WO2007093008A1

WO2007093008A1 - Antibodies to insulin-like growth factor i receptor

Info

Publication number: WO2007093008A1
Application number: PCT/AU2007/000168
Authority: WO
Inventors: Mehrnaz Keyhanfar; John Wallace; Grant Booker; Briony Forbes
Original assignee: Adelaide Research and Innovation Pty Ltd
Current assignee: Innovation and Commercial Partners Pty Ltd
Priority date: 2006-02-17
Filing date: 2007-02-16
Publication date: 2007-08-23
Anticipated expiration: 2008-08-17

Abstract

The present invention relates to an antibody to insulin-like growth factor I receptor, or an antigen-binding portion of the antibody. The antibody, or the antigen-binding portion, bind to an epitope located in the cysteine-rich domain of the α-subunit of the insulin-like growth factor I receptor, and the antibody or the antigen-binding portion modulates IGF-I mediated proliferation of an IGF-I dependent cell.

Description

ANTIBODIES TO INSULIN-LIKE GROWTH FACTOR I RECEPTOR

This application claims priority from Australian Provisional Patent Application No. 2006900785 filed on 17 February 2006, the contents of which are to be taken as incorporated herein by this reference.

Field of the Invention

The present invention relates to antibodies to insulin-like growth factor I receptor, to isolated cells expressing the antibodies and pharmaceutical compositions including the antibodies.

The present invention also relates to a method of detecting insulin-like growth factor receptor I using the antibodies, a method of modulating proliferation of an IGF-I dependent cell using the antibodies, and a method of preventing and/or treating an IGF-I dependent disease or condition using the antibodies.

Background of the Invention

The insulin-like growth factors, also known as somatomedins, include insulin-like growth factor-I (IGF-I) and insulin-like growth factor-II (IGF-II). These growth factors exert mitogenic activity on various cell types, including tumour cells, by binding to a common receptor named insulin-like growth factor I receptor (IGF-IR). Interaction of IGFs with IGF-IR activates the receptor by triggering autophosphorylation of the receptor on tyrosine residues. Once activated, IGF-IR in turn phosphorylates intracellular targets to activate various cellular signaling pathways.

There is considerable evidence that IGF-IR activation is critical for stimulation of tumour cell growth and survival. Several lines of evidence also indicate that IGF-I, IGF- II and IGF-IR are important mediators of the malignant phenotype. For example, over- expression of IGF-IR has been demonstrated in several cancer cell lines and tumour tissues. Increased IGF-I levels are also correlated with several non-cancerous pathological states, including acromegaly and gigantism, while abnormal IGF-I/IGF-I receptor function has been implicated in conditions such as psoriasis, atherosclerosis and smooth muscle restenosis of blood vessels following angioplasty. Increased IGF-I levels may also be problematic in diabetes, or in complications thereof, such as microvascular proliferation. Decreased IGF-I levels are also associated with neuropathy and osteoporosis.

There is a need for new agents and methods capable of modulating IGF-I binding to IGF-IR, particularly for the prevention and/or treatment of diseases and conditions in which the IGF-I receptor and/or IGF-I play a role. There is also a need for new agents capable of detecting IGF-IR.

The present invention relates to antibodies that specifically bind to IGF-IR and which have the capacity to modulate the binding of IGF-I to the receptor.

A reference herein to a patent document or other matter which is given as prior art is not to be taken as an admission that that document or matter was, in Australia or any other country, known or that the information it contains was part of the common general knowledge as at the priority date of any of the claims.

Summary of the Invention

The present invention provides an antibody to insulin-like growth factor I receptor, or an antigen-binding portion of the antibody, the antibody or the antigen-binding portion binding to an epitope located in the cysteine -rich domain of the α-subunit of the insulin- like growth factor I receptor, wherein the antibody or the antigen-binding portion modulates IGF-I mediated proliferation of an IGF-I dependent cell. The present invention also provides an antibody, or an antigen binding portion thereof, including the following CDR amino acid sequences:

(i) a V_H CDR-I sequence according to SEQ ID NO. 20, and a V_n CDR-2 sequence according to SEQ ID NO. 21, and a V_H CDR-3 sequence according to SEQ ID NO. 8; and/or

(ii) a V_L CDR-I sequence according to SEQ ID NO.22, and a V_L CDR-2 sequence according to SEQ ID NO. 23, and a V_L CDR-3 sequence according to SEQ ID NO.11; and/or an antibody, or an antigen-binding portion thereof, including a variant of one or more of the aforementioned sequences, that binds to IGF-I receptor.

The present invention also provides an antibody, or an antigen binding portion thereof, including the following amino acid sequences:

(i) a V_H CDR-I amino acid sequence according to SEQ ID NO.20; and (ii) a V_H CDR-2 amino acid sequence according to SEQ ID NO.21; and

(iii) a V_H CDR-3 amino acid sequence according to SEQ ID NO.8; and (iv) a V_L CDR-I amino acid sequence according to SEQ ID NO. 22; and (iv) a V_L CDR-2 amino acid sequence according to SEQ ID NO. 23; and (v) a V_L CDR-3 amino acid sequence according to SEQ ID NO. 11; and/or an antibody, or an antigen-binding portion thereof, including a variant of one or more of the aforementioned sequences, that binds to IGF-I receptor.

The present invention also provides an isolated compound including one or more of the amino acids sequences selected from the group consisting of SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.8, SEQ ID NO.22, SEQ ID NO.23, and SEQ ID NO.l 1.

The present invention also provides an isolated nucleic acid including a nucleotide sequence encoding a polypeptide including one or more of the amino acids sequences selected from the group consisting of SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.8, SEQ ID NO.22, SEQ ID NO.23, and SEQ ID NO.11. The present invention arises out of studies into the development of antibodies to soluble human insulin-like growth factor I receptor. In particular, it has been found that certain antibodies may be produced that bind to an epitope located in the cysteine-rich domain of the α-subunit of the receptor and which also modulate IGF-I dependent proliferation of a cancer cell line. These antibodies have either an IgGl or IgM isotype. Sequencing of the variable regions of these antibodies demonstrates that they contain unique antigen-binding sequences.

Various terms that will be used throughout the specification have meanings that will be well understood by a skilled addressee. However, for ease of reference, some of these terms will now be defined.

The term "variant" as used throughout the specification is to be understood to mean an amino acid sequence of a progenitor polypeptide or protein that is altered by one or more amino acids. The variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties to the replaced amino acid (e.g., replacement of leucine with isoleucine). A variant may also have "non-conservative" changes (e.g., replacement of a glycine with a tryptophan) or a deletion and/or insertion of one or more amino acids. The term also includes within its scope any insertions/deletions/fusions of amino acids to a particular polypeptide or protein.

Generally, the variant will be a functional variant, meaning that the variant substantially retains the functional capacity of the progenitor polypeptide or protein, such as an antibody including a variant of a particular CDR sequence that retains binding to the particular epitope of the progenitor antibody.

The term "antibody" as used throughout the specification means an entire antibody molecule or any antigen-binding portion of an antibody molecule. In this regard, the term "antigen binding portion" as used throughout the specification is to be understood to mean an antigen-binding portion of an antibody molecule including a Fab, Fab', F(ab')₂, Fv, a single-chain antibody (scFv), a chimeric antibody, a diabody or any polypeptide that contains at least a portion of an immunoglobulin (or a variant of an immunoglobulin) that is sufficient to confer specific antigen binding.

The term "nucleic acid" as used throughout the specification is to be understood to mean to any oligonucleotide or polynucleotide. The nucleic acid may be DNA or RNA and may be single stranded or double stranded. The nucleic acid may be any type of nucleic acid, including a nucleic acid of genomic origin, cDNA origin (ie derived from a mRNA), derived from a virus, or of synthetic origin.

In this regard, an oligonucleotide or polynucleotide may be modified at the base moiety, sugar moiety, or phosphate backbone, and may include other appending groups to facilitate the function of the nucleic acid. The oligonucleotide or polynucleotide may be modified at any position on its structure with constituents generally known in the art. For example, an oligonucleotide may include at least one modified base moiety which is selected from the group including 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5- iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyliydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1- methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2- methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5- methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta D- mannosylqueosine, S'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6- isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil5- oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3- (3- amino-3-N-2-carboxypropyl) uracil, (acp3) w, and 2,6-diaminopurine. The oligonucleotide or polynucleotide may also include at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2- fluoroarabinose, xylulose, and hexose. In addition, the oligonucleotide or polynucleotide may include at least one modified phosphate backbone, such as a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or any analogue thereof.

The term "isolated" as used throughout the specification is to be understood to mean an entity, for example a polypeptide, nucleic acid, antibody or a cell, which is purified and/or removed from its natural environment.

The term "polypeptide" as used throughout the specification is to be understood to mean any polypeptide comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds. "Polypeptide" refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than those normally encoded by a codon.

Polypeptides may also include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art. Modifications may occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini.

Brief Description of the Figures

Figure 1 shows fluorescence-activated cell-sorting analysis to determine the binding of various mAbs to the different receptors (IR-A, IR-B and IGF-IR). Cells over-expressed different receptors (IRA, IRB or IGF-IR). The cells were incubated with neat supernatant of the mAbs followed by incubation with sheep anti-mouse IgG FITC- conjugated, which was used as a secondary antibody. Figure 2 shows the isotype of various mAbs against IGF-IR. The mouse Typer™- Mouse Sub-isotyping panel (BIORAD) was used to determine the isotype of the antibodies. The neat supernatant from sub-clones of parental 9El 1, 7C2, 4C6, 5B6 and 5B2 were applied to the assay. The IgG2a mAb 24-60 was employed for this assay as a positive control.

Figure 3 shows purity of purified mAbs 7C2 and 9El 1. The purified mAbs were run on a 10% SDS-polyacrylamide gel under reducing conditions. The IgGl control purified Monoclonal Antibody (CHEMICON Cat. No. MABC002, 987710005, Australia) (containing 0.2% bovine serum albumin) was applied as a control.

Figure 4 shows epitope mapping by FACS. FACS analyses were performed on cells, which express chimeric receptors, incubated with the mAbs against IGF-IR. Competition between 7C2, 9El 1 and other previously characterised mAbs, 24-60 and αIR-3 is also shown.

Figure 5 shows the ability of 9El 1 and 7C2 mAbs to immunoprecipitate the IGF-IR from lysed P6 cells. The antibodies are able to detect IGF-IR on immunoblots of P6 lysates separated on SDS-polyacrylamide gels run under reducing conditions but not under non-reducing conditions.

Figure 6 shows blocking activity of the mAbs against the IGF-IR. The figure shows the blocking activity on Europium-IGF-I by mAbs 9El 1, 7C2, 24-60, unrelated IgGl, or ligand IGF-I. The solubilised IGF-IR was captured on the plate by mAb 24-31. Results are expressed as a percentage of Eu-IGF-I binding in the absence of competing MAb (Buffer). 10 nM of unlabelled IGF-I was used as control to compete with Eu-IGF-I respectively for binding to the receptor. The unrelated IgGl antibody was used as a negative control. The graph is the representative of three separate experiments. Figure 7 shows blocking activity of the mAbs against the IGF-IR. The figure shows the lack of blocking activity on Europium-IGF-II by mAbs 9El 1, 7C2, 24-60, unrelated IgGl, or ligand IGF-I. The solubilised IGF-IR was captured on the plate by mAb 24-31. Results are expressed as a percentage of Eu-IGF-II binding in the absence of competing MAb (Buffer). 10 nM of unlabelled IGF-II was used as control to compete with Eu-IGF-II respectively for binding to the receptor. The unrelated IgGl antibody was used as a negative control. The graph is the representative of three separate experiments.

Figure 8 shows the ability of different mAbs to inhibit the binding of Europium-IGF-I to the soluble receptor. The Europium binding assay in Figure 6 was used to evaluate the ability of the mAbs 9El 1, 7C2 and 24-60 to inhibit the binding of IGF-I to the solubilized receptor from P6 cells. Fluorescence was obtained and determined as a percentage of maximum binding (when there is no competitor) for four samples which were the average of triplicates of four separate experiments and plotted + SEM. EC50 values of mAb 9El 1, 24-60 and 7C2 inhibited Europium-IGF-I binding, generated from the curves. Non-linear regression was performed on GraphPad Prism.

Figure 9 shows the BIAcore sensograms of the interaction between 7C2 and ss-IGF-lR (recombinant soluble ectodomain of the IGF-IR, residues 1-906). A variety of s-IGF-lR concentrations were passed over the mAb 7C2 sensorsurface (50RU).

Figure 10 shows the effect of IGF-I on binding of the s-IGF-lR to the mAb 9El 1. The figure shows BIAcore sensograms of the interaction between the mAb 9El 1 sensorsurface and s-IGF-lR solution (12.5 nm), containing different concentrations of IGF-I (A), IGF-Il (B), BCIIAD which is IGF-II containing the IGF-I C domain (C) or BCIAD which is IGF-I containing the IGF-II C domain (D).

Figure 11 shows the ability of different mAbs to inhibit proliferation of the colon cancer cell line HT-29 in the presence of 5 mM sodium butyrate and 10 nM IGF-I (Figure HA) or 50 nM IGF-II (Figure HB). Proliferation was measured by the detection of ATP by the Cell-Titre GIo luminecent cell viability assay system. Mean luminescence was measured for triplicate samples for different concentrations of mAbs. The HT-29 cells were treated with butyrate (5mM) as chemotherapeutic reagent. 10 nM IGF-I (A) or 50 nM IGF-II (B) rescued the cells from death induced by butyrate.

Figure 12 shows histological analysis of binding of mAbs 9El 1, 7C2, 24-60, and an unrelated mAb to P6 cells.

Figure 13 shows the nucleotide and deduced amino acid sequences of the variable region of MAb 9El 1 and 7C2 genes. The V_H sequences for MAbs 9El 1 (A) and 7C2 (C) and the V_L sequences for MAbs 9El 1 (B) and MAb 7C2 (D) are shown. The sequences are 5' to 3' and the sequences complementary to primers used to amplify these regions are highlighted in grey. The CDR regions identified using the definition of IMGT/V-QUEST are shown in boxes.

Figure 14 shows the effects of the alanine mutations or chimeric IGF-1R/256-266IR on binding the Fab domains of MAbs 7C2 and 9El 1 to the IGF-IR. A) The binding to each mutant is shown as a percentage of binding Eu-7C2 and Eu-9E11 to 1:20 dilution of culture supernatants from cells secreting s-IGF-lR (containing 0.28 mg/ml s-IGF-lR). For each mutant the supernatant containing the receptor was diluted to give the same binding to 0.28 mg/ml s-IGF-lR in the ELISA test. The numbers in the x-axis relate to the amino acid number in the IGF-IR mutated to alanine. Chimeric refers to the chimeric IGF-1R/256-266IR. The graph shown is representative of three experiments and bars are means ± SD of triplicates. B) The Ca backbone of the IGF-IR cysteine- rich domain is shown as a ribbon and the amino acids mutated are shown in spacefilling representation. Alanine mutants of amino acids showed in black had disruptive effect on the Eu-7C2 and Eu-9E11 binding. Alanine mutants of those amino acids shown in white had no effect on binding the MAbs. The figure was created using the UCSF Chimera molecular graphics program.

Figure 15 shows the effect of different anti-IGF-lR MAbs on MCF-7 cells migration (A) and the effect of the MAbs on the IGF-IR down-regulation in MCF-7 cells (B). A) The MCF-7 cells migration was induced by different concentrations of IGF-I and a constant concentration (25 nM) of various MAbs were used to inhibit the induced migration. The unrelated IgGl antibody was applied as a negative control. The graph shown is a representative of three separate experiments and bars are means ± SD of triplicates. Symbols indicate statistically significantly different from control values at 0.01< P <0.05 = *, 0.001 < P < 0.01 = If, P < 0.001= #. The P values were calculated by comparing the data for no treatment (Buffer) with other data corresponding to relevant concentration of IGF-I using unpaired t-tests (Confidence Intervals 95%) by the Prism 3.03 software. B) MCF-7 cells were treated with IGF-I or different MAbs against the IGF-IR. Lane 1: No treatment, Lane 2: IGF-I treated, Lane 3: 9El 1 treated, Lane 4: 7C2 treated, Lane 5: 24-60 treated, Lane 6: αIR-3 treated, Lane 7: R^" cell lysates (no treatment), a) Pro-IGF-1R, b) IGF-IR β subunit, c) non-specific band (used here as loading control). Molecular weight was estimated in kilodaltons using the MagicMark™XP marker. A representative experiment is shown.

