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WO2009023266A1 - Production d'anticorps dirigés contre des récepteurs de surface cellulaire et des protéines associées au cancer comprenant des membres de la famille des récepteurs du facteur de croissance épidermique (egfr) - Google Patents

Production d'anticorps dirigés contre des récepteurs de surface cellulaire et des protéines associées au cancer comprenant des membres de la famille des récepteurs du facteur de croissance épidermique (egfr) Download PDF

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Publication number
WO2009023266A1
WO2009023266A1 PCT/US2008/009772 US2008009772W WO2009023266A1 WO 2009023266 A1 WO2009023266 A1 WO 2009023266A1 US 2008009772 W US2008009772 W US 2008009772W WO 2009023266 A1 WO2009023266 A1 WO 2009023266A1
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egfr
cysteine
antibodies
cancer
tumor
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PCT/US2008/009772
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English (en)
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Antony Burgess
Thomas Garrett
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Ludwig Institute For Cancer Research
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Publication of WO2009023266A1 publication Critical patent/WO2009023266A1/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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • 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
    • C07K16/3053—Skin, nerves, brain
    • 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
    • C07K16/3069—Reproductive system, e.g. ovaria, uterus, testes, prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • 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 generally to the generation of modulators including antibodies to cell-surface receptors and cancer-associated proteins, and particularly to such receptors or proteins with cysteine rich regions or cysteine loops.
  • Cell-surface receptors and cancer-associated proteins with cysteine rich regions or cysteine loops and suitable in this invention include EGFR and EGFR family members, insulin receptor, and tyrosine kinases such as IGFR, Ret and Ror.
  • a method is provided for the generation of modulators or binders, including antibodies, to cancer associated proteins, particularly cell-surface receptors, which may, particularly in cancers or on amplification or overexpression or other tumorigenic states, display epitopes not necessarily available in correctly folded and processed proteins (wild type or normal situations).
  • chemotherapeutic means often relies upon exploiting differences in target proliferating cells and other normal cells in the human or animal body.
  • many chemical agents are designed to be taken up by rapidly replicating DNA so that the process of DNA replication and cell division is disrupted.
  • Another approach is to identify antigens or specific structure or activity aspects on the surface of tumor cells or other abnormal cells which are not normally expressed in developed human tissue, such as tumorigenic forms, tumor antigens or embryonic antigens.
  • Such structures, activities or antigens can be targeted with modulators or binding proteins such as antibodies which can inhibit, block or neutralize the antigen or tumorigenic form, structure, or activity.
  • the modulators or binding proteins, including antibodies and fragments thereof may deliver a toxic agent or other substance which is capable of directly or indirectly activating a toxic agent at the site of a tumor.
  • the extracellular domains of tumor or cancer-associated proteins are appropriate targets for modulators and antibody therapies.
  • various such proteins have cysteine-rich domains or regions, including cysteine loops which provide target regions.
  • Cell-surface receptors and cancer-associated proteins with cysteine rich regions or cysteine loops include EGFR and EGFR family members (ErbBl, ErbB2, ErbB3, ErbB4), insulin receptor, and tyrosine kinases such as IGFR, Ret and Ror.
  • the stability of the cysteine loops is relevant or even critical for activity.
  • Insulin receptor (IR) family there are a number of mutations such as Leu233Pro (numbers from 1 in the mature polypeptide, see Klinkhamer et al, 1989) which would compromise the structural integrity of the cysteine-rich region and such mutations significantly affect the signalling competence of these receptors (Klinkhamer MP, et al (1989) EMBO J 8:2503-2507).
  • the ErbB family receptors are a promising target for antibody-based cancer therapies. Amplification/overexpression and mutant forms of the coding genes of these receptors have been found in many tumour types. The level of overexpression of the various ErbB members have been demonstrated by immunohistochemistry in many different types of cancer. In particular, ErbB2 is found 25% of breast cancer and ErbB3 is found 80% of tumours of the gastro-intestinal tract. Over expression and the ability of these receptors to heterodimerize with other family members is associated with aggressive disease and poor prognosis. [0005] The EGFR is an attractive target for tumor-targeted antibody therapy because it is over expressed in many types of epithelial tumors (Voldborg, B.
  • the corresponding EGFR protein has a 267 amino acid ⁇ deletion comprising residues 6-273 of the extracellular domain and a novel glycine residue at the fusion junction (Sugawa, N., et al. (1990) Proc Natl Acad Sci USA 87: 8602-6).
  • This deletion together with the insertion of a glycine residue, produces a unique junctional peptide at the deletion interface.
  • expression of this truncated receptor is restricted to tumor cells it represents a highly specific target for antibody therapy. Accordingly, a number of laboratories have reported the generation of both polyclonal and monoclonal antibodies specific to the unique peptide of de2-7 EGFR (Wikstrand, C.
  • de2-7 EGFR antibodies are particularly useful.
  • Modulators, including antibodies, which do not target normal tissues or normal protein forms but which particularly and specifically target tumor associated, tumorigenic, or amplified forms of tumor proteins are particularly needed and applicable. In particular, a general approach to deriving these modulators, including antibodies, would be extremely valuable and applicable.
  • One such antibody modulator an anti- EGFR antibody, monoclonal antibody mAb806 and its epitope
  • WO02092771 and WO05081854 Studies of this antibody and its EGFR epitope have revealed aspects of the structure and folding and forms of EGFR which now provide new approaches to generating modulators, including antibodies against EGFR and other tumor associated proteins.
  • certain structures and sequence aspects in the extracellular domain of these tumor and cancer-associated proteins provide a means and methods to generate novel therapies and therapeutics, including antibodies.
  • the invention provides a method for the generation of modulators, including antibodies, to cancer associated proteins, particularly cell-surface receptors, which may display epitopes not necessarily available for correctly folded and processed proteins.
  • modulators including antibodies
  • cancer associated proteins particularly cell-surface receptors
  • One appropriate such target is the cysteine-rich proteins such as EGFR and EGFR family members, including ErbB2, ErbB3, and ErbB4.
  • the mAb806 antibody binds to an epitope in EGFR, a cysteine rich cancer- associated protein, which is not available under wt normal conditions, but is exposed in tumorigenic EGFR mutants (e.g. de 2-7 EGFR) and upon amplification and/or overexpression of EGFR.
  • tumorigenic EGFR mutants e.g. de 2-7 EGFR
  • CGADS YEMEEDGVRKC cysteine loop 287-302 epitope
  • cysteine-rich or cysteine loop containing proteins and tyrosine kinases such as IGFR, Ret and Ror are particular potential targets.
  • Immunisation or screening to generate and identify modulators, including antibodies, would be with short disulfide- bonded modules, truncated proteins or mutants where a disulfide bond has been removed. More broadly, the generation of antibodies to transitional forms of growth factor receptors represents a novel way of reducing normal tissue targeting yet retaining anti- signaling activity.
  • the invention generally provides a method for generating immunogenic epitopes or target sites in a tumor-associated cysteine-containing protein comprising disrupting one or more cysteine loop or cysteine rich domain, whereby one or more cysteine is mutated to a different amino acid, such that an altered form of said tumor associated protein results that forms a target for modulators or an immunogenic peptide or protein for antibodies.
