma, or with cancers of bone, breast, colon, melanocytes, hepatocytes, epithelium, esophagus, brain, lung, prostate, or pancreas, or metastases thereof; bone loss associated with transplantation; or it is an osteosarcoma, prostate cancer, hepatocellular carcinoma (HCC), myeloma, including multiple myeloma, diabetes, obesity, muscular waste, Alzheimer's disease, osteoporosis, osteopenia, rheumatism, colitis, and / or unwanted hair loss.

35. The method according to claim 33, wherein the method further comprises administering a second therapeutic agent.

36. The method according to claim 35, wherein the chemotherapeutic agent is Zometa.

37. The pharmaceutical composition according to claim 35, wherein the additional therapeutic agent is selected from the group consisting of an anticancer agent; an anti-metabolic agent; an anti-diabetic agent; an anti-osteoporotic agent; an antibiotic; an anti-inflammatory agent; a growth factor; and a cytokine.

38. An isolated antibody, which comprises a first amino acid sequence that is a heavy chain selected from the group consisting of SEQ I D NOs: 2-20, and a

sequence having a sequence identity of at least 95 percent in the regions determining complementarity with the regions of complementary complementarity illustrated in SEQ I D NOs: 2-20; and a second amino acid sequence which is a light chain selected from the group consisting of SEQ ID NOs: 21 -39, and a sequence having a sequence identity of at least 95 percent in the determined regions. ners of complementarity with the complementarity determining regions illustrated in SEQ ID NOs: 21 -39.

39. An immunoconjugate, which comprises a first component that is an antibody or fragment thereof according to any of the preceding claims.

40. A kit comprising an antibody or fragment thereof according to any of the preceding claims.

41 The kit according to claim 40, which further comprises a pharmaceutically acceptable carrier or excipient for the same.

42. The kit according to claim 40, wherein the antibody is present in a unit dose.

43. The kit according to claim 40, which further comprises instructions for use in the administration to a subject.

44. A composition, which comprises a neutralizing anti-DKK1 / 4 antibody.

45. The antibody of claim 40, wherein the

antibody binds to DKK1 with a Kact? vada of less than 100 nM, 50 nM, 10 nM, 1.0 nM, 500 pM or 100 pM; and has a deactivation index for DKK1 of less than 10"2 per second, 10" 3 per second, 10"4 per second, or 10.5 per second,

46. The antibody of claim 40, wherein the antibody has an affinity of 102 to 106 higher for DKK1 or DKK4, compared to DKK2 or DKK3

47. The antibody of claim 40, wherein the antibody competes against a neutralizing anti-DKK1 / 4 antibody by binding to DKK1 or DKK4.

48. A method for the treatment of diseases with a composition comprising at least one polypeptide sequence of SEQ ID NO: 118, and a polypeptide sequence of SEQ ID NO: 119, or conservative or humaneered changes (humanized). ) thereof, wherein the composition is a neutralizing anti-DKK1 / DKK4 antibody

49. A method for preventing or treating proliferative diseases, or diseases such as cancer, in a mammal, in particular in a human being, with a combination of pharmaceutical agents icos comprising: (a) a neutralizing anti-DKK1 / 4 composition; and (b) one or more pharmaceutically active agents. The invention further relates to pharmaceutical compositions comprising: (a) a neutralizing anti-DKK1 / 4 composition;

(b) a pharmaceutically active agent; and (c) a pharmaceutically acceptable carrier. The present invention further relates to a commercial package or a product comprising: (a) a pharmaceutical formulation of a neutralizing anti-DKK1 / 4 composition; and (b) a pharmaceutical formulation of a pharmaceutically active agent for simultaneous, concurrent, separate, or sequential use.

50. The method of claim 49, wherein the pharmaceutically active agent is in particular any pharmaceutically active agent other than a neutralizing anti-DKK1 / 4 composition, or a derivative thereof, which agent is selected from: i. an aromatase inhibitor; ii. an anti-estrogen, an anti-androgen, or a gonadorelin agonist; iii. a topoisomerase I inhibitor or a topoisomerase II inhibitor; iv. an active agent in microtubules, an alkylating agent, an anti-neoplastic anti-metabolite, or a platinum compound; v. a compound that directs / reduces the activity of a protein or lipid kinase, or the activity of a protein or lipid phosphatase, an additional anti-angiogenic compound, or a

compound that induces cellular differentiation processes; saw. monoclonal antibodies; vii. a cyclo-oxygenase inhibitor, a bisphosphonate, a heparanase inhibitor, a biological response modifier; viii. an inhibitor of ras oncogenic isoforms; and ix. a telomerase inhibitor; x. a protease inhibitor, a matrix metalloproteinase inhibitor, a methionine aminopeptidase inhibitor, or a proteasome inhibitor; xi. agents used in the treatment of hematological malignancies, or compounds that direct, reduce, or inhibit the activity of Flt-3; xii. an inhibitor of HSP90; xiii. anti-proliferative antibodies; xiv. a histone deacetylase inhibitor (HDAC); xv. a compound that directs, reduces, or inhibits the activity / function of mTOR serine / threonine kinase; xvi. a somatostatin receptor antagonist; xvii. an anti-leukemic compound; xviii. approaches that damage tumor cells; xix. an EDG linker; xx. a ribonucleotide reductase inhibitor; xxi. an S-adenosyl-methionine decarboxylase inhibitor; xxii. a monoclonal antibody of VEGF or VEGFR;

xxiii. photodynamic therapy; xxiv. an angiostatic steroid; xxv. an implant that contains corticosteroids; xxvi. an AT1 receptor antagonist; and xxvii. an ACE inhibitor.

51 An antibody that binds to DKK1 with an affinity (KD) of 10"8 or less, and competes with any of the antibodies of claim 1.

52. The antibody according to claim 51, which does not detectably bind to DKK2 or DKK3

53. The antibody of claim 52, wherein the antibody binds to DKK1 or DKK4 with a (KD) of 10"3 or higher.