General Description of the Invention

As described above, in one embodiment the present invention provides an antibody to insulin-like growth factor I receptor, or an antigen-binding portion of the antibody, the antibody or the antigen-binding portion binding to an epitope located in the cysteine- rich domain of the α-subunit of the insulin-like growth factor I receptor, wherein the antibody or the antigen-binding portion modulates IGF-I mediated proliferation of an IGF-I dependent cell.

This embodiment of the present invention provides an antibody (or an antigen-binding portion thereof) that binds to an epitope (the cysteine -rich domain) located in the extracellular domain of the insulin-like growth factor I receptor (IGF-IR), and which has the capacity to modulate IGF-mediated proliferation of IGF-I dependent cells.

The modulation of IGF-I mediated proliferation in the various embodiments of the present invention may be an inhibition or promotion of proliferation.

In one embodiment, the antibody in the various embodiments of the present invention inhibits IGF-I mediated proliferation of a cell. In another embodiment, the antibody in the various embodiments of the present invention promotes IGF-I mediated proliferation of a cell.

The insulin-like growth factor I receptor is a transmembrane heterotetrameric protein, which has two extracellular alpha chains and two membrane-spanning beta chains in a disulfide-linked β-α-α-β configuration. The binding of insulin-like growth-factor-I

(IGF-I) and insulin-like growth factor-II (IGF-II) by the extracellular domain of IGF-I receptor activates the intracellular tyrosine kinase domain resulting in autophosphorylation of the receptor and substrate phosphorylation. The IGF-I receptor is homologous to insulin receptor, having a high sequence similarity in the α chain tyrosine kinase domain and a lower sequence similarity in the α chain.

The α-subunit of the receptor consists of a number of sub domains, designated 1, 2, 3' and 3", as defined in Schumacher et al. (1993) J. Biol. Chem. 268(2): 1087-1094. In the human receptor, sub domain 1 corresponds to amino acids 1 to 130, sub domain 2 corresponds to amino acids 131 to 315 and is referred to as the "cysteine -rich domain", sub domain 3' corresponds to amino acids 316 to 514, and sub domain 3" corresponds to amino acids 515 to 706. The insulin-like growth factor I receptor from other species has a similar arrangement of sub domains.

The IGF-I receptor and its ligands (IGF-I and IGF-II) play important roles in numerous physiological processes including growth and development during embryogenesis, metabolism, cellular proliferation and cell differentiation in adults.

The IGF-I receptor has also been implicated in promoting growth, transformation and survival of tumour cells. Several types of tumours express higher than normal levels of IGF-I receptor, including breast cancer, colon cancer, ovarian carcinoma, synovial sarcoma and pancreatic cancer. In vitro, IGF-I and IGF-II have been shown to be potent mitogens for several human tumour cell lines such as lung cancer, breast cancer, colon cancer, osteosarcoma and cervical cancer. Several of these tumours and tumour cell lines also express high levels of IGF-I or IGF-II, which may stimulate their growth in an autocrine or paracrine manner. Down-regulation of the IGF-I receptor level has also been shown to reduce the tumourigenicity of several tumour cell lines in vivo and in vitro.

Previous studies have also demonstrated that R^" cells (mouse 3T3-like cells with a targeted removal of the IGF-IR gene) could not be transformed by a number of cellular and viral oncogenes. Conversely IGF-IR overexpression or increased IGF-IR kinase activity is associated with a broad range of human cancers, including breast cancer and is seen in both primary and immortalized cervical cancer cell lines. A correlation has been made between high levels of IGF-IR expression and both higher grade and later stage of colorectal cancer. Activation of IGF-IR by IGFs increases the proliferation and migration of cancer cell lines derived from many cancer types, including breast, prostate and colon cancer. IGF-IR is also involved in protection of tumour cells from cytotoxic effects of chemotherapeutic agents. Overexpression of IGF-IR promotes cellular radioresistance and local breast cancer reappearance after radiation therapy and lumpectomy.

The primary amino acid of the human IGF-I receptor is provided in SEQ ID No. 1. This amino acid sequence is derived from GenBank Accession No: CAA28030. The cysteine -rich domain of the α-subunit is located between amino acids 131 to 315. IGF-I receptors from other species may be readily identified by a person skilled in the art, for example by comparison of the amino acid or nucleotide sequences.

The antibody according to the various embodiments of the present invention includes a monoclonal or polyclonal antibody, and which binds to the insulin-like growth factor I receptor. In one embodiment, the antibody binds to the human receptor.

The antibody according to the various embodiments of the present invention may also be an isolated antibody. Methods for producing and isolating polyclonal and monoclonal antibodies are known in the art. In this regard, an antibody is an intact immunoglobulin. An immunoglobulin is a tetrameric molecule, each tetramer being composed of two identical pairs of polypeptide chains, each pair having one light chain and one heavy chain. The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids that is 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, IgD, IgG, IgA, and IgE, 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. The variable regions of each light/heavy chain pair form the antibody binding site, with the result that an intact immunoglobulin has two binding sites.

The variable regions further include hypervariable regions that are directly involved in formation of the antigen binding site. These hypervariable regions are usually referred to as Complementarity Determining Regions (CDR). The intervening segments are referred to as Framework Regions (FR). In both light and heavy chains there are three CDRs (CDR-I to CDR-3) and four FRs (FR-I to FR-4).

In the various embodiments of the present invention, the antigen-binding portion of an antibody molecule includes for example a Fab, Fab', F(ab')₂, Fd, Fv, a single-chain antibody (scFv), a chimeric antibody, a diabody or a polypeptide that contains at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding.

A Fab fragment is a monovalent fragment consisting of the V_L, V_H, C_L and C_H I domains. A F(ab')₂ fragment is a bivalent fragment including two Fab fragments linked by a disulphide bridge at the hinge region. A Fd fragment consists of the V_H and C_H I domains. A Fv fragment consists of the V_L and V_H domains of a single arm of an antibody. A dAb consists of a V_H domain. A single chain antibody (scFv) is an antibody in which V_L and V_H regions are paired to form a monovalent molecule via a synthetic linker that enable them to be made as a single protein chain. Diabodies are bivalent, bispecific antibodies in which V_H and V_L domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites.

Methods for producing antigen-binding portions of antibodies are known in the art, for example as described in "Antibody Engineering: Methods and Protocols" (2004) ed. by B.K.C. Lo Humana Press, herein incorporated by reference; and "Antibody Engineering: A Practical Approach" (1996) ed. by J. McCafferty, H.R. Hoogenboom and DJ. Chriswell Oxford University Press, herein incorporated by reference.

In one embodiment, the antibody is a mouse or a human antibody, or a humanized antibody. Methods for humanizing antibodies are known in the art.

In this regard, it will be understood that the antibodies and antigen-binding portions in the various embodiments of the present invention include humanized antibodies and antigen-binding portions thereof, in which amino acids have been replaced in the non- antigen binding regions in order to more closely resemble a human antibody, while still retaining the original binding ability.

In the case where the antibody in the various embodiments of the present invention is produced by raising the antibody against a receptor antigen, the antibody may be raised against any IGF-IR receptor, provided that the receptor includes the cysteine -rich domain of the α-subunit of the receptor. In one embodiment, the antibody to insulin-like growth factor I receptor is an antibody raised against an animal or human insulin-like growth factor I receptor.

In this case, the antibody may also be raised against any form of the receptor, including a fragment of a receptor, a soluble form of the receptor or the receptor expressed on the surface of a cell. In one embodiment, the antibody is an antibody raised against a soluble form of the insulin-like growth factor I receptor. Once again, the form of the receptor will include the cysteine -rich domain of the α-subunit of the receptor.

As discussed previously herein, the antibody in the various embodiments of the present invention may be a polyclonal or a monoclonal antibody. In one embodiment, the antibody is a monoclonal antibody.

In one embodiment, the monoclonal antibody is 9El 1 or 7C2, as defined herein.

In this regard, the sequence of the V_H and V_L regions of 9E11 are as follows:

9E11 V_H (SEQ ID NO.2):

gggaattcatggagttcgggctaagctgggttttccttgtccttgttttaaaaggtgtcctg tgtgacgtgaagctcgtggagtctgggggaggcttagtgaagcttggagggtccctgaaa ctctcctgtgcagcctctggattcactttcagtaacttttacatgtcttgggttcgcctg actccagagaagaggctggaattggtcgcagccattaatagttatggtggtagtacctac tatccagacactgtgaagggccgattcaccatctccagagacaatgccaagagcaccctg tacctgcaaatgagcagtctgaagtctgaggacacagccttgtattactgtgtaagacag gcccccgattactacggtagtaaccgctggtacttcgatgtctggggcgcagggaccacg gtcaccgtctcctcagccaaaacgacacccccatccgtttatcccctggcccctggaagc ttggg

9EHV_L(SEQ ID NO.3):

actagtcgacatgaagttgcctgttaggctgttggtgctgatgttctggattcctgcttcg agcagtgatgttttgatgacccaaactccactctccctgcctgtcagtcttggagatcaa gcctccatctcttgcagatctagtcagaccattgtacatagtaatggaaacacctattta gaatggttcctgcagaaaccaggccagtctccaaagctcctgatctacaaagtttccaac cgattttctggggtcccagacaggttcagtggcagtggatcagggacagatttcacactc aagatcagcagagtggaggctgaggatctgggagtttattactgctttcaaggttcacat gttccgtggacgttcggtggaggcaccaagctggaaatcaaacgggctgatgctgcacca actgtatccatcttcccaccatccagtaagcttggg The amino acid sequence corresponding to the nucleotide sequence in the V_H region of 9El 1 is as follows:

9E11 V_H (SEQ ID NO.4):

EFMEFGLSWVFLVLVLKGVLCDVKLVESGGGLVKLGGSLKLSCAASGFTFSNF YMSWVRLTPEKRLELVAAINSYGGSTYYPDTVKGRFTISRDNAKSTL YLQMSS LKSEDTALYYCVRQAPDYYGSNRWYFDVWGAGTTVTVSSAKTTPPSVYPLAP GSLG

9EH V_L (SEQ ID NO.5):

LVDMKLPVRLLVLMFWIPASSSDVLMTQTPLSLPVSLGDQASISCRSSQTIVHSN GNTYLEWFLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDL GVYYCFQGSHVPWTFGGGTKLEIKRADAAPTVSIFPPSSKLG

The CDR sequences for the V_H region of 9El 1 are as follows:

CDR-I : GFTFSNFY (SEQ ID NO.6) CDR-2: INSYGGST (SEQ ID NO.7)

CDR-3: VRQAPDYYGSNRWYFDV (SEQ ID NO.8)

The CDR sequences for the V_L region of 9El 1 are as follows:

CDR-I : QTIVHSNGNTY (SEQ ID NO.9) CDR-2: KVS (SEQ ID NO.10) CDR-3: FQGSHVPWT (SEQ ID NO.11) The sequence of the V_H and V_L regions of 7C2 is as follows:

7C2 V_n (SEQ ID NO.12): actagtcgacatgaacttcgggctgagcttggttttccttgtccttgttttaaaaggtgtc ctgtgtgacgtgaagctcgtggagtctgggggaggcttagtgaagcttggagggtccctg aaactctcctgtgcagcctctggattcactttcagtagttattacatgtcttgggttcgc cagactccagagaagaggctggagttggtcgcagccgttaatagttatggtggtggcacc tactatccagacactgtgaagggccgattcaccatctccagagacaatgccaagaacacc ctgtacctgcaaatgagcagtctgaagtctgaggacacagccttgtatcactgtgtaaga caggcccccgattactacggtagtaaccgctggtacttcgatgtctggggcgcagggacc acggtcaccgtctcctcagccaaaacgacacccccatccgtctatcccttggcccctgga agcttggg

7C2 V_L(SEQ ID NO.13):

actagtcgacatgaagttgcctgttaggctgttggtgctgatgttctggattcctgcttcc agcagtgatgttttgatgacccaaagtccactctccctgcctgtcagtcttggagatcaa gcctccatctcttgcagatctagtcagagtattgtacatagtaatggaaacacctattta gaatggttcctgcagaaaccaggccagtctccaaagctcctgatctaccaagtttccaac cgattttctggggtcccagacaggttcagtggcagtggatcagggacagatttcacactc aagatcagcagagtggaggctgaggatctgggagtttattactgctttcaaggttcacat gttccgtggacgttcggtggaggcaccaagctggaaatcaagcgggctgatgctgcacca actgtatccatcttcccaccatccagtaagcttggg

The amino acid sequence corresponding to the nucleotide sequence in the V_H region of 7C2 is as follows:

7C2 V_n (SEQ ID NO.14):

LVDMNFGLSLVFLVLVLKGVLCDVKLVESGGGLVKLGGSLKLSCAASGFTFSS YYMSWVRQTPEKRLELVAAVNSYGGGTYYPDTVKGRFTISRDNAKNTLYLQM SSLKSEDTALYHCVRQAPDYYGSNRWYFDVWGAGTTVTVSSAKTTPPSVYPL APGSLG 7C2 V_L (SEQ ID NO.15):

LVDMKLPVRLLVLMFWIPASSSDVLMTQSPLSLPVSLGDQASISCRSSQSIVHSN GNTYLEWFLQKPGQSPKLLIYQVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDL GVYYCFQGSHVPWTFGGGTKLEIKRADAAPTVSIFPPSSKLG

The CDR sequences for the V_H region of 7C2 are as follows:

CDR-I : GFTFSSYY (SEQ ID NO.16) CDR-2 : VNSYGGGT (SEQ ID NO.17)

CDR-3: VRQAPDYYGSNRWYFDV (SEQ ID NO.8)

The CDR sequences for the V_L region of 7C2 are as follows:

CDR-I : QSIVHSNGNTY (SEQ ID NO.18) CDR-2: QVS (SEQ ID NO.19) CDR-3: FQGSHVPWT (SEQ ID NO.11)

The CDR sequences for the V_H region of 9El 1 and 7C2 share 6/8 identical amino acids in CDR-I and CDR-2, and are identical in the CDR-3, as follows:

CDR-I : GFTFSN/SF/YY (SEQ ID NO.20) CDR-2: 1/VNSYGGS/GT (SEQ ID NO.21)

The CDR sequences for the V_L region of 9El 1 and 7C2 share 10/11 identical amino acids in CDR-I, 2/3 amino acids in CDR- , and identical in the CDR-3, as follows:

CDR-I : QT/SIVHSNGNTY (SEQ ID NO.22) CDR-2: K/QVS (SEQ ID NO.23) Accordingly, in another embodiment the present invention provides an antibody, or an antigen binding portion thereof, the antibody or the binding portion thereof including the following CDR amino acid sequences:

In one embodiment, the antibody, or the antigen binding portion thereof, includes the following CDR amino acid sequences:

Antibodies to insulin-like growth factor I receptor may be generated using methods known in the art. For the production of antibodies, various hosts including goats, rabbits, rats, mice, humans, and others, may be immunized by injection with the receptor, or a fragment of the receptor that provides an epitope located between amino acids 131 to 315 of the receptor.

In one embodiment, the antibody is raised to an epitope in the cystein-rich domain of the α-subunit of the insulin-like growth factor I receptor that includes one or more amino acids selected from the group consisting of phenylalanine 241, phenylalanine 252 and phenylalanine 266. Depending on the host species, various adjuvants may be used to increase an immunological response. Such adjuvants include Freund's adjuvant, mineral gels such as aluminium hydroxide, and surface-active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.

A polyclonal antibody is an antibody that is produced among, or in the presence of one or more other, non-identical antibodies. Methods for producing and isolating polyclonal antibodies are known in the art. In general, polyclonal antibodies are produced from B- lymphocytes. Usually, polyclonal antibodies are obtained directly from an immunized subject, such as an immunized animal.

Monoclonal antibodies may be prepared using any technique that provides for the production of antibody molecules by continuous isolated cells in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique. Methods for the preparation of monoclonal antibodies are as generally described in Kohler et al. (1975) Nature 256:495-497, herein incorporated by reference; Kozbor et al. (1985) J. Immunol. Methods 81:31-42, herein incorporated by reference; Cote et al. (1983) Proc. Natl. Acad. ScL 80:2026-2030, herein incorporated by reference; and Cole et al. (1984) MoI. Cell Biol. 62: 109-120, herein incorporated by reference.

