WO2010138362A1 - Combination therapy using an igf-1r specific antibody with an anti-egfr specific antibody and/or a cytotoxic agent - Google Patents

Abstract

Disclosed is a synergistic therapeutic combination of anti-cancer compounds which comprises a) an IGF-IR inhibitor, and b) one of a cytotoxic agent or an EGFR inhibitor which binds to the epidermal growth factor receptor (EGFR) and blocks the ability of epidermal growth factor (EGF) to initiate receptor activities which results in tumor growth inhibition A preferred combination includes cetuximab MK-0646.

Description

TITLE OF THE TNVENTION

COMBINATION THERAPY USING AN IGF-IR SPECIFIC ANTIBODY WITH AN ANTI-

EGFR SPECIFIC ANTIBODY AND/OR A CYTOTOXIC AGENT

STATEMENT REGARDING FEDERALLY-SPONSORED R&D

Some part of this work was supported by NIH Grant No. 1 ROl DEOl 7982-01 Al .

FILED OF THE INVENTION

The present invention relates to methods and compositions for enhancing anti- tumor activity in a mammal. More particularly, the invention is concerned with combinations comprising an antibody that specifically binds to human Insulin Like-Growth Factor 1 receptor (IGF-IR) with an antibody that binds to Epidermal Growth Factor Receptor (EGFR) e.g., cetuximab or a cytotoxic compound such as methotrexate. In particular, the invention relates to combination therapy for treating EGFR mediated cancers via administration of an IGF-IR antibody and a tyrosine kinase inhibitor, particularly cetuximab and/or methotrexate. The methods and the pharmaceutical compositions comprising said combinations or agents can result in superior tumor cell proliferation inhibition than that observed relative to the use of each individual therapeutic agent, yielding more effective treatment than found by administering an individual component alone. A particular aspect provides for the treatment EGFR inhibitor resistant cancers.

BACKGROUND OF THE INVENTION

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer in developed countries, and of the 44,000 annual cases reported in the United States approximately 11 ,000 will result in an unfavorable outcome (Landis, S. H. et al., "Cancer statistics," CA Cancer J Clin. 49, 8-31 (1999); Parkin, D. M. et al., "Global cancer statistics," CA Cancer J Clin. 49, 33-64 (1999). They are characterized by early, predominantly lymphatic metastatic spread. The five year survival time of the patient drops dramatically if lymph node metastases are present at the time of diagnosis. Overexpression of the epidermal growth factor receptor (EGFR, ErbBl, HERl), a receptor tyrosine kinase, is a hallmark of many epithelial tumors and particularly of head and neck squamous cell carcinomas. Indeed, tumor progression, early metastatic spread and poor prognosis of HNSCC are all associated with EGFR expression.

After ligand-stimulation and internalization of EGFR, the receptor either recycles back to the plasma membrane, or is directed to the lysosomal compartment for degradation. Down regulation of EGFR due to internalization and subsequent lysosomal degradation is one mechanism that is targeted by diverse anti-EGFR strategies. Ligand-binding results in EGF receptor dimerization and autophosphorylation of its cytoplasmic COOH-terminal domain. The major phosphorylation site is tyrosine 1173. Activated, i.e. phosphorylated, EGFR can further activate different signaling cascades such as the MAPK (mitogen activated protein kinase) - or STAT (signal transducer and activator of transcription)~pathways, leading to transcription of EGFR-dependent genes. Since EGFR has this high impact on tumor progression and five-year survival of the patient, large branch of current research has been focused on the development of inhibitors that block EGFR function. The current protocol for treatment comprises the following two approaches: (1) blocking ligand binding to the extracellular domain using monoclonal antibodies (mAbs); and (2) inhibition of the intracellular domain TK activity. Small molecule inhibitors such as AG1478 or Gefitinib (Iressa^®) target the intracellular kinase domain of the receptor by competing with ATP, whereas monoclonal antibodies interact with the extracellular ligand binding site to, supposedly, block ligand stimulation. One of the most prominent biologic agents, approved for the treatment of advanced head and neck cancer is cetuximab (Erbitux®), a chimeric monoclonal antibody, directed against the extracellular ligand binding site of the EGF receptor. Binding of cetuximab to EGFR blocks binding of the natural ligands (TGF-α, EGF) and results in down regulation of EGFR due to antibody-induced receptor internalization and subsequent degradation. R. Mandic et al., FEBS Letters, 580:4793-4800 (2006); N. Kamata et al, Cancer Res., 46: 1648-1653 (1986).

The major chemotherapeutic agent used in the treatment of HNSCC is cisplatin. However, cisplatin resistance has been reported. Indeed, reduced cisplatin sensitivity of head and neck squamous cell carcinoma cell lines correlates with mutations affecting the COOH-terminal nuclear localization signal of p53

Likewise, when looking at the recent literature related to cetuximab, one can find examples of tumor cell lines particularly of NSCLC cell lines that after treatment with cetuximab do not show reduction but rather induction of receptor phosphorylation. As well, Raben et al., infra, while evaluating the effect of cetuximab on growth, cell cycle distribution, and downstream intracellular signaling molecules in a panel of NSCLC cancer cell lines observed that Cetuximab alone inhibited the in vitro growth of some but not all EGFR-expressing NSCLC cell lines in a dose-dependent manner. D Raben et al., Clinical Cancer Research, 11 :795-805 (2005). Several other studies have also reported failure of treatment with Cetuximab. For example, Rogerio C. Lilenbaum, Clinical Cancer Research, 12: 4432s-4435s (2006) expresses doubts relative to EGFR immunohistochemical expression as being an accurate predictor of benefit from cetuximab or tyrosine kinase inhibitors. See also Raez L, Lopes G, Lilenbaum R., Clinical responses to gefitinib after failure of treatment with cetuximab in advanced non-small cell lung cancer. J Clin Oncol 23:4244-5 (2005); and R. Mandic, supra.

Cancer is a generic name for a wide range of cellular malignancies characterized by unregulated growth, lack of differentiation, and the ability to invade local tissues and metastasize. Most agents that induce apoptosis in cancer cells (e.g. Doxorubicin and Vincristine) are extremely toxic and cause a number of undesirable side effects. The toxicity associated with these therapies is a result of the non-specific interaction of the drug with the DNA of noncancerous cells (e.g. intestinal and red blood cells). In order to circumvent such undesirable side effects, more selective compounds are being sought that inhibit one or more signaling proteins, growth factors and/or receptors involved in cancer cell proliferation. Current research demonstrates that not only are patient populations genetically heterogeneous, but even tumors from the same tissue or organ, are heterogeneous on the cellular and/or molecular level. As one consequence, the prognosis and/or responsiveness to therapy of each patient may differ. This unpredictability confounds treatment selection and may expose patients to the risks and discomforts of unneeded therapies. As such, a single selective therapy will not work in all cases. As a consequence of the above mentioned heterogeneity there is a continual need for improved cancer treatments, preferably combination therapeutics comprising a plurality of cancer drugs including cytotoxic agents. The present invention aim to provide an effective combination therapeutic that can be used to circumvent the EGFR blockade or treat patients that may be resistant to treatment with an EGFR inhibitor. The present invention aims to provide a combination treatment for HNSCC by combining an IGF-IR specific antagonist with an EGFR inhibitor and/or a cytotoxic agent that may allow for a more effective and less toxic therapeutical regimen than the conventional drugs used for such disorders or tumors.

SUMMARY OF THE INVENTION

Preventive or therapeutic agents of which the active ingredient is an anti-ΪGF-lR antibody such as MK-0646 is known. However, it has not been shown that synergistic effects can be obtained by the combination of MK-0646 antibody with a cytotoxic agent such as methotrexate (MTX) or an EGFR specific antibody such as Cetuximab in the treatment of EGFR mediated diseases. The proposed combination therapeutic agent(s) described herein can also be used to treat other cancers that exhibit EGFR inhibitor resistance or demonstrate EGFR blockade. Relative to the prior art, this invention provides combination therapeutic agents for the treatment of cancer, exemplified by HNSCC in mammalian subjects. The proposed combination therapeutic of the invention comprises the IGF-IR monoclonal antibody described in US Patent No.7, 241, 444 combined with an EGFR inhibitor, preferably a monoclonal antibody that is specific for EGFR such as Cetuximab, described is US Patent No. 6,217,866 or a chemotherapeutic agent, preferably methotrexate (MTX). The combination therapeutics detailed herein is more efficacious in treating HNSCC than the use of each of the EGFR or the chemotherapeutic being administered alone. As a result, the combination therapeutic agents of this invention show enhanced in vitro profiles when compared to compounds of the prior art. As well, the combination therapeutic agent show enhanced anti-tumor activity relative to the use of the individual compound by itself in treating HNSCC.

Therapy using an antibody of the present invention can be combined with therapy using another antibody. For example, an antibody to IGF-IR (MK-0646) can be administered to a subject in need thereof along with, prior to, or following administration of an antibody to EGFR. Such combined antibody therapy can be further combined with administration of one or more therapeutic agents (e.g., chemotherapeutics, taxanes, platinums, and/or methotrexate). . Radiotherapy can also be combined with the combination therapeutic agents of the invention. One skilled in the art would adapt the form of radiotherapy to the specific disorder. Suitable chemotherapeutic agents are described in REMINGTON'S

PHARMACEUTICAL SCIENCES₅ 19th Ed. (Mack Publishing Co. 1995), and in GOODMAN AND OILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th Ed. (MacMillan Publishing Co. 1985), as well as revised editions of these publications. Other suitable chemotherapeutic agents, such as experimental drugs, are known to those of skill in the art.

A combination therapeutic agent is a compound or composition effective in ameliorating a pathological condition attendant IGF-IR and/or EGFR expressing cells. An illustrative example of a combination therapeutic agent includes an anti-cancer compound such as an anti-IGF-lR antibody and an EGFR specific antibody such as Cetuximab. Another example includes the combination of an anti-IGF-lR antibody such as MK- 0646 and MTX. An anti-cancer or cytotoxic agent is a compound or composition effective in inhibiting or arresting the growth of an abnormally growing cell. A pharmaceutically effective amount of an anti-cancer compound is an amount administered to an individual sufficient to cause inhibition or arrest of the growth of an abnormally growing cell. Illustrative examples of anti-cancer compounds include: bleomycin, carboplatin, chlorambucil, cisplatin, colchicine, cyclophosphamide, daunorubicin, dactinomycin, diethylstilbestrol doxorubicin, etoposide, 5- fluorouracil, floxuridine, melphalan, methotrexate, mitomycin, 6-mercaptopurine, teniposide, 6- thioguanine, vincristine and vinblastine, preferably methotrexate. Further examples of anticancer compounds and therapeutic agents are found in The Merck Manual of Diagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway, NJ. and Sladek et al. Metabolism and Action of Anti-Cancer Drugs, 1987, Powis et al. eds., Taylor and Francis, New York, N. Y.

