WO2014150840A1 - Methods of treating breast cancer - Google Patents

Abstract

Methods of treating breast cancer in a subject by co-administering an anti-insulin-like growth factor I/II (anti-IGF-I/II) antibody or antigen-binding fragment thereof, and an aromatase inhibitor or an estrogen receptor antagonist. Pharmaceutical compositions for the treatment of breast cancer containing an anti-IGF-I/II antibody or antigen-binding fragment thereof, an aromatase inhibitor or an estrogen receptor antagonist, and a pharmaceutically acceptable carrier. Use of a composition containing an anti-IGF-I/II antibody or antigen-binding fragment thereof and an aromatase inhibitor or an estrogen receptor antagonist for the manufacture of a medicament for the treatment and/or prevention of breast cancer.

Description

METHODS OF TREATING BREAST CANCER

RELATED APPLICATION DATA

[0001] This application claims the benefit of U.S. Provisional Patent Application No.

61/791,003, filed March 15, 2013. The disclosure of which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

[0002] The invention relates to methods of treating breast cancer in a subject by

administering to a subject in need thereof a therapeutically effective amount of a fully human antibody that inhibits IGF signaling through both the IGF-1R and IR-A pathways in combination with an aromatase inhibitor or an estrogen receptor antagonist.

BACKGROUND

[0003] Breast cancer is one of the most common cancers among women in the Western world. Despite improvements in early diagnosis and clinical management, breast cancer kills more than 520,000 people worldwide each year. Most breast cancer deaths are due to recurrent and metastatic disease.

[0004] Breast cancer can be typed as to whether or not it is: endocrine receptor (estrogen or progesterone receptor) positive; HER2 (human epidermal growth factor receptor 2) positive; triple negative, not positive to receptors for estrogen, progesterone, or HER2; or triple positive, positive for estrogen receptors, progesterone receptors and HER2. These classifications provide doctors with valuable information about how the tumor acts and what kind of treatments may work best. Surgical and radiation treatments are similar for different types of breast cancer. But drug treatments, such as, chemotherapy, endocrine therapies, and other medications, are usually different. These treatments are targeted to the specific type of cancer.

[0005] About 75% of all breast cancers are estrogen receptor positive (ER-positive). They grow in response to the hormone estrogen. About 65% of these are also progesterone receptor positive (PR-positive), and they grow in response to another hormone, progesterone. If breast cancer cells have a significant number of receptors for either estrogen or progesterone, the cancer is considered hormone -receptor positive and likely to respond to endocrine therapies. Endocrine therapies for breast cancer are treatments usually taken after surgery, chemotherapy, and/or radiation are finished. They are designed to help prevent recurrence of the disease by blocking the effects of estrogen and include aromatase inhibitors and estrogen receptor antagonists, which are discussed in greater detail below.

[0006] Insulin- like Growth Factor Receptor I (IGF-IR) has been shown to play an important role in the growth of the primary tumor and metastasis formation in some cancers, including estrogen receptor positive breast cancers, among others. IGF-IR belongs to the family of transmembrane receptor tyrosine kinases and is expressed on the cell surface of most tissues. Physiologically, IGF-IR and its ligands play a key role in the regulation of growth and metabolism. Insulin-like growth factors bind to IGF-receptors (IGF-R), helping to regulate cellular functioning through activation of various intracellular signaling cascades. Ligands of IGF-R, Insulin-like growth factors I and II (IGF-I and IGF-II), play an integral role in the growth and development of somatic tissue, including bone and skeletal muscle. IGF-I and IGF-II are small polypeptides involved in regulating cell proliferation, survival,

differentiation and transformation. Expression of both Insulin- like growth factors and IGF-R increase during fetal development and in several types of cancers, including breast cancer.

[0007] Insulin-like growth factors circulate in serum mostly bound to IGF-binding proteins (primarily binding to the binding proteins IGFBP-1 to 6). IGF-I functions primarily by activating IGF-IR, whereas IGF-II can act through either the IGF-IR or through the insulin receptor-A isoform. Additionally, the interaction of both IGF-I and IGF-II with the IGF- binding proteins may affect the half-life and bioavailability of the Insulin-like growth factors, as well as their direct interaction with receptors in some cases.

[0008] Various research groups have developed antibodies to IGF-IR that inhibit receptor IGF-I-stimulated autophosphorylation, induce receptor internalization and degradation, and reduce proliferation and survival of diverse human cancer cell lines. Monoclonal antibodies targeting the IGF receptor have been the subject of many phase I and II clinical trials for different cancers. A number of agents that target the IGF-R pathway have been or are being studied in the treatment of breast cancer. Such agents include monoclonal antibodies that target IGF-IR, such as, ganitumab (AMG 479), figitumumab (CP-751,871), dalotuzumab (MK-0646, h7C10), and cixutumumab (IMCA12), among others.

[0009] Aromatase inhibitors (AIs) are a class of drugs used in the treatment of breast cancer and ovarian cancer. Aromatase inhibitors work by inhibiting the action of the enzyme aromatase, which converts androgens into estrogens. Estrogen is required for the growth of ovarian cancers and certain breast cancers. As breast tissue is stimulated by estrogens, decreasing their production is a way of suppressing recurrence of breast tumor tissue. AIs have proven an effective treatment for hormone-sensitive breast cancer in postmenopausal women. Aromatase inhibitors are generally not used to treat breast cancer in premenopausal women. AIs are taken to either block the production of estrogen or block the action of estrogen on receptors and are categorized as a hormonal therapy (endocrine treatment).

[0010] There are two types of aromatase inhibitors (AIs) approved to treat breast cancer: (a) irreversible steroidal inhibitors, such as exemestane (Aromasin), which form a permanent and deactivating bond with the aromatase enzyme and (b) non-steroidal inhibitors, such as anastrozole (Arimidex), which inhibit the synthesis of estrogen via reversible competition for the aromatase enzyme. Letrozole is an AI used in treating certain breast cancers.

[0011] Selective estrogen receptor modulators (SERMs) are an important class of hormonal therapy agents which act as antagonists of the estrogen receptor and are used primarily for the treatment and chemoprevention of breast cancer. Some members of this family, such as tamoxifen, are actually partial agonists, which can actually increase estrogen receptor signaling in some tissues, such as the endometrium.

[0012] Tamoxifen ((Z)-2-[4-(l,2-diphenylbut-l-enyl)phenoxy]-N,N-dimethylethanamine) is taken by some women for up to five years after initial treatment for breast cancer and acts as an antagonist of the estrogen receptor in breast tissue via its active metabolite, 4- hydroxytamoxifen. Tamoxifen is a standard endocrine (anti-estrogen) therapy for hormone receptor-positive breast cancer in pre-menopausal women, and is also a standard in postmenopausal women.

[0013] Fulvestrant ((7a,17 )-7-{9-[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl}estra-l,3,5 (10)-triene-3,17-diol) is an estrogen receptor antagonist with no agonist activity. Fulvestrant is categorized as an estrogen receptor downregulator (ERD) and works to down-regulate and degrade the estrogen receptor. Fulvestrant (FASLODEX, AstraZeneca) is used in the treatment of hormone receptor-positive metastatic breast cancer in postmenopausal women with disease progression following anti-estrogen therapy.

[0014] The effect of ganitumab (monoclonal antibody targeting IGF-IR) combined with hormonal therapy was studied in a randomized phase II double blind trial of postmenopausal women with estrogen receptor and/or progesterone receptor positive metastatic or locally advanced breast cancer who had received prior endocrine treatment. Study participants received either ganitumab or placebo, combined with exemestane (an aromatase inhibitor) or fulvestrant (an estrogen receptor antagonist). The study found that the combination of the monoclonal antibody targeting IGF-I receptor and the aromatase inhibitor, exemestane, or the estrogen receptor antagonist, fulvestrant, did not improve outcomes. Robertson et al., Lancet Oncol, 2013, 14: 228-235.

[0015] One phase III trial involving figitumumab, a monoclonal antibody that targets/inhibits IGF-IR, combined with chemotherapy (paclitaxel and carboplatin) in the treatment of non- small cell lung cancer, has been reported. The study was terminated early due to lack of clinical benefit. Jassem et al., J Clin Oncol, 2010, 28: abstr7500.

[0016] Improved treatments for estrogen/progesterone receptor positive breast cancers are desirable. Effective breast cancer treatments with fewer negative side-effects are desired.

SUMMARY

[0017] Certain embodiments are drawn to methods of treating breast cancer in a subject and include co-administering to a subject in need thereof a therapeutically effective amount of (a) an isolated antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain polypeptide having the amino acid sequence of SEQ ID NO: 6 and a light chain polypeptide, and (b) an aromatase inhibitor or an estrogen receptor antagonist. In one embodiment, the aromatase inhibitor is letrozole. In another embodiment, the estrogen receptor antagonist is fulvestrant or tamoxifen.

[0018] Some embodiments are drawn to methods of treating breast cancer in a subject that include co-administering to the subject in need thereof a therapeutically effective amount of (a) an isolated antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain polypeptide and a light chain polypeptide having the amino acid sequence of SEQ ID NO:8, and (b) an aromatase inhibitor or an estrogen receptor antagonist. In one embodiment, the aromatase inhibitor is letrozole. In another embodiment, the estrogen receptor antagonist is fulvestrant or tamoxifen.

[0019] Embodiments disclosed herein include methods of treating breast cancer in a subject that include co-administering to the subject in need thereof a therapeutically effective amount of (a) an isolated antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain polypeptide having the amino acid sequence of SEQ ID NO: 6 and a light chain polypeptide having the amino acid sequence of SEQ ID NO:8, and (b) an aromatase inhibitor or an estrogen receptor antagonist. In one embodiment, the aromatase inhibitor is letrozole. In another embodiment, the estrogen receptor antagonist is fulvestrant or tamoxifen.

[0020] Embodiments described herein include methods of treating breast cancer in a subject involving co- administering to the subject in need thereof a therapeutically effective amount of (a) an isolated antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain polypeptide and a light chain polypeptide, wherein the heavy chain polypeptide comprises a heavy chain complementarity determining region (CDR) 1 having the amino acid sequence of SEQ ID NO: 33, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:34, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:35, and wherein the light chain polypeptide comprises a light chain CDR1 having the amino acid sequence of SEQ ID NO:36, a light chain CDR2 having the amino acid sequence of SEQ ID NO:37, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:38, and (b) an aromatase inhibitor or an estrogen receptor antagonist. In one embodiment, the aromatase inhibitor is letrozole. In another embodiment, the estrogen receptor antagonist is fulvestrant or tamoxifen.

[0021] Some embodiments are drawn to methods of treating breast cancer in a subject by coadministering to the subject in need thereof a therapeutically effective amount of (a) an isolated antibody or antigen-binding fragment thereof, wherein the antibody has the amino acid sequence of the antibody produced by hybridoma cell line 7.159.2 (ATCC Accession Number PTA-7424), and (b) an aromatase inhibitor or an estrogen receptor antagonist. In one embodiment, the aromatase inhibitor is letrozole. In another embodiment, the estrogen receptor antagonist is fulvestrant or tamoxifen.

[0022] Certain embodiments are drawn to pharmaceutical compositions for the treatment of breast cancer that contain a therapeutically effective amount of (a) an isolated antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain polypeptide having the amino acid sequence of SEQ ID NO: 6 and a light chain polypeptide, and (b) an aromatase inhibitor or an estrogen receptor antagonist; and a pharmaceutically acceptable carrier. In one embodiment, the aromatase inhibitor is letrozole. In another embodiment, the estrogen receptor antagonist is fulvestrant or tamoxifen.