  • the tumor associated protein is a receptor with a cysteine loop or cysteine rich domain.
  • the tumor associated protein is selected from a member of the EGFR family, and a member of the insulin receptor family.
  • the EGFR family member is ErbB2, ErbB3 or ErbB4.
  • the insulin receptor family member is insulin receptor (ISNR) or insulin- like growth factor receptor (IGFlR).
  • Methods for screening modulators including antibodies, are provided to isolate or select those which are selective and specific for cysteine rich regions or loops of tumor-associated proteins, and in particular, which target or bind an epitope on the tumor-associated protein(s) which is hidden or is not readily exposed in the absence of overexpression, amplification, or such other tumorigenic alteration or activity.
  • the present invention relates to methods for identifying agents capable of modulating the expression or activity of proteins involved in the processes leading to cancer, cancer pathology, and tumors.
  • the present invention provides methods for identifying agents, including antibodies, which target cryptic or hidden cysteine loop or cysteine domains in cancer-associated proteins, particularly cell-surface receptors, and their use in the prevention and / or treatment of tumors and cancer.
  • the invention includes cysteine mutated tumor-associated proteins or mutant cysteine loop peptides.
  • isolated cysteine mutated peptides or proteins are contemplated and provided.
  • the cell surface receptor cysteine mutants may be prepared and/or expressed as soluble proteins, expressing only the extracellular domain. These soluble cysteine mutants are useful in screening or as immunogenic compositions.
  • aspects of the present method include the in vitro assay of compounds, including antibodies, using mutated cysteine modified polypeptide(s) of a cancer-associated protein, or fragments thereof. Cysteine-mutated fragments, peptides or proteins are modified at cysteine positions such that the fragments or proteins expose these cysteine bounded epitopes or targets. In some instances the cysteine modifie proteins are more tumorigenic, less tumorigenic or equally tumorigenic. Irrespective of their tumorigenicity however, the cysteine modified proteins or peptides are useful in effectively screening for and generating cancer-specific and anti-cancer agents, modulators, antibodies. Examplary cysteine mutant sequences, particularly wherein alanine replaces cysteine are described and exemplified herein.
  • Figure 1 Alignment of amino acid sequences for CDR' s from mAb806 and mAbl75. Sequence differences between the two antibodies are bolded.
  • FIG. 2 Immunohistochemical staining of cell lines and normal human liver with mAbl75.
  • A Biotinylated mAbl75 was used to stain sections prepared from blocks containing A431 cells (over-express the wtEGFR), U87MG. ⁇ 2-7 cells (express the ⁇ 2-7 EGFR) and U87MG cells (express the wtEGFR at modest levels).
  • B Staining of normal human liver (40Ox) with mAbl75 (left panel), isotype control (centre panel) and secondary antibody control (right panel). No specific sinusoidal or hepatocyte staining was observed.
  • Figure 3 Reactivity of mAb806 and mAbl75 with fragments of the EGFR displayed on yeast.
  • A Representative flow cytometry histograms depicting the mean fluorescence signal of mAbl75 and mAb806 labeling of yeast-displayed EGFR fragments. With yeast display a percentage of cells do not express protein on their surface resulting in 2 histogram peaks. The 9E10 antibody is used as a positive control as all fragments contain a linear C-terminal c-myc tag.
  • B Summary of antibody binding to various EGFR fragments.
  • C The EGFR fragments were denatured by heating yeast pellets to 80° C for 30 min. The c-myc tag was still recognized by the 9E10 anti-myc antibody in all cases, demonstrating that heat treatment does not compromise the yeast surface displayed protein.
  • the conformation sensitive EGFR antibody mAb 225 was used to confirm denaturation.
  • FIG. 4 Antitumor effects of mAbl75 on brain and prostate cancer xenografts.
  • B Cells were stained with two irrelevant antibodies ⁇ blue, solid and green, hollow), mAb 528 for total EGFR (pink, solid), mAb806 (light blue, hollow) and mAbl75 (orange, hollow) and then analyzed by FACS.
  • C DU145 cells were lysed, subjected to IP with mAb 528, mAb806, mAb 175 or two independent irrelevant antibodies and then immunoblotted for EGFR.
  • FIG. 5 Crystal structures of EGFR peptide 287-302 bound to the Fab fragments
  • A Cartoon of Fab 806, with the light chain, red; heavy chain, blue; bound peptide, yellow; and the superposed EGFR 287-302 from EGFR, purple.
  • B Cartoon of Fab 175 with the light chain, yellow; heavy chain, green; bound peptide, lilac; and EGFR 287- 302 from EGFR(D 1-3), purple.
  • C Detail from (B) showing the similarity Of EGFR 287-302 in the receptor to the peptide bound to FAb 175. Peptides backbones are shown as Ca traces and the interacting side chains as sticks.
  • O atoms are coloured red; N, blue; S, orange and C, as for the main chain.
  • D Superposition of EGFR with the Fabl75:peptide complex showing spacial overlap. Colouring as in (C) with the suface of EGFRl 87-286 coloued turquoise.
  • E Orthogonal view to (D) with EGFRl 87-286 shown in opaque blue and the surface of the light (orange) and heavy (green) chains transparent.
  • F Detailed stereoview of 175 Fab complex looking into the antigen-binding site. Colouring as in (C) and side chain hydrogen bonds dotted in black. Water molecules buried upon complex formation are shown as red spheres.
  • Figure 6 Influence of the 271-283 cystine bond on mAb806 binding to the EGFR.
  • A Cells transfected with wtEGFR, EGFR-C271A, EGFR-C283A or the C271 A/C283A mutant were stained with mAb528 (solid pink histogram), mAb806 (blue line) or only the secondary antibody (purple) and then analyzed by FACS. The gain was set up using a class-matched irrelevant antibody.
  • B BaF3 cells expressing the EGFR- C271A or or C271/283A EGFR were examined for their response to EGF in an MTT assay as described in Methods. EC 50 S were derived using the Bolzman fit of the data points. Data represent mean and sd of triplicate measurements
  • C BaF3 cells expressing the wt or the EGFR- C271A/C283A were IL-3 and serum starved, then exposed to EGF or vehicle control. Whole cell lyates were separated by SDS-PAGE and immunoblotted with anti-phosphotyrosine antibody (top panel) or anti-EGFR antibody (bottom panel).
  • Figure 7 A) Whole body gamma camera image of the biodistribution of 111 In- ch806 in a patient with metastatic squamous cell carcinoma of the vocal cord, showing quantitative high uptake in tumour in the right neck (arrow). Blood pool activity, and minor catabolism of free 1 11 In in liver, is also seen.
  • SPECT Single Photon Computed Tomography
  • Figure 8 A stereo model of the structure of the untethered EGFRl -621.
  • the receptor backbone is traced in blue and the ligand TGF- ⁇ in red.
  • the mAb806/175 epitope is drawn in turquoise and the disulfide bonds in yellow.
  • the atoms of the disulfide bond which ties the epitope back into the receptor are shown in space-filling format.