The present invention also provides isolated compounds including one or more of the CDR sequences identified for 9El 1 and 7C2. In this regard, the compounds may be polypeptides, or a compound having a non-polypeptide component and a polypeptide component associated with one or more of the CDR sequences. Methods for coupling one or more CDR sequences to polypeptide or non-polypeptide backbones are known in the art. Accordingly, in another embodiment the present invention provides an isolated compound including one or more of the amino acids sequences selected from the group consisting of SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.8, SEQ ID NO.22, SEQ ID NO.23, and SEQ ID NO.11. The compound may include a variant of one or more of the aforementioned sequences that binds to IGF-I receptor.

Such compounds that function as antibody mimetics are known in the art. In one embodiment, the compound is a polypeptide. In one specific embodiment, the polypeptide is an antibody or an antigen binding portion thereof.

The present invention also provides a cell expressing an antibody (or antigen-binding portion thereof) according to the various embodiments of the present invention, including isolated cells.

Accordingly, in another embodiment the present invention provides an isolated cell that expresses an antibody to insulin-like growth factor I receptor, or an antigen-binding portion of the antibody, the antibody or the antigen-binding portion binding to an epitope located in the cysteine-rich domain of the α-subunit of the insulin-like growth factor I receptor, wherein the antibody or the antigen-binding portion modulates IGF-I mediated proliferation of an IGF-I dependent cell.

In one embodiment, the isolated cell is a hybridoma cell.

For example, the isolated cell may be a hybridoma producing the monoclonal antibody 9El 1 or 7C2. The cell may further be an isolated cell.

In one embodiment, the present invention provides a cell expressing an antibody, or an antigen binding portion thereof, including the following CDR sequences:

(ii) a V_L CDR-I sequence according to SEQ ID NO.22, and a V_L CDR-2 sequence according to SEQ ID NO. 23, and a V_L CDR-3 sequence according to SEQ ID NO.11; and/or expressing an antibody, or an antigen-binding portion thereof, including a variant of one or more of the aforementioned sequences, that binds to IGF-I receptor.

In another embodiment, the present invention provides a cell expressing an antibody, or an antigen binding portion thereof, including:

(i) a V_H CDR-I amino acid sequence according to SEQ ID NO.20; and (ii) a V_H CDR-2 amino acid sequence according to SEQ ID NO.21; and (iii) a V_H CDR-3 amino acid sequence according to SEQ ID NO.8; and (iv) a V_L CDR-I amino acid sequence according to SEQ ID NO. 22; and (iv) a V_L CDR-2 amino acid sequence according to SEQ ID NO. 23; and

(v) a V_L CDR-3 amino acid sequence according to SEQ ID NO. 11; and/or expressing an antibody, or an antigen-binding portion thereof, including a variant of one or more of the aforementioned sequences that binds to IGF-I receptor.

In another embodiment, the present invention provides a cell that expresses a polypeptide including one or more of the amino acids sequences selected from the group consisting of SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.8, SEQ ID NO.22, SEQ ID NO.23, and SEQ ID NO.11, or expresses a polypeptide including a variant of one or more of the aforementioned sequences that binds to IGF-I receptor.

Examples of cells include prokaryotic and eukaryotic cells.

Humanized antibodies, or antibodies adapted for non-rejection by other mammals, may be produced by a suitable method known in the art, such as resurfacing or CDR grafting.

In resurfacing technology, molecular modeling, statistical analysis and mutagenesis are combined to adjust the non-CDR surfaces of variable regions to resemble the surfaces of known antibodies of the target host. Strategies and methods for the resurfacing of antibodies, and other methods for reducing immunogenicity of antibodies within a different host, are as described in US patent 5,639,641. Humanized forms of the antibodies may also be made by CDR grafting, by substituting the complementarity determining regions of, for example, a mouse antibody, into a human framework domain.

Methods for humanizing antibodies are known in the art. For example, the antibody may be generated as described in U.S. Pat. No. 6,180,370, herein incorporated by reference; WO 92/22653, herein incorporated by reference; Wright et al. (1992) Critical Rev. in Immunol. 12(3,4): 125-168, herein incorporated by reference; and Gu et al. (1997) Thrombosis and Hematocyst 77(4):755-759), herein incorporated by reference.

Humanized antibodies typically have constant regions and variable regions other than the complementarity determining regions (CDRs) derived substantially or exclusively from a human antibody and CDRs derived substantially or exclusively from the non- human antibody of interest.

Techniques developed for the production of "chimeric antibodies", for example the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, may be performed by a suitable method known in the art. For example, chimeric antibodies may be produced as described in Morrison, S. L. et al. (1984) Proc. Natl. Acad. ScL 81:6851-6855, herein incorporated by reference; Neuberger, M. S. et al. (1984) Nature 312:604-608, herein incorporated by reference; and Takeda, S. et al. (1985) Nature 314:452-454, herein incorporated by reference.

Antibody fragments that contain specific binding sites may be generated by methods known in the art. For example, F(ab')₂ fragments can be produced by pepsin digestion of the antibody molecule, and Fab fragments can be generated by reducing the disulfide bridges of the F(ab')₂ fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity, as described in Huse, W. D. et al. (1989) Science 254: 1275-1281, herein incorporated by reference. Included within the scope of the antibodies and antigen-binding portions of the various embodiments of the present invention are also variants of the antibodies and their antigen-binding portions of the present invention. For example, variants include polypeptides with amino acid sequences that are similar to the amino acid sequence of the variable or hypervariable regions of the antibodies of the present invention.

Inthis regard, the variant may have one or more "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties (eg hydrophobicity, hydrophilicity, charge) to the replaced amino acid (e.g., replacement of leucine with isoleucine). A variant may also have "non-conservative" changes (e.g., replacement of a glycine with a tryptophan) or a deletion and/or insertion of one or more amino acids.

In one embodiment, the variant has at least about 90%, such as having at least about 95% sequence identity to another amino acid sequence, as determined by the FASTA search method, as described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444-2448, herein incorporated by reference.

Various immunoassays may be used for screening to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies are known in the art.

The antibody molecules of the various embodiments of the present invention, and the antigen-binding portions thereof, may also be produced recombinantly by methods known in the art, for example by expression in E.colilTl expression systems. A suitable method for the production of recombinant antibodies is as described in US patent 4,816,567, herein incorporated by reference.

The antibodies of the present invention are highly specific for the insulin-like growth factor I receptor and bind to an epitope located in the cysteine-rich domain of the α- subunit of the receptor. The antibodies do not recognise the related insulin receptor type A (IR-A) or insulin receptor type B (IR-B). Accordingly, in another embodiment the present invention provides an antibody that specifically binds to insulin-like growth factor I receptor, or an antigen-binding portion of the antibody, the antibody or the antigen-binding portion binding to an epitope located in the cysteine -rich domain of the α-subunit of the insulin-like growth factor I receptor, wherein the antibody or antigen-binding portion does not specifically bind to insulin receptor and the antibody or the antigen-binding portion modulates IGF-I mediated proliferation of an IGF-I dependent cell.

Confirmation that an antibody binds to the desired epitope may be determined by a suitable method known in the art. For example, chimeric receptors of the insulin receptor and the insulin-like growth factor I receptor may be used. In this case, the chimeric receptor will contain amino acids 131 to 315 of IGF-IR, and the remainder of the receptor will be derived from the insulin receptor. The antibody will bind to IGF-IR, the chimeric receptor, but not to insulin-like receptor or a chimeric receptor of IGF-IR containing amino acids 131 to 315 from the insulin receptor.

In one embodiment, the isotype of the antibody is selected from the group consisting of IgGl, IgG2a, IgG2b, IgG3, IgM and IgA. In one specific embodiment, the antibody has an IgGl or an IgM isotype. Determination of the isotype of an antibody may be by a suitable method known in the art.

The antibody in the various embodiments of the present invention may also modulate binding of IGF-I to IGF-IR. Determination of the ability of an antibody to modulate binding of IGF-I to IGF-IR may be performed by a method known in the art.

In one embodiment, the antibody inhibits the binding of IGF-I to IGF-IR. In one specific embodiment, the antibody inhibits the binding of IGF-I to the IGF-IR by a maximum of at least 30%. In a further specific embodiment, the antibody inhibits the binding of IGF-I to IGF-IR by a maximum of at least 40%. The receptor may be for example a receptor expressed on the surface of a cell or a soluble form of the receptor. In one embodiment, the antibody does not substantially inhibit the binding of IGF-II to IGF-IR. In a specific embodiment, the antibody does not inhibit the binding of IGF-II to the IGF-IR by greater than 20%. In another specific embodiment, the antibody does not inhibit the binding of IGF-II to the IGF-IR by greater than 10%. In a further specific embodiment, the antibody does not inhibit the binding of IGF-II to IGF-IR by greater than 5%. For example, the antibody may not inhibit the binding of IGF-II to IGF-IR.

In one embodiment, the antibody has an affinity (K_D) for IGF-IR of at least 3x10 ⁹ M. In one specific embodiment, the antibody has an affinity (K_D) for IGF-IR of at least IxIO^"9 M. In a further specific embodiment, the antibody has an affinity (K_D) for IGF-IR of at least 5x10^"10 M.

The antibody and antigen binding portions of the present invention may also be used to modulate proliferation of an IGF-I dependent cell. In one embodiment, the antibody or antigen binding portion may be used to inhibit proliferation of an IGF-I dependent cell.

IGF-I dependent cells are known in the art, or their dependence on IGF-I for proliferation can be determined by a suitable method known in the art. An example of a cell that is IGF-I dependent is the colon cancer cell line HT-29.

Accordingly, in another embodiment the present invention provides a method of modulating IGF-I dependent proliferation of a cell, the method including binding an antibody, or an antigen-binding portion thereof, to insulin-like growth factor I receptor expressed on the cell, wherein the antibody, or the antigen-binding portion, binds to an epitope located in the cysteine-rich domain of the α-subunit of the insulin-like growth factor I receptor.

Generally, the antibody or antigen binding portion thereof will modulate IGF-I dependent proliferation by modulation binding of IGF-I to the IGF-I receptor. In one embodiment, the present invention provides a method of modulating IGF-I dependent proliferation of a cell, the method including binding an antibody, or an antigen-binding portion thereof, to insulin-like growth factor I receptor expressed on the cell, wherein the antibody includes the following amino acid sequences: (i) a V_H CDR-I sequence according to SEQ ID NO. 20, and a V_H CDR-sequence according to SEQ ID NO. 21, and a V_H CDR-3 sequence according to SEQ ID

NO. 8; and/or

(ii) a V_L CDR-I sequence according to SEQ ID NO.22, and a V_L CDR-2 sequence according to SEQ ID NO. 23, and a V_L CDR-3 sequence according to SEQ ID NO. l l; and/or binding an antibody, or an antigen-binding portion thereof, including a variant of one or more of the aforementioned sequences, that binds to IGF-I receptor.

In another embodiment, the present invention provides a method of of modulating IGF-I dependent proliferation of a cell, the method including binding an antibody, or an antigen-binding portion thereof, to insulin-like growth factor I receptor, wherein the antibody includes:

(i) a V_H CDR-I amino acid sequence according to SEQ ID NO.20; and (ii) a V_H CDR-2 amino acid sequence according to SEQ ID NO.21; and (iii) a V_H CDR-3 amino acid sequence according to SEQ ID NO.8; and

(iv) a V_L CDR-I amino acid sequence according to SEQ ID NO. 22; and (iv) a V_L CDR-2 amino acid sequence according to SEQ ID NO. 23; and (v) a V_L CDR-3 amino acid sequence according to SEQ ID NO. 11; and/or binding an antibody, or antigen binding portion thereof, including a variant of one or more of the aforementioned amino acid sequences, that binds to IGF-I receptor.

In another embodiment, the present invention provides a method of modulating IGF-I dependent proliferation of a cell, the method including binding a compound to insulin- like growth factor I receptor expressed on the cell, wherein the compound includes one or more of the amino acids sequences selected from the group consisting of SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.8, SEQ ID NO.22, SEQ ID NO.23, and SEQ ID NO.11, and/or binding a compound including a variant of one or more of the aforementioned sequences that binds to IGF-I receptor. Examples of such compounds are as previously described herein. In one embodiment, the compound is a polypeptide, such as an antibody or an antigen binding portion thereof.

In one embodiment, the antibodies (or antigen-binding portions thereof) and compounds of the present invention inhibit proliferation of an IGF-I dependent cell.

An IGF-I dependent cell is one in which the proliferation of the cell is modulated by the binding of IGF-I to IGF-IR. The cell may be present in vitro or in vivo.

For example, the cell may be an isolated cell or a cell present in a biological system.

In one embodiment, the cell is a cancerous cell or a pre-cancerous cell.

In this regard, the term "biological system means any cellular system. For example, the biological system may be a cell in tissue culture, a tissue or organ, or an entire animal or human subject, including a human or animal subject suffering the effects of an IGF-I dependent disease or condition.

In one embodiment, the biological system is a human or animal subject. More preferably, the biological system is a human or animal subject suffering from, or susceptible to, an IGF-I dependent disease or condition. For example, the biological system may be a human or animal subject suffering from, or susceptible to, one or more of the following IGF-I dependent diseases or conditions: acromegaly, ovarian cancer, pancreatic cancer, benign prostatic hyperplasia, breast cancer, prostate cancer, bone cancer, lung cancer, colorectal cancer, cervical cancer, synovial sarcoma, diarrhea associated with metastatic carcinoid, vasoactive intestinal peptide secreting tumours, gigantism, psoriasis, atherosclerosis, smooth muscle restenosis of blood vessels and inappropriate microvascular proliferation.

In one embodiment, the modulation of proliferation of the cell occurs in a human. The ability of an antibody (or an antigen-binding portion) or compound to modulate the proliferation of an IGF-dependent cell may be determined by a suitable method known in the art. For example, modulation of the proliferation of cells may be determined by cell counting, ³[H] thymidine incorporation, or immuno-histochemical staining.

The present invention also provides nucleic acids encoding the antibody, antibody fragments, or polypeptide compounds of the present invention, vectors including these nucleic acids, and prokaryotic (eg E. coli) or eukaryotic cells (eg a hybridoma cell) including the nucleic acids or vectors.

In one embodiment, the present invention also provides an isolated nucleic acid including a nucleotide sequence encoding a polypeptide including one or more of the amino acids sequences selected from the group consisting of SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.8, SEQ ID NO.22, SEQ ID NO.23, and SEQ ID NO.11, or encoding a polypeptide including a variant of one or more of the aforementioned sequences that binds to IGF-I receptor.

The antibody, antigen-binding portion thereof, or compounds of the present invention may also be used for detecting the presence of insulin-growth factor I receptor.

Accordingly, in another embodiment the present invention provides a method of detecting insulin-like growth factor I receptor, the method including binding an antibody, or an antigen-binding portion thereof, to insulin-like growth factor I receptor, wherein the antibody or the antigen-binding portion binds to an epitope located in the cysteine -rich domain of the α-subunit of the insulin-like growth factor I receptor, and the antibody, or the antigen-binding portion, modulates IGF-I mediated proliferation of an IGF-I dependent cell.

Various methods are also known in the art for using antibodies to detect proteins, and are as described generally in Sambrook, J, Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual 2nd. ed. Cold Spring Harbor Laboratory Press, New York. (1989), herein incorporated by reference. In one embodiment, the present invention provides a method of detecting insulin-like growth factor I receptor, the method including binding an antibody or an antigen- binding portion thereof to insulin-like growth factor I receptor, wherein the antibody includes the following amino acid sequences: (i) a V_H CDR-I sequence according to SEQ ID NO. 20, and a V_H CDR-2 sequence according to SEQ ID NO. 21, and a V_H CDR-3 sequence according to SEQ ID NO. 8; and/or

In another embodiment, the present invention provides a method of detecting insulin- like growth factor I receptor, the method including binding an antibody or an antigen- binding protion thereof to insulin-like growth factor I receptor, wherein the antibody includes:

(iv) a V_L CDR-I amino acid sequence according to SEQ ID NO. 22; and (iv) a V_L CDR-2 amino acid sequence according to SEQ ID NO. 23; and (v) a V_L CDR-3 amino acid sequence according to SEQ ID NO. 11; and/or binding an antibody, or an antigen-binding portion thereof, including a variant of one or more of the aforementioned sequences, that binds to IGF-I receptor.

