WO2007093008A1 - Anticorps dirigés contre le récepteur du facteur de croissance i analogue à l'insuline - Google Patents

Anticorps dirigés contre le récepteur du facteur de croissance i analogue à l'insuline Download PDF

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WO2007093008A1
WO2007093008A1 PCT/AU2007/000168 AU2007000168W WO2007093008A1 WO 2007093008 A1 WO2007093008 A1 WO 2007093008A1 AU 2007000168 W AU2007000168 W AU 2007000168W WO 2007093008 A1 WO2007093008 A1 WO 2007093008A1
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igf
antibody
seq
receptor
binding
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PCT/AU2007/000168
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English (en)
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Mehrnaz Keyhanfar
John Wallace
Grant Booker
Briony Forbes
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Adelaide Research & Innovation Pty Ltd
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Publication of WO2007093008A1 publication Critical patent/WO2007093008A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3015—Breast
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • 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.
  • 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.
  • IGF-I insulin-like growth factor-I
  • IGF-IR insulin-like growth factor I receptor
  • IGF-IR activation is critical for stimulation of tumour cell growth and survival.
  • IGF-I, IGF- II and IGF-IR are important mediators of the malignant phenotype.
  • 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.
  • 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.
  • 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 ⁇ -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:
  • 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:
  • 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 ⁇ -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.
  • 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.
  • 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.
  • antibody as used throughout the specification means an entire antibody molecule or any antigen-binding portion of an antibody molecule.
  • 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.
  • 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.
  • 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.
  • 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-
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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 TyperTM- 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 ⁇ IR-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).
  • ss-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).
  • the unrelated IgGl antibody was applied as a negative control.
  • 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: ⁇ IR-3 treated, Lane 7: R " cell lysates (no treatment), a) Pro-IGF-1R, b) IGF-IR ⁇ subunit, c) non-specific band (used here as loading control). Molecular weight was estimated in kilodaltons using the MagicMarkTMXP marker. A representative experiment is shown.
  • 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 ⁇ -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.
  • 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.
  • 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 ⁇ - ⁇ - ⁇ - ⁇ configuration. The binding of insulin-like growth-factor-I
  • IGF-I insulin-like growth factor-II
  • IGF-II insulin-like growth factor-II
  • the IGF-I receptor is homologous to insulin receptor, having a high sequence similarity in the ⁇ chain tyrosine kinase domain and a lower sequence similarity in the ⁇ chain.
  • the ⁇ -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.
  • 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
  • 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.
  • IGF-I receptor and its ligands 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.
  • 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.
  • 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 ⁇ -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.
  • 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 ⁇ light chains.
  • Heavy chains are classified as ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • 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.
  • 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).
  • CDR Complementarity Determining Regions
  • FR Framework Regions
  • CDR-I to CDR-3 Complementarity Determining Regions
  • FR-I to FR-4 Framework Regions
  • 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.
  • the antibody is a mouse or a human antibody, or a humanized antibody.
  • Methods for humanizing antibodies are known in the art.
  • 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.
  • 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 ⁇ -subunit of the receptor.
  • the antibody to insulin-like growth factor I receptor is an antibody raised against an animal or human insulin-like growth factor I receptor.
  • 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.
  • the antibody is an antibody raised against a soluble form of the insulin-like growth factor I receptor.
  • the form of the receptor will include the cysteine -rich domain of the ⁇ -subunit of the receptor.
  • the antibody in the various embodiments of the present invention may be a polyclonal or a monoclonal antibody.
  • the antibody is a monoclonal antibody.
  • the monoclonal antibody is 9El 1 or 7C2, as defined herein.
  • V H and V L regions of 9E11 are as follows:
  • 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:
  • amino acid sequence corresponding to the nucleotide sequence in the V H region of 7C2 is as follows:
  • 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:
  • 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:
  • 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 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.
  • the antibody is raised to an epitope in the cystein-rich domain of the ⁇ -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.
  • various adjuvants may be used to increase an immunological response.
  • 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.
  • 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.
  • 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.
  • the compound is a polypeptide.
  • 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.
  • 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 ⁇ -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 isolated cell is a hybridoma cell.
  • the isolated cell may be a hybridoma producing the monoclonal antibody 9El 1 or 7C2.
  • the cell may further be an isolated cell.
  • the present invention provides a cell expressing an antibody, or an antigen binding portion thereof, including the following CDR sequences:
  • 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.
