BRPI0707433A2 - method of inhibiting the growth of an androgen dependent cancer - Google Patents

Abstract

METHOD OF INHIBITING THE GROWTH OF AN ANDROGEN DEPENDENT CANCER The present invention relates to a method of treating prostate cancer with androgen deprivation therapy and an insulin-like growth factor receptor antagonist (IGF-IR). Although the rate of response of prostate cancer to androgen deprivation therapy (ADT) is high, surviving cancer cells invariably become androgen independent (AI) and tumor growth follows. The invention inhibits or delays the transition from androgen-dependent cancer to androgen-independent cancer, significantly decreases the risk of recurrence, and improves treatment outcome.

Description

"METHOD OF INHIBITING THE GROWTH OF AN ANDROGEN CANCER"

FEDERAL FINANCING

The present invention was made in part with the support of the United States Government under No. CA85859 of the National Institutes of Health and No. W81XWH-04-1-0912 of the Department of Defense. Accordingly, the United States Government has certain rights in this invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Application No. 60 / 765,072, filed February 3, 2006, which is incorporated herein by reference in its entirety.

FIELD OF INVENTION

The present invention relates to a method of treating deprostate cancer with androgen deprivation therapy and an insulin-like growth factor receptor antagonist (IGF-IR). The method inhibits our delaying the transition from androgen-dependent cancer to androgen-independent cancer and significantly decreases the risk of recurrence, and improves treatment outcome.

BACKGROUND OF THE INVENTION

Prostate cancer is the most common non-skin cancer and the second most common cause of cancer mortality in men in the U.S. Prostate cancer is mostly initially androgen dependent (AD). Prostate cancer cells initially require androgen for continued proliferation. Response to testosterone ablation by androgen deprivation therapy (TDA), either surgically (orchiectomy) or medically (GnRH estrogens or agonists), leads to rapid induction of sensitive prostate cancer cells. Positive response rate is about 86% based on decreased prostate specific antigen (PSA) and stabilization or decrease in tumor volume. The cell death that occurs usually happens within the first few days to a week. However, the positive response is followed by a period of growth in which the remaining cells tend not to die. After 18-36 months after hormone ablation, growth recurs in 90% of cases. Invariably, surviving cancer cells become independent of and unresponsive to androgen, and follow androgen-independent (AI) tumor growth. Since ADT is initially very effective, a therapy that could benefit from the benefits of ADT and prolong or intensify its effects would be of great benefit.

Androgen independence seems to stem from a variety of mechanisms. Mutations in the androgen receptor gene are diagnostic, but increase after exposure to anti-androgenflutamide. However, these mutations do not occur in most patients and do not explain most cases of hormone refractory disease. Levels of bcl-2 are seen more frequently in advanced disease compared with localized disease. Thus, the ability to induce apoptosis decreases as the disease progresses. Cell proliferation harboring p53 tumor suppressor gene mutations, loss of TGF-β receptors, and expression of peptide growth factors probably play a role in the development of an ahormonium refractory state. However, these processes do not explain the speed and frequency of development.

The insulin-like growth factor receptor (IGF-IR) is a ubiquitous transmembrane tyrosine kinase receptor that is essential for normal fetal and postnatal development and growth. IGF-IR can stimulate cell proliferation, cell differentiation, changes in cell size, and protects cells from apoptosis. It has also been considered almost mandatory for cell transformation (reviewed in Adamset al., Cell. Mol. Life Sci- 57: 1050-93 (2000); Baserga, Oncogene 19: 5574-81 (2000)). IGF-IR is located on the cell surface of most cell types and serves as the signaling molecule for IGF-I and IGF-II growth factors (collectively referred to as IGFs) .IGF-IR also binds to insulin, although in affinity of three orders of magnitude lower than it binds in IGFs. IGF-IR is a preformed hetero-tetramer containing two alpha chains and two beta chains covalently linked by disulfide bonds. Receptor subunits are synthesized as part of a single 180kd polypeptide chain, which is then proteolytically processed into alpha (130kd) and beta (95kd) subunits. Whole 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 required for cell-building transformation, but is unnecessary for mitogen signaling and apoptosis protection.

IGF-IR is highly similar to insulin receptor (IR), particularly within the beta chain sequence (70% homology). Because of this homology, recent studies have shown that these receptors can form hybrids containing an IR dimer and a IGF-IR dimer (Pandini et al., Clin. Canc. Res. 5: 1935-19 (1999)). Hybrid formation occurs in both normal and transformed cells and the hybrid content is dependent on the concentration of the two homodimeric receptors (IRe IGF-IR) within the cell. In a study of 39 breast cancer specimens, although both IR and IGF-IR were overexpressed in all tumor samples, hybrid receptor content consistently exceeded both homoreceptor levels by approximately 3 times (Pandini et al., Clin Canc Res 5: 1935-44 (1999)). Although hybrid receptors are composed of IR and IGF-IR pairs, hybrids selectively bind to IGFs, with similar affinity to that of IGF-IR, and only weakly bind to insulin (Siddle and Soos, The IGF System. HumanaPress. Pp. 199- 225. 1999). These hybrids can therefore bind to IGFs and transduce signals in both normal and transformed cells.

Endocrine expression of IGF-I is primarily regulated by growth hormone and produced in the liver, but recent evidence suggests that many other tissue types are also capable of expressing IGF-I. This ligand is therefore subject to endocrine and paracrine regulation as well as autocrine in this case. of many tumor cell types (Yu, H. and Rohan, J., J. Natl. Cancer Inst. 92: 1472-89 (2000)).

The androgen receptor (AR) consists of 3 functional and structural domains: an N-terminal (modulatory) domain; a DNA binding domain (Interpro Access No. No. IPR001628) that specifically targets specific DNA sequences (alligator responsive elements); and a hormone binding domain. The N-terminal domain (NTD) is unique to androgen receptors and covers approximately the first 530 residues; the highly conserved DNA binding domain is smaller (about 65 residues) and occupies the central portion of the protein; and the hormone ligand binding domain (LBD) is receptor-C termination. In the absence of ligand, steroid hormone receptors are considered to be weakly associated with nuclear components; Hormone binding greatly increases the affinity of the receptor. The interaction between androgen receptor (AR), androgen, prostate cancer is complex. RA distribution between nucleus and oxytoplasm is affected by androgen and androgen suppression. For example, RA immunoreactivity is observed in LuCaP 35 cell nuclei grown in intact male mice, but strong immunoreactivity is observed in cytoplasm and LuCaP 35 nuclei grown in intact and subsequently castrated male mice.

This invention relates to the treatment of androgen dependent tumors such as prostate cancer. Prostate tumors are typically androgen-stimulated such as testosterone, and exhibit androgen-dependent (AD) growth. Therefore, prostate cancer treatment typically involves therapy that deprives the prostate cancer cells of androgen. However, a large proportion of prostate cancers eventually transition to androgen independence (AI). Administration of an IGF-IR antagonist in combination with androgen deprivation therapy (ADT) has been found to inhibit or prevent the transition from AD tumors to AI tumors.

Accordingly, the invention provides a method for treating androgen dependent cancer by administering androgen deprivation therapy and an IGF-IR antagonist. In one embodiment of the invention, androgen dependent cancer is prostate cancer.

According to the invention, the IGF-IR antagonist may be an extracellular antagonist or an intracellular antagonist and more than one antagonist may be employed. More generally, the invention relates to the inhibition of IFG-IR signal transduction and the darota component modulation in order to inhibit the transition of AD to AI tumor cells.

Extracellular antagonists include, but are not limited to proteins or other biological molecules that bind to IGF-IR or its ligand (IGF). In certain embodiments of the invention, the extracellular antagonist inhibits IGF-IR binding in IGF. In one embodiment, the binding protein is an antibody, such as, for example, IMCA12. In another embodiment, the binding protein is IGF-IR soluble linker binding fragment. Intracellular IGF-IR antagonists may be biological molecules, but they are usually small molecules. In one embodiment of the invention, the IGF-IR antagonist is a small molecule selected from AG104, NVPAEW541, and BMS-554417.

The effectiveness of various antagonists to inhibit IGF-IR signal transduction can be observed, for example, by testing the IGF-IR signal transduction pathway state components. In one embodiment, IGF-IR inhibition is observed in reduced Akt phosphorylation. In another embodiment, IGF-IR signaling inhibition is observed in reduced expression of survivin or β-peptide tubulin (TUBB).

An IGF-IR antagonist of the invention is used with any form of ADT. In one embodiment of the invention, ADT comprises orchiectomy. In another embodiment of the invention, ADT comprises administering a hormone-luteinizing hormone-releasing hormone analogue. In another embodiment, ADT comprises administering an antiandrogen. In still another embodiment, an adrenal inhibitor is administered. According to the invention, two or more ADT methods may be combined.

The invention further provides for desalination inhibition through Akt. Accordingly, the invention includes the administration of signal transducing protein modulators that activate Akt. In one embodiment, such a modulator is an EGFR antagonist.

According to the invention, an IGF-IR antagonist is administered as an adjuvant to ADT. In one embodiment, ADT and administration of an IGF-IR antagonist are initiated at about the same time. In another embodiment, ADT is initiated first, and an IGF-IR antagonist is administered before androgen-independent cancer becomes androgen-independent. The invention further provides for the use of ADT antineoplastic agents and administration of deIGF-IR antagonist. In one embodiment of the invention, an IGF-IR antagonist and an ADT agent are used together as a neoadjuvant for surgical or radiation treatment of prostate cancer.

The invention also provides compositions comprising an IGF-IR antagonist and an ADT agent in a dosage form.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows a study in which LuCap35 subcutaneous grafts were observed in SCID mice. All mice were castrated when the average tumor size reached 400 mm. The mouse control group received only castration. In two other groups, IMCA12 was administered three times a week starting one or two weeks after castration.

Figure 2 shows PSA levels in castrated control mice and in IMC-Al2-treated castrated mice starting one (early) or two (late) weeks after castration.

Figure 3 shows the distribution of androgen receptor (RA) in response to IGF-IR stimulation with IGF and / or IGF-IR antagonism with IMCA12. Cytoplasm and nuclear AR RA levels were assessed by Western Blots.

Figure 4 shows the effect of an IGF-IR antagonist (IMC-A12) on androgen receptor (AR) distribution in androgen-dependent LuCaP 35 xenograft cell tumors in intact mice (left column) and LuCaP 35 dexenograft cell tumors androgen deficiency in castrated (columnar right) mice.

Figure 5 shows the correlation between RA score and tumor volume. R = 0.66, ρ <0.01. Castrated-only values are in open circles and late and early Castrated + A12 values are in closed circles. Values are the average value for 100 graded nuclei per tumor.

Figure 6 shows the changes in gene expression between two time periods for A12-treated subcutaneous tumors. Of the 3170 unique genes in the arrangement with sufficient data to test, there were 21 over-regulated (including many androgen-regulated, denoted by and 41 Q-downregulated in the late time period when all tumors began to recur compared to the initial time period). .

Figure 7 A shows the correlation between survivin copy number score and tumor volume (r = 0.66, ρ <0.01). Figure 7B shows the correlation between tubulinabeta peptide 3 copy number score and tumor volume (r = 0.59, ρ <0.01). Castrated-only values are in open circles and late and early Castrated + Al2 values are in closed circles. Each value is the average of three PCR runs.

DETAILED DESCRIPTION OF THE INVENTION

IGF-IR inhibitors have been found to be useful in therapies for treating prostate cancer. In particular, administration of an IGF-IR antagonist in combination with androgen deprivation therapy (ADT) results in improved treatment over ADT alone.

Androgens have been observed to over-regulate insulin-like growth factor-I receptor expression and may sensitize prostate cancer to the effects of IGF-I. Similarly, the transition to androgen independence that is observed in prostate cells may result from adaptations of the cell that increases dandrogen receptor signaling such as increased levels of AR that make the cell sensitive to low levels of circulating and orrogen mutations allowing for steroid activation -androgens. In fact, evidence demonstrates that IGF-I signaling can actually mediate RA translocation to tumor cell nucleus and leads to over-regulation of AR-dependent genes. Thus, it is proposed that IGF-I may promote the conversion of prostate cancer. androgen-dependent to androgen-independent after hormone ablation therapy by promoting AR signaling in the absence of circulating androgen levels. Recent data from human and male prostate xenografts have also shown that current androgen ablation methods fail to lower prostate androgens to levels that no longer result in androgen receptor activation. The prostate may actually be able to synthesize DHT from various precursor steroids and possibly acetate.

Therefore, it follows that inhibition of IGF-Icon signaling concurrent with hormone ablation therapy can prevent or prolong the time to conversion of prostate cancer into androgen-independent disease, significantly delaying the onset of recurrence. IGF-IR antagonists may therefore be adjuvant therapy. effective for androgen deprivation strategies to treat newly diagnosed and locally advanced or hormone-dependent prostate cancer.

The use of androgen suppressing IGF-IR antagonists also has the potential to block IGF-mediated recovery from daapoptosis. Mechanisms by which IGF-IR can abolish apoptosis include ras-raf-map kinase distribution, PI3 kinase including mTOR and forkhead signaling, and 14-3-3. Another mechanism by which IGF-IR inhibition may prolong the effects of androgen suppression is by maintaining the tumor in cell cycle arrest after initial apoptosis.