In another embodiment, the present invention provides a method of detecting insulin- like growth factor I receptor, the method including binding a compound to insulin-like growth factor I receptor, wherein the compound includes one or more of the amino acids sequences selected from the group consisting of SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.8, SEQ ID NO.22, SEQ ID NO.23, and SEQ ID NO. l l, and/or binding a compound including a variant of one or more of the aforementioned sequences that binds to IGF-I receptor. For example, assay methods such as Western Blot, ELISA, competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays may be utilized.

Such methods are as generally described in Zola (1987) "Monoclonal Antibodies: A Manual of Techniques" pp.147-158 (CRC Press, Inc.), herein incorporated by reference.

The antibody, antigen-binding portion thereof, or compound according to the various embodiments of the present invention may also be used as diagnostic agents to detect IGF-IR in vitro or in vivo. For example, the antibodies may be used in a conventional immunoassay, including an ELISA, an RIA, FACS, tissue immunohistochemistry, Western blot or immunoprecipitation.

In the case of detecting IGF-IR in vitro, the IGF-IR may be a purified or semi-purified form of the receptor, or be a receptor present in a biological sample. Examples of biological samples include a whole tissue, one or more cells derived from a tissue, one or more recombinant cells, or lysates of such cells or tissues. A biological sample for analysis may be prepared by a suitable method known in the art.

Accordingly, in another embodiment the present invention also provides a method of detecting IGF-IR in a biological sample, the method including contacting the biological sample with an antibody, antigen binding portion thereof, or antibody mimetic of the present invention and detecting the antibody bound to IGF-IR, thereby indicating the presence of IGF-IR in the biological sample.

For detection of IGF-IR in the various embodiments of the present invention, the antibody, antigen-binding portion or antibody mimetic may labelled with a detectable moiety and thereby detected directly.

Alternatively, the primary antibody or mimetic to IGF-IR may be unlabeled and a secondary antibody or other molecule that can bind to the anti-IGF-IR antibody or mimetic can be utilised. For example, the antibody may be used in immunohistochemical analysis of tissues or cells. The binding of antibody may be detected with a secondary antibody, such as a biotinylated IgG that recognises the primary antibody, and incubation with Streptavidin CY3/FITC used to detect the binding of the antibody to the receptor.

Examples of detectable moieties include radioisotopes, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹³¹I; fluorescent or chemiluminescent compounds, such as fluorescein isothiocyanate, rhodamine, or luciferin; or an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase. Methods for conjugating an antibody to a detectable moiety are known in the art.

The antibodies, antigen-binding portions thereof, and compounds of the present invention also are useful for in vivo imaging, wherein the antibody, antigen-binding portion or compound labelled with a detectable moiety such as a radio-opaque agent or radioisotope is administered to a subject, and the presence and location of the labelled antibody etc in the host is assayed. Such imaging techniques are useful in the staging and treatment of malignancies. The antibody etc may be labelled with any moiety that is detectable in a host, whether by nuclear magnetic resonance, radiology, or other detection means known in the art.

The present invention also provides the use of the antibody, antigen binding portion thereof, compounds or antibody mimetics of the various embodiments of the present invention as a therapeutic agent for preventing and/or treating an IGF-I dependent disease or condition in a subject.

Accordingly, in another embodiment the present invention provides a method of preventing and/or treating an IGF-I dependent disease or condition in a subject, the method including administering to the subject a therapeutically effective amount of an antibody to insulin-like growth factor I receptor, or administering an antigen-binding portion of the antibody, the antibody or the antigen-binding portion binding to an epitope located in the cysteine-rich domain of the α-subunit of the insulin-like growth factor I receptor. In another embodiment, the present invention provides a method of preventing and/or treating an IGF-I dependent disease or condition in a subject, the method including administering to the subject a therapeutically effective amount of an antibody to insulin- like growth factor I receptor, or administering an antigen-binding portion of the antibody, the antibody including the following amino acid sequences:

(i) a V_H CDR-I sequence according to SEQ ID NO. 20, and a V_H CDR-sequence according to SEQ ID NO. 21, and a V_H CDR-3 sequence according to SEQ ID

NO. 8; and/or

(ii) a V_L CDR-I sequence according to SEQ ID NO.22, and a V_L CDR-2 sequence according to SEQ ID NO. 23, and a V_L CDR-3 sequence according to

SEQ ID NO.11; and/or administering an antibody, or an antigen-binding portion thereof, including a variant of one or more of the aforementioned sequences, that binds to IGF-I receptor.

In another embodiment, the present invention provides a method of preventing and/or treating an IGF-I dependent disease or condition in a subject, the method including administering to the subject a therapeutically effective amount of an antibody to insulin- like growth factor I receptor, or administering an antigen-binding portion of the antibody, the antibody including: (i) a V_H CDR-I amino acid sequence according to SEQ ID NO.20; and

(ii) a V_H CDR-2 amino acid sequence according to SEQ ID NO.21; and (iii) a V_H CDR-3 amino acid sequence according to SEQ ID NO.8; and (iv) a V_L CDR-I amino acid sequence according to SEQ ID NO. 22; and (iv) a V_L CDR-2 amino acid sequence according to SEQ ID NO. 23; and (v) a V_L CDR-3 amino acid sequence according to SEQ ID NO. 11 ; and/or administering an antibody, or an antigen binding portion thereof, including a variant of one or more of the aforementioned amino acid sequences, that binds to IGF-I receptor.

In another embodiment, the present invention also provides a method of preventing and/or treating an IGF-I dependent disease or condition in a subject, the method including administering to the subject a therapeutically effective amount of a compound including one or more of the amino acids sequences selected from the group consisting of SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.8, SEQ ID NO.22, SEQ ID NO.23, and SEQ ID NO.11, and/or administering a compound including a variant of one or more of the aforementioned sequences that binds to IGF-I receptor.

Thus, the antibodies, antigen-binding fragments, compounds and polypeptides of the present invention may be used for treating and/or preventing an IGF-I dependent disease or condition in a subject (eg a disease or condition which is mediated by elevated activity of IGF-IR due to binding of IGF-I), and which may be treated or prevented by modulation of IGF-IR ligand binding.

In one embodiment, the disease or condition is a malignancy characterized by a tumour which expresses IGF-IR, such as bladder cancer, Wilm's cancer, bone cancer, prostate cancer, lung cancer, colorectal cancer, breast cancer, cervical cancer, synovial sarcoma, ovarian cancer, pancreatic cancer, benign prostatic hyperplasia (BPH), diarrhoea associated with metastatic carcinoid and vasoactive intestinal peptide secreting tumours (e.g., VIPoma or Werner-Morrison syndrome). Other non-malignant medical conditions which may also be treated include gigantism, psoriasis, atherosclerosis, smooth muscle restenosis of blood vessels or inappropriate microvascular proliferation, such as that found as a complication of diabetes, especially of the eye.

In one embodiment, the subject is an animal or human. For example, the subject may be a mammal, a primate, a livestock animal (eg. a horse, a cow, a sheep, a pig, or a goat), a companion animal (eg. a dog, a cat), a laboratory test animal (eg. a mouse, a rat, a guinea pig, a bird), an animal of veterinary significance, or an animal of economic significance.

In a specific embodiment, the subject is a human. For example, the human may be suffering from, or susceptible to, one or more of diseases or conditions selected from the group consisting of acromegaly, ovarian cancer, pancreatic cancer, benign prostatic hyperplasia, breast cancer, prostate cancer, bone cancer, lung cancer, colorectal cancer, cervical cancer, synovial sarcoma, diarrhea associated with metastatic carcinoid, vasoactive intestinal peptide secreting tumours, gigantism, psoriasis, atherosclerosis, smooth muscle restenosis of blood vessels and inappropriate microvascular proliferation.

The ability of an antibody, antigen-binding fragment, compound or polypeptide of the present invention to treat or prevent an IGF-I dependent disease or condition may be determined by a suitable method known in the art. For example, the ability of an antibody to inhibit cancer can be evaluated in an animal model system predictive of efficacy in human tumours. Alternatively, the antibody can be evaluated by examining the ability of the antibody or antigen-binding fragment of the invention to inhibit tumour cell growth in vitro.

When the antibody, fragment or compound is used as a therapeutic agent, the therapeutic agent may be either the antibody, fragment or compound itself, or be conjugated to another moiety. For example, an antibody or antibody fragment may be conjugated to a cytotoxic agent.

In the case of a conjugate, the conjugate can be prepared by in vitro methods known in the art. Suitable linking groups are known in the art and include disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups and esterase labile groups.

The antibodies, fragments and compounds of the invention may also be used for therapeutic purposes by administration to a subject in a pharmaceutically acceptable composition. Thus, the antibody, antigen-binding fragment or compound of the present invention can be incorporated into a pharmaceutical composition, generally along with a pharmaceutically acceptable carrier, suitable for administration to a subject in vivo.

Accordingly, in one embodiment the present invention provides a pharmaceutical composition including an antibody to insulin-like growth factor I receptor, or an antigen-binding portion of the antibody, the antibody or the antigen-binding portion binding to an epitope located in the cysteine-rich domain of the α-subunit of the insulin- like growth factor I receptor, wherein the antibody or the antigen-binding portion modulates IGF-I mediated proliferation of an IGF-I dependent cell. In another embodiment, the present invention provides a pharmaceutical composition including an antibody to insulin-like growth factor I receptor, or an antigen-binding portion of the antibody, the antibody including the following amino acid sequences: (i) a V_H CDR-I sequence according to SEQ ID NO. 20, and a V_H CDR-sequence according to SEQ ID NO. 21, and a V_H CDR-3 sequence according to SEQ ID

NO. 8; and/or

(ii) a V_L CDR-I sequence according to SEQ ID NO.22, and a V_L CDR-2 sequence according to SEQ ID NO. 23, and a V_L CDR-3 sequence according to SEQ ID NO. l l; and/or including an antibody, or an antigen-binding portion thereof, including a variant of one or more of the aforementioned sequences, that binds to IGF-I receptor.

In another embodiment, the present invention provides a pharmaceutical composition including an antibody to insulin-like growth factor I receptor, or an antigen-binding portion of the antibody, the antibody including:

(i) a V_H CDR-I amino acid sequence according to SEQ ID NO.20; and (ii) a V_H CDR-2 amino acid sequence according to SEQ ID NO.21; and (iii) a V_H CDR-3 amino acid sequence according to SEQ ID NO.8; and (iv) a V_L CDR-I amino acid sequence according to SEQ ID NO. 22; and

(iv) a V_L CDR-2 amino acid sequence according to SEQ ID NO. 23; and (v) a V_L CDR-3 amino acid sequence according to SEQ ID NO. 11; and/or including an antibody, or an antigen binding portion thereof, including a variant of one or more of the aforementioned amino acid sequences, that binds to IGF-I receptor.

In another embodiment, the present invention provides a pharmaceutical composition including a compound including one or more of the amino acids sequences selected from the group consisting of SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.8, SEQ ID NO.22, SEQ ID NO.23, and SEQ ID NO.11, and/or including a compound including a variant of one or more of the aforementioned sequences that binds to IGF-I receptor. The pharmaceutical composition according to the various embodiments of the present invention can be administered intravenously as a bolus or by continuous infusion over a period of time, by intramuscular, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. The antibody, antigen-binding fragment, compound or polypeptide of the present invention may also be administered by intratumoral, peritumoral, intralesional, or perilesional routes, to exert local as well as systemic therapeutic effects.

Suitable pharmaceutically acceptable carriers, diluents, and excipients are known in the art.

The preparation of pharmaceutical compositions is known in the art, for example Remington's Pharmaceutical Sciences, 18th ed., 1990, Mack Publishing Co., Easton, Pa., herein incorporated by reference; and U.S. Pharmacopeia: National Formulary, 1984, Mack Publishing Company, Easton, Pa., herein incorporated by reference.

Pharmaceutically acceptable carriers include aqueous and nonaqueous carriers, stabilizers, antioxidants, solvents, dispersion media, coatings, antimicrobial agents, buffers, serum proteins, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of suitable aqueous and nonaqueous carriers include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.

Suitable buffers which may be included in the pharmaceutical compositions of the invention include L-histidine based buffers, phosphate based buffers (e.g., phosphate buffered saline, pH. congruent.7), sorbate based buffers or glycine-based buffers. Serum proteins may also be included in the pharmaceutical composition, including human serum albumin. Isotonic agents, such as sugars, ethanol, polyalcohols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, mannitol or sorbitol), sodium citrate or sodium chloride (e.g., buffered saline) may also be included in the pharmaceutical compositions of the present invention.

Prolonged absorption of an injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminium monostearate and/or gelatin.

The antibody, antigen-binding fragment, compound or polypeptide of the present invention may also be orally administered. Pharmaceutical compositions for oral administration may contain, in addition to the binding composition, additives such as starch (e.g., potato, maize or wheat starch or cellulose), starch derivatives (e.g., microcrystalline cellulose or silica), sugars (e.g., lactose), talc, stearate, magnesium carbonate or calcium phosphate. In order to ensure that oral compositions including an antibody, antigen-binding fragment, compound or polypeptide of the present invention are well tolerated by the patient's digestive system, mucus formers or resins may be included. It may also be desirable to improve tolerance by formulating the antibody, antigen-binding fragment, compound or polypeptide of the present invention in a capsule which is insoluble in the gastric juices. An exemplary pharmaceutical composition of this invention in the form of a capsule is prepared by filling a standard two-piece hard gelatin capsule with the antibody or antigen-binding fragment of the invention in powdered form, lactose, talc and magnesium stearate.

An antibody, antigen-binding fragment, compound or polypeptide of the present invention may also be included in a pharmaceutical composition for topical administration. Formulations suitable for topical administration include liquid or semi- liquid preparations suitable for penetration through the skin to the site where treatment is required, such as liniments, lotions, creams, ointments or pastes, and drops suitable for administration to the eye, ear or nose. Drops according to the various embodiments of the present invention may comprise sterile aqueous or oily solutions or suspensions and may be prepared by dissolving the antibody, antigen-binding fragment, compound or polypeptide in a suitable aqueous solution of a bactericidal and/or fungicidal agent and/or any other suitable preservative, and preferably including a surface active agent. The resulting solution may then be clarified by filtration.

Lotions according to the various embodiments of the present invention include those suitable for application to the skin or eye. An eye lotion may comprise a sterile, aqueous solution optionally containing a bactericide and may be prepared by methods similar to those for the preparation of drops. Lotions or liniments for application to the skin may also include an agent to hasten drying and to cool the skin, such as an alcohol or acetone, and/or a moisturizer such as glycerol or an oil such as castor oil or arachis oil.

Creams, ointments or pastes according to the various embodiments of the present invention are semi-solid formulations of the active ingredient for external application. They may be made by mixing the antibody, antigen-binding fragment, compound or polypeptide of the present invention in finely-divided or powdered form, alone or in solution or suspension in an aqueous or non-aqueous fluid, with the aid of suitable machinery, with a greasy or non-greasy basis. The basis may comprise hydrocarbons such as hard, soft or liquid paraffin, glycerol, beeswax, a metallic soap; a mucilage; an oil of natural origin such as almond, corn, arachis, castor or olive oil; wool fat or its derivatives, or a fatty acid such as stearic or oleic acid together with an alcohol such as propylene glycol or macrogels. The formulation may incorporate any suitable surface active agent such as an anionic, cationic or non-ionic surface active such as sorbitan esters or polyoxyethylene derivatives thereof. Suspending agents such as natural gums, cellulose derivatives or inorganic materials such as silicaceous silicas, and other ingredients such as lanolin, may also be included.

The antibodies, antigen-binding fragments, compounds and polypeptides of the present invention may also be administered by inhalation. A suitable pharmaceutical composition for inhalation may be an aerosol. An exemplary pharmaceutical composition for inhalation of an antibody or antigen-binding fragment of the invention may include: an aerosol container with a capacity of 15-20 ml comprising the antibody or antigen-binding fragment of the invention, a lubricating agent, such as polysorbate 85 or oleic acid, dispersed in a propellant, such as freon, preferably in a combination of 1,2-dichlorotetrafluoroethane and difluorochloromethane. Typically, the composition is in an appropriate aerosol container adapted for either intranasal or oral inhalation administration.

