MX2007016228A - Receptor antagonists for treatment of metastatic bone cancer - Google Patents

Abstract

The invention provides methods of treating bone cancer, particularly metastatic bone cancer, by administering an IGF-IR antagonist and/or a PDGFRαantagonist. The invention also provides antibodies that bind to human PDGFRαand neutralize activation of the receptor. The invention further provides a methods for neutralizing activation of PDGFRα, and a methods of treating a mammal with a neoplastic disease using the antibodie alone or in combination with other agents.

Description

ANTAGONISTS OF THE RECEIVER FOR THE TREATMENT OF OSEUM CANCER IN METASTASIS Field of the Invention The invention provides methods for treating bone cancer, particularly bone cancer in metastasis, by administering an IGF-IR antagonist and / or a PDGFRa antagonist. The invention also provides antibodies that bind to human PDGFRα and that neutralize the activation of the receptor. The invention further provides methods for neutralizing the activation of PDGFRα, and methods for treating a mammal with a neoplastic disease using antibodies alone or in combination with other agents. Background of the Invention Prostate cancer is the most common cancer among men, with approximately 220,000 cases and 29,000 deaths annually in the United States of America. A significant number of men diagnosed with prostate cancer have the disease in metastasis. In addition, metastasis eventually develops in many other patients with prostate cancer despite treatment with surgery or radiation therapy. Bone is the most common site of prostate cancer metastasis, and it is also a site in which breast cancers and lung cancers often metastasize. Most prostate cancer metastases are androgen dependent, so there is a rapid response to surgical or medical castration, but in virtually all patients, the tumor eventually becomes independent of androgen, leading to morbidity and significant mortality. Once bone metastasis occurs, the therapies currently available have limited their effect. The most effective approved therapy that has been described for prostate cancer metastasis (administration of docetaxel) extends the average survival to approximately three months. (Petrylak et al., 2004, N. Engl. J. Med. 351: 1513, Tannock et al., 2004, N. Engl. J. Med. 351: 1502). Therefore, new therapies are urgently needed for bone cancers in metastases. The insulin-like growth factor receptor (IGF-IR) is a ubiquitous transmembrane tyrosine kinase receptor that is essential for normal and postnatal fetal growth and development. IGF-IR is located on the cell surface of most cell types and serves as the signaling molecule for growth factors IGF-I and IGF-II (collectively later called IGFs). IGF-IR can stimulate cell proliferation, cell differentiation, cell size changes, and the protection of cells against apoptosis. It has also been considered that cell transformation is almost mandatory (reviewed in Adams et al., Cell, Mol.Life Sd. 57: 1050-93 (2000), Baserga, Oncogen 19: 5574-81 (2000)). The high levels of IGF-IR expression have been reported in the tissue samples of the bone metastasis of prostate cancer. The bone contains the largest deposit of IGFs in the body. IGF-IR is a preformed hetero-tetramer that contains two beta chains and two alpha chains covalently linked by disulfide bonds. The receptor subunits are synthesized as part of a single 180kd polypeptide chain, which are then processed proteolytically into the alpha (130kd) and beta (95kd) subunits. The entire alpha chain is extracellular and contains the site for ligand binding. The beta chain possesses the transmembrane domain, the tyrosine kinase domain, and an extension of the C-terminal that is necessary for cell differentiation and transformation, but is dispensable for mitogen signaling and for protection against apoptosis. IGF-IR is highly similar to the insulin receptor (IR), particularly within the beta chain sequence (70% homology). Because of this homology, recent studies have shown that these receptors can form hybrids that contain an IR dimer and an IGF-IR dimer (Pandini et al., Clin. Canc. Res. 5: 1935-19 (1999)). The formation of hybrids occurs in normal and transformed cells and the hybrid content is dependent on the concentration of the two homodimer receptors (IR and IGF-IR) within the cell. Although the hybrid receptors are composed of IR and IGF-IR pairs, the hybrids selectively bind to IGFs, with an affinity similar to that of IGF-IR, and only binds weakly to insulin (Siddle and Soos, The IGF System Humana Press, pp. 199-225, 1999). These hybrids can therefore bind to IGFs and transduce signals into normal and transformed cells. A second IGF receptor, IGF-IIR receptor, or mannose-6-phosphate receptor (M6P), also binds to the IGF-II ligand with high affinity, but lacks tyrosine kinase activity (Oates et al. , Breast Cancer Res. Treat 47: 269-81 (1998)). Because it leads to the degradation of IGF-II, it is considered a sink for IGF-II, antagonizing the effects that promote the growth of this ligand. The loss of IGF-IIR in tumor cells can improve the growth potential through the release of its antagonistic effect on the binding of IGF-II with IGF-IR (Byrd et al.; J. Biol. Chem. 274: 24408-16 (1999)). The alpha and beta receptors of platelet-derived growth factor (PDGFRa and PDGFRß) are type III receptor tyrosine kinases. PDGFRa is essential for development and fulfills important functions in adulthood. For example, mice homozygous for a null mutation die during embryogenesis. In later stages of development, PDGFRa is expressed in many mesenchymal structures, while adjacent epithelial cells produce platelet-derived growth factors (PDGFs). Tissue samples from normal or hyperplastic prostate glands were tested negative for PDGFRα, while primary prostate tumors and skeletal masses from the same subjects expressed PDGFRα. In addition, of the prostate cell lines obtained from different metastatic sites, PDGFRα was found in PC3 cells derived from bone metastasis, but not in the cell lines obtained from lymph node (LNCaP) and brain metastases (DU- 145).

The platelet-derived growth factor family of growth factors consists of five different disulfide-linked dimers, PDGF-AA, -BB, -AB, -CC, and -DD, which act via PDGFRa and PDGFRβ. These growth factors are dimeric molecules composed of disulfide-linked polypeptide chains that bind to two receptor proteins simultaneously and induce dimerization, autophosphorylation, and intracellular receptor signaling. PDGFRa and PDGFR / 3 are structurally similar and can form heterodimers as well as homodimers. Because PDGFRβ does not bind to the PDGF-A chain with high affinity, PDGF-AA activates only the aa receptor dimers, while PDGF-AB and PDGF-CC activate the aa and aβ receptor heterodimers.

Brief Description of the Invention This invention relates to the treatment of primary and metastatic bone tumors, including tumors originating from the prostate, breast, or lung and expressing insulin-like growth factor receptor I (IGF-IR) and / or the platelet-derived growth factor-alpha receptor (PDGFRa). Tumors that will be treated may be hormone / androgen dependent or hormone / androgen independent, and may have originated, for example, from the prostate, breast, or lung. The invention provides methods for treating a subject having a bone tumor, and methods for inhibiting the growth of a bone tumor. The methods comprise administering an effective amount of an IGF-IR antagonist or an effective amount of a PDGFRa antagonist. Antagonists of the receptor include antibodies and antibody fragments as well as small molecule intracellular inhibitors. The invention provides anti-IGF-IR or anti-PDGFRα antibodies that bind to their target receptor and inhibit ligand binding. The invention also provides antibodies and other antagonists that neutralize the activation of IGF-IR or PDGFRα. Certain additional antibodies promote the down-regulation of their target receptor, for example by internalization and / or degradation. Accordingly, the small molecule antibodies and antagonists function to inhibit the activation of the downstream signaling molecules such as Akt, p42 / p44, and MAPK. The methods include the use of the IGF-IR or PDGFRa antagonists alone, in combination with each other or in combination with other cancer therapeutics, such as chemotherapeutics and radiation. The invention also provides the antibodies and antibody fragments that bind to PDGFRa as well as the nucleotides and the host cell for the production of the antibodies. Antibodies block ligand binding and neutralize receptor activation. The invention also provides the use of the antibodies alone, in combination with other receptor antagonists or anti-neoplastic agents, or as conjugates for the treatment of neoplastic disease. Anti-PDGFR antibodies are used to treat, for example, ovarian tumors, breast tumors, lung tumors, hepatocellular tumors, gastrointestinal stromal tumors, melanomas, renal cell carcinomas, prostate tumors, and soft tissue sarcomas. Description of the Figures Figure 1 depicts the growth of subcutaneous LuCaP 35V xenograft tumors in castrated SCID mice during a treatment period started when the tumors had reached 150-200 mm3. Panel A: untreated controls; Panel B: animals were treated for four weeks with docetaxel (10 mg / kg or 20 mg / kg) alone, or in combination with anti-IGF-IR antibodies (40 mg / kg IMC-A12); Panel C: Serum PSA levels in untreated and treated SCID mice result in subcutaneous LuCaP 35V xenograft tumors. The treated mice received docetaxel (20 mg / kg) alone or docetaxel (10 mg / kg or 20 mg / kg) in combination with anti-IGF-IR antibodies (40 mg / kg EV1C-A12). The treatment started when the tumors had reached 150-200 mm3 and ended after four weeks. Figure 2 shows unicellular suspensions of LuCaP 35V xenograft tumors treated with docetaxel (10 mg / kg) alone (panel A) or in combination with anti-IGF-IR antibodies (40 mg / kg IMC-A12) (panel B) . The field labeled R1 corresponds to the apoptotic cells with fragmented DNA (labeled with increased FITC). Figure 3 shows DNA synthesis (BrDu uptake) in tumor xenografts after the completion of docetaxel treatment (10 mg / kg or 20 mg / kg) alone, and in combination with anti-IGF-IR antibodies (40 mg / kg BVIC-A12). Figure 4 represents the differential expression of genes associated with aggressiveness of the prostate tumor (TUBB), resistance to anti-androgen therapy (BIRC 5), and induction of apoptosis (IGFBP3) in prostate tumor cells in response to treatment with docetaxel and A12 and docetaxel alone.

Figure 5 shows serum A12 levels after stopping treatment. Figure 6 shows body weight (a measurement of total cytotoxicity) of non-sick animals treated continuously with docetaxel (10 mg / kg or 20 mg / kg) alone, or in combination with anti-IGF-IR antibodies (40 mg / kg MC-A12).

Figure 7 shows the effect of treatment with an anti-IGF-IR antibody (MC-A12) on PSA produced by the xenograft in SCID mice grafted with LuCaP 23.1 cells. Figure 8 shows a series of radiographs of SCID mice grafted with LuCaP 23.1 cells. A12 mice received 40 mg / ml BVIC-A12 i.p. three times a week for six weeks. X-rays were taken at the time of sacrifice. Figure 9 shows PSA (a) levels and representative radiographs (b) of SCID mice with intratibial xenografts of human prostate cells LuCaP 23.1. Figure 10 depicts the effect of human marrow aspiration on Akt activity in prostate cancer cells. The cell lysates were subjected to SDS-PAGE. For Western Blot analysis, the membranes were stained with phospho-Akt antibody detection (Ser-473, Cell Signaling Technology), PDGFRa (R & D Systems) and actin (Sigma). The binding of the primary antibody was detected using protein A or protein G conjugated with HRP (sigma). Figure 11 depicts the induction and inhibition of AKT phosphorylation in PC3-ML cells. Panel A shows the dose-dependent inhibition of AG-1296 of Akt phosphorylation in cells exposed to 30 ng / ml PDFG-BB. Panel B shows the phosphorylation of Akt and the inhibition by 20 μM AG-1296 of the bone aspirate. Panel C shows the potency of the spinal cord aspirate to induce Akt phosphorylation with respect to the potency of a combination of 100 pg / ml PDGF-AA and 100 pg / ml PDGF-BB. Panel D compares the magnitudes of Akt phosphorylation induced by spinal cord aspiration, inhibition of Akt phosphorylation induced by the spinal cord by AG-1296, and Akt phosphorylation induced by PDFG-AA + PDFG-BB. Figure 12 depicts the inhibition of Akt phosphorylation in PC3-ML cells by PDGFRa antagonists. Panel A shows the dose-dependent effect of monoclonal antibody IMC-3G3 on Akt phosphorylation induced by 30 ng / ml PDGF-BB. Panels B and C provide a comparison of the effects of IMC-3G3 and AG1296 on Akt phosphorylation induced by the spinal cord. Panel D shows that the inhibition of Akt phosphorylation is dependent on the pre-incubation time of IMC-3G3. Figure 13 shows the binding of the antibody to PDGFRα, A: binding of the anti-PDGFRα antibody to the immobilized extracellular domain of PDGFRα, B: inhibition of the binding of [125I] PDGF-AA to PDGFRα immobilized. Figure 14 shows the specific inhibition of phosphorylation of PDGFRα and descending effector molecules. Figure 15 shows the inhibition of the incorporation of [3H] thymidine stimulated by PDGF-AA in PAE Ra cells by mAbs. Figure 16 shows the inhibition of PDGF-AA-induced downstream activation in SKl cells MS-1 (a) and U118 (b). Figure 17 shows the inhibition of the incorporation of [3H] thymidine stimulated by PDGF-AA in U118 (a) cells and SKLMS-1 (b) by mAbs. The inhibition of incorporation of [3H] Thymidine stimulated by PDGF-AA is also shown for SKLMS-1 (c) and U118 (d) cells. Figure 18 shows the dose-dependent effects for the treatment of established tumor xenografts of U118 (glioblastoma, panel A) and SKLMS-1 (leiomyosarcoma; panel B) in mice without hair. Figure 19 shows the reduction of phosphorylation of PDGFRα in vivo in tumors of UL 18 treated with the anti-PDGFRα antibody, with respect to the treatment with non-specific human IgG. The figure 20 represents the GS expression vectors used for the cloning of human VH and VK variable region genes derived from the hybridoma and for the expression of whole human heavy chain (IgG 1) and light chain chain proteins. The two vectors were recombined as explained in the examples and the combined vector was transfected into the NSO cells. Detailed Description of the Invention The present invention relates to the treatment of bone tumors with antibodies or antibody fragments that bind to the insulin-like growth factor I receptor., (IGF-IR). The endocrine expression of IGF-I is mainly regulated by growth hormone and is produced in the liver, but other tissue types are also capable of expressing IGF-I, including bone that contains a large reservoir of factors. increase. Depending on the type of tumor cell, IGF-I is involved in the regulation of endocrine, paracrine, and / or autocrine (Yu, H. and Rohan, J., J. Nati. Cancer Inst. 92: 1472-89 (2000 )). It has been found that antibodies that bind to IGF-IR are useful in therapies for the treatment of bone tumors that express IGF-IR. The antibodies can be used alone, or in combination with other cancer therapeutics, particularly chemotherapeutics. Anti-IGF-IR therapy, alone or in combination with therapy with one or more anti-neoplastic agents (such as, for example, radiotherapy or chemotherapy) has a significant therapeutic efficacy. The suppression of tumor growth is often accompanied by an increase in apoptosis and persists after all treatment is discontinued and tumors have started to grow again in animals treated with chemotherapy alone. It has also been discovered that PDGFRce plays an important role in the growth of bone tumors. For example, certain tumor cell lines expressing PDGFRa preferentially become metastases in the bone. Such display of cell lines increased the activation of PDGFRα and the phosphorylation of downstream signaling molecules in response to the soluble factors present in the spinal cord. Activation of PDGFRα by the spinal cord is reduced or completely inhibited by PDGFRα antagonists, and phosphorylation of the descending signaling molecules that are commonly activated by signaling via PDGFRα and other tyrosine systems is greatly reduced. receptor kinase. Some data suggest that the survival trajectory of PI3K / Akt is activated by the PDGFRα signaling and not only by the ligands that directly activate PDGFRα, but also by the factors present in the spinal cord that cause the transactivation of the receptor. Primary bone tumors to be treated according to the invention include, but are not limited to, osteosarcomas, chondrosarcomas, fibrosarcomas, and hemangiosarcomas.

