BRPI0622074B1 - ANTIBODY OR ISOLATED HUMAN ANTIBODY FRAGMENT SPECIFIC TO PDGFR-ALPHA, POLYNUCLEOTIDE AND EXPRESSION VECTOR - Google Patents

Abstract

PDGFRa ANTIBODIES. The invention provides uses of an IGF-IRa and/or PDGFRa antagonist in the manufacture of medicaments for treating bone cancer, in particular metastatic bone cancer. The invention also provides antibodies that bind to human PDGFRa and neutralize the activation of the receptors. The invention further provides a method for neutralizing the activation of PDGFRa, and a use of the antibody alone or in combination with other agents in the manufacture of medicines to treat a neoplastic disease.

Description

Field of Invention

[0001] The invention provides methods of treating bone cancer, in particular metastatic bone cancer, by administering an IGF-IR antagonist and/or a PDGFRα antagonist. The invention also provides antibodies that bind to human PDGFRα and neutralize the activation of the receptors. The invention further provides a method for neutralizing the activation of PDGFRα, and a method of treating a mammal with a neoplastic disease using the antibodies alone or in combination with other agents.

History of the Invention

[0002] Prostate cancer is more common among men, with about 220,000 cases and 29,000 deaths per year in the United States. A significant proportion of men diagnosed with prostate cancer have metastatic disease. Furthermore, metastasis eventually develops in many other prostate cancer patients despite treatment with surgery or radiation therapy. Bone is the most common site of prostate cancer metastasis, and is also a site to which lung cancer and breast cancer frequently metastasize. Most prostate metastases are androgen-dependent, so there is a rapid response to surgical or medical castration, but in almost all patients, the tumor eventually becomes androgen-independent, leading to significant morbidity and mortality. Once bone metastases occur, currently available treatments have a limited effect. The most effective approved therapy that has been described for metastatic prostate cancer (docetaxel administration) prolongs median survival by approximately three months. (Petrylak et al., 2004, N. Engl. J. Med. 351:1513; Tannock et al., 2004, N. Engl. J. Med. 351:1502) Thus, new therapies for metastatic bone cancer are urgently needed. needed.

[0003] The insulin-like growth factor receptor (IGF-IR) is a ubiquitous transmembrane tyrosine kinase receptor that is essential for normal fetal and postnatal growth and development. IGF-IR is located on the cell surface of most cell types and serves as a signaling molecule for the growth factors IGF-I and IGF-II (collectively referred to hereafter as IGFs). IGF-IR can stimulate cell proliferation, cell differentiation, changes in cell size, and protect cells from apoptosis. It has also been considered to be almost obligatory for cell transformation (reviewed in Adams et al., Cell. Mol. Life Sd. 57:1050-93 (2000); Baserga, Oncogene 19:5574-81 (2000)). High levels of IGF-IR expression have been reported in prostate cancer bone metastasis tissue samples. Bone contains the largest store of IGFs in the body.

[0004] IGF-IR is a preformed hetero-tetramer containing two alpha chains and a beta version covalently linked by disulfide loops. Receptor subunits are synthesized as part of a single 180kd chain polypeptide, which is then proteolytically transformed into alpha (130kd) and beta (95kd) subunits. The entire alpha chain is extracellular and contains the site for ligand binding. The beta chain has the transmembrane domain, the tyrosine kinase domain, and a C-terminal extension that is necessary for cellular differentiation and transformation but is dispensable for mitogen signaling and protection against apoptosis.

[0005] IGF-IR is very similar to the insulin receptor (IR), especially in the beta chain sequence (70% homology). Due to this homology, recent studies have demonstrated that these receptors can form hybrids containing an IR dimer and an IGF-IR dimer (Pandini et al., Clin. Canc. Res. 5:1935-19 (1999)). Hybrid formation occurs in both normal and transformed cells and the content of the hybrids is dependent on the concentration of the two receptor homodimers (IR and IGF-IR) within the cell. Although hybrid receptors are composed of IR and IGF-IR pairs, hybrids selectively bind IGFs, with similar affinity to IGF-IR, and bind insulin only weakly (Siddle and Soos, The IGF System. Human Press, pp. 199-225. 1999). These hybrids can therefore bind to IGFs and convert signals in both normal and transformed cells.

[0006] A second IGF receptor, IGF-IIR, or mannose-6-phosphate receptor (M6P), also binds the IGF-II ligand with high affinity, but does not exhibit tyrosine kinase activity (Oates et al., Breast Cancer Res Treat. 47:269-81 (1998)). Because it results in the degradation of IGF-II, it is considered a sink for IGF-II, antagonizing growth by promoting the effects of this ligand. Loss of IGF-IIR in tumor cells may increase growth potential by releasing its antagonistic effect on IGF-II binding to IGF-IR (Byrd et al., J. Biol. Chem. 274:24408- 16 (1999)).

[0007] Platelet-derived growth factor receptors alpha and beta (PDGFRα and PDGFRβ) are type III tyrosine kinase receptors. PDGFRα is crucial for development and fulfills important functions in adult life. For example, mice homozygous for a null mutation die during embryogenesis. At later stages of development, PDGFRα is expressed in many mesenchymal structures, while adjacent epithelial cells produce platelet-derived growth factors (PDGFs). Samples of normal tissue or hyperplastic prostate glands test negative for PDGFRα, whereas primary prostate tumors and skeletal masses from corresponding individuals express PDGFRα. Furthermore, of prostate cell lines obtained from different metastatic sites, PDGFRα is found in PC3 cells derived from bone metastases, but not in cell lines obtained from lymph node (LNCaP) and brain (DU-145) metastases. .

[0008] The platelet-derived growth factor family of growth factors consists of five different sulfide-linked dimers, PDGF-AA, -BB, -AB, -CC, and -DD, which act through PDGFRα and PDGFRβ. These growth factors are dimeric molecules composed of disulfide-linked polypeptide chains that bind to two protein receptors simultaneously and induce receptor dimerization, autophosphorylation, and intracellular signaling. PDGFRα and PDGFRβ are structurally similar and can form heterodimers as well as homodimers. Because PDGFRβ does not bind the PDGF-A chain with high affinity, PDGF-AA only activates αα dimer receptors, whereas PDGF-AB and PDGF-CC activate αα and αβ heterodimer receptors.

Brief Summary of the Invention

[0009] This invention relates to the treatment of primary tumors and bone metastases, including tumors originating from the prostate, breast, lung or insulin-like growth factor I receptor (IGF-IR) and/or growth factor receptor alpha derivative of platelets (PDGFRα).

[00010] The tumors to be treated can be hormone/androgen-dependent or hormone/androgen independent, and can originate, for example, from the prostate, breast, or lung.

[00011] The invention provides methods of treating an individual who has a bone tumor, as well as methods of 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 PDGFRα antagonist. Receptor antagonists include antibodies and antibody fragments, as well as small molecule intracellular inhibitors.

[00012] The invention provides anti-IGF-IR or anti-PDGFRα antibodies that bind to their target receptors and inhibit ligand binding. The invention also provides antibodies and other antagonists that neutralize the activation of IGF-IR or PDGFRα. Other certain antibodies promote downregulation of their target receptors, for example, by internalization and/or degradation. Therefore, antibodies and small molecule anatagonists work by inhibiting the activation of downstream signaling molecules such as Akt, p42/p44, and MAPK.

[00013] Methods include the use of IGF-IR antagonists or PDGFRαs alone, in combination with each other, or in combination with other cancer treatments, such as chemotherapy and radiotherapy.

[00014] The invention also provides antibodies and antibody fragments that bind to PDGFRα, as well as nucleotides and host cell for the production of antibodies. Antibodies block ligand binding and neutralize receptor activation. The invention also provides for the use of the antibodies alone, in combination with other receptor antagonists or antineoplastic agents, or as conjugates for the treatment of neoplastic disease. Anti-PDGFRα antibodies are used to treat, for example, ovarian tumors, breast tumors, lung tumors, hepatocellular tumors, gastrointestinal stromal tumors, melanomas, renal cell carcinoma, prostate tumors, and soft tissue sarcomas.

Description of Figures

[00015] Figure 1 shows the growth of LuCaP 35V engrafted subcutaneous tumors in castrated SCDD mice during a treatment period initiated when the tumors had reached 150-200 mm3. Panel A: untreated controls; Panel B: animals were treated for four weeks with docetaxel (either 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 carrying LuCaP 35V engrafted subcutaneous tumors. Treated mice received docetaxel (20 mg/kg) alone or docetaxel (either 10 mg/kg or 20 mg/kg) in combination with anti-IGF-IRs antibody (40 mg/kg EV1C-A12). Treatment was started when tumors had reached 150-200 mm3 and ended after four weeks.

[00016] Figure 2 shows single cell suspensions of LuCaP 35V engrafted tumors treated with docetaxel (10 mg/Kg) alone (Group A) or in combination with anti-IGF-IR antibodies (40 mg/Kg IMC-A12) (Panel B). The field called R1 corresponds to apoptotic cells with fragmented DNA (increased FITC labeling).

[00017] Figure 3 shows DNA synthesis (BrDu absorption) in grafted tumors after stopping treatment with docetaxel (10 mg/kg or 20 mg/kg) alone, and in combination with anti-IGF-IRs antibody (40 mg/kg BVIC-A12).

[00018] Figure 4 depicts the differential expression of genes associated with prostate tumor aggressiveness (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.

[00019] Figure 5 shows serum levels of A12 after stopping treatment.

[00020] Figure 6 shows body weight (a measure of general cytotoxicity) of healthy animals treated continuously with docetaxel (either 10 mg/kg or 20 mg/kg) alone, or in combination with anti-IGF-IRs antibody ( 40 mg/kg MC-A12).

[00021] Figure 7 shows the effect of treatment with an anti-IGF-IR antibody (MC-A12), on PSA produced in grafts in SCID mice engrafted with LuCaP 23.1 cells.

[00022] Figure 8 shows a series of X-ray photographs of SCID mice engrafted with LuCaP 23.1 Cells. A12 mice received 40 mg/mL BVIC-A12 IP three times a week for six weeks. X-ray photographs were taken at the time of sacrifice.

[00023] Figure 9 shows PSA levels (a) and representative radiographs (b) of SCID mice with intratibial grafts of LuCaP 23.1 human prostate cells.

[00024] Figure 10 illustrates the effect of human bone marrow aspiration on Akt activity in prostate cancer cells. Cell lysates were subjected to SDS-PAGE. For Western blot analysis, membranes were blotted with antibodies targeting phospho-Akt (Ser-473, Cell Signaling Technology), PDGFRα (R&D Systems), and actin (Sigma). Binding to the primary antibody was detected using protein A or protein G conjugated to HRP (Sigma).

[00025] Figure 11 illustrates induction and inhibition of AKT phosphorylation in PC3-ML cells. Panel A shows dose-dependent inhibition of Akt phosphorylation of AG-1296 in cells exposed to 30 ng/mL PDFG-BB. Panel B shows Akt phosphorylation and inhibition of 20 μM AG-1296 in bone aspirate. Panel C shows the potency of bone marrow aspirate to induce Akt phosphorylation compared to the potency of a combination of 100 pg/mL PDGF-AA and 100 pg/mL PDGF-BB. Panel D compares the magnitude of bone marrow aspirate-induced Akt phosphorylation, AG-1296 inhibition of bone marrow aspirate-induced Akt phosphorylation, and PDFG-AA + PDFG-BB-induced Akt phosphorylation.

[00026] Figure 12 illustrates the inhibition of Akt phosphorylation in PC3-ML cells by PDGFRαs antagonists. A panel shows the dose-dependent effect of EVIC-3G3 monoclonal antibody on Akt phosphorylation induced by 30 ng/mL PDGF-BB. Panels B and C provide a comparison of the effects of BVIC-3G3 and AG1296 on bone marrow-induced Akt phosphorylation. Panel D shows that inhibition of Akt phosphorylation is dependent on IMC-3G3 preincubation time.

[00027] Figure 13 shows the binding of the antibody to PDGFRα. A: direct binding of anti-PDGFRα antibodies to the immobilized extracellular domain of PDGFRα. B: inhibition of [125I] PDGF-AA bound to immobilized PDGFRα.

[00028] Figure 14 shows the specific inhibition of the phosphorylation of PDGFRα and effector molecules below.

[00029] Figure 15 shows inhibition of [3H] thymidine modality stimulated by PDGF-AA in PAE Ra cells by mAbs.

[00030] Figure 16 shows the inhibition of activation of effector molecules induced by PDGF-AA below in SKLMS-1 (A) and U118 (B) cells.