The present invention also provides the use of the antibodies, antigen-binding fragments, compounds and polypeptides in the preparation of a medicament for preventing and/or treating an IGF-I dependent disease or condition.

The present invention also provides an antibody selected from the group consisting of 4C6, 5B6, 7C2, 9El 1 and 5B2, or an antigen-binding portion thereof.

This embodiment of the present invention provides an antibody (or an antigen-binding portion thereof) selected from the group consisting of 4C6, 5B6, 7C2, 9El 1 and 5B2, as defined herein.

These antibodies are monoclonal antibodies that specifically bind to the insulin-like growth factor I receptor. The antibodies were raised in mice against the human insulin- like growth factor I receptor. The antibodies were each generated from a separate hybridoma.

Accordingly, in another embodiment the present invention also provides an isolated cell that expresses an antibody to insulin-like growth factor I receptor, or an antigen-binding portion of the antibody, the antibody selected from the group consisting of 4C6, 5B6, 7C2, 9E11 and 5B2.

These antibodies are highly specific for soluble insulin-like growth factor I receptor. The antibodies do not recognise the related insulin receptor type A (IR-A) or insulin receptor type B (IR-B).

Antibodies 4C6, 5B6, 7C2 and 9El 1 have an IgGl isotype. 5B2 has an IgM isotype. Antibodies 9El 1 and 7C2 also inhibit the binding of IGF-I to IGF-IR. They do not substantially inhibit the binding of IGF-II to IGF-IR.

9El 1 has an affinity (K_D) for IGF-IR of 2.OxIO^"9 M. 7C2 has an affinity (K_D) for IGF- IR of 7.3x10 ¹⁰ M.

9El 1 and 7C2 also inhibit proliferation of an IGF-I dependent cell, such as proliferation of HT29 colon cancer cells.

Accordingly, in another embodiment the present invention provides a method of inhibiting proliferation of an IGF-I dependent cell, the method including binding an antibody, or an antigen-binding portion thereof, to insulin-like growth factor I receptor expressed on the cell, wherein the antibody is 7C2 or 9El 1.

As discussed previously, an IGF-I dependent cell is one in which the proliferation of the cell is modulated by the binding of IGF-I to IGF-IR. The cell may be present in vitro or in vivo.

As discussed previously, the ability of an antibody or an antigen-binding portion to modulate the proliferation of an IGF-dependent cell may be determined by a suitable method known in the art. For example, modulation of the proliferation of cells may be determined by cell counting, ³[H] thymidine incorporation, or immuno-histochemical staining.

The present invention also provides nucleic acids including a nucleotide sequence encoding antibodies 4C6, 5B6, 7C2, 9El 1 and 5B2 (or an antigen binding portions thereof), vectors including these nucleic acids, and cells including the nucleic acids and vectors. These antibodies (and antibody fragments) may also be used as a diagnostic agent for detecting the presence of insulin-growth factor I receptor.

Accordingly, in another embodiment the present invention also provides a method of detecting insulin-like growth factor I receptor, the method including binding an antibody or an antigen-binding portion thereof to insulin-like growth factor I receptor, wherein the antibody is selected from the group consisting of 4C6, 5B6, 7C2, 9El 1 and 5B2.

Various methods are also known in the art for using antibodies or fragments thereof to detect proteins, as described previously herein.

In the case of detecting IGF-IR in vitro, the IGF-IR may be present in a purified or semi-purified form or be present in a biological sample. Examples of biological samples include a whole tissue, one or more cells derived from a tissue, one or more recombinant cells, or lysates of such cells or tissues. A biological sample for analysis may be prepared by a suitable method known in the art.

Accordingly, in another embodiment the present invention provides a method of detecting IGF-IR in a biological sample, the method including contacting the biological sample with any one of the antibodies or antibody fragments of the present invention and detecting the antibody or fragment bound to IGF-IR, thereby indicating the presence of IGF-IR in the biological sample.

The antibodies are useful for in vivo imaging, wherein an antibody labelled with a detectable moiety such as a radio-opaque agent or radioisotope is administered to a subject, and the presence and location of the labelled antibody in the host is assayed.

The present invention also contemplates the use of 4C6, 5B6, 7C2, 9El 1 and 5B2 as therapeutic agents for preventing and/or treating an IGF-I dependent disease or condition in a subject. Accordingly, in another embodiment the present invention also provides a method of preventing and/or treating an IGF-I dependent disease or condition in a subject, the method including administering to the subject a therapeutically effective amount of an antibody to insulin-like growth factor I receptor, or administering an antigen-binding portion of the antibody, wherein the antibody is selected from the group consisting of 4C6, 5B6, 7C2, 9El 1 and 5B2.

Thus, the antibodies or antigen-binding fragments of the present invention, and pharmaceutical compositions including the antibodies or antigen-binding fragments of the present invention, may be used for treating and/or preventing a disease or condition in a subject which is mediated by elevated activity of IGF-IR due to binding of IGF-I, and which may be treated or prevented by modulation of IGF-IR ligand binding.

Examples of IGF-I dependent diseases and conditions are as previously discussed herein.

Antibodies 4C6, 5B6, 7C2, 9El 1 and 5B2 may also be used for therapeutic purposes by administration to a subject in a pharmaceutically acceptable composition. Thus, the antibodies or antigen-binding fragments can be incorporated into a pharmaceutical composition, preferably along with a pharmaceutically acceptable carrier, suitable for administration to a subject in vivo.

Accordingly, in another embodiment the present invention also provides a pharmaceutical composition including an antibody selected from the group consisting of 4C6, 5B6, 7C2, 9El 1 and 5B2, and/or including an antigen-binding portion thereof.

Finally, standard techniques may be used for recombinant DNA technology, oligonucleotide synthesis, and tissue culture and transfection (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be 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. See e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), herein incorporated by reference.

Description of Specific Embodiments

Reference will now be made to experiments that embody the above general principles of the present invention. However, it is to be understood that the following description is not to limit the generality of the above description.

Example 1

Cell lines and cell culture conditions

P6 cells, BALB/c-3T3 cells overexpressing the human IGF-IR (Pietrzkowski et al (1992) MoI Cell Biol 12: 3883-3889), and R^" cells, mouse 3T3-like cells with a targeted deletion of the IGF-IR gene (Sell et al (1994) MoI Cell Biol 14: 3604-3612, herein incorporated by reference), were kindly provided by Professor Renato Baserga (Philadelphia, USA). RlR-A cells (R^" cells expressing insulin receptor isoform-A, IR- A) and R IR-B cells (R^" cells expressing insulin receptor isoform-B, IR-B) were provided by Dr. Eric R. Bonython (The University of Adelaide, Australia) (Denley et al. (2004) MoI Endocrinol 18: 2502-2512, herein incorporated by reference). BHK21 cells recombinantly producing the extracellular part of the human IGF-IR (amino acids 1-906) (s-IGF-lR) were generated by Dr. Kathy Surinya (The University of Adelaide, Australia) as generally described in Hoyne et al (2000) FEBS Lett 479: 15-18 (herein incorporated by reference) and Cosgrove et al. (1995) Protein Expr Purif β: 789-798 (herein incorporated by reference) for the production of recombinant soluble insulin receptor extracellular domain.

Breast cancer cell lines, MCF-7 and colon cancer cell line, HT-29, were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). 293 EBNA cells were from Invitrogen (CA, USA). MCF 7 cells, R^" cells and P6 cells were cultured in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10 % (v/v) FBS and 1% (v/v) penicillin/streptomycin whereas HT-29 cells were in 47% (v/v) DMEM, 47% (v/v) F 12 Nutrient Mixture (HAM), 1% (v/v) dilution of penicillin/streptomycin and 5% (v/v) FCS at 37° C in 5% CO2 atmosphere. The 293 EBNA cells and NIH 3T3 cells expressing the chimeric receptors were cultured in the same medium as MCF-7 cells but containing 0.5% (v/v) geneticin (G418). BHK21 cells producing s-IGF-lR were grown in GMEM-S medium containing 10% (v/v) Dialyzed fetal bovine serum, 2% (v/v) glutamine synthetase supplement (5Ox) and 25 μM methionine sulfoximine.

Example 2

Materials

Unless otherwise mentioned, the chemicals were from Sigma. Lipofectamine 2000 was purchased from Life Technologies Inc. BSA (BovoStar grade) was from Bovogen, The αIR-3 MAb was purchased from Calbiochem. MAb 24-60 was a kind gift from Prof. K. Siddle (Cambridge, UK). MAbs 24-55, 16-13 and receptor grade IGF-I and IGF-II were from GroPep Ltd (Adelaide, SA, Australia). The chimeric ligands (IGF-ICII) and (IGF- IICI) were made by Dr. A. Denley (The University of Adelaide, Australia). Biotinylated MAb 16-13 (Bt-16-13) was provided by F. Occhiodoro (The University of Adelaide, Australia). Recombinant cDNAs that encode secreted alanine mutants of the IGF-IR, which were constructed in the plasmid pcDNA3 zeo (+) for expression, were kindly provided by Prof. J. Whittaker (Case Western Reserve University, Cleveland, Ohio, USA) (Whittaker et al. (2001) J Biol Chem 276: 43980-43986, herein incorporated by reference).

Example 3

s-IGF-lR (recombinant soluble IGF-IR) purification

Recombinant cells that secrete into the medium the extracellular part of the receptor, referred to as the soluble receptor (s-IGF-lR) were used. These cells are the BHK 21 cell line (Syrian hamster kidney), which has been stably transfected with a plasmid expressing the extracellular part of the IGF-IR. The expression plasmid contained only the human IGF-IR ectodomain. A 2.8kb EcoRl-BclI fragment cDNA encoding the ectodomain of the human IGF-IR was isolated from the plasmid pECE/IGF-IR cDNA (Steele-Perkins et al. (1988) J Biol Chem. 263(23): 11486-92, herein incorporated by reference), blunted with Klenow polymerase and then ligated into the blunted Xbal linearized pCelltech vector pEEL to produce pEEl-IGF-IR-E. The s-IGF-lR was purified by an affinity column: mAb 24-55 against the IGF-IR was coupled to Affi- Gel® 10 Gel (BIO-RAD Cat. No: 153-6046). The mAb 24-55 was obtained from Gropep (Cat.No: MADl) as described in Soos, M.A. et al. (1992) J. Biol. Chem. 267: 12955 - 12963 (herein incorporated by reference). The column was washed with filtered PBS [(PBS: 0.137M NaCl, 2.7mM KCl, 1.46mM KH2PO4, 8. ImM Na2HPO4 pH: 7.4)] for 40-50 minutes (Flow rate: I ml/min) and the filtered supernatant loaded onto the column. The column was washed with filtered PBS for 40-50 minutes again and then using pH 2.6 Glycine 0.1M, NaCl 0.15m solution, s-IGF-lR was eluted. The eluate, containing s-IGF-lR, was collected in different tubes in 1 ml fractions and neutralized immediately by adding 80 μl IM Tris solution, pH 9. The elution profile was recorded at OD280nm. The fractions, which contained most of the purified s-IGF- IR, were then collected and dialysed against PBS buffer in Regenerated Cellulose Tubular Membrane from Cellu.Sep®T2.

Example 4

Production of monoclonal antibodies

A female BALB/C mouse was given 3 intraperitoneal injections of 30 or 50 μg s-IGF- IR plus 50 μg adjuvant, 3 to 5 weeks apart. Then, a test bleed was carried out showing that the mouse had an acceptable serum antibody response. After four weeks, the mouse was boosted with 20 μg s-IGF-lR and fusion was carried out between mouse spleen cells and SP2/0 mouse myeloma cells (ATCC CRL 1581, used in developing hybridomas (J. Immunol. 126: 317-321, 1981, herein incorporated by reference).

Another female BALB/C mouse was immunized with P6 cells and s-IGF-lR. P6 cells are BALB/c3T3 cells transfected with only an IGF -1 receptor expression plasmid, as described in Pietrzkowski et al. (1992) Cell Growth Differ 3: 199- 205 (herein incorporated by reference). This mouse was given 6 intraperitoneal injections of (1-5) xlO⁶ P6 cells 3 to 12 weeks apart followed by two injections of 30-40 μg of s-IGF-lR plus 50 μg adjuvant, 3 weeks apart. After 4 weeks the mouse was boosted with 30 μg adjuvant and fusion carried out between the mouse spleen cells and SP2/0 mouse myeloma cells.

Antibodies in the hybridoma culture supernatant or in the mice sera were detected by an ELISA test (as described below), which showed the reaction of the antibody with the s- IGF-IR.

Example 5

ELISA test for mouse sera or hybridoma culture supernatants

Each well of a 96 well Maxisorp (NuncTM) plate (Cat: 442404) was coated with 100 μ 1 of s-IGF-lR solution (0.5 μg/ml) in PBS and blocked with 200 μl of BSA 2% solution. For the ELISA test the plate was washed once with PBS - Tween20 (0.05%) (SIGMA, Cat No: P-1379). Then 100 μl of the mouse sera in different dilutions in PBS+3% BSA or hybridoma culture supernatants (neat) was added to each well of the plate. The positive control was mAb 24-60 (Soos et al. (1992) J. Biol. Chem. 267(18): 12955- 12963, June 1992, herein incorporated by reference) and the negative control was mouse IgGl negative control (CHEMICON Australia Cat No: MABC002, 987710005). After incubation the plate at room temperature for 2 hours, the plate was washed three times with PBS-Tween followed by adding 100 μl secondary mAb, which was sheep anti-mouse immunoglobulin (gamma and light chain specific), affinity isolated HRP Conjugated (CHEMICON Australia Cat.No: 985033), diluted 1:500 in PBS-1% BSA. The plate was incubated at room temperature for one hour. After washing the plate three times with PBS-Tween20 (0.1%) [(PBS: 0.137M NaCl, 2.7mM KCl, 1.46mM KH2PO4, 8.ImM Na2HPO4 pH: 7.4), (Tween20, from SIGMA, Cat No: P-1379)] the bound mAbs were visualized with ABTS reagents (ABTS ^R tablets cat. No: 1204521, Roche, Germany) dissolved in citric acid buffer (0.06M Citric Acid, 0.08M di-Sodium hydrogen orthophosphate pH=4.0) and the plate was read with a microplate reader (Molecular Device E_Max precision) at 405nm after 20-30 minutes. Example 6

FACS (fluorescence - activated cell sorting) analysis for studying the specific binding of the mAbs

Binding of the mAbs to IGF-IR, IR-A or IR-B was studied by FACS analysis. IR-A and IR-B are two isoforms of insulin receptor. Exon 11 of the IR gene codes for 12 amino acids that in the mature protein are the C-terminal most amino acids in the extracellular α subunits of the IR-B (or IR exon 11+) isoform. The IR-A (or IR exon H-) isoform lacks these 12 amino acids.

For IGF-IR, the P6 cells (BALB/c3T3 cells over expressing the human IGF-IR) were used. R^" cells (mouse 3T3-like cells with a targeted ablation of the IGF-IR gene) were used as a negative control, as described Sell et al. (1994) MoI. Cell Biol. 14:3604-3612 (herein incorporated by reference). The cDNA encoding the human IR-A and IR-B isoforms were generated as described in Ellis et al. (1986) Cell 45: 721-32 and Hoyne et al. (2000) FEBS Lett 479: 15-18, herein incorporated by reference.

The pECE: hIR-A and hIR-B plasmids were restricted with SaR and Xbal to release a 2.9kb fragment containing the insulin receptor and ligated to XhoVXbal cut pEFIRESneo, as described in Hobbs et al. (1998) Biochem Biophys Res Commun 252:368-372, herein incorporated by reference.