Notably, malignant secondary tumors (in metastases) are much more common than primary bone tumors. Bone tumors in metastases to be treated according to the invention may be presented from a variety of sources, the most common of which are prostate, breast, or lung cancers. The source of a bone cancer in metastasis will usually be evident from the medical history of the patients. Tumors can be osteoblastic or osteolytic. Tumors may depend on the stimulus of IGF-IR when they occur, or may transcend the dependence of IGF-IR. For example, prostate cancers or metastases from prostate cancers that are initially dependent on the hormone / androgen and are controllable by physical or chemical treatments that suppress the production of androgen or hormone, may become independent of the hormone / androgen at through increased sensitivity to stimulation with IGF-IR. In addition to providing treatment of the hormone / androgen independent tumors, the invention may be useful for treating hormone / androgen dependent bone tumors without relying on the suppression of androgen and hormone production, for example, by co-administration of IGF-IR antibodies with anti-neoplastic agents. Such tumors would include bone tumors in metastases that are stimulated through IFG-IR in the bone-rich IGF environment, which may be sensitive to hormone stimulation but not sensitive enough to develop without the involvement of IGF. The elimination of the hormone may not be necessary for such tumors. Bone tumors that are dependent on PDGF can also be treated according to the invention, as well as tumors that are dependent on the "spinal cord". Spinal cord-dependent tumors exhibit the activation of PDGFRα in response to soluble factors present in the spinal cord. For example, as exemplified herein, a human cancer cell line in metastasis expressing PDGFRα undergoes activation of PDGFRα and phosphorylation of Akt + during exposure to spinal cord aspiration. An anti-PDGFRα antibody and a small molecule PDGFRα antagonist each inhibit the activation of PDGFRa and the phosphorylation of Akt + in the cell line. Soluble factors of the spinal cord that activate PDGFRa include, but are not limited to, PDGF-AA and -BB. While such dependence on the spinal cord implies signaling with PDGFRa; may not only imply joining PDGFRα of a ligand of PDGFRα. For example, as exemplified herein, it is observed that the activation of PDGFRa by the defined ligands (PDGF-AA or -BB) is weaker than activation by the spinal cord aspirate. In addition, it is observed that in the presence of the spinal cord aspirate, the phosphorylation of Akt + decreases with the increase of the incubation time. Taken together, these results suggest that in addition to responding to PDGFα binding, PDGFRα can be transactivated (phosphorylated) by other elements of signal transduction (eg, other receptor tyrosine kinases) sensitive to other components of the spinal cord. In any case, in a cell line adapted for growth in bone metastasis (ie, a cell line that preferentially metastasizes to the bone), activation of PDGFRα dependent on the spinal cord is observed, which is inhibited by the PDGFRa antagonists. In addition, treatment with a PDGFRa antagonist inhibits the stimulation induced by the spinal cord of the anti-apoptotic pathway of PI3K / Akt and mitogen-activated protein kinase (MAPK). Bone tumors that will be treated with a PDGFRa antagonist may present as metastases to prostate cancer cells, and, as previously, may depend on the hormone / androgen, or have become independent of the the hormone / androgen. Such tumors may also present as metastases from cancers other than prostate cancers. The person skilled in the art could easily diagnose such conditions and disorders using conventional known tests. Treatment means any treatment of a disease in an animal and includes: (1) preventing the disease from occurring in a mammal that may be predisposed to the disease but still does not experience or exhibit the symptoms of the disease; for example, the prevention of the onset of clinical symptoms; (2) inhibit the disease, for example, stop its development; or (3) alleviate the disease, for example, by causing the symptoms of the disease to recede. Inhibition of tumor growth includes slowing or stopping growth, as well as causing the tumor to regress. An effective amount for the treatment of a disease means the amount that, when administered to a mammal in need thereof, is sufficient to effect the treatment, as defined above, of such a disease. The IGF-IR antagonists and the PDGFRα antagonist of the invention can be administered alone, in combination with each other, or in combination with one or more anti-neoplastic agents such as, for example, a chemotherapeutic or radiological agent. In one embodiment of the invention, it may be desirable to determine the level of expression of IGF-IR and / or PDGFRa in a tumor to be treated. In such cases, tumor biopsies can be collected and analyzed by methods well known in the art. In another embodiment of the invention, an IGF-IR antagonist or PDGFRa antagonist is administered on the basis that the corresponding receptor is commonly expressed or activated in a particular type of tumor or that it is invariably expressed as activated as the disease progresses. An IGF-IR antagonist can be an extracellular antagonist or an intracellular antagonist and more than one antagonist can be used. Extracellular antagonists include, but are not limited to proteins or other biological molecules that bind IGF-IR or one or more of its ligands (eg, IGF-I and IGF-II are natural ligands of IGF-IR). In one embodiment of the invention, an extracellular antagonist inhibits the binding of IGF-IR to its ligands. In one embodiment, the antagonist is an anti-IGF-IR antibody, such as, for example, IMC-A12. In another embodiment, the antagonist is a soluble ligand binding fragment of IGF-IR. IGF-IR intracellular antagonists can be biological molecules, but they are commonly small molecules. Examples include, but are not limited to, the tyrosine kinase inhibitor AG1024 (Calbiochem), inhibitor of the insulin-like growth factor-kinase receptor I NVP-AEW541 (Novartis), and the insulin receptor inhibitor / insulin-like growth factor I BMS-554417 (Bristol Myers Squibb). It will be appreciated that the useful small molecules to be used in the invention are IGF-IR inhibitors, but do not need to be totally specific for IGF-IR. Anti-IGF-IR Antibodies to be used according to the present invention exhibit one or more of the following characteristics: 1) Antibodies bind to the external domain of IGF-IR and inhibit the binding of IGF-I or IGF-II to IGF-IR. Inhibition can be determined, for example, by direct binding analysis using the membrane bound or purified receptor. In this embodiment, the antibodies of the present invention, or fragments thereof, preferably bind to IGF-IR at least as strongly as the natural IGF-IR ligands (IGF-I and IGF-II). 2) The antibodies neutralize IGF-IR. The binding of a ligand, for example, IGF-I or IGF-II, to an extracellular external domain of IGF-IR, stimulates the autophosphorylation of the beta subunit and of the descending signaling molecules, including MAPK, Akt, and IRS- 1. The neutralization of IGF-IR includes the inhibition, decrease, inactivation and / or interruption of one or more of these activities normally associated with signal transduction. Neutralization can be determined in vivo, ex vivo, or in vitro using, for example, tissues, cultured cells, or purified cellular components. Neutralization includes the inhibition of IGF-IR / IR heterodimers as well as IGF-IR homodimers. Thus, the neutralization of IGF-IR has several effects, including inhibition, decrease, inactivation and / or growth arrest (proliferation and differentiation), angiogenesis (selection, invasion, and metastasis of the blood vessel), and motility and cell metastasis ( adhesion and cellular invasion). A measurement of the neutralization of IGF-IR is the inhibition of receptor tyrosine kinase activity. Inhibition of tyrosine kinase can be determined using well-known methods; for example, by measuring the level of autophosphorylation of the recombinant kinase receptor, and / or the phosphorylation of natural or synthetic substrates. Thus, phosphorylation assays are useful in determining the neutralization of the antibodies in the context of the present invention. Phosphorylation can be detected, for example, by using an antibody specific for phosphotyrosine in an ELISA or in a Western Blot. Some assays for tyrosine kinase activity are described in Panek et al., J. Pharmacol. Exp. Thera. 283: 1433-44 (1997) and Batley et al., Life Sci. 62: 143-50 (1998). The antibodies of the invention cause a decrease in tyrosine phosphorylation of IGF-IR of at least about 75%, preferably at least about 85%, and more preferably at least about 90% in cells that respond to the ligand. Another measure of the neutralization of IGF-IR is the inhibition of the phosphorylation of the descending substrates of IGF-IR. Accordingly, the level of phosphorylation of MAPK, Akt, or IRS-I can be measured. The decrease in phosphorylation is at least about 40%, and can be at least about 60%, or at least about 80%. In addition, methods for the detection of protein expression can be used to determine the neutralization of IGF-IR, where the proteins that are measured are regulated by the activity of the tyrosine kinase of IGF-IR. These methods include immunohistochemistry (IHC, for its acronym in English) for the detection of protein expression, in situ hybridization of fluorescence (FISH, for its acronym in English) for the detection of genetic amplification, competitive radioligand binding analysis, spotting techniques solid matrix, such as Northern and Southern Blot, reverse transcriptase polymerase chain reaction (RT-PCR) and ELISA. See, for example, Grandis et al., Cancer, 78: 1284-92 (1996); Shimizu et al., Japan J. Cancer Res., 85: 567-71 (1994); Sauter et al., Am. J. Path., 148: 1047-53 (1996); Collins, GHa 15: 289-96 (1995); Radinsky et al., Clin. Cancer Res. 1: 19-31 (1995); Petrides et al., Cancer Res. 50: 3934-39 (1990); Hoffmann et al., Anticancer Res. 17: 4419-26 (1997); Wikstrand et al., Cancer Res. 55: 3140-48 (1995). Ex vivo analyzes can also be used to determine the neutralization of IGF-IR. For example, inhibition of the tyrosine kinase receptor can be observed by mitogenic analysis using the cell lines stimulated with the receptor ligand in the presence and absence of the inhibitor. The MCF7 cell line of breast cancer (American Type Culture Collection (ATCC), Rockville, MD) is the cell line that expresses IGF-IR and is stimulated by IGF-I or IGF-II. Another method involves testing the inhibition of growth of tumor cells expressing IGF-IR expressing or of the cells transfused to express IGF-IR. Inhibition can also be observed using tumor models, for example, human tumor cells injected into a mouse. The antibodies of the present invention are not limited by any particular mechanism of neutralization of IGF-IR. The anti-IGF-IR antibodies of the present invention can be externally bound to the IGF-IR cell surface receptor, block ligand binding (eg, IGF-I or IGF-II) and then signal transduction mediated via the tyrosine kinase associated with the receptor, and prevent the phosphorylation of IGF-IR and other descending proteins in the signal transduction cascade. 3) The antibodies inframodulate IGF-IR. The amount of IGF-IR present on the surface of a cell depends on the production, internalization, and degradation of the receptor protein. The amount of IGF-IR present on the surface of a cell can be measured indirectly, by detecting the internalization of the receptor or molecule bound to the receptor. For example, internalization of the receptor can be measured by contacting the cells expressing IGF-IR with a labeled antibody. The antibody bound to the membrane was then treated, collected and counted. The internal antibody is determined by treating the cells with Usinas and detecting the label in the lysates. Another way is to directly measure the amount of the receptor present in the cell after treatment with an anti-IGF-IR antibody or another substance, for example, by fluorescence-activated cell sorting analysis of stained cells for surface expression of IGF -GO. The stained cells are incubated at 37 ° C and the fluorescence intensity was measured over a period of time. As a control, part of the stained population can be incubated at 4 ° C (conditions under which internalization of the receptor is stopped). IGF-IR of the cell surface can be detected and measured using a different antibody that is specific to IGF-IR and that does not block or compete with the binding of the antibody being tested. (Burtrum, et al Cancer Res. 63: 8912-21 (2003)). The treatment of a cell expressing IGF-IR with an antibody of the invention results in the reduction of cell surface IGF-IR. In a preferred embodiment, the reduction is at least about 70%, more preferably at least about 80%, and even more preferably at least about 90% in response to treatment with an antibody of the invention. A significant decrease can be observed at only four hours elapsed. Another measure of infra-modulation is the reduction of the total receptor protein present in a cell, and reflects the degradation of internal receptors. Accordingly, treatment of the cells (particularly cancer cells) with the antibodies of the invention results in a reduction of total cellular IGF-IR. In a preferred embodiment, the reduction is at least about 70%, more preferably of at least about 80%, and even more preferably of at least about 90%. For the treatment of human subjects, the antibodies according to the invention are preferably human. Alternatively, the antibodies may be from non-human primates or other mammals, or be humanized or chimeric antibodies. In one embodiment of the invention, an anti-IGF-IR antibody comprises one, two, three, four, five, and / or six complementary determination regions (CDRs) selected from the group consisting of SEQ ID NO: 35, SEC ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 45, SEQ ID NO: 47, and SEQ ID NO: 49 (CDRIH, CDR2H, CDR3H, CDR1L, CDR2L, CDR3L, respectively). In another embodiment, the anti-IGF-IR antibody comprises one, two, three, four, five, and / or six regions of complementarity determination (CDRs) selected from the group consisting of SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 55, SEQ ID NO: 57, and SEQ ID NO: 59 (CDR1H, CDR2H, CDR3H, CDR1L, CDR2L, CDR3L, respectively). Preferably, the antibodies (or fragments thereof) of the present invention have heavy chain CDRs of SEQ ID NO: 35, SEQ ID NO: 37 and SEQ ID NO: 39. Alternatively and preferably also, the present antibodies include the fragments thereof, have light chain CDRs of SEQ ID NO: 45, SEQ ID NO: 47 and SEQ ID NO: 49 or SEQ ID NO: 55, SEQ ID NO: 57 and SEQ ID NO: 59. Such anti-HIV antibody. IGF-IR is the human IgG 1 antibody IMC-A12 (WO2005016970), which has a heavy chain variable domain represented by SEQ ID NO: 41 and a light chain variable domain represented by SEQ ID NO: 51. Other The preferred human antibody is IMC-2F8 (WO2005016970), which has a heavy chain variable domain identical to IMC-A 12 and a light chain variable domain represented by SEQ ID NO: 61. Antibodies useful in addition include anti- body antibodies. IGF-IR that compete with MC-A12 or IMC-2F8 to bind to IGF-IR, as well as the antibodies that are bind to other epitopes (ie, antibodies that bind to other epitopes and exhibit the properties as previously described, such as ligand block, receptor internalization, etc., but do not compete with BVIC-A12 or IMC-2F8 ). According to the invention, PDGFRα antagonists can also be used for the treatment. A PDGFRα antagonist can be an extracellular antagonist or an intracellular antagonist and more than one antagonist can be used. Extracellular antagonists include, but are not limited to proteins or other biological molecules that bind PDGFRα or one or more of its ligands (eg, PDGF-AA, -AB, -BB, -CC). In one embodiment of the invention, an extracellular antagonist inhibits the binding of PDGFRα to its ligands. In one embodiment, the antagonist is an anti-PDGFRα antibody, such as, for example, IMC-3G3. In another embodiment, the binding protein is a soluble ligand binding fragment of PDGFRα. IGF-IR intracellular antagonists can be biological molecules, but they are usually small molecules. In one embodiment, the intracellular PDGFRα antagonist is AG1296. AG1296 (Calbiochem) is an inhibitor of PDGFa, PDGFIß, and c-KIT, and also reacts with Flt3. The other small molecules that detect PDGFRα include STI-571 (imatinib mesylate, Gleevec®, Novartis) and SU11248 (sunitinib malate, SUTENT®, Pfizer).