[00031] Figure 17 shows the inhibition of [3H] thymidine modality stimulated by PDGF-AA in U118 (A) and SKLMS-1 (B) cells by mAbs. Inhibition of PDGF-AA-stimulated [3H]thymidine modality is also shown for SKLMS-1 (C) and U118 (D) cells.

[00032] Figure 18 shows dose-dependent treatment effects of established U118 (glioblastoma; panel A) and SKLMS-1 (leiomyosarcoma; panel B) tumors engrafted in hairless mice.

[00033] Figure 19 shows the reduction of PDGFRα phosphorylation in vivo in U118 tumors treated with anti-PDGFRα antibody, compared to treatment with nonspecific human IgG.

[00034] Figure 20 illustrates GS expression vectors used for cloning human hybridoma-derived VH and VK variable region genes and expression of complete human heavy (IgGl) and light chain proteins. The two vectors were recombined as explained in the Examples and the combined vector was transfected into NSO cells.

Detailed Description of the Invention

[00035] 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 primarily regulated by growth hormone and produced in the liver, but other tissue types are also capable of expressing IGF-I, including bone, which contains a large store of growth factors. Depending on the tumor cell type, IGF-I is involved in endocrine, paracrine, and/or autocrine regulation (Yu, H. and Rohan, J., J. Natl. Cancer Inst. 92:1472-89 (2000)).

[00036] It has been discovered that antibodies that bind to IGF-IR are useful in therapies for the treatment of bone tumors that express IGF-IR. Antibodies can be used alone, or in combination with other cancer treatments, particularly chemotherapy. Anti-IGF-IR therapy, alone or in combination with therapy with one or more antineoplastic agents (such as, for example, chemotherapy or radiotherapy), has significant therapeutic efficacy. Suppression of tumor growth is often accompanied by an increase in apoptosis and persists after all treatment is stopped and tumors begin to grow again in animals treated with chemotherapy alone.

[00037] It has also been discovered that PDGFRα plays an important role in the growth of bone tumors. For example, some tumor cell lines that express PDGFRα preferentially metastasize to bone. These cell lines exhibit increased activation and phosphorylation of PDGFRα on downstream signaling molecules in response to soluble factors present in the bone marrow. Activation of PDGFRα by the bone marrow is reduced or completely inhibited by antagonists of PDGFRαs, and phosphorylation of downstream signaling molecules that are commonly activated by signals through PDGFRα and other tyrosine kinase receptor systems is greatly reduced. Some data suggest that the PI3K/Akt survival pathway is activated by PDGFRα signaling, not only by ligands that directly activate PDGFRα, but also by factors present in the bone marrow that cause transactivation of the receptor.

[00038] Primary bone tumors to be treated according to the invention include, but are not limited to, osteosarcomas, chondrosarcomas, fibrosarcomas, and hemangiosarcomas. Notably, secondary malignant (metastatic) tumors are much more common than primary bone tumors. The metastatic bone tumors to be treated in accordance with the invention may arise from a variety of sources, the most common of which are prostate, breast, or lung cancers. The origin of a metastatic bone cancer will usually be apparent from the patient's history. Tumors can be osteoblastic or osteolytic. Tumors may be dependent on IGF-IR stimulation when they arise, or may transition to IGF-IR dependence. For example, prostate cancer or prostate cancer metastases that are initially hormone/androgen-dependent and controllable by physical or chemical treatments that suppress androgen or hormone production, can become hormone/androgen-independent through increased sensitivity to stimulation through IGF-IR. Furthermore, in addition to providing hormone/androgen-independent treatment of tumors, the invention may be useful in treating hormone/androgen-dependent bone tumors without dependence on androgen repression or hormone production, for example, by co-administering IGF- IR with antibodies anti-neoplastic agents. Such metastatic tumors would include bone tumors that are stimulated through IFG-IR in the IGF-rich environment of bone, which may be sensitive to sensitive hormonal stimulation, but not sensitive enough to grow without IGF involvement. Hormone ablation may not be necessary for these tumors.

[00039] Bone tumors that are PDGF-dependent can also be treated according to the invention, as well as tumors, which are "bone marrow"-dependent. Bone marrow-dependent tumors exhibit activation of PDGFRα in response to soluble factors present in the bone marrow. For example, as exemplified here, a metastatic cancer cell line expressing human PDGFRα undergoes PDGFRα activation and Akt+ phosphorylation following exposure to bone marrow aspirate. An anti-PDGFRα antibody and a small molecule PDGFRα antagonist each inhibit PDGFRα activation and Akt+ phosphorylation in the cell line. Bone marrow soluble factors that activate PDGFRα include, but are not limited to, PDGF-AA and -BB.

[00040] Although such bone marrow dependence involves signaling through PDGFRα, it cannot concern only the binding of PDGFRα to a PDGFRα ligand. For example, as exemplified here, it should be noted that PDGFRα activation defined by ligands (PDGF-AA- or BB), is weaker than activation by bone marrow aspirate. Furthermore, it was observed that in the presence of bone marrow aspirate, Akt+ phosphorylation decreases with increasing incubation time. Taken together, these results suggest that in addition to responding to the binding of PDGFs, PDGFRα can be transactivated (phosphorylated) by other signal translation elements (e.g., other tyrosine kinase receptors) sensitive to other bone marrow components. In any case, in a cell line suitable for growth of metastatic bone (i.e., a cell line that preferentially metastasizes to bone), bone marrow-dependent activation of PDGFRα is observed, which is inhibited by PDGFRα antagonists. Furthermore, treatment with a PDGFRα antagonist inhibits bone marrow-induced stimulation of anti-apoptotic PI3K/Akt and mitogen-activated protein kinase (MAPK) pathways.

[00041] Bone tumors to be treated with a PDGFRα antagonist may arise as metastases from prostate cancer cells, and, as mentioned, may be hormone/androgen-dependent, or have transferred to hormone/androgen-independence. Such tumors can also arise as metastases from cancer other than prostate cancer. One skilled in the art would easily be able to diagnose these conditions and illnesses using known conventional tests.

[00042] 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 does not yet experience or exhibit symptoms of the disease; for example, preventing the emergence of clinical symptoms; (2) inhibiting the disease, such as stopping its development; or (3) alleviating the disease, such as causing regression of disease symptoms. Tumor growth inhibition includes slowing or stopping growth as well as causing tumor regression. An effective amount for the treatment of a disease means that amount which, when administered to a mammal in need thereof, is sufficient for the purpose of treatment, as defined above, for that disease. The IGF-IR antagonists and the PDGFRα antagonist of the invention can be administered as monotherapy, in combination with one another, or in combination with one or more antineoplastic agents, such as, for example, a chemotherapeutic or radiotherapeutic agent.

[00043] In one embodiment of the invention, it may be desirable to determine the level of expression of IGF-IR and/or PDGFRα 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, the IGF-IR antagonist or PDGFRα antagonist is administered on the basis that the corresponding receptor is commonly expressed or activated in a given tumor type or invariably becomes expressed and activated as the disease progresses. .

[00044] The 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 to IGF-IR or one or more of its ligands (e.g., 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 ligand-binding soluble IGF-IR fragment. Antagonists of intracellular IGF-IRs can be biological molecules, but are generally small molecules. Examples include, but are not limited to, tyrosine kinase inhibitor AGl 024 (Calbiochem), insulin-like growth factor-I kinase receptor inhibitor NVP-AEW541 (Novartis), as well as insulin-like growth factor-I receptor inhibitor insulin/insulin BMS-554417 (Bristol Myers Squibb). It will be appreciated that a useful small molecule for use in the invention are IGF-IR inhibitors, but they need not be fully specific for IGF-IR.

[00045] The anti-IGF-IR antibodies to be used according to the present invention have one or more of the following properties: 1) The antibodies bind to the external domain of the IGF-IR and inhibit the binding of IGF-I or II of IGF-IGF-IR. Inhibition can be determined, for example, through a direct binding assay using purified or membrane-bound receptors; in the present 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) Antibodies neutralize IGF-IR. Binding of a ligand, e.g., IGF-I or IGF-II, to an external extracellular domain of IGF-IR stimulates autophosphorylation of the beta subunit and downstream signaling molecules, including MAPK, Akt, and IRS-I.

[00046] Neutralization of IGF-IR includes inhibition, decrease, inactivation and/or disruption of one or more of these activities normally associated with signal translation. Neutralization can be determined in vivo, ex-vivo, in vitro or using, for example, tissues, cultured cells, or purified cellular components. Neutralization includes inhibition of IGF-IR/IR heterodimers as well as IGF-IR homodimers. Thus, neutralization of IGF-IR has several effects, including inhibition, decrease, inactivation and/or disruption of growth (proliferation and differentiation), angiogenesis (blood vessel selection, invasion and metastasis), and cell motility and metastasis (adhesion and cellular invasiveness).

[00047] One measure of neutralization of IGF-IR is the inhibition of receptor tyrosine kinase activity. Tyrosine kinase inhibition can be determined using well-known methods, for example, by measuring the level of recombinant kinase receptor autophosphorylation, and/or phosphorylation of natural or synthetic substrates. Thus, phosphorylation assays are useful in determining neutralizing antibodies in the context of the present invention. Phosphorylation can be detected, for example, using an antibody specific for phosphotyrosine in an ELISA assay or in a Western blot. Some assays of tyrosine kinase activity are described in Panek et al., J. Pharmacol. Exp. Them. 283: 1433-44 (1997) and Batley et al., Life Sd. 62:143-50 (1998). The antibodies of the invention cause a decrease in IGF-IR tyrosine phosphorylation of at least about 75%, preferably at least about 85%, and more preferably at least about 90% in cells that respond to the ligand.

[00048] Another measure of IGF-IR neutralization is the inhibition of the phosphorylation of IGF-IR substrates below. Consequently, the level of phosphorylation of MAPK, Akt, or IRS-I can be measured. The decrease in phosphorylation is at least about 40%, and may be at least about 60%, or at least about 80%.

[00049] Additionally, protein expression detection methods can be used to determine IGF-IR neutralization, where the proteins being measured are regulated by the tyrosine kinase activity of IGF-IR. These methods include immunohistochemistry (IHC) for the detection of protein expression, fluorescence in situ hybridization (FISH) for the detection of gene amplification, competitive radioligand binding assays, solid matrix blot techniques such as Northern and Southern blots, 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).

[00050] Ex vivo assays can also be used to determine IGF-IR neutralization. For example, inhibition of tyrosine kinase receptors can be observed by mitogenic assays using cell lines stimulated with receptor ligand in the presence and absence of inhibitor. The MCF7 breast cancer cell line (American Type Culture Collection (ATCC), Rockville, MD) is a cell line that expresses IGF-IR and is stimulated by IGF-IGF-I or II. Another method involves testing inhibition of the growth of tumor cells expressing IGF-IR or cells transfected to express IGF-IR. Inhibition can also be observed using tumor models, for example, human tumor cells injected into a mouse.

[00051] The antibodies of the present invention are not limited by any special IGF-IR neutralization mechanism. The anti-IGF-IR antibodies of the present invention can externally bind to cell surface IGF-IR receptors, blocking ligand binding (e.g., IGF-IGF-I or II) and subsequent signal translation mediated by associated receptors. tyrosine kinase, and prevent the phosphorylation of IGF-IR and other proteins downstream in the signal translation cascade. 3) Antibodies infra-modulate IGF-IR. The amount of IGF-IR present on the surface of a cell depends on protein receptor production, internalization and degradation. The amount of IGF-IR present on the surface of a cell can be measured indirectly, by detecting the internalization of the receptor or a molecule bound to the receptor. For example, receptor internalization can be measured by contacting IGF-IR expressing cells labeled with an antibody. The antibody bound to the membrane is then removed, collected and counted. Internalized antibody is determined by lysing the cells and detecting the marker in the lysate.

[00052] Another way is to directly measure the amount of receptor present in the cell after treatment with an anti-IGF-IR antibody or other substance, for example, by fluorescence-activated cell sorting analysis of cells marked by surface expression of IGF -GO. The labeled cells are incubated at 37°C and the fluorescence intensity is measured over time. As a control, a part of the labeled population can be incubated at 4°C (conditions under which receptor internalization is stopped).

[00053] Cell surface IGF-IR can be detected and measured using a different antibody that is specific for 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)) 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 seen in less than four hours.

[00054] Another measure of infra-modulation is the reduction in the total receptor protein present in a cell, and reflects the degradation of internal receptors. Consequently, treatment of cells (particularly cancer cells) with antibodies of the invention results in a reduction in total IGF-IR cells. 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%.