  • the model was constructed by docking the EGFR-ECD CR2 domain from the tethered conformation ⁇ 5) onto the structure of an untethered EGFR monomer in the presence of its ligand (14).
  • Figure 9 Reactivity of mAb806 with fragments of the EGFR. Lysates from 293T cells transfected with vectors expressing the soluble 1-501 EGFR fragment or GH/EGFR fragment fusion proteins (GH-274-501, GH-282-501, GH-290-501 and GH- 298-501) were resolved by SDS-PAGE, transferred to membrane and immunoblotted with mAb806 (left panel) or the anti-myc antibody 9Bl 1 (right panel).
  • Figure 10 depicts the ErbB2 amino acid sequence (SEQ ID NO: 6) with the C277/C289 806 homologous region noted.
  • Figure 11 depicts the ErbB3 protein structure and domains.
  • Figure 12-1 through 12-9 provides the ErbB3 amino acid sequence (SEQ ID NO: 7) and encoding nucleic acid sequence (SEQ ID NO: 8). The cysteine region homologous to the 806 epitope is highlighted.
  • Figure 13 depicts the ErbB4 protein structure and domains.
  • Figure 14 shows the ErbB4 amino acid sequence (SEQ ID NO: 9) and the homologous region to the 806 epitope is highlighted.
  • Figure 15 A and 15B depict the generation of plasmid constructs for soluble ErbB protein expression.
  • Figure 16 provides the insulin-like growth factor receptor (IGFlR) amino acid sequence (SEQ ID NO: 10), with the cysteines 282 and 303 underlined.
  • Figure 17 provides the insulin receptor (INSR) amino acid sequence (SEQ ID NO: 11), with the cysteines 286 and 311 underlined.
  • IGFlR insulin-like growth factor receptor
  • INSR insulin receptor
  • the term "aberrant expression” in its various grammatical forms may mean and include any heightened or altered expression or overexpression of a protein in a tissue, e.g. an increase in the amount of a protein, caused by any means including enhanced expression or translation, modulation of the promoter or a regulator of the protein, amplification of a gene for a protein, or enhanced half-life or stability, such that more of the protein exists or can be detected at any one time, in contrast to a non-overexpressed state.
  • Aberrant expression includes and contemplates any scenario or alteration wherein the protein expression or post-translational modification machinery in a cell is taxed or otherwise disrupted due to enhanced expression or increased levels or amounts of a protein, including wherein an altered protein, as in mutated protein or variant due to sequence alteration, deletion or insertion, or altered folding is expressed.
  • abnormal quantities of protein may result from overexpression of the protein in the absence of gene amplification, which is the case e.g. in many cellular/tissue samples taken from the head and neck of subjects with cancer, while other samples exhibit abnormal protein levels attributable to gene amplification.
  • antibody should be construed as covering any specific binding member or substance having a binding domain with the required specificity.
  • this term covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023 and U.S. Patent Nos. 4,816,397 and 4,816,567.
  • binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CHl domains; (ii) the Fd fragment consisting of the VH and CHl domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward, E.S.
  • an "antibody combining site” is that structural portion of an antibody molecule comprised of light chain or heavy and light chain variable and hypervariable regions that specifically binds antigen.
  • antibody molecule in its various grammatical forms as used herein contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule.
  • Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contains the paratope, including those portions known in the art as Fab, Fab 1 , F(ab') 2 and F(v), which portions are preferred for use in the therapeutic methods described herein.
  • Antibodies may also be bispecific, wherein one binding domain of the antibody is a specific binding member of the invention, and the other binding domain has a different specificity, e.g. to recruit an effector function or the like.
  • Bispecific antibodies of the present invention include wherein one binding domain of the antibody is a specific binding member of the present invention, including a fragment thereof, and the other binding domain is a distinct antibody or fragment thereof, including that of a distinct anti- EGFR antibody, for instance antibody 528 (U.S. Patent No. 4,943,533), the chimeric and humanized 225 antibody (U.S. Patent No. 4,943,533 and WO/9640210), an anti-de2-7 antibody such as DH8.3 (Hills, D.
  • the other binding domain may be an antibody that recognizes or targets a particular cell type, as in a neural or glial cell-specific antibody.
  • the one binding domain of the antibody of the invention may be combined with other binding domains or molecules which recognize particular cell receptors and/or modulate cells in a particular fashion, as for instance an immune modulator (e.g., interleukin(s)), a growth modulator or cytokine (e.g. tumor necrosis factor (TNF), and particularly, the TNF bispecific modality demonstrated in U.S.S.N. 60/355,838 filed February 13, 2002 incorporated herein in its entirety) or a toxin (e.g., ricin) or anti-mitotic or apoptotic agent or factor.
  • an immune modulator e.g., interleukin(s)
  • a growth modulator or cytokine e.g. tumor necrosis factor (TNF)
  • TNF tumor necrosis factor
  • Fab and F(ab') 2 portions of antibody molecules may be prepared by the proteolytic reaction of papain and pepsin, respectively, on substantially intact antibody molecules by methods that are well-known. See for example, U.S. Patent No. 4,342,566 to Theofilopolous et al.
  • Fab' antibody molecule portions are also well-known and are produced from F(ab') 2 portions followed by reduction of the disulfide bonds linking the two heavy chain portions as with mercaptoethanol, and followed by alkylation of the resulting protein mercaptan with a reagent such as iodoacetamide.
  • An antibody containing intact antibody molecules is preferred herein.
  • the phrase "monoclonal antibody” in its various grammatical forms refers to an antibody having only one species of antibody combining site capable of immunoreacting with a particular antigen.
  • a monoclonal antibody thus typically displays a single binding affinity for any antigen with which it immunoreacts.
  • a monoclonal antibody may also contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different antigen; e.g., a bispecific (chimeric) monoclonal antibody.
  • an antigen binding domain describes the part of an antibody which comprises the area which specifically binds to and is complementary to part or all of an antigen. Where an antigen is large, an antibody may bind to a particular part of the antigen only, which part is termed an epitope.
  • An antigen binding domain may be provided by one or more antibody variable domains. Preferably, an antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).
  • VL antibody light chain variable region
  • VH antibody heavy chain variable region
  • Post-translational modification may encompass any one of or combination of modification(s), including covalent modification, which a protein undergoes after translation is complete and after being released from the ribosome or on the nascent polypeptide cotranslationally.
  • Post-translational modification includes but is not limited to phosphorylation, myristylation, ubiquitination, glycosylation, coenzyme attachment, methylation and acetylation.
  • Post-translational modification can modulate or influence the activity of a protein, its intracellular or extracellular destination, its stability or half- life, and/or its recognition by ligands, receptors or other proteins
  • Post-translational modification can occur in cell organelles, in the nucleus or cytoplasm or extracellularly.
  • the term "specific” may be used to refer to the situation in which one member of a specific binding pair will not show any significant binding to molecules other than its specific binding partner(s).
  • the term is also applicable where e.g. an antigen binding domain is specific for a particular epitope which is carried by a number of antigens, in which case the specific binding member carrying the antigen binding domain will be able to bind to the various antigens carrying the epitope.