Previous studies have shown that IGF-IR antagonists may have a positive effect when used to treat both androgen-dependent and androgen-independent prostate cancers. Xenograft growth, although slower, has not been interrupted or reversed. It has now been shown that IGF-IR antagonists are particularly useful for treating prostate cancer when administered with androgen deprivation therapy (ADT). Typically, prostate tumors that transition to androgen dependence become insensitive to ADT. As has been previously observed, such androgen-insensitive tumors tend not to show strong responses to IGF-IR antagonists. However5 as shown, the time to progression of AD to AI prostate tumors is significantly prolonged by therapy that combines ADT with the administration of an IGF-IR antagonist. During that prolonged period, the tumors shrink, and PSA levels are reduced. Combination therapy reduces the high risk of recurrence that is seen with ADT alone, and reduces the risk that metastatic cancer will develop.

Treatment with an IGF-IR antagonist is also advantageous for the treatment of advanced prostate cancer in which metastases are potentially present or have been diagnosed.

In models incorporating prostate cancer cells, translocation of RA from the cytoplasm to the nucleus is observed to be induced not only by androgen stimulation but also, albeit to a lesser extent, by IGF-IR stimulation. Even in the presence of androgen, translocation of RA in the presence of androgen and IGF is reduced by an IGF-IR antagonist.

In the prostate, after castration, low levels of androgens are still detectable. It has also been reported that IGF-IR expression, which signals through Akt, first decreases in response to castration, but then increases, and furthermore that Akt growth factor stimulation intensifies AR signaling for low androgen levels.

As demonstrated herein, treatment with a deIGF-IR antagonist significantly delays tumor regrowth in castrated mice. Moreover, there is a good correlation between decreased nuclear RA and decreased tumor volume. This suggests that IGF-IR de-signaling inhibition plays a considerable role in inhibiting AR-triggered tumor regression. In the experiments described herein, IGF-IR signaling is inhibited using an antibody called A12, which binds on IGF-IR. Previous experiments with A12 and similar antibodies show that there is decreased phosphorylation (i.e. activation) of various signal transducing molecules, including ERK and MAPK, and particularly Akt. The effect of IGF-IR inhibition has been observed in a variety of tumor cell types, including Ml2 prostate cancer lineage (Wu, JD etal., 2005, Clin. Cancer Res. 11: 3065-74) and cancer cells. breast cancer (Burtrum, D. et al., 2003, Cancer Res. 63: 8912-21). Thus, it should be appreciated that the same or similar adjuvant activity observed herein for an IGF-IR antagonist would be observed for agents that exerted a similar effect on Akt activation.

Common IGF-IR antagonist treatment is observed resulting in inhibition of RA translocation to the nucleus. Inhibition may be observed histochemically or by fluorescence microscopy, as well as reduced expression levels of AR-induced genes. Two castration resistance-associated genes, survivin and β-peptide tubulin are regulated by IGF-IR through Akt activation. Gene expression is suppressed in castrated mice treated with an IGF-IR antagonist compared to castration alone. Similar inhibitory effects of AR overtranslocation and Akt-activated gene expression would be observed in response to an Akt-specific inhibitor or antagonist of another signal transduction pathway involving Akt to a significant degree.

A variety of IGF-IR antagonists may be used according to the invention. IGF-IR antagonists may be extracellular antagonists or intracellular antagonists. Extracellular and intracellular IGF-IR antagonists can be biological molecules, small molecules, or any other substance that inhibits IGF-IR activation, for example by interaction with the receptor extracellular binding region (ie, extracellular antagonist), by inhibiting phosphorylation. IGF-IR intracellular tyrosine kinase domain, or by inhibiting interaction activation of any other cellular component involved in the IGF-IR signaling pathway, thereby ultimately inhibiting gene activation or cell proliferation.

In one embodiment of the present invention, an extracellular IGF-IR antagonist interacts with the receptor extracellular ligand binding region through sufficient chemical or physical interaction between the antagonist and the extracellular receptor binding region, such that cheligation of IGF-IR and its receptor. ligand (IGF) is blocked and receptor tyrosine kinase activity is inhibited. One skilled in the art will recognize that examples of such chemical interactions, which include association or bonding, are known in the art and include covalent bonding, ionic bonding, hydrogen bonding, and the like between the antagonist and the extracellular bonding region. In one embodiment of the invention, the extracellular antagonist deIGF-IR is a biological molecule. Biological molecules include, but are not limited to, IGF-IR binding antibodies or antibody fragments. In another embodiment, the IGF-IR antagonist may be a small molecule that blocks ligand binding in IGF-IR. In another embodiment, the extracellular antagonist is a substance that sequesteres or degrades IGF-IR binders. An example is a soluble extracellular IGF-IR fragment that binds to IGF. Another example of such a substance is an IGF binding protein (IGFBP) which can be IGF binding such as IGF receptor limiting activation, such as, for example, IGFBP-1, IGFBP-2, and IGFBP-3. In another embodiment of the invention, a small molecule inhibitor binds in the IGF-IR ligand binding domain and blocks allocation and receptor activation by an IGF-IR ligand.

Although there is no desire to be bound by theory, it is considered that the extracellular IGF-IR antagonist inhibits all signal transduction cascades initiated by conformational changes in the extracellular region of IGF-IR following activation of IGF-IR. This inhibition includes surface IGF-IR as well as those IGF-IR that have been incorporated within a cell. For example, it is considered that activated receptor tyrosine kinases (RTKs) can be incorporated via a clathrin-coated tip into an endosome while still maintaining its signaling activity. Upon incorporation, such receptors are either recycled back to the cell surface or degraded in the endosome or lysosome.

Another way to inhibit IGF-IR mediated signal transduction is by downregulating IGF-IR expression. In one embodiment of the invention, an IGF-IR antagonist binds to the receptor and promotes receptor uptake and degradation. In another embodiment, an IGF-IR antagonist reduces receptor expression.

Biological molecules, in the context of the present invention, include all amino acids, nucleotides, lipids and demonosaccharide polymers that generally have a molecular weight greater than 650 D. Thus, biological molecules include, for example, oligopeptides, polypeptides, peptides, and proteins, oligonucleotides. and polynucleotides such as, for example, DNA and RNA, and oligosaccharides and polysaccharides. Biological molecules further include other derivatives of any of the molecules described above. For example, derivatives of biological molecules include lipids and glycosylation or oligopeptide derivatives, polypeptides, peptides, peptides, and proteins. Derivatives of biological molecules additionally include lipid derivatives of epolysaccharide oligosaccharides, e.g. lipopolysaccharides. More typically, biological molecules are antibodies or their functional derivatives.

Small molecules include organic compounds such as heterocycles, peptides, saccharides, steroids, and the like, organometallic compounds, salts of organic compounds and organometallic compounds, and inorganic compounds. Atoms in a small molecule are linked together via covalent and ionic bonds; the former are typically for small organic compounds such as small molecule detirosine kinase inhibitors and the latter are typical of small inorganic compounds. The arrangement of atoms in a small organic molecule may represent a chain, eg a carbon-carbon chain or a carbon-heteroatom chain, or may represent a ring containing carbon atoms, eg benzene or a polycyclic system, or a combination of carbon and heteroatoms, ie, heterocycles such as a pyrimidine or quinazoline. Although small molecules may have any molecular weight, they generally include molecules that would otherwise be considered biological molecules, except that their molecular weight is no greater than 650 D. Small molecules include both naturally occurring compounds such as hormones, neurotransmitters, nucleotides, amino acids, sugars. lipids and their derivatives as well as synthetically made compounds, either by traditional organic synthesis, biomediated synthesis, or a combination thereof. See e.g. Ganesan, DrugDiscov. Today 7 (1): 47-55 (Jan. 2002); Lou, Drug Discov. Today, 6 (24): 1288-1294 (Dec. 2001). The compounds may be modified to enhance efficacy, stability, pharmaceutical compatibility, or the like.

Intracellular IGF-IR antagonists may be biological molecules, such as mutant receptor subunits, intracellular binding proteins (e.g., intracellularly expressed antibody fragments), or the like. In a preferred embodiment, intracellular antagonists are small molecules. Small molecule inhibitors include but are not limited to small molecules that modify or block the ATP deletion domain, substrate binding regions, or deIGF-IR kinase domain. Small molecule inhibitors also include substances that inhibit other components of the IGF-IR signal transduction pathway, including, but not limited to, ras-mitogen-activated protein kinase (MAPK) pathway, and the phosphatidyl-pathway. inositol-3 kinase (PI3K) -Akt.

To identify antagonists, small molecule libraries can be screened for inhibitory activity using high throughput cell-based, enzymatic or biochemical assays. Assays can be formulated to detect the ability of a test compound to inhibit IGF-IR binding in IGF-IR binders or IRS-I substrate or to inhibit formation of functional receptors of IGF-IR dimers. The intracellular IGF-IR antagonist may inhibit IGF-IR tyrosine kinase activity by binding or inhibiting activation of the intracellular region having a kinase domain or by binding or inhibiting activation of any intracellular protein in the IGF-IR signaling pathway. Small molecule IGF-IR antagonists include, for example, NVP-AEW541 insulin-like growth factor-I receptor selective kinase inhibitors (Garcia-Echeverria, C. et al., 2004, Cancer Cell5: 231-9) and NVP-ADW742 (Mitsiades, C. et al., 2004, Cancer Cell 5: 221-30), INSM-18 (Insmed Incorporated), which selectively inhibits IGF-IR and HERZ, AG1024 and AG1034 tyrosine kinase inhibitor triphosphates ( Párrizas, M. et al, 1997, Endocrinology 138: 1427-33) which inhibit substrate-binding blockade phosphorylation and have a significantly greater IC 50 for inhibition of IFG-IR phosphorylation than for IR phosphorylation.

The cyclolignan picropodophylline (PPP) derivative is another IGF-IR antagonist that inhibits IGF-IR phosphorylation without interfering with IR activity (Girnita, A. et al., 2004, Cancer Res. 64: 236-42) . Other small IGF-IR demolecule antagonists include benzimidazole derivatives BMS-536924 (Wittman, M. et al., 2005, J. Med. Chem. 48: 5639-43) and BMS-554417 (Haluska P. et al., 2006, CancerRes 66: 362-71), which inhibit IGF-IR eIR almost equipotently. For compounds that inhibit receptors in addition to IGF-IR, it should be noted that IC50 values measured in vitro in direct binding assays may not reflect IC50 values measured ex vivo or inventive (i.e., in intact organisms or cells). For example, where it is desired to avoid IR inhibition, a compound that inhibits IR in vitro may not significantly affect receptor activity when used in vivo at a concentration that effectively inhibits IGF-IR.

Antisense oligodeoxynucleotides, antisense RNAs and small inhibitory eRNAs (siRNAs) provide for selected demRNA degradation, thus preventing protein translation. Consequently, expression of receptor tyrosine kinases and other critical proteins for IGF signaling may be inhibited. The ability of antisense oligonucleotides to suppress gene expression was described over 25 years ago (Zamecnik and Stephenson, 1978, Proc. Natl. Acad. Sci. USA- 75: 280-84). Antisense oligonucleotides pair bases with mRNA and pre-mRNAs can potentially interfere with various processing steps and RNA message translation, including editing, polyadenylation, export, stability, and protein translation (Sazani and Kole, 2003, J. Clin.Invest- 112: 481- 86). However, the two most powerful and widely used antisense strategies are the degradation of fmRNA or pre-mRNAvia RNaseH and alteration of editing via selection of aberrant editing junctions. RNaseH recognizes DNA / RNA heteroduplicates and cleaves RNA approximately in the middle between the 5 'and 3' ends of the DNA oligonucleotide. IGF-IR inhibition by antisense oligonucleotides is exemplified in Wraight, Nat-Biotechnol. 18: 521-6.

Innate RNA-mediated mechanisms may regulate mRNA stability, message translation, and chromatin organization (Mello and Conte, 2004, Nature. 431: 338-42). In addition, exogenously introduced long double-stranded RNA (dsRNA) is an effective gene parasitization tool in a variety of lower organisms. However, in mammals, long dsRNAs generate highly toxic responses that are related to the effects of viral infection and interferon production (Williams, 1997, Biochem. Soc. Trans. 25: 509-13). To avoid this, echolegic Elbashir (Elbashir, et al., 2001, Nature. 411: 494-98) began using desiRNAs composed of 19-mer duplexes with 5 'phosphates and 2 base3' projections at each end, which selectively degrade. Selected mRNAs under introduction into cells.

The action of interfering dsRNA in mammals usually involves two enzymatic steps. First, Dicer, a type III RNase enzyme, cleaves dsRNA into 21-23-mer siRNA segments. Then, RNA-induced deletion complex (RISC) unwinds the RNA duplex, resembles a strand with a complementary region in a cognate mRNA, and initiates cleavage at a 10 nucleotide site upstream of the 5 'end of the desiRNA strand (Hannon, 2002, Nature 418: 244-51). Short chemically synthesized siRNAs in the 19-22 mer range do not require the Dicer step and can enter the RISC machinery directly. It should be noted that any strand of an RNA duplex may potentially be loaded onto the RISC complex, but the oligonucleotide composition may choose the strand choice. Thus, to achieve selective degradation of a particular mRNA target, the duplex must favor loading of the antisense tape component by having relatively weak base pairing at its 5 'end (Khvorova, 2003, Cell 115: 209-16). Exogenous siRNAs may be provided as synthesized or expressed oligonucleotides of viral or plasmid vectors (Paddison and Hannon, 2003, Curr.Opin. Mol. Ther. 5: 217-24). In the latter case, precursor molecules are normally expressed as short hairpin RNAs (shRNAs) containing 4-8 nucleotide loops and 19-30 nucleotide trunks; these are then cleaved by Dicer to form functional siRNAs.

Anti-IGF-IR antibodies to be used according to the present invention exhibit one or more of the following properties:

1) Antibodies bind to the external domain of IGF-IR and inhibit IGF-I or IGF-II binding to IGF-IR. Inhibition may be determined, for example, by a direct binding assay used for purified or membrane bound receptor. In this embodiment, the antibodies of the present invention, or fragments thereof, preferably bind IGF-IR at least as tightly as natural IGF-IR (IGF-I and IGF-II) ligands.