In one embodiment of the invention, an anti-PDGFR antibody comprises one, two, three, four, five, and / or six complementary determination regions (CDRs) selected from the group consisting of SEQ ID NO: 2, SEQ ID NO. : 4, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 14 (CDR1H, CDR2H, CDR3H, CDR1L, CDR2L, CDR3L, respectively). Preferably, the antibodies (or fragments thereof) of the present invention have CDRs of the SEC ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6. Alternatively and preferably also, the present antibodies, or fragments thereof, have CDRs of SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 10. NO: 14. The amino acid sequences of the CDRs are set forth below in Table 1.

Table 1 - CDRs of IMC-3G3 In another embodiment, the anti-PDGFRα antibody, or fragments thereof, has a human heavy chain variable region of SEQ ID NO: 8 and / or a human light chain variable region of SEQ ID NO: 16. IMC-3G3 is such an antibody and is exemplified in the present invention. Preferably, the antibodies, or fragments thereof, of the present invention neutralize PDGFRα. Binding of a ligand, eg, PDGF-AA, PDGF-AB, PDGF-BB or PDGF-CC, to an extracellular domain of PDGFRα, stimulates receptor dimerization, autophosphorylation, activation of the receptor cytoplasmic internal tyrosine kinase domain , and the initiation of the multiple transduction and signal transactivation trajectories involved in the regulation of DNA synthesis (gene activation) and in the progress or division of the cell cycle. Anti-PDGFRα antibodies commonly block ligand binding and / or dimerization of the receptor, and inhibit one or more of autophosphorylation, activation of tyrosine kinase activity and signal transduction. The anti-PDGFRα antibodies of the present invention may be specific for the extracellular ligand binding region of PDGFRα and for preventing binding of a PDGFRα ligand. Preferably, such anti-PDGFRα antibodies, or fragments thereof, bind to PDGFRα at least as strong as the natural ligands of PDGFRα Alternative or additionally, the antibodies may be specific for a region of the receptor monomer that would otherwise form an interface of the receiver's dimer. Such antibodies block the formation of the dimer, although binding of the ligand to a monomer of the receptor may or may not be blocked. As described above for anti-IGF-IR antibodies, neutralization of the receptor can be determined by a variety of methods in vivo, in vitro, and ex vivo. In one embodiment of the invention, anti-PDGFRα antibodies reduce the phosphorylation of PDGFRα to at least about 75%. In other embodiments, the phosphorylation is reduced to at least about 85% or at least about 90%. In one embodiment of the invention, as a result of the inhibition of PDGFRα signal transduction, phosphorylation or of a downstream signal transduction path component (eg, Akt, p42 / p44, etc.) is reduced to at least about 40%, at least about 60%, or at least about 80%. The neutralization of the receptor can be determined using defined ligands (eg, PDGF-AA, -AB, -BB, -CC), mixtures of such ligands, or preparations such as spinal cord aspirates comprising PDGFs as well as other factors of growth stimulants. Neutralization of PDGFRa includes the inhibition, decrease, inactivation and / or interruption of one or more of these activities normally associated with signal transduction. Thus, the neutralization of PDGFRa has several effects, including inhibition, decrease, inactivation and / or growth arrest (proliferation and differentiation), angiogenesis (selection, invasion, and metastasis of the blood vessel), and motility and cell metastasis (adherence and cellular invasion). The ex vivo analyzes, as described above, can also be used to determine the neutralization of PDGFRα. For example, human leiomyosarcoma cells from SKLMS-1 (American Type Culture Collection (ATCC), Rockville, MD; ATCC HTB-88 ™) or glioblastoma cells fromU118 (ATCC HTB-15 ™) stimulated with PDGF-AA can be used to analyze the inhibition of PDGFRα. Growth inhibition can be checked using human tumor cells expressing PDGFRα injected into a SCID mouse. The present invention is not limited by any particular PDGFRa neutralization mechanism. The anti-PDGFRα antibodies of the present invention bind externally to the PDGFRα cell surface receptor, block ligand binding (eg, PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC), inhibit phosphorylation of PDGFRα inhibit receptor-mediated tyrosine kinase-mediated signal transduction and modulate the activity of downstream signal transduction components. The receptor-antibody complex can also internalize and degrade, resulting in infra-regulation of the cell surface receptor. Matrix metalloproteinases, which function in the invasion and metastasis of tumor cells, may also be under-regulated by the antibodies of the present invention. On the other hand, antibodies of the present invention may exhibit inhibition of growth factor angiogenesis and production. As described above, the PDGFRα antagonists of the invention are useful for treating bone tumors, including bone tumors in metastases. Other types of tumors that express PDGFRα and that can be treated according to the invention include, but are not limited to, ovarian tumors, breast tumors, lung tumors, hepatocellular tumors, gastrointestinal stromal tumors, melanoma, renal cell carcinoma, tumors of prostate, and soft tissue sarcomas. Soft tissue sarcomas originate in tissues such as fat, muscles, nerves, tendons, and blood and lymph vessels. Commonly, tumor cells overexpress PDGFRa. The expression of PDGFRα can be determined, for example, by histochemistry or RNA analysis. For example, a Scatchard analysis of radiolabeled IMC-3G3 binding to U118 cells and SKLMS-1 tumor cells indicates that the number of PDGFRα molecules in the cells will be approximately 500 and 2500, respectively. PDGFRα antagonists function by inhibiting signal transduction by PDGFRα expressed on tumor cells by themselves, or by inhibiting PDGFRα expressed on surrounding stromal cells that otherwise undergo paracrine stimulation by expressed PDGFα from tumor cells. Thus, antibodies such as IMC-3G3 and other PDGFRa antagonists are useful for treating tumors characterized by the autocrine and / or paracrine stimulation of PDGFRα. Antibody fragments according to the invention can be produced by dividing an entire antibody, or by expressing the DNA encoding the fragment. Antibody fragments can be prepared by the methods described by Lamoyi et al., J. Immunol. Methods, 56: 235-243 (1983) and by Parham, J. Immunol. 131: 2895-2902 (1983). Such fragments may contain one or both Fab fragments or F (ab ') 2 fragments. Such fragments may also contain the variable region antibodies of the single chain fragment, ie scFv, antibodies, or other fragments of the antibody. Methods for producing such functional equivalents are described in PCT Application WO 93/21319, European Patent Application No. EP 239400; PCT Application WO 89/09622; Application European Patent EP 338745; and European Patent Application EP 332424. Preferred host cells for the transformation of the vectors and for the expression of the antibodies of the present invention are mammalian cells, for example, COS-7 cells, Chinese hamster ovarian cells (CHO, for its acronym in English), and cell lines of lymphatic origin such as lymphoma, myeloma (for example NSO), or hybridoma cells. Other eukaryotic hosts, such as yeasts, can be used alternatively. Where it is desired to express a genetic construct in the yeast, a suitable selection gene for use in yeast is the trp 1 gene present in the yeast plasmid YRp7. Stinchcomb et al. Nature, 282: 39 (1979); Kingsman et al., Gene, 7: 141 (1979). The trp 1 gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1. Jones, Genetics, 85: 12 (1977). The presence of the trp 1 lesion in the genome of the yeast host cell then provides an effective environment for detecting transformation by development in the absence of tryptophan. Similarly, Leu2 deficient yeast strains (ATCC 20,622 or 38,626) are complemented by the known plasmids containing the Leu2 gene. The transformed host cells are cultured by methods known in the art in a liquid medium containing the assimilable sources of carbon (carbohydrates such as glucose or lactose), nitrogen (amino acids, peptides, proteins or their degradation products such as peptones, salts of ammonium or the like), and inorganic salts (sulphates, phosphates and / or carbonates of sodium, potassium, magnesium and calcium). The medium further contains, for example, growth stimulating substances, such as trace elements, for example iron, zinc, manganese and the like. High affinity anti-PDGFRα and anti-IGF-IR antibodies according to the present invention can be isolated from a bacteriophage display library constructed of heavy and light chain variable region human genes. For example, a variable domain of the invention can be obtained from a peripheral blood lymphocyte containing a changed variable region gene. Alternatively, variable domain portions, such as CDR and FW regions, can be obtained from different sources and recombined. In addition, portions of the variable domains (e.g., FW regions) may be synthetic consensus sequences.

The antibodies and antibody fragments of the present invention can be obtained, for example, from natural antibodies or Fab or scFv bacteriophage display libraries. It is understood that, in order to make a single domain antibody from an antibody comprising a VH and VL domain, certain amino acid substitutions outside of CDRs may be desired to improve binding, expression or solubility. For example, it may be desirable to modify the amino acid residues that would otherwise be hidden at the VH-VL interface. In addition, antibodies and antibody fragments of the invention can be obtained by standard hybridoma technology (Harlow &Lane, ed., Antibodies: A Laboratory Manual, Cold Spring Harbor, 211-213 (1998), which is incorporated by reference herein) using the transgenic mice (e.g., KM mice from Medarex, San Jose, Calif.) that produce the light chain and gamma heavy chains of human immunoglobulin. In a preferred embodiment, a substantial portion of the human antibody that produces the genome is inserted into the mouse genome, and are deficient in the production of the endogenous murine antibodies. Such mice can be immunized subcutaneously (s.c.) with PDGFRα (generally in a complete Freund's adjuvant) with stimulations as needed. Immunization methods are well known in the art.

The protein used to identify the IGF-IR binding antibodies of the invention are preferably IGF-IR and, more preferably, it is the extracellular domain of IGF-IR. The protein used to identify the PDGFRα binding antibodies of the invention is preferably PDGFRα and, more preferably, it is the extracellular domain of PDGFRα. Such extracellular domains may be free or conjugated to other molecules.

The present invention also provides the isolated polynucleotides encoding the antibodies, or fragments thereof, previously described. Details of the anti-IGF-IR IMC-A12 antibody are described in WO2005016970. Table 2 sets forth the nucleic acid sequences for MC-3G3.