  • the present invention provides a cell expressing an antibody, or an antigen binding portion thereof, including:
  • 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.
  • 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.
  • 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.
  • CDRs complementarity determining regions
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ - subunit of the receptor.
  • the antibodies do not recognise the related insulin receptor type A (IR-A) or insulin receptor type B (IR-B).
  • 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 ⁇ -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.
  • chimeric receptors of the insulin receptor and the insulin-like growth factor I receptor may be used.
  • 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.
  • the isotype of the antibody is selected from the group consisting of IgGl, IgG2a, IgG2b, IgG3, IgM and IgA.
  • 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.
  • 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%.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ -subunit of the insulin-like growth factor I receptor.
  • the antibody or antigen binding portion thereof will modulate IGF-I dependent proliferation by modulation binding of IGF-I to the IGF-I receptor.
  • 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
  • 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.
  • 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:
  • 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.
  • 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.
  • the compound is a polypeptide, such as an antibody or an antigen binding portion thereof.
  • 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.
  • the cell may be an isolated cell or a cell present in a biological system.
  • the cell is a cancerous cell or a pre-cancerous cell.
  • biological system means any cellular system.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • prokaryotic eg E. coli
  • eukaryotic cells eg a hybridoma cell
  • 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.
  • 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 ⁇ -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.
  • 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
  • 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.
  • 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:
  • V L CDR-I amino acid sequence according to SEQ ID NO. 22 (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.
  • 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.
  • assay methods such as Western Blot, ELISA, competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays may be utilized.
  • 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.
  • the antibodies may be used in a conventional immunoassay, including an ELISA, an RIA, FACS, tissue immunohistochemistry, Western blot or immunoprecipitation.
  • the IGF-IR may be a purified or semi-purified form of the receptor, or be a receptor present in a biological sample.
  • 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.
  • 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.
  • the antibody, antigen-binding portion or antibody mimetic may labelled with a detectable moiety and thereby detected directly.
  • 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.
  • 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.
  • detectable moieties include radioisotopes, such as 3 H, 14 C, 32 P, 35 S, or 131 I; fluorescent or chemiluminescent compounds, such as fluorescein isothiocyanate, rhodamine, or luciferin; or an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase.
  • radioisotopes such as 3 H, 14 C, 32 P, 35 S, or 131 I
  • fluorescent or chemiluminescent compounds such as fluorescein isothiocyanate, rhodamine, or luciferin
  • an enzyme such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase.
  • 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.
  • a detectable moiety such as a radio-opaque agent or radioisotope
  • 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.
  • 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 ⁇ -subunit of the insulin-like growth factor I receptor.
  • 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:
  • 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.
  • 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
  • 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.
  • 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.
  • an IGF-I dependent disease or condition eg a disease or condition which is mediated by elevated activity of IGF-IR due to binding of IGF-I
  • 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.
  • the subject is an animal or human.
  • 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.
  • the subject is a human.
  • 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.
  • 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
  • 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.
  • the ability of an antibody to inhibit cancer can be evaluated in an animal model system predictive of efficacy in human tumours.
  • 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.
  • the therapeutic agent may be either the antibody, fragment or compound itself, or be conjugated to another moiety.
  • an antibody or antibody fragment may be conjugated to a cytotoxic agent.
  • 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.
  • 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.
  • 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 ⁇ -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 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
  • 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.
  • 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:
  • V L CDR-2 amino acid sequence according to SEQ ID NO. 23 (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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • mucus formers or resins may be included.
  • 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.
  • a suitable aqueous solution of a bactericidal and/or fungicidal agent and/or any other suitable preservative, and preferably including a surface active agent 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 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.
  • 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.
  • 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.
  • 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.
  • IR-A insulin receptor type A
  • IR-B insulin receptor type 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.
  • 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.
  • 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.
  • the cell may be an isolated cell or a cell present in a biological system.
  • 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.
  • 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.
  • 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.
  • the IGF-IR may be present in a purified or semi-purified form or be present in a biological sample.
  • 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.
  • 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.
  • a detectable moiety such as a radio-opaque agent or radioisotope
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • R IR-B cells R " cells expressing insulin receptor isoform-B, IR-B
  • 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 Sydney, 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 ⁇ : 789-798 (herein incorporated by reference) for the production of recombinant soluble insulin receptor extracellular domain.