[0023] Some embodiments are drawn to pharmaceutical compositions for the treatment of breast cancer comprising a therapeutically effective amount of (a) an isolated antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain polypeptide and a light chain polypeptide having the amino acid sequence of SEQ ID NO:8, and (b) an aromatase inhibitor or an estrogen receptor antagonist; and a pharmaceutically acceptable carrier. In embodiments, the heavy chain polypeptide can have the amino acid sequence of SEQ ID NO:6. In certain embodiments, the aromatase inhibitor is letrozole. In some embodiments, the estrogen receptor antagonist is fulvestrant or tamoxifen.

[0024] Embodiments disclosed herein include pharmaceutical compositions for the treatment of breast cancer comprising a therapeutically effective amount of (a) an isolated antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain polypeptide having the amino acid sequence of SEQ ID NO: 6 and a light chain polypeptide having the amino acid sequence of SEQ ID NO:8, and (b) an aromatase inhibitor or an estrogen receptor antagonist; and a pharmaceutically acceptable carrier. In some embodiments, the aromatase inhibitor is letrozole. In certain embodiments, the estrogen receptor antagonist is fulvestrant or tamoxifen.

[0025] Certain embodiments are drawn to pharmaceutical compositions for the treatment of breast cancer comprising a therapeutically effective amount of (a) an isolated antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain polypeptide and a light chain polypeptide, wherein the heavy chain polypeptide comprises a heavy chain complementarity determining region (CDR) 1 having the amino acid sequence of SEQ ID NO:33, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:34, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:35, and wherein the light chain polypeptide comprises a light chain CDR1 having the amino acid sequence of SEQ ID NO:36, a light chain CDR2 having the amino acid sequence of SEQ ID NO:37, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:38, and (b) an aromatase inhibitor or an estrogen receptor antagonist; and a pharmaceutically acceptable carrier. In one embodiment, the aromatase inhibitor is letrozole. In another embodiment, the estrogen receptor antagonist is fulvestrant or tamoxifen.

[0026] Embodiments described herein include pharmaceutical compositions for the treatment of breast cancer comprising a therapeutically effective amount of (a) an isolated antibody or antigen-binding fragment thereof, wherein the antibody has the amino acid sequence of the antibody produced by hybridoma cell line 7.159.2 (ATCC Accession Number PTA-7424), and (b) an aromatase inhibitor or an estrogen receptor antagonist; and a pharmaceutically acceptable carrier. In one embodiment, the aromatase inhibitor is letrozole. In another embodiment, the estrogen receptor antagonist is fulvestrant or tamoxifen. [0027] Described herein is the use of a composition for the treatment and/or prevention of breast cancer, wherein the composition comprises a therapeutically effective amount of (a) an isolated antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain polypeptide having the amino acid sequence of SEQ ID NO: 6 and a light chain polypeptide, and (b) an aromatase inhibitor or an estrogen receptor antagonist. In one embodiment, the aromatase inhibitor is letrozole. In another embodiment, the estrogen receptor antagonist is fulvestrant or tamoxifen.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain embodiments, and together with the written description, serve to explain certain principles of the antibodies and methods disclosed herein.

[0029] FIG. 1 shows results of treating MCF-7/AC-1/IGF-I cells with letrozole, 4- hydroxytamoxifen, fulvestrant, MEDI-573 or combinations of MEDI-573 with the various chemical therapies. FIG. 1A shows the results of treatments of MCF-7/AC-1/IGF-I cells including 4-hydroxytamoxifen, FIG. IB shows the results of treatments including fulvestrant (ICI), and FIG. 1C shows the results for treatments including letrozole. FIG. ID shows the combination index for the combination of MEDI-573 with each of 4-hydroxytamoxifen, fulvestrant and letrozole as tested in MCF-7/AC-1/IGF-I cells.

[0030] FIG. 2 shows results of treating MCF-7/AC-l/IGF-II cells with letrozole, 4- hydroxytamoxifen, fulvestrant, MEDI-573 or combinations of MEDI-573 with the various chemical therapies. FIG. 2A shows the results of treatments of MCF-7/AC-l/IGF-II including 4-hydroxytamoxifen, FIG. 2B shows the results of treatments including fulvestrant (ICI), and FIG. 2C shows the results for treatments including letrozole. FIG. 2D shows the combination index for the combination of MEDI-573 with each of 4-hydroxytamoxifen, fulvestrant and letrozole as tested in MCF-7/AC-l/IGF-II cells.

[0031] FIG. 3 shows results demonstrating that the antiproliferative activity of MEDI-573 is not overcome by physiologically relevant levels of insulin in MCF-7/AC-1/IGF-I and MCF- 7/AC-l/IGF-II cells in MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium) cell proliferation assays. FIG. 3A is a graph with results from experiments in MCF-7/AC-1/IGF-I cells with 4-hydroxytamoxifen and MEDI-573. FIG. 3B is a graph with results from experiments in MCF-7/AC-l/IGF-II cells with 4- hydroxytamoxifen and MEDI-573. FIG. 3C is a graph with results from experiments in MCF-7/AC-1/IGF-I cells with letrozole and MEDI-573. FIG. 3D is a graph with results from experiments in MCF-7/AC-l/IGF-II cells with letrozole in combination with MEDI-573. FIG. 3E is a graph with results from experiments in MCF-7/AC-1/IGF-I cells with fulvestrant in combination with MEDI-573. FIG. 3F is a graph with results from experiments in MCF- 7/AC-l/IGF-II cells with fulvestrant in combination with MEDI-573.

[0032] FIG. 4 is a graph of data for % tumor volume change using MCF-7/AC-1/IGF-I xenografts in experiments conducted in vivo with a treatment of letrozole, or a treatment of MEDI-573 in combination with letrozole over time.

[0033] FIG. 5 is a graph of data for % tumor volume change using MCF-7/AC-l/IGF-II xenografts in experiments conducted in vivo with a treatment of letrozole, or a treatment of MEDI-573 in combination with letrozole over time.

DETAILED DESCRIPTION

[0034] Reference will now be made in detail to various exemplary embodiments, examples of which are illustrated in the accompanying drawings. It is to be understood that the following detailed description is provided to give the reader a fuller understanding of certain embodiments, features, and details of aspects of the invention, and should not be interpreted as a limitation of the scope of the invention.

[0035] Embodiments include methods of co-administering (a) a binding protein that specifically binds to IGF-II with cross reactivity to IGF-I (referred to herein as "IGFI/Π" binding proteins or anti-IGF-I/II antibodies or fragments thereof) and (b) an aromatase inhibitor or estrogen receptor antagonist, for treating breast cancer, such as estrogen receptor positive breast cancer.

[0036] Embodiments herein include binding proteins that specifically bind to IGF-II with cross reactivity to IGF-I (referred to herein as "IGFI/II"). In some embodiments, the binding proteins are antibodies, or antigen-binding fragments thereof, and bind to IGF-II with cross- reactivity to IGF-I and inhibit the binding of these proteins to their receptor, IGF-IR. Other embodiments include fully human neutralizing anti-IGF-I/II antibodies, and antibody preparations that are therapeutically useful and bind both Insulin-like growth factors. Such anti-IGF-I/II antibody preparations can have desirable therapeutic properties, including strong binding affinity for IGF-I/II, the ability to neutralize IGF-I/II in vitro, and the ability to inhibit IGF-I/II-induced cell proliferation in vivo. [0037] Certain embodiments also include isolated antigen-binding fragments of anti-IGF-I/II antibodies. The antigen-binding fragments can be derived from fully human anti-IGF-I/II antibodies. Exemplary fragments include Fv, Fab' or other well know antibody fragments, as described in more detail below.

Sequence Listing

[0038] Embodiments include the specific anti-IGF-I/II antibodies listed below in Table 1. This table indicates the identification number of each anti-IGF-I/II antibody, along with the SEQ ID number of the corresponding heavy chain and light chain genes. Further, the germline sequences from which each heavy chain and light chain derive are also provided below in Table 1.

[0039] Each antibody was given an identification number that includes either two or three numbers separated by one or two decimal points. In some cases, several clones of one antibody were prepared. Although the clones have the identical nucleic acid and amino acid sequences as the parent sequence, they may also be listed separately, with the clone number indicated by the number to the right of a second decimal point. Thus, for example, the nucleic acid and amino acid sequences of antibody 7.159.2 are identical to the sequences of antibody 7.159.1.

[0040] As can be seen by comparing the sequences in the sequence listing, SEQ ID NOs. : 1- 20 differ from SEQ ID NOs.: 39-58 because SEQ ID NOs. : 39-58 include the untranslated, signal peptide, and constant domain regions for each sequenced heavy or light chain. Sequences in the sequence listing are described in U.S. Patent No. 7,939,637, "Insulin-Like Growth Factor Antibodies and Uses Thereof," to Raeber et al., issued May 10, 2011, and in U.S. Patent Pub. No. 2011/0200607, "Binding Proteins Specific for Insulin-Like Growth Factors and Uses Thereof," to Raeber et al., published August 18, 2011, which are hereby incorporated by reference in their entirety.

TABLE 1

TABLE 1 -CONTINUED

Definitions

[0041] Unless otherwise defined, scientific and technical terms used herein shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well known and commonly used in the art.

[0042] Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g. , electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures can be performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g. , Sambrook et al. Molecular Cloning: A Laboratory Manual (3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001)), which is incorporated herein by reference. The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0043] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0044] The term "IGF-I" refers to the molecule Insulin-like growth factor-I, and the term "IGF-II" refers to the molecule Insulin-like growth factor-II. The term "IGF-1/Π" refers to both molecules Insulin-like growth factors-I and -II, and relates to the preferential binding to IGF-II with cross-reactivity to IGF-I. Thus, an antibody that binds to IGF-I/II will preferentially bind to IGF-II, but cross-reacts with IGF-I, binding to IGF-II with higher affinity than to IGF-I. For example, the antibody can bind to IGF-II with 2.5 times greater affinity than to IGF-I. In certain embodiments, the antibody can bind to IGF-II with at least 5, at least 10, at least 25, at least 50 or at least 150 times greater affinity than to IGF-I.

[0045] The term "neutralizing" when referring to an antibody relates to the ability of an antibody to eliminate, or significantly reduce, the activity of a target antigen. Accordingly, a "neutralizing" anti-IGF-I/II antibody is capable of eliminating or significantly reducing the activity of IGF-I/II. A neutralizing IGF-I/II antibody may, for example, act by blocking the binding of IGF-I/II to its receptor IGF-IR. By blocking this binding, the IGF-IR mediated signal transduction is significantly, or completely, eliminated. Ideally, a neutralizing antibody against IGF-I/II inhibits cell proliferation.

[0046] The term "isolated polynucleotide" as used herein shall mean a polynucleotide that has been isolated from its naturally occurring environment. Such polynucleotides may be genomic, cDNA, or synthetic. Isolated polynucleotides may in some instances not be associated with all or a portion of the polynucleotides they associate with in nature. The isolated polynucleotide may be operably linked to another polynucleotide that it is not linked to in nature. In addition, isolated polynucleotides may in some instances not occur in nature as part of a larger sequence.

[0047] The term "isolated protein" refers to a protein that has been isolated from its naturally occurring environment. Such proteins may be derived from genomic DNA, cDNA, recombinant DNA, recombinant RNA, or a synthetic origin or some combination thereof, which by virtue of its origin, or source of derivation, the "isolated protein" (1) is not associated with proteins found in nature, (2) is free of other proteins from the same source, e.g. , free of murine proteins, (3) is expressed by a cell from a different species, or (4) does not occur in nature.

[0048] The term "polypeptide" is used herein as a generic term to refer to native protein, fragments, or analogs of a polypeptide sequence. Hence, native protein, fragments, and analogs are species of the polypeptide genus. Polypeptides in accordance with certain embodiments comprise the human heavy chain immunoglobulin molecules and the human kappa light chain immunoglobulin molecules, as well as antibody molecules formed by combinations comprising the heavy chain immunoglobulin molecules with light chain immunoglobulin molecules, such as the kappa or lambda light chain immunoglobulin molecules, and vice versa, as well as fragments and analogs thereof. Polypeptides in accordance with certain embodiments may also comprise solely the human heavy chain immunoglobulin molecules or fragments thereof.