  • the term "consisting essentially of” refers to a product, particularly a peptide sequence, of a defined number of residues which is not covalently attached to a larger product.
  • a product particularly a peptide sequence
  • minor modifications to the N- or C- terminal of the peptide may however be contemplated, such as the chemical modification of the terminal to add a protecting group or the like, e.g. the amidation of the C-terminus.
  • isolated refers to the state in which antibodies of the invention, or nucleic acid encoding such antibodies or CDRs thereof will be, in accordance with the present invention.
  • Antibodies and nucleic acid will be free or substantially free of material with which they are naturally associated such as other polypeptides or nucleic acids with which they are found in their natural environment, or the environment in which they are prepared (e.g. cell culture) when such preparation is by recombinant DNA technology practised in vitro or in vivo.
  • Antibodies and nucleic acid may be formulated with diluents or adjuvants and still for practical purposes be isolated - for example the members will normally be mixed with gelatin or other carriers if used to coat microtitre plates for use in immunoassays, or will be mixed with pharmaceutically acceptable carriers or diluents when used in diagnosis or therapy.
  • Antibodies may be glycosylated, either naturally or by systems of heterologous eukaryotic cells, or they may be (for example if produced by expression in a prokaryotic cell) unglycosylated.
  • glycoproteins include and encompasses the post-translational modification of proteins, termed glycoproteins, by addition of oligosaccarides. Oligosaccharides are added at glycosylation sites in glycoproteins, particularly including N-linked oligosaccharides and O-linked oligosaccharides. N-linked oligosaccharides are added to an Asn residue, particularly wherein the Asn residue is in the sequence N-X-S/T, where X cannot be Pro or Asp, and are the most common ones found in glycoproteins.
  • a high mannose type oligosaccharide (generally comprised of dolichol, N- Acetylglucosamine, mannose and glucose is first formed in the endoplasmic reticulum (ER). The high mannose type glycoproteins are then transported from the ER to the Golgi, where further processing and modification of the oligosaccharides occurs. O- linked oligosaccharides are added to the hydroxyl group of Ser or Thr residues. In O- linked oligosaccharides, N-Acetylglucosamine is first transferred to the Ser or Thr residue by N-Acetylglucosaminyltransferase in the ER.
  • amino acid residues described herein are preferred to be in the "L" isomeric form.
  • residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the desired fuctional property of immunoglobulin-binding is retained by the polypeptide.
  • NH 2 refers to the free amino group present at the amino terminus of a polypeptide.
  • COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide.
  • amino-acid residue sequences are represented herein by formulae whose left and right orientation is in the conventional direction of amino- terminus to carboxy-terminus. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino-acid residues.
  • the above Table is presented to correlate the three-letter and one-letter notations which may appear alternately herein.
  • a "replicon” is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control.
  • a "vector” is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.
  • a "DNA molecule” refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form, or a double- stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes.
  • linear DNA molecules e.g., restriction fragments
  • viruses e.g., plasmids, and chromosomes.
  • sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).
  • An "origin of replication" refers to those DNA sequences that participate in DNA synthesis.
  • a DNA "coding sequence” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5 1 (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus.
  • a coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences.
  • a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.
  • Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
  • a "promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • a transcription initiation site (conveniently defined by mapping with nuclease Sl), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT” boxes.
  • Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the -10 and -35 consensus sequences.
  • An "expression control sequence” is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence.
  • a coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.
  • a "signal sequence” can be included before the coding sequence. This sequence encodes a signal peptide, N-terminal to the polypeptide, that communicates to the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the media, and this signal peptide is clipped off by the host cell before the protein leaves the cell. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes.
  • oligonucleotide as used herein in referring to the probe of the present invention, is defined as a molecule comprised of two or more ribonucleotides, preferably more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of the oligonucleotide.
  • primer refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH.
  • the primer may be either single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon many factors, including temperature, source of primer and use of the method.
  • the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.
  • Primers are selected to be "substantially" complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template.
  • a non- complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the strand.
  • non- complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the strand to hybridize therewith and thereby form the template for the synthesis of the extension product.
  • restriction endonucleases and “restriction enzymes” refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.
  • a cell has been "transformed” by exogenous or heterologous DNA when such DNA has been introduced inside the cell.
  • the transforming DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.
  • the transforming DNA may be maintained on an episomal element such as a plasmid.
  • a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA.
  • a "clone” is a population of cells derived from a single cell or common ancestor by mitosis.
  • a "cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.
  • Two DNA sequences are "substantially homologous" when at least about 75% (preferably at least about 80%, and most preferably at least about 90 or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Maniatis et al., supra; DNA Cloning, VoIs. I & II, supra; Nucleic Acid Hybridization, supra.
  • DNA sequences encoding peptides, proteins of use in the invention and antibodies of the invention which code for e.g. a peptide region of a tumor-associated protein, particularly a cysteine-rich region or cysteine loop region of a tumor associated protein, having or comprising the amino acid sequences described, referred to, and/or set out herein, such as as SEQ ID NOS: 1- but which are degenerate to SEQ ID NOS: 1-.
  • degenerate to is meant that a different three-letter codon is used to specify a particular amino acid. It is well known in the art that the following codons can be used interchangeably to code for each specific amino acid:
  • codons specified above are for RNA sequences.
  • the corresponding codons for DNA have a T substituted for U.
  • Mutations can be made in nucleic acid sequences encoding the antibody domains set out herein such that a particular codon is changed to a codon which codes for a different amino acid. Such a mutation is generally made by making the fewest nucleotide changes possible.
  • a substitution mutation of this sort can be made to change an amino acid in the resulting protein in a non-conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to another grouping) or in a conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to the same grouping).
  • Such a conservative change generally leads to less change in the structure and function of the resulting protein.
  • a non-conservative change is more likely to alter the structure, activity or function of the resulting protein.
  • the present invention should be considered to include seguences containing conservative changes which do not significantly alter the activity or binding characteristics of the resulting protein.
  • Amino acids with charged polar R groups negatively charged at Ph 6.0
  • Aspartic acid Glutamic acid Basic amino acids (positively charged at pH 6.0) Lysine, Arginine, Histidine (at pH 6.0)
  • Another grouping may be those amino acids with phenyl groups: Phenylalanine, Tryptophan, Tyrosine
  • Another grouping may be according to molecular weight (i.e., size of R groups):
  • Amino acid substitutions may also be introduced to substitute an amino acid with a particularly preferable property.
  • a Cys may be introduced as a potential site for disulfide bridges with another Cys.
  • a Cys may be replaced by any of various amino acids.
  • Alanine is used as an exemplary amino acid herein, however other amino acids may also be utilized. Similar amino acids to alanine, for example may be utilized. Other such non-polar amino acids might be substituted, or amino acids of similar size, structure or molecular weight.
  • a His may be introduced as a particularly "catalytic" site (i.e., His can act as an acid or base and is the most common amino acid in biochemical catalysis).
  • Pro may be introduced because of its particularly planar structure, which induces, ⁇ -turns in the protein's structure.