[00055] For the treatment of human beings, the antibodies according to the invention are preferably human. Alternatively, the antibodies may be from non-human primates or other mammals, i.e. humanized or chimeric antibodies. In one embodiment of the invention, an anti-IGF-IR antibody comprises one, two, three, four, five, and/or six complementarity determining regions (CDRs) selected from the group consisting of SEQ ID NO: 35, SEQ ID NO : 37, SEQ ID NO: 39, SEQ ID NO: 45, SEQ ID NO: 47, and SEQ ID NO: 49 (CDR1H, CDR2H, CDR3H, CDR1L, CDR2L, CDR3L, respectively). In another embodiment, the anti-IGF-IR antibody comprises one, two, three, four, five, and/or six complementarity determining regions (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 also preferably, the present antibodies including fragments, 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. One of these anti-IGF-IR antibodies is the human IgG1 antibody IMC-A12 (WO2005016970), with a heavy chain variable domain represented by SEQ ID NO: 41 and a light chain variable domain represented by SEQ ID NO: 51. Another preferred human antibody is IMC-2F8 ( WO2005016970), with a heavy chain variable domain identical to IMC-A12 and a light chain variable domain represented by SEQ ID NO: 61. Useful antibodies further include anti-IGF-IRs antibodies that compete with MC-A12 or IMC-2F8 for binding to IGF-IR, as well as antibodies that bind to other epitopes (i.e., antibodies that bind to other epitopes and exhibit properties as previously described such as ligand blocking, receptor internalization, etc., but do not compete with BVIC- A12 or BMI-2F8).

[00056] According to the invention, PDGFRαs antagonists can also be used for treatment. The PDGFRα antagonist can be an extracellular antagonist or an intracellular antagonist and more than one antagonist can be employed. Extracellular antagonists include, but are not limited to, proteins or other biological molecules that bind to PDGFRα or one or more of its ligands (e.g., PDGF-AA, -AB, -BB, -CC). In one embodiment of the invention, an extracellular antagonist is inhibited by 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 ligand is a soluble protein binding to the PDGFRα fragment. Intracellular antagonists of IGF-IRs can be biological molecules, but they are generally small molecules. In one embodiment, the intracellular PDGFRα antagonist is AG1296. AG1296 (Calbiochem) is an inhibitor of PDGF α, PDGF1Ss, and c-KIT, and also reacts with Flt3. Other small molecules that target PDGFRs include STI-571 (imatinib mesylate, Gleevec®, Novartis) and SUl 1248 (sunitinib malate, SUTENT®, Pfizer).

[00057] In one embodiment of the invention, an anti-PDGFRα antibody comprises one, two, three, four, five, and/or six complementarity determining 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 SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6. Alternatively, and also preferably, the present antibodies, or fragments thereof, have CDRs of SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14. The amino acid sequences of the CDRs are defined below in Table 1. Table 1-IMC-3G3 CDRs

[00058] In another embodiment, the anti-PDGFRα antibody, or fragment 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 one such antibody and is exemplified in the present invention.

[00059] Preferably, the antibodies, or fragments thereof, of the present invention neutralize PDGFRα. Binding of a ligand, e.g., PDGF-AA, PDGF-AB, PDGF-BB, or PDGF-CC, to an extracellular domain of PDGFRα stimulates receptor dimerization, autophosphorylation, activation of the internal cytoplasmic domain of the receptor tyrosine kinase, and initiation of several signal translation and transactivation pathways involved in the regulation of DNA synthesis (gene activation) and cell cycle progression or division. Anti-PDGFRα antibodies typically block ligand binding and/or receptor dimerization, and inhibit one or more autophosphorylation, activation of tyrosine kinase activity, and signal translation. The anti-PDGFRα antibodies of the present invention can be specific for the extracellular binding region of PDGFRα and prevent the binding of a PDGFRα ligand. Preferably, such anti-PDGFRα antibodies, or fragments thereof, bind PDGFRα at least as strongly as natural PDGFRα ligands. Alternatively or additionally, antibodies may be specific to a region of the receptor monomer that would otherwise interface a receptor dimer. These antibodies block dimer formation, although ligand binding of a receptor monomer may or may not be blocked.

[00060] As described above for anti-IGF-IRs antibody, receptor neutralization can be determined by a variety of in vivo, in vitro, and ex vivo methods. In one embodiment of the invention, anti-PDGFRα antibodies reduce PDGFRα phosphorylation by at least about 75%. In other embodiments, phosphorylation is reduced by at least about 85% or at least about 90%. In one embodiment of the invention, as a result of inhibition of PDGFRα signal translation, phosphorylation or a component of the downstream signal translation pathway (e.g., Akt, p42/p44, etc.) is reduced by at least about 40%, at least about 60%, or at least about 80%. Receptor neutralization can be determined using defined ligands (e.g., PDGF-AA, -AB, -BB, -CC), mixtures of such ligands, or preparations such as bone marrow aspirate that comprise PDGFs, as well as other factors. growth stimulators.

[00061] Neutralization of PDGFRα includes the inhibition, decrease, inactivation and/or disruption of one or more of these activities normally associated with signal translation. Thus, neutralizing PDGFRα has several effects, including inhibition, decrease, inactivation and/or disruption of growth (proliferation and differentiation), angiogenesis (blood vessel selection, invasion and metastasis), cell motility and metastasis (cell adhesion and invasiveness).

[00062] Ex vivo assays, as described above, can also be used to determine the neutralization of PDGFRα. For example, SKLMS-1 leiomyosarcoma cells (American Type Culture Collection (ATCC), Rockville, MD; ATCC HTB-88™), or human U118 glioblastoma cells (ATCC HTB-15™) stimulated with PDGF-AA can be used. for measuring PDGFRα inhibition. Growth inhibition can be determined using human tumor cells expressing PDGFRα injected into a SCID mouse.

[00063] The present invention is not limited by any special PDGFRα neutralization mechanism. The anti-PDGFRα antibodies of the present invention externally bind to the PDGFRα cell surface receptor, block ligand binding (e.g., PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC), inhibit phosphorylation of PDGFRα, inhibit signal translation mediated by the receptor-associated tyrosine kinase, and modulate the activity of downstream translation signal components. The receptor-antibody complexes can also be internalized and degraded, resulting in downregulation of the cell surface receptor. Matrix metalloproteinases, which function in tumor cell invasion and metastasis, which can also be downregulated by the antibodies of the present invention. Furthermore, the antibodies of the present invention may exhibit inhibition of growth factor production and angiogenesis.

[00064] As described above, the PDGFRαs antagonists of the invention are useful for the treatment of bone tumors, including metastatic bone tumors. Other types of tumors that express PDGFRα and can be treated in accordance with the invention include, but are not limited to, ovarian tumors, breast tumors, lung tumors, hepatocellular tumors, gastrointestinal stromal tumors, melanoma, renal cell carcinoma, tumors prostate, and soft tissue sarcomas. Soft tissue sarcomas originate in tissues such as fat, muscles, nerves, tendons, and blood and lymphatic vessels. Typically, tumor cells overexpress PDGFRα. Expression of PDGFRα can be determined, for example, through histochemical or RNA analysis. For example, a scatchard analysis of binding of U118 IMC-3G3 cells and radiolabeled SKLMS-1 tumor cells indicates that the number of PDGFRα molecules in the cells is about 500 and 2500, respectively.

[00065] PDGFRα antagonists function by inhibiting signal translation by PDGFRα expressed on tumor cells, or by inhibiting PDGFRα, expressed in surrounding stromal cells that otherwise undergo paracrine stimulation by PDGFs expressed from tumor cells. Thus, antibodies such as IMC-3G3 and other PDGFRα antagonists are useful for the treatment of tumors characterized by autocrine and/or paracrine stimulation of PDGFRα.

[00066] Antibody fragments according to the invention can be produced by cleaving an entire antibody, or by expressing DNA encoding the fragment. Antibody fragments can be prepared by methods described by Lamoyi et al., J. Immunol. Methods, 56: 235-243 (1983) and by Parham, J. Immunol. 131:28952902 (1983). These fragments may contain one or both Fab fragment or F(aB')2 fragment. These fragments may also contain single chain fragment variable region antibodies, i.e., scFv, dibodies, or other antibody fragments. Methods of producing such functional equivalents are disclosed in PCT Application WO 93/21319, European Patent Application No. EP 239400; PCT Application WO 89/09622; European Patent Application No. EP 338745; and European Patent Application No. EP 332424.

[00067] Preferred host cells for vector transformation and expression of the antibodies of the present invention are mammalian cells, for example, COS-7 cells, Chinese hamster ovary (CHO) cells, and cell lines of lymphoid origin, such as of lymphoma, myeloma (ex NSO), or hybridoma. Other eukaryotic hosts, such as yeast, can be used as an alternative.

[00068] Whenever it is desired to express a gene constructed in yeast, a suitable genetic selection for use in yeast is the trpl gene present on the YRp7 yeast plasmid. Stinchcomb et al. Nature, 282: 39 (1979); Kingsman et al., Gene, 7: 141 (1979). The trpl gene provides a selection marker for a mutant strain of yeast lacking the ability to grow on tryptophan, e.g., ATCC No. 44076 or PEP4-1. Jones, Genetics, 85: 12 (1977). The presence of the trpl lesion in the yeast host cell genome then provides an effective means to detect transformation by growth in the absence of tryptophan. Likewise, Leu2-deficient yeast strains (ATCC 20622 or 38626) are complemented by known plasmids containing the Leu2 gene.

[00069] The transformed host cells are cultivated by methods known in the art in a liquid medium containing assimilable sources of carbon (carbohydrates, such as glucose or lactose), nitrogen (amino acids, peptides, proteins or their degradation products, such as peptones , ammonium salts or similar), and inorganic salts (sulfates, phosphates and/or carbonates of sodium, potassium, magnesium and calcium). The medium still contains, for example, growth-promoting substances such as trace elements, for example, iron, zinc, manganese and the like.

[00070] The high affinity anti-PDGFRα and anti-IGF-IRs antibodies according to the present invention can be isolated from a phage display library constructed from human heavy and light chain variable region genes. For example, a variable domain of the invention can be obtained from a peripheral blood lymphocyte that contains a rearranged variable region gene. Alternatively, variable domain portions, such as CDR and FW regions, can be obtained from different sources and recombined. Furthermore, portions of the variable domain (e.g., FW regions) may be consensus synthetic sequences.

[00071] The antibodies and fragments of the antibodies of the present invention can be obtained, for example, from naturally occurring antibodies, or Fab or scFv phage display libraries. It is understood that, to make a single-domain antibody from an antibody that includes an MP and VL domain, certain amino acid substitutions outside of the CDRs may be desired to enhance binding, expression or solubility. For example, it may be desirable to modify amino acid residues that would otherwise be buried at the VH-VL interface.

[00072] Furthermore, 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 herein document by reference), using transgenic mice (e.g., KM mice from Medarex, San Jose, California), which produce human gamma heavy and kappa light chain immunoglobulin, in a preferred embodiment, a substantial portion of the antibody-producing genome human is inserted into the mouse genome, and becomes deficient in the production of endogenous murine antibodies. These mice can be immunized subcutaneously (sc), with PDGFRα (usually in complete Freund's adjuvant), with pulses as needed. Immunization methods are well known in the art. The proteins used to identify the binding of IGF-IR to the antibodies of the invention are preferably IGF-IR and, even more preferably, the extracellular domain of the IGF-IR. The proteins used to identify the binding of PDGFRα to the antibodies of the invention are preferably PDGFRα and, even more preferably, the extracellular domain of PDGFRα. These extracellular domains can be free or conjugated with other molecules.

[00073] The present invention also provides isolated polynucleotides encoding the antibodies, or fragments thereof, described above. Details of IMC-A12 anti-IGF-IRs antibodies are disclosed in WO2005016970. Table 2 sets out the nucleic acid sequences for MC-3G3. Table 2 - Nucleotide Sequences Encoding IMC-3G3 CDRs

[00074] DNA encoding human antibodies can be obtained by recombining human DNA encoding constant regions and variable regions, other than CDRs, derived substantially or exclusively from the corresponding human antibody regions and DNA encoding CDRs resulting from a human (SEQ ID NOS : 1, 3, and 5 for heavy chain variable domain CDRs and SEQ ID NOS: 9, 11, and 13 for light chain variable domain CDRs).

[00075] Suitable sources of DNAs encoding antibody fragments include any cell, such as hybridoma and spleen cells, that express the full-length antibody. The fragments can be used alone as equivalent antibodies, or can be recombined into equivalents, as described above. The DNA deletions and recombinations described in this section can be carried out by known methods, such as those described in the publications listed above regarding antibody equivalences and/or other standard DNA recombinant techniques, such as those described below. Another source of DNAs are single-chain antibodies produced from a phage display library, as is known in the art.