[0049] The term "naturally-occurring" as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory or otherwise is naturally-occurring.

[0050] The term "operably linked" as used herein refers to positions of components so described that are in a relationship permitting them to function in their intended manner. For example, a control sequence "operably linked" to a coding sequence is connected in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

[0051] The term "polynucleotide" refers to a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide, or RNA-DNA hetero-duplexes. The term includes single and double stranded forms of DNA.

[0052] The term "oligonucleotide" referred to herein includes naturally occurring, and modified nucleotides linked together by naturally occurring, and non-naturally occurring linkages. Oligonucleotides are a polynucleotide subset comprising a length of 200 bases or fewer. Oligonucleotides can be 10 to 60 bases in length and in some embodiments 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases in length. Oligonucleotides may be single stranded, e.g. for probes; although oligonucleotides may be double stranded, e.g. , for use in the construction of a gene mutant. Oligonucleotides can be either sense or antisense oligonucleotides.

[0053] The term "naturally occurring nucleotides" referred to herein includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" referred to herein includes nucleotides with modified or substituted sugar groups and the like. The term "oligonucleotide linkages" referred to herein includes oligonucleotides linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate, phosphoroamidate, and the like. See e.g. , LaPlanche et al. Nucl. Acids Res. 14:9081 (1986); Stec et al. J. Am. Chem. Soc. 106:6077 (1984); Stein et al. Nucl. Acids Res. 16:3209 (1988); Zon et al. Anti-Cancer Drug Design 6:539 (1991); Zon et al. Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford University Press, Oxford England (1991)); Stec et al. U.S. Pat. No. 5,151 ,510; Uhlmann and Peyman Chemical Reviews 90:543 (1990), the disclosures of which are hereby incorporated by reference. An oligonucleotide can include a label for detection, if desired.

[0054] The term "selectively hybridize" refers to detectably and specifically bind. Polynucleotides, oligonucleotides and fragments thereof selectively hybridize to nucleic acid strands under hybridization and wash conditions that minimize appreciable amounts of detectable binding to nonspecific nucleic acids. High stringency conditions can be used to achieve selective hybridization conditions as known in the art and discussed herein.

[0055] The term "corresponds to" is used herein to mean that a polynucleotide sequence is homologous (i.e., is identical, not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is identical to a reference polypeptide sequence.

[0056] The term "polypeptide fragment" as used herein refers to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the naturally-occurring sequence deduced, for example, from a full-length cDNA sequence. Fragments can be at least 5, 6, 8, 10, 14, 20, 50 or 70 amino acids long. The term "analog" as used herein refers to polypeptides which are comprised of a segment of at least 25 amino acids that has substantial identity to a portion of a deduced amino acid sequence and which has at least one of the following properties: (1) specific binding to IGF-I/II, under suitable binding conditions, (2) ability to block appropriate IGF-I/II binding, or (3) ability to inhibit IGF-I/II activity. Polypeptide analogs can comprise a conservative amino acid substitution (or addition or deletion) with respect to the naturally-occurring sequence. Analogs can be at least 20 amino acids long or at least 50 amino acids long or longer, and can often be as long as a full-length naturally-occurring polypeptide.

[0057] Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compound are termed "peptide mimetics" or "peptidomimetics". Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber and Freidinger TINS p. 392 (1985); and Evans et al. J. Med. Chem. 30: 1229 (1987), which are incorporated herein by reference. Such compounds are often developed with the aid of computerized molecular modeling. Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce an equivalent therapeutic or prophylactic effect. Peptidomimetics are structurally similar to a paradigm polypeptide (i.e. , a polypeptide that has a biochemical property or pharmacological activity), such as human antibody, but have one or more peptide linkages optionally replaced by a linkage selected from the group consisting of: ~CH₂NH~, ~CH₂S~, ~CH₂~CH₂~, — CH=CH~(cw and trans), ~COCH₂~, ~CH(OH)CH₂~, and -CH₂SO~, by methods well known in the art. Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g. , D-lysine in place of L- lysine) may be used to generate more stable peptides. In addition, constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variation may be generated by methods known in the art (Rizo and Gierasch Ann. Rev. Biochem. 61 :387 (1992), incorporated herein by reference); for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges which cyclize the peptide.

[0058] As used herein, the term "antibody" refers to a polypeptide or group of polypeptides that are comprised of at least one binding domain that is formed from the folding of polypeptide chains having three-dimensional binding spaces with internal surface shapes and charge distributions complementary to the features of an antigenic determinant of an antigen. An antibody can have a tetrameric form, comprising two identical pairs of polypeptide chains, each pair having one "light" and one "heavy" chain. The variable regions of each light/heavy chain pair form an antibody binding site. [0059] "Antigen-binding fragments" of an antibody are produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include Fab, Fab', F(ab)₂, Fv, and single-chain antibodies. An antibody other than a "bispecific" or "bifunctional" antibody is understood to have each of its binding sites identical. An antibody substantially inhibits adhesion of a receptor to a counterreceptor when an excess of antibody reduces the quantity of receptor bound to counterreceptor by at least about 20%, 40%, 60%, 80%, or 85% (as measured in an in vitro competitive binding assay).

[0060] As used herein, a "binding protein" or a "specific binding protein" is a protein that specifically binds to a target molecule. Antibodies, and antigen-binding fragments of antibodies, are binding proteins.

[0061] The term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitopic determinants can consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and may, but not always, have specific three-dimensional structural characteristics, as well as specific charge characteristics. An antibody is said to specifically bind an antigen when the dissociation constant is <1 μΜ, <100 nM or <10 nM.

[0062] The term "agent" is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials.

[0063] "Active" or "activity" in regard to an IGF-I/II polypeptide refers to a portion of an IGF-I/II polypeptide that has a biological or an immunological activity of a native IGF-I/II polypeptide. "Biological" when used herein refers to a biological function that results from the activity of the native IGF-I/II polypeptide. IGF-I/II biological activity can include, for example, IGF-I/II induced cell proliferation.

[0064] "Mammal" when used herein refers to any animal that is considered a mammal. The mammal can be a human.

[0065] Digestion of antibodies with the enzyme, papain, results in two identical antigen- binding fragments, known also as "Fab" fragments, and a "Fc" fragment, having no antigen- binding activity but having the ability to crystallize. Digestion of antibodies with the enzyme, pepsin, results in the a F(ab')₂ fragment in which the two arms of the antibody molecule remain linked and comprise two-antigen binding sites. The F(ab')₂ fragment has the ability to crosslink antigen. [0066] "Fv" when used herein refers to the minimum fragment of an antibody that retains both antigen-recognition and antigen-binding sites.

[0067] "Fab" when used herein refers to a fragment of an antibody that comprises the constant domain of the light chain and the CHI domain of the heavy chain.

[0068] The term "mAb" refers to monoclonal antibody.

[0069] "Liposome" when used herein refers to a small vesicle that may be useful for delivery of drugs that may include the IGF-I/II polypeptide of embodiments or antibodies to such an IGF-I/II polypeptide to a mammal.

[0070] "Label" or "labeled" as used herein refers to the addition of a detectable moiety to a polypeptide, for example, a radiolabel, fluorescent label, enzymatic label chemiluminescent labeled or a biotinyl group. Radioisotopes or radionuclides may include ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ^mIn, ¹²⁵I, ¹³¹I, fluorescent labels may include rhodamine lanthanide phosphors or FITC and enzymatic labels may include horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase.

[0071] The term "pharmaceutical agent or drug" as used herein refers to a chemical compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient. Other chemistry terms herein are used according to conventional usage in the art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms (Parker, S., Ed., McGraw-Hill, San Francisco (1985)), (incorporated herein by reference).

[0072] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" means solvents, dispersion media, coatings, antibacterial agents and antifungal agents, isotonic agents, and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art.

[0073] The term "subject" includes human and veterinary subjects.

[0074] The terms "treatment" or "treating" and the like refer to any treatment of any disease or condition in a subject, e.g. particularly a human, and includes inhibiting a disease, condition, or symptom of a disease or condition, e.g., arresting its development and/or delaying its onset or manifestation in the patient or relieving a disease, condition, or symptom of a disease or condition, e.g., causing regression of the condition or disease and/or its symptoms. [0075] It should be noted that, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. The terms "a" (or "an"), as well as the terms "one or more," and "at least one" can be used interchangeably herein.

[0076] Furthermore, "and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and/or" as used in a phrase such as "A and/or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0077] It is also understood that wherever embodiments are described herein with the language "comprising," otherwise analogous embodiments described in terms of "consisting of and/or "consisting essentially of are also provided.

Methods of Treatment

[0078] Certain embodiments are drawn to methods of treating breast cancer in a subject comprising co-administering to the subject in need thereof a therapeutically effective amount of (a) an isolated antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain polypeptide having the amino acid sequence of SEQ ID NO: 6 and a light chain polypeptide, and (b) an aromatase inhibitor or an estrogen receptor antagonist. The light chain polypeptide can have the amino acid sequence of SEQ ID NO: 8 in some embodiments. In one embodiment, the aromatase inhibitor is letrozole. In another embodiment, the estrogen receptor antagonist is fulvestrant or tamoxifen.

[0079] Embodiments disclosed herein include methods of treating breast cancer in a subject comprising co-administering to the subject in need thereof a therapeutically effective amount of (a) an isolated antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain polypeptide and a light chain polypeptide having the amino acid sequence of SEQ ID NO:8, and (b) an aromatase inhibitor or an estrogen receptor antagonist. In one embodiment, the aromatase inhibitor is letrozole. In another embodiment, the estrogen receptor antagonist is fulvestrant or tamoxifen.

[0080] Some embodiments are drawn to methods of treating breast cancer in a subject comprising co-administering to the subject in need thereof a therapeutically effective amount of (a) an isolated antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain polypeptide and a light chain polypeptide, wherein the heavy chain polypeptide comprises a heavy chain complementarity determining region (CDR) 1 having the amino acid sequence of SEQ ID NO: 33, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:34, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:35, and wherein the light chain polypeptide comprises a light chain CDR1 having the amino acid sequence of SEQ ID NO:36, a light chain CDR2 having the amino acid sequence of SEQ ID NO:37, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:38, and (b) an aromatase inhibitor or an estrogen receptor antagonist. In one embodiment, the aromatase inhibitor is letrozole. In another embodiment, the estrogen receptor antagonist is fulvestrant or tamoxifen.

[0081] Certain embodiments are drawn to methods of treating breast cancer in a subject comprising co-administering to the subject in need thereof a therapeutically effective amount of (a) an isolated antibody or antigen-binding fragment thereof, wherein the antibody has the amino acid sequence of the antibody produced by hybridoma cell line 7.159.2 (ATCC Accession Number PTA-7424 or MEDI-573), and (b) an aromatase inhibitor or an estrogen receptor antagonist. In one embodiment, the aromatase inhibitor is letrozole. In another embodiment, the estrogen receptor antagonist is fulvestrant or tamoxifen.

[0082] Pharmaceutical compositions for the treatment of breast cancer are described herein, the compositions comprising a therapeutically effective amount of (a) an isolated antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain polypeptide having the amino acid sequence of SEQ ID NO: 6 and a light chain polypeptide, and (b) an aromatase inhibitor or an estrogen receptor antagonist; and a pharmaceutically acceptable carrier. The light chain polypeptide can have the amino acid sequence of SEQ ID NO:8 in some embodiments. In one embodiment, the aromatase inhibitor is letrozole. In another embodiment, the estrogen receptor antagonist is fulvestrant or tamoxifen.