PCR analysis confirmed the exon 11 status of the constructs. R^" cells were infected with the constructs using Lipofectamine+™ (Gibco/BRL Life Technologies) and stably transfected cells were screened for the IR cDNA by PCR analysis and for IR expression by FACS analysis via the monoclonal anti-IR antibody 83-7. For isolating cells expressing similar levels of receptors, cells expressing human IR underwent single-cell sorting. These clonal cell lines were used in all subsequent experiments. R^" cells expressing the human IR-A were designated RIR-A and R cells expressing the human IR-B were designated R-IR-B. For FACS analysis, the cells were trypsinized, washed and suspended in PBS, 10⁵ to 10⁶ cells per FACS tube. The cells were centrifuged, the supernatant aspirated off and the cells resuspended in 100 μl supernatant primary mAbs, which were the mAbs against IGF-IR, positive (mAb 24-60) and negative controls (unrelated mouse IgGl). The tubes were incubated on ice for 1-2 hours then washed three times with 3 mis wash solution (pBS- l%BSA-0.01% Na-Azide). Anti-mouse IgG FITC conjugated antibody (Sheep, anti-mouse immunoglobulin, IgG fraction, fluorescein conjugated, CHEMICON Australia Cat No: 985021020) was diluted 1:50 in PBS-10% normal rat serum and used as a secondary antibody (50 μl). After incubating of the tubes on ice for one hour, the cells were washed three times in wash solution by centrifugation and aspiration. The cells were fixed in 500 μl of 1% paraformaldehyde in PBS and stored in the dark on ice until FACS acquisition.

Example 7

Isotyping the monoclonal antibodies

To determine the isotype of various monoclonal antibodies, the Mouse Typer Sub- Isotyping Panel, (BIO-RAD Cat No: 172-3055) was used. The Maxisorp (Nunc™) plate was coated with s-IGF-lR and blocked with 200 μl of 2-3% BSA in PBS. The primary antibody, which was the mAbs in the neat supernatant, was added 100 μl/well and incubated at room temperature for 1 hour. After washing the plate 3 times with PBS- Tween 20 (0.1%), 50 μl of the rabbit anti-isotype was added to each well neat and incubated at room temperature for 30 minutes. The plate was then washed three times with PBS-Tween. The donkey anti-rabbit-HRP was diluted 1:400 in PBS+1% BSA and 50 μl was added to each well of the plate. After one hour incubation at room temperature, the plate was washed three times with PBS-Tween and the isotype of antibodies detected by using ABTS reagents and reading the plate at 405nm after 20- 30 minutes. Example 8

Purification of IgGl monoclonal antibodies by Protein G column

Hybridoma cells were grown in DMEM [(Dulbecco's Modified Eagle Medium) Gibco Invitrogen Corporation cat. No: 12430-54] and 2.5% FCS (Foetal Bovine Serum, JRH Biosciences, USA) and 1% penicillin/streptomycin in a spinner (cell culture Bioreactor manufactured by the New Brunswick Scientific Co., Inc., U.S.A.) for production of a large amount of monoclonal antibodies. 15 mis of Protein G column (Sigma Protein G Fast Flow) was washed with filtered PBS at 1 ml/min flow rate for 30 minutes. The filtered mAb supernatant was loaded onto the column at 1 ml/min flow rate. After washing the column with filtered PBS for 30-40 minutes, the bound antibodies were eluted with 20 ml Glycine-NaCl solution pH=2.6. Each 1 ml fraction was collected in a tube and neutralised by 80 μl of Tris IM pH=9.0. The fractions with most antibodies were found by the elution profile and then dialysed against HEPES buffer.

Example 9

SDSPAGE of mAbs

For mAbs 9El 1, 7C2 and IgGl negative control (CHEMICON Australia), 2.5 μg of purified mAb was loaded onto a SDS-polyacrylamide gel under reducing conditions and analysed using 5% stacking gel and a 10% separating gel. Coomassie Brilliant Blue staining was used to detect the heavy and light chains of the mAbs.

Example 10

Spectrophotometry for determination of the concentration of mAbs

The concentration of mAbs in buffer was determined using UV absorbance at 280 nm: OD 1.35 at 280 nm equals 1.0 mg/ml of IgG. Example 1 1

Epitope mapping

We selected two antibodies, which bound to the whole receptor on P6 cells. We conducted FACS analysis, for testing their reaction with chimeric constructs in which portions of IR were replaced by the corresponding IGF-IR sequences or vice versa. We had three chimeras, as described in Schumacher et al. (1993) J. Biol. Chem. 268(2): 1087-1094, herein incorporated by reference.

The three chimeric receptors were:

1) IR/IGF1RC12, Cell bank number 629, Specification: NIH3T3 cl7 expressing IR from which the Cys-rich region derives from IGFR-IR IR/IGFIRC12 stable (C 12)

2) N7/IR/IGF-IRC2cI7, Cell bank number 163, Specification: NIH3T3 expressing IR from which the Ll and Cys-rich regions are derived from IGFR-IR (CI7)

3) N7/HIR1, Cell bank number 624, Specification: NIH3T3 cl7 expressing the IGF-IR from which the Ll region derives from the IR IGFIR/IR cl (HIRl)

The method was exactly the same as other FACS analysis, however the purified forms of the primary mAbs were used at a concentration of 4 μg/ml.

Example 12

Europium-ligand binding assays

Europium-IGF-I and Europium-IGF-11 were labelled using Wallac Kit 1244-302 and purified with Superdex™ HR 10/30 (Pharmacia, Sweden).

Competition of Binding Europium-IGF-I or Europium-IGF-11 with different concentrations of mAbs to the solubilized IGF-IR was studied as follows: 96 well Greiner plate was coated by mAb 24-31 which is a mAb against the IGF-IR (abeam, Cat. No: ab 4065), as described in Soos et al (1992) J. Biol. Chem. 267, 12955-12963 (herein incorporated by reference). The plate was then blocked with 0.5% BSA. A T- 175 flask of P6 cells were starved in serum-free medium for 5 hours at 37°C and 5% CO₂. Then, after washing the cells in PBS, 11 mis of lysate buffer (2OmM HEPES, 15OmM NaCl,1.5mM MgC12,10°/oGlycerol,l°/oTriton X-IOO, ImMEDTA) containing 1 : 1000 protease inhibitor, was added to the flask and incubated on ice for 30-60 minutes. The flask was shaken well and the lysate centrifuged at 1200 rpm for 2 minutes. The supernatant containing IGF-IR was taken. The plate was then washed once with TBST (Tris 50 mM, NaCl 15mM, Tween- 20 (0.05%), pH 8.0) solution and 100 μl of the lysate was added to each well of the plate and incubated overnight at 4°C. Next day, the solutions of IGF-I or IGF-Il and monoclonal antibodies in different concentrations in HEPES buffer-Tween 0.05% were made. Also, a solution of Europium-IGF-I or II containing approximately 30000 to 50000 fluorescence counts per 5 μl of the solution (5 μl +100 μl Enhancement solution) was prepared. The plate was then washed once with TBST and incubated with 50 μl of the Europium-IGF-1 or II plus 50 μl of antibody or ligand solution. The plate was incubated at 4°C overnight in the dark. Next day, the plate was washed three times with TBST and three times with MQ water and after adding 100 μl Enhancement solution (DELFIA@/AutoDELFIATM, 1244-105, Finland), time -resolved fluorescence was read by microplate reader (BMG FluoStar Galaxy).

Example 13

BIACORE analysis

The binding kinetics of the soluble IGF-IR (s-IGF-lR) to the mAbs were determined by using a BIACORE 2000. (BIACORE) Rabbit anti-mouse IgGl antibody (Biacore AB

Sweden Code No: BR- 1000-55) immobilized onto a CM5 sensor chip and mAb was injected at the same concentrations followed by injecting different concentrations of the soluble IGF-IR. The IgGl surface was regenerated with 1OmM glycine-HCl, pH 1.7 for

4 minutes. Sensorgrams for different mAbs were obtained and evaluated using BIAevaluation 3.2 software provided by BIACORE to determine rate constants and the affinity constants (K_D), which was calculated from the ratio of the rate constants koff/kon. The data were evaluated using 1 : 1 (Langmuir) binding model. The ability of IGF-I or IGF-II to inhibit the binding of s-IGF-lR to the mAbs was determined by pre-incubation of different concentrations of the ligands with 12.5 nM of the soluble receptor for at least 3 hours at room temperature. By using an anti-mouse IgG 1 coupled chip, a constant concentration of mAb was injected followed by injection of the different complex solutions of ligands and the receptor. Plotting the RU (Resonance Units) immediately after injection of the complex against the log concentration of ligands provides the EC50 for the interaction of the ligands and the receptor.

Example 14

Cell proliferation assay

Cell proliferation was measured using a Cell-Titer Glo™ Luminescent Cell Viability Assay Kit: For the proliferation assay, 12000 HT-29 cells (colon cancer tumour cells) were seeded into 96 well flat bottom plate in complete medium. After 48 hours incubation at 37°C and 5% CO₂, the medium was replaced with the FCS free medium and the plate was left in the incubator for 5 hours to starve the cells. Different concentrations of mAbs were made in assay buffer were prepared ((47%)DMEM +(47%) F- 12 Nutrient Mixture (HAM) GIBCO™ Cat.No.11765-054 Invitrogen Corporation + 1% penicillin/streptomycin+0.5% BSA+ 5 mM n-Butyric Acid sodium salt (Sigma Cat No: B05887)+10 mM IGF-I or 50 nM IGF-II) filtered by 0.2 μl filters before use. The control solutions were (1) assay buffer without mAbs and IGFs (2) just assay buffer. The plate was incubated for 48 hours at 37°C and 5% CO₂ and then left at room temperature for 2-3 hours. The Cell-Titer Glo™ reagents (Cell-Titer Glo™ Luminescent Cell Viability Assay Kit Promega, USA, Cat No: G7571) were mixed and then 100 μl of the mixture was added to each well of the plate. After shaking at 960 rpm for 2 minutes, the luminescence was read by a microplate reader (BMG FluoStar Galaxy). Example 15

Immunohistochemistry

The ability of the mAbs for use in immunohistochemistry was determined by using three layers Biotin/Streptavidin-Fluorescent method. Cytospin cell preparations of cells were dried under vacuum for 5 min followed by fixation in different fixatives [Acetone, Ethanol and 10% BFS (Buffered formalin 10%)] for 5 minutes. Sections were then rinsed in hypertonic phosphate -buffered saline (10 mM sodium/potassium phosphate with 0.274 M NaCl, 5 mM KCl; pH 7.2; hPBS), 3 times for 5 minutes each time. Normal donkey serum (Sigma Cat No: D9663) was applied in 1: 10 dilution in antibody diluent [containing 0.55 M sodium chloride and 10 mM sodium phosphate (pH 7.1)] and incubated at room temperature in a humid chamber for 30 minutes. Then the slides were incubated with the primary antibodies against the IGF-IR [(mAbs: 24-60,7C2, 9El 1, 5B6, 4C6), diluted in 1 : 10 dilution of the normal donkey serum (NDS)] at room temperature, overnight and in a humid chamber. As negative controls, no primary antibody and an unrelated Ab (3-beta-HSD, mouse anti human, FD0600Q, Flinders technology) were used. Next day, the slides were washed with hPBS 3 times for 5 minutes for each time followed by incubation with the biotinylated donkey anti-mouse IgG (Jackson Immunoresearch code: 715-066-151) at 1 : 100 dilutions in antibody diluent for two hours at room temperature and in a humid chamber. After washing the slides 3 times for 5 minutes each time with hPBS, they were incubated with Streptavidin CY3/FITC (CYTM3-conjugated streptavidin Jakson Immunoresearch Lab Code:016- 160-084) 1 : 100 dilutions in antibody diluent for one hour at room temperature and in a humid chamber. The slides were washed with hPBS 3 times for 5 minutes each time and wet mounted in mounting medium for fluorescence (Cat. No: S3023; Dako Corporation, Carpinteria, CA, USA). The slides were examined with The Olympus B X 51 microscope with epifluorescence attachment (Olympus Australia) and images captured with a stop RT digital camera (Diagnostic Instruments Inc. Sterling heights, MI, USA). Example 16

Sequencing the variable chains ofMAbs

The sequences of MAbs variable regions were determined using the Mouse Ig-Primer Set kit (Novagen). The sequence of V_H or V_L for each MAb was analysed by IMGT/V QUEST (Giudicelli et al (2004) Nucleic Acids Res 32: W435-40). This software numbers the translated amino acids of the immunoglobulin variable region on the basis of the IMGT unique numbering (Lefranc et al (2003) Dev Comp Immunol 27: 55-77, herein incorporated by reference) and shows the region's structurally important features including the three frameworks and the three CDRs (Pommie et al (2004) J MoI Recognit YV. 17-32, herein incorporated by reference).

Example 17

Epitope Mapping by Competition Assay

Purified MAbs 7C2 and 9El 1 were digested with papain in the presence of the reducing agent cysteine as described in Raychaudhuri et al. (1985) MoI Immunol 22: 1009-1019 (herein incorporated by reference )and Lutomski et al (1995) J Chromatogr B Biomed Appl 664: 79-82 (herein incorporated by reference). Then the Fab domain of the MAbs were purified using protein A column as described in Harlow, E., and Lane, D. (1999) Using Antibodies: a laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (herein incorporated by reference) and labeled with europium following the instruction provided by the DELFIA^® Eu-labelling kit.

To determine the antibodies with overlapping epitopes on the IGF-IR a competition assay was carried out between antibodies for binding to the IGF-IR. The method was as described in the europium competition assay as described above with some modifications. The capturing antibody on the plate was the MAb 24-55 because its epitope does not overlap with the MAb 24-60 and αIR-3 (Gustafson, T. A., and Rutter, W. J. (1990) J Biol Chem 265: 18663-18667, herein incorporated by reference; Soos et al (1992) J Biol Chem 267: 12955-12963, herein incorporated by reference). Then the plate was washed with TBS-T, 100 μl of cultured media containing s-IGF-lR was added to each well of the plate and incubated for overnight at 4°C. After rewashing approximately 300,000 fluorescent counts of Europium-labelled Fab domain of the MAbs (Eu-7C2 or Eu-9E11) in 50 μl (each contains ~ 16.5 ng MAb (Fab)) were added to each well of the plate along with 50 μl of 25 nM solution of unlabelled competitor and incubated for 16 h at 4°C. Then, wells were washed with TBS-T and milli-Q H₂O and after adding lOOμl/well DELFIA^® enhancement solution, the time -resolved fluorescence was measured.

Example 18

Epitope Mapping Using Alanine Mutants of the IGF-IR

Recombinant cDNAs that encode secreted alanine mutants of the IGF-IR or chimeric IGF-1R/256-266IR (The amino acids 256-266 of the IGF-IR replaced with the corresponding amino acids 262-277of IR-A) were expressed transiently in 293 EBNA cells (an adenovirus-transformed human kidney cell line expressing Epstein-Barr virus nuclear antigen) by transfection using lipofection 2000 reagent according to the manufacturer's instructions (Mynarcik et al (1997) J Biol Chem TVt. 18650-18655, herein incorporated by reference). The culture supernatants were harvested after 72 h of culturing the cells at 37°C and 5% (v/v) CO₂. The production of the secreted recombinant receptors was assessed by ELISA as follows. A 96-well MaxiSorp plate was coated with MAb 24-55 (0.25 μg/well) and blocked with 2% (w/v) BSA (BovoStar, Bovogen) in PBS (0.137 M NaCl, 2.7 mM KCl, 1.46 mM KH₂PO4, 8.1 mM Na₂HPO₄, pH 7.4). For the secreted alanine mutants and chimeric IGF-1R/256-266IR, 100 μl of the harvested culture supernatant were added to each well of the plate. In this experiment, 100 μl of culture supernatants from s-IGF-lR cells secreting (containing 0.28 mg/ml receptor) and the harvested supernatant of untransfected cells were used as positive and negative controls respectively. The plate was then incubated overnight at 4°C, and after washing the plate with PBS-T (PBS with 0.1% (v/v) Tween-20), 100 μl of Bt- 16- 13 in 0.005 mg/ml concentration in PBS-T containing 1% (w/v) BSA were added to each well of the plate, and incubated for 2 h at RT. Bt- 16- 13 was used to detect the secreted receptors from the transfected cells. The epitope for the MAb 16-13 is near the N-terminus of the IGF-IR (between amino acids 62-184) which is intact in all of the recombinant constructs. The plate was then washed and the binding was detected with Streptavidin-HRP (CHEMICON) (1 :200 (v/v) diluted) and ABTS reagent (Roche) as manufacture's instruction.