2) Antibodies neutralize IGF-IR. Binding of a ligand, e.g., IGF-I or IGF-II, to an extracellular, external domain of IGF-IR stimulates beta subunit autophosphorylation and phosphorylation of IFG-IR substrates including MAPK, Akt, and IRS-1.

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

One measure of IGF-IR neutralization is inhibition of receptor tyrosine kinase activity. Tyrosine kinase inhibition can be determined using well known methods; for example by measuring recombinant kinase receptor autophosphorylation, and / or phosphorylation of natural or synthetic substrates. Thus, dephosphorylation assays are useful in determining the context-neutralizing antibodies of the present invention. Phosphorylation can be detected, for example, using a phosphoritosin-specific antibody in an ELISA or western blot. Some assays for tyrosine kinase activity are described in Panek et al., 1997, J. Pharmacol. Exp. Thera. 283: 1433-44 eBatley et al., 1998, Life Sci. 62: 143-50. 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 90% in ligand responsive cells.

Another measurement of IGF-IR neutralization is inhibition of phosphorylation of downstream IGF-IR substrates. Consequently, the level of phosphorylation of MAPK, Akt, or IRS-I can be measured. The decrease in substrate phosphorylation is at least about 50%, preferably at least about 65%, more preferably at least about 80%.

In addition, methods for protein expression detection can be used to determine IGF-IR neutralization, in which proteins being measured are regulated by IGF-IR tyrosine kinase activity. An example of such a protein that is associated with cancer progression and drug resistance is survivin, which is a member of the apoptosis inhibitor (IAP) family. Although survivin regulation is complex mediated by more than one route, Akt-mediated and IGF-I enhanced regulation has been demonstrated. See, e.g., Zhang et al., 2005, Oncogene, 24: 2474-82. Methods for analyzing gene expression include immunohistochemistry (IHC) for protein expression detection, in situ hybridization fluorescence (FISH) for degene amplification detection, competitive radioligand binding assays, solid matrix blotting techniques such as Northern and Southern blots , reverse transcriptase polymerase chain reaction (RT-PCR) and ELISA. See, e.g., Grandis et al., 1996, Cancer, 78: 1284-92; Shimizu et al., 1994, Japan J. Cancer Res., 85: 567-71; Sauter et al., 1996, Am. J. Path., 148: 1047-53; Collins, 1995, Glia15: 289-96; Radinsky et al., 1995, Clin. Cancer Res. 1: 19-31; Petrides et al., 1990, Cancer Res. 50: 3934-39; Hoffmann et al., 1997, Anticancer Res. 17: 4419-26; Wikstrand et al., 1995, Cancer Res. 55: 3140-48.

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

The antibodies of the present invention are not limited by any particular mechanism of IGF-IR neutralization. The anti-IGF-IR antibodies of the present invention may bind externally at the IGF-IR cell surface receptor, block ligand binding (eg, IGF-I or IGF-II) and subsequent receptor-tyrosine kinase-mediated signal transduction. prevent phosphorylation of IGF-IR and other downstream signal transduction nacascata proteins.

3) Antibodies infra-modulate IGF-IR. The amount of GF-IR present on a cell surface depends on the production, incorporation, and degradation of receptor protein. The amount of IGF-IR present on the surface of a cell can be measured indirectly by detecting receptor incorporation or a receptor-bound molecule. For example, receptor incorporation can be measured by counting IGF-IR expressing cells with an antibody. marked. Membrane bound antibody is then extracted, collected and counted. Incorporated antibody is determined by cell lysis and detection of labeling in lysates. Another way is to directly measure the amount of receptor present on the cell after treatment with an anti-IGF-IR antibody or other substance, for example, by fluorescence-activated cell classification analysis. stained cells for surface expression of IGF-IR.

Stained cells are incubated at 37 ° C and fluorescence intensity is measured over time. As a control, part of the stained population may be incubated at 4 ° C (conditions under which receptor uptake is interrupted).

Cell surface IGF-IR can be detected by measuring 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, etal., 2003, Cancer Res. 63: 8912-21 ). Treatment of an IGF-IR expressing cell with an antibody of the invention results in reduction of IGF-IR cell surface. In a preferred embodiment, the reduction is at least about 70%, more preferably at least about 80%, and even more preferably at least about 90% in response to treatment with an antibody of the invention. A significant decrease can be observed in as little as four hours.

Another measure of infra-modulation is reduction of the total protein receptor 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 cell. 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%.

For treatment of human subjects, the antibodies are preferably human antibodies, but may also be chimeric or humanized antibodies. A preferred human antibody that binds IGF-IR is Al2 (see, W02005016970). Another preferred human antibody is 2F8 (see, W02005016970). Useful antibodies additionally include anti-IGF-IR antibodies that compete with IMCA12 or IMC-2F8 for IGF-IR binding, as well as antibodies that bind on other epitopes (ie, antibodies that bind on other epitopes and exhibit properties as previously described such as blockade of binder, receptor uptake, etc., but do not compete with IMCA1 or IMC-2F8). Other non-limiting examples of anti-IGF-IR neutralizing antibodies useful in accordance with the invention are described by Wang et al. (WO2003 / 1000008; US 2004/0018191) and Singh et al. (WO 2003/106621; US2003 / 0235582). The nucleotide and amino acid sequences of various antibodies mentioned herein are indexed in Table 1.

Table 1. SEQ DD NOS for CDRs and Antibody Variable Domains (nucleotide / amino acid)

<table> table see original document page 23 </column> </row> <table>

Antibodies which may be used according to the invention include whole immunoglobulins, immunoglobulin antigen binding fragments, as well as antigen binding proteins comprising immunoglobulin antigen binding domains. Immunoglobulin antigen binding fragments include, for example, Fab, Fab ', and F (ab'). 2. Other antibody formats have been developed which retain binding specificity, but have other characteristics which may be desirable, including, for example. , bispecificity, multivalence (more than two binding sites), compact size (eg, binding domains only).

Single chain antibodies comprise two variable domains missing from some or all of the constant domains of the antibody antibodies from which they are derived. Therefore, they may overcome some of the problems associated with the use of whole antibodies. For example, single chain antibodies tend to be free of certain unwanted interactions between heavy chain constant regions and other biological molecules. In addition, single chain antibodies are considerably smaller than whole antibodies and may have higher permeability than whole antibodies, allowing single chain antibodies are localized and bind at target antigen binding sites more efficiently. Moreover, the relatively small size of single chain antibodies makes them less likely to elicit an unwanted immune response in one container than whole antibodies.

Multiple single stranded antibodies, each single strand having a Vh domain and a Vl domain covalently linked by a first peptide linker, may be covalently linked by at least one or more peptide linkers to form multivalent single chain antibodies, which may be monospecific or multispecific. Each of a multivalent single chain antibody includes a variable light chain fragment and a variable heavy chain fragment, and is linked by a peptide linker to at least one other chain. The peptide linker is comprised of at least fifteen amino acid residues. The maximum number of amino acid residues is about one hundred.

Two single chain antibodies may be combined to form a diabody, also known as a bivalent dimer. Bodies have two chains and two binding sites, and may be either monospecific or bispecific. Each chain of the body includes a Vh domain connected to a VL domain. Domains are connected with linkers that are short enough to prevent pairing of domains within the same chain, thus conducting pairing between complementary domains over different chains to recreate the two antigen binding sites. Similarly, three single chain antibodies may be combined to form a tri-antibody, also known as a tri-equivalent. Triabodies are constructed with the amino acid terminus of a V1 or Vh domain directly fused to the carboxyl terminus of a V1 or Vh domain (i.e., without any linker sequence). Triabodies may be monospecific, bispecific or trypecific. Bispecific antibodies that are bivalent to each antigen binding site also have been developed. For example, Zhu (WO 01/90192) describes an antibody with four binding sites that otherwise has the structure of and retains effector functions of a naturally occurring antibody. Zhu (WO2006 / 020258) describes a bispecific antibody that incorporates two Ig constant bodies and regions.

Thus, antibodies of the invention and fragments thereof include, but are not limited to, naturally occurring antibodies, divalent fragments such as (Fab ') 2, monovalent fragments such as Fab, single chain antibodies, single chain Fv (scFv), single domain antibodies multivalent single chain antibodies, diabodies, triacibodies, and the like that specifically bind with antigens.

IGF-IR antagonists are exemplified herein by IMCA12, a human monoclonal antibody that binds in the extracellular domain of IGF and blocks IGF binding. Properties of IMCA12 and a similar human antibody may be provided in International Publication WO2005 / 016970.

Effects of IGF-IR antagonists of the invention on androgen dependent prostate cancer cells include one or more of the following. 1) IGF can mediate activation or translocation of RA in the absence of androgen. IGF-IR antagonists of the invention block IGF-mediated translocation. 2) IGF-IR antagonists mediate intensified cell killing or inhibition of tumor cell proliferation. 3) AR-mediated androgen receptor-activated degene expression is reduced. Genes demonstrating AR-mediated expression include, for example, PSA and TMPRSS2 (a transmembrane serine protease).

According to the invention, an IGF-IR antagonist is administered to an individual having prostate cancer coinciding with androgen deprivation therapy (ADT; also called hormone therapy). The goal of ADT is to lower the levels of male hormones (androgens such as testosterone) in the body. Androgens, produced mainly in the testicles, can actually stimulate prostate cancer cell growth. Lowering androgen levels can actually cause prostate cancers to shrink or grow slower.

ADT is used in several situations: as first-time (initial) therapy for patients unable to have surgery or radiation or who cannot be cured by these treatments because cancer has already been spread beyond the prostate gland; after initial treatment, such as surgery or radiation therapy, if the cancer remains or returns; as an (adjuvant) addition to radiation therapy as initial treatment in certain groups of men at high risk of cancer recurrence; and before surgery or radiation (neoadjuvant therapy) in an attempt to retract cancer and make the other treatment more effective. According to the invention, an IGF-IR antagonist is administered in conjunction with ADT in any setting where ADT would otherwise be employed. IGF-IR antagonist is an adjuvant that enhances and / or prolongs the effect of ADT.

There are several methods used for ADT. Orchiectomy involves removal of the testicles, where more than 90% of androgens, in their majority testosterone, are produced. With this source removed, most prostate cancers retract. While permanent and resulting in a variety of unwanted side effects usually related to changing hormone levels in the body, orchiectomy is probably the least expensive and simplest way to reduce androgen production and can be done as a simple procedure outside the patient.

Luteinizing hormone (LHRH) release analogs (also called LHRH agonists) lower testosterone levels effectively when orchiectomy by the decrease in androgens, especially testosterone, produced by the testes. LHRH analogs are injected or positioned as small implants under the skin and are delivered either monthly or every 3, 4, 6, or 12 months. Examples of LHRH analogs include leuprolide, goserelin, and triptorelin. Possible side effects of LHRH are similar to those of orchiectomy, and are largely due to changes in hormone levels.

Antiandrogens block the body's ability to use any androgens. Even after orchiectomy or during treatment with LHRH analogs, a small amount of androgens is still produced by the adrenal glands. Drugs of this type include flutamide, bicalutamide, and nilutamide. These drugs are usually taken daily as pills.

Antiandrogen treatment is often combined with orchiectomy or LHRH analogs. This combination is called combined androgen blocking (CAB). In addition, an antiandrogen may be added if treatment with orchiectomy or an LHRH analogue no longer works on its own. Several recent studies have compared the effectiveness of antiandrogens with that of LHRH agonists. Most found no difference in survival rates, but a few found that antiandrogens are slightly less effective.

Antiandrogen side effects in patients already treated for orchiectomy or LHRH agonists are not usually serious. Diarrhea is the major side effect, although nausea, liver problems, and tiredness may also occur. The major difference in LHRH agonists is that antiandrogens have fewer sexual side effects and allow maintenance of libido and potency if used alone.

Adrenal androgen inhibitors may be administered because the low level of androgens produced by the adrenal glands may be sufficient to provide continued stimulation. Following androgen ablation, a subset of prostate cancer cells may become hypersensitive to androgens and the adrenal gland is the source of 5 to 10% of peripheral testosterone. The two agents most commonly used to inhibit adrenal androgen production are aminoglutethimide and ketoconazole.

Other examples of androgen suppressor drugs include diethyl stilbestrol (DES), megesterol acetate, cyproterone acetate, andprednisone. Estrogens were once the main alternative to orchiectomy for men with advanced prostate cancer, but because of their possible side effects, including blood clots and breast enlargement, estrogens have been largely replaced by LHRH and 15 antiandrogen analogues.

According to the invention, a course of treatment with an IGF-IR antagonist is administered starting before, at, or after the initiation of ADT. The course of administration of an IGF-IR antagonist should coincide with ADT, but the coincidence need not be complete. For example, the IGF-IR antagonist may be administered any time during remission resulting in androgen suppression. In one embodiment of the invention, the IGF-IR antagonist is administered within 24 months of androgen suppression for the treatment of primary or metastatic tumors. In another embodiment, the IGF-IR antagonist is administered within 18 months of androgen suppression. In one embodiment of the invention, the IGF-IR antagonist is administered during or near the end of the cell death period observed under ADT treatment, and will further prevent or retard the subsequent growth of AI cells. In one embodiment of the invention, administration of the IGF-IR antagonist is initiated within two weeks of androgen suppression. In another embodiment, administration is begun within one week of androgen suppression.