In one embodiment of the invention, a pharmaceutically effective amount of an antibody of the invention (MK-0646) inhibits cell proliferation or induces apoptosis by contact with a target cell or with target cells expressing the EGFR. A pharmaceutically effective amount or quantity of an antibody recognizing IGF-IR or the hybrid receptor is an amount administered to an individual sufficient to cause a desired effect. As used herein, the terms "pharmaceutically effective amount" and "therapeutic quantity" are synonymous. Desired effects of administration of a pharmaceutically effective amount of IGF-IR recognizing antibodies or EGFR antibodies include death of one or more target cells, growth inhibition of one or more target cells etc. A target cell is a cell that expresses EGFR and/or IGF-IR either separately on the same cell and illustratively includes abnormally growing cells and tumor cells expressing IGF-IR and/or EGFR. In vivo, the target cell is a cell of an individual with a pathological condition, including those where cell proliferation is abnormal or dysreguiated such as malignant or benign cancer or any other condition mediated by one of IGF-IR and/or EGFR expressing cells.

Thus, a broad aspect the invention employs an anti-IGF-lR antibody of the present invention (MK-0646) combined with other therapies or compounds in the treatment of malignancy, wherein therapeutic efficacy can be enhanced by apoptosis-inducing compounds, such as methotrexate or blocking the ability of epidermal growth factor (EGF) to initiate receptor activities that ultimately results in tumor growth inhibition.

An embodiment of the invention thus proposes the use of an IGF-IR antibody of the present invention in combination with an EGFR-specific antibody - Cetuximab. In yet another embodiment, the combination may further comprise a chemotherapeutic (e.g., methotrexate). It is noted that the IGF-I antibody need not be limited to a chimeric or humanized antibody. It may include any antibody or a fragment there of that is specific for IGF-I receptor protein. The same holds true for Cetuximab, which is a chimeric antibody.

In another embodiment, the IGF-IR antibody or an antigen binding fragment thereof of the present invention (MK-0646) can be combined with Cetuximab or another EGFR inhibitor, e.g., small molecule, alone or in combination thereof.

In another embodiment, the combination therapeutic agent or compounds described herein includes combining MK-0646 with methotrexate, or platinums or a taxanes.

It should be noted that while the discussion herein exemplifies HNSCC that is resistant to treatment with cetuximab or another EGFR agent when administrated alone (EGFR Inhibitor resistant), other disorders which exhibit similar resistant to an EGFR inhibitor are also within the scope of the present invention, e.g., colorectal cancer.

Other uses and objectives of this invention will be apparent to those of ordinary skill in the art in view of the Detailed Description which follows. Such other uses and objectives are deemed to be within the scope of the claims which follow.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Cetuximab and MK0646 dose response curve using four head and neck cancer cell lines in vitro. Cells were treated with the indicated concentration of Cetuximab and MK0646 for 7 days and growth inhibition rate was measured by MTS assay. Combination treatment with cetuximab and MK0646 showed additive effect in 2 cetuximab-sensitive cell lines, SCCl and SCC25. Figure 2. Cetuxiraab and MK0646 response determination in vivo using SCCl and 1 CC8. Xenografted mice (n=5) were treated with 50 mg/kg of cetuximab thrice a week for 3 weeks and the size of tumor was measured serially. 2mg/kg of MK0646 was injected once a week for 3 weeks. The control group was xenografted mice (n~5) without the treatment. Figure 3. Total RNA was isolated from each cell line using Qiagen RNeasy Mini kit The labeled RNA was fragmented and loaded on to the Affymetrix Human Genome U 133 plus 2.0 GeneChip (Affymetrix). The raw microarray data was normalized using Perfect Match software for further statistical analyses. 900 genes that were differentially expressed between SCCl and 1CC8 were determined with greater than 2 fold. This gene signature was analyzed by Ingenuity Pathway Analysis. The genes associated with MAPK pathway were closely related to cetuximab sensitivity.

Figure 4. The cells were serum starved for 16 hours. MK-0646 was added and incubated for 24 hours. Six hours prior to cell lysis, 1OnM of IGFl was added to the culture for IGFl R activation. Figure 5. Methotrexate dose response was examined to detect the MK-0646 effects given as a combination. Cells were treated with the indicated concentration of MK-0646 for 7 days and MTX for 24 hours; growth inhibition rate was measured by MTS assay. Interestingly, methotrexate sensitivity is inversely correlated with cetuximab sensitivity and MK-0646 enhanced the effect of MTX in SCCl and 1 Cc8. Figure 6. Determination of MK-0646, cetuximab and a combination of MK-0646 and cetuximab sensitivities in HNSCC cell lines (SCCl, 1CC8, SCC25 and JHUO 12) in vitro. A) MK-0646 and/or cetuximab dose response curve of HNSCC cell lines in vitro measured by MTS assay. B) MK-0646 and/or cetuximab response determination by colony formation assay in matrigel. C) Graphical presentation of the colony formation assay in matrigel. All colony sizes in the ruled cross-sectional area of SCC 1 and 1 Cc8 were measured and analyzed.

Figure 7. Determination of MK-0646, cetuximab and a combination of MK-0646 and cetuximab sensitivities in HNSCC cell lines (SCCl and 1CC8) in vivo. A) Measurement of xenografted tumor volumes over 28 days. B) Bar graph of relative tumor volumes at the end of the treatment on Day 28.

Figure 8. Western blot analyses of IGF-IR, EGFR and MET receptor tyrosine kinases, AKT and MAPK expression upon stimulation with IGF-I in the presence and absence of MK-0646.

DETAILED DESCRIPTION OF THE INVENTION Definitions and General Techniques

The reference works, patents, patent applications, and scientific literature, including accession numbers to GenBank database sequences that are referred to herein establish the knowledge of those with skill in the art and are hereby incorporated by reference in their entirety to the same extent as if each was specifically and individually indicated to be incorporated by reference. Any conflict between any reference cited herein and the specific teachings of this specification shall be resolved in favor of the latter. Likewise, any conflict between an art-understood definition of a word or phrase and a definition of the word or phrase as specifically taught in this specification shall be resolved in favor of the latter. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. It must be noted that as used herein and in the appended claims, the singular forms

"a", "and", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a genetic alteration" includes a plurality of such alterations and reference to "a probe" includes reference to one or more probes and equivalents thereof known to those skilled in the art, and so forth. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. Publications cited herein are cited for their disclosure prior to the filing date of the present application. Nothing here is to be construed as an admission that the inventors are not entitled to antedate the publications by virtue of an earlier priority date or prior date of invention. Further the actual publication dates may be different from those shown and require independent verification.

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention 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 used in connectioa with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. The methods and techniques of the present invention are generally 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 unless otherwise indicated. See, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990), which are incorporated herein by reference. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclatures used 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. The following terms, unless otherwise indicated, shall be understood to have the following meanings:

For the purposes herein a "section" of a tissue sample is meant a single part or piece of a tissue sample, e.g. a thin slice of tissue or cells cut from a tissue sample. It is understood that multiple sections of tissue samples may be taken and subjected to analysis according to the present invention.

"Cancer" or "malignancy" are used as synonymous terms and refer to any of a number of diseases that are characterized by uncontrolled, abnormal proliferation of cells, the ability of affected cells to spread locally or through the bloodstream and lymphatic system to other parts of the body (i.e., metastasize) as well as any of a number of characteristic structural and/or molecular features. A "cancerous" or "malignant cell" is understood as a cell having specific structural properties, lacking differentiation and being capable of invasion and metastasis. Examples of cancers are kidney, colon, breast, prostate and liver cancer, (see DeVita, V. et al. (eds.), 200I₉ Cancer Principles And Practice Of Oncology, ό.sup.th Ed., Lippincott Williams & Wilkins, Philadelphia, Pa.; this reference is herein incorporated by reference in its entirety for all purposes). More specifically, while the examples detail the treatment of NSCLC using the combination therapeutic detailed herein, the term "cancer" is not so limited. It includes any and all tumours that are IGF-IR dependent as well as EGFR-dependent. Exemplary cancers if this type includes for example pancreatic cancer.

A feature of cancer cells is the tendency to grow in a manner that is uncontrollable by the host, but the pathology associated with a particular cancer cell may take any form.

Primary cancer cells (that is, cells obtained from near the site of malignant transformation) can be readily distinguished from non-cancerous cells by well-established pathology techniques, particularly histological examination. The definition of a cancer cell, as used herein, includes not only a primary cancer cell, but any cell derived from a cancer cell ancestor. This includes metastasized cancer cells, and in vitro cultures and cell lines derived from cancer cells.

Cell line— A "cell line" or "cell culture" denotes higher eukaryotic cells grown or maintained in vitro. It is understood that the descendants of a cell may not be completely identical (either morphologically, genotypically, or phenotypically) to the parent cell. Cells described as "uncultured" are obtained directly from a living organism, and have been maintained for a limited amount of time away from the organism: not long enough or under conditions for the cells to undergo substantial replication.

"Diagnosing" a disease as used in the application is intended to include, for example, diagnosing or detecting the presence of a pathological hyperproliferative oncogenic disorder associated with or mediated by expression of IGF-IR, monitoring the progression of the disease, and identifying or detecting cells or samples that are indicative of a disorder associated wit expression of IGF-IR. The terms diagnosing, detecting, identifying etc. are used interchangeably herein. "Pathology" as used herein — The "pathology" caused by cancer cells within a host is anything that compromises the well-being or normal physiology of the host. This may involve, but is not limited to abnormal or uncontrollable growth of the cancer cell, metastasis, increase in expression levels of IGF-IR bearing cells, or other products at an inappropriate level, manifestation of a function inappropriate for its physiological milieu, interference with the normal function of neighboring cells, aggravation or suppression of an inflammatory or immunological response, or the harboring of undesirable chemical agents or invasive organisms. "Treatment" of an individual or a cell is any type of intervention in an attempt to alter the non-treated course of the individual or cell. For example, treatment of an individual may be undertaken to decrease or limit the pathology caused by a cancer harbored in the individual. Treatment includes but is not limited to a) administration of a composition or a combination therapeutic, such as a pharmaceutical composition comprising an IGF-IR specific mAb a nd a tyrosine kinase inhibitor. The term "treating" refers to having a therapeutic effect and at least partially alleviating or abrogating an abnormal condition in the organism. Treating includes inhibition of tumor growth, maintenance of inhibited tumor growth, and induction of remission.

The term "preventing" refers to decreasing the probability that an organism contracts or develops an abnormal condition.