[0083] Certain embodiments are drawn to pharmaceutical compositions for the treatment of breast cancer comprising a therapeutically effective amount of (a) an isolated antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain polypeptide and a light chain polypeptide having the amino acid sequence of SEQ ID NO:8, and (b) an aromatase inhibitor or an estrogen receptor antagonist; and a pharmaceutically acceptable carrier. The heavy chain polypeptide has the amino acid sequence of SEQ ID NO: 6, in some embodiments. In one embodiment, the aromatase inhibitor is letrozole. In another embodiment, the estrogen receptor antagonist is fulvestrant or tamoxifen.

[0084] Certain embodiments are drawn to pharmaceutical compositions for the treatment of breast cancer comprising a therapeutically effective amount of (a) an isolated antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain polypeptide and a light chain polypeptide, wherein the heavy chain polypeptide comprises a heavy chain complementarity determining region (CDR) 1 having the amino acid sequence of SEQ ID NO:33, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:34, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:35, and wherein the light chain polypeptide comprises a light chain CDR1 having the amino acid sequence of SEQ ID NO:36, a light chain CDR2 having the amino acid sequence of SEQ ID NO:37, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:38, and (b) an aromatase inhibitor or an estrogen receptor antagonist; and a pharmaceutically acceptable carrier. In one embodiment, the aromatase inhibitor is letrozole. In another embodiment, the estrogen receptor antagonist is fulvestrant or tamoxifen.

[0085] Some embodiments are drawn to methods of treating breast cancer in a subject comprising co-administering to the subject in need thereof a therapeutically effective amount of (a) an isolated antibody or antigen-binding fragment thereof, wherein the antibody has the amino acid sequence of the antibody produced by hybridoma cell line 7.159.2 (ATCC Accession Number PTA-7424), and (b) an aromatase inhibitor or an estrogen receptor antagonist. In some embodiments, the aromatase inhibitor is letrozole. In certain embodiment, the estrogen receptor antagonist is fulvestrant or tamoxifen.

[0086] Some embodiments are drawn to use of a composition for the treatment and/or prevention of breast cancer, wherein the composition comprises a therapeutically effective amount of (a) an isolated antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain polypeptide having the amino acid sequence of SEQ ID NO: 6 and a light chain polypeptide, and (b) an aromatase inhibitor or an estrogen receptor antagonist. The light chain polypeptide can have the amino acid sequence of SEQ ID NO: 8 in some embodiments. In one embodiment, the aromatase inhibitor is letrozole. In another embodiment, the estrogen receptor antagonist is fulvestrant or tamoxifen.

[0087] Embodiments described herein include use of a composition for the treatment of breast cancer, wherein the composition comprises a therapeutically effective amount of (a) an isolated antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain polypeptide and a light chain polypeptide, wherein the heavy chain polypeptide comprises a heavy chain complementarity determining region (CDR) 1 having the amino acid sequence of SEQ ID NO:33, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:34, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:35, and wherein the light chain polypeptide comprises a light chain CDR1 having the amino acid sequence of SEQ ID NO: 36, a light chain CDR2 having the amino acid sequence of SEQ ID NO:37, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:38, and (b) an aromatase inhibitor or an estrogen receptor antagonist for the manufacture of a medicament for the treatment and/or prevention of breast cancer. In one embodiment, the aromatase inhibitor is letrozole. In another embodiment, the estrogen receptor antagonist is fulvestrant or tamoxifen.

[0088] Some embodiments are drawn to use of a composition for the treatment of breast cancer, wherein the composition comprises a therapeutically effective amount of (a) an isolated antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain polypeptide and a light chain polypeptide having the amino acid sequence of SEQ ID NO: 8, and (b) an aromatase inhibitor or an estrogen receptor antagonist for the manufacture of a medicament for the treatment and/or prevention of breast cancer. The heavy chain polypeptide has the amino acid sequence of SEQ ID NO:6, in some embodiments. In one embodiment, the aromatase inhibitor is letrozole. In another embodiment, the estrogen receptor antagonist is fulvestrant or tamoxifen.

[0089] Certain embodiments are drawn to use of a composition for the treatment of breast cancer, wherein the composition comprises a therapeutically effective amount of (a) an isolated antibody or antigen-binding fragment thereof, wherein the antibody has the amino acid sequence of the antibody produced by hybridoma cell line 7.159.2 (ATCC Accession Number PTA-7424), and (b) an aromatase inhibitor or an estrogen receptor antagonist for the manufacture of a medicament for the treatment and/or prevention of breast cancer. The heavy chain polypeptide has the amino acid sequence of SEQ ID NO:6, in some embodiments. In one embodiment, the aromatase inhibitor is letrozole. In another embodiment, the estrogen receptor antagonist is fulvestrant or tamoxifen.

[0090] The breast cancer treated in embodiments can be an estrogen receptor positive breast cancer or an estrogen receptor unknown breast cancer (breast cancer where it is unknown whether the cancer cells are expressing the estrogen receptor). The breast cancer treated in embodiments can also be a progesterone receptor positive breast cancer in some embodiments. The breast cancer can be typed as endocrine receptor (estrogen or progesterone receptor) positive; or triple positive, positive for estrogen receptors, progesterone receptors and HER2, in some embodiments. The subject that treatment is administered to can be a postmenopausal woman, a premenopausal woman, a man with breast cancer, or another mammal with breast cancer. In some embodiments, the subject is a postmenopausal woman. In certain embodiments, the subject is a postmenopausal woman and the breast cancer is of an estrogen receptor-positive type.

[0091] The treatment methods described herein may be applied as a sole therapy or may involve (in addition to the co-administration of the isolated antibody or antigen-binding fragment thereof and an aromatase inhibitor or an estrogen receptor antagonist) conventional surgery or radiotherapy or chemotherapy.

[0092] As discussed above, a treatment where a monoclonal antibody targeting IGF-IR (ganitumab) was co-administered with exemestane (an aromatase inhibitor) or fulvestrant (an estrogen receptor antagonist) to postmenopausal women with estrogen receptor and/or progesterone receptor positive metastatic or locally advanced breast cancer who had received prior endocrine treatment did not improve outcomes. Robertson et al., Lancet Oncol, 2013, 14: 228-235. In Robertson's study co-administering the monoclonal antibody targeting IGF- IR (ganitumab) with an aromatase inhibitor or an estrogen receptor antagonist did not enhance the effects of the aromatase inhibitor or estrogen receptor antagonist alone.

[0093] The antibodies, aromatase inhibitors and estrogen receptor antagonists of embodiments are discussed greater detail below.

[0094] The co-administering step of embodiments can be achieved by way of the simultaneous, sequential or separate dosing of (a) the isolated antibody or antigen-binding fragment thereof and (b) the aromatase inhibitor or the estrogen receptor antagonist. Such combinations of embodiments employ the compounds ((a) and (b)) within their approved dosage ranges. By co-administering (a) the isolated antibody or antigen-binding fragment thereof and (b) the aromatase inhibitor or the estrogen receptor antagonist can both be present in the subject at the same time, although they are not administered simultaneously.

Therapeutic Administration and Formulations

[0095] Embodiments can include sterile pharmaceutical formulations of anti-IGF-I/II antibodies or anti-IGF-I/II antibodies with an aromatase inhibitor or an estrogen receptor antagonist that are useful as treatments for breast cancer. Such formulations can inhibit the binding of IGF-I/II to its receptor IGF-IR, thereby effectively treating breast cancer where, for example, IGF-I/II is abnormally elevated. Anti-IGF-I/II antibodies can possess adequate affinity to potently neutralize IGF-I/II, and in some embodiments can have an adequate duration of action to allow for infrequent dosing in humans. A prolonged duration of action will allow for less frequent and more convenient dosing schedules of the pharmaceutical formulation comprising anti-IGF-I/II antibodies by alternate parenteral routes such as subcutaneous or intramuscular injection.

[0096] The aromatase inhibitor and estrogen receptor antagonist can be used at dosages known in the art using methods of administering them known in the art. They may be administered in pharmaceutical formulations separate from the antibody or they may also be part of a pharmaceutical formulation containing the anti-IGF-I/II antibody.

[0097] Sterile formulations can be created, for example, by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution of the antibody or drug. The antibody ordinarily will be stored in lyophilized form or in solution. Therapeutic formulations can be placed into a container having a sterile access port, for example, an intravenous solution bag or vial having an adapter that allows retrieval of the formulation, such as a stopper pierceable by a hypodermic injection needle.

[0098] The route of antibody administration is in accord with known methods, e.g. , injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, intrathecal, inhalation or intralesional routes, or by sustained release systems as noted below. The antibody is can be administered continuously by infusion or by bolus injection. Similarly, the aromatase inhibitor or estrogen receptor antagonist can be administered using known methods.

[0099] An effective amount of the pharmaceutical formulation (e.g. , antibody composition) to be employed therapeutically will depend, for example, upon the therapeutic objectives, the route of administration, and the condition of the patient. Accordingly, the therapist can titer the dosage and modify the route of administration as required to obtain the optimal therapeutic effect. The clinician can administer pharmaceutical formulation until a dosage is reached that achieves the desired effect. The progress of this therapy is easily monitored by conventional assays or by the assays described herein. [0100] Antibodies, as described herein, can be prepared in a mixture with a pharmaceutically acceptable carrier. Antibodies together with an aromatase inhibitor or estrogen receptor antagonist can be prepared in a mixture with a pharmaceutically acceptable carrier. These therapeutic compositions can be administered intravenously or through the nose or lung, such as, as a liquid or powder aerosol (lyophilized). These compositions may also be administered parenterally or subcutaneously as desired. When administered systemically, the therapeutic compositions can be sterile, pyrogen-free and in a parenterally acceptable solution having due regard for pH, isotonicity, and stability. These conditions are known to those skilled in the art. Briefly, dosage formulations of the compounds described herein are prepared for storage or administration by mixing the compound (e.g. , antibody and/or aromatase inhibitor or estrogen receptor antagonist) having the desired degree of purity with physiologically acceptable carriers, excipients, or stabilizers. Such materials are non-toxic to the recipients at the dosages and concentrations employed, and include buffers such as TRIS HC1, phosphate, citrate, acetate and other organic acid salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) peptides such as polyarginine, proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidinone; amino acids such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium and/or nonionic surfactants such as TWEEN, PLURONICS or polyethyleneglycol.

[0101] Sterile compositions for injection can be formulated according to conventional pharmaceutical practice as described in Remington: The Science and Practice of Pharmacy (20th ed., Lippincott Williams & Wilkens Publishers (2003)). For example, dissolution or suspension of the active compound in a vehicle such as water or naturally occurring vegetable oil like sesame, peanut, or cottonseed oil or a synthetic fatty vehicle like ethyl oleate or the like may be desired. Buffers, preservatives, antioxidants and the like can be incorporated according to accepted pharmaceutical practice.

[0102] Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, which matrices are in the form of shaped articles, films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g. , poly(2-hydroxyethyl-methacrylate) as described by Langer et al., J. Biomed Mater. Res., (1981) 15: 167-277 and Langer, Chem. Tech., (1982) 12:98-105, or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L- glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers, (1983) 22:547- 556), non-degradable ethylene-vinyl acetate (Langer et al., supra), degradable lactic acid- glycolic acid copolymers such as the LUPRON Depot™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3- hydroxybutyric acid (EP 133,988).

[0103] While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated proteins remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for protein stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S-S bond formation through disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.

[0104] Sustained-released compositions also include preparations of crystals of the antibody or antibody combined with an aromatase inhibitor or an estrogen receptor antagonist suspended in suitable formulations capable of maintaining crystals in suspension. These preparations when injected subcutaneously or intraperitoneally can produce a sustained release effect. Other compositions also include liposomally entrapped antibodies. Liposomes containing such antibodies are prepared by methods known per se: U.S. Pat. No. DE 3,218,121 ; Epstein et al., Proc. Natl. Acad. Sci. USA, (1985) 82:3688-3692; Hwang et al., Proc. Natl. Acad. Sci. USA, (1980) 77:4030-4034; EP 52,322; EP 36,676; EP 88,046; EP 143,949; 142,641 ; Japanese patent application 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324.