  • Two amino acid sequences are "substantially homologous" when at least about 70% of the amino acid residues (preferably at least about 80%, and most preferably at least about 90 or 95%) are identical, or represent conservative substitutions.
  • a "heterologous" region of the DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature.
  • the gene when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism.
  • Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.
  • phrases "pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
  • terapéuticaally effective amount is used herein to mean an amount sufficient to prevent, and preferably reduce by at least about 30 percent, preferably by at least 50 percent, preferably by at least 70 percent, preferably by at least 80 percent, preferably by at least 90%, a clinically significant change in the growth or progression or mitotic activity of a target cellular mass, group of cancer cells or tumor, or other feature of pathology.
  • the degree of EGFR activation or activity or amount or number of EGFR positive cells, particularly of antibody or binding member reactive or positive cells may be reduced.
  • a DNA sequence is "operatively linked" to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that DNA sequence.
  • the term "operatively linked” includes having an appropriate start signal (e.g., ATG) in front of the DNA sequence to be expressed and maintaining the correct reading frame to permit expression of the DNA sequence under the control of the expression control sequence and production of the desired product encoded by the DNA sequence. If a gene that one desires to insert into a recombinant DNA molecule does not contain an appropriate start signal, such a start signal can be inserted in front of the gene.
  • standard hybridization conditions refers to salt and temperature conditions substantially equivalent to 5 x SSC and 65 0 C for both hybridization and wash.
  • standard hybridization conditions are dependent on particular conditions including the concentration of sodium and magnesium in the buffer, nucleotide sequence length and concentration, percent mismatch, percent formamide, and the like.
  • Also important in the determination of “standard hybridization conditions” is whether the two sequences hybridizing are RNA- RNA, DNA-DNA or RNA-DNA.
  • standard hybridization conditions are easily determined by one skilled in the art according to well known formulae, wherein hybridization is typically 10-20 0 C below the predicted or determined T m with washes of higher stringency, if desired.
  • the term 'treating' means an intervention performed with the intention of preventing the development or altering the pathology of, and thereby ameliorating a disorder, disease or condition, including one or more symptoms of such disorder or condition. Accordingly, 'treating' refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treating include those already with the disorder as well as those in which the disorder is to be prevented.
  • the related term 'treatment,' as used herein, refers to the act of treating a disorder, symptom, disease or condition, as the term 'treating' is defined above.
  • therapeutically effective dose means that amount of protein, polynucleotide, peptide, or its antibodies, agonists or antagonists, which ameliorate the symptoms or condition.
  • Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, ED 50 (the dose therapeutically effective in 50% of the population) and LD 50 (the dose lethal to 50% of the population).
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD 50 /ED 50 .
  • Pharmaceutical compositions that exhibit large therapeutic indices are particular.
  • the data obtained from cell culture assays and animal studies are used in formulating a range of dosage for human use.
  • the dosage of such compounds lies particularly within a range of circulating concentrations that include the ED 5O with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
  • the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs.
  • the animal model is also used to achieve a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • the exact dosage is chosen by the individual physician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Additional factors which may be taken into account include the severity of the disease state, age, weight and gender of the patient; diet, desired duration of treatment, method of administration, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long acting pharmaceutical compositions might be administered every 3 to 4 days, every week, or once every two weeks depending on half- life and clearance rate of the particular formulation.
  • compositions according to this invention may be administered to a subject by a variety of methods. They may be added directly to targeted tissues, complexed with cationic lipids, packaged within liposomes, or delivered to targeted cells by other methods known in the art. Localized administration to the desired tissues may be done by direct injection, transdermal absorption, catheter, infusion pump or stent. Alternative routes of delivery include, but are not limited to, intravenous injection, intramuscular injection, subcutaneous injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, intraperitoneal and/or intrathecal delivery. Examples of ribozyme delivery and administration are provided in Sullivan et al. WO 94/02595.
  • Antibodies according to the invention may be delivered as a bolus only, infused over time or both administered as a bolus and infused over time.
  • Those skilled in the art may employ different formulations for polynucleotides than for proteins.
  • delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
  • the EGFR exists in two well-defined conformers - tethered and untethered.
  • the tethered conformer which has only been observed in ligand-free (and partly ligated) forms of the receptor, can be induced by a ligand to form the untethered, back-to-back dimer.
  • mAb806 recognizes an epitope on some truncated, overexpressed or activated forms of the EGFR on the cell surface, but it does not recognize the EGFR on normal unstimulated cells.
  • Another related antibody, mAbl75 also recognizes this unusual epitope.
  • This epitope has been identified as the loop 287-302 epitope (CGADSYEMEEDGVRKC) (SEQ ID NO: 1).
  • CGADSYEMEEDGVRKC SEQ ID NO: 1
  • mAb806 fails to inhibit the in vitro growth of cells expressing wild-type EGFR, mAb806 inhibits completely, ligand associated stimulation of BaF/3 cells expressing EGFR C27IA/C 2 83A -
  • Our results indicate that the mechanisms of binding of antibodies mAb806 and mAbl75 requires a form of the EGFR where the epitope is preferentially exposed either during receptor activation or through truncation or overexpression. Consequently, and in contrast to other EGFR antibodies, mAb806 preferentially localizes to the tumor in cancer patients overexpressing the EGFR.
  • the mechanism of action suggests new approaches to the generation of antibodies for detection of tumors and for improving antibody/inhibitor killing of cancer cells with over-expressed, truncated or activated forms of receptors in the EGFR family
  • Fab 806 and 175 The structures of Fab 806 and 175 showed that these antibodies bind the peptide antigen previously identified and that it bound the antigen in the same conformation as it is found in both the tethered and untethered conformations of the EGF receptor. As these antibodies do not bind well to inactive EGFR, it was perhaps not surprising was that the mode of binding was inconsistent with a monomer (or dimer) of EGFR being in any of the conformations characterised so far. However, the structures show how the antibodies recognised the epitope and lead to a hypothesis that 806 and 175 must open up or partially unfold in the neighbourhood of the epitope to allow access to the antigen.
  • the epitope in a cryptic epitope is not generally available in the folded wild-type receptor. However, it can be made more accessible by reducing the structural rigidity of the receptor, such as during the transition from tethered to untethered which occurs upon activation, or by incorrect post-translational modifications, such as transient breaking of disulfide bonds or disulfide scrambling. Theoretically, access to such an epitope could also be facilitated by incorrect or immature glycosylation.
  • the invention provides a method for the generation of antibodies to cancer associated proteins, particularly cell-surface receptors, which may display epitopes not necessarily available for correctly folded and processed proteins.
  • cysteine-rich proteins such as EGFR and EGFR family members, including ErbB2, ErbB3, and ErbB4.
  • antibodies targeted to the equivalent cysteine loop in ErbB3/B4 by similar cysteine mutations wherein the disulfide bond is removed may have similar properties to mAbs 806 and 175 (i.e. specificity restricted to tumors and the ability to block receptor activation).
  • TABLE 1 provides a comparison of the loop sequence of EGF family members EGFR, ErbB2, ErbB3 and ErbB4.
  • the cysteine loop sequences for ErbB2, ErbB3, and ErbB4 are provided in SEQ ID NOS: 3, 4, and 5 respectively.