As used herein, the term "about" refers to an approximation of a stated value within an acceptable range. Preferably the range is +/-5% of the stated value. The term "or" is used herein to mean, and is used interchangeably with, the term

"and/or", unless context clearly indicates otherwise.

The terms "IGFlR", "IGFRl", "Insulin-like Growth Factor Receptor-I" and "Insulin-like Growth Factor Receptor, type I" are well known in the art. Although IGF-IR may be from any organism, it is preferably from an animal, more preferably from a mammal (e.g., mouse, rat, rabbit, sheep or dog) and most preferably from a human. The nucleotide and amino acid sequence of a typical human IGF-IR precursor is available at Genbank , eg. Gene ID 3480 or NM000875. Cleavage of the precursor (e.g., between amino acids 710 and 71 1) produces an α-subunit and a β-subunit which associate to form a mature receptor.

An "immunoglobulin" is a tetrameric molecule. In a naturally-occurring immunoglobulin, each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" chain (about 50 70 kDa). The amino- termϊnal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as K and λ light chains. Heavy chains are classified as μ, δ, γ, α, or ε, and define the antibody's isotype as IgM, IgD₅ IgG, IgA, and IgE₅ respectively. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N. Y. (1989)) (incorporated by reference in its entirety for all purposes). The variable regions of each light/heavy chain pair form the antibody ^'binding site such that an intact immunoglobulin has two binding sites.

An "antibody" refers to an intact immunoglobulin or to an antigen-binding portion thereof that competes with the intact antibody for specific binding. Antigen-binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding portions include, inter alia, Fab, Fab¹, F(ab')2, Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, diabodies and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide. There are several anti-IGFlR antibodies that are known in the art (see e.g., WO 03/100008; WO 2002/53596; WO 04/71529; WO 03/106621; US2003/235582; WO 04/83248; WO 03/59951; WO 04/87756 or WO 2005/16970). Other small molecule IGF IR inhibitors are also known in the art

As used in the application, the term "anti-IGF-lR antibody" is collectively referred to as an anti-IGF-lR antibody disclosed in U.S patent No. 7,241,444, filed Dec. 16, 2003, the entire content of which is incorporated by reference herein in its entirety. The amino acid sequences of the various CDRs, light and heavy chain as well as the nucleotide sequences encoding the entire antibody claimed therein area also incorporated in their entirety by reference herein. Likewise, the disclosure of Serial No. 11/801,080 is also incorporated by reference herein in its entirety.

The term "patient" includes human and veterinary subjects.

Antibodies - IGF- 1 R (h7C 10)

As detailed herein, an aspect of the present invention is directed to a method of improving the anti-tumor efficacy of an anti-cancer agent by co-administering a tyrosine kinase inhibitor - EGFR, e.g., cetuximab (EGFR specific antibody) and an antibody which specifically binds to human Insulin-like growth factor -1 receptor (IGF-IR)-I to a patient with cancer. As a consequence, the IGF-IR antibody for use in the proposed combination therapeutic is one that specifically binds insulin-like growth factor 1 receptor (IGF-IR). Exemplary anti-IGF-lR antibodies for use in the combination therapeutic and methods of use thereof are described in U.S Patent No. 7,241,444 ('444 patent) the content of which is incorporated by reference herein in its entirety. See for example Claim 1 of the '444 patent.

"h7C10" or "MK-0646" (Dalotuzumab) are used interchangeably to describe a humanized antibody that is characterized as binding IGF-IR as well as binding the IR/IGF-1 hybrid receptor. Such an antibody preferably includes the antibody described, for example, in the '444 patent, wherein the antibody is a humanized antibody or a fragment thereof and comprises a light chain and/or a heavy chain in which the skeleton segments FRl to FR4 of said light chain and/or heavy chain are respectively derived from skeleton segments FRl to FR4 of human antibody light chain and/or heavy chain. The humanized antibody may comprise at least one light chain that comprises at least one or more complementary determining regions derived from a non-human source and having the amino acid sequence selected from the group consisting of SEQ ID NOs: 1 , 2, or 3 and at least one heavy chain comprising at least one or more complementary determining regions having an amino acid sequence selected from the group consisting of SEQ ID NOs 4, 5 or 6. The light chain may comprise one or more of the amino acid sequences as set forth in one of SEQ ID NOs. 7 or 8, or a sequence having at least 80% identity after optimum alignment with the sequence SEQ ID Nos: 7 or 8. Likewise, the heavy chain comprises one or more amino acid sequences as set forth in one of SEQ ID No. 9, 10 or 11 , or a sequence having at least 80% identity after optimum alignment with the sequence SEQ ID Nos 9, 10 or 11. In certain embodiments, the methods of treatment include administering an antibody that binds the same epitope on IGF-IR as that bound by MK-0646 (Dalotuzumab).

Nucleic acid molecule for expressing the recombinant antibodies (IGF-IR specific mAbs - Dalotuzumab) are described in- the '444 patent, the content of which is incorporated by reference herein in its entirety.

"Nucleic acid" or a "nucleic acid molecule" as used herein refers to any DNA or RNA molecule, either single- or double-stranded and, if single-stranded, the molecule of its complementary sequence in either linear or circular form. In discussing nucleic acid molecules, a sequence or structure of a particular nucleic acid molecule may be- described herein according to the normal convention of providing the sequence in the 5' to 3' direction. In some embodiments of the invention, nucleic acids are "isolated." This term, when applied to DNA, refers to a DNA molecule that is separated from sequences with which it is immediately contiguous in the naturally occurring genome of the organism in which it originated. For example, an "isolated nucleic acid" may comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a prokaryotic or eukaryotic cell or host organism. When applied to RNA, the term "isolated nucleic acid" refers primarily to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that has been sufficiently separated from other nucleic acids with which it would be associated in its natural state (i.e., in cells or tissues). An isolated nucleic acid (either DNA or RNA) may further represent a molecule produced directly by biological or synthetic means and separated from other components present during its production.

Nucleic acids of the invention also include fragments of the nucleic acids of the invention. A "fragment" refers to a nucleic acid sequence that is preferably at least about 10 nucleic acids in length, more preferably about 40 nucleic acids, and most preferably about 100 nucleic acids in length. A "fragment" can also mean a stretch of at least about 100 consecutive nucleotides that contains one or more deletions, insertions, or substitutions. A "fragment" can also mean the whole coding sequence of a gene and may include 5' and 3' untranslated regions. The antibodies , Dalotuzurnab or cetuximab, for use in the present invention include, but are not limited to, monoclonal antibodies, synthetic antibodies, polyclonal antibodies, multispecific antibodies (including bi-specifϊc antibodies), human antibodies, humanized antibodies, chimeric antibodies, single-chain Fvs (scfv) (including bi-specifϊc scFvs), single chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv), and epitope-binding fragments of any of the above. In particular, antibodies for use in the present invention include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain a IGF-IR binding site that immunospecifically binds to IGF-IR. The immunoglobulin molecules for use in the invention can be of any type (e.g. IgG, IgE, IgM₅ IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and Ig A2) or subclass of immunoglobulin molecule. Preferably, the antibodies for use in the invention are IgG, more preferably, IgGl.

The antibodies for use in the invention may be from any animal origin. Preferably, the antibodies are humanized monoclonal antibodies. Alternatively, the antibodies may be fully human so long as they bind the same epitope of the antibody claimed in the '444 patent. As used herein, "human" antibodies include antibodies having the amino acid sequence of -a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from mice or other animals that express antibodies from human genes.

The antibodies for use in the present invention may be monospecific, bispecific, trispecifϊc or of greater multispecificity. Multispecific antibodies may immunospecificaπy bind to different epitopes of a polypeptide or may immunospecifically bind to both a polypeptide as well a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g., International Publication Nos. WO 93/17715, WO 92/08802, WO 91/00360, and WO 92/05793; Tutt, et al, 1991, J. Immunol. 147:60-69; U.S. Pat Nos. 4,474,893, 4,714,681, 4,925,648, 5,573,920, and 5,601,819; and Kostelny et al., 1992, J. Immunol. 148:1547-1553. The antibodies for use in the invention include derivatives of the antibodies.

Standard techniques known to those of skill in the art can be used to introduce mutations in the nucleotide sequence encoding an antibody to be used with the methods for use in the invention, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis which result in amino acid substitutions. Preferably, the derivatives include less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the original molecule. In a preferred embodiment, the derivatives have conservative amino acid substitutions axe made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed and the activity of the protein can be determined.

The antibodies for use in the present invention include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the antibody. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylatton, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, synthesis in the presence of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.

The present invention also provides antibodies for use in the invention that comprise a framework region known to those of skill in the art. In certain embodiments, one or more framework regions, preferably, all of the framework regions, of an antibody to be used in the compositions and methods for use in the invention are human. In certain other embodiments for use in the invention, the fragment region of an antibody for use in the invention is humanized. In certain embodiments, the antibody to be used with the methods for use in the invention is a synthetic antibody, a monoclonal antibody, an intrabody, a chimeric antibody, a human antibody, a humanized chimeric antibody, a humanized antibody, a glycosylated antibody, a multispecific antibody, a human antibody, a single-chain antibody, or a bispecific antibody.

In certain embodiments, an antibody for use in the invention has a high binding affinity for IGF-IR.

In certain embodiments, an antibody for use in the invention has a half-life in a subject, preferably a human, of about 12 hours or more, about 1 day or more, about 3 days or more, about 6 days or more, about 10 days or more, about 15 days or more, about 20 days or more, about 25 days or more, about 30 days or more, about 35 days or more, about 40 days or more, about 45 days or more, about 2 months or more, about 3 months or more, about 4 months or more, or about 5 months or more. Antibodies with increased in vivo half-lives can be generated by techniques known to those of skill in the art. For example, antibodies with increased in vivo half-lives can be generated by modifying (e.g., substituting, deleting or adding) amino acid residues identified as involved in the interaction between the Fc domain and the FcRn receptor (see, e.g., International Publication No. WO 97/34631 and U.S. patent application Ser. No. 10/020,354, entitled "Molecules with Extended Half-Lives, Compositions and Uses Thereof, filed Dec. 12, 2001, by Johnson et al.; and U.S. Publication Nos. 2005/003700 and 2005/0064514, which are incorporated herein by reference in their entireties). Such antibodies can be tested for binding activity to antigens as well as for in vivo efficacy using methods known to those skilled in the art, for example, by immunoassays described herein.