  • 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.
  • DMEM Dulbecco's Modified Eagle's medium
  • HAM Nutrient Mixture
  • HAM Nutrient Mixture
  • 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 ⁇ M methionine sulfoximine.
  • s-IGF-lR soluble receptor
  • BHK 21 cell line Syrian hamster kidney
  • 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.
  • 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 ⁇ l IM Tris solution, pH 9.
  • a female BALB/C mouse was given 3 intraperitoneal injections of 30 or 50 ⁇ g s-IGF- IR plus 50 ⁇ g 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 ⁇ g 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).
  • 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) xlO 6 P6 cells 3 to 12 weeks apart followed by two injections of 30-40 ⁇ g of s-IGF-lR plus 50 ⁇ g adjuvant, 3 weeks apart. After 4 weeks the mouse was boosted with 30 ⁇ g 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.
  • mice IgGl negative control 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 ⁇ l 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.
  • FACS fluorescence - activated cell sorting
  • 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 ⁇ subunits of the IR-B (or IR exon 11+) isoform. The IR-A (or IR exon H-) isoform lacks these 12 amino acids.
  • 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.
  • 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.
  • R " cells were infected with the constructs using Lipofectamine+TM (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.
  • the cells were trypsinized, washed and suspended in PBS, 10 5 to 10 6 cells per FACS tube.
  • the cells were centrifuged, the supernatant aspirated off and the cells resuspended in 100 ⁇ l 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 ⁇ l). 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 ⁇ l of 1% paraformaldehyde in PBS and stored in the dark on ice until FACS acquisition.
  • the Mouse Typer Sub- Isotyping Panel (BIO-RAD Cat No: 172-3055) was used.
  • the Maxisorp (NuncTM) plate was coated with s-IGF-lR and blocked with 200 ⁇ l of 2-3% BSA in PBS.
  • the primary antibody which was the mAbs in the neat supernatant, was added 100 ⁇ l/well and incubated at room temperature for 1 hour. After washing the plate 3 times with PBS- Tween 20 (0.1%), 50 ⁇ l 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 ⁇ l 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.
  • 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.
  • mAbs 9El 1, 7C2 and IgGl negative control CHEMICON Australia
  • 2.5 ⁇ g 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.
  • 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.
  • the three chimeric receptors were:
  • 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)
  • 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)
  • 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 ⁇ g/ml.
  • Europium-IGF-I and Europium-IGF-11 were labelled using Wallac Kit 1244-302 and purified with SuperdexTM HR 10/30 (Pharmacia, Sweden).
  • the plate was then washed once with TBST (Tris 50 mM, NaCl 15mM, Tween- 20 (0.05%), pH 8.0) solution and 100 ⁇ l 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 ⁇ l of the solution (5 ⁇ l +100 ⁇ l Enhancement solution) was prepared.
  • TBST Tris 50 mM, NaCl 15mM, Tween- 20 (0.05%), pH 8.0
  • the plate was then washed once with TBST and incubated with 50 ⁇ l of the Europium-IGF-1 or II plus 50 ⁇ l 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 ⁇ l Enhancement solution (DELFIA@/AutoDELFIATM, 1244-105, Finland), time -resolved fluorescence was read by microplate reader (BMG FluoStar Galaxy).
  • s-IGF-lR soluble IGF-IR
  • mAbs 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
  • Cell proliferation was measured using a Cell-Titer GloTM 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% CO 2 , the medium was replaced with the FCS free medium and the plate was left in the incubator for 5 hours to starve the cells.
  • 12000 HT-29 cells colon cancer tumour cells
  • assay buffer Different concentrations of mAbs were made in assay buffer were prepared ((47%)DMEM +(47%) F- 12 Nutrient Mixture (HAM) GIBCOTM 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 ⁇ l 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% CO 2 and then left at room temperature for 2-3 hours.
  • the Cell-Titer GloTM reagents (Cell-Titer GloTM Luminescent Cell Viability Assay Kit Promega, USA, Cat No: G7571) were mixed and then 100 ⁇ l 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 The Cell-Titer GloTM reagents (Cell-Titer GloTM Luminescent Cell Viability Assay Kit Promega, USA, Cat No: G7571) were mixed and then 100 ⁇ l 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).
  • 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.
  • hypertonic phosphate -buffered saline 10 mM sodium/potassium phosphate with 0.274 M NaCl, 5 mM KCl; pH 7.2; hPBS
  • 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.