[00076] Furthermore, the present invention provides expression vectors containing the previously described polynucleotide sequences operably linked to an expression sequence, a promoter sequence and an enhancer. A variety of expression vectors for the effective synthesis of polypeptide antibodies in prokaryotes such as bacteria and eukaryotic systems, including, but not limited to, yeast cell culture and mammalian systems have been developed. The vectors of the present invention may include segments of chromosomal, non-chromosomal and synthetic DNA sequences.

[00077] Any suitable expression vector can be used. For example, prokaryotic cloning vectors include E. Coli plasmids, such as colEl, pCR1, pBR322, pMB9, pUC, pKSM, and RP4. Prokaryotic vectors also include phage DNA derivatives such as Ml 3 and other single-stranded filamentous DNA phages. An example of a useful vector in yeast is a 2 μ plasmid. Suitable expression vectors in mammalian cells include well-known S V40 derivatives, adenoviruses, DNA sequences derived from retroviruses, and initiation vectors derived from combinations of mammalian functional vectors, such as those described above, and functional plasmids and phage DNA.

[00078] Additional eukaryotic expression vectors are known in the art (e.g., PJ. Southern and P. Berg, J. Mol. Appl. Genet., I, 327-341 (1982); Subramani et al., MoI. Cell Biol., 1: 854-864 (1981); Kaufmann and Sharp, "Amplification And Expression of Sequences Cotransfected with a Modular Dihydrofolate Reductase Complementary DNA Gene," J. Mol. Biol. 159, 601-621 (1982); Kaufmann and Sharp, MoI. Cell. Biol. 159, 601-664 (1982);Scahill et al., "Expression And Characterization Of The Product OfA Human Immune Interferon DNA Gene In Chinese Hamster Ovary Cells," Proc. Nat'l Acad. Sci. USA 80, 4654-4659 (1983);Urlaub and Chasin, Proc. Nat'l Acad. Sci. USA 77, 4216-4220, (1980).

[00079] The expression vectors useful in the present invention contain at least one control expression sequence that is operatively linked to the DNA sequence or fragment to be expressed. The control sequence is inserted into the vector in order to control and regulate the expression of the cloned DNA sequence. Examples of useful expression control sequences are the lac system, the trp system, the tac system, the trc system, phage lambda major operator and promoter regions, the coat protein fd control region, the yeast glycolytic promoters, e.g. example, the promoter for 3-phosphoglycerate kinase, the yeast acid phosphatase promoters, e.g., Pho5, the yeast alpha mating factor promoters, polyoma, adenovirus, retrovirus, and simian virus promoters and derivatives, e.g., promoters early and late or SV40, and other sequences known to control the expression of genes from prokaryotes or eukaryotic cells and their viruses or combinations thereof.

[00080] The present invention also provides recombinant host cells containing the previously described expression vectors. Antibodies of the present invention can be expressed in cell lines other than hybridomas. Nucleic acids, which comprise a sequence encoding a polypeptide according to the invention, can be used for transformation of a suitable mammalian host cell.

[00081] Cell lines of special preference are selected based on high level of expression, constitutive expression of the protein of interest and minimal contamination of host proteins. Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines, such as, but not limited to, NSO cells, Chinese Hamster Ovary (CHO) cells, Baby Hamster Kidney (BHK) cells, ) and many others. Additional suitable eukaryotic cells include yeast and other fungi. Useful prokaryotic hosts include, for example, E. Coli, such as E. Coli SG-936, E. Coli MP 101, E. Coli W3110, E. Coli X1776, E. Coli X2282, E. Coli DHI, and E. Coli Coli MRCl, Pseudomonas, Bacillus, such as Bacillus subtilis, and Streptomyces.

[00082] These present recombinant host cells can be used to produce an antibody, or fragment thereof, by culturing the cells in conditions that allow expression of the antibody or fragment thereof and purifying the antibody or fragment thereof from the host cell or medium surrounding the host cell. Targeting of the expressed antibody or fragment for secretion into recombinant host cells can be facilitated by insertion of a sequence encoding a signal or secretory leader peptide (see, Shokri et al., Appl Microbiol Biotechnol. 60(6):654-64 (2003 ), Nielsen et al., Prot. [Epsilon]ng. 10:1-6 (1997) and von Heinje et al., Nucl. Acids Res. 14:4683-4690 (1986)), at the 5' end of the gene coding for the Antibody of interest. These secretory leader peptide elements can be obtained from either prokaryotic or eukaryotic sequences. Therefore, secretory leader peptides are used, with amino acids attached to the N-terminal end of a polypeptide to direct the movement of the polypeptide out of the host cell cytosol and secretion into the medium.

[00083] The antibodies of this invention can be fused to additional amino acid residues. Such amino acid residues could be a marker peptide, perhaps to facilitate isolation. Other amino acid residues for harboring organ or tissue-specific antibodies are also contemplated.

[00084] 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, antibodies can be expressed in a tissue-specific manner that facilitates recovery and purification. In one such embodiment, an antibody of the invention is expressed in the secreting mammary gland during lactation. Transgenic animals include, but are not limited to, mice, goats, and rabbits.

[00085] Antibodies that can be used according to the invention include complete immunoglobulins, antigen-binding immunoglobulin fragments, as well as antigen-binding proteins that make up antigen-binding domains of immunoglobulins. Antigen-binding immunoglobulin fragments include, for example, Fab, Fab', and F(aB')2. Other antibody formats have been developed that maintain binding specificity but have other characteristics, which may be desirable, including, for example, bispecificity, multivalency (more than two binding sites), compact size (e.g., binding domains alone).

[00086] Single chain antibodies do not have some or all of the constant domains of the set of antibodies that they are derived from. Therefore, they can overcome some of the problems associated with the use of any antibodies. For example, single-chain antibodies tend to be free from certain unwanted interactions between heavy chain constant regions and other biological molecules. Furthermore, single-chain antibodies are considerably smaller than any antibody and can have greater permeability than any antibody, allowing single-chain antibodies to localize and bind to target antigen binding sites more efficiently. Furthermore, the relatively small size of single-chain antibodies makes them less likely to provoke an unwanted immune response in a whole antibody recipient.

[00087] Various single-chain antibodies, each with a single VH chain and a VL domain covalently linked by a first linker peptide, can be covalently linked by at least one or more linker peptides to form a multivalent single-chain antibody , which can be monospecific or multispecific. Each chain of a multivalent single-chain antibody includes a variable light chain fragment and a variable heavy chain fragment, and is linked by a linker peptide to at least one other chain. The linker peptide is composed of at least fifteen amino acid residues. The maximum number of amino acid residues is about one hundred. Two single-chain antibodies can be combined to form a diabody, also known as a bivalent dimer. Diabodies have two chains and two binding sites, and can be monospecific or bispecific. Each diabody chain includes a VH domain connected to a VL domain. The domains are related to linkers that are short enough to prevent pairing between domains on the same chain, thus directing pairing between complementary domains on different chains to recreate the two antigen-binding sites.

[00088] Three single-chain antibodies can be combined to form triabodies, also known as trivalent trimers. Triabodies are constructed with the amino acid end of a VL or VH domain directly or fused to the carboxyl end of a VL or VH domain, i.e. without any linker sequence. The triabody has three Fv heads with the polypeptides arranged in a cyclic, head-to-tail fashion. One possible conformation of the triabody is planar with the three binding sites located in one plane at an angle of 120 degrees to each other. Triabodies can be monospecific, bispecific or trispecific.

[00089] Thus, antibodies of the invention and fragments thereof include, but are not limited to, naturally occurring antibodies, such as bivalent (Fab')2 fragments, monovalent ones, such as Fab fragments, single chain antibodies, single chain Fv (scFv), single domain antibodies, multivalent single chain antibodies, diabodies, triabodies, as well as those that specifically bind with antigens.

[00090] Anti-IGF-IR and anti-PDGFRα antibodies or antibody fragments, which can be internalized upon binding to cells carrying IGF-IR (WO2005016970) or PDGFRα, can be chemically or biosynthetically linked to anti-tumor agents. Antitumor agents linked to this type of antibodies include any agents that destroy or damage a tumor to which the antibody is bound or in the cell environment to which the antibody is bound. For example, an antitumor agent is a toxic agent, such as a chemotherapy agent or a radioisotope. Suitable chemotherapeutic agents are known to those skilled in the art and include anthracyclines (ex daunomycin and doxorubicin), methotrexate, vindesine, neocarzinostatin, cisplatin, chlorambucil, cytosine arabinoside, 5-fluorouridine, melphalan, ricin and calicheamicin. Chemotherapeutic agents are conjugated to the antibody using conventional methods (See, for example, Hermentin and Seiler, Behring Inst. Mitt. 82:197-215 (1988)).

[00091] Radioisotopes suitable for use as anti-tumor agents are also known to those skilled in the art. For example, I 131 or 211At is used. These isotopes are attached to the antibody using conventional techniques (See, for example, Pedley et al., Br. J. Cancer 68, 69-73 (1993)).

[00092] Alternatively, the anti-tumor agent that is linked to the antibody is an enzyme that activates a prodrug. In this way, a prodrug is administered to remain 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 allowed to localize in the region of the tissue to be treated. The prodrug is then administered to the patient, so that conversion to the cytotoxic drug occurs in the region of the tissue to be treated.

[00093] Other anti-tumor medicinal agents include cytokines such as interleukin-2 (IL-2), interleukin-4 (IL-4) or tumor necrosis factor alpha (TNF-α). The antibody targets the cytokine to the tumor, so that the cytokine mediates the damage or destruction of tumor tissues without affecting others. The cytokine can be conjugated to the antibody on DNA using conventional recombinant DNA techniques.

[00094] In certain embodiments of the invention, 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 antineoplastic agents); WO 99/60023 (Waksal et al.) (Anti-EGFR antibodies in combination with radiation). Any suitable antineoplastic agent can be used, such as a chemotherapeutic agent, radiation or combinations thereof. The antineoplastic agent may be an alkylating agent or an antimetabolite. 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.

[00095] Useful antineoplastic agents also include mitosis inhibitors such as taxanes, docetaxel and paclitaxil. Topoisomerase inhibitors are another class of antineoplastic drugs that can be used in combination with antibodies of the invention. These include inhibitors of topoisomerase I or topoisomerase II. Topoisomerase I inhibitors include irinotecan (CPT-11), aminocamptothecin, camptothecin, DX-8951f, topotecan. Topoisomerase II inhibitors include etoposide (VP-16), and teniposide (VM-26). Other substances are currently being evaluated for topoisomerase inhibitory activity and efficacy as antineoplastic agents. In a preferred embodiment, the topoisomerase inhibitor is irinotecan (CPT-Il). 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.

[00096] When the right antineoplastic agent is radiation, the radiation source can be either external (external beam radiation therapy-EBRT) or internal (brachytherapy-BT), for the patient to be treated. The dose of antineoplastic agent administered depends on several factors, including, for example, the type of agent, the type and severity of the tumor being treated, and the route of administration of the agent. It should be emphasized, however, that the present invention is not limited to a certain dose.

[00097] Antibody treatments (anti-IGF-IR or anti-PDGFRα antibodies) and antibodies plus anti-neoplastic agent can also be used for patients receiving adjuvant hormonal therapy (for example, for breast cancer) or androgen deprivation (e.g. prostate cancer).

[00098] Anti-IGF-IR and anti-PDGFRα antagonists of the invention can be co-administered, or administered with receptor antagonists that neutralize other receptors involved in tumor growth or angiogenesis. For example, in one embodiment of the invention, an anti-IGF-IRs antibody and an anti-PDGFRα antibody are co-administered; in one embodiment, in which a target tumor cell expresses both IGF-IR and PDGFRα, common elements of signal translation are activated by signal translation through each receiver. Although inhibition of one receptor generally results in decreased activation of the common components below, inhibition of both receptor activation will decrease it further. In another embodiment, certain tumor cells or surrounding tissue express significant amounts of one receptor, and other cells express significant amounts of the second receptor. Co-administration of antagonists reduces tumor cell growth and paracrine stimulation of surrounding cells.

[00099] A bispecific antibody can be provided as an alternative to co-administration. The variety of bispecific antibodies that exist 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 anti-binding characteristics that are similar to those of the corresponding natural antibodies. Certain regions of bispecific antibodies incorporate Fc, thereby maintaining effector functions (e.g., complete dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC)) of natural antibodies. WO 01/90192 describes tetravalent antibodies similar to IgG. WO2006/020258 describes a tetravalent antibody that integrates two diabodies and maintains effector functions.