Further, antibodies with increased in vivo half-lives can be generated by attaching to the antibodies polymer molecules such as high molecular weight polyethyleneglycol (PEG). PEG can be attached to the antibodies with or without a multifunctional linker either through site-specific conjugation of the PEG to the N- or C-terminus of the antibodies or via epsilon- amino groups present on lysine residues. Linear or branched polymer derivatization that results in minimal loss of biological activity will be used. The degree of conjugation will be closely monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation of PEG molecules to the antibodies. Unreacted PEG can be separated from antibody-PEG conjugates by, e.g., size exclusion or ion-exchange chromatography. PEG-derivatized antibodies can be tested for binding activity to antigens as well as for in vivo efficacy using methods known to those skilled in the art, for example, by immunoassays described herein.

In certain embodiments, an antibody for use in the present invention includes antigen-binding portions of an intact antibody that retain capacity to bind IGF-IR and/or EGFR. Examples include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHl domains; (ii) a F(ab')2 fragment, ambivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHl domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341 :544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); See, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad Sci. USA 85:5879-5883). Such single chain antibodies are included by reference to the term "antibody."

Methods of Producing Antibodies to IGF-IR are well known. See for example, the '444 patent. Screening for Antibody Specificity - Techniques for generating antibodies have been described above. One may further select antibodies with certain biological characteristics, as desired. Thus, once produced, the antibodies may be screened for their binding affinity for IGF-IR. Screening for antibodies that specifically bind to IGF-IR may be accomplished using an enzyme-linked immunosorbent assay (ELISA) in which microtϊter plates are coated with IGF- IR. In some embodiments, antibodies that bind IGF-IR from positively reacting clones can be further screened for reactivity in an ELISA-based assay to other IGF-IR isoforms, for example, IGF-IR using microliter plates coated with the other IGF-IR isoform(s). Clones that produce antibodies that are reactive to another isoform of IGF-IR are eliminated, and clones that produce antibodies that are reactive to IGF-IR only may be selected for further expansion and development. Confirmation of reactivity of the antibodies to IGF-IR may be accomplished, for example, using a Western Blot assay in which protein from ovarian, breast, renal, colorectal, lung, endometrial, or brain cancer cells and purified IGF-IR and other IGF-IR isoforms are run on an SDS-PAGE gel, and subsequently are blotted onto a membrane. The membrane may then be probed with the putative anti-IGF-lR antibodies. Reactivity with IGF-IR and not another insulin-like receptor isoform confirms specificity of reactivity for IGF-IR.

General methods for detecting IGF-IR or its Derivatives - The assaying method for detecting IGF-IR using the antibodies of the invention or binding fragments thereof are not particularly limited. Any assaying method can be used, so long as the amount of antibody, antigen or antibody-antigen complex corresponding to the amount of antigen (e.g., the level of IGF-IR) in a fluid to be tested can be detected by chemical or physical means and the amount of the antigen can be calculated from a standard curve prepared from standard solutions containing known amounts of the antigen. Representative immunoassays encompassed by the present invention include, but are not limited to, those described in U.S. Pat. Nos. 4,367,110 (double monoclonal antibody sandwich assay); Wide et al., Kirkham and Hunter, eds.

Radioimmunoassay Methods, E. and S. Livingstone, Edinburgh (1970); U.S. Pat. No. 4,452,901 (western blot); Brown et al., J. Biol. Chem. 255: 4980-4983 (1980) (immunoprecipitation of labeled ligand); and Brooks et al., Clin. Exp. Immunol. 39:477 (1980) (irnmunocytochemistry); immunofluorescence techniques employing a fiuorescently labeled antibody, coupled with light microscopic, flow cytometric, or fiuorometric detection etc. See also Immunoassays for the 80's, A. Voller et al., eds., University Park, 1981, Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).

(1) Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein to be detected. In a sandwich assay, the test sample analyte is bound by a first antibody which is immobilized on a solid support, and thereafter a second antibody binds to the analyte, thus forming an insoluble three-part complex. See, e.g., U.S. Pat. No. 4,376,110. The second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an antiimmunoglobulin antibody that is labeled with a detectable moiety (indirect sandwich assay). For example, one type of sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme.

In the sandwich assay, the immobilized antibody of the present invention is reacted with a test fluid (primary reaction), then with a labeled form of antibody of the present invention (secondary reaction), and the activity of the labeling agent on the immobilizing carrier is measured, whereby the IGF-IR level in the test fluid can be quantified. The primary and secondary reactions may be performed simultaneously or with some time intervals. The methods of labeling and immobilization can be performed by modifications of those methods described above. In the immunoassay by the sandwich assay, the antibody used for immobilized or labeled antibody is not necessarily from one species, but a mixture of two or more species of antibodies may be used to increase the measurement sensitivity, etc. In the method of assaying IGF-IR by the sandwich assay, for example, when the antibodies used in the primary reaction recognize the partial peptides at the C-terrainal region of IGF-IR, the antibodies used in the secondary reaction are preferably those recognizing partial peptides other than the C-terminal region (i.e., the N- terminal region). When the antibodies used for the primary reaction recognize partial peptides at the N-terminal region of IGF-IR, the antibodies used in the secondary reaction, antibodies recognizing partial peptides other than the N-terminal region (i.e., the C-terminal region) are preferably employed.

Other types of "sandwich" assays, which can also be useful for detecting IGF-IR, are the so-called "simultaneous" and "reverse" assays. A simultaneous assay involves a single incubation step wherein the antibody bound to the solid support and labeled antibody are both added to the sample being tested at the same time. After the incubation is completed, the solid support is washed to remove the residue of fluid sample and uncomplexed labeled antibody. The presence of labeled antibody associated with the solid support is then determined as it would be in a conventional "forward" sandwich assay.

In the "reverse" assay, stepwise addition first of a solution of labeled antibody to the fluid sample followed by the addition of unlabeled antibody bound to a solid support after a suitable incubation period, is utilized. After a second incubation, the solid phase is washed in conventional fashion to free it of the residue of the sample being tested and the solution of unreacted labeled antibody. The determination of labeled antibody associated with a solid support is then determined as in the "simultaneous" and "forward" assays. In one embodiment, a combination of antibodies of the present invention specific for separate epitopes can be used to construct a sensitive three-site immunoradiometric assay.

This type of assays may also be used to quantify IGF-IR expression in whatever "sample" it may present itself. Thus, in certain aspects, the sandwich assay includes:

(i) a method for quantifying expression levels of IGF-IR in a test fluid, comprising reacting the antibody specifically reacting with a partial peptide at the N-terminal region of the IGF-IR immobilized on a carrier, a labeled form of the antibody specifically reacting with a partial peptide at the C-terminal region and the test fluid, and measuring the activity of the label; or

(ii) a method for quantifying IGF-IR expression in a test fluid, comprising reacting the antibody specifically reacting with a partial peptide at the C-terminal region of the IGF-I R immobilized onto a carrier, the antibody specifically reacting with a partial peptide at the N-terminal region of a labeled form of the IGF-IR and the test fluid, and measuring the activity of the label; etc.

(2) Competitive binding assays rely on the ability of a labeled standard to compete with the test sample analyte for binding with a limited amount of antibody. The amount of IGF-IR protein in the test sample is inversely proportional to the amount of standard that becomes bound to the antibodies. To facilitate determining the amount of standard that becomes bound, the antibodies generally are insolubilized before or after the competition, so that the standard and analyte that are bound to the antibodies may conveniently be separated from the standard and analyte which remain unbound. For quantifying the level of IGF-I R expression, one skilled in the art may combine and/or competitively react antibodies of the invention or fragments thereof, a test fluid and a labeled form of IGF-IR, measure a ratio of the labeled IGF-IR bound to the antibodies or fragments thereof b to thereby quantify the IGF-IR in the test fluid.

(3) Immunometric Assay In the immunometric assay, an antigen in a test fluid and a solid phase antigen are competitively reacted with a given amount of a labeled form of the antibody of the present invention followed by separating the solid phase from the liquid phase; or an antigen in a test fluid and an excess amount of labeled form of the antibody of the present invention are reacted, then a solid phase antigen is added to bind an unreacted labeled form of the antibody of the present invention to the solid phase and the solid phase is then separated from the liquid phase. Thereafter, the labeled amount of any of the phases is measured to determine the antigen level in the test fluid.

Typical, and preferred, immunometric assays include "forward" assays in which the antibody bound to the solid phase is first contacted with the sample being tested to extract the IGF- 1 R from the sample by formation of a binary solid phase antibody-IGF- 1 R complex. After a suitable incubation period, the solid support is washed to remove the residue of the fluid sample, including unreacted IGF-IR₅ if any, and then contacted with the solution containing a known quantity of labeled antibody (which functions as a "reporter molecule"). After a second incubation period to permit the labeled antibody to complex with the IGF-IR bound to the solid support through the unlabeled antibody, the solid support is washed a second time to remove the unreacted labeled antibody. This type of forward sandwich assay can be a simple "yes/no" assay to determine whether IGF-IR is present or can be made quantitative by comparing the measure of labeled antibody with that obtained for a standard sample containing known quantities of IGF- IR. Such "two-site" or "sandwich" assays are described by Wide (Radioimmune Assay Method,- Kirkham, ed., Livingstone. Edinburgh, 1970, pp. 199 - 206). (4) Nephrometry

In the nephrometry, the amount of insoluble sediment, which is produced as a result of the antigen-antibody reaction in a gel or in a solution, is measured. Even when the amount of an antigen in a test fluid is small and only a small amount of the sediment is obtained, a laser nephrometry utilizing laser scattering can be suitably used.

Examples of labeling agents, which may be used in the above referenced assay methods (1) to (4) using labeling agents, include radioisotopes (e.g., 1251, 1311, 3H, 14C, 32P, 33P, 35S, etc., fluorescent substances, e.g., cyanine fluorescent dyes (e.g., Cy2, Cy3, Cy5, Cy5.5, Cy7), fluorescamine, fluorescein isothiocyanate, etc., enzymes (e.g., β-galactosidase, β~ glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase, etc.), luminescent substances (e.g., luminol, a luminol derivative, luciferin. lucigenin, etc.), biotin, lanthanides, etc. In addition, a biotin-avidin system may be used as well for binding an antibody to a labeling agent.

In the immobilization of antigens or antibodies, physical adsorption may be used. Alternatively, chemical binding that is conventionally used for immobilization of proteins, enzymes, etc. may be used as well. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, cellulose, etc.; synthetic resins such as polystyrene, polyacrylamide, silicone, etc.; or glass; and the like.