IGF-IR antagonists of the invention may be administered with antagonists that neutralize other receptors involved in tumor growth. Of particular interest are the receivers involved in a signal transduction pathway that Akt. For example, signal transduction via EGFR or HER2 (erbB2) is considered to involve Akt activation. Accordingly, IGF-IR antagonists of the invention may be combined with intracellular or extracellular EGFR or HER2 antagonists.

EGFR or HER2 antagonists include antigenic ligand proteins that bind to the extracellular domain of EGFR or HER2 and block the one or more ligands and / or neutralize ligand-induced activation. Antagonists also include antibodies or other binding proteins that bind to an EGFR ligand and inhibit noligant EGFR binding. EGFR binders include, for example, EGF, TGF-Î ±, anfiregulin, heparin-binding EGF (HB-EGF) and betacellulin. EGF and TGF-α are considered to be the major endogenous ligands that result in EGFR-mediated stimulation, although TGF-α has been shown to be more potent in promoting angiogenesis. EGFR antagonists also include substances that inhibit EGFR dimerization with other EGFR receptor subunits (i.e., EGFR homodimers) or heterodimerization with other growth factor receptors (e.g., HER2). EGFR antagonists additionally include biological molecules and small molecules, such as synthetic kinase inhibitors that act directly on the EGFR cytoplasmic domain to inhibit EGFR-mediated desinal transduction. Erbitux® (cetuximab; C225) is an example of an EGFR antagonist antibody that binds to EGFR and blocks deleterious binding. Erbitux® is an IgG1 chimeric antibody having murine M225 variable domains (see, e.g., WO 96/40210) and human constant domains; an anti-EGFR human antibody designated 11F8 is described by Zhu (WO 2005/090407). Other anti-EGFR antibodies include EMD 72000 (matuzumab), Vectibix ™ (panitumumab; ABX-EGF), TheraCIM (nimotuzumab), and Hu-Max-EGFR (zalutumumab). 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 U.S. Patent No. 5,616,582 (Zeneca Limited). Another example of a small molecule EGFR antagonist is TARCEVA ™ (OSI-774), which is a 4- (substituted-phenyl-amino) -quinazoline derivative [6,7-bis (2-methoxy-ethoxy) -hydrochloride. quinazolin-4-yl] - (3-ethynyl-phenyl) -amine] EGFR inhibitor. See WO 96/30347 (Pfizer Inc.); Moyer et al., Cancer Res., 57: 4838-48 (1997); Pollack et al., J. Pharmacol., 291: 739-48 (1999). TARCEVA ™ may function by inhibiting phosphorylation of EGFR and its downstream PI3 / Akt and MAP kinase (pormitogen-activated protein) signal transduction pathways resulting in p27-mediated cell cycle arrest. See Hidalgo et al., Abstract 281 presented at the 37th Annual Meeting of ASCO, San Francisco, CA, 12-15 May 2001.

Although antagonists may be administered separately, on occasion it may be desirable to combine the antagonistic finger functions into a single molecule, such as a bispecific antibody or a dual inhibitor. Bispecific antibodies can be engineered to match IGF-IR specificity with specificity by a different RTK or other cell surface molecule. IGF-IR specificity combinations with EGFR specificity of HER2 specificity are of particular interest. An example of a bispecific antibody that binds IGF-IR and EGFR is provided by Zhu (WO 2006/020258). Similarly, small IGF-IR inhibiting molecules and a second cellular component are available, or may be selected. For example as mentioned above, INSM-18 (Insmed / University of California San Francisco) inhibits IGF-IR and HER2 / neu.

Another aspect of the present invention relates to pharmaceutical compositions containing the antagonists of the present invention or a pharmaceutically acceptable salt, hydrate, or prodrug thereof, in combination with a pharmaceutically acceptable carrier. Such compositions may be separate compositions of the IGF-IR antagonist and the ADT agent or a single composition containing both.

The compositions of the present invention may be in solid or liquid form, in solution or in suspension. Routes of administration include, for example, oral, parenteral (intravenous, intraperitoneal, subcutaneous, or intramuscular), topical, transdermal and inhalation.

For oral administration, the IGF-IR antagonist may be administered, for example, in liquid form with an ingestible or inertible diluent, or incorporated into a solid dosage form.

Examples of liquid and solid dosage forms include, for example, solutions, suspensions, syrups, emulsions, tablets, lozenges, capsules (including soft gelatin capsules), and the like. Oral dosage forms may be formulated as sustained release products using, for example, a coating to retard disintegration or to control diffusion of the active compound. Where necessary, the compositions may also include a solubilizing agent.

Examples of injectable dosage forms include sterile injectable liquids, including, for example, solutions, emulsions and suspensions.

Injectable dosage forms additionally include solids such as postinjectables that are reconstituted, dissolved or suspended in an injection liquidant. Sterile injectable solutions are prepared by incorporating the EGF-IR antagonist and / or the ADT agent in the required amount in the appropriate solvent with various other ingredients listed above, as required, followed by filter sterilization. Vehicles typically include, for example, sterile water, saline, injectable organic esters, peanut oil, vegetable oil, and the like. Buffering agents, preservatives, and the like may be included in the administrable forms. Sterile formulations may be prepared by heating, irradiating, microfiltration, and / or by adding various antifungal antibacterial agents, such as, for example, parabens, chlorobutanol, phenol, acidic , thimerosal, and the like.

For topical administration, IGF-IR antagonists and ADT agents of the present invention may be administered separately or together, for example, in the form of gels, creams, or ointments, or inks. Typical carriers for such application include hydrophobic or hydrophilic bases, oily or alcoholic liquids, and dry powders. IGF-IR antagonists and ADT agents may also be incorporated into a gel or matrix base for application in a bandage, optionally providing controlled release of compound through a transdermal barrier. IGF-IR antagonists and ADT agents may also be formulated by known methods for rectal administration.

For inhalation administration, IGF-IR antagonists and ADT agents of the present invention may be dissolved or suspended in, or adsorbed onto, a vehicle suitable for use in a nebulizer, aerosol, or dry powder inhaler.

Appropriate dosages may be determined by a physician or qualified medical professional, and depend on factors such as the nature of the disease being treated, the route of administration, and the condition of the patient. IGF-IR antagonists and ADT agents may be administered as often as necessary for the purpose of obtaining the desired therapeutic effect. Frequency of administration will depend, for example, on the nature of the dosage form used. One skilled in the art will understand that dosages and frequency of treatment depend on the individual patient's tolerance and the pharmacological and pharmacokinetic properties of the blocking or inhibitory agent used. Ideally, it is desired to achieve saturable pharmacokinetics for the agent used. A loading dose for an anti-IGF-IR antibody may range within, for example, from about 10 to about 1000 mg / m, preferably from about 200 to about 400 mg / m. This may be followed by various daily or weekly additional dosages ranging, for example, from about 200 to about 400

mg / m. An exemplary dosage of IGF-IR antibody is loading 400

mg / m and weekly infusion of 250 mg / m. (For conversions between mg / kg and

mg / m for humans and other mammals, see Freireich, E.J. et al., 1966,

Chemother cancer. Rep. 50: 219-44). The patient is monitored for side effects and treatment is discontinued when such side effects are serious. Effective dosages of ADT agents are well known in the art.

One of ordinary skill in the art will also know how to monitor treatment progress in order to determine an effective dose. For prostate cancer, one way is to monitor PSA levels. Another is to monitor prostatic acid phosphatase (PAP). Other ways to monitor prostate cancers include ultrasound, computed tomography (CT), magnetic resonance imaging (MRI), and the like. Tissue samples may also be examined for expression and cellular distribution of RA, as well as expression of survivin and / or TUBB.

In certain embodiments of the invention, treatments combining administration of IGF-IR antagonists with ADT may be employed with one or more antineoplastic agents. For example, as noted above, ADT is often employed as a neoadjuvant for the treatment of prostate tumor radiation. When the antineoplastic agent is radiation, the radiation source may be either external (external beam radiation therapy -EBRT) or internal (brachytherapy - BT) to the patient being treated. The antineoplastic agent may be an alkylating agent or an anti-metabolite. . Examples of alkylating agents include, but are not limited to, cisplatin, cyclophosphamide melfalan, and dacarbazine. Examples of anti-metabolites include, but are not limited to, doxorubicin, daunorubicin, and paclitaxel, gemcitabine.

Useful antineoplastic agents also includemitotic inhibitors such as docetaxel and paclitaxil taxanes. Detopoisomerase inhibitors are another class of antineoplastic agents that may be used in combination with antibodies of the invention. These include topoisomerase I or topoisomerase II inhibitors. Topoisomerase inhibitors include irinotecan (CPT-11), aminocamptothecin, camptothecin, DX-8951f, topotecan. Topoisomerase II inhibitors include etoposide (VP-16), eteniposide (VM-26). Other substances are currently being evaluated with respect to topoisomerase inhibitory activity and effectiveness as antineoplastic agents. In a preferred embodiment, the detopoisomerase inhibitor is irinotecan (CPT-11).

Throughout this application, various publications, reference texts, textbooks, technical manuals, patents, and patent applications are referred to. The teachings and descriptions of these publications, patents, patent applications, and other documents in their entirety are hereby incorporated. as a reference in this application to further describe the state of the art to which the present invention belongs.

And to be understood and expected that variations in the principles of the invention described herein may be made by a person skilled in the art, it is intended that such modifications be included within the scope of the present invention.

The following examples further illustrate the invention, but in no way should be construed as limiting the scope of the invention. Detailed descriptions of conventional methods, such as those employed in vector and plasmid construction, and expression of antibodies and antibody fragments can be obtained from numerous publications, including Sambrook, J et al., (1989) Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press; Coligan, J. et al. (1994) Current Protocols in Immunology, Wiley & Sons, Incorporated; Enna, S.J. et al. (1991) Current Protocols in Pharmacology, Wiley & Sons, Bonifacino, J.S. et al. (1999) Current Protocols in Cell Biology, Wiley & Sons. All references mentioned herein are incorporated in their entirety.

EXAMPLES

IGF-IR antagonism inhibits tumor regrowth after

The DT.

A preclinical model was developed to test the effectiveness of IGF-IR signaling inhibition using an IGF-IR human monoclonal antibody (IMC-A12) with castration on recurrence of prostate cancer after castration. For the study, a LuCaP 35 xenograft, an androgen-responsive human prostate cancer cell line, was implanted subcutaneously into the flank of male SCID mice. LuCaP 35 may transition to an independent state of androgen and may be used to assess molecular changes associated with this process. First, PSA levels fall and tumor volume decreases, but after a period of 60-120 days, tumor regrowth is observed. LuCaP 35 has metastatic potential and results in bone lesions. Growth of LuCaP 35 in intact male mice is androgen sensitive and responds to androgen suppression in the manner commonly seen in patients.

LuCaP 35 cells were subcutaneously implanted in the flank of male SCID mice. When the tumors reached a volume of ca. 400 mm, the mice were castrated and divided into three groups of 20 animals each. Control group 1 received castration only, Group 2 received castration and IMC-Al2 intraperitoneally three times a week for 14 days starting 7 days after castration and Group 3 received IMCA12 for 14 days starting 14 days after castration. After 14 days of IMCA12 no additional therapy was given. The 2-week Al2 administration time starting either 1 or 2 weeks after castration was based on data published with the LuCaP 35 cell line indicating that maximal castration-induced apoptosis occurs within four days of castration (Corey, E. et al., 2003, Prostate 99: 392-401). Since IGF-IR de-signaling inhibition can cause cell cycle arrest and prevent cells from apoptosis, it was decided to initiate A12 administration when apoptosis was "complete" after castration (Corey et al., 2003; Tennant, M. et al., 2003, Prostate, 56: 115-22).

Blood samples were collected from the bell orbitally weekly. Serum was separated and PSA levels were determined using IMx Total PSA Assay (Abbott Laboratories, Abbott Park, IL). Tumors were measured twice a week and tumor volume was estimated by the formula: volume = LX W2 / 2. Mice were killed and tumors reached 20% of initial body weight. BrdU was injected i.p. in mice 1 h before animals were killed to determine the rate of tumor cell proliferation in vivo.

With castration, tumor growth was initially disrupted in all mice. (Fig. 1) In mice treated with IMCA12, tumor volume decreased during the course of the study and there were no specific tumor-related deaths. In the untreated group, an increase in mean tumor volume was evident by week 5, with specific tumor deaths (sacrificed) starting at week four and continuing through the study. Note that the mean tumor volume plot is artificially depressed for mice not receiving IMC-Al 2 because each kill removed a large tumor from the medium tumor pool.

PSA levels were monitored in LuCaP 35 grafted mice. All mice initially responded to hormone ablation and a similar drop in PSA levels was observed within the first week after castration (Fig. 2). In mice treated only for castration, after initial drop, PSA levels then increased during the course of the study beginning around the second week. In contrast, PSA levels in castrated mice that were treated with IMCA12 did not increase but remained close to baseline.

This study demonstrates that blocking IGF-IR signaling and expression after castration with IGF-IR antibody, IMCA12, results in a significantly greater decrease in tumor volume than just castration, p <0.001, and significantly prolongs the time to AI tumor growth. determined by tumor volume and an increase in PSA, p <0.001.

In control animals treated by castration alone, tumor growth stopped for about four weeks, but increased thereafter. Of the castrated animals, only more than half of them were killed due to tumor growth at 9 weeks after castration and most of the animals were killed at the end of 16 weeks. In contrast, all animals receiving IMCA12 remained alive after 16 weeks.

The in vivo results presented demonstrate the effectiveness of IGF-IR signal transduction inhibition. Notably, the IGF-IR antagonist was administered during the 14-day course, and was then discontinued. In a separate study in which A12 was administered in a similar manner, some tumor regrowth was observed late in the study following administration of A12. Two of the 40 animals from Groups 2 and 3 had to be killed because of tumor volume at the end of the study. Maintenance doses of an IGF-IR antagonist would prolong the time to tumor growth indefinitely.