To determine the binding of Eu-7C2 and Eu-9E11 to different alanine mutants of the IGF-IR or the chimeric IGF-1R/256-266IR, the europium binding assay was conducted generally as described for the epitope mapping by competition assay. The supernatant for each mutant of IGF-IR was diluted to give the same absorbance as s-IGF-lR (0.28 mg/ml) as detected in the ELISA. This allowed the same concentrations of secreted receptors to be applied in the europium binding assay for the different secreted receptors. Then, 100 μl of each diluted supernatant were added to each well of the MAb 24-55 coated plate, and incubated overnight at 4°C. After washing the plate, approximately 300,000 fluorescent counts per well of Eu-7C2 or Eu-9E11 in 50 μl (each ~ 16.5 ng MAb (Fab)) were added and incubated for 16 h at 4°C. After washing the plates DELFIA^® enhancement solution was added and the time-resolved fluorescence was measured.

Example 19

Migration Assay

For the migration assay, chemotaxis was measured in a Modified Boyden chamber. Framed polycarbonate filters with a pore size of 12 μm coated with 25 μg/ml collagen type I (Sigma) in 10 mM acetic acid. The lower wells of an AC96 NeuroProbe A Series 96-Well Chamber (NeuroProbe) were filled with RPMI-1640 containing 0.5% (w/v) BSA and different concentrations of IGF-I (1,10 or 10OnM) or no IGF-I. Cell suspensions in serum-free medium (RPMI-1640) containing 0.5% BSA were pre-incubated with calcein-AM (1 μg/ml of final concentration) (Eugene, OR, USA) for 30 mins and then with various MAbs 7C2, 9El 1, αIR-3 or IgGl (negative control) all at a final concentration of 25 nM for 1 h at 37°C, 5% (v/v) CO₂. A series of cells was also incubated in no MAb. The upper wells of the chamber were loaded with 40 μl/ml cell suspension (60,000 cells/well). After the chamber was incubated for 5.5 h at 37 ⁰C and 5% (v/v) CO₂, the membrane was taken out and cells on the upper surface were removed. The transmigrated cells on the lower surface were measured by their fluorescent intensity using Molecular Imager ® Fx (BioRad Laboratories, USA) and expressed as a migration index, representing the fluorescent signals of stimulated cells compared with those of non-stimulated cells.

Example 20

Down-regulation Analysis

To determine the down-regulation of the IGF-IR in human breast cancer cells (MCF-7) in vitro, the method described in Hailey et al. (2002) MoI Cancer Ther J_: 1349-1353 (herein incorporated by reference) was utilised with modifications. MCF-7 cells were used due to the overexpression of the IGF-IR in malignant breast tissue compared with its level in normal breast tissue (Resnik et al (1998) Cancer Res 58: 1159-1164, herein incorporated by reference). These cells were treated with different MAbs against the IGF-IR as well as IGF-I and no MAb and IGF-IR levels were measured by western blot analysis. The cells (7x10⁵) were seeded into each well of 6-well plates in MCF-7 growth medium, and incubated at 37°C in 5% (v/v) CO₂. Then the cells were incubated in serum-free growth medium for 20 h in the same conditions. Subsequently, the treatment solutions, which were IGF-I [50 nM final concentration] or one of the MAbs 9El 1, 7C2 and αIR-3 [all at 25 nM final concentration], were added to each well of the plate (in triplicate) and the cells were incubated for 24 h at 37°C in 5% (v/v) CO2. The effect of the MAbs treatment on the IGF-IR down-regulation was studied by detecting IGF-IR with Western blot analysis. The various treated cells were lysed and centrifuged as described in Denley et al (2004) MoI Endocrinol J_8: 2502-2512 (herein incorporated by reference) and supernatants were collected. Total protein was determined using the BCA assay (Pierce Biotechnology Inc.). 15μg of total protein for each treated MCF-7 cell lysates and 15 μg of R^" cell lysates (as a negative control for IGF-IR) were loaded into a 10% SDS-PAGE gel and electrophoresed under reducing condition and transferred to a nitrocellulose membrane (Hybond TM P, Amersham Pharmacia Biotech). Then the membranes were blocked with 5% (w/v) skim milk in PBS for 2 h at RT and the IGF-IR was detected using anti-IGF-lR antibody C-20 (polyclonal) in a 1: 1,000 (v/v) dilution directed to the β subunit of the IGF-IR by incubating overnight at 4°C. The binding was detected with donkey anti-rabbit HRP-conjugated antibody (Rockland) (1: 10,000 v/v diluted) and the HRP was visualized with enhanced chemiluminescence detection solutions (in house made) and exposed to X-Ray film. PBS-T was used for washing steps and making antibody dilutions.

Example 21

Initial characterization of 5 independent antibodies

ELISA test with s-IGF-lR coated on the plate detected four independent mAbs (4C6, 5B6, 7C2 and 9E11) from a mouse immunized against the s-IGF-lR and one mAb (5B2) from the mouse immunized against P6 cells followed by immunization against the s- IGF-IR.

By FACS analysis, all mAb supernatants were tested against the whole IGF-IR, Insulin receptor type A (IR-A) and Insulin receptor type B (IR-B) and only two of them (7C2 and 9E11) detected the whole receptor on the surface of P6 cells and none of them was positive against the IR-A or IR-B, as shown in Figure 1. The mAb 5B2 (IgM) was also negative in this assay (data not shown).

MAbs were cloned and ELISA isotyping on the sub-clones showed that all sub- clones of the mAbs (4C6, 5B6, 7C2 and 9E11) were IgGl and all sub-clones of 5B2 were IgM, as shown in Figure 2.

Example 22

Purification of mAbs

All hybridoma cells were grown in DMEM+ 2.5% (FCS)+ 1% Penicillin/Streptomycin and secreted IgGl mAbs were purified on a Protein-G column. Running the purified mAbs 9El 1 and 7C2 on SDS-polyacrylamide 10% gel showed two single bands of -47.5 kDa for the heavy chain and -27 kDa for the light chain, as shown in Figure 3. Example 23

Epitope mapping: MAbs 7C2 and 9El 1 bind to the cysteine-rich domain of the IGF-IR

mAbs 9El 1 and 7C2 were selected for epitope mapping because they were positive against the whole receptor on FACS analysis. The chimeras of IGF-1R/IR were utilised to determine the anti-IGF-lR binding region on the IGF-IR. The MAbs 24-60 and 24-55 were used as positive controls. It showed that MAbs 7C2, 9El 1 and 24-60 bound to all three chimeras of IGF-IR/IR but the MAb 24-55 only binds to the IGF-1R/IR Cl expressing cells. It is consistent with the reported epitope for MAbs 24-60 and 24-55 which are between amino acids 184-283 and 440-586 of the IGF-IR respectively. The results are summarised and compared to the results obtained for IGF-IR or IR and shown in Figure 4a. It can be concluded that the epitope for the MAbs 24-60, 7C2 and 9El 1 are in the cysteine-rich domain of the IGF-IR between amino acids 137-315.

Example 24

Competition ofEu-7C2 and Eu-9E11 with other MAbs

Because the flow cytometry analysis on chimeric receptors showed that the epitope for

MAbs 7C2 and 9El 1 is between residues 137-315 of IGF-IR, the MAb 24-55, which binds to different epitope (440-586), was used to capture s-IGF-lR. This assay revealed that Eu-7C2 and Eu-9E11 both significantly compete with 24-60, αIR-3 and each other for binding to the IGF-IR (Fig. 4b). Hence, it can be concluded that, the epitope for MAbs 7C2 and 9El 1 is overlapping with the MAbs 24-60 and αIR-3. It suggests that MAbs 7C2 and 9El 1 bind to the cysteine-rich domain of the receptor because the epitope of both MAbs 24-60 and αIR-3 are in the cysteine-rich domain of the IGF-IR. This is consistent with the results of epitope mapping using chimeric IGF-IR/IR receptors. Example 25

Immunoprecipitation and immunoblotting Analysis

The IGF IR from lysed P6 cells (Denley et al. (2004) MoI Endocrinol 18: 2502-2512) was immunoprecipitated as previously described (Soos et al (1992) J Biol Chem 267: 12955-63) with protein G-agarose beads (Santa Cruz Biotechnology) from 250 μl P6 lysate containing 850 μg total protein. Protein concentration was determined with BCA protein assay reagent (Pierce). Immune complexes were eluted by boiling in 30 μl reducing SDS-PAGE loading buffer and were then subjected to 10% SDS polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli) and transferred to BioTraceTM nitrocellulose membrane (Pall Gelman Laboratory). Blots were probed with MAb IGFR1-2 (1 : 1000) and developed with with anti-mouse, HRP coupled secondary antibody and enhanced chemiluminescence (ECL) using a standard Western immunodetection method (Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K, L.M. A, Coen DM, Varki A, and Chanda VB: Current protocols in molecular biology John Wiley & Sons, Inc., New York, 1995).

For immunoblotting cell lysates (16 or 8 μg) were subjected to 7.5% SDS-PAGE and transferred to Hybond P PVDF membrane (Amersham, Biosciences). Immunoblots membranes were probed with the new MAbs (7C2 and 9El 1), MAb IGFRl 2 (positive control) and mouse IgGl MAb (negative control) at a concentration of 0.4 μg/ml. Stripped blots (methanol, 30 seconds) were reprobed with an anti β actin MAb (1:20,000 dilution, Sigma) to assess uniform loading.

The IGF-IR β subunit and pro-IGF-lR (αβ) were detected in immunoprecipitates with the MAb IGFR1-2. Both MAbs 7C2 and 9El 1 immunoprecipitated the IGF-IR from P6 cell lysates (Fig. 4) as did the positive control MAb 24-55 (Fig. 5a). Immunoblotting experiments showed neither the MAb 9El 1 nor 7C2 detected the α or β subunits of the reduced IGF-IR on immunoblots (Fig. 5). In contrast two bands were detected with IGFRl -2 (Fig. 5b). The ~ 95 kDa band corresponds to the β subunit of the receptor whereas the larger protein detected is a non-specific band also appearing in R^" cell lysates (data not shown). In contrast, immunoblotting experiments under non-reducing conditions showed both 7C2 and 9El 1 reacted with whole receptor under these conditions (Fig. 5 c).

Example 26

Effect of the mAbs on IGF-I or II binding in Europium binding assay

MAbs 9El 1 and 7C2 inhibited the binding of Europium-IGF-I to the soluble receptor by a maximum of about 40% for mAb 7C2 and about 30% for mAb 9El 1, as compared to about 20% for mAb 24-60 at 200 nM concentration, as shown in Figure 6.

However, it seemed that they had no effect on the binding of Europium-IGF-II to the IGF-IR, as shown in Figure 7.

MAb 7C2 showed the highest ligand (Europium-IGF-I) blocking activity (ECso= 0.8526 nM), followed by 9El 1 (EC₅₀=I.15 nM) and finally 24-60

nM), as shown in Figure 8.

Example 27

MAbs 7C2 and 9EII Show High Affinity to the IGF-IR

Figure 9 shows the association and dissociation phases of binding different concentrations of purified s-IGF-lR over the same concentration of mAb 7C2 captured on a IgGl sensor surface. By fitting the binding curves globally to a 1: 1 Langmuir binding model, the association and dissociation kinetics of the interaction between the MAbs and s-IGF-lR were initially assessed. The s-IGF-lR exhibited a fast association rate with both MAbs 7C2 and 9El 1 (k_a shown in Table 1). The resultant complexes between the MAbs and s-IGF-lR were stable as illustrated by the slow dissociation rates (kd shown in Table 1). Kinetic analysis of the biosensorgram curves suggests 7C2 and 9El 1 both have slightly better affinity than αIR-3 for binding to the receptor. However, the KD values for these three MAbs are not significantly different (P>0.05). Table 1 : BIAcore kinetic analysis of binding of MAbs to s-IGF-lR.

MAb Ka kd KD (M)

7C2 (6 .6+1 8)x 10⁴ (3 .l±0.2)x lo-⁵ (0.5+1.6)x lo ⁹

9E11 (0 .8+0 2)x 10⁵ (1 .7+0.1)x lo-⁴ (2.1+0.4)x lo ⁹ αIR-3 (1 .0+0 3)x 10⁵ (1 2 +0.5)x lo ⁴ (1.3+0.5)x lo ⁹

The results generated are from three separate runs and the average of ka, kd and KD for each MAb is shown. Values are the means + SD from three independent experiments. The dissociation constant (KD)=kd/ka, kd= dissociation rate and ka=association rate.

Example 28

Effect of IGF-I, II and the chimeras of the IGF-I and II on binding s-IGF-lR to the mAbs

IGF-I caused changes in binding of s-IGF-lR to the mAbs 9El 1, 7C2 and α-IR3. With more concentration of this ligand in the complex of ligand and receptor there was less binding of the s-IGF-lR to the mAb (Fig 10a showing 9El 1 which behaves the same as 7C2, data not shown). However, over the same range of concentrations (0.01-25OnM) IGF-II did not change the binding ability of s-IGF-lR to the mAbs comparison with no ligand (Fig 10b). These results suggest that the IGF-I inhibited the binding of the mAbs to the receptor but the IGF-II did not.

By plotting the sensorgram RU immediately after injection of the soluble receptor together with different concentrations of IGF-I against the concentration of IGF-I, EC50 for the binding of different concentrations of IGF-I to the soluble IGF-IR, passed over the captured mAbs, was determined. For each mAb the experiment was repeated three times and the EC50 determined. The EC50 for the other mAbs was as follows: EC₅₀ for IGF-I (mAb7C2): 36.11 ± 2.96 (nM)

EC₅₀ for IGF-I (mAb9El 1): 53.46 + 2.42 (nM)

EC₅₀ for IGF-I (mAbαIR-3): 54.58 ± 8.72 (nM)

Example 29

The IGF-I and the IGF-I C Domain Compete with the MAbs 7C2 and 9EII

The BIAcore results showed that the IGF-IICI also concentration dependently caused a reduction in the Resonance Units (RU) for binding the s-IGF-lR to MAbs 7C2, 9El 1 and αIR-3 (Fig 10c). Interestingly, IGF-ICII did not cause any reduction in RU (Fig 1Od). Hence, it can be concluded that the IGF-I C domain is responsible for competition of IGF-I with MAbs 7C2 and 9E 11 for binding to the s-IGF- 1 R.

Example 30

Cell proliferation assay

Cell proliferation assays were determined using a Cell Titer-Glo™ Luminescent Cell Viability Assay Kit. The data is shown in Figure 11.

The results of this assay revealed that the mAbs 9El 1, α-IR3 and 7C2 inhibited the effect of IGF-I (by maximum about 80%) on survival of colon cancer cells (HT-29) from death induced by sodium butyrate (5mM) as a chemotherapeutic reagent.

However, the mAbs were not able to inhibit the effect of IGF-II. Example 31

Immunohistochemical analysis ofmAbs on P6 cell slides

The data is shown in Figure 12.

P6 slides were fixed in different fixatives [Acetone, Ethanol and 10% BFS (Buffered formalin 10%)] and different mAbs were used as a primary mAb followed by incubation with the biotinylated donkey anti-mouse IgG as a secondary antibody and then incubation with Streptavidin CY3/FITC. This experiment showed that all the slides fixed in different fixatives, stained positive by mAbs 24-60 (O. lμg/ml), 9El l(0.2μg/ml), and 7C2(0.2μg/ml). However, there was a high back ground for ethanol-fixed compared to the others. The P6 cells slides were negative in all fixations and unfixed for the no mAb and unrelated mAb, which were the negative controls. MAb 5B6 was also negative in all fixatives.

Example 32

Sequencing Results

Amplification of PCR products for the V_H region of cDNA was achieved using

MuIgV_H5'-F and MuIgG V_H3 '-2 primers for MAb 7C2 and MuIgV_H5'-A and MuIgGV_H3'-2 primers for MAb 9El 1. MuIgκV_L5'-G and MuIgκV_L3'-l primers amplified the V_L region of cDNA for both MAbs. These primers were part of the Mouse Ig-Primer Set kit.