[000100] In another embodiment, an anti-IGF-IR antibody or an anti-PDGFRα antibody or other antagonist is used in combination with a receptor antagonist that specifically binds to an epidermal growth factor receptor (e.g., EGFR, Her2/erbB2, erbB3, erbB4). Especially preferred are antigen-protein bonds that bind to the extracellular domain of the EGFR and block the binding of one or more of its ligands and/or neutralize ligand-induced activation of the EGFR. EGFR antagonists also include antibodies that bind to an EGFR ligand and inhibit the binding of EGFR to its ligand. EGFR ligands include, for example, EGF, TGF-α, amphiregulin, heparin-binding EGF (HB-EGF), and betacellulin. EGF and TGF-α are thought to be the main endogenous ligands that result in EGFR-mediated stimulation, although TGF-α has been shown to be more potent in promoting angiogenesis. Antagonist of EGFRs also include substances that inhibit dimerization of EGFR with other EGFR receptor subunits (i.e., EGFR homodimers) or heterodimerization with other growth factor receptor (e.g., HER2). Antagonists of EGFRs include novel biological molecules and small molecules such as synthetic kinase inhibitors that act directly on the cytoplasmic domain of the EGFR to inhibit EGFR-mediated signal translation. Erbitux ® (cetuximab) is an example that binds to the EGFR Antagonist EGFR and blocks binding to the ligand. An example of a small molecule EGFR antagonist is IRESSA™ (ZD1939), which is a quinozaline derivative that functions as an ATP-mimetic to inhibit EGFR. See US Patent no. 5,616,582 (Zeneca Limited); WO 96/33980 (Zeneca Limited) at p. 4; see also, Rowinsky et al, Abstract 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, 2001. Another example of a small molecule EGFR antagonist is Tarceva® (OSI-774), which is a derivative of 4- (substituted-phenylamine)quinozaline [6,7-Di(2-methoxy-ethoxy)-quinazolin-4-yl]-(3-ethynyl-phenyl)amine hydrochloride] EGFR inhibitor. See WO 96/30347 (Pfizer Inc.) at, for example, on page 2, line A12, through page 4, line 34, 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® may work by inhibiting the phosphorylation of EGFR and its signal translation pathways downstream PI3/Akt and MAP (mitogen activated protein) kinase resulting in p27-mediated cell-cycle arrest. See Hidalgo et al-Abstract 281 presented at the 37th Annual Meeting of ASCO, San Francisco, CA, May 12, 2001.

[000101] Other small molecules have also been reported to inhibit EGFR, many of which are thought to be specific for the tyrosine kinase domain of an EGFR. Some examples of such small molecule EGFR 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). Specific examples of small molecule EGFR antagonists include Cl-1033 (Pfizer), which is a quinozaline (N-[4-(3-chloro-4-fluoro-phenylamino)-7-(3-morpholin-4-yl- propoxy)-Quinazolin-6-yl]-acrylamide) inhibitor of tyrosine kinase, particularly EGFR and is described in WO 00/31048 on page 8, lines 22-6; PKIl 66 (Novartis), which is a pyrrolopyrimidine inhibitor of EGFR and is described in WO 97/27199 at pages 10-12; GW2016 (GlaxoSmithKline), which is an EGFR and HER2 inhibitor; EKB569 (Wyeth), which is reported to inhibit the growth of tumor cells that overexpress EGFR or HER2 in vitro and in vivo; AG-1478 (Trifostin), which is a small molecule quinazoline that inhibits both EGFR and erbB-2 signaling; AG-1478 (Sugen), which is an inhibitory bisubstrate that also inhibits the CK2 protein kinase; PD 153035 (Parke-Davis), which is reported to inhibit EGFR kinase activity and tumor growth, induce apoptosis in cells in culture, and enhance the cytotoxicity of cytotoxic chemotherapeutic agents; SPM-924 (Schwarz Pharma), which is a tyrosine kinase inhibitor targeted for the treatment of prostate cancer; CP- 546,989 (OSI Pharmaceuticals), which is reportedly an angiogenesis inhibitor for the treatment of solid tumors; ADL-681, which is an EGFR kinase inhibitor targeted for the treatment of cancer; PD 158780, which is a pyridopyrimidine that is reported to inhibit the growth rate of A4431 tumor engrafted in mice; CP-358774, which is a quinzoline that is reported to inhibit autophosphorylation in HN5 engrafted mice; ZD 1839, which is a quinzoline that is reported to have antitumor activity in engrafted mouse models including vulvar, NSCLC, prostate, ovarian, and colorectal cancers; CGP 59326A, which is a pyrrolopyrimidine that is reported to inhibit the growth of EGFR-positive grafted mice; PD 165557 (Pfizer); CGP54211 and CGP53353 (Novartis), which are dianylnophthalimides. Naturally derived EGFR tyrosine kinase inhibitors include genistein, herbimycin A, quercetin, and erbstatin.

[000102] Other small molecules reported to inhibit EGFR and which are therefore within the scope of the present invention are tricyclic compounds such as the compounds described in US Patent no. 5679683; quinazoline derivatives, such as the derivatives described in US Patent no. 5616582; and indole compounds such as the compounds described in US Patent no. 5196446.

[000103] Another receptor that can be targeted along 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 is used that specifically binds to the VEGFR-I/FIt-I receptor. In another embodiment, the EGFR antagonist specifically binds to the VEGFR-2/KDR receptor. Especially preferred are antigen-protein bonds that bind to the extracellular domain of VEGFR-I or VEGFR-2 and block binding by their ligands (VEGFR-2 is more strongly stimulated by VEGF, VEGFR-I is more strongly stimulated by PlGF, but also By VEGF) and/or neutralize ligand-induced activation. For example, IMC-1121 is an antibody that binds to humans and neutralizes VEGFR-2 (WO 03/075840; Zhu). Another example is MAb 6.12 which binds to soluble VEGFR-I expressed on the surface of cells and ScFv 6.12 comprises the VL and VH domains of the mouse monoclonal antibody MAb 6.12. The MAb 6.12 producing hybridoma cell line has been deposited with the ATCC number PTA-3344 under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and the corresponding regulations (Budapest Treaty) . In another embodiment, the EGFR antagonist binds to a VEGFR ligand and blocks activation of a VEGFR by the ligand. For example, Avastin (bevacizumab) ® is an antibody that binds to VEGF.

[000104] Other examples of growth factor receptors involved in tumorigenesis are nerve growth factor (NGFR), and fibroblast growth factor (FGFR).

[000105] In yet another alternative modality, anti-IGF-IR and anti-PDGFRα antibodies can either be administered in combination with one or more suitable adjuvants, such as, for example, cytokines (IL-10 and IL-13, for Example) as well as other immune stimulators, such as, but not limited to, chemokine, tumor-associated antigens, and peptides. See, for example, Larrivee et al., cited above. It should be appreciated, however, that administration of just one anti-IGF-IR or anti-PDGFRα antibody is sufficient to prevent, inhibit, or reduce tumor progression in a therapeutically effective manner.

[000106] In a combination therapy, the anti-IGF-IR or anti-PDGFRα antibody is administered before, during or after therapy with another agent, as well as any combination, that is, before and during, before and after, During and after, or before, during and after anti-neoplastic agent therapy begins. For example, the antibody may be administered between 1 and 30 days, preferably 3 and 20 days, more preferably between 5 and 12 days before initiating radiation therapy. In a preferred embodiment of the invention, it is administered concomitantly with chemotherapy or, even more preferably, for subsequent antibody therapy.

[000107] In the present invention, any suitable method or route can be used to administer antibodies of the invention and, optionally, to co-administer anti-neoplastic agents and/or other receptor antagonists. The antineoplastic agent regimens used in accordance with the invention include any regimen believed to be best suited for treating the patient's neoplastic condition. Different malignancies may require use of specific anti-tumor antibodies and specific anti-neoplastic agents, which will be determined on a patient-to-patient basis. Routes of administration include, for example, orally, intravenously, intraperitoneally, subcutaneously, or intramuscularly. The dose administered depends on several factors, including, for example, the type of antagonists, the type and severity of the tumor being treated and the route of administration of the antagonists. It should be emphasized, however, that the present invention is not limited to a specific method or route of administration.

[000108] One skilled in the art would understand that the doses and frequency of treatment will depend on the tolerance of the individual patient and on the pharmacological and pharmacokinetic properties of the blocking or inhibitory agent used. Ideally, one will want to achieve saturable pharmacokinetics for the agent used. The dose for both anti-IGF-IR and anti-PDGFRα antibodies can vary, for example, from about 10 to about 1000 mg V/m2, preferably from about 200 to about 400 mg V /m2. This may be followed by several other daily or weekly dosages ranging, for example, from about 200 to about 400 mg V/m2. The patient is monitored for side effects and treatment is stopped when such side effects are severe.

[000109] Someone skilled in the art is also necessary to know how to monitor the progress of the treatment in order to determine an effective dose. For bone metastases from prostate cancer, one such way is to monitor PSA levels. Other ways to track bone metastases include bone scans and MRI.

[000110] For patients for whom cancer treatment induced bone loss (CTIBL) is a risk or problematic (e.g., patients receiving adjuvant hormone therapy for breast cancer or androgen-deprivation therapy for prostate cancer) , any aforementioned treatment can be completed by the administration of CTEBL prevention agents, such as bisphosphonates. Bisphosphonates include, for example, clodronate, risedronate, and zoledronic acid.

[000111] Throughout this application, various publications, reference texts, books, technical manuals, patents, patents and applications were referred to. The teachings and disclosures of these publications, patents, patent applications and other documents in their entirety are incorporated by reference into this application to more fully describe the prior art to which the present invention pertains.

[000112] It should be understood and expected that variations in the principles of the invention disclosed herein can be made by one skilled in the art and it is intended that such modifications should be included within the scope of the present invention.

[000113] The following examples further illustrate the invention, but should not be interpreted to limit the scope of the invention in any way. Detailed descriptions of conventional methods, such as those working on the construction of vectors and plasmids, and expression of antibodies and antibody fragments, can be obtained from several publications, including Sambrook, J et al., (1989) Molecular Cloning : A Laboratory Manual, 2nd edition., Cold Spring Harbor Laboratory Press; Coligan, J. et al. (1994) Current Protocols in Immunology, Wiley & Sons, Incorporated; Enna, SJ. Al ET. (1991) Current Protocols in Pharmacology, Wiley & Sons, Bonifacino, JS Al ET. (1999) Current Protocols in Cell Biology, Wiley & Sons. The references mentioned here are incorporated in full.

Examples Example 1 Effects of IMC-A12 and docetaxel on tumor growth.

[000114] Tumor pieces (20 to 30 mm3), of LuCaP 35V androgen-independent (AI) were implanted subcutaneously (sc) in SCID mice castrated at 32 six weeks of age respectively as previously described (4). When tumor implantation was observed to reach a volume of 150-200 mm3, animals were randomly assigned to four treatment study groups. The animals in group 1 received treatment with docetaxel at a dose of 20 mg/kg. Animals in Group 2 received treatment with docetaxel at a dose of 10 mg/kg. Animals in Group 3 received a combined treatment of 10 mg/kg docetaxel and 40 mg/kg A12. Animals in Group 4 received a combined treatment of 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 an additional four weeks before sacrifice. Tumors were measured twice a week and tumor volume was calculated using the formula: Volume = L x P2/2. Following our IACUC-approved University of Washington animal protocol, some animals were sacrificed one hour early when the tumor reached a volume of 1000 mm3 or when the animals' body weight loss exceeded 20% of initial body weight. The animals were weighed twice a week. Blood samples were collected from the orbital sinus weekly. Serum was separated and the PSA level was determined using the EVIx Total PSA Assay (Abott Laboratories, Abott Park, IL). BrdU was injected into the tumors 1 hour before the animals were sacrificed to evaluate the proliferation rate of tumor cells in vivo.

[000115] After sacrifice, the tumors were collected and halved. A portion of the tumors were fixed in 10% neutral formalin buffer (NFB), and embedded in paraffin. Five-micron sections were prepared for immunohistochemical (IHC) staining. The remaining part of the tumor was mechanically divided into single cells by grinding and filtered through 70 μm Nylon sieves.