[0105] The dosage of the pharmaceutical formulation (e.g. , antibody formulation) for a given patient will be determined by the attending physician taking into consideration various factors known to modify the action of drugs including severity and type of disease, body weight, sex, diet, time and route of administration, other medications and other relevant clinical factors. Therapeutically effective dosages may be determined by either in vitro or in vivo methods. [0106] An effective amount of the antibodies, aromatase inhibitors, and estrogen receptor antagonists, described herein, to be employed therapeutically will depend, for example, upon the therapeutic objectives, the route of administration, and the condition of the patient. Accordingly, the therapist can titer the dosage and modify the route of administration as required to obtain the optimal therapeutic effect. A typical daily dosage for the antibody might range from about 0.001 mg/kg to up to 100 mg/kg or more, depending on the factors mentioned above. Thus, an effective amount of antibody may include, but is not limited to, dosage ranges of about 0.1 mg/kg to about 100 mg/kg; 0.1 mg/kg to about 100 mg/kg; 0.1 mg/kg to about 10 mg/kg; about 0.5 mg/kg to 75 mg/kg; 1 mg/kg to about 50 mg/kg; 1 mg/kg to about 10 mg/kg; 0.5 mg/kg to about 25 mg/kg; or about 1 mg/kg to about 5 mg/kg. The clinician can administer the therapeutic antibody until a dosage is reached that achieves the desired effect. The progress of this therapy is easily monitored by conventional assays or as described herein. Appropriate dosages for the aromatase inhibitor or estrogen receptor antagonist used are known in the art.

[0107] It will be appreciated that administration of therapeutic entities in accordance with the compositions and methods herein will be administered with suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as Lipofectin™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semisolid gels, and semi-solid mixtures containing carbowax. Any of the foregoing mixtures may be appropriate in treatments and therapies in accordance with certain embodiments, provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and tolerable with the route of administration. See also Baldrick P. "Pharmaceutical excipient development: the need for preclinical guidance." Regul. Toxicol. Pharmacol. 32(2):210-8 (2000), Wang W. "Lyophilization and development of solid protein pharmaceuticals." Int. J. Pharm. 203(1-2): 1-60 (2000), Charman WN "Lipids, lipophilic drugs, and oral drug delivery-some emerging concepts." J Pharm Sci. 89(8):967-78 (2000), Powell et al. "Compendium of excipients for parenteral formulations" PDA J Pharm Sci Technol. 52:238-311 (1998) and the citations therein for additional information related to formulations, excipients and carriers well known to pharmaceutical chemists.

Binding Proteins/ Antibodies and Methods of Producing Such [0108] Antibodies (binding proteins) used in certain embodiments described herein can be recombinant monoclonal antibodies. In certain embodiments, the binding proteins are fully human monoclonal antibodies, or binding fragments thereof that specifically bind to insulinlike growth factors. Embodiments include binding proteins that specifically bind to insulinlike growth factors and that can reduce tumor growth. Mechanisms by which this can be achieved can include and are not limited to either inhibition of binding of IGF-I/II to its receptor IGF-IR, inhibition of IGF-I/II-induced IGF-IR signaling, or increased clearance of IGF-I/II, therein reducing the effective concentration of IGF-I/II. Certain binding proteins for use in embodiments herein are disclosed in U.S. Patent No. 7,939,637, "Insulin-Like Growth Factor Antibodies and Uses Thereof," to Raeber et al., issued May 10, 2011, and in U.S. Patent Pub. No. 2011/0200607, "Binding Proteins Specific for Insulin-Like Growth Factors and Uses Thereof," to Raeber et al., published August 18, 2011, which are hereby incorporated by reference in their entireties.

[0109] Some embodiments provide a fully human isolated specific binding protein that preferentially binds to insulin-like growth factor-II (IGF-II) with cross-reactivity to insulinlike growth factor I (IGF-I) and neutralizes IGF-I and IGF-II activity. In certain aspects, the binding protein binds to IGF-II with at least 2.5 times greater affinity than to IGF-I. In other aspects, the binding protein binds to with at least 3, at least 4, at least 5, at least 7, at least 10, at least 50, at least 60, at least 100 or at least 150 times greater affinity than to IGF-I.

[0110] In some embodiments, the specific binding protein has an EC5₀ of no more than 15 nM for inhibiting IGF- 1 -dependent IGF-I receptor phosphorylation in NIH3T3 cells expressing IGF-IR ectopically. In some aspects, the specific binding protein has an ECso of no more than 15 nM, no more than 10 nM, or no more than 8 nM for inhibiting IGF-1- dependent IGF-I receptor phosphorylation in NIH3T3 cells expressing IGF-IR ectopically.

[0111] In some embodiments, the specific binding protein has an EC 50 of no more than 5 nM, no more than 4 nM, or no more than 3 nM for inhibiting IGF-II-dependent IGF-I receptor phosphorylation in NIH3T3 cells expressing IGF-IR ectopically.

[0112] In other embodiments, the specific binding protein inhibits greater than 70% of IGF-II dependent proliferation of NIH3T3 cells that express recombinant hIGF-IR with an EC5₀ of no more than 25 nM, no more than 20 nM, no more than 15 nM, or no more than 10 nM. [0113] In other embodiments, the specific binding protein inhibits greater than 70% of IGF-I dependent proliferation of NIH3T3 cells that express recombinant hIGF-IR with an EC5₀ of no more than 40 nM, no more than 30 nM, or no more than 25 nM.

[0114] In certain embodiments, the specific binding protein competes for binding with a monoclonal antibody comprising a variable heavy chain sequence selected from the group consisting of SEQ ID NO.: 2, SEQ ID NO.: 6, SEQ ID NO.: 10, SEQ ID NO. : 14 and SEQ ID NO.: 18, and comprising a variable light chain sequence selected from the group consisting of SEQ ID NO.: 4, SEQ ID NO. : 8, SEQ ID NO. : 12 and SEQ ID NO.: 16.

[0115] Embodiments can include a fully human antibody that binds to IGF-I with a ¾ less than 500 picomolar (pM). In certain embodiments, the antibody can bind with a ¾ less than 450 picomolar (pM). The antibody can, in some embodiments, bind with a ¾ less than 410 picomolar (pM). The antibody can bind with a ¾ of less than 350 μΜ, in certain embodiments. In some embodiments, the antibody can bind with a K_d of less than 300 μΜ. Affinity and/or avidity measurements can be measured by BIACOR®, as described herein.

[0116] Certain embodiments can include a fully human monoclonal antibody that binds to IGF-II with a ¾ of less than 175 picomolar (pM). In some embodiments, the antibody can bind with a ¾ less than 100 picomolar (pM), a ¾ less than 50 picomolar (pM), a ¾ less than 5 picomolar (pM), or a ¾ of less than 2 μΜ.

[0117] In certain embodiments, the specific binding protein can be a fully human monoclonal antibody or an antigen-binding fragment of a fully human monoclonal antibody. The antigen- binding fragment can include fragments such as Fab, Fab' or F(ab')₂ and Fv.

[0118] Certain embodiments comprise fully human monoclonal antibodies 7.251.3 (ATCC Accession Number PTA-7422), 7.34.1 (ATCC Accession Number PTA-7423) or 7.159.2 (ATCC Accession Number PTA-7424), or antigen-binding fragments thereof, which specifically bind to IGF-I/II, as discussed in more detail below.

[0119] In some embodiments the specific binding protein that binds to insulin-like growth factor-II (IGF-II) with cross-reactivity to insulin-like growth factor-I (IGF-I), or antigen- binding fragment thereof can include a heavy chain polypeptide having the sequence of SEQ ID NO.: 6, and a light chain polypeptide having the sequence of SEQ ID NO.: 8.

[0120] In some embodiments, the specific binding protein can include a heavy chain polypeptide having the sequence of SEQ ID NO.: 10, and a light chain polypeptide having the sequence of SEQ ID NO.: 12. [0121] The specific binding protein of some embodiments can include heavy chain polypeptide having the sequence of SEQ ID NO.: 14 and a light chain polypeptide having the sequence of SEQ ID NO.: 16.

[0122] In certain embodiments, the specific binding protein can be in a mixture with a pharmaceutically acceptable carrier.

[0123] In certain embodiments the specific binding protein that binds to insulin-like growth factor-II (IGF-II) with cross-reactivity to insulin-like growth factor-I (IGF-I), or antigen- binding fragment thereof does not bind specifically to IGF-II or IGF-I proteins when said proteins are bound to Insulin Growth Factor Binding Proteins. In certain embodiments the antibody or antigen-binding fragment thereof does not bind specifically to IGF-I or IGF-II ligands when said ligands are bound to insulin growth factor binding protein-3.

[0124] In other embodiments, the fully human monoclonal antibodies, or antigen-binding fragment thereof, bind to insulin-like growth factor-II (IGF-II) with cross-reactivity to insulin-like growth factor-I (IGF-I), and comprise a heavy chain complementarity determining region 1 (CDRl) having the amino acid sequence of "Ser Tyr Tyr Trp Ser" (SEQ ID NO: 21); a heavy chain complementarity determining region 2 (CDR2) having the amino acid sequence of "Tyr Phe Phe Tyr Ser Gly Tyr Thr Asn Tyr Asn Pro Ser Leu Lys Ser" (SEQ ID NO: 22); and a heavy chain complementarity determining region 3 (CDR3) having the amino acid sequence of "He Thr Gly Thr Thr Lys Gly Gly Met Asp Val" (SEQ ID NO: 23).

[0125] In certain embodiments, the fully human monoclonal antibody, or antigen-binding fragment thereof, has a light chain complementarity determining region 1 (CDRl) having the amino acid sequence of "Thr Gly Ser Ser Ser Asn He Gly Ala Gly Tyr Asp Val His" (SEQ ID NO: 24). Antibodies herein can also include a light chain complementarity determining region 2 (CDR2) having the amino acid sequence of "Gly Asn Asn Asn Arg Pro Ser" (SEQ ID NO: 25); and a light chain complementarity determining region 3 (CDR3) having the amino acid sequence of "Gin Ser Phe Asp Ser Ser Leu Ser Gly Ser Val" (SEQ ID NO: 26).

[0126] In some embodiments, the fully human monoclonal antibody, or antigen-binding fragment thereof, can bind to insulin-like growth factor-II (IGF-II) with cross-reactivity to insulin- like growth factor-I (IGF-I), and comprises a heavy chain complementarity determining region 1 (CDRl) having the amino acid sequence of "Ser Tyr Tyr Trp Ser" (SEQ ID NO: 27); a heavy chain complementarity determining region 2 (CDR2) having the amino acid sequence of "Tyr Phe Phe Tyr Ser Gly Tyr Thr Asn Tyr Asn Pro Ser Leu Lys Ser" (SEQ ID NO: 28); and a heavy chain complementarity determining region 3 (CDR3) having the amino acid sequence of "He Thr Gly Thr Thr Lys Gly Gly Met Asp Val" (SEQ ID NO: 29).

[0127] Certain embodiments can include a fully human monoclonal antibody, or antigen- binding fragment thereof, having a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of "Thr Gly Arg Ser Ser Asn He Gly Ala Gly Tyr Asp Val His" (SEQ ID NO: 30); a light chain complementarity determining region 2 (CDR2) having the amino acid sequence of "Gly Asn Ser Asn Arg Pro Ser" (SEQ ID NO: 31); and a light chain complementarity determining region 3 (CDR3) having the amino acid sequence of "Gin Ser Tyr Asp Ser Ser Leu Ser Gly Ser Val" (SEQ ID NO: 32).

[0128] Some embodiments include a fully human monoclonal antibody, or antigen-binding fragment thereof, that binds to insulin-like growth factor-II (IGF-II) with cross-reactivity to insulin- like growth factor-I (IGF- 1), and comprises a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of "Ser Tyr Asp He Asn" (SEQ ID NO: 33); a heavy chain complementarity determining region 2 (CDR2) having the amino acid sequence of "Trp Met Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gin Lys Phe Gin Gly" (SEQ ID NO: 34); and a heavy chain complementarity determining region 3 (CDR3) having the amino acid sequence of "Asp Pro Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val" (SEQ ID NO: 35).