In another embodiment, the present invention assists in the diagnosis of cancers and tumors by the identification and measurement of the IGF-IR levels in body fluids, such as blood, serum, plasma, sputum and the like. If IGF-IR is normally present, and the development of the oncogenic disorder is caused by an abnormal quantity of the cell surface receptor (IGF- IR), e.g., expression relative to normal, the assay should compare IGF-IR levels in the biological sample to the range expected in normal, non-oncogenic tissue of the same cell type. Thus, a statistically significant increase in the amount of IGF-IR bearing cells or IGF-IR expression level in the subject relative to the control subject or subject's baseline, can be a factor that may lead to a diagnosis of an oncogenic disorder that is progressing or at risk for such a disorder. Likewise, the presence of high levels of IGF-IR indicative of cancers likely to metastasize can also be detected. For those cancers that express the antigen recognized by the antibodies of the invention, e.g., IGF-IR, the ability to detect the antigen provides early diagnosis, thereby affording the opportunity for early treatment. Early detection is especially important for cancers difficult to diagnose in their early stages. Moreover, the level of antigen detected and measured in a body fluid sample such as blood provides a means for monitoring the course of therapy for the cancer or tumor, including, but not limited to, surgery, chemotherapy, radiation therapy, the therapeutic methods of the present invention, and combinations thereof. By correlating the level of the antigen in the body fluid with the severity of disease, the level of such antigen can be used to indicate successful removal of the primary tumor, cancer, and/or metastases, for example, as well as to indicate and/or monitor the effectiveness of other therapies over time. For example, a decrease in the level of the cancer or tumor-specific antigen over time indicates a reduced tumor burden in the patient. By contrast, no change, or an increase, in the level of antigen over time indicates ineffectiveness of therapy, or the continued growth of the tumor or cancer.

Detection of the antibody in the specimen can be accomplished using techniques known in the art such as immunoenzymatic techniques, e.g., immunoperoxidase staining technique, or the avidin-biotin technique, or immunofluorescence techniques (see, e.g., Ciocca et aL, 1986, "Immunohistochemical Techniques Using Monoclonal Antibodies", Meth. EnzymoL, 121:562 79 and Introduction to Immunology, Ed. Kimball, (2,sup.nd Ed), Macmillan Publishing Company, 1986, pp. 113 117). Those skilled in the art can determine operative and optimal assay conditions by routine experimentation.

A typical in vitro immunoassay for detecting IGF-IR comprises incubating a biological sample in the presence of a detectably labeled anti-IGF-lR antibody or antigen binding fragment of the present invention capable of selectively binding to IGF-IR₅ and detecting the labeled fragment or antibody which is bound in a sample. The antibody is bound to a label effective to permit detection of the cells or portions (e.g., IGF-IR or fragments thereof liberated from hyperplastic, dysplastic and/or cancerous cells) thereof upon binding of the antibody to the cells or portions thereof. The presence of any cells or portions thereof in the biological sample is detected by detection of the label.

The biological sample may be brought into contact with, and immobilized onto, a solid phase support or carrier, such as nitrocellulose, or other solid support or matrix, which is capable of immobilizing cells, cell particles, membranes, or soluble proteins. The support may then be washed with suitable buffers, followed by treatment with the detectably-labeled anti-IGF- IR antibody. The solid phase support may then be washed with buffer a second time to remove unbound antibody. The amount of bound label on the solid support may then be detected by conventional means. Accordingly, in another embodiment of the present invention, compositions are provided comprising the monoclonal antibodies, or binding fragments thereof, bound to a solid phase support, such as described herein.

By "solid phase support" or "carrier" is intended any support capable of binding peptide, antigen or antibody. Well-known supports or carriers, include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material can have virtually any possible structural configuration so long as the coupled molecule is capable of binding to IGF-IR or an Anti-IGF-lR antibody. Thus, the support configuration can be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface can be Sat, such as a sheet, culture dish, test strip, etc. Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody, peptide or antigen, or can ascertain the same by routine experimentation. In vitro assays in accordance with the present invention also include the use of isolated membranes from cells expressing a recombinant IGF-IR, soluble fragments comprising the ligand binding segments of JGF-IR, or fragments attached to solid phase substrates. These assays allow for the diagnostic determination of the effects of either binding segment mutations and modifications, or ligand mutations and modifications, e.g., ligand analogues. Assays For Efficacy of Combination Immunotherapy in In vivo Models - Tumor burden can be assessed at various time points after tumor challenge using techniques well known in the art. Assays for monitoring anti-tumor response and determining the efficacy of combination immunotherapy are described below. While an improved or enhanced anti-tumor response may be most dramatically observed shortly following administration of the immunotherapy, e.g. within 5-10 days, the response may be delayed in some instances, depending on factors such as the expression level of the IGF-IR, the dosage and dosing frequency of the anti-IGF-lR antibody, and the relative timing of administration of the anti-IGF- IR-I antibody relative to the timing of administration of the tyrosine kinase inhibitor- Erlotinib. Thus, any of the well known assays may be performed on biological samples harvested at various time points following treatment or administration of the combination therapeutic in order to fully assess the anti-tumor response following immunotherapy.

Monitoring Treatment - One skilled in the art is aware of means to monitor the therapeutic outcome and/or the systemic immune response upon administering a combination treatment of the present invention. In particular, the therapeutic outcome can be assessed by monitoring attenuation of tumor growth and/or tumor regression and or the level of tumor specific markers. The attenuation of tumor growth or tumor regression in response to treatment can be monitored using one or more of several end-points known to those skilled in the art including, for instance, number of tumors, tumor mass or size, or reduction/prevention of metastasis. The EGF and IGF receptor tyrosine kinase (RTK) families are well-recognized mediators of tumor cell proliferation and survival, acting principally via phosphoinositide 3- kinase (PDK)-AKT and RAS-RAF-MAPK signaling. As a consequence, much research has focused on inhibiting EGFR and its specific TK activity.

The EGF receptor family of type 1 receptor tyrosine kinases is comprised of four monomelic receptors, the EGFR (ErbB I/Her 1 ), ErbB2/Her2, ErbB3/Her3, and ErbB4/Her4. EGFR is a 170-kd transmembrane glycoprotein, composed of an extra cellular ligand-binding domain, a transmembrane lipophilic segment and an intracellular protein kinase domain with a regulatory carboxyl terminal segment. It is well-recognized that ligand binding induces EGFR homo- and hetero-dimerisation, which leads to subsequent-activation of EGFR tyrosine kinase (TK) activity, initiating receptor-mediated signal transduction, cell mϊtogenesis and cell transformation (Thomson and Gill 1985; Schlessinger 2000; Carpenter and Cohen 1990; UMch and Schlessinger 1990; Olayioye et al 2000; Schleissinger 2000; Yarden and Sliwkowski 2001; Mendelsohn and Baselga 2003; Hynes and Lane 2005).

Ensulin-like growth factor receptor 1 (IGF-IR), is a tyrosine kinase receptor of the insulin receptor family. It is a transmembrane heterotetrameric protein, encoded by the IGFR-I gene located on chromosome 15q25-q26, promoting oncogenic transformation, growth and survival of cancer cells. The binding of insulin-like growth factor (IGF) I and IGF-EE to the extracellular subunit domain of IGFR-I activates the tyrosine kinase activity of IGFR-I and triggers a cascade of reactions involving the Raf/MEK/MAPK and the PI3K/PKB pathways (LeRoith et al, 1995; Jones and Clemmons, 1995). IGR-IR and its ligand IGF-2 are over expressed in many types of advanced cancer, and ligand-stimulated receptor signaling promotes the proliferation of cancer cells in vitro. Significantly, IGF-IR signaling is closely linked to the PBK axis. IGF-IR inhibition has shown potent anti-cancer effects in preclinical studies, and a number of IGF-IR inhibitors are currently in clinical development. See, Baserga, R, et al., "Mini Review: The IGF-IR receptor in cancer biology," Int. J. Cancer., 107: 873-77 (2003); Baserga et al, 1997; Blakesley etal, 1997; Dufourny et al, 1997; Khandwala et al, 2000).

Antibodies and kinase inhibitors targeting the EGFR have shown significant anti- cancer activity. A representative EGFR antagonist is Cetuximab (Erbitux™) which as a single agent is indicated for the treatment of patients with recurrent or metastatic squamous cell carcinoma of the head and neck whose tumor has returned in the same location or spread to other parts of the body and who have failed platinum-based chemotherapy. Cetuximab, in combination with radiation therapy, is indicated for the treatment of locally or regionally advanced squamous cell carcinoma of the head and neck.

Cetuximab is a chimeric human- murine immunoglobulin Gl (IgGl) monoclonal antibody that binds the epidermal growth factor receptor (EGFR) with high specificity and competitively inhibits endogenous ligand binding (Sato et al 1983; Gill et al 1984; Goldstein et al 1995). Cetuximab binds specifically to the EGFR with an affinity that is approximately 5-10- fold higher than that of endogenous ligands. Thus, Cetuximab blocks binding of endogenous EGFR ligands (particularly of EGF and TGF-α) resulting in an inhibition of the function of the receptor. Furthermore, Cetuximab induces an internalization of the receptor leading to down- regulation of EGFR and it targets cytotoxic immune effector cells towards EGFR-expressing tumor cells (antibody dependent cell-mediated cytotoxicity). The link between Cetuximab and EGFR induces receptor dimerization, internalization and degradation (Fan et al 1994; Schlessinger 2000; Herbst and Shin 2002). Methods of making and using Cetuximab are detailed in U. S Patent No. 6,217,866, the content of which is incorporated by reference in its entirety. MK-0646 is a humanized IGF-IR specific antibody. It comprises at least one heavy chain complementary determining region (CDR) of non-human origin and at least one light chain complementary determining region (CDR) derived from a non-human source, wherein the antibody that binds to IGF-IR has at least one of the following properties selected from the group consisting of: a) binding IGF-IR but not IR; (b) binds a hybrid receptor comprising an insulin receptor and insulin growth factor receptor (IR/IGF-1R hybrid-R) but not IR alone; c) inhibiting the binding between a human IGF-IR and IGF-I and/or IGF-2 ; (d) binding the hybrid- R and its native ligand, preferably designated herein as IGFl and/or IGF2 and/or insulin, with an inhibition constant and/or IC50 of less than 100 nM; (e) specifically inhibiting the tyrosine kinase activity of said IGF-IR; (f) specifically inhibiting the tyrosine kinase activity of said hybrid-R; (g) having a binding affinity of 10 nM or less for said hybrid-R; (h) down-regulating IGF-IR expression; (i) down-regulating hybrid-R expression; (j) inhibiting in vivo tumor growth. Methods for making and using said anti-IGF-lR antibody are described in U.S. Patent No. 7,214,444, which is hereby incorporated by reference in its entirety. Methotrexate abbreviated MTX and formerly known as arnethopterin, is an antimetabolite and antifolate drug used in treatment of cancer and autoimmune diseases. It acts by inhibiting the metabolism of folic acid.