Table 2 - CDRs that encode the Nucleotide Sequences of IMC-3G3 The human antibodies encoding the DNA can be prepared by recombining the human constant regions and the variable regions encoding the DNA, other than CDRs, derived substantially or exclusively from the corresponding human regions of the antibody and the CDRs encoding the DNA derived from a human (SEQ ID NO: 1, 3, and 5 for heavy chain variable domain CDRs and SEQ ID NO: 9, 11, and 13 for light chain variable domain CDRs). Suitable sources of DNA encoding those of the antibodies include any cells, such as hybridomas and spleen cells, that express the integral antibody. The fragments can be used by themselves as antibody equivalents, or they can be recombined in equivalents, as described above. The deletions and DNA recombinations described in this section can be performed by known methods, such as those described in the publications listed above with respect to the equivalents of the antibodies and / or other standard recombinant DNA techniques, such as those described below. . Another source of DNA is the single chain antibodies produced from a bacteriophage display library, as is known in the art. In addition, the present invention provides expression vectors containing the previously described polynucleotide sequences operably linked to an expression sequence, promoter and an enhancer sequence. A variety of expression vectors have been developed for efficient synthesis of the antibody polypeptide in prokaryotes, such as bacteria and eukaryotic systems, including but not limited to yeast and mammalian cell culture systems. The vectors of the present invention may comprise segments of chromosomal DNA sequences, not chromosomal and synthetic. Any suitable expression vector can be used. For example, prokaryotic cloning vectors include E. coli plasmids, such as colEI, pCRI, pBR322, pMB9, pUC, pKSM, and RP4. Prokaryotic vectors also include DNA derivatives of bacteriophages such as MI3 and other filamentous bacteriophages of single-stranded DNA. An example of a vector useful in yeast is the 2μ plasmid. Suitable vectors for expression in mammalian cells include well-known derivatives of SV40, adenoviruses, DNA sequences derived from retroviruses and transfer vectors derived from the combination of functional mammalian vectors, such as those described above, and functional plasmids and bacteriophage DNA. . Additional eukaryotic expression vectors are known in the art (eg, P.J. Southern and P. Berg, J. Mol.Appl. Genet., 1, 327-341 (1982).; Subramani et al., Mol. Cell. Biol., 1: 854-864 (1981); Kaufmann and Sharp, "Amplification And Expression of Sequences Cotransfected with a Modular Dihydrofolate Reduced Complementary DNA Gene, "J. Mol. Biol. 159.601-621 (1982), Kaufmann and Sharp, Mol.Cell. Biol. 159.601-664 (1982); col., "Expression and Characterization of the Product of Human Immune Interferon DNA Gene In Chínese Hamster Ovary Cells, "Proc. Nat'l Acad. Sci. USA 80, 4654-4659 (1983); Uriaub and Chasin, Proc. Nat'l Acad. Sci. USA 77, 4216-4220, (1980). Expression vectors useful in the present invention contain at least one expression control sequence that is operably linked to the DNA sequence or fragment to be expressed.The control sequence is inserted into the vector to control and regulate expression of the cloned DNA sequence Examples of useful expression control sequences are the lac system, trp system, tac system, trc system, principal operator regions and promoter of bacteriophage lambda, the control region of the protein coated with fd , yeast glycolic promoters, for example, the promoter for 3-phosphoglycerate kinase, acid phosphatase promoters, for example, Pho5, promoters of yeast alpha mating factors, and promoters derived from polyoma, adenovirus, retroviruses , and simian virus, for example the anterior and posterior promoters or SV40, and other sequences known to control the expression of the genes of prokaryotic or eukaryotic cells and their viruses or combinations thereof. The present invention also provides recombinant host cells that contain the expression vectors described previously. The antibodies of the present invention can be expressed in cell lines other than hybridomas. The nucleic acids, comprising a sequence that codes for a polypeptide according to the invention, can be used for the transformation of a suitable mammalian host cell. The cell lines of particular preference are selected based on the high level of expression, constitutive expression of the protein of interest and minimal contamination of the host proteins. Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines, for example but not limited to, NSO cells, Chinese hamster ovarian cells (CHO), baby hamster kidney cells ( BHK, for its acronym in English) and many others. Additional convenient eukaryotic cells include yeast and other fungi. Useful prokaryotic hosts include, for example, E. coli, such as E. coli SG-936, E. coli HB 101, E. coli W3110, E. co // 'X1776, E. co // X2282, E. co // 'DHI, and E. co // MRCI, Pseudomonas, Bacilus, such as Bacilus subtilis, and Streptomyces. These present recombinant host cells can be used to produce an antibody, or fragments thereof, by culturing the cells under conditions that allow the expression of the antibody or fragment thereof and purifying the antibody or fragment thereof from the host cell or medium that surrounds the host cell. Detection of the antibody or fragment expressed for secretion in the recombinant host cells can be facilitated by inserting a signal-secretory or secretory signal peptide coding sequence (see, Shokri et al., Appl Microbiol Biotechnol.60 (6): 654-64). (2003), Nielsen et al., Prot. Eng 10: 1-6 (1997) and von Heinje et al., Nucí Acids Res. 14: 4683-4690 (1986)) at the 5 'end of the coding gene of interest antibody. These major secretory peptide elements can be derived from prokaryotic or eukaryotic sequences. Accordingly, the secretory major peptides, which are amino acids attached to the N-terminal end of a polypeptide, are used conveniently to direct the movement of the polypeptide out of the cytosol of the host cell and the secretion into the medium. The antibodies of this invention can be fused to the additional amino acid residues. Such amino acid residues may be a peptide tag, maybe to facilitate the isolation. Other amino acid residues are also contemplated for locating the antibodies in specific organs or tissues. In another embodiment, an antibody of the present invention is made by expressing a nucleic acid encoding the antibody in a transgenic animal, such that the antibody is expressed and can be recovered. For example, the antibody can be expressed in a tissue in a specific manner that facilitates recovery and purification. In such an embodiment, an antibody of the invention is expressed in the mammary gland for secretion during lactation. Transgenic animals, include but are not limited to, mice, goats, and rabbits. Antibodies which can be used according to the invention include the complete immunoglobulins, immunoglobulin antigen binding fragments, as well as the antigen binding proteins comprising the antigen binding domains of immunoglobulins. Immunoglobulin antigen binding fragments include, for example, Fab, Fab ', and F (ab') 2. Other antibody formats have been developed, which retain the binding specificity, but have other characteristics that may be desirable, including for example, bispecificity, multivalency (more than two binding sites), compact size (eg, binding domains). alone). The single chain antibodies lack some or all of the constant domains of the whole antibodies from which they are derived. Therefore, they can overcome some of the problems associated with the use of whole antibodies. For example, single chain antibodies tend to be free of certain undesired interactions between the heavy chain constant regions and other biological molecules. In addition, single chain antibodies are considerably smaller than whole antibodies and may have higher permeability than whole antibodies, allowing single chain antibodies to be localized and bound to the target antigen binding sites more efficiently. In addition, the relatively small size of the single chain antibodies makes them less likely to cause an undesired immune response in a recipient of the whole antibodies. Single chain single antibodies, each single chain has a V H and V L domain linked by a first peptide bond, can be covalently linked by at least one or more peptide bonds to form single chain polyvalent antibodies, which can be mono-specific or multi-specific. Each chain of a single chain multivalent antibody includes a variable light chain fragment and a variable heavy chain fragment, and is linked by a peptide bond to at least one other chain. The peptide bond is composed of at least fifteen amino acid residues. The maximum number of amino acid residues is approximately one hundred. Two single chain antibodies can be combined to form a diabody, also known as a divalent dimer. Diabodies have two chains and two binding sites, and can be mono-specific or bi-specific. Each chain of the diabody includes a VH domain connected to a VL domain. The domains are connected to the links that are short enough to avoid pairing between the domains in the same chain, thus leading the pairing between the complementary domains in different chains to recreate the two antigen binding sites. Three single chain antibodies can be combined to form triabodies, also known as trivalent trimers. The triabodies are constructed with the amino acid terminal of a VL or VH domain fused directly to the carboxyl terminal of a V or VH domain, ie, without any linking sequence. The triabody has three Fv principles with the polypeptides located in a cyclical manner, from start to finish. A possible form of the triabody is flat with three bonding sites located in a plane at an angle of 120 degrees to each other. Triabodies can be mono-specific, bi-specific or tri-specific. Thus, antibodies of the invention and fragments thereof include, but are not limited to, natural antibodies, bivalent fragments such as (Fab ') 2, monovalent fragments such as Fab, single chain antibodies, single chain Fv ( scFv), single domain antibodies, multivalent single chain antibodies, diabodies, triabodies, and the like that specifically bind antigens. Anti-IGF-IR and anti-PDGFRα antibodies or antibody fragments, which can be internalized during binding to cells containing IGF-IR (WO2005016970) or PDGFRα, can be chemically or biosynthetically linked to anti-tumor agents. Anti-tumor agents linked to such an antibody include any agent that removes or damages a tumor to which the antibody has bound or the environment of the cell to which the antibody has bound. For example, an anti-tumor agent is a toxic agent such as a chemotherapeutic agent or a radioisotope. Suitable chemotherapeutic agents are known to those skilled in the art and include anthracyclines (for example daunomycin and doxorubicin), methotrexate, vindesine, neocarzinostatin, cis-platinum, chlorambucil, cytosine arabinoside, 5-fluorouridine, melphalan, ricin and calicheamicin. The chemotherapeutic agents are conjugated to the antibody using conventional methods (see, for example, Hermentin and Seiler, Behring, Inst. Mitt. 82: 197-215 (1988)). Suitable radioisotopes for use as anti-tumor agents are also known to those skilled in the art. For example, 131l or 211At is used. These isotopes are bound to the antibody using conventional techniques (see, for example, Pedley et al., Br J. Cancer 68, 69-73 (1993)). Alternatively, the anti-tumor agent that binds to the antibody is an enzyme that activates a prodrug. In this way, a prodrug is administered which remains in its inactive form until it reaches the target site where it is converted to its cytotoxin form. In practice, the antibody-enzyme conjugate is administered to the patient and allows localization in the region of the tissue to be treated. The prodrug is then administered to the patient for the conversion of the cytotoxic drug to occur in the region of the tissue to be treated. Other anti-tumor agents include cytokines such as interleukin-2 (IL-2), interleukin-4 (I L-4) or tumor necrosis factor alpha (TNF-a). The antibody detects the cytokine in the tumor so that the cytokine mediates damage to or destruction of the tumor without affecting other tissues. The DNA cytokine can conjugate the antibody at the DNA level using conventional recombinant techniques. In certain embodiments of the invention, the anti-IGF-IR or anti-PDGFRα antibodies are administered in combination with one or more anti-neoplastic agents. For examples of combination therapies, see, for example, US Patent No. 6,217,866 (Schlessinger et al.) (Anti-EGFR antibodies in combination with anti-neoplastic agents); WO 99/60023 (Waksal et al.) (Anti-EGFR antibodies in combination with radiation). Any suitable anti-neoplastic agent can be used, such as a chemotherapeutic agent, radiation or combinations thereof. The anti-neoplastic agent can be an alkylating agent or an anti-metabolite. Examples of alkylating agents include, but are not limited to, cisplatin, cyclophosphamide, melphalan, and dacarbazine. Examples of anti-metabolites include, but are not limited to, doxorubicin, daunorubicin, and paclitaxel, gemcitabine. Useful anti-neoplastic agents also include mitotic inibitors, such as taxane docetaxel and paclitaxyl.

Topoisomerase inhibitors are another class of antineoplastic agents that can be used in combination with the antibodies of the invention. These include topoisomerase I or topoisomerase II inhibitors. Topoisomerase I inhibitors include irinotecan (CPT-11), aminocamptothecin, camptothecin, DX-8951f, topotecan. Topoisomerase II inhibitors include etoposide (VP-6), and teniposide (VM-26). Other substances are currently being evaluated with respect to topoisomerase inhibitory activity and effectiveness as anti-neoplastic agents. In a preferred embodiment, the topoisomerase inhibitor is irinotecan (CPT-11). In a particular embodiment of the invention, an anti-IGF-IR antibody is administered in combination with docetaxel. In another embodiment of the invention, an anti-PDGFRα antibody is administered in combination with doxorubicin. When the anti-neoplastic agent is radiation, the source of the radiation can be external (external laser radiation therapy -BRTT) or internal (brachytherapy-BT) to the patient being treated. The dose of the anti-neoplastic agent administered depends on numerous factors, including, for example, the type of agent, type and severity of which it is treated and route of administration of the agent. It should be emphasized, however, that the present invention is not limited to any particular dose. The antibody (anti-IGF-IR or anti-PDGFRa) and antibody plus treatments with anti-neoplastic agents can also be used for patients receiving adjuvant hormone therapy (for example, for breast cancer) or deprivation therapy of androgen (for example, for prostate cancer). The anti-IGF-IR and anti-PDGFRα antagonists of the invention can be co-administered, or administered with the receptor antagonists that neutralize other receptors involved in tumor growth or angiogenesis. For example, in one embodiment of the invention, an anti-IGF-IR antibody and an anti-PDGFRα antibody are co-administered. In one embodiment, in which an object tumor cell expresses IGF-IR and PDGFRa, the common elements of signal transduction are activated by signal transduction through each receptor. Although inhibition of a receptor will generally result in decreased activation of the common downstream components, inhibition of both receptors will further decrease activation. In another embodiment, certain cells in a surrounding tumor or tissue express significant amounts of a receptor, and other cells express significant amounts of the second receptor. The co-administration of the antagonists reduces the growth of the tumor cell and the stimulation of the paracrine of surrounding cells. A bi-specific antibody can be provided as an alternative to co-administration. A variety of bispecific antibodies exist, which are designed to incorporate several desirable characteristics. For example, bispecific diabodies have a minimum size. Bispecific antibodies with four antigen-binding sites (two for each binding specificity) have binding purposes that are similar to those of the corresponding natural antibodies.