[0129] Certain embodiments include a fully human monoclonal antibody, or antigen-binding fragment thereof, having a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of "Ser Gly Ser Ser Ser Asn He Glu Asn Asn His Val Ser" (SEQ ID NO: 36); a light chain complementarity determining region 2 (CDR2) having the amino acid sequence of "Asp Asn Asn Lys Arg Pro Ser" (SEQ ID NO: 37); and a light chain complementarity determining region 3 (CDR3) having the amino acid sequence of "Glu Thr Trp Asp Thr Ser Leu Ser Ala Gly Arg Val" (SEQ ID NO: 38).

[0130] Some embodiments include a fully human monoclonal antibody, or antigen-binding fragment thereof, that binds to insulin-like growth factor-II (IGF-II) with cross-reactivity to insulin- like growth factor-I (IGF- 1), and comprises a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of "Ser Ser Ser Tyr Tyr Trp Gly" (SEQ ID NO: 81); a heavy chain complementarity determining region 2 (CDR2) having the amino acid sequence of "Gly He Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser" (SEQ ID NO: 82); and a heavy chain complementarity determining region 3 (CDR3) having the amino acid sequence of "Gin Arg Gly His Ser Ser Gly Tip Tip Tyr Phe Asp Leu" (SEQ ID NO: 83).

[0131] Certain embodiments include a fully human monoclonal antibody, or antigen-binding fragment thereof, having a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of "Arg Ala Ser Gin Gly lie Ser Ser Tyr Leu Ala" (SEQ ID NO: 84); a light chain complementarity determining region 2 (CDR2) having the amino acid sequence of "Ala Ala Ser Ser Leu Gin Ser" (SEQ ID NO: 85); and a light chain complementarity determining region 3 (CDR3) having the amino acid sequence of "Gin Gin Ala Asn Asn Phe Pro Phe Thr" (SEQ ID NO: 86).

[0132] Some embodiments include a fully human monoclonal antibody, or antigen-binding fragment thereof, that binds to insulin-like growth factor-II (IGF-II) with cross-reactivity to insulin- like growth factor-I (IGF- 1), and comprises a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of "Ser Ser Ser Asn Tyr Tip Gly" (SEQ ID NO: 87); a heavy chain complementarity determining region 2 (CDR2) having the amino acid sequence of "Gly He Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Arg Ser" (SEQ ID NO: 88); and a heavy chain complementarity determining region 3 (CDR3) having the amino acid sequence of "Gin Arg Gly His Ser Ser Gly Tip Tip Tyr Phe Asp Leu" (SEQ ID NO: 89).

[0133] Certain embodiments include a fully human monoclonal antibody, or antigen-binding fragment thereof, having a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of "Arg Ala Ser Arg Gly He Ser Ser Tip Leu Ala" (SEQ ID NO: 90); a light chain complementarity determining region 2 (CDR2) having the amino acid sequence of "Thr Ala Ser Ser Leu Gin Ser" (SEQ ID NO: 91); and a light chain complementarity determining region 3 (CDR3) having the amino acid sequence of "Gin Gin Ala Asn Ser Phe Pro Phe Thr" (SEQ ID NO: 92).

[0134] Some embodiments provide the use of the specific binding proteins described herein in the preparation of a medicament for the treatment of a breast cancer. In some aspects, the specific binding protein can be a fully human monoclonal antibody. In certain aspects, the binding protein can be mAb 7.251.3 (ATCC Accession Number PTA-7422) or mAb 7.34.1 (ATCC Accession Number PTA-7423) or mAb 7.159.2 (ATCC Accession Number PTA- 7424). In some aspects, the medicament is for use in combination with an aromatase inhibitor or an estrogen receptor antagonist. In some aspects, the medicament is for use in conjunction with or following a conventional surgery.

[0135] Embodiments can include monoclonal antibodies that bind IGF-I/II and affect IGF- I II function. Certain embodiments include fully human anti-IGF-I/II antibodies and anti- IGF-I/II antibody preparations with desirable properties from a therapeutic perspective, including high binding affinity for IGF-I/II, the ability to neutralize in vitro and in vivo, and the ability to inhibit IGF-I/II induced cell proliferation.

[0136] Human antibodies avoid some of the problems associated with antibodies that possess murine or rat variable and/or constant regions. The presence of such murine or rat derived proteins can lead to the rapid clearance of the antibodies or can lead to, the generation of an immune response against the antibody by a subject. In order to avoid the utilization of murine or rat derived antibodies, fully human antibodies can be generated through the introduction of functional human antibody loci into a rodent, other mammal or animal so that the rodent, other mammal or animal produces fully human antibodies.

[0137] Methods of generating fully human antibodies are known in the art and may be through the use of XenoMouse® strains of mice that have been engineered to contain up to but less than 1000 kb-sized germline configured fragments of the human heavy chain locus and kappa light chain locus. See Mendez et al. Nature Genetics 15: 146-156 (1997) and Green and Jakobovits J. Exp. Med. 188:483-495 (1998). The XenoMouse® strains are available from Abgenix, Inc. (Fremont, California, US).

[0138] Human antibodies can also be derived by in vitro methods. Suitable examples include but are not limited to phage display (CAT, Morphosys, Dyax, Biosite/Medarex, Xoma, Symphogen, Alexion (formerly Proliferon), Affimed) ribosome display (CAT), yeast display, and the like.

[0139] Antibodies, as described herein, can be prepared through the utilization of the XenoMouse® technology, as described below. Such mice, then, are capable of producing human immunoglobulin molecules and antibodies and are deficient in the production of murine immunoglobulin molecules and antibodies. An embodiment of transgenic production of mice and antibodies therefrom is disclosed in U.S. patent application Ser. No. 08/759,620, filed Dec. 3, 1996 and International Patent Application Nos. WO 98/24893, published Jun. 11 , 1998 and WO 00/76310, published Dec. 21, 2000, the disclosures of which are hereby incorporated by reference. See also Mendez et al. Nature Genetics 15 : 146- 156 (1997), the disclosure of which is hereby incorporated by reference.

[0140] Through the use of such technology, fully human monoclonal antibodies to a variety of antigens have been produced. Essentially, XenoMouse® lines of mice are immunized with an antigen of interest (e.g. IGF-I/II), lymphatic cells (such as B -cells) are recovered from the hyper-immunized mice, and the recovered lymphocytes are fused with a myeloid-type cell line to prepare immortal hybridoma cell lines. These hybridoma cell lines are screened and selected to identify hybridoma cell lines that produced antibodies specific to the antigen of interest. Antibodies used in embodiments can be produced by multiple hybridoma cell lines that produce antibodies specific to IGF-I/II. The nucleotide and amino acid sequences of the heavy and light chains of such antibodies are included herein.

[0141] In general, antibodies produced by the fused hybridomas can be human IgG2 heavy chains with fully human kappa or lambda light chains. Antibodies described herein possess human IgG4 heavy chains as well as IgG2 heavy chains. Antibodies can also be of other human isotypes, including IgGl . The antibodies can possess high affinities, typically possessing a K_d of from about 10^~6 through about 10^~12 M or below, when measured by solid phase and solution phase techniques. Antibodies possessing a ¾ of at least 10^"11 M can inhibit the activity of IGF-I/II.

Aromatase Inhibitors

[0142] Aromatase inhibitors (AIs) for treatment of breast cancer known in the art can be used in embodiments. Certain embodiments include AIs known to be effective in treatment of breast cancer in postmenopausal women.

[0143] The aromatase inhibitor can be (a) an irreversible steroidal inhibitor, such as exemestane (Aromasin) or (b) a non-steroidal inhibitor, such as anastrozole (Arimidex). The AI can be a non-selective AI, a selective AI, a natural AI or another type of AI. Nonselective AIs include: aminoglutethimide and testolactone (Teslac). Selective AIs include: anastrozole (Arimidex), letrozole (Femara), exemestane (Aromasin), vorozole (Rivizor), formestane (Lentaron), and fadrozole (Afema). Natural AIs include: resveratrol, nicotine, myosmine, zinc, catechin, chalcones, apigenin, eriodictyol, isoliquiritigenin, and mangostin, and other AIs include: 4-hydroxyandrostenedione, l ,4,6-androstatrien-3, 17-dione (ATD), and 4-androstene-3,6, 17-trione ("6-OXO"). In some embodiments, the aromatase inhibitor can comprise anastrozole, letrozole, exemestane, vorozole, formestane, or fadrozole. The aromatase inhibitor can be letrozole (4,4'-((lH-l ,2,4-triazol-l-yl)methylene)dibenzonitrile) in certain embodiments. AIs can be administered by any suitable route of administration to a patient. Doses are administer so as to achieve levels in the blood serum known in the art to be effective.

Estrogen Receptor Antagonists

[0144] Estrogen receptor antagonists for treatment of breast cancer known in the art can be used in embodiments. Certain embodiments include estrogen receptor antagonists known to be effective in treatment of breast cancer in postmenopausal women.

[0145] The estrogen receptor antagonist can be a selective estrogen receptor modulator (SERM) or an estrogen receptor downregulator (ERD) in embodiments. Tamoxifen ((Z)-2- [4-(l ,2-diphenylbut-l-enyl)phenoxy]-N,N-dimethylethanamine) can be used in some embodiments as an estrogen receptor antagonist. Tamoxifen acts as an antagonist of the estrogen receptor in breast tissue via its active metabolite, 4-hydroxytamoxifen. Raloxifene (EVISTA) is another partial agonist SERM that can be used in embodiments. Toremifene (FARESTON) is a SERM with little or no agonist activity that can be used in certain embodiments. Other estrogen receptor antagonists that can be used in certain embodiments include droloxifene and iodoxyfene.

[0146] Fulvestrant ((7a,17 )-7-{ 9-[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl}estra-l,3,5 (10) -triene-3,17-diol) is a estrogen receptor antagonist (with no agonist activity) that can be used in certain embodiments. Fulvestrant is categorized as an estrogen receptor downregulator (ERD) and works to down-regulate and degrade the estrogen receptor.

[0147] In certain embodiments, the estrogen receptor antagonist can be toremifene, raloxifene, fulvestrant or tamoxifen. The estrogen receptor antagonist can be fulvestrant or tamoxifen in some embodiments.

[0148] Routes of administration include oral, parenteral (e.g., intravenous, subcutaneous, intramuscular), intranasal, buccal, topical, rectal, aerosol, ophthalmic, pulmonary, transmucosal, transdermal, vaginal, otic, nasal, and topical administration. In addition, by way of example only, parenteral delivery includes intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intralymphatic, and intranasal injections. In certain embodiments, a compound as described herein is administered in a systemic manner. In certain other embodiments, a compound as described herein is administered in a local rather than systemic manner. EXAMPLES

[0149] All references cited herein, including patents, patent applications, papers, text books, and the like, and the references cited therein, to the extent that they are not already, are hereby incorporated herein by reference in their entirety. While this invention has been particularly shown and described with references to embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Example 1 - In Vitro Testing

Cell Culture

[0150] MCF-7/AC-1 cells were used for in vitro testing and in xenografts in vivo. MCF- 7/AC-l cells are a recognized model for estrogen dependent breast cancer, and the cells stably express human aromatase and synthesize estrogens upon supplementation with the androgen precursor, androstenedione. The MCF-7/AC-1 cells were employed as a model because of their responsiveness to estrogen and sensitivity to clinical estrogen-targeting agents, including the selective estrogen receptor modulator, tamoxifen (in its active form of 4-hydroxy tamoxifen), the aromatase inhibitor, letrozole, and the estrogen receptor antagonist (having no agonist activity), fulvestrant.