  • 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).
  • 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).
  • 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.
  • Recombinant cDNAs that encode secreted alanine mutants of the IGF-IR or chimeric IGF-1R/256-266IR 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) CO 2 .
  • 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 ⁇ g/well) and blocked with 2% (w/v) BSA (BovoStar, Bovogen) in PBS (0.137 M NaCl, 2.7 mM KCl, 1.46 mM KH 2 PO4, 8.1 mM Na 2 HPO 4 , pH 7.4).
  • BSA BovoStar, Bovogen
  • 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.
  • 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 ⁇ l of each diluted supernatant were added to each well of the MAb 24-55 coated plate, and incubated overnight at 4°C.
  • chemotaxis was measured in a Modified Boyden chamber. Framed polycarbonate filters with a pore size of 12 ⁇ m coated with 25 ⁇ g/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.
  • 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.
  • MCF-7 human breast cancer cells
  • the cells (7x10 5 ) were seeded into each well of 6-well plates in MCF-7 growth medium, and incubated at 37°C in 5% (v/v) CO 2 . 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 ⁇ IR-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 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 ⁇ 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.
  • 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.
  • 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.
  • 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.
  • 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, ⁇ IR-3 and each other for binding to the IGF-IR (Fig. 4b).
  • the epitope for MAbs 7C2 and 9El 1 is overlapping with the MAbs 24-60 and ⁇ IR-3.
  • the IGF IR from lysed P6 cells 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 ⁇ l P6 lysate containing 850 ⁇ g total protein. Protein concentration was determined with BCA protein assay reagent (Pierce).
  • Immune complexes were eluted by boiling in 30 ⁇ l 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).
  • SDS-PAGE SDS polyacrylamide gel electrophoresis
  • ECL enhanced chemiluminescence
  • the IGF-IR ⁇ subunit and pro-IGF-lR ( ⁇ ) 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 ⁇ or ⁇ subunits of the reduced IGF-IR on immunoblots (Fig. 5). In contrast two bands were detected with IGFRl -2 (Fig. 5b).
  • ⁇ 95 kDa band corresponds to the ⁇ subunit of the receptor whereas the larger protein detected is a non-specific band also appearing in R " cell lysates (data not shown).
  • immunoblotting experiments under non-reducing conditions showed both 7C2 and 9El 1 reacted with whole receptor under these conditions (Fig. 5 c).
  • 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.
  • 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.
  • 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.
  • IGF-I caused changes in binding of s-IGF-lR to the mAbs 9El 1, 7C2 and ⁇ -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.
  • 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 ⁇ IR-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.
  • the results of this assay revealed that the mAbs 9El 1, ⁇ -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.
  • 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. l ⁇ g/ml), 9El l(0.2 ⁇ g/ml), and 7C2(0.2 ⁇ g/ml).
  • 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.
  • MuIg ⁇ V L 5'-G and MuIg ⁇ V L 3'-l primers amplified the V L region of cDNA for both MAbs. These primers were part of the Mouse Ig-Primer Set kit.
  • 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.
  • 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).
  • 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).
  • the ⁇ 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 ⁇ lR- 3.
  • the treatment with IGF-I 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.
  • IGF-II/IGF-II ligands IGF-ICII and IGF-IICI represented similar effects to IGF-II and IGF-I, respectively.
  • 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.
  • 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.
  • the positively charged residues, R36 and R37 bind to the N-terminal 283 amino acids of the IGF-IR ⁇ -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.
  • MAbs 7C2 and 9El 1 inhibited MCF-7 cells migration induced by IGF-I, indicating the potential anti-metastatic effects of these MAbs.

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Abstract

La présente invention concerne un anticorps dirigé contre le récepteur du facteur de croissance I analogue à l'insuline ou une partie de liaison antigénique de cet anticorps. L'anticorps ou la partie de liaison antigénique se lie à un épitope situé dans le domaine riche en cystéine de la sous-unité α du récepteur du facteur de croissance I analogue à l'insuline, et l'anticorps ou la partie de liaison antigénique module la prolifération d'une cellule IGF-I-dépendante médiée par l'IGF-I.