To investigate whether there was a relationship between tumor volume reduction in Al2-treated tumors and RA translocation, RA immunohistochemistry was performed on tumors from each of the three groups, as shown in Fig. 5. Nuclear RA was determined in 100 nuclei of each tumor. Nuclei were blindly classified by two individuals and the average of the two results was counted as the result of that tissue. There was a significant positive correlation between tumor volume and nuclear RA intensity, r = 0.66, ρ <0.01.

IGF-IR antagonism inhibits RA translocation.

The effect of an IGF-IR stimulation and antagonism on androgen receptor overlocation was evaluated. LuCaP 35 cells were cultured with or without IGF-1 stimulation in the presence of absence of IMCA12. (Fig. 3) Cytoplasmic and nuclear extracts were prepared from the treated cells and evaluated by PAGE. ERK level was used to equalize track loading. In IGF-1 stimulated cells, IMCA1 2 caused a reduction in the proportion of androgen receptor observed in the nucleus.

Androgen receptor translocation was also evaluated by immunohistochemistry. (Fig. 4). LuCaP 35 (AD) xenograft tumors were grown in intact male mice and LuCaP 35V (AI) graft tumors were grown in castrated mice. Test mice were treated with IMCA12. Serum double sections were prepared and stained with AR-specific antibody. In intact control mice, RA in tissue-dependent LuCaP 35 tissue was predominantly located in the nucleus. In test tissue treated with IMCA12, RA staining was observed nocitoplasma. In castrated control mice, AR in androgen independent LuCaP35v cells was distributed between nucleus and oxytoplasm. In tissue from test animals treated with IMCA12, AR staining was predominantly in the cytoplasm.

In a similar experiment, RA localization was studied by fluorescence microscopy in tissue culture. Treatment with 10-8M DHT resulted in a significant redistribution of cytoplasm RA to the nucleus. IGF-I treatment only resulted in a partial redistribution of RA to the nucleus, and IMCA12 completely reversed that effect.

IGF-IR antagonism inhibits AR dependent gene expression.

Survivin, which is an apoptosis inhibitor, is strongly expressed in several human prostate cancer cell lines. In cell lines with intact androgen receptors, DHT androgen stimulation increases survival of survivin. Desurvivin expression is also observed to be mediated by AKT because IGF-induced AKT signaling enhances survivin expression even in RA negative cell lines. A gene chip experiment to detect survivin differential expression indicates that survivin expression is reduced under treatment with IMCA12.

Custom cDNA microarrays were constructed as previously described [ref] using clones derived from ProstateExpression Data Base (PEDB), a publicly available human prostate expressed sequence label (EST) repository sequence. (Nelson, P.S. et al., 2002, Nucl. Acids Res. 30: 218-20). Methods of labeling with Cy3 and Cy5 fluorescent dyes, microarray slide hybridization, and array processing were as described (Tusher, V. et al., 2001, Proc. Natl. Acad. Sci. U.S.A. 98: 5116-21).

Three tumors were pooled in each experimental group. To provide a reference RNA standard for use in cDNA microarrays, equal amounts of total RNA were isolated and pooled from LNCaP, DU145, PC3, and CWR22rVl (American TypeCulture Collection, Manassas, VA) growing in the Phase Iog in dye free RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS; LifeTechnologies, Rockville, MD). Total RNA was isolated from pooled tumors and cell lines using Trizol (Invitrogen, SanDiego, CA). mRNA was amplified one round using the Ambion MessageAmpTM II Amplification Kit (Ambion Inc5 Austin, TX), and sample quality and quantity were evaluated by agarose gel electrophoresis and A260 absorbance. Hybridization probes were labeled and quality control of the array experiments was performed as previously described (Tusher, V. et al., 2001). Gene expression differences associated with treatment groups were determined using the SAM procedure (Chu, G., Narasimhan , B., Tibshirani, R. & Tusher, V., 2002, Significance analysis of microarrays (sam) software, Stanford University) with a false discovery rate (FDR) of <10% considered significant (37). Similarities between samples assessed by unsupervised, hierarchical clustering of samples and samples using the Cluster 3.0 computer program (de Hoon et al., 2004, Bioinformatics 20: 1453-4) and views by TreeView (Page, RD, 1996, Comput.Appl Biosci- 12: 357-8).

Survivin and TUBB were also assayed by PCR using previously described initiators and methods (Wu, J. et al., 2006, Clin. Cancer Res. 12: 6153-60). A standard PCR fragment from the target cDNA was purified. A series of standard dilutions from 10 ng / µL to 10 3 pg / µL was used for real-time RT-PCR to generate standard curves. One µg of total RNA RNA from each pooled tumor group was used for decDNA synthesis. first strand using Superscript First Strand Synthesis System (Invitrogen) Real-time RT-PCR was performed on 20 μL of reaction mix consisting of 1 μΐ of first fitad and cDNA, specific primer sets, and Lightcycler FastStart DNA Master Plus SYBRGreen using a Roche Lightcycler following manufacturer's protocol (Roche, Nutley, NJ) RT-PCR products were subjected to fusion analysis in the Lightcycler v3.5 computer program.Deamplicon sizes were confirmed by agarose gel electrophoresis. tested in duplicate.

Castration combined with an IGF-IR antagonist is associated with a decrease in AR gene expression until tumor recurrence. Tumor RNA samples collected in each group at the time periods noted in Table 2 were analyzed on cDNA microarrays. No significantly altered genes were found between time periods for group 1 (castration only) when tested by the two-sample t-test in SAM (q-value> 100%). In addition, unsupervised, hierarchical grouping of known androgen-regulated genes did not segregate the two time periods. This may not be surprising because many of the animals in this group had increased PSA recurrence and increased nuclear AR scores compared with Groups 2 and 3 by day 40. In contrast, there were significant gene expression changes between the two time periods of Al2-treated tumors. Of the 3170 unique genes on the array with sufficient data to test, there were 21 over-regulated (including many androgen-regulated) and 41 down-regulated with 0% q-value in the late time period when tumors began to recur compared with the period. In addition, the unsupervised, hierarchical clustering of known androgen-regulated genes clearly differentiated the two time periods treated with Al2 into two separate clusters. These data indicate that nuclear AR expression is associated with RA transcription activity and prostate cancer progression through RA activation.

Table 2. cDNA Arrays at Each Time Instant

<table> table see original document page 41 </column> </row> <table> <table> table see original document page 42 </column> </row> <table>

Expression of survivin and β Tubulin is significantly decreased by an IGF-IR antagonist. Microarray studies determined that survivin expression was decreased in tumors treated with Al2 antibody. As shown in Fig. 7A, Qt-RT PCR in tumor-extracted RRNA demonstrates a significant positive correlation between survivin copy number and tumor volume, r = 0.66, ρ <0.01. A second gene recently implicated in formation IGF-IR and β-tubulin-induced tumor tumor, TUBB (O, Connor, R., 2003, Horm. Metab. Res.35: 771-7; Geller, J-et al., 1984, J. Urol. 132: 693 -700). TUBB has been shown to be decreased in microarrays and as shown in Fig. 7B, it has been shown in tumor specimens that correlates positively with tumor volume, r = 0.59, ρ <0.01, and is significantly decreased in groups 2 and 3. compared to group 1. A third gene that was not different over time in group 1 microarray but was decreased in the initial two time periods in groups 2 and 3 of 3 animals was PSA. The change in PSA expression was confirmed by a similar pattern in serum PSA levels.

Proliferation and Apoptosis

Apoptosis was determined by terminal deoxynucleotityl transferase-mediated failed labeling assay (TUNEL) and propidium staining (PI) using the Apop-Direct kit (BDBioScience) as previously described (Wu, JD et al., 2005, Clin. CancerRes ., 11: 3065-74). Briefly, 1x10 6 cells of the single cell suspension were fixed with 10% neutral buffered formalin (NBF) followed by 70% ethanol at -20 ° C for 30 min. After several washes, cells were permeabilized with 0.1% Triton X-100 and incubated with FITC-conjugated dUTP and terminal deoxynucleotidyl transferase - (TdT) at 37 ° C for 1 h, followed by an incubation with PI / RNase buffer (100 µl). (µg / mL PI, 50 μ§ / ηιL RNase) at room temperature for 60 min. Samples were analyzed by flow cytometry using a BD FACscan. Data were analyzed with the CellQuestPRO computer program. Apoptosis was also determined using the TUNEL formalin-fixed tissue assay using the Apop-Tag kit (Millipore Co, MA) following the manufacturer's recommendations. Apoptotic cells were determined per 300 cells per tissue slide.

As shown in Table 3, proliferation was significantly higher in Group 1 tumors compared with Groups 2 and 3, ρ <0.01. In contrast, apoptosis as determined by TUNEL staining was higher in Group 1 compared with Groups 2 and 3, Table 3.

<table> table see original document page 43 </column> </row> <table> SEQUENCE LIST

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<400> 1. ·., · '

gag gtc cag ecg gtg cag -tcfc ggg gct gag gtg aag aag cct ggg EccGlu goes Gln Iieu Val 'Gln Ser' Gly Allah Glu Val Lys »ys Pro Gly Sar1 5 10 15

tcg gtg aag gtc tcc fcgc aag gcfc tct gga ggc acc tfcc age act tafcSee will Eiys Val Ser Cy s Lys Ala Ser Gly Har Phe Ser Ser Tyr20 25 30

cag ggc aga gtc acg afefc acc gcg gae aaa tcc acg age ac gcc tacGln Gly Arg Val Thr Xle Thr Allah Asp Lys Ser Thr Seir Thr Ala Tyr65 70 75 80

48

96

gcfc till age fcgg gtg cga cag gcc çet gga caa ggg Ctt gag fcgg afcg 144

Ala lie Ser Trp Val Arg Oln Pro wing Gly Oln Gly Read Ola Trp Met35 40 45

gga ggg afcc to cct till fctt ggt aca gea aac tac gea cag aag tte 192

Oly Oly He Ile Px-Ile Phe Gly Tlir Wing Asn Tyr Wing Gln Lya Phe50 55 € 0

240

afcg gag ctg age age ctg aga tet gag gac acg gcc gtg tafc tac tgt 288

Met Glw Read Be Ser Read Ar Be Glu Asp Thr Allah Val Tyr Tyr Cys85 90 ^ S

gcg aga gcg cca tta cga ttfc fctg gag fcgg fcce aee caa gac cac tac 336

Arg Wing Pro Wing Read Arg Phe Read Glu Trp Ser Thr Gln Asp His TyrISO 105 110

tac tac tac tac afcg gac gtc tgg ggc aaa ggg acc acg gtc acc gtc 384

Tyr Tyr Tyr Tyr Mefc Asp Val Trp Oly Lye Qly Thr Thr Val Thr vai115 120 125tca age 390

Ser Ser30

<210> 2 <211> 130 <212> PRT <213> Homo sapiens <400> 2

Glu Val Gln Leu Val Gln Ser Gly Wing Glu Val Lys Lys Pro Gly Ser1 S 10 15

Be Val Lys Will Be Cys Lys Wing Be Gly Gly Thr Phe Be Be Tyr20 25 30

Wing Ile Ser Trp Val Arg Gln Pro Wing Gly Gln Gly Leu Glu Trp Met35 40 45

Gly Gly Ile Ile Pro Ile Phe Gly Thr Allah Asn Tyr Wing Gln Lys Phe50 55 60

Gln Gly Arg Val Thr Ile Thr Asp Wing Lys Be Thr Be Thr Wing Tyr65 70 75 80

Met Glu Read Be Ser Read Ar Be Glu Asp Thr Wing Val Tyr Tyr Cys85 90 95

Arg Wing Pro Wing Read Arg Phe Read Glu Trp Be Thr Gln Asp His Tyr100 105 110

Tyr Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val115 120 125

Ser Ser30

<210> 3 <211> 1440 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1) (1440) <400> 3

atg gga tgg tca tgt till ctt ttt cta gta gea act gea act gga 4 8

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Wing Thr Wing Gly15 10 15

gta cat tca gag gtc cag ctg gtg cag tct ggg

Val His Ser Glu Val Gln Leu Val Gln Ser Gly Wing Glu Val Lys Lys20 25 30

cct ggg tcc tcg gtg aag gtc tcc tgc aag gct tct gga ggc acc ttc 144

Pro Gly Ser Be Val Lys Val Ser Cys Lys Wing Be Gly Gly Thr Phe35 40 45

age age tat gct till age tgg gtg cga cag gcc cct gga caa ggg ctt 192

Be Ser Tyr Wing Ile Ser Trp Val Arg Gln Pro Wing Gly Gln Gly Leu50 55 60

gag tgg atg gga ggg till cct till ttt ggt aca gea aac tac gea 240

Glu Trp Met Gly Gly Xle Ile Pro Ile Phe Gly Thr Wing Asn Tyr Ala65 70 75 80

cag aag ttc cag ggc aga gtc acg att acc gcg gac aaa tcc acg age 2 88

Gln Lys Phe Gln Gly Arg Val Ile Thr Thr Wing Asp Lys Ser Thr Ser85 90 '95

aca gcc tac atg * gag ctg age age ctg aga tct gag gac acg gcc gtg 336

Thr Wing Tyr Met Glu Read Be Ser Read Arg Be Glu Asp Thr Wing Val100 105 110

tat tgt gcg "aga gcg cca tta cga ttt ttg gag tgg tcc acc caa 384.