  • cysteine-rich or cysteine loop containing proteins and tyrosine kinases such as IGFR, Ret and Ror are particular potential targets. Immunisation would be with short disulfide-bonded modules, truncated proteins or mutants where a disulfide bond has been removed. More broadly, the generation of antibodies to transitional forms of growth factor receptors represents a novel way of reducing normal tissue targeting yet retaining anti-signaling activity.
  • the invention generally provides a method for generating immunogenic epitopes or target sites in a tumor-associated cysteine-containing protein comprising disrupting one or more cysteine loop or cysteine rich domain, whereby one or more cysteine is mutated to a different amino acid, such that an altered form of said tumor associated protein results that forms a target for modulators or an immunogenic peptide or protein for antibodies.
  • the tumor associated protein is a receptor with a cysteine loop or cysteine rich domain.
  • the tumor associated protein is selected from a member of the EGFR family, and a member of the insulin receptor family.
  • the EGFR family member is ErbB2, ErbB3 or ErbB4.
  • the insulin receptor family member is insulin receptor (ISNR) or insulin- like growth factor receptor (IGFlR).
  • Methods for screening modulators including antibodies, are provided to isolate or select those which are selective and specific for cysteine rich regions or loops of tumor-associated proteins, and in particular, which target or bind an epitope on the tumor-associated protein(s) which is hidden or is not readily exposed in the absence of overexpression, amplification, or such other tumorigenic alteration or activity.
  • the present invention relates to methods for identifying agents capable of modulating the expression or activity of proteins involved in the processes leading to cancer, cancer pathology, and tumors.
  • the present invention provides methods for identifying agents, including antibodies, which target cryptic or hidden cysteine loop or cysteine domains in cancer-associated proteins, particularly cell-surface receptors, and their use in the prevention and / or treatment of tumors and cancer.
  • the invention includes cysteine mutated tumor-associated proteins or mutant cysteine loop peptides.
  • isolated cysteine mutated peptides or proteins are contemplated and provided.
  • the cell surface receptor cysteine mutants may be prepared and/or expressed as soluble proteins, expressing only the extracellular domain. These soluble cysteine mutants are useful in screening or as immunogenic compositions.
  • aspects of the present method include the in vitro assay of compounds, including antibodies, using mutated cysteine modified polypeptide(s) of a cancer-associated protein, or fragments thereof.
  • Cysteine-mutated fragments, peptides or proteins are modified at cysteine positions such that the fragments or proteins expose these cysteine bounded epitopes or targets, hi some instances the cysteine modifie proteins are more tumorigenic, less tumori genie or equally tumorigenic. Irrespective of their tumorigenicity however, the cysteine modified proteins or peptides are useful in effectively screening for and generating cancer-specific and anti-cancer agents, modulators, antibodies.
  • Exemplary cysteine mutant sequences, particularly wherein alanine replaces cysteine are described and provided herein.
  • the EGFR exists in two well-defined conformers - tethered and untethered.
  • the tethered conformer which has only been observed in ligand-free (and partly ligated) forms of the receptor, can be induced by a ligand to form the untethered, back-to-back dimer.
  • mAb806 recognizes an epitope on some truncated, overexpressed or activated forms of the EGFR on the cell surface, but it does not recognize the EGFR on normal unstimulated cells.
  • Another related antibody, mAbl75 also recognizes this unusual epitope.
  • the cystine mutant EGFR C27IA/C283A not only binds mAb806 and mAbl75, but the stoichiometry is 1 :1 (i.e. equivalent to mAb528 which recognizes the EGFR L2 ligand binding domain).
  • mAb806 fails to inhibit the in vitro growth of cells expressing wild-type EGFR
  • mAb806 inhibits completely, ligand associated stimulation of BaF/3 cells expressing EGFRC27IA /C 2 83 A-
  • Our results indicate that the mechanisms of binding of antibodies mAb806 and mAbl75 requires a form of the EGFR where the epitope is preferentially exposed either during receptor activation or through truncation or overexpression.
  • mAb806 preferentially localizes to the tumor in cancer patients overexpressing the EGFR.
  • the mechanism of action suggests new approaches to the generation of antibodies for detection of tumors and for improving antibody/inhibitor killing of cancer cells with over-expressed, truncated or activated forms of receptors in the EGFR family SIGNIFICANCE
  • the EGFR is involved in stimulating the growth of many human tumors. Although inhibitiors and antagonists have been used as therapeutic agents, success has been limited, in part by interfering with the EGFR on normal tissues and in part by the limited temporal action of some of the agents, ie Abs have longer action.
  • the antibodies Mab806 and Mabl75 recognize an unusual conformation of the receptor, which often occurs on tumor cells, but not normal cells. The three dimensional binding site of these antibodies on the EGFR identifies the unusual conformation which explains their tumor specificity. These antibodies synergize with other anti-EGFR agents to induce profound tumor killing in mice. The intitial results in cancer patients using radiolabeled forms of the antibodies confirm the tumor selectivity.
  • the EGFR is activated by autocrine ligands(/P;27;22) and, in a high proportion of advanced gliomas, the EGFR receptor extracellular domain is truncated(2J;2- ⁇ ) and consequentially activated. Often the activation of the EGFR is required for the maintenance of the malignant state. Conversely, except for a small number of cells in hair follicles and Brunner's gland, in adult organisms the EGFR is expressed at low levels and is inactive in adult life.
  • TKI's tyrosine kinase inhibitors
  • mAb's monoclonal antibodies
  • TKJ's such as gefitinib (ZDl 839) and erlotinib (OSI-774)
  • ZDl 839) tyrosine kinase inhibitors
  • OSI-774 monoclonal antibodies
  • TKJ's such as gefitinib (ZDl 839) and erlotinib (OSI-774)
  • OSI-774 erlotinib
  • antibodies against EGFR such as cetuximab (C225) and panitumumab (ABX-EGFR) competitively inhibit ligand binding and thereby prevent receptor activation.
  • cetuximab C225
  • ABX-EGFR panitumumab
  • Both classes of the inhibitors and antibodies display significant antitumor activity in a range of EGFR-dependant mouse xenograft models(25-2P) and both have been approved in select cancers including NSCL, pancreatic, head & neck and colon (30-32). While response rates to these EGFR therapeutics are modest, it is hoped that successful identification of patient sub-sets likely to respond to EGFR blockade will be able to improve on outcomes for the patients. In glioma for example, response to Tarceva appears largely restricted to a sub-set of patients who are double positive for ⁇ 2- 7EGFR (also called EGFRvIII), the extra-cellular truncation of the EGFR commonly expressed in glioma, and PTEN (33). While these therapeutics show promise, their use is restricted by dose limiting toxicities such as skin rash, which results from significant uptake of these agents in normal skin where EGFR expression is significant.
  • gliomas over-express EGFR(23;34), predominantly due to amplification of the EGFR gene.
  • EGFR gene amplification in glioma is also associated with a mutation event that leads to the excision of exons 2-7 (34) and the subsequent expression of a truncated, partially activated ⁇ 2-7 EGFR form of the EG ⁇ R(35;36) mentioned above.