A brief interrogation of recent publications detailing the effects and mode of action relative to EGFR antagonists reveals a growing number of patients becoming resistant to therapy with an EGFR inhibitor. Such EGFR blockade is attributable to numerous factors, including, for example, cross talk between the EGFR and IGF-IR signaling pathway. (Johann Riedemann , Biochemical and Biophysical Research Communications, 335: 707-714 (2007)). Scharf et al., The Role of the IGF axis in hepatocarcinogenesis, Horm. Metab. Res. 35: 685-693 (2003) suggest that resistance to EGFR targeting may arise from the fact that alternative signaling pathways of the growth factor receptors can compensate for a blocked primary EGFR pathway. One such pathway is the IGf-IR signaling pathway and the authors conclude that the insulin-like growth factor 1 receptor (IGF-IR) is known to be involved in EGFR resistance.

Huether et al. suggest that signaling through the IGFR-I represents at least one mechanism by which tumor cells can become resistant to anti-EGFR therapy. According to their data, combining Cetuximab with the IGF-IR inhibitor TKI AG 1024 (small molecule) exhibited an additive and especially in Huh-7 cells a synergistic effect. The reference, however, is limited to treating hepatocellular cancer with the combination of a small molecule IGF-IR inhibitor with Cetuximab. Missing from the reference is any suggestion for combining two antibodies for treating HNSCC, let alone combining MK-0646 and cetuximab. Huether et al, EGFR blockade by Cetuήmab alone or as combination therapy for growth control of hepatocellular cancer, Biochemical Pharmacology, 70: 1568-1578 (2005). Chakravarti, et al., Cancer Res 62:200-207 (2002) suggest that IGF-IR mediates resistance to anti-epidermal growth factor receptor therapy in primary human glioblastoma cells through continued activation of phosphoinositide 3-kinase signaling, The investigators caution however, that simple measurement of absolute EGFR expression levels may be inadequate by itself in predicting which tumors will respond most favorably to anti-EGFR therapy. The teaching continues that IGFR-I signaling through PI3-K represents one mechanism of treatment resistance in response to EGFR antagonism. The authors conclude that other mechanisms of resistance exist, which requires additional studies, (emphasis supplied)

Several deletions and mutations within EGFR have also been described that may contribute to EGFR antagonism resistance. For example, the most common is the type III mutation (EGFRvIII which leads to a deletion of the extracellular domain of EGFR thereby influencing Cetuximab binding. See N. Sugawa et al., Identical splicing of aberrant epidermal growth factor receptor transcripts from amplified rearranged genes in human glioblastomas, Proc Natl Acad Sci USA. 87:8602-8606 (1990). As late as 2008, investigators were still unclear about the mechanism of intrinsic resistance to Cetuximab. G. Finocchiaro et al, J Clin Oncol 26: 2008, suggest that KRAS mutations cannot account for all resistant cases. Their preclinical data from studies conducted in colorectal cancer patients suggested that MET could interfere with anti-EGFR strategies and IGFlR mediated resistance to anti-EGFR therapies through continued activation of PI3K-Akt pathway. The investigators concluded by noting that the rarity of MET and IGF-IR gene amplification suggested a marginal role in intrinsic resistance to anti-EGFR resistance therapy in metastatic colorectal cancer patients. According to the investigators, the potential prognostic implications of IGF-IR expression warranted additional evaluation. (Emphasis supplied) As a result of assiduous studies, the present inventors have found that a synergistic anticancer activity can be achieved by using an IGF-IR inhibitor, e.g., MK-0646 or a pharmaceutically acceptable salt thereof, in combination with an EGFR-specific antibody or a cytotoxic agent selected from the group consisting of platinums, taxanes or methotrexate or a combination thereof

The invention is especially useful in the treatment of a cancer mediated by EGFR receptor mediated pathways or responsive to treatment with an EGFR inhibitor. In certain embodiments, the disorders are NHSCC. However, the instant invention could prove useful in the treatment of various other cancers, which are EGFR inhibitor resistant.

Tumor burden can be assessed at various time points after tumor challenge using techniques well known in the art. Assays for monitoring anti-tumor response and determining the efficacy of combination immunotherapy are described below. While an improved or enhanced anti-tumor response may be most dramatically observed shortly following administration of the immunotherapy, e.g. within 5-10 days, the response may be delayed in some instances, depending on factors such as the expression level of the IGF-IR, the dosage and dosing frequency of the anti-IGF-lR antibody, and the relative timing of administration of the anti-IGF-lR-1 antibody relative to the timing of administration of the EGFR antibody (Cetuximab). Thus, any of the well known assays may be performed on biological samples harvested at various time points following treatment or administration of the combination therapeutic in order to fully assess the anti-tumor response following immunotherapy.

Monitoring Treatment - One skilled in the art is aware of means to monitor the therapeutic outcome and/or the systemic immune response upon administering a combination treatment of the present invention. In particular, the therapeutic outcome can be assessed by monitoring attenuation of tumor growth and/or tumor regression and or the level of tumor specific markers. The attenuation of tumor growth or tumor regression in response to treatment can be monitored using one or more of several end-points known to those skilled in the art including, for instance, number of tumors, tumor mass or size, or reduction/prevention of metastasis.

Dose and Route of Administration The combination therapeutic comprising IGF-IR specific antibodies and EGFR specific antibody or chemotherapeutic agents of the invention are administered to a human patient, in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. Intravenous or subcutaneous administration of the antibody is preferred. Three distinct delivery approaches are expected to be useful for delivery of the antibodies in accordance with the invention. Conventional intravenous delivery will presumably be the standard delivery technique for the majority of tumours. However, in connection with some tumours, such as those in the peritoneal cavity exemplified by tumours of the ovaries, biliary duct, other ducts, and the like, intraperitoneal administration may prove favorable for obtaining high dose of antibody at the tumour and to minimize antibody clearance. In a similar manner certain solid tumours possess vasculature that is appropriate for regional perfusion. Regional perfusion will allow the obtention of a high dose of the antibody at the site of a tumour and will minimize short term clearance of the antibody. As with any protein or antibody infusion based therapeutic, safety concerns are related primarily to (i) cytokine release syndrome, i.e., hypotension, fever, shaking, chills, (ii) the development of an immunogenic response to the material (i.e., development of human antibodies by the patient to the antibody therapeutic, or HAHA or HACA response), and (iii) toxicity to normal cells that express EGFR, e.g., hepatocytes which express EGFR and/or IGF-IR. Standard tests and follow up will be utilized to monitor each of these safety concerns. In particular, liver function will be monitored frequently during clinical trails in order to assess damage to the liver, if any. For the prevention or treatment of disease, the appropriate dosage of antibody will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments. In a combination therapy regimen, the compositions of the present invention are administered in a therapeutically effective or synergistic amount. As used herein, a therapeutically effective amount is such that co-administration of anti-IGF-lR antibody and one or more other therapeutic agents - Cetuximb or MTX, or administration of a composition of the present invention, results in reduction or inhibition of the targeting disease or condition. A therapeutically synergistic amount is that amount of anti-IGF-lR antibody and one or more other therapeutic agents necessary to synergistically or significantly reduce or eliminate conditions or symptoms associated with a particular disease.

In a broad embodiment, the treatment of the present invention involves the combined administration of an anti-IGF-lR antibody and one or more chemotherapeutic agents or an EGFR antibody such as Cetuximab. The combined administration includes co administration, using separate formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein preferably there is a time period while both (or all) active agents simultaneously exert their biological activities. Preparation and dosing schedules for such chemotherapeutic agents may be used according to manufacturers' instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for chemotherapy are also described in Chemotherapy Service Ed., M. C. Perry, Williams Sc Wilkiαs, Baltimore, Md. (1992). The chemotherapeutic agent may precede, or follow administration of the antibody or may be given simultaneously therewith. The clinical dosing of therapeutic combination of the present invention are likely to be limited by the extent of adverse reactions skin rash as observed with monoclonal anti-IGF-lR or EGFR antibodies or a cytotoxic agent used in the clinic today.

The term "therapeutically effective amount" or "therapeutically effective dosage" means that amount or dosage of a composition of the invention (e.g., IGFlR inhibitor, such as an anti-IGFlR antibody) that will elicit a biological or medical response of a tissue, system, subject or host that is being sought by the administrator (such as a researcher, doctor or veterinarian) which includes any measurable alleviation of the signs, symptoms and/or clinical indicia of cancer, such as non-small cell lung cancer or any other Erlotinib or IGF-IR resistant cancer (e.g., tumor growth) and/or the prevention, slowing or halting of progression or metastasis of the cancer to any degree.

Suitable dosages are known to medical practitioners and will, of course, depend upon the particular disease state, specific activity of the composition being administered, and the particular patient undergoing treatment. In some instances, to achieve the desired therapeutic amount, it can be necessary to provide for repeated administration, i.e., repeated individual administrations of a particular monitored or metered dose, where the individual administrations are repeated until the desired daily dose or effect is achieved. Further information about suitable dosages is provided in the Example below. For example, in one embodiment, a "therapeutically effective dosage" of any anti-

IGFlR antibody; for example, an antibody or antigen-binding fragment thereof corresponding to Dolutuzumab or any other anti-IGFlR antibody mentioned herein is between about 40 and about 1000 mg/m² (e.g., about 50 mg/m², 60 mg/m² _s 70 mg/m², 80 mg/m², 90 mg/m², 100 mg/m², about 200 mg/m², about 300 mg/m², about 400 mg/m²,, about 500 mg/m², about 600 mg/m² or about 700 mg/m²) or 1-20 mg/kg of body weight (e.g., about 1 mg/kg of body weight, about 2 mg/kg of body weight, about 3 mg/kg of body weight, about 4 mg/kg of body weight, about 5 mg/kg of body weight, about 6 mg/kg of body weight, about 7 mg/kg of body weight, about 8 mg/kg of body weight, about 9 mg/kg of body weight, about 10 mg/kg of body weight, about 11 mg/kg of body weight, about 12 mg/kg of body weight, about 13 mg/kg of body weight, about 14 mg/kg of body weight, about 15 mg/kg of body weight, about 16 mg/kg of body weight, about 17 mg/kg of body weight, about 18 mg/kg of body weight, about 19 mg/kg of body weight, about 20 mg/kg of body weight), once per week.

Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single dose may be administered or several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by exigencies of the therapeutic situation. For example, dosage may be determined or adjusted, by a practitioner of ordinary skill in the art (e.g., physician or veterinarian) according to the patient's age, weight, height, past medical history, present medications and the potential for cross-reaction, allergies, sensitivities and adverse side-effects. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.