[0151] A line of MC-7/AC-1 cells was engineered to express human IGF-I (MCF-7/AC- 1/IGF-I cells), and another line was engineered to express human IGF- II (MCF-7/AC-1/IGF- II cells) by infecting cells with self-inactivation lentiviruses carrying the human genes IGF1 and IGF2, respectively and then selecting for the stable overexpression of the ligands. Expression of human IGF-I/II ligands in the cell lines was used to mimic the autocrine environment of breast cancer cells. The cells were maintained in DMEM (Dulbecco's Modified Eagle Medium) with 10% FBS (fetal bovine serum), 1% penicillin/streptomycin solution, and 750 μg/mL G418 (geneticin). The culture medium was changed twice weekly.

[0152] MEDI-573, a monoclonal antibody (mAb) 7.159.2 (ATCC Accession Number PTA- 7424), is a fully human \gG2k monoclonal antibody molecule composed of two identical heavy chains and two identical light chains, with an overall molecular weight of approximately 151 kDa.

[0153] The effects of MEDI-573 alone and in combination with letrozole, 4- hydroxytamoxifen (for in vitro use from Sigma), or fulvestrant were determined in the postmenopausal cell model using MTS cell proliferation assays and the effects on the IGF pathway were analyzed by western blotting by quantifying proteins being expressed in the pathway. (Results not shown.) (It should be noted that 4-hydroxytamoxifen is for in vitro use and is the equivalent to tamoxifen for in vivo use; 4-Hydroxytamoxifen is tamoxifen's active metabolite.)

Antibody

[0154] MEDI-573 (mAb 7.159.2, ATCC Accession Number PTA-7424) was generated previously as a fully human ¾02λ monoclonal antibody using XenoMouse® (Abgenix) technology. MEDI-573 was isolated from mice immunized alternately with soluble recombinant human IGF-I and IGF-II coupled to keyhole limpet hemocyanin. Hybridoma supernatants were screened for binding to IGF-I and IGF-II and the lack of cross-reactivity to human insulin.

[0155] As reported in Gao et al., Cancer Res, 2011, 71(3): 1029-40, MEDI-573 is a fully human antibody that neutralizes both IGF-I and IGF-II and inhibits IGF signaling through both the IGF-1R and IR-A pathways. MEDI-573 was shown to block the binding of IGF-I and IGF-II to IGF-1R or IR-A, leading to the inhibition of IGF- induced signaling pathways and cell proliferation. MEDI-573 was shown to significantly inhibit the in vivo growth of IGF-I- or IGF-II-driven tumors. Pharmacodynamic analysis demonstrated inhibition of IGF- 1R phosphorylation in tumors in mice dosed with MEDI-573, indicating that the antitumor activity was mediated, at least in part, via inhibition of IGF-1R signaling pathways. Finally, MEDI-573 significantly decreased 18F-fluorodeoxyglucose (18F-FDG) uptake in IGF-driven tumor models.

MTS Proliferation Assay

[0156] The effect of the various drugs (MEDI-573, letrozole (Femara, CGS 20267, Novartis Pharma), fulvestrant, or 4-hydroxytamoxifen) and hormones (androstenedione and insulin) on MCF-7/AC-1/IGF-I and MCF-7/AC-l/IGF-II cellular growth was examined using a MTS (3- (4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetra-zolium) proliferation assay (CellTiter 96 Aqueous; Promega). Briefly, cells that were grown in regular media were transferred to IMEM (Improved minimum essential media) containing 5% charcoal-stripped serum and 1 % penicillin/streptomycin (charcoal-stripped serum in medium, CSSM) for 72 hours. This medium was used for all cell growth assays. The cells were detached from their flask using trypsin and seeded in 96-well plates at 1.0 x 10³/mL (100 μΕ) on day 0. Twenty-four hours later, several concentrations of the drugs were added at doses indicated in the Figures 1-3. The cells were incubated for 6 days in the absence and presence of the tested drugs and hormones. At the end of the treatment, the MTS dye reduction was assessed as per the product information label. Proliferation was calculated as a percentage of the nondrug-treated controls. Experiments were done in at least triplicate.

[0157] To evaluate the effect of combination treatment with MEDI-573 and 4- hydroxytamoxifen, letrozole or fulvestrant, the method of Chou and Talalay was used to determine synergy as described previously (Chou and Talalay, Adv Enzyme Regul, 1984, 22:27-55). Median effect analysis was done using CalcuSyn software (Biosoft). Mean values of the combination index (CI) at the effected fractions of 50% (Fa50) were determined. A CI value significantly less than 1 indicated synergism, a CI not significantly different from 1 indicated addition, and a CI significantly higher than 1 indicated antagonism.

[0158] In both MCF-7/AC-1/IGF-I and MCF-7/AC-l/IGF-II cell lines, hormonal therapy (letrozole, fulvestrant, or 4-hydroxytamoxifen) in combination with MEDI-573 had enhanced antitumor activity in vitro. Surprisingly, the anti-proliferative effects of MEDI-573 in MCF- 7/AC-l/IGF-I or MCF-7/AC-l/IGF-II cells were profoundly synergistic in combination with letrozole, 4-hydroxytamoxifen or fulvestrant, with combination index (CI) values less than 0.05 at the 50% fraction affected by median effect analysis. See Figures ID and 2D.

[0159] FIG. 1 shows results of MTS proliferation assays with treatment of MCF-7/AC- 1/IGF-I cells with letrozole, 4-hydroxytamoxifen, fulvestrant, MEDI-573 or combinations of MEDI-573 with the various chemical therapies. FIG. 1A shows the results of MTS proliferation assays with treatments of MCF-7/AC-1/IGF-I cells including 4- hydroxytamoxifen, FIG. IB shows the results of MTS proliferation assays with treatments including fulvestrant (ICI), and FIG. 1C shows the results of MTS proliferation assays with treatments including letrozole. It is clear in all of Figures 1A-1C that the combination of the antibody and the drug, whether 4-hydroxytamoxifen, fulvestrant, or letrozole, provided significantly improved results as compared to MCF-7/AC-1/IGF-I cells treated with drug alone or with MEDI-573 alone. FIG. ID shows the calculated combination indices for the combinations of MEDI-573/letrozole, MEDI-573/4-hydroxytamoxifen, and MED- 573/fulvestrant in treated MCF-7/AC-1/IGF-I cells, indicating that the observed effects of the combinations were synergistic. [0160] Experiments that yielded the results shown in FIG. 1 were carried out as follows. Cells were cultured in IMEM steroid-reduced medium without phenol red for 2 days before plating. MTS cell proliferation was measured using an MTS assay after 6 days of treatment, as described above. Cell proliferation was expressed as the percentage of the cells compared with the control wells (1 nM androstenedione treated cells).

[0161] MCF-7/AC-1/IGF-I cell proliferation was assessed following treatment with various concentrations of MEDI-573, 4-hydroxytamoxifen, letrozole and fulvestrant either as single agents or in combination with MEDI-573 at a fixed ratio. Treatments with single agents had only modest antiproliferative effects, the combination treatments had significant antiproliferative effects (FIGS. 1A-1C). In comparison to the single agent anti-proliferative effects, the combination of MEDI-573 with letrozole, 4-hydroxytamoxifen or fulvestrant was strongly synergistic at the 50% fraction affected (P < 0.001 ; Fig. ID); columns, mean; bars, SD (standard deviation). A CI value significantly less than 1 indicated synergism, a CI not significantly different from 1 indicated addition, and a CI significantly higher than 1 indicated antagonism.

[0162] FIG. 2 shows results of MTS proliferation assays with treatment of MCF-7/AC- 1/IGF-II cells with letrozole, 4-hydroxytamoxifen, fulvestrant, MEDI-573 or combinations of MEDI-573 with the various chemical therapies. FIG. 2A shows the results of MTS proliferation assays with treatments of MCF-7/AC-l/IGF-II including 4-hydroxytamoxifen, FIG. 2B shows the results of MTS proliferation assays with treatments including fulvestrant (ICI), and FIG. 2C shows the results of MTS proliferation assays with treatments including letrozole. It is clear in all of Figures 2A-2C that the combination of the antibody and the drug, whether 4-hydroxytamoxifen, fulvestrant, or letrozole, provided significantly improved results as compared to MCF-7/AC-l/IGF-II cells treated with drug alone or MEDI-573 alone. FIG. 2D shows the calculated combination indices for the combinations of MEDI- 573/letrozole, MEDI-573/4-hydroxytamoxifen, and MED-573/fulvestrant in treated MCF- 7/AC-l/IGF-II cells indicating there was a synergistic effect.

[0163] Experiments that yielded the results shown in FIG. 2 were carried out as follows. Cells were cultured in IMEM steroid-reduced medium without phenol red for 2 days before plating. Cell proliferation was measured using the MTS proliferation assay, described above.

[0164] MCF-7/AC-l/IGF-II cell proliferation was assessed by treatment with various concentrations of MEDI-573, 4-hydroxytamoxifen, letrozole and fulvestrant either as single agents or in combination with MEDI-573 at a fixed ratio. Treatments with single agents had modest antiproliferative effects, the combination treatments had significant anti-proliferative effects (FIGS. 2A-2C). In comparison to the single agent anti-proliferative effects, the combination of MEDI-573 with letrozole, 4-hydroxytamoxifen or fulvestrant was strongly synergistic at the 50% fraction affected (P < 0.001 ; Fig. 2D); columns, mean; bars, standard deviation (SD). A CI value significantly less than 1 indicated synergism, a CI not significantly different from 1 indicated addition, and a CI significantly higher than 1 indicated antagonism.

[0165] As shown in FIG. 3, the antiproliferative activity of MEDI-573 could not be overcome by physiologically relevant doses of insulin in MCF-7/AC-1/IGF-I and MCF- 7/AC-l/IGF-II cells in MTS cell proliferation assays. FIG. 3A is a graph with results of MTS proliferation assays performed with MCF-7/AC-1/IGF-I cells treated with the combination of 4-hydroxytamoxifen and MEDI-573; FIG. 3C with the combination of letrozole and MEDI- 573; and FIG. 3E with the combination of fulvestrant and MEDI-573. Similarly, FIG. 3B is a graph with results of MTS proliferation assays performed with MCF-7/AC-l/IGF-II cells treated with the combination of 4-hydroxytamoxifen and MEDI-573; FIG. 3D with the combination of letrozole and MEDI-573; and FIG. 3F with the combination of fulvestrant and MEDI-573.

[0166] Experiments that yielded the results shown in FIG. 3 were carried out as follows. Cells were cultured in IMEM steroid-reduced medium without phenol red for 2 days before plating. Then different concentrations of combined MEDI-573 and hormonal agents were added to MCF-7/AC-1/IGF-I and MCF-7/AC-l/IGF-II cells along with physiological (basal systemic concentrations of 40-100 pM in humans) or superphysiological relevant concentrations. After 6 days of treatment, cell proliferation was measured using an MTS assay, as described above.

[0167] It is known that increase in synthesis and/or bioavailability of IGFs can be mediated by hyperinsulinemia. These tests were used to determine if the observed effects of MEDI-573 in cell proliferation could be overcome by physiologically relevant doses of insulin. The results demonstrated that insulin signaling through the insulin receptor was not sufficient for the cells to demonstrate resistance to hormonal therapy. As such, MEDI-573, functions well in combination with the hormonal therapies.

Example 2 - In Vivo Testing [0168] To examine the antiproliferative effects of MEDI-573, letrozole, or letrozole in combination with MEDI-573, in vivo xenograft studies were conducted in athymic nude mice bearing MCF-7/AC-1/IGF-I or MCF-7/AC-l/IGF-II xenografts (FIG. 4 and FIG. 5).