[000116] As shown in the figure. 1, LuCaP 35V grafts grew aggressively in mice with a mean growth rate of 362.0±72.0 mm3/week without any treatment. All animals in the non-treated group had to be sacrificed within three weeks after the start of treatment in experimental groups due to tumor volumes exceeding 1000 mm3. When animals were treated with 40 μg/kg A12 alone, the tumor growth rate was reduced to 192.7±35.6 mm3/week during treatment. When docetaxel was administered to animals at a dose of 10 mg/kg, the LuCaP 35V tumor growth rate was reduced to an average of 29.6±6.1 mm3/week. When docetaxel was administered in combination with A12 treatment, the LuCaP 35V tumor growth rate was further reduced to an average of 7.9± 1.0 mm3/week (Fig. Ib). The inhibitory effect of docetaxel combined with A12 persisted for more than four weeks after the end of treatment. When a higher dose of docetaxel (20mg/kg) was given to animals independently, with or without combined A12 treatment, tumor volume did not increase during the four-week treatment period; on the other hand, a tendency towards a reduction in tumor volume was observed. However, in the four weeks following the end of treatment, the reduction in tumor volume was maintained in the group of animals treated with docetaxel combined with A12. In contrast, tumor volumes were increasing at a mean rate of 27.0±16.1 mm3/week in the group of animals treated with docetaxel monotherapy. These results have suggested that, at a certain dose of docetaxel, combination treatment with A12 may enhance the inhibitory effect of docetaxel on tumor growth during treatment or after treatment follow-up.

[000117] PSA is a clinical parameter commonly used to assess prostate tumor growth. Serum PSA levels were measured in animals during and after treatment. As shown in the figure. Ic, in animals treated with A12 and docetaxel or 20 mg/kg docetaxel alone, no significant change was observed in serum PSA levels during the four weeks of treatment, consistent with repressed tumor growth. After the end of treatment, serum PSA levels were shown to be increased in animals treated only with docetaxel and, on the other hand, coherent or even decreased in animals treated with docetaxel in association with A12. These data are consistent with continued treatment post-inhibition of tumor growth in animals treated with docetaxel and A12.

Induction of apoptosis by docetaxel combined with anti-IGF-IRs antibody

[000118] The combined in vivo effect of docetaxel and A12 treatment on cell cycle and cell survival at experimental endpoints was measured by deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay and propidium labeling (PI) using Apop -Direct Kit (BioScience BD), as previously described. Briefly, 1x106 cells of single cell suspension were fixed with 10% neutral formalin buffer (NBF), followed by 70% ethanol in 20°C alcohol for 30 minutes. After several washes, cells were permeabled with 0.1% Triton buffer (100 μg/mL PI, 50 μg/mL RNase) at room temperature for 60 minutes. Samples were analyzed by flow cytometry using a BD FACscan. Data were analyzed with CellQuestPRO software.

[000119] Four weeks after the end of treatment, apoptosis was detected in a significant percentage of tumors from 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 20mg/kg), in combination with A12 (Fig. 2b and Table 1), regardless of the dose of docetaxel being used. The average number of apoptotic events in these tumors occurred at a rate of 15.0±4.3%. No apoptosis in tumors was detected in animals that were treated with docetaxel alone. Instead, the majority (88% in the group treated with Docetaxel 10 mg/kg and 100% in the group treated with Docetaxel 20 mg/kg) of the tumors proceeded to the normal cell cycle (Fig. 2a and Table 3). Table 3 - Tumor cell cycle and survival activities at the time of sacrifice

[000120] To further evaluate the proliferation capacity of tumor cells after a different end of treatment, paraffin section stained anti-BrDu antibodies. Tumor samples were fixed in 10% NBF, embedded in paraffin, and sectioned into 5-μm slides. After paraffin removal and rehydration, antigens were recovered with 0.01 M citric acid (pH 6.0) at 95°C for 2 X 5 minutes. Slides were allowed to cool for 30 minutes, followed by sequential washing with PBS. Endogenous peroxidase activity was stopped by 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 hour, slides were incubated with the measured mouse anti-BrdU antibody (1 μμ g/mL) for 1 hour. followed by sequential incubation with biotin goat anti-mouse IgG for 30 minutes, peroxidase-labeled avidin for 30 minutes (Santa Cruz Biotechnology) and diaminobenzidine (DAB)/chromogen hydrogen peroxide substrate (Vector Laboratories, Burlingame, CA) for 5-10 minutes. The incubation steps were carried out at room temperature. Slides were costained with hematoxylin (Sigma), and mounted with permount (Fisher Scientific, Fair Lawn, New Jersey). For negative control, mouse IgG (Vector Laboratories) was used instead of primary anti-BrdU antibody. The slides were examined under a Zeiss Microscope and digital images were obtained. Numbers of BrdU-labeled core and total core were collected from 10 random views of each section. The proliferation index was calculated as the number of BrdU-positive nuclei divided by the total number of nuclei. Ten fields were counted by slip. H&E labeling was performed using hematoxylin and eosin (Richard Allen, Kalamazoo, MI).

[000121] In animals that were treated with docetaxel and A12, BrDu uptake was significantly lower than those treated with the same dose of docetaxel in monotherapy (Fig. 3). These BrDu incorporation data are consistent with the above cell cycle and apoptosis observations, suggesting that A12 significantly enhances the cytotoxicity of docetaxel.

Differential regulation of gene expression in tumors treated with docetaxel combined with anti-IGF-IRs antibody versus docetaxel alone

[000122] To determine the potential mechanisms for the markedly enhanced effect of docetaxel by A12, IGF-IR expression was analyzed in all harvested tumors by immunological analysis and flow cytometry. There was no difference in surface IGF-IR expression between all treatment groups or compared to the control group (data not shown). Post-treatment gene expression was analyzed using cDNA microarray analyzes on tumors from animals that had received 20 mg/kg docetaxel and 20 mg/kg docetaxel combined with A12. Based on SAM analysis, 49 genes were identified as differentially expressed in tumors that received the combination treatment of docetaxel and A12 compared to those received docetaxel alone, with more than 2-fold change and less than 10% false discovery rate (FDR) ( data not shown). Thirteen genes were identified that are potentially involved in cell cycle regulation or apoptosis (Table 4). All 13 genes were at least 2-fold different 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 monotherapy. Table 4 - Differentiated post-treatment gene expression in tumors treated with docetaxel + A12, compared to tumors treated with docetaxel alone.

[000123] For selected genes, the results were confirmed by real-time RT-PCR. A standard PCR fragment of target cDNA was purified. A series of standard dilutions from 10 ng/μL to 10-3 pg/μL were used for real-time RT-PCR to generate standard curves. One μg of total RNA from each pooled tumor group was used for first-strand cDNA synthesis using Superscript First Strand Synthesis System (Invitrogen). Real-time RT-PCR was performed on 20 μl of reaction mixture containing 1 μl of first-strand cDNA, cd-specific primer sets, and Lightcycler FastStart DNA Master Plus SYBR Green using a Roche Lightcycler following the manufacturer's protocol (Roche, Nutley , NJ). The RT-PCR products were subjected to melting curve analysis using the Lightcycler v3.5 program. Amplicon sizes were confirmed by agarose gel electrophoresis. Each sample was dosed twice. The results are presented in Fig. 4.

[000124] Of the down-regulated genes, TUBB has been shown to result in resistance to docetaxel (Tanaka et al. 2004, Int. J. Cancer 111, 617-26), and increased expression of BIRC 5 (survivin) has shown to be associated with aggressive prostate cancer and resistance to antiandrogen therapy (de Angelis et al. 2004, Int. J. Oncol. 24, 1279-88; Zhang et al. 2005, Oncogene 24, 2474-82) Additionally , TUBB is an IGF-IR regulated gene that is involved in IGF-IR-mediated transformation (Loughran et al. 2005, Oncogene 24, 6185-93). Of the four upregulated genes, IGFBP3 has been shown to inhibit ligand-IGF signaling as well as to induce apoptosis in prostate tumor cells in a ligand-dependent manner (Grimberg et al. 2000, J. Cell. Physiol. 183 , 1-9).

Post-treatment serum levels of A12

[000125] Serum levels of A12 were measured in animals that received docetaxel combined with A12. Serum A12 levels decreased 100-fold two weeks after stopping treatment, and were detected at a very low level four weeks after stopping treatment (Fig. 5).

General Cytotoxicity

[000126] The cytotoxicity of co-administration of docetaxel and IMC-A12 was examined. Although A12 has greater than 95% cross-reactivity with murine IGF-IR, no activity or abnormal daily changing behavior was observed in animals treated with docetaxel alone or combined reagents, compared to control tumor-bearing animals. No significant effects on renal cells were observed in any treatment group by either cell cycle or apoptosis assays (data not shown). No significant changes in body weight were observed between treatment groups (Fig. 6).

Anti-IGF-IR Antibody Therapy for Bone Metastasis

[000127] The effectiveness of anti-IGF-IR antibody treatment on metastatic growth of prostate cancer in bone cells was evaluated using prostate cells injected directly into the tibia of SCID mice. By this method, metastatic tumors are established directly, without reliance on chemotaxis-dependent invasion of the circulation. A variety of tumor lines are available to establish bone metastases. These include PC-3, LuCaP35, and LnCaP cells that produce osteolytic lesions and LuCaP 23.1 cells that produce osterblastic lesions.

[000128] LuCaP 23.1 cells, which express IGF-IR, have an index of -80% in the bone environment and result in osteoblastic reactions, in preliminary experiments, LuCaP 23.1 samples showed a significant increase in quantity vs volume bone tissue (BV% /TV) in tumor versus control tibias (254-503% control; p = 0.024). AU LuCaP 23.1 tumors in tibias exhibited new bony trabeculae, which were not present in the normal samples, and a high number of tumor foci, which had replaced the normal bone marrow. In some specimens the tumor growth and bone extended beyond the original bone. Increased % BV/TV of LuCaP 23.1 samples was also observed after castration; The %BV/TV of tibias with tumor was 212-354% than that of tibias without tumor (p = 0.024). The results observed for intra-tibia grafts of LuCaP 23.1 indicate the existence of new bone formation stimulated by tumor cells. Furthermore, the tumors show many similarities to human samples of osteoblastic bone metastases, including a large number of tumor foci and increased amounts of mineralized bone.

[000129] To evaluate the efficacy of treatment with IMC-A12, LuCaP 23.1 tumor grafts were engrafted into SCID mice, and serum PSA levels were measured biweekly to assess tumor growth. tibia tumor cell graft. Administration of EVIC-A12 to test mice was initiated when serum PSA levels reached 5-10 ng/mL (indicating established tumors). 40 mg/kg IMC-A12 was injected ip three times a week for six weeks.

[000130] Bone mineral density (BMD) of tibias with tumors and contralateral tibias without tumor was measured by double X-ray absorptiometry (PIXImus Lunar densitometer) performed in an area of 2.5 mm x 2.5 mm in the tumor cell injection, or the corresponding location of the contra-tibia at the time of grafting. Every two weeks assessment of the lesions was made by serum PSA measurements. All animals were sacrificed when the bone lesions in the control group had recurred after castration based on serum PSA levels (LuCaP 35 >60 ng/mL, LuCaP 23.1 >500 ng/mL), radiographic appearance of the bone lesions or when animals have become compromised. One hour before being sacrificed, the animals were injected with BrdU to control tumor cell proliferation. Radiographs were taken before sacrifice (FaxitronX-rays-MX-20), and BMD of both tibias were measured at the time of sacrifice. Table 5 - Bone Mineral Density (BMD)

[000131] Serum PSA levels were significantly lower in mice treated with IMC-A12 (Fig. 7), and the increase in BMD associated with the growth of osteoblastic metastatic tumors was significantly reduced as well (Table 5). BMD measurements of tumor-free legs indicated that treatment with IMC-A12 did not cause a loss of bone density (osteoporosis). Radiographs of IMC-A12-treated and untreated mice show that tumor progression was significantly reduced or prevented in treated mice (Fig. 8).

[000132] Combination of anti-IGF-IRs antibody and docetaxel for bone metastases. SCID mice are castrated 2 weeks prior to tibia tumor injectables. Bone metastases are generated by direct injection of LuCaP 23.1 prostate cancer cells into the tibia of mice, giving rise to osteoblastic lesions. The grafts express IGF-IR. Serum PSA levels are measured every two weeks to assess tumor growth. When serum PSA levels reach 5-10 ng/mL indicating tumor establishment, the animals are randomly distributed into four groups.

[000133] In two groups, 40 mg/kg of BVIC-A12 are injected IP three times a week for six weeks with one group receiving BVI-A12 + Docetaxel 20 mg/kg IP once a week for 6 weeks and a second group, IMC-A12 + Docetaxel 10 mg IP Three times a week for 6 weeks. Control groups receive either 20 mg IP docetaxel Without BMI-A12.

[000134] Animals are monitored weekly with PSA measurements. After the end of treatment, animals continue to be monitored weekly with PSA measurements until tumors in docetaxel-only groups show tumor resurgence. As PSA values increase in docetaxel-only groups (albeit at a slower rate than in untreated animals), PSA levels in mice treated with IMC-A12 + docataxel level off and, in some animals, begin to decrease. Reductions in PSA levels are seen to continue even after treatment ends at six weeks.