Certain bispecific antibodies incorporate the Fe regions, thus preserving the effector functions (eg, complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC)) of the natural antibodies. WO 01/90192 discloses tetravalent IgG-like antibodies, WO2006 / 020258 discloses a tetravalent antibody that incorporates two diabodies and retains effector functions. In another embodiment, an anti-IGF-IR antibody or an anti-PDGFRα antibody or another antagonist is used in combination with a receptor antagonist that specifically binds to an epidermal growth factor receptor EGFR, (e.g., Her2 / erbB2 , erbB3, erbB4). Particularly preferred are antigen binding proteins that bind to the extracellular domain of EGFR and block the binding of one or more of their ligands and / or neutralize the activation induced by the EGFR ligand. EGFR antagonists also include antibodies that bind to an EGFR ligand and inhibit the binding of EGFR to its ligand. Ligands for EGFR include, for example, EGF, TGF-α, amfiregulin, heparin-binding EGF (HB-EGF) and betacellulin. It is thought that EGF and TGF-a are major endogenous ligands that give rise to EGFR-mediated stimulation, although TGF-a has been shown to be more potent in promoting angiogenesis. EGFR antagonists also include substances that inhibit the dimerization of EGFR with other EGFR receptor subunits (ie, EGFR homodimers) or heterodimerization with other growth factor receptors (eg, HER2). EGFR antagonists further include biological molecules and small molecules, such as synthetic kinase inhibitors that act directly in the cytoplasmic domain of EGFR to inhibit EGFR-mediated signal transduction. Erbitux® (cetuximab) is an example of an EGFR antagonist that binds to EGFR and blocks ligand binding. An example of a small molecule EGFR antagonist is IRESSA ™ (ZD1939), which is a quinoxaline derivative that functions as an ATP mimic to inhibit EGFR. See the Patent North American No. 5,616,582 (Zeneca Limited); WO 96/33980 (Zeneca Limited) on p. 4; see also, Rowinsky et al., extract 5 presented at the 37th Annual Meeting of ASCO, San Francisco, CA, May 12-15, 2001; Anido et al., Abstract 1712 presented at the 37th Annual Meeting of ASCO, San Francisco, CA, May 12-15, 2001. Another example of a small molecule EGFR antagonist is Tarceva® (OSI-774), which is an EGFR inhibitor [6,7-bis (2-methoxy-ethoxy) -quinazolin-4-yl] -3-ethynyl-phenyl) amine] -hydrochloride derivative of 4- (substituted phenylamino) quinoxaline. See WO 96/30347 (Pfizer Inc ..) in, for example, page 2, line 12 to page 4, line 34 and page 19, lines 14-17. See also Moyer et al., Cancer Res., 57: 4838-48 (1997); Pollack et al., J. Pharmacol, 291: 739-48 (1999). Tarceva® can function by inhibiting the phosphorylation of EGFR and its downstream pathways of PI3 / Akt and MAP kinase signal transduction (mitogen-activated protein) resulting in cell cycle arrest mediated by p27. See Hidalgo et al., Extract 281 presented at the 37th Annual Meeting of ASCO, San Francisco, CA, May 12-15, 2001. It is also reported that other small molecules inhibit EGFR, many of which are believed to be specific to tyrosine kinase domain of an EGFR. Some examples of such EGFR small molecule antagonists are described in WO 91/116051, WO 96/30347, WO 96/33980, WO 97/27199 (Zeneca Limited). WO 97/30034 (Zeneca Limited), WO 97/42187 (Zeneca Limited), WO 97/49688 (Pfizer Inc.), WO 98/33798 (Warner Lambert Company), WO 00/18761 (American) Cyanamid Company), and WO 00/31048 (Warner Lambert Company). Examples of small molecule EGFR-specific antagonists include CI-1033 (Pfizer), which is a quinoxaline inhibitor (N- [4- (3-chloro-4-fluoro-phenylamino) -7- (3-morpholin-4). -yl-propoxy) -quinazolin-6-yl] -acrylamide) tyrosine kinases, particularly EGFR and are described in WO 00/31048 on page 8, lines 22-6; PKII 66 (Novartis), which is a pyrrolopyrimidine inhibitor of EGFR and is described in WO 97/27199 on pages 10-12; GW2016 (GlaxoSmithKIine), which is an inhibitor of EGFR and HER2; EKB569 (Wyeth), which is reported to inhibit the growth of tumor cells that overexpress EGFR or HER2 in vitro and in vivo; AG-1478 (Tryfostin), which is a small molecule of quinoxaline that inhibits the signaling of EGFR and erbB-2; AG-1478 (Sugen), which is a bisubstrate inhibitor that also inhibits the protein kinase CK2; PD 153035 (Parke-Davis) which is reported to inhibit EGFR kinase activity and tumor growth, which induces apoptosis in cells in culture, and improves the cytotoxicity of cytotoxic chemotherapeutic agents; SPM-924 (Schwarz Pharma), which is a tyrosine kinase inhibitor targeted at the treatment of prostate cancer; CP-546,989 (OSI Pharmaceuticals), which is reported to be an inhibitor of angiogenesis for the treatment of solid tumors; ADL-681, which is an EGFR kinase inhibitor targeted at the treatment of cancer; PD 158780, which is a pyridopyrimidine that is reported to inhibit the tumor growth index of A4431 xenografts in mice; CP-358,774, which is a quinazoline that is reported to inhibit autophosphorylation in HN5 xenografts in mice; ZD1839, which is a quinozolin that is reported to have anti-tumor activity in mouse xenograft models including vulvar, NSCLC, prosthetic, ovarian, and colorectal cancers; CGP 59326A, which is a pyrrolopyrimidine that is reported to inhibit the growth of EGFR-positive xenografts in mice; PD 165557 (Pfizer); CGP54211 and CGP53353 (Novartis), which are dianilnophthalimides. EGFR naturally-occurring tyrosine kinase inhibitors include genistein, herbimycin A, quercetin, and erbstatin. Other small molecules that reported inhibiting EGFR and which are therefore within the scope of the present invention are tricyclic compounds such as the compounds described in US Pat. No. 5,679,683; quinazoline derivatives such as the derivatives described in U.S. Patent No. 5,616,582; and indole compounds such as the compounds described in US Patent No. ,196,446. Another receptor that can be detected together with IGF-IR or PDGFRα is a vascular endothelial growth factor receptor (VEGFR). In one embodiment of the present invention, an anti-IGF-IR antibody or anti-PDGFRα antibody is used in combination with a VEGFR antagonist. In one embodiment, an antagonist used binds specifically to the VEGFR-1 / Flt-1 receptor. In another embodiment, the VEGFR antagonist binds specifically to the VEGFR-2 / KDR receptor. Particularly preferred are antigen binding proteins that bind to the extracellular domain of VEGFR-1 or VEGFR-2 and block binding via their ligands (VEGFR-2 is more strongly stimulated by VEGF; VEGFR-1 is stimulated in a stronger by PIGF, but also by VEGF) and / or neutralizes the activation induced by the ligand. For example, IMC-1121 is a human antibody that binds to and neutralizes VEGFR-2 (WO 03/075840; Zhu). Another example is MAb 6.12 which binds to soluble VEGFR-1 and expressed on the cell surface. ScFv 6.12 comprises the VL and VH domains of the mouse monoclonal antibody MAb 6.12. A hybridoma cell line producing MAb 6.12 has been deposited as ATCC number PTA-3344 under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and under its regulations (Budapest Treaty). In another embodiment, the VEGFR antagonist binds to a VEGFR ligand and blocks the activation of a VEGFR by the ligand. For example, Avastin® (bevacizumab) is an antibody that binds to VEGF. Other examples of growth factor receptors involved in tumorigenesis are nerve growth factor (NGFR), and fibroblast growth factor (FGFR). In a further alternative embodiment, the anti-IGF-IR and anti-PDGFRα antibodies can be administered in combination with one or more convenient adjuvants, such as, for example, cytokines (IL-10 and IL-13, for example) or others. immune stimulators, such as, but not limited to, chemokine, tumor-associated antigens, and peptides. See, for example, Larrivee et al., Supra. It should be appreciated, however, that the administration of only one anti-IGF-IR or anti-PDGFRα antibody is sufficient to prevent, inhibit, or reduce the progress of the tumor in a therapeutically effective manner. In a combination therapy, the anti-IGF-IR or anti-PDGFRα antibody is administered before, during, or after beginning therapy with another agent, as well as any combination thereof, ie, before and during, before and after after, during and after, or before, during and after beginning therapy with the anti-neoplastic agent. For example, the antibody can be administered between 1 and 30 days, preferably 3 and 20 days, more preferably between 5 and 12 days before starting radiotherapy. In a preferred embodiment of the invention, chemotherapy is currently administered with or, more preferably, after therapy with the antibody. In the present invention, any convenient method or route can be used to administer the antibodies of the invention, and optionally, to co-administer the anti-neoplastic agents and / or antagonists of other receptors. The regimens of the anti-neoplastic agent used according to the invention include any regimen that is believed to be optimally convenient for the treatment of the neoplastic condition of the patient. Different diseases may require the use of specific anti-tumor antibodies and specific anti-neoplastic agents, which will be determined in a patient according to the patient. Administration routes include, for example, oral, intravenous, intraperitoneal, subcutaneous, or intramuscular administration. The dose of the antagonist administered depends on numerous factors, including, for example, the type of antagonists, type and severity of the tumor being treated and the route of administration of the antagonists. It must be emphasized, however, that the present invention is not limited to any particular method or route of administration. One skilled in the art will understand that dosages and frequency of treatment depend on the tolerance of the individual patient and the pharmacological and pharmacokinetic properties of blocking or inhibitory agent. Ideally, one wants to achieve saturable pharmacokinetics for the agent used. A loading dose for the anti-IGF-IR and anti-PDGFRa antibodies may range, for example, from about 10 to about 1000 mg / m2, preferably from about 200 to about 400 mg / m2. This can be followed by several additional daily or weekly dosages ranging, for example, from about 200 to about 400 mg / m2. The patient's side effects are monitored and the treatment is stopped when such side effects are severe. A person skilled in the art would also know how to monitor the progress of the treatment to determine an effective dose. For bone metastasis of prostate cancer, one way is to monitor PSA levels. Other ways to monitor bone metastasis include bone scans and MRI. For patients, cancer-induced bone loss (CTIBL) is a risk or problem (for example, patients receiving adjuvant hormone therapy for breast cancer or androgen deprivation therapy for prostate cancer), any aforementioned treatment can be supplemented by the administration of agents for the prevention of CTEBL, such as bisphosphonates. Bisphosphonates include, for example, clodronate, risedronate, and zoledronic acid. Through this application, several publications, reference texts, textbooks, technical manuals, patents, and patent applications have been referred to. The teachings and descriptions of these publications, patents, patent applications and other documents in their entireties are hereby incorporated by reference in this application to more fully describe the state of the art to which the present invention pertains. It should be understood and expected that variations in the principles of the invention described herein may be made by the person skilled in the art and it is planned that such modifications should be included within the scope of the present invention. The following examples further illustrate the invention, but in no way should they be construed as limiting the scope of the invention. Detailed descriptions of conventional methods, such as those used in the construction of vectors and plasmids, and the expression of antibodies and antibody fragments, can be obtained from numerous publications, including Sambrook, J et al., (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press; Coligan, J. et al. (1994) Current Protocols in Immunology, Wiley & Sons, Incorporated; Enna, SJ. and col. (1991) Current Protocols in Pharmacology, Wiley & Sons, Bonifacino, J.S. and col. (1999) Current Protocols in Cell Biology, Wiley & Sons. All references mentioned herein are incorporated in their entirety. EXAMPLES Example 1 Effects of IMC-A12 and Docetaxel on Tumor Growth.

Tumor fractions (20-30 mm3) of androgen-independent LuCaP 35V (Al) were implanted subcutaneously (s.c.) in 32 castrated SCID mice at six weeks of age respectively as previously described (4). When it was observed that the implanted tumor reached a volume of 150-200 mm3, the animals were randomly selected into four groups for the treatment studies. Animals of group 1 were treated with docetaxel at a dose of 20 mg / kg. Animals of group 2 were treated with docetaxel at a dose of 10 mg / kg. The animals of group 3 received the combined treatment with 10 mg / kg docetaxel and 40 mg / kg A12. The animals of group 4 received the combined treatment with 20 mg / kg docetaxel and 40 mg / kg A12. All treatments were administered intraperitoneally (ip). Docetaxel was administered once a week. A12 was administered three times a week. All animals were treated for four weeks and monitored for four additional weeks before slaughter. The tumors were measured twice a week and the volume of the tumor was calculated by the formula: volume = L X W2 / 2. After the University of Washington IACUC approved the animal protocol, some animals were sacrificed at an earlier time when the tumor reached a volume of 1000 mm3 or when the weight loss of the animal exceeded 20% of the initial body weight. The animals were weighed twice a week. Blood samples were collected from the orbital sinus weekly. The serum was separated and the PSA level was determined using the IMx Total PSA analysis (Abott Laboratories, Abott Park, IL). BrdU was injected into the tumors 1 h before the animals were sacrificed for evaluation of the tumor cell proliferation index in vivo. After sacrifice, the tumors were collected and divided in two. A portion of the tumors were placed in 10% neutral formalin buffer (NFB) and embedded in paraffin. Five sections of micron were prepared for staining by immunohistochemistry (IHC). The remaining portion of the tumors was mechanically separated into single cells by grinding and filtering through 70 μm nylon sieves. As shown in Figure 1, the LuCaP 35V xenograft grew aggressively in the mice at an average growth rate of 362.0 ± 72.0 mm3 / week without any treatment. All animals in the untreated group had to be sacrificed within a period of three weeks after the initiation of treatment in the experimental groups, because the tumor volume exceeded 1000 mm3. When animals were treated with 40 μg / kg A12 alone, the rate of tumor growth was reduced to 192.7 ± 35.6 mm3 / week during treatment. When docetaxel was administered to the animals at a dose of 10 mg / kg, the tumor growth rate of LuCaP 35V was reduced to an average of 29.6 ± 6.1 mm3 / week. When docetaxel was administered in combination with the A12 treatment, the tumor growth rate of LuCaP 35V was further reduced to an average of 7.9 ± 1.0 mm3 / week (Fig. 1 B). The inhibition effect of docetaxel combined with A12 continued for more than four weeks after the completion of the treatments. When a higher dose of docetaxel (20 mg / kg) was administered to the animals, regardless of with or without the combination treatment of A12, the volume of the tumor did not increase during a period of four weeks of treatment; In contrast, a trend of reduced tumor volumes was observed. However, in the four weeks after finishing treatment, the reduction in tumor volumes continued in the group of animals treated with docetaxel combined with A12. In contrast, tumor volumes increased by an average of 27.0 ± 16.1 mm3 / week in the group of animals treated with docetaxel alone. These results have suggested that, in a administered dose of docetaxel, the combination treatment with A12 may improve the inhibitory effect of docetaxel on tumor growth during treatment or after completion of treatment. PSA is a clinical parameter commonly used to determine the growth of the prostate tumor. Serum PSA levels were measured in animals during and after treatments. As shown in Figure 1c, in animals treated with A12 and docetaxel or 20 mg / kg docetaxel alone, no significant change in serum PSA levels was observed during the four-week treatment, in accordance with the growth of the suppressed tumor. After the end of treatment, the serum PSA level showed an increase in the animals treated with docetaxel alone and, in contrast, was constant or even decreased in animals treated with docetaxel in combination with A12. These data are consistent with the continuous inhibition post-treatment of tumor growth in animals treated with docetaxel and A12. Induction of Apoptosis by Docetaxel Combined with the Anti-IGF-IR Antibody. The combined in vivo effect of docetaxel and A12 treatment on the cell cycle and cell survival at the end of the experiment was measured by the terminal deoxinucleotidyl transferase-mediated abbreviation and labeling (TUNNEL) analysis and staining with propidium (Pl) using the Apop-Direct kit (BD BioScience) as previously described. Briefly, 1x106 cells of the single cell suspension were placed fixed with 10% neutral buffer formalin (NBF) followed by 70% ethanol alcohol at -20 ° C for 30 minutes. After several washes, the cells were permeabilized with 0.1% Triton X-100 and incubated with FITC-conjugated dUTP and terminal deoxynucleotidyl transferase (TdT) enzyme at 37 ° C for 1 h, followed by incubation with PI / buffer. RNase (100 μg / ml Pl, 50 μg / ml RNase) at room temperature for 60 minutes. The samples were analyzed by flow cytometry using a BD FACscan. The data was analyzed with the software CellQuestPR0.

Four weeks after the end of treatment, apoptosis was detected in a significant percentage of tumors of the animals that had been treated with docetaxel (66.7% in the group treated with docetaxel 10 mg / kg and 77.8% in the group treated with docetaxel). mg / kg docetaxel) in combination with A12 (figure 2b and table 1), regardless of the dosage of docetaxel that is used. The average apoptotic events in these tumors occurred at an index of 15.0 ± 4.3%. No apoptosis was detected in tumors in the animals that were treated with docetaxel alone. In fact, the majority (88% in the group treated with 10 mg / kg docetaxel and 100% in the group treated with 20mg / kg docetaxel) of the tumors proceeded to the normal cell cycle (figure 2a and table 3).

Table 3 - Tumor Cell Cycle and Survival Activities at the Sacrifice Moment To further evaluate the cellular proliferation capacity of the tumor after the completion of a different treatment, paraffin section of staining with the anti-BrDu antibody. The tumor samples were fixed in 10% NBF, embedded in paraffin, and sectioned at 5 μm on the slides. After deparaffinization and rehydration, the antigens were recovered with 0.01 M citric acid (pH 6.0) at 95 ° C for 2 x 5 minutes. The slides were allowed to cool for 30 minutes, followed by sequential rinsing with PBS. The activity of the endogenous peroxidase was stopped by an incubation with 0.3% H2O2 in methanol for 15 minutes. After blocking with 1.5% normal goat serum in PBS containing 0.05% Tween 20 (PBST) for 1 h, the slides were incubated with mouse anti-BrdU antibody (1 μg / ml) for 1 h followed by incubation sequential with biotinylated goat anti-mouse IgG for 30 minutes, avidin labeled with peroxidase for 30 minutes (Santa Cruz Biotechnology) and diaminobenzidine (DAB) / hydrogen peroxide chromogen substrate (Vector Laboratories, Burlingame, CA) for 5-10 minutes. All incubation steps were performed at room temperature. The slides were counter-stained with hematoxylin (Sigma), and mounted with Permount (Fisher Scientific, Fair Lawn, New Jersey). For the negative control, mouse IgG (Vector Laboratories) was used in place of the primary anti-BrdU antibody. The slides were examined under a Zeiss Microscope and the digital images were obtained. The numbers of nuclei labeled with BrdU and total nuclei were collected from 10 random observations of each section. The proliferation index was calculated by the number of positive nuclei to BrdU divided by the total number of nuclei. Ten fields were counted per slide. The H & E stain was performed using hematoxylin and eosin (Richard Alien, Kalamazoo, Ml). In the animals that were treated with docetaxel and A12, the absorption of BrDu was significantly lower than in those treated with the same dose of docetaxel alone (figure 3). These data of the incorporation of BrDu are constant with the previous observations of the cell cycle and apoptosis, suggesting that A12 significantly improved the cytotoxicity of docetaxel. Differential Regulation of Genetic Expression in Tumors Treated with Docetaxel Combined with the Anti-IGF-IR Antibody Against Docetaxel Solo. To determine the potential mechanisms of the significantly enhanced effect of docetaxel by A12, expression of IGF-IR was examined in all harvested tumors by immunohistochemistry and flow cytometric analysis. There was no difference in surface expression of IGF-IR between all treatment groups or compared to the control group (data not shown). Post-treatment gene expression was examined using cDNA microarray analyzes in tumors from animals that had received 20 mg / kg docetaxel and 20 mg / kg docetaxel combined with A12. According to SAM analysis, 49 genes were identified as differentially expressed in the tumors that received the combined treatment of docetaxel and A12 compared to those that received docetaxel alone, with a change of more than 2 times and less than 10% of the false discovery rate ( FDR, for its acronym in English) (data not shown). Thirteen genes that are potentially involved in the regulation of apoptosis or cell cycle were identified (table 4). All 13 genes were at least 2 different times between the two treatments and had an FDR of less than 0.02%. Nine genes were down-regulated and four genes were up-regulated in tumors treated with docetaxel and A12, compared to tumors treated with docetaxel alone. Table 4 - Differential Genetic Expression Post-Treatment in Tumors Treated with Docetaxel + A12 compared to Tumors Treated with Docetaxel Solo.