Tyr Tyr Cys Arg Wing Pro Wing Read Arg Phe Read Glu Trp Ser Thr Gln115 120 125

gac cac tac tac tac tac tac atg gac gtc tgg ggc aaa ggg acc acg 432

Asp His Tyr Tyr Tyr Tyr Met Asp Val Trp 'Gly Bys Gly Thr Thr130 135 140

gtc acc gtc tca age gcc tcc acc aag ggc cca tcg gtc ttc ccc ctg 480

Val Thr Val Be Ser Ala Be Thr Lys Gly Pro Ser Val Phe Pro Leu145 150 155 160

gea ccc tcc tcc aag age acc tct ggg ggc aca gcg gcc ctg

Wing Pro Be Be Lys Be Thr Be Gly Gly Thr Wing Wing Read Gly Cys165 170 175

ctg gtc aag gac ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca 576

Read Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser180 185 190

ggc gcc ctg acc age ggc gtg cac acc ttc ccg gct gtc cta cag tcc 624

Gly Wing Leu Thr Be Gly Val His Thr Phe Pro Wing Val Leu Gln Ser195 200 205

tca gga etc tac tcc ctc age age gtg gtg acc gtg ccc age tc 672

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser210 215 220

ttg ggc acc cag acc tac up to tgc aac gtg aat cac aag ccc age aac 720

Read Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn225 230 235 240

acc aag gt gac aag aaa gtt gag ccc aaa tct tgt gac aaa act cac 768

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His245 '250 255aca tgc cca ccg tgc cca gca cct ga ctc ctg ggg gga ccg tca gtcThr Cys Pro Cys Pro Wing Glu Leu Gly Gly Pro Ser Val260 2 65 270

ttc ctc ttc ccc cca aaa ccc aag gac acc ctc atg to tcc cgg acc

Phe Leu Phe Le Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr275 280 285

cct gag gtc aca tgc gtg gtg gtg gac gtg age cac gaa gac cct gag

Pro Glu Goes Thr Cys Val Val Val Asp Val Be His Glu Asp Pro Glu290 295 300

gtc aag ttc aac tgg tac gtg gac ggc gtg gag gtg cat aat gcc aag

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

305 310 315 320

aca aag ccg gg gag gag cag tac aac age acg tac cgg gtg gtc

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser325 330 335

gtc ctc acc gtc ctg cac cag gac tgg ctg

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys340 345 350

tgc aag gtc tcc aac aaa gcc ctc cca gcc ccc until gag aaa acc until

Cys Lys Val Ser Asn Lys Wing Leu Pro Wing Pro Ile Glu Lys Thr Ile355 360. 365 ·

tcc aaa gcc aaa ggg cag ccc cga gaa cca cag gtg tac acc ctg ccc

Ser Lys Ala Lys Gly Gn Pro Gln Pro Gln Pro Gln Val Tyr Thr Leu Pro 370 375 380cca tcc cgg gag gag atg acc aag aac cag gtc

Pro Be Arg Glu Glu Met Thr Lys Asn Gln Val Be Leu Thr Cys Leu

38'5 390 395 400

gtc aaa ggc ttc tat ccc age gac till gcc gtg gag tgg gag age aat

Val Lys Gly Phe Tyr Pro Asp Ile Wing Val Glu Trp Glu Ser Asn405 410 415

ggg cag ccg gag aac aac tac aag acc acg cct ccc gtg ctg gac tcc

Gly Gln Pro Asu Gln Asn Tyr Lys Thr Thr Pro Valu Le Asp Ser420 425 430

gac ggc tcc ttc ttc ctc tac age aag ctc acc gtg

Asp Gly Ser Phe. .Phe Read Tyr Ser Lys Read Thr Val Asp Lys Ser Arg435 440 445

tgg cag cag ggg aac gtc ttc tca tgc tcc gtg

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Wing Leu450 455 460

cac aac cac tac ag cag aag age ctc tee ctg tet ccg ggt aaa tgaHis Asn His Tyr Thr Gln Lys Seru Leu Ser Pro Gly Lys465 470 475

<210> 4

<211> 479

<212> PRT

<213> Homo sapiens <400> 4

Met Gly Trp Being Cys Xle Xle Read Phe Read Val Val Wing Thr Wing Gly15 10 15

Val His Ser Glu Val Gln Leu Val Gln Ser Gly Wing Glu Val Lys Lys20 25 30

Pro Gly Ser Be Val Lys Val Ser Cys Lys Wing Be Gly Gly Thr Phe35 40 45

Be Ser Tyr Wing Ile Ser Trp Val Arg Gln Pro Wing Gly Gln Gly Leu50 55 60

Glu Trp Met Gly Gly Ile Ile Pro Ile Phe Gly Thr Wing Asn Tyr Ala65 70 75 80

Gln Lys Phe Gln Gly Arg Val Ile Thr Thr Wing Asp Lys Ser Thr Ser85 90 95

Thr Wing Tyr Met Glu Read Be Ser Read Arg Be Glu Asp Thr Wing Val100 105 110

Tyr Tyr Cys Arg Wing Pro Wing Read Aarg Phe Read Glu Trp Ser Thr Gln115 120 125

Asp His Tyr Tyr Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr130 135 140

Val Thr Val Be Ser Ala Be Thr Lys Gly Pro Ser Val Phe Pro Leu145 150 155 160

Wing Pro Be Be Lys Be Thr Be Gly Gly Thr Wing Wing Read Gly Cys165 170 175

Valu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asix Ser180 185 190

Gly Wing Leu Thr Be Gly Val His Thr Phe Pro Wing Val Leu Gln Ser195 200 205

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser210 215 220

Read Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Be Asn225 230 235 240Thr Lys Val Asp Lys Val Glu Pro Lys Be Cys Asp Lys Thr His245 - 250 255

Thr Cys Pro Pro Cys Pro Wing Pro Glu Read Leu Gly Gly Pro Ser Val260 265 270

Phe Leu Phe Le Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr275 280 285

Pro Glu Val Thr Cys Val Val Val Asp Val Be His Glu Asp Pro Glu290 295 300

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Wing Lys305 310 315 320

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser325 330 335

Val Leu TJir Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys340 345 350

Cys Lys Val Ser Asn Lys Wing Pro Leu Pro Wing Ile Glu Lys Thr Ile355 360 365

Ser Lys Wing Lys Gly Gln Pro Gln Pro Gln Gln Val Tyr Thr Leu Pro370 375 380

Pro Be Arg Glu Glu Met Thr Lys Asn Gln Val Be Leu Thr Cys Leu385 · 390 395 400

Val Lys Gly Phe Tyr Pro Asp Ile Wing Val Glu Trp Glu Ser Asn405 410 415

Gly Gln Pro Asu Gln Asn Tyr Lys Thr Thr Pro Valu Le Asp Ser420 425 430

Asp Gly Be Phe Phe Read Tyr Be Lys Read Val Thr Asp Lys Be Arg435 440 445

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Wing Leu450 455 460

His Asn His Tyr Thr Gln Lys Be Read Be Read Be Pro Gly Lys465 470 475

<210> 5 <211> 327 <212> DNA

<213> Homo sapiens

<220>

<221> CDS <222> (X) .. (327)

<400> 5

tct tct gag ctg act cag gac cct gtc gtg tct gtg gcc ttg gga cag 4 8

Be Ser Glu Leu Thr Gln Asp Pro Val Wing Be Val Wing Leu Gly Gln15 10 15

aca gtc agg till aca tgc caa gga gac age ctc aga age tat tat gea 96

Thr Val Arg Ile Thr Cys Gln Gly Asp Be Read Arg Be Tyr Tyr Ala20 25 30

age tgg tac eag cag aag hollow gga cag gcc cct gta ctt gtc until tat 144

Ser Trp Tyr Gln Lys Pro Gly Gln Pro Wing Val Leu Val Ile Tyr35 40 45

ggt aaa aac aac cgg ccc tca ggg up to cea gac cga ttc tct ggc tcc 192

Gly Lys Asn Asn Arg Pro Be Gly Ile Asn Arg Pro Phe Be Gly Ser50 55 60

age tca gga aac aca gct tcc ttg acc till act ggg gct cag gcg gaa 240

Be Ser Gly Asn Thr Wing Be Read Thr Ile Thr Gly Wing Gln Wing Glu65 70 75 80

gat gag gct gac tat tac tgt aac tcc cgg gac aac agt gat aac cgt. 288

Asp Glu Wing Asp Tyr Tyr Cys Asn Be Arg Asp Asn Be Asp Asn Arg85 90 95

ctg ata ttt ggc ggc ggg acc aag ctg acc gtc ctc agt 327.

Leu Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ser100 105

<210> 6

<211> 109

<212> PRT

<213> Homo sapiens

<400> 6

Be Ser Glu Leu Thr Gln Asp Pro Val Wing Be Val Wing Leu Gly Gln15 10 15

Thr Val Arg Ile Thr Cys Gln Gly Asp Be Read Arg Be Tyr Tyr Ala20 25 30

Ser Trp Tyr Gln Lys Pro Gly Gln Pro Wing Val Leu Val Ile Tyr35 40 45

Gly Lys Asn Asn Arg Pro Be Gly Ile Asn Arg Pro Phe Be Gly Ser50 55 60

Be Ser Gly Asn Thr Wing Be Read Thr Ile Thr Gly Wing Gln Wing Glu65 70 75 80

Asp Glu Wing Asp Tyr Tyr Cys Asn Be Arg Asp Asn Be Asp Asn Arg85 90 95

Leu Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ser100 105

<210> 7

<211> 702

<212> DNA.

<213> Homo sapiens

<220>

<221> CDS

<222> (1). . (702)

<400> 7

atg gga tgg tca · tgt till ctt ttt cta gta gea act gea act gga. 48

Met Gly Trp Being Cys Xle Xle Read Phe Read Val Val Wing Thr Wing Gly15 10 15

gta cat tca tet tet gag ctg act cag gac cct gct gtg tet "gtg gcc 96 ·.

Val His Ser Ser Ser Glu Leu Thr Gln Asp Pro Wing Val Ser Ser Val Wing20 25 30

ttg gga cag aca gtc agg until aca tgc caa gga gaç age etc aga age 144 ·

Read Gly Gln Thr Val Arg Ile Thr Cys Gln Gly Asp Be Read Arg Ser35 40 45

tat tat gea age tgg tac cag cag aag cea gga cag gcc cct gta ctt -192

Tyr Tyr Wing Ser Trp Tyr Gln Gln Lys Pro Gly Gln Wing Pro Val Leu50 55 60

gtc up to tat ggt aaa aae aae cgg ccc tca ggg up to ceagac cga ttc 240

Val Ile Tyr Gly Lys Asn Arg Asn Pro Be Gly Ile Pro Asp Arg Phe65 70 75 80

tet ggc tcc age tca gga aae aca gct tcc ttg acc till act ggg gct 288

Be Gly Ser Be Ser Gly Asn Thr Wing Be Read Thr Ile Thr Gly Ala85 90 95

cag gcg gaa gat gag gct gac tat tac tgt aae tcc cgg gac aae agt 3 36

Gln Wing Glu Asp Glu Wing Asp Tyr Tyr Cys Asn Ser Arg Asp Asn Ser100 105 110

gat aae cgt ctg ata ttt ggc ggc ggg acc aag ctg acc gtc etc agt 384

Asp Asn Arg Leu Ile Phe Gly Gly Gly Gly Thr Lys Leu Thr Will Leu Ser115 120 125

cag ccc aag gct gcc ccc tcg gtc act ctg ttc ceg ccc tcc tet gag 432

Gln Pro Lys Wing Pro Wing Be Val Thr Leu Phe Pro Wing Be Glu130 135 140

gag ctt caa gcc aae aag gcc aa ctg gtg tgt etc ata agt gac ttc 4 80

Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe145 150 155 160tac ccg gga gcc gtg acg gcc tgg aag gca gat age act ccc gtc 528

Tyr Pro Gly Wing Val Thr Val Wing Trp Lys Wing Asp Ser Ser Pro Val165 170 175

aag gcg gga gtg gag acc acc aca ccc tcc aaa caa acts aac aac aag 576

Lys Wing Gly Val Glu Thr Thr Thr Pro Be Lys Gln Ser Asn Asn Lys180 185 190

tac gcg gcc age act tat ctg age ctg acg cct gag cag tgg aag tcc 624

Tyr Ala Wing To Be Tyr Read To Be Read Thr Pro Glu Gln Trp Lys Ser195 200 205

cac aga age tac tac tgc cag gtc acg cat gaa ggg age acc gtg gag 672

His Arg Be Tyr Be Cys Gln Val Thr His Glu Gly Be Thr Val Glu210 215 220

aag aca gtg gcc cct gca gaa tgc tet tga 702

Lys Thr Val Wing Pro Wing Glu Cys Ser225 230

<210> 8

<211> 233

<212> PRT

<213> Homo sapiens

; <400> 8

Met Gly Trp Being Cys Xle Ile Leu1 5

Phe Leu Val Wing Thr Wing Wing Gly10 · 15

Val His Ser Ser Ser Glu Leu Thr Gln Asp Pro Wing Val Ser Ser Val Wing20 25 30

Read Gly Gln Thr Val Arg Ile Thr Cys Gln Gly Asp Be Read Arg Ser35 40 45

Tyr Tyr Wing Ser Trp Tyr Gln Gln Lys Pro Gly Gln Wing Pro Val Leu50 55 60

Val Ile Tyr Gly Lys Asn Arg Asn Pro Be Gly Ile Pro Asp Arg Phe65 70 75 80

Be Gly Ser Be Ser Gly Asn Thr Wing Be Read Thr Ile Thr Gly Ala85 90 95

Gln Wing Glu Asp Glu Wing Asp Tyr Tyr Cys Asn Ser Arg Asp Asn Ser100 105 110

Asp Asn Arg Leu Ile Phe Gly Gly Gly Gly Thr Lys Leu Thr Val Leu Ser115 120 125

Gln Pro Lys Wing Pro Wing Be Val Thr Leu Phe Pro Pro Be Glu130 135 140Glu Leu Gln Wing Asn Lys Wing Thr Valu Cys Leu Ile Be Asp Phe145 150 155 160