[000135] As indicated above, BMD measurements are taken at the time of grafting and at sacrifice, and radiographs are taken only before sacrifice. The IMC-A12 + docataxel- treated groups show little or no increase in BMD, and radiographs show little or no sign of osteoblastic activity.

Combination of anti-IGF-IRs antibody and docetaxel for bone metastases

[000136] Pieces of human LuCaP 23.1 prostate tumor (20 to 30 mm3) were mechanically digested. 2-5 x 105 of viable LuCaP 23.1 cells were injected into the tibias of 6-8 week old SCID mice. 21 mice randomly distributed into three groups were used for the study. After tumor injection, serum PSA was monitored weekly. Treatment began when serum PSA level reached 5-10 ng/mL, an indication of tumor growth. Group 1 received control buffer saline vehicle. Group 2 received 20 mg/kg IP docetaxel once a week for 4 weeks. Group 3 received 20mg/kg of docetaxel once a week and 40mg/kg of A12 IP three times a week for 4 weeks. To determine whether the response to treatment was osteoblastic or osteolytic, BMD was measured by Dexa-scan and X-rays of the animals at the end point of all treatments.

[000137] Only docetaxel or docetaxel combined with A12 significantly inhibited LuCaP 23.1 tumor growth as reflected by the suppression of serum PSA levels (Fig. 9), with no significant difference between the two treatments. However, after stopping treatment, serum PSA levels began to increase in animals that had been treated with docetaxel monotherapy, indicating tumor regrowth; Continued suppression of serum PSA levels was observed in the animals that received the combined treatment, indicating a prolonged period of post-treatment tumor quiescence. Serum PSA levels have been shown to correlate with bone density (BMD) and radiographed tumor bone sizes (Fig. 9b). At week five, the mean bone density in control, docetaxel 20, and docetaxel 20 combined with A12 treated animals was 0.112±0.01, 0.09±0.02, and 0.05±0.009 (mean±SEM), respectively. There was an apparent trend toward a decrease in bone density with treatment.

Example 2 Akt phosphorylation induced by bone marrow aspirate

[000138] Bone marrow samples from normal male donors (ages 18-45), were provided by Cambrex (Poietics ™ Donor Program). The samples were centrifuged at 1500 RPM, in order to separate the soluble and cellular phases. The supernatant was filtered using 0.8 μm and 0.22 μm filters in succession. 50 μg/mL bone marrow aspirate was administered to cells in 1 mL of medium (1:20 final dilution).

[000139] For experiments performed in the presence of serum, cells were cultured in DMEM supplemented with 10% FBS and 50 μg/mL gentamicin for 24 hours before exposure to bone marrow. For experiments in the absence of serum (starved cells), cells were washed twice with PBS, growth medium was replaced with serum-free DMEM, and cells were incubated for 4 hours before exposure to the bone marrow preparation. When used, AG-1296, a specific inhibitor of the PDGF receptor (Rice et al., 1999, Amer. J. Path. 155, 213-21) was added to the cultures for 30 minutes. Before exposure to marrowaspirate bone. IMC-3G3 antibodies were administered as described at pre-treatment times as indicated below.

[000140] Akt activation by bone marrow was detected in PC3-ML cells, which express PDGFRα, but not in DU-145 cells, which lack the receptor. In one experiment, to minimize the effect of serum components on Akt activation cells were preincubated for 4 hours in serum-free medium. Addition of bone marrow extracts resulted in robust phosphorylation of Akt in PC3-ML cells but not DU-145 cells. (Fig. 10A). To assess the significance of the response, a second experiment was performed with serum. A robust stimulation of Akt phosphorylation in PC3-ML by bone marrow aspirate cells was also observed in the presence of serum. (Fig. 10B). Only a small response was elicited in DU-145 airframes.

PDGFRα-mediated Akt phosphorylation.

[000141] It is imagined that osteoblasts and osteoclasts, which secrete both PDGF-AA and PDGF-BB, provide the growth of these soluble factors in the bone marrow medium. To determine whether the response of PC3-ML cells to bone marrow extracts was related to signal translation through PDGFRα, PC3-ML cells were exposed to bone marrow aspirate in the absence or presence of 20 μM AG-1296. This concentration of AG-1296 completely inhibits Akt activation. induced by PDGF-BB (Fig. 1 IA) AG-1296 inhibited bone marrow aspirate-induced Akt activation by more than 40%. (Fig. 1 IB and D). This indicates that PDGFRα signaling is responsible for a significant proportion of bone marrow-induced Akt activation.

[000142] The direct contribution of PDGF-AA and BB-to PDGFRα signaling in relation to other components of bone marrow aspirates was also evaluated. It was decided that the concentrations of PDGF-AA and BB-in bone marrow aspirates from three different donors ranged from 400 pg/mL to 2 ng/mL. Given the 20-fold dilution of the marrow aspirate, test cells were actually being exposed to PDGF-AA and BB at concentrations between 20 and 100 pg/mL. Therefore, PC3-ML cells were treated with 100 pg/mL of each of PDGF-AA and BB-. Akt phosphorylation was less than 10% of that obtained with bone marrow aspirates. Fig. 3 C and D). Therefore, it appears that Akt activation through PDGFRα signaling may involve PDGFRαs ligands with the exception of PDGF-AA and BB-and/or other mechanisms of PDGFRα activation by direct ligand binding.

Inhibition of Akt phosphorylation by an anti-PDGFRα antibody.

[000143] The neutralizing 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 of simulant neutralized the effects of 30 ng/mL of PDGF-BB. (Fig. 12A) Antibody treatment also resulted in about 40% inhibition of bone marrow-induced Akt phosphorylation (Fig. 12B and C). It was also observed that the inhibitory effect of IMC-3G3 on Akt phosphorylation was dependent on the duration of preincubation, with 120 minutes of incubation time being significantly more effective (Fig. 12D) than the 30 minutes of incubation time (Fig. 12 B, C). One possible explanation is that IMC-3G3 induces the internalization of PDGFRα, and that its inhibitory effect is related not only to blocking ligand binding, but also to the removal of the receptor from the plasma membrane.

Example 3 Isolation of Human Anti-PDGFRα Antibodies

[000144] Human anti-PDGFRα monoclonal antibodies were generated by standard hybridoma technology (Harlow & Lane, ed., Antibodies: A Laboratory Manual, Cold Spring Harbor, 211213 (1998), which is incorporated herein by reference), using Transgenic mice (Medarex Inc., Sunnyvale, CA) expressing human gamma heavy and light kappa immunoglobulin chains. Human PDGFRα extracellular domain (ECD) was purchased from R&D Systems (Minneapolis, MN). KM mice were immunized subcutaneously (sc) with 3 x 107 porcine aortic endothelial cells stably expressing PDGFRα (PAE Ra). After 4 weeks, mice were injected sc with 50 μg of PDGFRα ECD in Freund's complete adjuvant plus 3 x 107 PAE Ra cells. by Freund.

[000145] Splenocytes from mice with high serum levels of binding and blocking titers were isolated and fused with myeloma cells. Hybridoma cultures exhibiting blocking activity were subcloned and antibodies from these hybridomas were purified by protein G chromatography.

[000146] IgGs were evaluated for binding to PDGFRα in a direct binding assay. PDGFRα ECD in PBS was immobilized in a 96-well plate (100 ng/well). 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 the immobilized PDGFRα ECD for 1 h at 25 °C, and the plates were washed with PBST. A secondary antibody (goat anti-human F(aB')2 IgG-horseradish peroxidase conjugate; BioSource International, Camarillo, CA) diluted 1:5000 in PBSM was introduced 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 of 1 mol/L H2SO4. Plates were read on an A450 run using a microplate reader (Molecular Devices, Sunnyvale, CA).

[000147] PDGF blockade was evaluated using a solid phase PDGF blockade assay (see Duan et al.-1991, J. Biol. Chem. 266:413-8, which will be incorporated by reference). PDGFRα ECD was diluted in PBS and coated in 96-well microtiter plates (Immulon 2HB Flat-Bottom 1 x 12 Removawell Strips from Irradiated Polystyrene Protein Binding; Dynex Technologies, Chantilly, VA). Each well was coated with 60 ng PDGFRα for 3 hours at 25°C in a total volume of 100 μL. 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. Plates were then heated. at 25°C for 20 minutes and then washed with binding buffer (25 mmol/L HEPES (pH 7.45), 0.3% gelatin, 100 mmol/L NaCl, 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 1 nmol/L solution) to each well. 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. The cell-based blocking assay was performed as described in Heldin et al., 1988, EMBO J. 7, 1387-93.

[000148] The kinetics of antibody binding to PDGFRα was measured using a BIAcore 3000 instrument (BIAcore, Inc., Piscataway, NJ). PDGFRαECD was immobilized on a sensor chip and the antibody was injected at various concentrations. Sensograms were obtained at each concentration and evaluated using the BIA Evaluation 2.0 program to determine rate constants. The affinity constant, Kd, was calculated from the ratio of the Koff/Kon rate constants.

[000149] Fig. 13 shows the dose-dependent binding of the human monoclonal antibody DVIC-3G3 to PDGFRα ECD immobilized in ELISA. The antibody concentration required for 50% maximum binding to PDGFRα ECD was 0.06 nmol/L (Table 6). The ED50 is consistent with the Kd for the antibody as determined by surface plasmon resonance on a BIAcore instrument (Table 1). The monoclonal antibody also blocks [125I]PDGF-BB binding to immobilized receptor, with an IC50 of 0.43 nmol/L. The binding sites for PDGF-PDGF-AA and BB on PDGFRα are structurally not coincident. The data suggest that the epitope for 3G3 spatially overlaps both growth factor binding sites. Table 6 - anti-PDGFRα antibody binding characteristics

Inhibition of receptor activation and phosphorylation of downstream effector molecules.

[000150] The effects on intracellular signaling induced by PDGF by IMC-3G3 were determined using PAE Ra cells. Cells were seeded in six-well Falcon tissue culture plates (250,000 cells per well), and allowed to grow overnight. Wells were washed and then incubated in serum-free medium. After overnight incubation to render cells quiescent, cells were treated with antibodies for 30 minutes at 37°C followed by addition of PDGF-AA or PDGF-BB and incubation for an additional 10 minutes at 37°C. Cells were then detached and lysed in 200 μL lysis (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 enhanced chemiluminescence and Hyperfilm reagents (Amersham Biosciences).

[000151] The antibody was tested for the ability to inhibit ligand-induced tyrosine receptor phosphorylation. PDGF-AA and PDGF-BB increase PDGFRα tyrosine phosphorylation by about 5-fold at concentrations of 1 and 3 nmol/L, respectively. Higher ligand concentrations (10 nmol/L) resulted in less phosphorylated receptor possibly due to ligand-induced degradation. The antibody inhibited PDGF-BB-induced receptor to near background levels (Fig. 14A, top row). Similar data were obtained using PDGF-AA to induce receptor phosphorylation.

[000152] PDGFs translate mitogenic signals and exert antiapoptotic effects on receptor expressing proteins through effector cells below. Consequently, the monoclonal antibody was tested for its ability to inhibit the activation of MAPKs p44/p42 and Akt (involved in cell growth and antiapoptotic precursors, respectively). Anti-PDGFRα antibodies inhibited phosphorylation of both MAPKs and Akt in response to PDGF-BB (Fig. 2A) and PDGF-AA (not shown). Inhibition of PDGFRα phosphorylation was dose dependent, with 50% inhibition obtained at 0.25 nmol/L (Fig. 14B).

Antimitogenic activity

[000153] The anti-PDGFRα monoclonal antibody was tested for its ability to block PDGFAA-induced mitogenesis of PAE Ra cells. Cells were seeded in 96-well tissue culture plates (1 x 104 cells per well) and grown rapidly in 100 μL of medium per well. The wells were then washed with serum-free medium and cells were serum-free overnight with 75 μL of serum-free medium 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) was 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 [3H]thymidine (25 μL/well). Antibody, PDGF, and [3H]thymidine were all diluted in serum-free medium. Cells were then washed with PBS plus 1% bovine serum albumin and separated by trypsin treatment (100/xL/well). Cells were collected on a thrice filter and washed with double-distilled water using a Harves MACH III cell (Tomtec, Inc., Hamden, CT). After filter processing, the radioactivity of the incorporated DNA was determined in a scintillation counter (Wallac Microbeta, model 1450).

[000154] When MC-3 G3 was added to serum-starved PAE Ra cells, PDGF-AA-induced thymidine incorporation was specifically inhibited (Fig. 15), with an EC50 of 8.3 nmol/L. The antibody also inhibited the 3 nmol/L PDGF-BB-induced mitogenesis of PAE Ra cells with an EC50 of 1.25 nmol/L (data not shown).