PCT/AU2007/000168 2006-02-17 2007-02-16 Anticorps dirigés contre le récepteur du facteur de croissance i analogue à l'insuline WO2007093008A1 (fr)

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WO2008098115A2 (fr) * 2007-02-07 2008-08-14 Xencor, Inc. Anti-corps igf-1r optimisés et procédés utilisant ceux-ci
US7667021B2 (en) 2002-05-24 2010-02-23 Schering Corporation Neutralizing human anti-IGFR antibody
EP2216344A1 (fr) * 2007-11-14 2010-08-11 Forerunner Pharma Research Co., Ltd. Diagnostic et traitement du cancer à l'aide d'un anticorps anti-gpr49
WO2010105302A1 (fr) * 2009-03-19 2010-09-23 Queensland University Of Technology Cibles pour la signalisation des facteurs de croissance et procédés thérapeutiques
US7811562B2 (en) 2004-12-03 2010-10-12 Schering Corporation Biomarkers for pre-selection of patients for anti-IGF1R therapy
WO2010146059A2 (fr) 2009-06-16 2010-12-23 F. Hoffmann-La Roche Ag Biomarqueurs pour une thérapie par inhibiteur d'igf-1r
US8017735B2 (en) 2003-11-21 2011-09-13 Schering Corporation Anti-IGFR1 antibody therapeutic combinations
US8062886B2 (en) 2003-11-12 2011-11-22 Schering Corporation Plasmid system for multigene expression

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Cited By (18)

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Publication number Priority date Publication date Assignee Title
US7667021B2 (en) 2002-05-24 2010-02-23 Schering Corporation Neutralizing human anti-IGFR antibody
US7847068B2 (en) 2002-05-24 2010-12-07 Schering Corporation Neutralizing human anti-IGFR antibody
US8062886B2 (en) 2003-11-12 2011-11-22 Schering Corporation Plasmid system for multigene expression
US8017735B2 (en) 2003-11-21 2011-09-13 Schering Corporation Anti-IGFR1 antibody therapeutic combinations
US7811562B2 (en) 2004-12-03 2010-10-12 Schering Corporation Biomarkers for pre-selection of patients for anti-IGF1R therapy
WO2008098115A3 (fr) * 2007-02-07 2008-10-02 Xencor Inc Anti-corps igf-1r optimisés et procédés utilisant ceux-ci
WO2008098115A2 (fr) * 2007-02-07 2008-08-14 Xencor, Inc. Anti-corps igf-1r optimisés et procédés utilisant ceux-ci
AU2008321840B2 (en) * 2007-11-14 2014-02-06 Chugai Seiyaku Kabushiki Kaisha Diagnosis and treatment of cancer using anti-GPR49 antibody
EP2216344A4 (fr) * 2007-11-14 2010-12-29 Forerunner Pharma Res Co Ltd Diagnostic et traitement du cancer à l'aide d'un anticorps anti-gpr49
JPWO2009063970A1 (ja) * 2007-11-14 2011-03-31 株式会社 未来創薬研究所 抗ｇｐｒ４９抗体を用いる癌の診断および治療
EP2216344A1 (fr) * 2007-11-14 2010-08-11 Forerunner Pharma Research Co., Ltd. Diagnostic et traitement du cancer à l'aide d'un anticorps anti-gpr49
US8680243B2 (en) 2007-11-14 2014-03-25 Chugai Seiyaku Kabushiki Kaisha Diagnosis and treatment of cancer using anti-GPR49 antibody
CN102112492B (zh) * 2007-11-14 2015-02-25 中外制药株式会社 使用抗gpr49抗体的癌症的诊断和治疗
JP5676107B2 (ja) * 2007-11-14 2015-02-25 中外製薬株式会社 抗ｇｐｒ４９抗体を用いる癌の診断および治療
JP2015134757A (ja) * 2007-11-14 2015-07-27 中外製薬株式会社 抗ｇｐｒ４９抗体を用いる癌の診断および治療
US9296823B2 (en) 2007-11-14 2016-03-29 Chugai Seiyaku Kabushiki Kaisha Diagnosis and treatment of cancer using anti-GPR49 antibody
WO2010105302A1 (fr) * 2009-03-19 2010-09-23 Queensland University Of Technology Cibles pour la signalisation des facteurs de croissance et procédés thérapeutiques
WO2010146059A2 (fr) 2009-06-16 2010-12-23 F. Hoffmann-La Roche Ag Biomarqueurs pour une thérapie par inhibiteur d'igf-1r

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