  • the ⁇ 2-7 EGFR contains a unique fusion peptide at the N-terminus resulting from the splicing together of exons 1 and 8 and the insertion of an unique glycine.
  • ⁇ 2-7 EGFR over-expression or activation of the receptor.
  • EGFR over-expression there appears to be increased untethering as a result of both ligand-independent EGFR activation and changes in glycosylation(JP).
  • JP glycosylation
  • mAb806 has shown robust antitumor activity against U87MG glioma cells expressing the ⁇ 2-7 EGFR, as well as a range of other models that over-express the wtEGFR in absence of the this mutation(28; 40). Furthermor, mAb806 shows synergistic anti-tumor activity in animal models when used in combination with other EGFR therapeutics, including EGFR kinase inhibitors(27) and antibodies( ⁇ i) with unrelated epitopes
  • mAb806 could not bind to either the tethered EGFR as observed in the full length ECD structure ⁇ 5) or to the ligand-bound, untethered, back-to-back dimer seen with the EGFR-ECD I -50 I (14) or EGFR-ECDi -62I (42) constructs. Therefore, we have proposed that mAb806 binds to a partially untethered form of the wtEGFR that exist between the inactive and active states. The inability of mAb806 to bind to the ligated, untethered EGFR was further confirmed by pre-incubating wtEGFR expressing BaF/3 cells with EGF under conditions that prevented receptor internalization.
  • MAb806 also binds to EGFR immobilized on plastic and surface plasmon resonance chips(57).
  • the mAbl75 recognized the yeast fragment 273-621, which corresponds to the extracellular domain of the ⁇ 2-7 EGFR, but not to fragments 1-176, 1- 294, 294-543 or 475-621 ( Figure 3A and 35). Thus, at least part of the mAbl75 epitope must be contained within the region between amino acids 274-294, agreeing with our immunoblotting data using EGFR fragments. Since mAbl75 binds to the denatured fragment of the 273-621 ( Figure 3C), the epitope must be linear in nature (Supplemental Figure 9). It is clear that mAb 806 and mAbl75 recognize a similar region and conformation of the EGFR.
  • mAb806 and mAb 175 have similar affinities for EGFR 287-302
  • mAb 175 appears to display a higher affinity for the extra-cellular domain of the EGFR (Table 1).
  • the mAb 175 epitope is contained within the EGFR 287-302 and, like mAb806, the binding affinity to extra-cellular domain of the EGFR is dependent on conformation. [0123] Table 2: BIAcore determination of antibody affinities for mAb806 and mAbl75 binding to EGFR epitopes
  • mAbl75 appeared moderately more sensitive to mutations V299 and D297 but mAb806 also showed reduced binding to some mutations at these sites (Table 3). Again, the mAbl75 epitope appears to be essentially the same as the epitope recognized by mAb806.
  • Table 3 Display of EGFR Epitope 287-302 mutations on yeast and the binding scores for mAb806 and mAbl75
  • the average tumor volume at this time was 1530, 300 and 100 mm 3 for the vehicle, mAb806 and mAbl75 treatment groups, respectively ( Figure 4A), confirming mAbl75 is antitumor activity against xenografts expressing the ⁇ 2-7 EGFR.
  • the prostate cell line DU 145 expresses the wtEGFR at levels similar to that observed in U87MG cells, however unlike the U87MG cells, the DU 145 cells contain an amplification of the TGF- ⁇ gene and thus exhibit an EGFR/TGF- ⁇ autocrine loop. Both mAb 175 and 806 bind to DU 145 cells as determined by FACS analysis ( Figure 4B) and both are able to immunoprecipitate a small proportion of the EGFR extracted from these cells ( Figure 4C).
  • the control group was sacrificed on day 61 and had a mean tumor volume of 1145 mm 3 compared with 605 and 815 mm 3 for the mAb806 and mAbl75 groups respectively (p ⁇ 0.007 and 0.02 respectively) ( Figure 4D).
  • the epitope adopts a ⁇ -ribbon structure, with one edge of the ribbon pointing towards the Fab and V299 buried at the centre of the antigen-binding site (Figure 5C-E). Both ends of the epitope are exposed to solvent, consistent with these antibodies binding much longer polypeptides.
  • mAb806 and mAbl75 contact residues are: light-chain S30, S31, N32, Y49, H50, Y91, F94, W96 and heavy-chain D32, Y33, A34, Y51, S53, Y54, S55, N57, R59, A99, GlOO, RlOl; the mAb806 contact residues are the same, with sequence differences for the light-chain, N30 and heavy-chain, F33.
  • EGFR 287-3 02 binds to the Fab through close contacts between peptide residues 293-302, with most of the contacts being between residues 297 and 302.
  • the only hydrogen bonds between main chain atoms of EGFR 28 7- 30 2 and the Fab are for residues 300 and 302 ( Figure 5F).
  • Recognition of the epitope sequence occurs through side-chain hydrogen bonds to residues E293 (to H50 and RlOl of the Fab), D297 (to Y51 and N57), R300 (to D32) and K301 (via water molecules to Y51 and W96). Hydrophobic contacts are made at G298, V299 and C302.
  • the conformation of the EGFR 287-302 peptide in contact with the antibodies is quite closely related to the EGFR 287-302 conformation observed in the backbone of the tethered or untethered EGFR structures (Li et al., 2005; Garrett et al., 2002).
  • the rms deviations in Ca positions are 0.66 and 0.75 A, respectively ( Figure 5).
  • Disulfide bonds in proteins provide increased structural rigidity but in some cell surface receptors, particularly those for cytokines and growth factors, transient breaking of disulfide bonds and disulfide exchange can control the receptor's function(4P).
  • transient breaking of disulfide bonds and disulfide exchange can control the receptor's function(4P).
  • mAb806 and mAbl75 could gain access to their binding site, we attempted to increase the accessibility of the epitope by mutating either or both of the cysteine residues at positions 271 and 283 to alanine residues (C271A/C283A).
  • the vectors capable of expressing full length C271A-, C283A- or C271A/C283A- EGFR were transfected into the IL-3 dependent Ba/F3 cell line.
  • Stable Ba/F3 clones, which expressed the C271A- and C271A/C283A- EGFR mutant at levels equivalent to the wtEGFR were selected ( Figure 6A). Ba/F3 cells expressing high levels of mutant C283A-EGFR were not observed.
  • the wtEGFR reacts poorly with mAb806; however, the mutant receptors reacted equally strongly with mAb528, mAb806 and the anti-FLAG antibody, suggesting that the receptor is expressed at the cell surface, is folded correctly and that the epitope for mAb806 is completely accessible in such cases.
  • mAb806 recognizes the C271A/C283A mutant more efficiently than the wtEGFR.
  • mAb806 only recognized a small proportion of the total wtEGFR expressed on the surface of Ba/F3 cells (the mAb806/528 binding ratio is 0.08) (Table 4). In contrast, mAb806 recognized virtually all of the C271A/C283A mutant EGFR expressed on the cell surface (an mAb806/528 binding ratio of 1.01) ( Figure 6A and Table 4).