For the selected genes, the results were confirmed by RT-PCR in real time. A standard PCR fragment of cDNA object was purified. A series of dilutions of the standards from 10 ng / μl to 10"3 pg / μl was used for real-time RT-PCR to generate the standard curves, One μg of total RNA from each assembled tumor group was used for the synthesis of CDNA of a first strand using the Superscript First Strand Synthesis System (Invitrogen) Real-time RT-PCR was performed in 20 μl of reaction mixture containing 1 μl of the first cDNA strand, sets of specific primers, and Lightcycler FastStart DNA Master Plus SYBR Green using a Roche Lightcycler after the manufacturer's protocol (Roche, Nutley, NJ) The RT-PCR products were subjected to the fusion curve analysis using the Lightcycler v3.5 software.The sizes of the amplicon were confirmed by Agarose gel electrophoresis Each sample was analyzed in duplicate.The results are shown in Figure 4. Of the infra-regulated genes, TUBB has been shown to give rise to resistance to docetaxel (Tanaka et al. l., 2004, Int. J. Cancer 111, 617-26), and increased expression of BIRC 5 (survivin) has been shown to be associated with aggressive prostate cancer and resistance to anti-androgen therapy (Angelis et al., 2004, Int. J. Oncol. 24, 1279-88; Zhang et al., 2005, Oncogene 24, 2474-82). In addition, TUBB is a gene regulated by IGF-IR that is involved in the transformation mediated by IGF-IR (Loughran et al., 2005, Oncogene 24, 6185-93). Of the four over-regulated genes, IGFBP3 has been shown to inhibit signaling of the IGF ligand as well as to induce apoptosis in prostate tumor cells in a ligand-dependent manner (Grimberg et al., 2000. J. Cell. Physiol. , 1-9). Levels in Serum Post-Treatment of A12. Serum levels of A12 were measured in the animals that had received docetaxel combined with A12. Serum levels of A12 decreased 100-fold two weeks after the cessation of treatment and were detected at a very low level four weeks after cessation of treatment (FIG. 5). Total cytotoxicity. The cytotoxicity of co-administration of docetaxel and IMC-A12 was examined. Although A12 has more than 95% cross-reactivity with murine IGF-IR, no abnormal daily changes in activity or behavior were observed in the animals treated with the combined reagents or docetaxel alone compared to animals with control tumor. No significant effect on kidney cells was observed in any treatment group by cell cycle and apoptosis analyzes (data not shown). No significant change in body weight was observed between the treatment groups (figure 6). Anti-IGF-IR Antibody Therapy for Bone Metastasis. The effectiveness of treatment with anti-IGF-IR antibodies in the growth of prostate cancer cell metastasis in bone was evaluated using prostate cancer cells injected directly into the tibia of SCID mice. With this method, tumors in metastases are established directly without being based on invasion dependent on the chemotaxis of the circulation. A variety of tumor lines are available to establish bone metastases. These include the PC-3, LuCaP35, and LnCaP cells that produce the osteolytic lesions and the LuCaP 23.1 cells that produce osteoblastic lesions. LuCaP 23.1 cells, which express IGF-IR, have an absorption index of -80% in the bone environment and give rise to osteoblastic reactions. In the preliminary experiments, the LuCaP 23.1 samples exhibited a significant increase in bone volume versus tissue volume (% BV / TV) in the tumor against the control tibia (254-503% control, p = 0.024). All luCaP 23.1 tumors in the tibias exhibited the new bone trabicules, which were not present in the normal samples, and a high number of tumor foci, which had replaced the normal spinal cord. In some specimens the growth of the tumor and bone spread out of the original bone. % BV / TV increased from LuCaP 23.1 samples was also observed after castration; the% BV / TV of the tibias with tumor was 212-354% that of the tibias without tumor (p = 0.024). The results observed for the intra-tibial xenografts of LuCaP 23.1 are indicative of the formation of new bone stimulated by the tumor cells. In addition, tumors show many similarities with human bone osteoblastic metastases, including a large number of tumor foci and increased amounts of mineralized bone.

To assess the effectiveness of treatment with IMC-A12, LuCaP 23.1 xenograft tumors were grafted onto the SCID mice, and serum PSA levels were measured every two weeks to evaluate tumor growth. All the animals were castrated two weeks before the tibial graft of the tumor cell. The administration of IMC-A12 to test the mice started when serum levels of PSA reached 5-10 ng / ml (indicating established tumors). 40 mg / kg IMC-A12 were injected i.p. three times a week for six weeks. Bone mineral density (BMD) of tibias with tumors and contralateral tibias without tumors was measured by Dual X-ray absorptiometry (PlXImus Lunar densitometer) performed in an area of 2.5 mm x 2.5 mm at the site of cell injection tumoral, or in the corresponding site of the contralateral tibia at the time of grafting. Every two weeks the analysis of the lesions was made by measuring serum PSA. All animals were sacrificed when the bone lesions in the control group had been repeated after castration based on serum PSA levels (LuCaP 35> 60 ng / ml, LuCaP 23.1> 500 ng / ml), Radiographic appearance of bone lesions or when animals were compromised. One hour before the sacrifice of the animals they were injected with BrdU to monitor tumor cell proliferation. Radiographs were taken before slaughter (Faxitron X-ray MX-20), and BMD from both tibias were measured at the time of sacrifice. Table 5 - Bone Mineral Density (BMD) Serum PSA levels were significantly lower in mice treated with IMC-A12 (Figure 7), and the increase in BMD associated with tumor growth in osteoblastic metastases was also significantly reduced (Table 5). The BMD measurements of the legs without tumor indicated that treatment with BMI-A12 did not cause a loss of bone density (osteoporosis). Radiographs of mice treated with IMC-A12 and untreated mice show that the progress of the tumor was reduced or significantly prevented in the treated mice (FIG. 8). Combination of Anti-IGF-IR Antibody and Docetaxel for Bony metastases. SCID mice are castrated 2 weeks before tibial tumor injections. Bone metastases are generated by the direct injection of LuCaP 23.1 prostate cancer cells into the tibia of the mice, giving rise to osteoblastic lesions. Xenografts express IGF-IR. Serum PSA levels are measured every two weeks to evaluate tumor growth. When the serum PSA levels reached 5-10 ng / ml indicating that the tumor was established, the animals are randomly selected into four groups. In two groups, 40 mg / kg of BVIC-A12 are injected i.p. three times a week for six weeks with a group receiving IMC-A12 + docetaxel 20 mg / kg i.p once a week for 6 weeks and a second group IMC-A12 + docetaxel 10 mg i.p. three times a week for 6 weeks. The control groups received 10 or 20 mg docetaxel i.p. without IMC-A12. The animals are monitored with weekly measurements of PSA. After the end of treatment, the animals continue to be monitored with weekly PSA measurements until the tumors in the docetaxel groups only show the tumor growth again. While PSA values rise in docetaxel groups alone (albeit at a slower rate than in untreated animals), PSA levels in mice treated with IMC-A12 + docataxel decreased, and in some animals, they began to decrease. It is noted that reductions in PSA levels continue, even after the end of treatment six weeks ago. As indicated above, BMD measurements are made at the time of grafting and at sacrifice, and radiographs are taken just before slaughter. Groups treated with IMC-A12 + docataxel show little or no increase in BMD, and radiographs show little or no evidence of osteoblastic activity. Combination of Anti-IGF-IR Antibody and Docetaxel for Bony metastases. Human prostate tumor fractions of LuCaP 23.1 (20 to 30 mm3) were mechanically digested. 2-5 x 105 viable LuCaP 23.1 cells were injected into the tibias of the SCID mice with an age of 6-8 weeks. 21 mice randomly selected in three groups were used for the study. After tumor injection, serum PSA was monitored weekly. Treatment started when the serum PSA level reached 5-10 ng / ml, an indication of tumor growth. Group 1 received the saline buffer of the control vehicle. Group 2 received 20 mg / kg of docetaxel i.p once a week for 4 weeks. Group 3 received 20mg / kg of docetaxel once a week and 40 mg / kg of A12 i.p. three times a week for 4 weeks. To determine whether the response to treatment was osteoblastic or osteolytic, BMD was measured by Pro Dexa scan and x-rays of the animals were taken at the end of all treatments. Docetaxel alone or docetaxel combined with A12, significantly inhibited the tumor growth of LuCaP 23.1 as reflected by the suppression of serum PSA levels (Figure 9a), with no significant difference between the two treatments. However, after cessation of treatment, serum PSA began to increase in animals that had been treated with docetaxel alone, indicating a new tumor growth; While the suppression of serum PSA levels continued, it was observed in animals that combined treatment received indicates a prolonged period of post-treatment tumor stability. Serum PSA levels were shown correlated to bone density (BMD) and the sizes of the bones with tumor were radiographed (Figure 9b). At week five, the average bone density in the control animals treated with docetaxel 20 and docetaxel 20 combined with A12 was 0.112 ± 0.01, 0.09 ± 0.002, and 0.05 ± 0.009 (mean ± SEM), respectively. There was an evident trend towards a decrease in bone density with the treatment. Example 2 Phosphorylation of Akt Induced by the Spinal Cord Aspirate. Spinal cord samples from normal male donors (ages 18-45) were provided by Cambrex (Poietics ™ Donor Program). The samples were centrifuged at 1,500 rpm to separate the soluble and cellular phases. The supernatant was filtered using filters of 0.8 μm and 0.22 μm in succession. 50 μl of spinal cord aspirate was administered to the cells in 1 ml of medium (1:20 final dilution). For experiments performed in the presence of serum, the cells were cultured in DMEM supplemented with 10% FBS and 50 μg / ml gentamicin for 24 hours before exposure to the spinal cord. For the experiments in the absence of serum (hungry cells), the cells were washed twice with PBS, the growth medium was replaced by DMEM without serum, and the cells were incubated for 4 hours before exposure to the spinal cord preparations. . When used, AG-1296, a specific inhibitor of PDGF receptors (Rice et al., 1999, Amer. J. Path. 55, 213-21) were added to the cultures 30 min before exposure to the aspirate. spinal cord. The IMC-3G3 antibodies were administered as described at the time of pre-treatment as indicated below. Activation of the spinal cord of Akt was detected in PC3-ML cells, which expressed PDGFRa but not in DU-145 cells, which lack the receptor. In one experiment, to minimize the effect of serum components on Akt activation, the cells were pre-incubated for 4 hours in medium without serum. The addition of the spinal cord extracts resulted in the phosphorylation of Akt robusta in PC3-ML cells, but not in DU-145 cells. (Figure 10a). To evaluate the importance of the response, a second experiment was conducted with serum. The robust stimulation of Akt phosphorylation in PC3-ML cells by the spinal cord aspirate was also observed in the presence of serum (Figure 10b). Only a small response was produced in the DU-145 cells. Phosphorylation of Akt mediated by PDGFRα. Osteoblasts and osteoclasts, which secrete PDGF-AA and PDGF-BB, are intended to provide these growth factors in the soluble environment of the spinal cord. To determine if the sensitivity of PC3-ML cells to spinal cord extracts was related to signal transduction via PDGFRa, PC3-ML cells were exposed to spinal cord aspiration in the absence or presence of 20 μm AG- 1296 This concentration of AG-1296 totally inhibits Akt activation induced by PDGF-BB (Figure 11A). AG-1296 inhibited Akt activation induced by spinal cord aspiration by more than 40% (Figure 11 B and D). This indicates that the PDGFRα signaling is responsible for a significant proportion of Akt activation induced by the spinal cord.

The direct contribution of PDGF-AA and -BB to the signaling of PDGFRa relative to other components of spinal cord aspirates. It was determined that the concentrations of PDGF-AA and -BB in the spinal cord aspirates of three different donors ranged from 400 pg / ml to 2 ng / ml. Due to the 20-fold dilution of the spinal cord aspirate, the test cells were actually exposed to the PDGF-AA and -BB concentrations between 20 and 100 pg / ml. Accordingly, PC3-ML cells were treated with 100 pg / ml of each PDGF-AA and -BB. Phosphorylation of Akt was less than 10% of that obtained with the spinal cord aspirate (Figure 3c and D). Accordingly, it appears that activation of the Akt path by PDGFRα signaling may involve PDGFRα ligands other than PDGF-AA and -BB and / or mechanisms other than PDGFRα activation by direct binding of a ligand.