A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the antibody or antigen-binding fragment of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. The effectiveness of a given dose or treatment regimen of an antibody or combination of the invention can be determined, for example, by determining whether a tumor being treated in the subject shrinks or ceases to grow. The size of tumor can be easily determined, for example, by X-ray, magnetic resonance imaging (MRI) or visually in a surgical procedure. Tumor size and proliferation can also be measured by use of a thymidine PET scan (see e.g., Wells etal., Clin. Oncol. 8: 7-14 (1996)). Generally, the thymidine PET scan includes the injection of a radioactive tracer, such as [2-^uC]-thyrnidine, followed by a PET scan of the patient's body (Vander Borght et al, Gastroenterology 101 : 794-799, 1^"99I ; Vander Borght et ah, J. Radiat. Appl. Instrum. Part A₅ 42: 103-104 (1991)). Othertracers that can be used include [¹⁸F]-FDG (-18-fluorodeoxyglucose), [¹²⁴I]IUdR (5-[124I]iodo-2'-deoxyuridine)_J [⁷⁶Br]BrdUrd (Bromodeoxyuridine), [¹⁸F]FLT (3'-deoxy-3'fluorothymidine) or [¹¹C]FMAU (2'-fluoro-5- methyl- 1 -β-D-arabmofuranosyluracil) .

For example, NSCLC progress can be monitored, by the physician or veterinarian by a variety of methods, and the dosing regimen can be altered accordingly. Methods by which to monitor progress include, for example, CT scan (e.g., to monitor tumor size), MRI scan (e.g., to monitor tumor size), chest X-ray (e.g. , to monitor tumor size), bone scan, bone marrow biopsy, hormone tests, complete blood test (CBC), testing for NSCLC tumor markers in the urine or blood.

Depending on the type and severity of the disease, about 1 μg/kg to 100 mg/kg, preferably 50 mg/kg (e.g. 0.1-20 mg/kg) of te IGF-IR or EGFR antibody is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dosage might range from about 1 μg/kg to about 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. In certain embodiments, the IGF-IR antibody is dosed at 2 mg/kg once a week for three weeks. Likewise, Cetuximab may be dosed at 50 mg/kg thrice weekly alone or in combination with the IGF-IR antibody.

For parenteral administration, the antibody can be formulated as a solution, suspension, emulsion or lyophilized powder in association, or separately provided, with a pharmaceutically acceptable parenteral vehicle. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 1-10% human serum albumin. Liposomes and nonaqueous vehicles such as fixed oils can also be used. The vehicle or lyophilized powder can contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives). The formulation is sterilized by known or suitable techniques. The administration of the combination therapeutic may continue until disease progression.

While having described the invention in general terms, the embodiments of the invention will be further disclosed in the following examples, which are not to be construed as limiting the invention.

EXAMPLES

Experimental Design

In summary, the cell lines, SCCl and its cetuximab-resistant clone 1CC8, were obtained from Dr. Paul Harari at the University of Wisconsin. A cetuximab-sensitive cell line, SCC25, was purchased from ATCC. A cetuximab-resistant cell line, JHUO 12, was obtained from Dr. David Sidransky at the Johns Hopkins University. The cell lines were treated with MK- 0646, cetuximab or MTX, and a combination of MK-0646 and each anticancer drag. Also, to investigate the effect of MK-0646, SCC25 which has high expression of IGFl R and JHU12 which has no detectable expression of IGFl R were used for the experiment. The effects of treatments on cell proliferation and anti-tumor activity were determined using MTS assay in vitro and in vivo using mouse xenografts generated from the cell lines. Overall changes in the gene and protein expressions with the treatments were determined by DNA microarrays and western blots.

In vitro MTS assay

Four cell lines, SCCl , 1CC8 which is cetuximab resistant clone of SCCl, SCC25 and JHUO 12, were used for drug sensitivity assays. Cetuximab (ImClone Systems, Branchburg, NJ) and MTX (Bedford Labs, Bedford, OH) were purchased from the Vanderbilt Pharmacy. MK- 0646 was supplied by Merck & Co., Inc. The drugs were diluted in culture medium prior to each experiment. Cells were seeded in flat-bottom 96 well culture plates with 5x102 cells per well in quadruplicate at each dose level on day 0. The drugs were added on day 1, cetuximab or MK0646 at 1, 10, 100, 1000 nM which are physiological dose levels as previously described. All of the media and the drug are changed on day 4. The drugs were kept in the media through the subsequent culturing for the total of 7 days, and the measurement was obtained on the day 7, For the combination treatment with cetuximab and MK-0646, 10OnM of MK-0646 and 1 to 1000 nM of cetuximab were added on day 1, and the media and the drugs were changed on day 4. For the combination treatment with MK-0646 and MTX, 10OnM of MK-0646 was added to each well on day 1, and the media and the drug were changed on day 4 while each concentration (10 nM, 100 nM, 1 pM or 10 pM) of MTX was added on day 6. The measurement was obtained on the day 7. For the investigation of MTX alone sensitivity, 1x104 cells per well were seeded on day 0, each concentration of MTX was added on day 1, and the intensity were measured on the day 2. Growth inhibition was measured using the CellTiter 96 Aqueous One Solution Reagent (Promega, Madison, WI) as recommended by the manufacturer. This assay uses bioreduction of MTS to a formazan product that is soluble hi culture medium and can be detected by spectrophotometry. Optical density was determined at 490nm within the linear range of the assay (0.5-1.5 OD units) using a BioTek Synergy HT instrument. Zerodose treated cells were measured in 4 independent wells for each cell line. Data were expressed as a percentage of growth relative to the zerodose-treated cells. Each data point represented the mean of four wells.

In vivo HNSCC mouse xenograft model studies

Athymic nude mice (4 to 6-week-old females) were obtained from Harlan Laboratories {Indianapolis, IN). A suspension of 4 x 106 each HNSCC cells in 100 pL volume of HBSS was injected subcutaneously into the right flank of mice using a 1-cc syringe with 27%- gauge needle. Tumor volumes were measured in length and width twice a week. Tumor volumes were calculated using the formula; length x width 2xn/6. All planted tumors were grown for 6 to 8 days until average tumor volume reached 30 mm3 before treatment. For the treatment group in cetuxiraab alone, 5 mice were treated with intraperitoneal (IP) injections of 50 mg/kg cetuximab thrice weekly for 3 weeks. In MK0646 alone, 5 mice were treated with IP injections of 2 mg/kg MK-0646 weekly for 3 weeks, hi the combination of cetuximab and MK- 0646, 5 mice were treated with IP injections of 50 mg/kg cetuximab thrice weekly and 2 mg/kg MK0646 once a week for 3 weeks. Five mice in control group were not treated. All mice were sacrificed 24 days after the first treatment, and the tumors were surgically excised for measurements.

Detection of protein expression by western blotting

The cells were serum starved for 16 hours. MK-0646 was added and incubated for 24 hours. Six hours prior to cell lysis, IGFl (Cell Signaling Technology) 10 nM was added to the culture for IGFlR activation. The cells were lysed with RIPA lysis buffer (ImM NaV03,lmM DTT, ImM PMSF, phosphatase inhibitor cocktail and protease inhibitor cocktail mini tablet; Roche, Indianapolis, IN), and sonicated. Protein was quantified with a standard Bradford absorbance assay. Twenty micrograms of protein from each sample were fractionated by SDS- PAGE. Proteins were transferred to nitrocellulose membrane and incubated with the appropriate primary antibodies (total- and phosphoEGFR, -AkI, -IGFl R and -MAPK and (3-Actin; Cell Signaling Technology, Boston, MA) followed by secondary antibody. Signal intensity was determined by the Odyssey system (Li-cor Bioscience, Lincoln, NE, USA).

RNA isolation and DNA microarray analysis

Total RNA was isolated from each cell line using Qiagen RNeasy Mini kit according to the manufacturer's recommendations (Qiagen, Valencia, CA). The quality and quantity of the RNA was determined using the Agilent RNA 6000 NanoLabChip kit and Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA). The RNA was labeled with GeneChipR One-Cycle Target Labeling and Control Reagents according to the manufacturer's recommendation (Affymetrix, Santa Clara, CA). The labeled RNA was fragmented and loaded on to the Affymetrix Human Genome U133 plus 2.0 GeneChip (Affymetrix, Santa Clara, CA). After hybridization, the GeneChip was washed, stained with streptavidin/phycoerythrin conjugate and biotinylated antibody, and scanned according to the manufacturer's recommendations. The raw microarray data was normalized using Perfect Match software for further statistical analyses. The normalized microarray data were imported to Genespring 7.2 (Silicon Genetics, Redwood City, CA). The genes that were differentially expressed between cetuximab sensitive and resistant cell lines were selected using one-way ANOVA with FDR<1 %. Results

The effects of combined inhibition of EGFR and IGFI R pathways in cell growth: SCCl and SCC25 were sensitive, and 1CC8 and JHUO 12 were resistant to cetuximab. SCC25 showed moderate sensitivity to the IGFl R inhibitor, MK-0646, as a monotherapy in vitro (IC5o approximately 1 pM), but other 3 cell lines were resistant (IC5o > 1 pM). While addition of MK- 0646 did not have significant effects in the cetuximab resistant cell lines, MK-0646 had an additive effect in the inhibition of cell proliferation in cetuximab-sensitive cell lines (SCCl and SCC25; Figure 1). MK-0646 also enhanced the cetuximab sensitivity in the mouse xenograft model of SCCl, but not in 1CC8 the mouse xenograph model (Figure 2).

DNA microarray and western blot analyses of cetuximab-sensitive and -resistant cell lines

Using the DNA microarray, we compared the expression profiles of SCCl and 1 CC8. Approximately 900 genes were differentially expressed between SCCl and 1CC8 with greater than 2-fold differences (Table 1). This gene signature included the genes that are associated with EGFR and IGFR pathways (Figure 3). Western blot analyses showed that inhibition of IGFlR with MK-0646 treatment induced EGFR pathway activation in SCCl and SCC25 (Figure 4). In SCCl, the downstream proteins of EGFR pathway, Akt and MAPK, were also activated after MK-0646 treatment. This suggests that these two receptor pathways crosstalk and compensate for each other when one of the two pathways is inhibited. However, the baseline activation levels of EGFR and IGFlR were much lower in cetuximab- and MK0646-resistant cell lines, 1CC8 and JHU 12, compared to SCCl and SCC25, and the compensatory effects of EGFR activation seen in SCCl and SCC25 were not seen in these cell lines. SCC25 has a very high expression of IGFl R at baseline, but activation of IGFl R was inhibited completely by MK- 0646. After the treatment with MK0646, expression of phosphorylated AKT was only slightly decreased in SCC25 suggesting that the resistance to MK-0646 as a monotherapy may be due to persistent activation of Akt through EGFR signaling. JHU 12 did not have detectable IGFIR expression at baseline; therefore, the expression of downstream protein of EGFR and IGFlR pathway were not affected after the treatments with IGFl and MK-0646.