Tyr Pro Gly Wing Val Thr Val Wing Trp Lys Wing Asp Ser Ser Pro Val165 170 175

Lys Wing Gly Val Glu Thr Thr Thr Pro Be Lys Gln Ser Asn Asn Lys180 185 190

Tyr Ala Wing To Be Tyr Read To Be Read Thr Pro Glu Gln Trp Lys Ser195 200 205

His Arg Be Tyr Be Cys Gln Val Thr His Glu Gly Be Thr Val Glu210 215 220

Lys Thr Val Wing Pro Wing Glu Cys Ser225 230

<210> 9

<211> 327

<212> DNA

<213> Homo sapiens

<22'0>

<221> CDS

<222> (1) .. (327)

<4 0 ò> 9

tct tct gag ctg act cag gac cct gctSer Ser Glu Leu Thr Gln Asp

gtg tct gtg gcc ttg gga cag 48 '

Val Ser Val Wing Read Gly Gln10 15

aca gtc agg till aca tgc caa gga gac age ctc aga age tat tat gea 96

Thr Val Arg Ile Thr Cys Gln Gly Asp Be Read Arg Be Tyr Tyr Ala20 25 30

acc tgg tac cag cag aag cca gga cag gcc cct att ctt gtc until tat 144

Thr Trp Tyr Gln Gln Lys Pro Gly Gln Wing Pro Ile Read Val Ile Tyr35 40 45

ggt gaa aat aag cgg ccc tca ggg until cca gac cga ttc tct

Gly Glu Asn Lys Arg Pro Be Gly Ile Pro Asp Arg Phe Be Gly Ser

50 55 60

age tca gga aac aca gct tcc ttg acc till act ggg gct cag gea gaa 240

Be Ser Gly Asn Thr Wing Be Read Thr Ile Thr Gly Wing Gln Wing Glu65 70 75 80

gat gag gct gac tac tat tgt aaa tct cgg gat ggc agt ggt caa cat 288

Asp Glu Wing Asp Tyr Tyr Cys Lys Be Arg Asp Gly Be Gly Gln His85 90 95

ctg gtg ttc ggc gga ggg acc aag "ctg acc gtc cta ggt 327Leu Val Phe Gly Gly Gly Tlir

<210> 10

<211> 109

<212> PRT

<213> Horao sapiens

<400> 10

Be Ser Glu Leu Thr Gln Asp Pro Val Wing Be Val Wing Leu Gly Gln15 10 15

Thr Val Arg Xle Thr Cy3 Gln Gly Asp Be Read Arg Be Tyr Tyr Ala20 25 30

Thr Trp Tyr Gln Gln Lys Pro Gly Gln Wing Pro Ile Read Val Ile Tyr35 40 45

Gly Glu Asn Lys Arg Pro Be Gly Ile Pro Asp Arg Phe Be Gly Ser50 55 60

Be Ser Gly Asn Thr Wing Be Read Thr Ile Thr Gly Wing Gln Wing Glu65 70 75 80

Asp Glu Wing Asp Tyr Tyr Cys Lys Ser Arg Asp Gly Ser · Gly Gln His85 90 95

Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly100 105

<210> 11

<211> 702

<212> DNA

<213> Homo saplens

<220>

<22l> CDS

<222> (1) .. (702)

<400> 11

atg gga tgg tca tgt till cttMet Gly Trp Ser Cys Ile Ile Leu1 5

.gta cat tca tet tet gag ctg actVal His Ser Ser Ser Glu Leu Thr20

ttt cta gta gea act gea act ggaPhe Leu Val Ala Thr Ala. Thr Gly10 15

cag gac cct gct gtg tet gtg gccGln Asp Pro Ala Val Ser Val Ala25 30

ttg gga cag aca gtc agg till aca tgc caa gga gac age etc aga ageLeu Gly Gln Ttir Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser35 40 45tat tat gca acc tgg tac cag aag cca gga cag gcc cct afct ctt 192

Tyr Tyr Wing Thr Trp Tyr Gln Lys Pro Gly Gln Wing Pro Xle Leu50 55 60

gtc till tat ggt gaa aat aag cgg ccc tca ggg till cca gac cga ttc 240

Val Ile Tyr Gly Glu Asn Lys Arg Pro Be Gly Ile Pro Asp Arg Phe65 70 75 80

tet ggc tcc age tca gga aac aca gct tcc ttg acc till act ggg get 2 8a

Be Gly Ser Be Ser Gly Asn Thr Wing Be Read Thr Ile Thr Gly Ala85 90 95

cag gca gaa gat gag gct gac tac tat tgt aaa tet cgg gat ggc agt 336

Gln Wing Glu Asp Glu Wing Asp Tyr Tyr Cys Lys Ser Arg Asp Gly Ser100 105 110

ggt caa cat ctg gtg ttc ggc gga ggg acc aag ctg acc gtc cta ggt 384

Gly Gln His Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly115 120 125

cag ccc aag gct gcc ccc tcg gtc act ctg ttc ccg ccc tcc tet gag 432

Gln Pro Lys Wing Pro Wing Be Val Thr Leu Phe Pro Wing Be Glu130 135 140

gag ctt càa gcc aac aag ag gcc aea ctg gtg tgt ctc ata agt gac ttc 480

Glu Leu Gln Wing Asn Lys Wing Thr Read Le Val Cys Leu Ile Ser Asp Phe145 150 155 160

tac · ccg gga gcc gtg aca gtg gcc tgg aag gca gat age act ccc gtc 528

Tyr Pro- Gly Wing Val Thr Val Wing Trp Lys Wing Asp Ser Ser Pro Val165 170 175

aag gcg gga gtg gag acc aca ccc tcc aaa tiaa acts aac aac aag · 576Lys Glly Val Glu Wing Thr Thr Pro Ln Gln Ser Asn Asn Lys180 185 190

tac gcg gcc age act tat ctg age ctg acg cct gag cag tgg aag tcc · 624

Tyr Ala Wing To Be Tyr Read To Be Read Thr Pro Glu Gln T.rp Lys Ser195 200 205

cac aga age tac tac tgc cag gtc acg cat gaa ggg age acc gtg gag · 672

His Arg Be Tyr Be Cys Gln Val Thr His Glu Gly Be Thr Val Glu210 215 220

aag aca gtg gcc cct gca gaa tgc tet tga 702

Lys Thr Val Wing Pro Wing Glu Cys Ser225 230

<210> 12

<211> 233

<212> PRT

<213> Hoir.o sapiens

<400> 12

Met Gly Trp Being Cys Ile Ile Leu Phe Leu Val Val Wing Thr Wing Gly1 5 10 15

Val His Be Ser Be Glu Leu Thr Gln Asp Pro Wing Val Ser Val Vala20 25 30Leu Gly Gln Thr Val Arg Xle Thr Cys Gln Gly Asp Ser Leu Arg Ser35 40 45

Tyr Tyr Wing Thr Trp Tyr Gln Gln Lys Pro Gly Gln Wing Ile Leu50 55 60

Val Ile Tyr Gly Glu Asn Lys Arg Pro Be Gly Ile Pro Asp Arg Phe65 70 75 80

Be Gly Ser Be Ser Gly Asn Thr Wing Be Read Thr Ile Thr Gly Ala85 90 95

Gln Wing Glu Asp Glu Wing Asp Tyr Tyx Cys Lys Be Arg Asp Gly Ser100 105 110

Gly Gln His Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly115 120 125

Gln Pro Lys Wing Pro Wing Be Val Thr Read Phe Pro Ero. Being Ser Glu130 135 140

Glu Leu Gln Wing Asn Lys Wing Thr Read Le Val Cys Leu Ile Ser Asp Phe145 150 155 160

Tyr Pro Gly Wing Val Thr Val Wing Trp Lys Wing Asp Ser Ser Pro Val165 170 175

Lys Wing Gly Val Glu Thr Thr Thr Pro Be Lys Gln Ser Asn Asn Lys180 185 190

It's

Tyr Ala Wing To Be Tyr Read To Be Read Thr Pro Glu Gln Tirp Lys Ser195 200 205

His Arg Be Tyr Be Cys Gln Val Thr His Glu Gly Be Thr Val Glu210 215 220

Lys Thr Val Wing Pro Wing Glu Cys Ser225 230

<210> 13

<211> 15

<212> DNA

<213> Homo sapiens

<220> <221>

CDS <222> (X) .. (15) <400> 13

age tat gct till ageSer Tyr Ala Ile Ser1 5

<210> 14

<211> 5

<212> PRT

<213> Homo sapiens

<4 00> 14

Ser Tyr Wing Xle Ser1 5

<210> 15

<211> 51

<212> DNA

<213> Homo sapiens

<220> • <221> CDS

<222> (1) .. (51)

<400> 15

ggg.ate till cct till ttt ggt aca geaGly: Xle Ile Pro Ile Phe Gly Thr Ala1 5

gge 'gly

ae tac gea cag aag tte cag. 48

Asn Tyr Wing Gln Lys Phe Gln. . i-10 15

51

<210> 16

<211> 17

<212> PRT

<213> Homo sapiens

<400> 16

Gly Ile Ile Pro Ile Phe Gly Thr Wing Asn Tyr Wing Gln Lys Phe Gln1 5 10 15

Gly

<210> 17

<211> 63

<212> DNA

<213> Homo sapiens

<220>

<221> CDS <222> (1) .. (63)

<400> 17

gcg cca tta cga ttt ttg gag tgg tcc acc

Pro Wing Read Arg Phe Read Glu Trp Ser Thr Gln Asp

1 5 ίο 15

tac tac afcg gac gtcTyr Tyr Met Asp Val20

63

<210> 18

<211> 21

<212> PRT

<213> Homo sapiens

<400> 18

Pro Wing Read Arg Phe Read Glu Trp Ser Thr Gln Asp His Tyr Tyr Tyr15 10 15

Tyr Tyip Met .Asp Val20

<210> 19

<211> 33

<2 1.2> DNA

•• <213> Homo sapiens-

<220> <221> <222?

CDS

(1) - (33)

<400> 19

hunting gga gac age ctc aga age tat tat gea age

Gln Gly Asp Ser Read Arg Ser Tyr Tyr Ala Ser15 IO

33

<210> 20

<211> 11

<212> PRT

<213> Homo sapiens

<400> 20

Gln Gly Asp Be Read Arg Be Tyr Tyr Wing Ser15 10

<210> 21

<211> 21

<212> DNA

<213> Hotiio sapiens

<220> <221>

CDS <222> (1) .. (21) <4OO> 21

ggt aaa aac aac cgg ccc Cca 21

Gly Lys Asn Asn Arg £> ro Ser1 5

<210> 22

<211> 7

<212> PRT

<213> Homo sapiens

<400> 22

Gly Lys Asn Asn Arg Pro Ser1 5

<210> 23

<211> 33

<212> DNA

<213> Homo sapiens

<220>

<221> CDS <222> (1) .. (33)

<400> 23

aac tcc cgg gac aac: agt gat aac cgt

Asn Be Arg Asp Asn Be Asp Asn Arg1 5

ctg ata 33 ··

Read Xle10

<210> 24

<211> 11

<212> PRT

<213> Homo sapiens

<400> 24

Asn Be Arg Asp Asn Be Asp Asn Arg Leu Xle15 10

<210> 25

<211> 33

<212> DNA

<213> Homo sapiens

<220>

<221> CDS <222> (1). . (33)

<400> 25

ca gga gac age ctc aga age tat tatGln Gly Asp Ser Leu Arg Ser Tyr Tyr1 5

gea acc 33

Thr10 wing <210> 26

<211> 11

<212 »PRT

<213> Homo sapiens

<400> 26

Glri Gly Asp Be Read Arg Be Tyr Tyr Wing Thr15 10

<210> 27

<211> 21

<212> DNA

<213> Homo sapiens

<220>

<221> CDS <222> (1) .. Χ21)

<400> 27

ggt gaa aat aag cgg ccc fccaGly Glu asn

<210> 28

<211> 7

<212> PRT

<213> Homo s'.apiens

<400> 28

Gly Glu Asn. Lys Axg. Pro Ser1 5

<210> 29

<211> 33

<212> DNA

<213> Homo sapiens

<220>

<221> CDS <222> (1) .. (33)

<400> 29

aaa tct cgg gat ggc agfc ggt caa cat ctg gtgLys Ser Arg Asp Gly Ser Gly Gln His Leu Val1 5 10

<210> 30

<211> 11

<212> PRT

<213> Homo sapiens

<400> 30

Lys Ser Arg Asp Gly Ser Gly Gln His Leu Val <210> 31

<211> 24

<212> DNA

<213> Homo sapiens

<220>

<221> CDS <222> (1). . (24)

<4 00> 31age agt ggt gatSer Ser Gly Asp1

tac tac tgg agtTyr Tyr Trp Ser5

<210> 32

<211> 8

<212> PRT

<213> Homo sapiens

<400> 32

Ser Ser Gly Asp Tyr Tyr Trp Ser1 5

<210> 33

<211> 48

<212> DNA ·

<213> Homo sapiens

<220>

<221> CDS <222> (1) .. (48)

<400> 33

tac up tat tac agg ggg age acc gac tac aac ccg tcc ctc aag agtTyr Xle Tyr Tyr Ser Gly Ser Thr Asp Tyr Asn Pro Ser Leu Lys Ser15 10 15