Sequencing of these PCR products revealed the MAbs 7C2 and 9El 1 have different sequences. Nucleotide sequence of the MAbs 9El 1 and 7C2 variable region genes and the deduced amino acid sequences are shown in Fig. 13. Thus, although the biochemical characteristics of the MAbs 7C2 and 9El 1 were similar, each one displayed a unique sequence. Example 33

Identification of the IGF-IR Residues Involved in the MAbs Binding

For the fine epitope mapping of MAbs 7C2 and 9El 1, the binding of the Fab domain to single alanine mutants of the receptor was investigated. Also, the effect of the receptor mobile loop, residues 255-265 on the MAbs binding was tested with chimeric IGF-1R/256-266IR. For all recombinant receptors except the IGF-IR 1255 A, the expression was successful and the ELISA detected secreted receptors in the supernatants. The tested alanine mutants are shown in Figure 14a. After an optimising ELISA on the supernatants containing the recombinant receptors, the supernatants were diluted to give the same absorbances as s-IGF-lR in 0.28 mg/ml concentration using a standard curve for serial dilutions of the s-IGF-lR in the ELISA test. The results for the ELISA on diluted supernatants showed that the recombinant receptors have almost the same absorbances as the culture supernatants from cell secreting s-IGF-lR (containing 0.28 mg/ml of the receptor) indicating the presence of a similar concentrations of expressed receptors. Among all alanine mutants only the mutation of only 3 amino acids, phenylalanine 241, phenylalanine 251 and phenylalanine 266 to alanine have major effects on the binding of either Eu-7C2 or Eu-9E11 to the IGF-IR (Fig. 14a). Also, the chimeric secreted receptor IGF-1R/256-266IR, which is IGF-IR with replacement of the IR amino acids 262-277 into amino acids 256-266 of the IGF-IR, showed dramatic reduction in binding to either Eu-7C2 or Eu-9E11 (Fig. 14a) and this was consistent with the effect of the single alanine substitution at position 266. Residues phenylalanine 241, phenylalanine 251 and phenylalanine 266 map to a similar region of the CR domain (Fig 14b).

Example 34

MAbs 7C2 and 9El 1 Inhibit Cancer Cells Migration

In this study, the anti-migration effects of the generated MAbs were studied in breast cancer MCF-7 cell line as a model due to the overexpression of the IGF-IR (up to 14-fold) in malignant breast tissue compared with its level in normal breast tissue. This assay revealed that MAbs 9El 1 (25v nM) and 7C2 (25 nM) inhibited the IGF-I induced migration significantly in the highest concentration of IGF-I (100 nM) as shown in (Fig. 15 a). MAbs αIR-3 (25 nM) and 24-60 (25nM) did not inhibit the migration induced by 100 or 10 nM IGF-I however, they both inhibited the effect of 1 nM IGF-I (Fig.15a).

Example 35

MAbs 7C2 and 9El 1 Down-Regulate IGF-IR

In the lysate extracted from the P6 cells, the β subunit of the IGF-IR was detected with Western blot analysis and anti-IGF-lR antibody. Prolonged treatment of the MCF-7 cells with the MAbs revealed that compared to no treatment, the level of IGF-IR was dramatically reduced after 24 h treatment with 25 nM MAbs 7C2, 9El 1, 24-60 or αlR- 3. In contrast, the treatment with IGF-I (50 nM) did not down-regulate the IGF-IR compared to no treatment (Fig. 15 B). A non-specific band was detected in P6 cells lysate as well as R^" cells lysate for the anti-IGF-lR antibody (C -20) at 55 kDa. These bands could be considered as loading controls.

General Discussion

In this study we report the binding characterisation and epitope mapping of two high affinity anti-IGF-lR MAbs 7C2 and 9El 1. The europium competition assay revealed MAbs 7C2 and 9El 1 efficiently inhibit IGF-I binding to the IGF-IR. The inhibitory effect (IC50) of the MAbs on IGF-I binding is comparable with other MAbs to IGF-IR. Moreover, the kinetic studies using BIAcore showed the affinity of MAbs 7C2 and 9El 1 to IGF-IR to be high and similar to αIR-3 (see Table 1). The binding analysis experiments revealed that MAbs 7C2 and 9El 1 only inhibited IGF-I binding to the receptor and not IGF-II. The same results have been obtained for the MAb αIR-3. It can be concluded that this reflects the presence of two distinct binding sites for IGF-I and IGF-II on IGF-IR. Fine epitope mapping showed that for both MAbs 7C2 and 9El 1, the three amino acids F241, F251 and F266 which are in the cysteine-rich domain of IGF-IR are involved in epitope. These amino acids are distinct from each other in the sequence, but mapping of these amino acids on the model of the IGF-IR domains from the crystal structure of the IGF-IR Ll -cysteine -rich-L2 domains revealed that they are actually very close together in the folded form of the receptor. The observation that the disruptive mutants in the cysteine-rich domain of the receptor form a patch on the protein surface surrounded by non-disruptive mutations suggests that they are contact sites for MAbs 7C2 and 9El 1. In addition, it has been shown in other studies that the mutation of residues F241 and F251 to alanine caused a major reduction in binding IGF-I to the receptor. However, mutation of residues F241, F251 and F266 to alanine had no significant effect on binding IGF-II to the receptor. Hence, it can be concluded that binding of MAbs 7C2 and 9El 1 particularly to amino acids F241 and F251 is responsible for specific inhibition of binding IGF-I to the receptor. Moreover, although it is possible that these three mutants of the IGF-IR could cause indirect intramolecular perturbation of the structure of the binding site for MAbs, this seems unlikely. Particularly for F266, because its mutation to alanine had no effect on IGF-I or IGF-II binding, the direct binding of the MAbs to F266 is very likely.

In this study it was shown that chimeric IGF-I/IGF-II ligands, IGF-ICII and IGF-IICI represented similar effects to IGF-II and IGF-I, respectively. It can be concluded that the C domain of the IGF-I interferes with the binding of MAbs 7C2 and 9El 1 to IGF- IR. This finding also implies that out of all domains of the IGF-I, it is the C domain, which binds to the epitope for these MAbs or to nearby residues which were sterically affected by the presence of MAbs binding to the receptor. This binding is related to residues F241 and F251 of IGF-IR as mentioned earlier.

In the C domain of IGF-I, the amino acids R36 and R37 and also Y31 have been recognized as critical for binding to IGF-IR. Therefore, it suggests that the binding of the IGF-I C domain to the IGF-IR cysteine-rich domain involves these three amino acids (R36, R37 and Y31) binding to or in close proximity residues F241 and F251. In addition, it has been shown that the positively charged residues, R36 and R37, bind to the N-terminal 283 amino acids of the IGF-IR α-subunit and more specifically to the cysteine -rich region 217-283 of the IGF-IR. Electrostatic interactions could be involved in the IGF-I C domain binding to the receptor.

Biological Effects of the MAbs was tested in vitro. The cell proliferation experiment showed that MAbs 7C2 and 9El 1 inhibited IGF-I induced HT-29 cell proliferation. This effect of the MAbs was consistent with their inhibitory effects on IGF-I binding. It suggests that the inhibitory effect of MAbs 7C2 and 9El 1 on cell proliferation was due to their inhibiting effect on the IGF-I binding to the IGF-IR. In addition, the proliferation assay revealed that MAbs 7C2 and 9El 1 inhibited IGF-II induced HT-29 cell proliferation. However, this inhibitory effect could not be considered as potent as the effect of MAbs 7C2 and 9El 1 on IGF-I induced proliferation. Because these MAbs did not inhibit IGF-II binding to the receptor, their effects on IGF-II induced cell proliferation could be due to an indirect effect of the MAbs on the IGF-IR, such as inactivation or down-regulation of the receptor. This is consistent with the result for the receptor down-regulation experiment on MCF-7 cells, which showed that 24 h incubation with MAbs 7C2 and 9El 1, reduced IGF-IR levels dramatically. So, it implies that in HT-29 cells, the IGF-IR is down-regulated resulting in indirect inhibition of the IGF-II induced proliferation.

In this study we also showed that MAbs 7C2 and 9El 1 inhibited MCF-7 cells migration induced by IGF-I, indicating the potential anti-metastatic effects of these MAbs.

Finally, it will be appreciated that various modifications and variations of the described methods and compositions of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are apparent to those skilled in the art are intended to be within the scope of the present invention.

Claims

Claims:

1. An antibody to insulin-like growth factor I receptor, or an antigen-binding portion of the antibody, the antibody or the antigen-binding portion binding to an epitope located in the cysteine-rich domain of the α-subunit of the insulin-like growth factor I receptor, wherein the antibody or the antigen-binding portion modulates IGF-I mediated proliferation of an IGF-I dependent cell.

2. An antibody according to claim 1, wherein the antibody has an affinity (K_D) to insulin-like growth factor I receptor of at least 3x 10 ⁹M.

3. An antibody according to claim 1, wherein the antibody has an affinity (K_D) to insulin-like growth factor I receptor of at least IxIO ⁹M.

4. An antibody according to claim 1, wherein the antibody has an affinity (K_D) to insulin-like growth factor I receptor of at least 5xl0^"10M.

5. An antibody according to any one of claims 1 to 4, wherein the antibody binds to an epitope in the cystein-rich domain of the α-subunit of the insulin-like growth factor I receptor that includes one or more amino acids selected from the group consisting of phenylalanine 241, phenylalanine 252 and phenylalanine 266.

6. An antibody according to any one of claims 1 to 5, wherein the antibody inhibits IGF-I mediated proliferation of an IGF-I dependent cell.

7. An antibody according to any one of claims 1 to 6, wherein the antibody does not substantially inhibit binding of IGF-II to the insulin growth factor receptor I.

8. An antibody according to any one of claims 1 to 7, wherein the antibody has an isotype selected from the group consisting of IgGl, IgG2a, IgG2b, IgG3, IgM and IgA.

9. An antibody according to any one of claims 1 to 8, wherein the antibody is a monoclonal antibody.

10. An antibody according to any one of claims 1 to 9, wherein the antibody is a human antibody or a humanized antibody.

11. An antibody according to any one of claims 1 to 10, wherein the antibody includes the following amino acid sequences:

(i) a V_H CDR-I sequence according to SEQ ID NO. 20, and a V_n CDR-2 sequence according to SEQ ID NO. 21, and a V_H CDR-3 sequence according to SEQ ID NO. 8; and/or (ii) a V_L CDR-I sequence according to SEQ ID NO.22, and a V_L CDR-2 sequence according to SEQ ID NO. 23, and a V_L CDR-3 sequence according to SEQ ID NO.11; and/or an antibody, or an antigen-binding portion thereof, including a variant of one or more of the aforementioned sequences, that binds to IGF-I receptor.

12. An antibody, or an antigen binding portion thereof, including the following CDR amino acid sequences:

13. An antibody, or an antigen binding portion thereof, including the following amino acid sequences:

14. A hybridoma cell expressing an antibody according to any one of claims 1 to 13.

15. A method of detecting insulin-like growth factor receptor 1, the method including the step step of binding an antibody according to any one of claims 1 to 13, or an antigen-binding portion thereof, to insulin-like growth factor I receptor.

16. A method of modulating IGF-dependent proliferation of a cell, the method including binding an antibody according to any one of claims 1 to 13, or an antigen- binding portion thereof, to insulin-like growth factor I receptor expressed on the cell.

17. A method according to claim 16, wherein the binding of the antibody or the antigen binding portion thereof inhibits proliferation of the cell.

18. A method according to claims 16 or 17, wherein the cell is a cancerous cell or a pre-cancerous cell.

19. A method according to any one of claims 16 to 18, wherein the modulation of proliferation of the cell occurs in a human.

20. A method according to claim 19, wherein the human is suffering from, or susceptible to, one or more of diseases or conditions selected from the group consisting of acromegaly, ovarian cancer, pancreatic cancer, benign prostatic hyperplasia, breast cancer, prostate cancer, bone cancer, lung cancer, colorectal cancer, cervical cancer, synovial sarcoma, diarrhea associated with metastatic carcinoid, vasoactive intestinal peptide secreting tumours, gigantism, psoriasis, atherosclerosis, smooth muscle restenosis of blood vessels and inappropriate microvascular proliferation.

21. A pharmaceutical composition including an antibody to insulin-like growth factor I receptor according to any one of claims 1 to 13, and/or including an antigen- binding portion thereof.

22. A method of preventing and/or treating an IGF-I dependent disease or condition in a subject, the method including administering to the subject a therapeutically effective amount of an antibody to insulin-like growth factor I receptor according to any one of claims 1 to 13, and/or administering an antigen-binding portion thereof.

23. A method according to claim 22, wherein the subject is a human subject.

24. A method according to claim 23, wherein the human is suffering from, or susceptible to, one or more of diseases or conditions selected from the group consisting of acromegaly, ovarian cancer, pancreatic cancer, benign prostatic hyperplasia, breast cancer, prostate cancer, bone cancer, lung cancer, colorectal cancer, cervical cancer, synovial sarcoma, diarrhea associated with metastatic carcinoid, vasoactive intestinal peptide secreting tumours, gigantism, psoriasis, atherosclerosis, smooth muscle restenosis of blood vessels and inappropriate microvascular proliferation.

25. An isolated compound including one or more of the amino acids sequences selected from the group consisting of SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.8, SEQ ID NO.22, SEQ ID NO.23, and SEQ ID NO.11.

26. An isolated compound according to claim 25, wherein the compound is a polypeptide.

27. An isolated compound according to claims 24 or 25, wherein the compound binds to an epitope located in the cysteine-rich domain of the α subunit of the insulin- like growth factor receptor.

28. An isolated compound according to claim 27, wherein the compound binds to an epitope in the cystein-rich domain of the α-subunit of the insulin-like growth factor I receptor that includes one or more amino acids selected from the group consisting of phenylalanine 241, phenylalanine 252 and phenylalanine 266.

29. An isolated compound according to any one of claims 25 to 28, wherein the compound modulates binding of IGF-I to IGF-I receptor.

30. An isolated compound according to claim 29, wherein the compound inhibits binding of IGF-I to IGF-I receptor.

31. An isolated compound according to any one of claims 25 to 30, wherein the compound inhibits IGF-I mediated proliferation of an IGF-I dependent cell.

32. An isolated compound according to any one of claims 25 to 29, wherein the compound promotes binding of IGF-I to IGF-I receptor.

33. An isolated compound according to any one of claims 25 to 32, wherein the compound is an antibody or a fragment thereof.

34. An isolated compound according to claim 33, wherein the antibody fragment is selected from the group consisting of a Fab, a Fab', a F(ab')₂, a Fv, a Facb, and a single- chain antibody.

35. A cell expressing a compound according to any one of claims 26 to 34.

36. A method of detecting insulin-like growth factor receptor 1, the method including binding a compound according to any one of claims 25 to 34 to insulin-like growth factor receptor 1.

37. A method of modulating proliferation of an IGF-I dependent cell, the method including binding a compound according to any one of claims 25 to 34 to insulin-like growth factor I receptor expressed on the cell.

38. A method according to claim 37, wherein the binding of the compound inhibits proliferation of the cell.

39. A method according to claims 37 or 38, wherein the cell is a cancerous cell or a pre-cancerous cell.

40. A method according to any one claims 37 to 39, wherein the modulation of proliferation of the cell occurs in a human.

41. A method according to claim 40, wherein the human is suffering from, or susceptible to, one or more of diseases or conditions selected from the group consisting of acromegaly, ovarian cancer, pancreatic cancer, benign prostatic hyperplasia, breast cancer, prostate cancer, bone cancer, lung cancer, colorectal cancer, cervical cancer, synovial sarcoma, diarrhea associated with metastatic carcinoid, vasoactive intestinal peptide secreting tumours, gigantism, psoriasis, atherosclerosis, smooth muscle restenosis of blood vessels and inappropriate microvascular proliferation.

42. A pharmaceutical composition including a compound according to any one of claims 25 to 34.

43. A method of preventing and/or treating an IGF-I dependent disease or condition in a subject, the method including administering to the subject a therapeutically effective amount of a compound according to any one of claims 25 to 34.

44. A method according to claim 43, wherein the subject is a human subject.

45. A method according to claim 44, wherein the human is suffering from, or susceptible to, one or more of diseases or conditions selected from the group consisting of acromegaly, ovarian cancer, pancreatic cancer, benign prostatic hyperplasia, breast cancer, prostate cancer, bone cancer, lung cancer, colorectal cancer, cervical cancer, synovial sarcoma, diarrhea associated with metastatic carcinoid, vasoactive intestinal peptide secreting tumours, gigantism, psoriasis, atherosclerosis, smooth muscle restenosis of blood vessels and inappropriate microvascular proliferation.

46. An isolated nucleic acid including a nucleotide sequence encoding a polypeptide including one or more of the amino acids sequences selected from the group consisting of SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.8, SEQ ID NO.22, SEQ ID NO.23, and SEQ ID NO.l 1.

47. An expression vector including the nucleic acid according to claim 46.

48. A cell including the nucleic acid according to claim 44 and/or the expression vector according to claim 47.