The effects of combined inhibition of IGFI R and DHFR by MTX in cell growth

One of the genes that were differentially expressed between SCCl and 1 CC8 in the DNA microarray analyses was dihydrofolate reductase (DHFR), a target of MTX. Overexpression of DHFR has been associated with MTX resistance and oncogenic signalling. Interestingly, the cetuximab-sensitive cell lines, SCCl and SCC25, were resistant, and the cetuximab-resistant cell lines, 1CC8 and JHUOl 2, were sensitive to MTX by the MTS assay. In the MTX-resistant cell lines, addition of MK-0646 to MTX had additive effects in the inhibition of cell proliferation determined by the MTS assay (Figure 5). Determination of MK-0646 sensitivities in HNSCC cell lines in vitro

To assess the inhibitory effects on cell growth, each of MK-0646. cetuximab and the combination therapeutic agent (MK-0646 and cetuximab) were utilized in MTS assays performed with S CC 1 , 1 Cc8 (a derived clone of SCC 1 with acquired resistance to cetuximab), SCC25 and JHUO 12. Four cell lines were treated with MK-0646 and/or cetuximab for seven days (Figure 6A). MK-0646 showed moderate growth inhibition as a monotherapy only in SCC25 (IC50, >100 nM). While SCCl and SCC25 showed high sensitivity to cetuximab (SCCl - IC50, 8.0 nM; SCC25 - IC50, 6.0 nM), 1CC8 and JHUΘ12 showed resistance to cetuximab (IC50, >1 JVl). Increased anti-proliferation effects of a combination of MK-0646 and cetuximab was observed in the cetuximab-sensitive cell lines, SCCl and SCC25, relative to cetuximab treatment alone. Colony formation assay on matrigel (Figure 6B) was used to confirm the MTS assay results. Again, growth inhibition properties attendant MK-0646 was most prominent in SCC25. Increased anti-proliferation effects relative to the combination therapiric agent (MK-0646 and cetuximab) was observed in SCC25 and SCCl compared to cetuximab alone (Figure 6C). In JHUO 12 with de novo resistance to cetuximab, no effects were seen with MK-0646 and/or cetuximab treatments suggesting that JHUO 12 is not dependent on IGF-IR or EGFR for its growth signal.

Determination of MK-0646 and cetuximab sensitivities in HNSCC cell lines in vivo

To confirm the MK-0646 and/or cetuximab sensitivity in vivo, mouse xenografts were generated using SCCl and 1CC8. The mouse xenografts were treated with MK-0646 and/or cetuximab for 28 days. When the growth inhibitory effect of MK-0646 was only suggestive when assessed by the in vitro assays, MK-0646 was effective in inhibiting tumor growth in vivo in SCCl (Figures 7A and 7B). The in vivo results in 1CC8 were consistent with the in vitro assays being resistant to MK-0646 and relatively resistant to cetuximab compared to SCCl although overall it was sensitive to cetuximab. Treatment with MK-0646 enhanced anti-tumor effects of cetuximab given together in SCCl. In 1 CC8, the MK-0646 and cetuximab combination suggested to increase tumor growth compared to cetuximab alone as also seen in the in vitro assays although it did not meet the statistical cutoff to be significant (Figures 6C and 7B). Considering both in vitro and in vivo data, it appeared that cetuximab sensitive cell lines were also sensitive to MK-0646 while cetuximab was significantly more effective in growth inhibition than MK- 0646.

Activation of EGFR as a mechanism of resistance to MK-0646

To determine the role of receptor crosstalk as a mechanism of resistance to MK- 0646 inHNSCC cell lines, the investigators examined EGFR and MET activations that are known to crosstalk with IGF-IR in NSCLC and HNSCC. Refer to Wheeler et al., Mechanisms of acquired resistance to cetuximab: role of HER (ErbB) family members.; Oncogene 2008 Feb 25; Engelman et al., MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling.; Science 2007 May 18;316(5827): 1039-43; Guix et al.; Acquired resistance to EGFR tyrosine kinase inhibitors in cancer cells is mediated by loss of IGF-binding proteins.; J Clin Invest 2008 JuI; 118(7):2609-l 97-9). According to the data, MK-0646 treatment was the most effective in SCC25 completely inhibiting activation of IGF-IR after IGF-I stimulation (Figure 8). This was consistent with the results that SCC25 was the most sensitive cell line to MK-0646 among the four cell lines (Figures 6 A-C) indicating that IGF-I Ractivation is important m SCC25 proliferation. The significant increases in IGF-IR activation upon stimulation with IGF-I were also reflected in the increased activation of downstream proteins, AKT and MAPK, in MK-0646 sensitive cell lines, SCC25 and SCCl; however, inhibition of IGF-IR appeared to be compensated by further activation of EGFR, and decreased but persistent activation of AKT and MAPK. This, the present investigators may be attributed to the inability of MK-0646 to induce complete inhibition of cell growth even in the sensitive cell lines and being less effective than cetuximab. In 1CC8, the compensatory EGFR activation seen in its parent line SCCl was not observed with MK-0646 treatment, and activation status of all three receptors upon IGF-I stimulation had very little effects on activation of AKT and MAPK suggesting other receptors or pathways may be involved in the MK-0646 resistance. JHXJO 12 which was the most resistant to both MK-0646 and cetuximab had the lowest detectable levels of GF-I R and EGFR₅ but had the highest level of phospho-MET at baseline suggesting that MET may be an important signaling pathway for de novo resistance to MK-0646 and cetuximab. Interestingly, when activation levels of all three receptors were decreased upon MK-0646 treatment in JHUOl 2, activation of AKT was increased, again suggesting other receptors may be involved in the MK-0646 resistance.

Conclusion

The IGFlR inhibitors appear to enhance Cetuximab response in cell lines that are already sensitive to cetuximab, and to enhance MTX response by modulating genes associated with MTX resistance; thereby providing alternative regimens for recurrent or refractory HNSCC patients who have developed resistance to cetuximab and MTX therapies. SEQUENCE LISTING

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Asp VaI GIn Leu GIn GIu Ser GIy Pro GIy Leu VaI Lys Pro Ser GIn Ser Leu Ser Leu Thr Cys Ser VaI Thr GIy Tyr Ser He Thr GIy GIy Tyr Leu Trp Asn Trp He Arg GIn Phe Pro GIy Asn Lys Leu GIu Trp Met GIy Tyr He Ser Tyr Asp GIy Thr Asn Asn Tyr Lys Pro Ser Leu Lys Asp Arg De Ser He Thr Arg Asp Thr Ser Lys Asn GIn Phe Phe Leu Lys Leu Asn Ser VaI Thr Asn GIu Asp Thr Ala Thr Tyr Tyr Cys Ala Arg Tyr GIy Arg VaI Phe Phe Asp Tyr Trp GIy GIn GIy Thr Thr Leu Thr VaI Ser Ser - SEQ. DD. NO. 13

Claims

WHAT IS CLAIMED:

1. A method for screening potential combination cancer therapeutic which comprises: growing a transformed eukaryotic host cell with an EGFR gene under conditions favoring said cell to hyper proliferate and become cancerous in the presence of a combination therapeutic comprising Cetuximab and MK-0646; growing said transformed eukaryotic host cell in the absence of said combination therapeutic, determining the rate of growth of said host cell in the presence of said combination therapeutic and the rate of growth of said host cells in the absence of said combination therapeutic; and comparing the growth rate of said host cells, wherein a slower rate of growth of said host cell in the presence of said combination therapeutic compound is indicative of a cancer therapeutic.

2. A method of treating recurrent or metastatic HNSCC comprising administering to a patient in need of such treatment an effective amount of a combination therapeutic agent that inhibits cell proliferation sufficient to effect said treatment, wherein the combination therapeutic agent comprises an anti-IGF-lR antibody or an antigen binding fragment thereof and an EGFR-specific antibody or an antigen binding fragment thereof, wherein the IGF-IR antibody is a humanized antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises at least one CDR comprising an amino acid sequence selected from the group consisting of SEQ ED NOs. 4, 5 or 6 and the light chain comprises at least one CDR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 1 , 2 or 3.

3. The method according to Claim 2, wherein the humanized antibody, or one of its functional fragments, comprises a light chain comprising the amino acid sequence selected from the group consisting of SEQ ID No. 7 or 8, or a heavy chain comprising the amino acid sequence selected from the group consisting of SEQ ID NOs.: 9, 10 or 11.

4. The method according to claim 2, further comprising a cytotoxic agent selected from the group consisting of platinums, taxanes or methotrexate.

5. The method according to one of claims 2 or 3, wherein one of the individual components of the combination therapeutic agent is administered before, after or concomitantly with the other member of the combination therapeutic agent.

6. A combination therapeutic effective to treat recurrent or metastatic Head and Neck squamous cell carcinoma comprising a therapeutically effective amount of an IGF-IR antibody or an antigen binding fragment thereof and an EGFR-specific antibody or an antigen binding fragment thereof and a pharmaceutically effective carrier, wherein said IGF-IR antibody is Dalotuzumab and comprises a heavy chain comprising the amino acid sequence selected from the group consisting of SEQ ID NOs.: 9, 10 or 11 and the EGFR antibody is Cetuximab.

7. The combination therapeutic according to claim 6 further comprising a cytotoxic agent.

8. The combination therapeutic according to claim 6, wherein said each of said IGF-IR antibody and said EGFR-specific antibody is administered together or separately.

9. A synergistic pharmaceutical combination of anti-cancer compounds which comprises a) an epidermal growth factor receptor antibody, Cetuximab, and b) an insulin- like growth factor -1 receptor antibody, MK-0646 (Dalotuzumab), in which the active ingredients are present in each case in free form or as a pharmaceutically acceptable salt, solvate or ester.

10. A synergistic pharmaceutical combination of anti-cancer compounds which comprises a) an orally administered methotrexate compound and b) an IGF-IR specific antibody, MK-0646, in which the active ingredients are present in each case in free form or as a pharmaceutically acceptable salt, solvate or ester.

11. The method according to claim 2, wherein each of the IGF-IR antibody and the EGFR-specific antibody are administered separately.

12. The method according to claim 3, wherein each of the IGF-IR antibody and the cytotoxic agent are administered separately.

13. The method according to claim 2, wherein the IGF-IR antibody is administered at a dose of 10 mg/kg weekly.

14. The method of Claim 13 wherein the IGF-IR antibody is administered once a week.

15. The method of Claim 13 wherein the dalotuzumab is administered once every other week.