Inhibition of Akt Phosphorylation by an Anti-PDGFRα Antibody. The neutralization antibody IMC-3G3, which is specific for human PDGFRα was also tested for its ability to inhibit Akt phosphorylation in PC3-ML cells. A pre-incubation time of 30 minutes and a concentration of 20 μg / ml neutralized the stimulating effect of 30 ng / ml of PDGF-BB (FIG. 12A). Treatment with the antibody also resulted in approximately 40% inhibition of Akt phosphorylation induced by the spinal cord (Figure 12B and C). It was also observed that the inhibitory effect of IMC-3G3 on Akt phosphorylation was dependent on the duration of the pre-incubation, with an incubation duration of 120 minutes which is considerably more effective (Figure 12D) than the incubation duration of 30 minutes (Figure 12B and C). One possible explanation is that IMC-3G3 induces the internalization of PDGFRα and that its inhibitory effect is related not only to the blocking of ligand binding, but also to the elimination of the plasma membrane receptor. Example 3 Isolation of Human Anti-PDGFR Antibodies. Human anti-PDGFRα monoclonal antibodies were generated by a standard hybridoma technology (Harlow &Lane, ed., Antibodies: A Laboratory Manual, Cold Spring Harbor, 211-213 (1998), which is incorporated by reference herein) that uses transgenic mice (Medarex Inc., Sunnyvale, CA) that express human immunoglobulin light chains and heavy kappa gammas. The human extracellular domain of PDGFRα (ECD) was purchased from R &D Systems (Minneapolis, MN). KM mice were immunized subcutaneously (s.c.) with 3x107 porcine aortic endothelial cells stably expressing PDGFRα (PAE Ra). After 4 weeks, the mice were stimulated s.c. with 50 μg PDGFRa ECD in complete Freund's adjuvant plus 3 x 107 PAE Ra cells administered i.p. Mice were stimulated twice more, 3 additional weeks, with 25 μg PDGFRα ECD in the incomplete Freund's adjuvant. Splenocytes from mice with high serum binding and blocking titers were isolated and fused with the myeloma cells. The hybridoma cultures that exhibited the blocking activity were subcloned and the antibodies of these hybridomas were purified by protein G chromatography. IgGs were evaluated for binding to PDGFRα in a direct binding assay. PDGFRa ECD in PBS was immobilized in a 96-well plate (100 ng / well). The plates were then washed with PBST (PBS + 0.05% Tween 20) and blocked with PBSM (3% milk in PBS), 200 μl / well) for 2 hours at 25 ° C. IgGs diluted in PBSM were incubated with immobilized PDGFRa ECD for 1 hour at 25 ° C, and the plates were washed with PBST. A secondary antibody (conjugate of horseradish peroxidase-IgG anti-human F (ab ') 2 goat; BioSource International, Camarillo, CA) diluted 1: 5,000 in PBSM was added for 1 hour at 25 ° C. After the plates were washed with PBST, a TMB peroxidase substrate (KPL, Gaithersburg, MD) was added and the reaction was stopped with 100 μl 1 mol / L H2SO. Plates were read at A450 nm using a microplate reader (Molecular Devices, Sunnyvale, CA). Blockade of PDGF was evaluated using a solid-phase PDGF blocking assay (see Duan et al., 1991, J. Biol.

Chem. 266: 413-8, which is incorporated by reference). PDGFRa ECD was diluted in PBS and coated in 96-well microtiter plates (Removawell 1 x 12 round-bottomed Immulon 2HB strips of irradiated protein binding polystyrene, Dynex Technologies, Chantilly, VA). Each well was coated with 60 ng PDGFRa for 3 hours at 25 ° C in a total volume of 100 μl. The plates were then washed twice and blocked overnight at 4 ° C with 25 mmol / L HEPES (pH 7.45), 0.5% gelatin, 100 mmol / l NaCl, and 0.1% Tween 20. The plates were then heated to 25 ° C. ° C for 20 minutes and washed once with binding buffer (25 mmol / l HEPES (pH 7.45), 0.3% gelatin, 100 mmol / INaCl, 0.01% Tween 20). Fifty microliters of IgGs were added to each well and incubated at 25 ° C for 30 minutes. Iodinated PDGF was diluted in binding buffer and added (50 μl of a solution of 1 nmol / l) to each well. The plates were incubated for 2 hours at 25 ° C and then washed five times with binding buffer. Each well was counted in a gamma counter. A cell-based blocking analysis was performed as described in Heldin et al., 1988, EMBO J. 7, 1387-93. The kinetics of antibody binding to PDGFRα was measured using a BIAcore 3000 instrument (BIAcore, Inc., Piscataway, NJ). PDGFRa ECD was immobilized on a sensor chip and the antibody was injected at various concentrations. The sensograms were obtained at each concentration and evaluated using the BIA evaluation 2.0 program to determine the rate constants. The affinity constant, d, was calculated from the ratio of the velocity constants Figure 13 shows the dose-dependent binding of human monoclonal antibody IMC-3G3 to immobilized PDGFRa ECD in ELISA. The concentration of the antibody required for 50% maximum binding to PDGFRa ECD was 0.06 nmol / l (Table 6). ED50 is constant with Ka for the antibody as determined by plasmon surface resonance in a BIAcore instrument (Table 1). The monoclonal antibody was also blocked the binding of [125I] PDGF-BB to the immobilized receptor, with an Cl50 of 0.43 nmol / l. The binding sites for PDGF-AA and PDGF-BB in PDGFRa do not coincide structurally. The data suggest that the epitope for 3G3 spatially overlaps both growth factor binding sites. Table 6 - Union Characteristics of the Anti-PDGFRα Antibody Inhibition of Receptor Phosphorylation and Activation of Descending Effector Molecules. The effects on intracellular signaling induced by PDGF by IMC-3G3, were determined using PAE cells Ra. Cells were seeded in six-well Falcon tissue culture plates (250,000 cells per well) and allowed to develop overnight. The wells were then rinsed and incubated in medium without serum. After an overnight incubation to produce the inactive cells, the cells were treated with antibodies for 30 minutes at 37 ° C followed by the addition of PDGF-AA or PDGF-BB and by incubation for an additional 10 minutes at 37 ° C. The cells were then separated and subjected to lysines in 200 μl of lysis buffer (50 mmol / l Tris-HCl (pH 8.0), 1% Triton X-100, 150 mmol / l NaCl, 1 mmol / l EDTA, 0.1% SDS, 1 mmol / l sodium orthovanadate, and protease inhibitors (Complete Mini, Roche, Mannheim, Germany)). Cell lysates were analyzed by SDS-PAGE and Western Blot using the enhanced chemiluminescence reagents and Hyperfilm (Amersham Biosciences). The antibody was tested for the ability to inhibit tyrosine receptor phosphorylation induced by the ligand. PDGF-AA and PDGF-BB increase the tyrosine phosphorylation of PDGFRα approximately five times at concentrations of 1 and 3 nmol / l, respectively. Higher concentrations of ligand (10 nmol / l) resulted in the less phosphorylated receptor possibly due to degradation induced by the ligand. The antibody inhibited the receptor induced by PDGF-BB almost at the base levels (Figure 14A, upper row).

Similar data were obtained using PDGF-AA to induce phosphorylation of the receptor. PDGFs transduce mitogenic signals and exert anti-apoptotic effects on cells expressing the receptor through the descending effector protein. Therefore, the monoclonal antibody was tested for its ability to inhibit the activation of MAPKs p44 / p42 and Akt (involved in cell growth and anti-apoptotic trajectories, respectively). The anti-PDGFRα antibody inhibited the phosphorylation of MAPKs and Akt in response to PDGF-BB (Figure 2A) and to PDGF-AA (not shown). The inhibition of PDGFRα phosphorylation was dose dependent, with a 50% inhibition reached at 0.25 nmol / l (Figure 14B). Anti-mitogenic activity. The anti-PDGFRα monoclonal antibody was tested for its ability to block the mitogenesis induced by PDGFAA from PAE Ra cells. The cells were seeded in 96-well tissue culture plates (1 x 10 4 cells per well) and grown overnight at 100 μl of medium per well. The wells were then rinsed with serum-free medium and the cells were starved overnight with 75 μl medium without serum added to each well. IgG was added (25 μl / well) and the plates were incubated for 30 minutes at 37 ° C. PDGF-AA or PDGF-BB (25 μl / well) were then added and the plates were incubated for 18 to 20 hours at 37 ° C. The plates were incubated for an additional 4 hours after each well received 0.25 μCi [3 H] thymidine (25 μl / well). The antibody, PDGF, and [3 H] thymidine, were all diluted in medium without serum. The cells were then washed with PBS plus 1% bovine serum albumin and separated by treatment with trypsin (100 μl / well). The cells were harvested on a filter and washed three times with distilled water twice using a MACH III cell splitter (Tomtec, Inc., Hamden, CT). After processing the filter, the radioactivity incorporated into the DNA was determined in a scintillation counter (Wallac Microbeta, model 1450). When MC-3 G3 was added to serum PAE Ra cells deprived of serum, the thymidine incorporation induced by PDGF-AA was specifically inhibited (Figure 15) with an EC5O of 8.3 nmol / L. The antibody also inhibited mitogenesis induced by 3 nmol / l of PDGF-BB from PAE Ra cells with an EC50 of 1.25 nmol / l (data not shown). Inhibition of Cell Growth of Human Tumor Cell Lines Expressing PDGFRa. The human tumor cell lines that express PDGFRa were tested to determine the effects of the anti-human PDGFRα antibody on malignant growth in in vitro ß 'systems in vivo. Two lines of tumor cells expressing PDGFRa as determined by flow cytometry are SKLMS-1 (leiomyosarcoma) and U118 (glioblastoma). These cell lines also respond to the ligand in mitogenic analyzes and form tumors in mice. SKLMS-1 has the potential not only to stimulate paracrine but also to stimulate autocrine. SKLMS-1 was shown to express the PDGF-AA protein when grown in the culture using a quantitative sandwich enzyme immunoassay technique (R &D Systems). As can be seen in Figure 16A, IMC-3G3 inhibited the phosphorylation of Akt and MAPKs in response to the stimulation of PDGF-AA of SKLMS-1 cells. The inhibition of Akt phosphorylation was 100% and that of MAPKs was approximately 80%. The antibody is also an effective inhibitor of phosphorylation in U118 cells (Figure 16B). The ligand-induced mitogenesis of the tumor cells was also blocked. When the anti-PDGFRα antibody was added to the serum-deprived U118 cells, the thymidine incorporation induced by PDGF-AA was specifically inhibited (Figure 17A) with an EC50 of 3.4 nmol / L. The antibody also inhibited the mitogenetic response induced by PDGF-AA of SKLMS-1 cells with an EC50 of 5 nmol / l (Figure 17B), as well as the mitogenetic response stimulated by PDGF-BB (Figure 17C). Only partial inhibition (40% at 66 nmol / l; Figure 17D) of the mitogenetic response stimulated by PDGF-BB was observed for U118 cells. This is attributed to the expression of PDGFRα and PDGFRβ in such cells (data not shown). Inhibition of Tumor Xenograft Growth. IMC-3G3 was tested in vivo in subcutaneous xenograft (s.c.) models of glioblastoma (U118) and leiomyosarcoma (SKLMS-1) in athymic nude mice. The tumor xenografts s.c. they were established by injecting 10 x 106 cells SKLMS-1 or U118 mixed in Matrigel (Collaborative Research Biochemicals, Bedford, MA) in female nude nude mice (Crl: NU / NU-nuBR, Charles River Laboratories, Wilmington, MA). The tumors were allowed to reach an average tumor volume (p / 6 x longest length x perpendicular width2) of approximately 400 mm3. The mice were selected randomly in five groups (n = 12) and treated by i.p. twice a week for the duration of the study. Group 1 mice were treated with vehicle control (0.9% NaCl, USP for irrigation, B / Braun). Mice from groups 2 to 4 were treated with 6, 20, and 60 mg / kg of the current anti-PDGFRα antibody. The mice of group 5 were treated with 60 mg / kg of human IgG (Sigma). Groups treated with 6, 20, or 60 mg / kg of anti-PDGFRα antibody or human IgG were administered 21.4, 71.4, and 214 mg / kg loading dose, respectively. Charge doses were calculated to achieve a steady state plasma concentration of the first dose (elimination period, 7 days) using a dosing regimen twice a week. The tumor volumes were evaluated twice a week and the tumor growth in the treatment groups was compared with the repeated ANOVA measurements. As shown in Figure 18A, human IgG had no effect on glioblastoma growth compared to mice treated with saline (P = 0.74), while anti-PDGFRα antibody significantly inhibited tumor growth at 6 (P = 0.06), 20 (P = 0.03), and 60 (P = 0.0004) mg / kg dose. At the end of the U118 study, the values of% T / C [(average tumor volume for the group treated with 3G3 at the conclusion of the study / average tumor volume at the beginning of treatment) / (average tumor volume for the group treated with the control at the conclusion of the study / average tumor volume at the beginning of the treatment) x 100] were 67%, 63%, and 35% for the dose groups treated with 6, 20, and 60 mg / kg of 3G3, respectively. In addition, tumor regression was observed in 4 of 12, 5 of 11, and 10 of 12 animals in the treatment groups of 6, 20, and 60 mg / kg. There were no setbacks in any control group. Figure 18B shows that the growth of leiomyosarcoma was also significantly inhibited by the treatment at 6 (P = 0.02), 20 (P = 0.003), and 60 (P <0.0001) mg / kg. The final values of% T / C were 66%, 57%, and 31% for the treatment groups of 6, 20, and 60 mg / kg, respectively without tumor regressions. The histological examination of the xenografts at the end of the treatment showed marked differences in the tumors of treated animals with respect to the tumors of animals that received the control therapy. The resected tumors were fixed in QDL fixative at 4 ° C for 24 hours. After paraffin embedding and sectioning at 4 μm, the formalin-fixed sections were stained with H &E from Mayer (Richard Alien, Kalamazoo, Ml). In the group treated with U118 at the highest dose (60 mg / kg), few viable tumor cells were found and there were substantially more sparse cell regions compared to the control group with saline (Figure 18C). The xenografts treated with SKLMS-1 on day 25 also showed a reduction in the amount of viable tumor cells and cell packing compared to the control group with saline (FIG. 18D). In Vitro Inhibition of Stimulus Mediated by PDGFRα of a Glioblastoma Line. The phosphotyrosine level of the receptor in U118 tumors was evaluated one week after treatment with the anti-PDGFRα antibody or human IgGt. Mice with established U118 tumors (500 mm3) were treated with a loading dose of 214 mg / kg followed by a maintenance dose of 60 mg / kg of antibody after 72 hours. The tumors were harvested from the mice one week (168 hours) after the first injection of the antibody (at the time that the regression of the tumor was observed in the average, see Figure 18A) and they were homogenized in lysis buffer of the analysis of phosphorylation (see above). The lysates were centrifuged twice at 14,000 rpm and the concentration of the protein for the supernatant harvested was determined (protein analysis Bio-Rad, Bio-Rad, Hercules, CA). The lysate (4 mg) of each sample was subjected to immunoprecipitation using the anti-PDGFRα antibody. The immunoprecipitated human PDGFRa was then subjected to immunoblotting with an anti-PDGFR or anti-phosphotyrosine antibody. Figure 19 shows that administration of the anti-PDGFRα antibody resulted in the reduction of the phosphotyrosine level of PDGFRα related to a control of human IgG in these tumors. Cell Line Engineering. First, the genes encoding the heavy and light chain variable domains of the human anti-PDGFRα antibody were cloned and sequenced. A series of primers was obtained from MEDAREX which subjected the 5 'and 3' flanking sequences of the human immunoglobulin variable region sequences within the hybridomas derived from MEDAREX to anelous. The heavy chain variable region amplified with primer pair AB88 (front) and AB90 (back) (table 7). The light chain products were amplified with pairs of primers containing the forward primer AB182 and the rear primer AB16 (table 7). The 0.4kb products of these reactions were cloned into the ZeroBlunt vector (Invitrogen) to produce AB88-1 (VH) and AB 182-3 (VK), and the inserts were sequenced with the universal primers T7 and M13R. Table 7 - Primers for the MEDAREX Hybridomas To generate the plasmid vectors to express the entire IgG1 antibody, the cloned variable regions were amplified by PCR and ligated in two steps into the expression vectors containing the constant region genes. The PCR heavy chain primary amplification used 25 ng of plasmid AB88-1 as standard for primers IPHF5 (forward) and IPHR5 (rear). The PCR heavy chain secondary amplification used 5 μl of primary reaction as standard and primers OPSIF and IPHR5. The combination of the two forward primers adds a sequence of base 57 pairs to the 5 'end of the immunoglobulin genes encoding a mouse heavy chain gene signal sequence of the amino acid 19 (MGWSCIILFLVATATGVHS; SEQ ID NO: 24) for the efficient processing and secretion of immunoglobulin.