Growth inhibition of human tumor cell lines expressing PDGFRα

[000155] Human tumor cell lines expressing PDGFRα were tested to determine the influence of human anti-PDGFRα antibodies on growth in malignant systems, in vitro and in vivo. Two such tumor cell lines that express PDGFRα as determined by flow cytometry are SKLMS-1 (leiomyosarcoma) and U118 (glioblastoma). These cell lines also respond to ligands in mitogenic assays and form tumors in mice. SKLMS-1 has the potential for not only paracrine but also autocrine stimulation. SKLMS-1 has been shown to express PDGF-AA protein when grown in culture using a quantitative enzyme sandwich immunoassay technique (R&D Systems).

[000156] As can be seen in the figure. 16 A, IMC-3G3 inhibited the phosphorylation of both Akt and MAPKs in response to PDGF-AA stimulation of SKLMS-1 cells. The inhibition of Akt phosphorylation was 100% and that of MAPKs was around 80%. The antibody is also an effective inhibitor of phosphorylation in U118 cells (Fig. 16B). Ligand-induced mitogenesis of tumor cells was also blocked. When anti-PDGFRα antibody was added to serum-starved U118 cells, PDGF-AA-induced thymidine incorporation was specifically inhibited (Fig. 17A), with an EC50 of 3.4 nmol/L. The antibody also inhibited the PDGF-AA-induced mitogenic response of SKLMS-1 cells with an EC50 of 5 nmol/L (Fig. 17B), as well as the PDGF-BB-induced mitogenic response (Fig. 17C). Only partial inhibition (40% at 66 nmol/L; Fig. 17D) of PDGF-BB-induced mitogenic response was observed for U118 cells. This is attributed to the expression of both PDGFRα and PDGFRβ in these cells (data not shown).

Inhibition of grafted tumor growth

[000157] IMC-3G3 was tested in vivo in glioblastoma (U118) and leiomyosarcoma (SKLMS-1) via subcutaneous (sc) models grafted into hairless athymic mice. Sc tumor grafts were established by injecting 10 x 106 SKLMS-1 or U118 mixed cells in Matrigel (Collaborative Research Biochemicals, Bedford, MA) into female athymic hairless mice (Crl: NU/NU-nuBR, Charles River Laboratories, Wilmington , MA). Tumors were allowed to reach an average tumor volume (π/6 x longest length x perpendicular depth2) of approximately 400 mm3. The mice were randomly distributed into five groups (N = 12) and treated by ip injection twice a week during the study period. Group 1 mice were treated with vehicle control (0.9% NaCl, USP for Irrigation, B/Braun). Mice in Groups 2 to 4 were treated with 6, 20, and 60 mg/kg of instantaneous anti-PDGFRα antibodies. Group 5 mice were treated with 60 mg/kg human IgG (Sigma). Groups treated with 6, 20, or 60 mg/kg anti-PDGFRα antibodies or human IgG were given 21.4, 71.4, and 214 mg/kg loading dose, respectively. Loading doses were calculated to achieve a steady-state plasma concentration of the first dose (elimination half-life, 7 days) using a twice-weekly dosing schedule. Tumor volumes were assessed twice weekly and tumor growth in the treatment groups was compared with a repeated measures ANOVA.

[000158] As shown in the figure. 18A, human IgG, had no effect on glioblastoma growth compared to saline-treated mice (P = 0.74), whereas anti-PDGFRα antibody significantly inhibited tumor growth in 6 (P = 0.06). ), 20 (P = 0.03), and 60 (P = 0.0004) mg/kg dose. In the final study of U118, the %T/C values [(mean tumor volumes for 3G3-treated group at study completion/mean tumor volume at start of treatment)/(mean tumor volume for control-treated group at study completion /mean tumor volume at the start of treatment) x 100] were 67%, 63%, and 35% for groups treated with 3G3 doses of 6, 20, and 60 mg/kg, respectively. Furthermore, tumor regression was observed in 4 of 12, 5 of 11, and 10 of 12 animals in the 6, 20, and 60 mg/kg treatment groups. There were no regressions in any control group.

[000159] Fig. 18 B shows that leiomyosarcoma growth was also significantly inhibited by treatment at 6 (P=0.02), 20 (P=0.003), and 60 (P<0.0001) mg/Kg. The final % T/C values were 66%, 57%, and 31% for treatment groups 6, 20, and 60 mg/kg, respectively, without tumor regressions.

[000160] Histological examination of grafts at the end of treatment showed marked differences in tumors from treated animals, compared to tumors from animals that received control therapy. Resected tumors were fixed in QDL fixative at 4°C for 24 hours. After paraffin embedding and sectioning at 4 μm, formalin-fixed sections were stained with Mayer’s H&E (Richard Allen, Kalamazoo, MI).

[000161] In the U118 group treated with the highest dose (60 mg/kg), fewer viable tumor cells were found and there were substantially more regions with sparse cells, compared to the serum control group (Fig. 18C). SKLMS-1 grafts treated on day 25 also showed a reduction in the amount of viable tumor cells and cell packaging compared to the serum control group (Fig. 18D).

In vitro inhibition of PDGFRα-mediated stimulation of a glioblastoma cell line

[000162] The level of the phosphotyrosine receptor in U118 tumors was evaluated one week after treatment with anti-PDGFRα antibody or human IgGt. Mice with stabilized U118 tumors (500 mm3) were treated with a loading dose of 214 mg/kg followed 72 hours later by a maintenance dose of 60 mg/kg of antibodies. Tumors were harvested from the mice one week (168 hours) after the first antibody injection (at a time before tumor regression was observed on average; see Fig. 18A) and homogenized in phosphorylation assay lysis buffer (see above). Lysates were centrifuged twice at 14000 RPM and the protein concentration for the harvested supernatant was determined (Bio-Rad protein assay, Bio-Rad, Hercules, CA). Lysate (4 mg) from each sample was immunoprecipitated using anti-PDGFRα antibodies. Immunoprecipitated human PDGFRα were then immunoblotted with either an anti-PDGFR or anti-phosphotyrosine antibody. Fig. 19 shows that administration of anti-PDGFRα antibody resulted in a reduction in the level of PDGFRα phosphotyrosine relative to a human IgG control in these tumors.

Cell Lineage Engineering

[000163] 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 were obtained from MEDAREX that anneals the 5' and 3' flanking sequences of human immunoglobulin variable region sequences in MEDAREX-derived hybridomas. The heavy chain variable region amplified with primer pair AB88 (forward) and AB90 (reverse) (Table 7). The light chain products were amplified with primer pairs containing the forward primer AB 182 and the reverse primer AB 16 (Table 7). The 0.4-kb 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 T7 and M13R universal primers. Table 7 - Primers for MEDAREX hybridomas

[000164] In order to generate plasmid vectors to express complete IgGl antibodies, the cloned variable regions were amplified with PCR and ligated in two steps into constant region genes containing expression vectors. Heavy chain primary PCR amplification used 25 ng of AB88-1 plasmids as template for primers IPHF5 (front) and IPHR5 (back). Amplification with heavy chain secondary PCR used 5 μl of primary reaction as a template and the primers OPSIF and IPHR5. The combination of the two forward primers add a 57 basic pair sequence to the 5' end of the immunoglobulin genes encoding a 19 amino acid mouse heavy chain gene signal sequence (MGWSCIILFLVATATGVHS; SEQ ID NO: 24) for efficient processing and secretion. of immunoglobulin. Furthermore, the OPSIF primary balance adds a consensus "Kozak" sequence (J MoI. Biol. 196:947) for efficient translation initiation of these genes in mammalian cells and a 5' HindIII endonuclease restriction site for cloning of the amplified product. in the appropriate expression vector. The heavy chain reverse primer contains an inframe NheI site for cloning into the constant region of the vector. PCR was performed in two steps using the Expand PCR kit (Boehringer Mannheim Inc.), according to manufacturer specifications using the Expand Buffer #3 system in 50 μl reactions with the following cycle conditions:

[000165] After two rounds of PCR, the product was purified following agarose gel electrophoresis and cloned as a HindIII-NheI digested fragment into pDFc vector (Fig. 8), which contains the human gamma 1 constant region.

[000166] Light chain primary PCR amplification used 25 ng of plasmid pAB182-3 as model primers IPLF4 (front) and IPLR2 (back). The light chain secondary PCR amplification used 5 μl of primary reaction as template and the primers OPSIF and IPLR2. As for the heavy chain, the two forward primers provide a secretion signal sequence. The primary reverse light chain contains an inframe BsiWI site for cloning into the kappa constant region vector pLck (Fig. 8). PCR reactions were performed as the heavy chain above. After two rounds of PCR, the product was purified following agarose gel electrophoresis and cloned into pLck, which contains the human kappa constant light chain region. Table 8 - Primers for VH and VK expression vectors

[000167] In order to generate a single plasmid vector for stable transfection, the heavy chain expression strand, containing the CMV promoter, heavy chain coding region, and polyA element were cloned into the light chain vector as a NotI- fragment. Sa1I (Fig. 20).

[000168] This construct was then used to generate a stable production line of myeloma cell line from NSO cells. NSO cells were transfected with the expression plasmid through electroporation using the BioRad Gene Pulser II. Before transfection, plasmid DNA was linearized with PvuI, precipitated with ethanol, and resuspended at a concentration of 0.4 mg/mL (40 μg in 100 μl dH2O). Cells were electroporated with 40 μg of DNA in a final volume of 800 μL by a single pulse of 250 V, 400 μFd. Electroporated cells were dispersed in 50 μL aliquots in DMEM medium (JRH Biosciences Inc.) containing 10% fetal dialysate (dFCS) calf serum (Hyclone, Lot #: AHA7675) and 2 mM glutamine (Invitrogen/Life Technologies) in wells of approximately eighteen 96-well plates at a density of 5000-10000 cells per well. Selection of glutamine synthetase (GS) positive transfectants was initiated 24 hours later by adding glutamine-free DMEM containing 10% dFCS and supplemented with 1x GS supplement (JRH Biosciences Inc.). Cells were cultured for 2-4 weeks at 37°C, 5% CO2 to allow colony growth and expansion. More than 300 colonies were selected using an anti-human Fc (gamma) ELISA (horseradish peroxidase detection at A450nm). Clones expressing antibodies (58%) were expanded and reanalyzed for productivity during 3-5 days of cultivation. To adapt cells to serum-free medium, positive cell lines were expanded by adding an equal volume of serum-free GS-OS culture medium at each passage. Strong positives, producing 25 μg/mL or more in subconfluent 24-well cultures within 3 days, were expanded for further analysis to fully adapt to serum-free medium.

[000169] It is understood and expected that variations in the principles of the invention disclosed herein can be made by someone skilled in the art and it is intended that these modifications should be included within the scope of application of the present invention.

Claims (9)

1. Isolated human antibody or antibody fragment specific for PDGFRα, characterized by the fact that it comprises 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. 2. Antibody or antibody fragment according to claim 1, characterized in that it comprises the heavy chain variable region having SEQ ID no.: 8 or a light chain variable region having SEQ ID no.: 16. 3. Antibody or antibody fragment according to claim 1, characterized in that it comprises a heavy chain variable region having SEQ ID no.: 8 and a light chain variable region having SEQ ID no.: 16. 4. Antibody or antibody fragment according to claim 3, characterized in that it comprises a heavy chain having SEQ ID no.: 31 and a light chain having SEQ ID no.: 33. 5. Antibody or antibody fragment according to any one of claims 1 to 4, characterized in that it inhibits the binding of PDGFRα to a PDGFRα ligand or that neutralizes PDGFRα. 6. Isolated polynucleotide, characterized in that it comprises a nucleotide sequence that encodes an antibody or antibody fragment, as defined in any one of claims 1 to 5, wherein the nucleotide sequence consists of SEQ ID no.: 1, 3, 5, 9, 11 and 13, or degenerate nucleotide sequences thereof encoding the same amino acid sequences. 7. Expression vector, characterized by the fact that it comprises the polynucleotide as defined in claim 6 operably linked to expression control elements so that the antibody or encoded fragment can be expressed. 8. Antibody or antibody fragment according to any one of claims 1 to 5, characterized in that it is for use as a medicament to inhibit the growth of a primary tumor, metastatic tumor, refractory tumor or vascularized tumor in a mammal . 9. Antibody or antibody fragment according to claim 8, characterized in that the tumor is selected from the group consisting of an ovarian tumor, a breast tumor, a lung tumor, a hepatocellular tumor, a gastrointestinal stromal tumor, a melanoma, a renal cell carcinoma, a prostate tumor, a bone tumor, a soft tissue sarcoma, a leiomyosarcoma, and a glioblastoma.