<210> 34

<211> 16

<212> PRT

<213> Homo sapiens

<400> 34

Tyr Ile Tyr Tyr Be Gly Be Thr Asp Tyr Asn Pro Be Read Lys Ser15 10 15

<210> 35

<211> 33

<212> DNA

<213> Homo sapiens <22 0>

<221> CDS <222> (X) .. (33)

<4 00> 35

gtg tcg att ttt gga gtg ggg aca ttt gac tac 33

Val Ser Xle Phe Gly Val Gly Thr Phe Asp TyrX 5 10

<210> 36

<211> IX

<212> PRT

<213> Homo sapiens

<400> 36

Val Ser Ile Phe Gly Val Gly Thr Phe Asp Tyr15 10

<210> 37

<211> 363

<212> DNA

<213> Ηοττιο sapiens

<220>

<221> CDS <222> (1) .. (363)

<400> 37

cag gtg cag ctg cag gag tcg cgg cca gga ctg gtg aag cct tca cag 48

Gln Val Gln Leu Gln Glu. Ser Gly Pro Gly Read Val Lys Pro Ser GlnX 5 10 IS

acc ctg tcc ctc acc tgc act gtc tct ggt ggc tcc until age agt ggt 96

Thr Read Be Read Thr Cys Thr Val Be Gly Gly Be Ile Be Gly20 25 30

gat tac tac tgg agt. tgg till cgc cag ccc cca ggg aag ggc ctg gag 144

Asp Tyr Tyr Trp Being Trp Ile Arg Gln Pro Gly Lys Gly Leu Glu35 40 45

tgg att ggg tac till tat tac agt ggg age acc gac tac aac ccg tcc 192

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asp Tyr Asn Pro Ser50 55 60

ctc aag agt cga gtc acc atg tcc gta gac acg tcc aag aat cag ttt 240

Leu Lys Be Arg Val Thr Met Be Val Asp Thr Be Lys Asn Gln Phe65 70 75 80

tcc ctg aag gtc aac tct gtg acc gcc gea gac acg gct gtg tat tac 288

Ser Leu Lys Val Asn Ser Val Thr Wing Asp Thr Wing Val Tyr Tyr

85 90 95

tgt gcg aga gtg tgg att ttt gga gtg ggg

Cys Wing Arg Val Ser Ile Phe Gly Val Gly Thr Phe Asp Tyr Trp Gly100 105 no

cag ggc acc ctg gtc acc gtc tca age 363Gln Gly Thr Read Val Val Val Ser Ser 120

<210> 38 <211> 121 <2 12> PRT <213> Homo sapiens <400> 38 Gln Val Gln Leu Gln

1 5 10 15

Thr Read Be Read Thr Cys Thr Val Be Gly Gly Be Ile Be Gly20 25 30

Asp Tyr Tyr Trp Being Trp Ile Arg Gln Pro Gly Lys Gly Leu Glu35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asp Tyr Asn Pro Ser50 55 60

Leu Lys Be Arg Val Thr Met Be Val Asp Thr Be Lys Asn Gln Phe65 70 75 80

Ser Leu Lys Val Asn Ser Val Thr Wing Asp Wing Wing Thr Va3 Wing Tyr Tyr85 90 95

Cys Wing Arg Val Ser Ile Phe Gly Val Gly Thr Phe Asp Tyx Trp Gly100 105 110

Gln Gly Thr Read Val Val Val Ser Ser 120 120

<210> 39

<211> 30

<212> DNA

<213> Homo eapiens

<220>

<221> CDS <222> (1) .. (30)

<400> 39

agg gcc agt cag agt gtt age age tac ttaArg Ala Ser Gln Ser Val Ser Ser Tyr Leu1 5 10 15

<210> 40 <211> 10 <212> PRT <213> Homo sapiens <400> 40

Arg Wing Be Gln Be Val Be Ser Tyr Leu15 10

<210> 41

<211> 21

<212> DNA

<213> Homo sapiens

<220> <221> CDS <222> (1) .. (21)

<400> 41

gat gca tcc aac agg gcc actAsp Ala Ser Asn Arg Ala Thrx1 5

<210> 42

<211> 7

<212> PRT

<213> Homo sapiens

<40 0> 42

Asp.- Ala Ser Asn Arg Ala Thr f1 5

<210> 43

<211> 24

<212> DNA

<213> Homo sapiens

<22 0> <221> CDS <222> (1) .. (24)

<400> 43cac cag tat ggtHis Gln Tyr Gly1

age aca cct ctcSer Thr Pro Leu5

<210> 44

<211> 8

<212> PRT

<213> Homo .sapiens

<400> 44

His Gln Tyr Gly Being Thr Pro Leu1 5 <210> 45

<211> 321

<212> DNA

<213> Homo sapiens

<220>

<221> CDS <222> (1) .. (321)

<4 0 0> 45

gaa att gtg atg aca cag tct cca gcc acc ctg tct ttg tct cca gggGlu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Pro Gly15 10 15

gaa aga gcc acc ctc tcc tgc agg gcc agt cag agt gtfc age age tacGlu Arg Wing Thr Leu Ser Cys Arg Wing Ser Gln Ser Val Ser Ser Tyr20 25 30

tta gcc tgg tac caa cag aaa cct ggc cag gct ccc agg ctc ctc atteLeu Ala Trp Tyr Gln Lys Pro Gly Gln Ala Pro Arg

tat gaC gea tcc aac agg gcc act ggc till cca gcc agg ttc agt ggcTyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly50 55 60

agt ggg tct ggg aca gac ttc act ctc acc up age act cta gag cctSer Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro65 70 75 80

gaa gat ttt gea gtg tat tac tgt cac cag tat agt age aca cct ctcGlu Asp Phe Ala Val Tyr Tyr Cys His Gln Tyr Gly Ser Thr Pro Leu85 90 95

act ttc ggc gga ggg acc aag gcg gag till aaaThr Phe Gly Gly Gly Thr Lys Ala Glu Ile LysIOO 105

<210> 46

<211> 107

<212> PRT

<213> Homo sapiens

<400> 46

Glu Ile Val Met Thr Gln Be Pro Wing Thr Read Be Read Be Pro Gly15 10 15

Glu Arg Wing Thr Read Le Be Cys Arg Wing Be Gln Be Val Ser Be Tyr20 25 30

Leu Trp Wing Tyr Gln Gln Lys Pro Gly Gln Wing Pro Arg Leu Leu Ile35 40 45

Tyr Asp Wing Be Asn Arg Wing Wing Thr Gly Ile Pro Wing Wing Arg Phe Wing Gly50 55 60Ser Gly Be Gly Wing Wing Asp Phe Thr Wing Read.Thr Ile Wing Wing Be Glu Pro65 70 75 80

Glu Asp Phe Wing Val Tyr Cyr His Gln Tyr Gly Ser Thr Thr Leu85 90 95

Thr Phe Gly Gly Gly Thr Lys Wing Glu Ile Lys100 105

<210> 47

<211> 357

<212> DNA

<213> Mus rausculus

<220>

<221> CDS <222> (1) -. (357)

<400> 47

cag gtg cag ctg aag cag tca gga cct ggc cta

Gln Val Gln Leu Lys Gln Be Gly Pro Gly Leu Val Gln Pro Be Gln1 '5 10 15

• age ctg tcc until acc tgc aca gtc tet ggt ttc t "ca tta act aac tat · 96

Be "Read Be Ile Thr Cys Thr Val Be Gly Phe Be Read Thr Asn Tyr - · · _20 25 30

ggt .gta cac tgg gtt cgc cag tet cca gga aag g§t ctg

Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu35 40 45

gga gtg ata tgg agt ggt gga aac aca gac tat aat aca cct ttc aca 192

Gly Val Ile Trp Being Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr50 55 60

tcc aga ctg age till aac aag gac aat tcc aag age caa gtt ttc ttt 240

Ser Arg Read Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe65 70 75 80

aaa atg aac agt ctg caa tet aat gac aca gcc ata tat tac tgt gcc 2 88

Lys Met Asn Be Read Gln Be Asn Asp Thr Wing Ile Tyr Tyr Cys Ala85 90 95

ag gcc ctc acc tac tat gat tac gag ttt gct tac tgg ggc caa ggg 336

Arg Wing Read Thr Tyr Tyr Asp Tyr Glu Phe Wing Tyr Trp Gly Gln Gly100 105 110

act ctg gtc act gtc tet gea 357

Thr Leu Val Thr Val Ser Ala115

<210> 48

<211> 119

<212 => PRT

<213> Mus rausculus <400> 48

GXn VaX GXn Read Lys GXn Be GXy Pro GXy Read VaX GXn Pro Be GXn15 10 15

Be Read Be Ile Thr Cys Thr VaX Be GXy Phe Be Read Thr Asn Tyr20 25 30

GXy VaX His Trp VaX Arg GXn Ser Pro GXy Lys GXy Leu GXu Trp Leu35 40 45

GXy Val IXe Trp Being Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr50 55 60

Ser Arg Read Ser IXe Asn Lys Asp Asn Be Lys Ser GXn VaX Phe Phe65 70 75 80

Lys Met Asn Be Leu GXn Be Asn Asp Thr AXa Ile Tyr Tyr Cys AXa85 90 95

Arg Wing Read Thr Tyr Tyr Asp Tyr GXu Phe AXa Tyr Trp Gly Gln Gly100 105 110

Thr Leu Val Thr Val Ser AlaIlH

<210> 49

<211> 321

<2-12> DNA

<213> Mus musculus

<220>

<221> CDS

<222> (1). . (321) ^

<400> 49

gac by ttg ctg act by cag tet cca gtc by ctg tet gtg by agt cca gga 48

Asp Ile Leu Read Thr Gln Be Pro VaX Ile Leu Be Val Ser Pro Gly15 10 15

gaa aga gtc agt ttc tec tgc agg gcc agt cag agt att ggc aca aac 96

Glu Arg Val Be Phe Be Cys Arg Wing Be Gln Be Ile Gly Thr Asn20 25 30

aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 144

Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Rrg

aag tat gct tet gag tet till tet ggg till cct tcc agg ttt agt ggc 192

Lys Tyr Wing Be Glu Be Ile Be Gly Ile Pro Be Arg Phe Be Gly50 55 60

agt gga tca ggg aca gat ttt act ctt age till aac agt gtg gag tet 24 0Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Xle Asn Ser Val Glu Ser65 70 75 80

gaa gat att gca gafc tat tac tgt caa caa aat aat aac tgg cca acc 288Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Asn Asn Asn Tirn Pro Thr85 90 95

acg ttc ggt gct ggg acc aag ctg gag ctg aaa 321Thx Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys100 105

<210> 50

<211> 107

<212> PRT

<2X3> Mus musculus

<400> 50

Asp Xle Leu Leu Thr Gln Be Pro Val Ile Leu Be Val Ser Pro Gly1 5 10 15

Glu Arg Val Be Phe Be Cys Arg Wing Be Gln Be Ile Gly Thr Asn20 25 30

Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Read Leu Ile35 40 45

Lys Tyr Wing Be Glu Be Ile Be Gly Ile Pro Be Arg Phe Be Gly50 "55 60

Ser Gly Ser Gly Thr Asp Phe Thr Read Ser Ile Asn Ser Val Glu Ser65 70 75 80

Glu Asp Ile Asp Wing Tyr Tyr Cys Gln Asn Asn Asn Asp Trp Pro Thr85 90 95

Thr Phe Gly Wing Gly Thr Lys Leu Glu Leu Lys100 105

Claims (21)

1. Method of inhibiting the growth of an androgen-dependent cancer, characterized in that it comprises administering androgen deprivation therapy and an IGF-IR antagonist. Method according to claim 1, characterized by the fact that androgen dependent cancer is prostate cancer. Method according to Claim 1, characterized in that the androgen deprivation therapy pellet and the IGF-IR antagonist are started at approximately the same time. Method according to Claim 1, characterized in that the IGF-IR antagonist is initiated after androgen deprivation therapy and before androgen-dependent cancer becomes androgen-independent. Method according to claim 1, characterized in that the IGF-IR antagonist is an extracellular antagonist. The method according to claim 5, characterized in that the extracellular antagonist is an IGF-IR binding antibody. Method according to claim 5, characterized in that the extracellular antagonist binds to an IGF-IR ligand. Method according to claim 1, characterized in that the IGF-IR antagonist downregulates IGF-IR. The method according to claim 8, characterized in that the IGF-IR antagonist is an siRNA or antisense RNA. A method according to claim 5, characterized in that the extracellular antagonist is IMCA1. A method according to claim 1, characterized in that the IGF-IR antagonist is an intracellular antagonist. A method according to claim 11, characterized in that the intracellular antagonist is a small molecule. A method according to claim 11, characterized in that the intracellular antagonist is selected from the group consisting of AG1024, NVP-AEW541, and BMS-554417. A method according to claim 1, characterized in that androgen deprivation therapy comprises administering a luteinizing hormone releasing hormone (LHRH) analog. A method according to claim 1, characterized in that androgen deprivation therapy comprises administering anti-androgen treatment. A method according to claim 1, characterized in that androgen deprivation therapy comprises administering an adrenal androgen inhibitor. Method according to claim 1, characterized in that the androgen deprivation therapy is orchiectomy. A method according to claim 1, further comprising administering an Akt antagonist. A method according to claim 18, characterized in that the Akt antagonist is an EGFR antagonist. A method according to claim 1, characterized in that androgen deprivation therapy and the IGF-IR antagonist are administered with an antineoplastic agent. Method according to claim 20, characterized in that the anti-neoplastic agent is radiation.