In addition, the forward primer OPSIF adds a consensus "Kozak" sequence (J Mol. Biol. 196: 947) for the efficient initiation of the translation of these genes into mammalian cells and a 5 'Hindlll restriction endonuclease site for cloning the amplified product into the convenient expression vector. The heavy chain rear primer contains a Nhel site within the structure to be cloned into the constant region vector. The PCR was performed in two stages using the Expand PCR kit (Boehringer Mannheim Inc.) according to the manufacturer's specifications using the Expand Buffer # 3 buffer system in 50 μl of reactions with the following cycle conditions: 1 cycle 94 ° , 2 minutes 5 cycles 94 °, 20 seconds 48 °, 60 seconds 68 °, 2 minutes 20 cycles 94 °, 20 seconds 65 °, 60 seconds 68 °, 2 minutes 1 cycle 68 °, 5 minutes After two phases of PCR , the product was purified after agarose gel electrophoresis and cloned as an indigestible H fragment in lN hel in the vector pDFc (figure 8), which contains the gamma 1 human constant region. Primary amplification of light chain PCR used 25 ng of plasmid, pAB182-3 as standard primers IPLF4 (forward) and IPLR2 (rear). Secondary amplification of light chain PCR used 5 μl of primary reaction as the standard and primers OPSIF and IPLR2. As for the heavy chain, the two forward primers provide a secretion signal sequence. The light chain rear primer contains a BsiWI site within the structure to be cloned into the kappa constant region vector pLck (Figure 8). PCR reactions were performed according to the previous heavy chain. After two phases of PCR, the product was purified after agarose gel electrophoresis and was cloned into pLck, which contains the human constant region of light chain kappa. Table 8 - Primers for Expression Vectors VH and VKTo generate a single plasmid vector for stable transfection, the heavy chain expression module, containing the CMV promoter, the heavy chain coding region, and the poIyA element, was cloned into the light chain vector as the fragment aNotl-Sall (figure 20). This construct was then used to generate a stable production line in NSO cells of the myeloma cell line. The NSO cells were transfected with the expression plasmid via electroporation using the BioRad Gene Pulser II. Before transfection, the plasmid DNA was linearized with Pvul, precipitated ethanol, and suspended again at a concentration of 0.4 mg / ml (40 ug in 100 ul dH2O). The cells were electroporated with 40 ug of DNA in a final volume of 800 ul by a single pulse of 250 volts, 400 μFd. Electroporated cells were dispersed in 50 ul of aliquots in DMEM medium (JRH Biosciences Inc.) containing 10% dialyzed fetal calf serum (dFCS) (Hyclone, lot #: AHA7675) and 2 mM glutamine (Invitrogen / Life Technologies) in wells of approximately eighteen 96-well plates at a density of 5,000-10,000 cells per well. The selection of glutamine synthetase (GS) positive transfectants was initiated 24 hours later by the addition of DMEM without glutamine containing 10% dFCS and is supplemented with 1x GS supplement (JRH Biosciences Inc.). The cells were cultured during 2-4 weeks at 37 ° C, 5% CO2 to allow the growth and increase of the colonies. More than 300 colonies were analyzed using an anti-human Fe (gamma) ELISA (detection of horseradish peroxidase at 450 nm). The clones expressing the antibody (58%) were augmented and retested to determine productivity during cultivation for days 3-5. To adapt the cells in medium without serum, the positive cell lines were increased by the addition of an equal volume of GS-OS culture medium without serum at each step. Strong positives, which produced 25 ug / ml or more in cultures of 24 sub-confluent wells of 3 days, were augmented for further analysis in order to complete the adaptation to the medium without serum. It is understood and expected that the variations to the principles of the invention described herein may be elaborated by the person skilled in the art and it is thought that such modifications should be included within the scope of the present invention.

Claims (80)

1. CLAIMS 1. A method for treating a subject having a bone tumor, comprising administering to an effective amount of an IGF-IR antagonist.
2. 2. A method for inhibiting the growth of a bone tumor, comprising administering an effective amount of an IGF-IR antagonist.
3. 3. A method for treating a subject having a bone tumor, comprising administering an effective amount of a PDGFR3a antagonist.
4. 4. A method for inhibiting the growth of a bone tumor, comprising administering an effective amount of a PDGFR3a antagonist. The method of any of claims 1 to 4, wherein the bone tumor is a primary tumor. 6. The method of any of claims 1 to 4, wherein the bone tumor is a secondary tumor. The method of any of claims 1 to 4, wherein the growth of tumor cells is androgen dependent. The method of any of claims 1 to 4, wherein the growth of tumor cells is independent of androgen. The method of any of claims 1 to 4, wherein the tumor is in metastasis from a prostate cancer. The method of any of claims 1 to 4, wherein the tumor is in metastasis from a breast cancer. The method of any of claims 1 to 4, wherein the tumor is in metastasis from a lung cancer. The method of claim 1 or 2, wherein the IGF-IR antagonist is an antibody or antibody fragment. The method of claim 12, wherein the antibody or antibody fragment competes for binding to IGF-IR with an antibody comprising a heavy chain variable domain having SEQ ID NO: 41 and a variable chain domain light having SEQ ID NO: 51. 14. The method of claim 1 or 2, wherein the IGF-IR antagonist is an intracellular IGF-IR inhibitor. The method of claim 14, wherein the intracellular IGF-IR inhibitor is selected from the group consisting of AG1024, NVP-AEW541, and BMS-554417. 16. The method of claim 3 or 4, wherein the PDGFR3a antagonist is an antibody or antibody fragment. The method of claim 16, wherein the antibody or antibody fragment competes for binding to PDGFRα with an antibody comprising a heavy chain variable domain having SEQ ID NO: 8 and a light chain variable domain that has SEQ ID NO: 16. 18. The method of claim 3 or 4, wherein the PDGFRa antagonist is an intracellular inhibitor of PDGFR3a. 19. The method of claim 18, wherein the intracellular PDGFR3a inhibitor is selected from the group consisting of AG1296, STI-571 and SU11248. The method of claim 12 or 16, wherein the antibody or antibody fragment is human. 21. The method of claim 12 or 16, wherein the antibody or antibody fragment is humanized. 22. The method of claim 12 or 16, wherein the antibody or antibody fragment is chimeric. 23. The method of claim 1 or 2, wherein the IGF-IR antagonist inhibits the binding of IGF-I or IGF-II to IGF-IR. 24. The method of claim 1 or 2, wherein the IGF-IR antagonist neutralizes IGF-IR activation by IGF-I or IGF-II. 25. The method of claim 1 or 2, wherein the IGF-IR antagonist reduces the concentration of the IGF-IR surface receptor. 26. The method of claim 1 or 2, wherein the IGF-IR antagonist is an antibody that binds IGF-IR with a Kd of about 3 x 10 ~ 10 M "1 or less. claim 1 or 2, wherein the IGF-IR antagonist inhibits the phosphorylation of a downstream signaling molecule of IFG-IR 28. The method of claim 3 or 4, wherein the PDGFR3a antagonist inhibits the binding of a platelet-derived growth factor to PDGFR3a 29. The method of claim 3 or 4, wherein the PDGFR3a antagonist neutralizes the activation of PDGFR3a. 30. The method of claim 3 or 4, wherein the PDGFR3a antagonist reduces the surface receptor concentration of PDGFRα. The method of claim 3 or 4, wherein the PDGFR3a antagonist is an antibody that binds IGF-IR with a Kd of about 10 ~ 9 M "1 or less 32. The method of claim 3 or 4, wherein the PDGFR3a antagonist inhibits phosphorylation of a downstream PDGFRα signaling molecule 33. The method of claim 3 or 4, wherein the PDGFR3a antagonist inhibits the spinal cord-induced activation of Akt. The method of any one of claims 1 to 4, further comprising the co-administration of a second receptor tyrosine kinase antagonist 35. The method of claim 1 or 2, wherein the IGF-IR antagonist is a bispecific antibody. 36. The method of claim 35, wherein the bispecific antibody is specific for IGF-IR and PDGFR3a 37. The method of claim 35, wherein the bispecific antibody is specific for IGF-IR and EGFR. method d e claim 3 or 4, wherein the PDGFR3a antagonist is a bispecific antibody. 39. The method of claim 38, wherein the bispecific antibody is specific for PDGFR3a and EGFR. 40. The method of any of claims 1 to 4, further comprising administering an effective amount of an anti-neoplastic agent. 41. The method of claim 1, wherein the anti-neoplastic agent is docetaxel. 42. The method of claim 1, wherein the anti-neoplastic agent is doxorubicin. 43. The method of claim 1, wherein the anti-neoplastic agent is radiation. 44. An isolated human antibody or antibody fragment specific for PDGFR3a, comprising one or more complementary determination regions selected from the group consisting of SEQ ID NO: 2 in CDRH1; SEQ ID NO: 4 in CDRH2; SEQ ID NO: 6 in CDRH3; SEQ ID NO: 10 in CDRL1; SEQ ID NO: 12 in CDRL2; and SEQ ID NO: 14 in CDRL3. 45. The antibody or antibody fragment of claim 44, comprising SEQ ID NO: 2 in CDRH1; SEQ ID NO: 4 in CDRH2; and SEQ ID NO: 6 in CDRH3. 46. The antibody or antibody fragment of claim 44, comprising SEQ ID NO: 8. 47. The antibody or antibody fragment of claim 44, comprising SEQ ID NO: 10 in CDRL1; SEQ ID NO: 12 in CDRL2; and SEQ ID NO: 14 in CDRL3. 48. The antibody or antibody fragment of claim 44, comprising SEQ ID NO: 16. 49. The antibody or antibody fragment of claim 44, comprising SEQ ID NO: 2 in CDRH1; SEQ ID NO: 4 in CDRH2; SEQ ID NO: 6 in CDRH3; SEC ID NO: 10 in CDRL1; SEQ ID NO: 12 in CDRL2; and SEQ ID NO: 14 in CDRL3. 50. The antibody or antibody fragment of claim 44, comprising SEQ ID NO: 8 and SEQ ID NO: 16. 51. The antibody or antibody fragment of any of claims 44 to 50, which selectively binds to PDGFR3a. 52. The antibody or antibody fragment of any of claims 44 to 50, which inhibits the binding of PDGFR3a to a PDGFR3a ligand. 53. The antibody or antibody fragment of any of claims 44 to 50, which neutralizes PDGFR3a. 54. The antibody fragment of any one of claims 44 to 50, which is selected from the group consisting of a single chain antibody, Fab, single chain Fv, diabody, and a triabody. 55. An antibody conjugate or antibody fragment of any of claims 44 to 50. 56. The conjugate of claim 55, comprising an anti-neoplastic agent, an object radical or a reporter moiety. 57. An isolated polynucleotide that encodes an antibody or antibody fragment and comprises one or more nucleotide sequences selected from the group consisting of SEQ ID NO: 1 in CDRH1; SEQ ID NO: 3 on CDRH2; SEQ ID NO: 5 in CDRH3; SEQ ID NO: 9 in CDRL1; SEQ ID NO: 11 in CDRL2; and SEQ ID NO: 13 in CDRL3. 58. The isolated polynucleotide of claim 57, comprising SEQ ID NO: 7. 59. The isolated polynucleotide of claim 57, comprising SEQ ID NO: 15. 60. An expression vector comprising the polynucleotide of either of claims 57 to 59. 61. A recombinant host cell comprising the expression vector of claim 60. 62. The recombinant host cell of claim 61, which produces a polypeptide comprising SEQ ID NO: 8 and a polypeptide. comprising SEQ ID NO: 16. 63. The recombinant host cell of claim 61, which produces a polypeptide comprising SEQ ID NO: 8 and SEQ ID NO: 16. 64. A method for neutralizing the activation of PDGFR3a in a mammal comprising the administration of an effective amount of The antibody of any one of claims 44 to 54. 65. A method for inhibiting tumor growth in a mammal, comprising administering a therapeutically effective amount of the antibody of any of claims 44 to 54. 66. The method of claim 65, where the tumor expresses PDGFRα. 67. The method of claim 65, wherein the tumor over-expresses PDGFR3a. 68. The method of claim 65, wherein the tumor is a primary tumor. 69. The method of claim 65, wherein the tumor is a tumor in metastasis. 70. The method of claim 65, wherein the tumor is a refractory tumor. 71. The method of claim 65, wherein the tumor is a vascularized tumor. 72. The method of claim 65, wherein the tumor is selected from the group consisting of an ovarian tumor, breast tumor, lung tumor, hepatocellular tumor, gastrointestinal stromal tumor, melanoma, renal cell carcinoma, prostate tumor, and a soft tissue sarcoma 73. The method of claim 65, wherein the antibody or antibody fragment is administered in combination with an anti-neoplastic agent. 74. The method of claim 73, wherein the anti-neoplastic agent is a chemotherapeutic agent. 75. The method of claim 73, wherein the anti-neoplastic agent is doxorubicin. 76. The method of claim 73, wherein the anti-neoplastic agent is radiation. 77. The method of claim 65, wherein the antibody or antibody fragment is administered with a second PDGFR3a antagonist. 78. The method of claim 77, wherein the PDGFRa antagonist is an intracellular PDGFR3a antagonist. 79. The method of claim 65, further comprising administering a therapeutically effective amount of an epithelial growth factor receptor (EGFR) antagonist. 80. The method of claim 65, further comprising administering a therapeutically effective amount of an insulin-like growth factor receptor (IGF-IR) antagonist.