[0169] As a postmenopausal intratumoral aromatase mice xenograft model, female ovariectomized BALB/c athymic nude mice 5 to 6 weeks of age were obtained from Harlan Laboratories. The animals were housed in a pathogen-free environment under controlled conditions of light and humidity and received food and water. All animal studies were carried out according to the guidelines approved by the Animal Care Committee of the Mayo Clinic, Rochester, MN. Animals were allowed to acclimatize for 48 hours after shipment before tumor inoculation was done. For inoculation, subconfluent MCF-7/AC-1/IGF-I or MCF- 7/AC-l/IGF-II cells were suspended in Matrigel (10 mg/mL) at 2.5 x 10⁷ cells/mL. Each mouse was injected subcutaneously with 100 μΕ of cell suspension on each flank. Tumors were measured weekly with calipers, and volumes were calculated with the formula 4/3π x ri² x r₂ (ri < r₂), in which ri is the smaller radius. Treatments began when the tumors reached a measurable size (250-300 mm³ at about 4-5 weeks). Mice were randomized to treatment groups using JMP (SAS) so that the mean tumor volumes between the groups were not significantly different at the start of treatment.

[0170] Mice received subcutaneous injection with vehicle and nonsteroidal aromatase inhibitor letrozole (10 μg/day), which was prepared as a suspension in 0.3% hydroxypropyl cellulose; MEDI-573 was administered at 60 mg/kg twice weekly intraperotineally (IP) and was prepared in water. The mice treated with the combination of letrozole and MEDI-573 received each at their full doses. All animal groups were supplemented with androstenedione (100 μg/day) for the duration of the experiment. All other treatments were given once daily for 28 days continuously.

[0171] After 28 days of treatment, one of the two flank tumors was surgically excised from each tumor-bearing animal. Animals were then maintained and observed until the study end (300% tumor growth from treatment baseline), at which point the remaining tumor was resected at the time of animal sacrifice. After each resection, tumors were carefully excised from mouse tissue with portions frozen in OCT medium (Tissue-Tek) or placed in formalin for further analyses. Differences between the groups were analyzed by ANOVA with multiple comparison posttests using Prism (Graph Pad). For comparisons between multiple samples, ANOVA with Tukey's multiple comparisons posttest was used. For P values, differences less than 0.001 were considered extremely significant (***); 0.001 to 0.01 very significant (**); 0.01 to 0.05 significant (*). P values more than 0.05 were considered not significant (ns).

[0172] At the end of 28 days of treatment, the tumors in all of the control group had at least doubled in size, while the tumors in the groups treated with letrozole alone (p<0.005 vs. control) or the combination of letrozole and MEDI-573 group (p<0.05) demonstrated significant growth inhibition when compared to the controls.

[0173] FIG. 4 is a graph of data for % tumor volume change with MCF-7/AC-1/IGF-I xenografts in experiments conducted in vivo with a treatment of letrozole or MEDI-573 and letrozole over time. Tumor volume was measured at the intervals indicated and expressed as percentage change in tumor volume relative to start of treatment (baseline, day 0). FIG. 5 is a graph of data for % tumor volume change with MCF-7/AC-l/IGF-II xenografts in experiments conducted in vivo with a treatment of letrozole or MEDI-573 and letrozole over time.

[0174] Treatment with letrozole in combination with MEDI-573 was observed to have positive effects in MCF-7/AC-l/IGF-II xenografts in vivo.

[0175] In comparison to MCF-7/AC-l/IGF-II, the MCF-7/AC-1/IGF-I tumor model showed only modest response to letrozole/MEDI-573 in vivo. Not to be bound by theory, but this may be have been due to the low cross-reactivity of MEDI-573 to mouse IGF-I. Mouse IGF-I is normally expressed in the mouse model that was used for in vivo testing, and the mouse IGF-I is known to have nearly equivalent biological activity to human IGF-I with respect to the activation of human IGF-IR and is not inhibited in this activity by MEDI-573 (MEDI-573 binding affinity for mIGF-I is KD=2,000 pmol/L compared to its binding affinity for hIGF-I which is KD=294 pmol/L).

[0176] The combination of letrozole with MEDI-573 had significant anti-tumor growth effects compared to untreated MCF-7/AC-1/IGF-I or MCF-7/AC-l/IGF-II tumors.

[0177] To the extent that the terms "containing," "including," "includes," "having," "has," "with," or variants thereof are used in either the detailed description and the claims, such terms are intended to be inclusive in a manner similar to the term "comprising."

[0178] Further, in the discussion and claims herein, the term "about" indicates that the values listed may be somewhat altered, as long as the alteration does not result in nonconformance of the process or structure to the illustrated embodiment. [0179] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the present teachings are approximations, the numerical values set forth in the specific examples are reported as precisely as possible, any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Moreover, all ranges disclosed herein are to be understood to encompass any and all sub-ranges subsumed therein. For example, a range of "less than 10" can include any and all sub-ranges between (and including) the minimum value of zero and the maximum value of 10, that is, any and all sub-ranges having a minimum value of equal to or greater than zero and a maximum value of equal to or less than 10, e.g., 1 to 5. In certain cases, the numerical values as stated for the parameter can take on negative values. In this case, the example value of range stated as "less than 10" can assume values as defined earlier plus negative values, e.g., -1, -1.2, -1.89, -2, -2.5, -3, -10, -20, and -30, etc.

Claims

What is claimed:

1. A method of treating breast cancer in a subject comprising:

co-administering to the subject in need thereof a therapeutically effective amount of

(a) an isolated antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain polypeptide having the amino acid sequence of SEQ ID NO: 6 and a light chain polypeptide, and

(b) an aromatase inhibitor or an estrogen receptor antagonist.

2. The method of claim 1 , wherein the light chain polypeptide has the amino acid sequence of SEQ ID NO:8.

3. The method of claim 1, wherein the breast cancer is an estrogen receptor positive breast cancer or an estrogen receptor unknown breast cancer.

4. The method of claim 1, wherein the subject is a postmenopausal woman.

5. The method of claim 1, wherein the aromatase inhibitor comprises aminoglutethimide, testolactone, anastrozole, letrozole, exemestane, vorozole, formestane, fadrozole, resveratrol, nicotine, myosmine, zinc, catechin, chalcones, apigenin, eriodictyol, isoliquiritigenin, mangostin, 4-hydroxyandrostenedione, l,4,6-androstatrien-3,17-dione, or 4-androstene-3,6,17-trione.

6. The method of claim 1, wherein the aromatase inhibitor comprises anastrozole, letrozole, exemestane, vorozole, formestane, or fadrozole.

7. The method of claim 1, wherein the aromatase inhibitor is letrozole.

8. The method of claim 1, wherein the estrogen receptor antagonist is toremifene, raloxifene, fulvestrant or tamoxifen.

9. The method of claim 1, wherein the estrogen receptor antagonist is fulvestrant or tamoxifen.

10. The method of claim 1 , wherein the antibody is a recombinant monoclonal antibody.

11. The method of claim 1 , wherein the antibody is a human monoclonal antibody.

12. The method of claim 1, wherein the antibody does not bind specifically to IGF-I or IGF- II ligands when said ligands are bound to insulin growth factor binding protein-3.

13. A method of treating breast cancer in a subject comprising:

co-administering to the subject in need thereof a therapeutically effective amount of

(a) an isolated antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain polypeptide and a light chain polypeptide, wherein the heavy chain polypeptide comprises a heavy chain complementarity determining region (CDR) 1 having the amino acid sequence of SEQ ID NO:33, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:34, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:35, and wherein the light chain polypeptide comprises a light chain CDR1 having the amino acid sequence of SEQ ID NO:36, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 37, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:38, and

(b) an aromatase inhibitor or an estrogen receptor antagonist.

14. The method of claim 13, wherein the aromatase inhibitor is letrozole.

15. The method of claim 13, wherein the estrogen receptor antagonist is fulvestrant or tamoxifen.

16. A method of treating breast cancer in a subject comprising:

co-administering to the subject in need thereof a therapeutically effective amount of

(a) an isolated antibody or antigen-binding fragment thereof, wherein the antibody has the amino acid sequence of the antibody produced by hybridoma cell line 7.159.2 (ATCC Accession Number PTA-7424), and

(b) an aromatase inhibitor or an estrogen receptor antagonist.

17. The method of claim 16, wherein the aromatase inhibitor is letrozole.

18. The method of claim 16, wherein the estrogen receptor antagonist is fulvestrant or tamoxifen.

19. A pharmaceutical composition for the treatment of breast cancer comprising:

a therapeutically effective amount of (a) an isolated antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain polypeptide having the amino acid sequence of SEQ ID NO: 6 and a light chain polypeptide, and (b) an aromatase inhibitor or an estrogen receptor antagonist; and

a pharmaceutically acceptable carrier.

20. The pharmaceutical composition of claim 19, wherein the light chain polypeptide has the amino acid sequence of SEQ ID NO:8.

21. The pharmaceutical composition of claim 19, wherein the aromatase inhibitor comprises aminoglutethimide, testolactone, anastrozole, letrozole, exemestane, vorozole, formestane, fadrozole, resveratrol, nicotine, myosmine, zinc, catechin, chalcones, apigenin, eriodictyol, isoliquiritigenin, mangostin, 4-hydroxyandrostenedione, l,4,6-androstatrien-3,17-dione, or 4- androstene-3,6,17-trione.

22. The pharmaceutical composition of claim 19, wherein the aromatase inhibitor is letrozole.

23. The pharmaceutical composition of claim 19, wherein the estrogen receptor antagonist is fulvestrant or tamoxifen

24. A pharmaceutical composition for the treatment of breast cancer comprising:

a therapeutically effective amount of (a) an isolated antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain polypeptide and a light chain polypeptide having the amino acid sequence of SEQ ID NO:8, and (b) an aromatase inhibitor or an estrogen receptor antagonist; and

a pharmaceutically acceptable carrier.

25. The pharmaceutical composition of claim 24, wherein the heavy chain polypeptide has the amino acid sequence of SEQ ID NO: 6.

26. A pharmaceutical composition for the treatment of breast cancer comprising:

a therapeutically effective amount of (a) an isolated antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain polypeptide and a light chain polypeptide, wherein the heavy chain polypeptide comprises a heavy chain complementarity determining region (CDR) 1 having the amino acid sequence of SEQ ID NO:33, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:34, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:35, and wherein the light chain polypeptide comprises a light chain CDR1 having the amino acid sequence of SEQ ID NO:36, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 37, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:38, and (b) an aromatase inhibitor or an estrogen receptor antagonist; and

a pharmaceutically acceptable carrier.

27. A pharmaceutical composition for the treatment of breast cancer comprising:

a therapeutically effective amount of (a) an isolated antibody or antigen-binding fragment thereof, wherein the antibody has the amino acid sequence of the antibody produced by hybridoma cell line 7.159.2 (ATCC Accession Number PTA-7424), and (b) an aromatase inhibitor or an estrogen receptor antagonist; and

a pharmaceutically acceptable carrier.

28. A method of treating breast cancer in a subject comprising:

co-administering to the subject in need thereof a therapeutically effective amount of

(a) an isolated antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain polypeptide and a light chain polypeptide having the amino acid sequence of SEQ ID NO:8, and

(b) an aromatase inhibitor or an estrogen receptor antagonist.

29. The method of claim 28, wherein the heavy chain polypeptide has the amino acid sequence of SEQ ID NO:6.

30. A method of treating breast cancer in a subject comprising:

co-administering to the subject in need thereof a therapeutically effective amount of

(a) an isolated antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain polypeptide having the amino acid sequence of SEQ ID NO: 6 and a light chain polypeptide having the amino acid sequence of SEQ ID NO:8, and

(b) an aromatase inhibitor or an estrogen receptor antagonist.

31. Use of a composition for the treatment of breast cancer, wherein the composition comprises a therapeutically effective amount of (a) an isolated antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain polypeptide having the amino acid sequence of SEQ ID NO: 6 and a light chain polypeptide, and (b) an aromatase inhibitor or an estrogen receptor antagonist for the manufacture of a medicament for the treatment and/or prevention of breast cancer.