  • Table 4 mAb806 reactivity with cells expressing the wt or C271 A/C283A EGFR
  • mAb806 has no effect on the in vitro EGF-induced proliferation of Ba/F3 cells expressing the wtEGFR, while the ligand blocking mAb 528 completely inhibits the EGF-induced proliferation of these cells ( Figure 6D, left panel). In contrast, mAb806 totally ablated the EGF-induced proliferation in BaF3 cells expressing the C271/283A mutant ( Figure 6D, right panel). When the 271-283 cysteine loop is disrupted, not only does mAb806 bind more effectively, but once bound, mAb806 prevents ligand induced proliferation.
  • Total liver uptake was a maximum of 14.45 ⁇ 2.43 %ID immediately post infusion, and declined to 8.45 ⁇ 1.63 %ID by 72 hours, and 3.18 ⁇ 0.87 %ID by one week post infusion. This is in marked contrast to the uptake of antibodies to wtEGFR (eg 225), which have been shown to reach over 30 %ID in liver (for a 40mg dose) for over 3 days post infusion(50).
  • wtEGFR eg 225
  • Peak ch806 uptake in tumor ranged from 5.21 to 13.73 x 10 '3 %ID/gm tumor tissue.
  • EGFR epidermal growth factor receptor
  • EGFR kinase inhibitors can decrease the growth and survival of the tumor cells.
  • Antibodies directed to the unique junctional peptide in the ⁇ 2-7 EGFR have the potential to target several tumors(57) without the difficulties associated with normal tissue uptake.
  • the expression of the ⁇ 2-7 EGFR is accompanied by over-expression of the wtEGFR which would not be inhibited by other ⁇ 2-7 EGFR antibodies, but should be inhibited by mAb806 or mAbl75.
  • mAb806 which was raised against cells expressing ⁇ 2-7 EGFR. Not only does mAb806 bind this truncated receptor, but also binds to over-expressed wtEGFR. Mab806 recognizes an epitope contained within a cysteine loop (amino acids 287-302) that is accessible in the ⁇ 2-7 EGFR, but not in the wtEGFR when expressed at low to moderate levels on cells and in the absence of ligands. Similarly, purified, full-length extracellular domain of EGFR (EGFRi -62 ]).
  • the epitope for this antibody was found to be near the hinge region of the EGFR extracellular domain that undergoes at change conformation during the formation of the active state. Furthermore, not only is the epitope buried in the inactive conformation, it also appeared to be inaccessible in the ligand bound back-to-back, untethered EGFR dimer.
  • the interesting properties of mAb806 prompted us to reanalyze other hybridomas expressing the monoclonal antibodies isolated from the initial fusion(5#). In preliminary screens, one of these mAbl75, appeared to have similar EGFR binding properties to mAb806.
  • the amino acid sequences within their CDR loops are remarkably similar (90% sequence identity), and these differences preserve the size and charge of the relevant side chain.
  • the mAbl75 stains tumor cells which over-express the EGFR or which express the ⁇ 2-7 EGFR, but not cells with moderate levels of the wtEGFR, e.g. human liver.
  • Detailed epitope mapping showed that not only does mAbl75 bind the same cysteine loop as mAb806, but it also has a near identical binding profile to a series of mutants containing point mutations in this loop. Furthermore, neither antibody required the epitope disulfide bond to be intact for binding.
  • Both mAb806 and mAbl75 possess anti-tumor activity against human glioma xenografts that express the ⁇ 2-7 EGFR and both induce a significant delay in tumor growth, although mAbl75 appeared slightly more potent in this model.
  • mAb806 and mAbl75 bind to the EGFR expressed on DU 145 prostate cells, a cell line that expresses modest levels of EGFR but secretes significant amount of TGF- ⁇ (52) in an autocrine fashion.
  • both antibodies only bind a small proportion of the surface EGFR on DU 145 cells.
  • the EGFR 287-3O2 epitope hangs from a second disulfide bonded loop (amino acids 271-283) and disruption of this disulfide bond should allow access to the EGFR 287-302 loop without changing the backbone conformation of the epitope (see Figure 8).
  • Our results with the C271A/C283A EGFR mutant indicate that the CRl domain must open up to allow mAb806 and 175 to bind stoichiometrically to the mutant receptor.
  • This mutant receptor can still adopt a native conformation as it is fully responsive to EGF stimulation but, unlike the wtEGFR, is fully inhibited by mAb806.
  • Targeting the EGFR 2 g 7-302 epitope with antibodies derived from mAb806 or mAbl75 is a way of attacking the activated EGFR in cancer cells with minimal uptake in normal tissue. Activation of the receptor can result from many of the mechanisms associated with cancer. Also, and possibly most importantly, these antibodies may be used to target cytotoxics, therapeutic nanoparticle, siRNA and radioisotopes directly to the tumor site. Finally, these studies confirm that mAb806 and mAbl75 are valuable tools for helping map those events associated with EGFR activation on the cell surface.
  • the ⁇ 2-7 EGFR transfected U87MG. ⁇ 2-7(5 ⁇ ) and the A431 cell lines(2) have been described previously.
  • the hormone-independent prostate cell line DU 145(55) was obtained from the ATCC (atcc.org). See Supplemental Data for growth conditions of the cell lines.
  • mAb806 and mAbl75 were produced and purified in the Biological Production Facility (Ludwig Institute for Cancer Research, Melbourne). For preparation and characterization of the antibodies, antibody fragments and peptide epitope see Supplemental Data
  • a BIAcore 3000 was used for all experiments.
  • the peptides containing the putative mAb806 epitope were immobilized on a CM5 sensor chip using amine, thiol or Pms coupling at a flow rate of 5 ⁇ l/min( ⁇ 7).
  • the mAb806 and mAbl75 were passed over the sensor surface at a flow rate of 5 ⁇ l/min at 25°C.
  • the surfaces were regenerated between runs by injecting 10 mM HCl at a flow rate of lO ⁇ l/min.
  • Cells were lysed with lysis buffer (1% Triton X-100, 30 mM HEPES, 150 mM NaCl, 500 mM 4-(2-aminoethyl) benzenesulfonylfluoride, 150 nM aprotinin, 1 mM E-64 protease inhibitor, 0.5 mM EDTA, and 1 mM leupeptin, pH 7.4) for 20 minutes, clarified by centrifugation at 14,000 x g for 30 minutes, immunoprecipitated with the relevant antibodies at a final concentration of 5 ⁇ g/ml for 60 minutes and captured by Sepharose- A beads overnight.
  • lysis buffer 1% Triton X-100, 30 mM HEPES, 150 mM NaCl, 500 mM 4-(2-aminoethyl) benzenesulfonylfluoride, 150 nM aprotinin, 1 mM E-64 protease inhibitor,
  • U87MG. ⁇ 2-7 cells (3xlO 6 ) in 100 ⁇ L of PBS were inoculated s.c. into both flanks of 4- to 6-week-old, female Balb/c nude mice (Animal Research Centre, Perth, Australia). All studies were conducted using established tumor models as reported previously( ⁇ i). Treatment commenced once tumors had reached the mean volume indicated in the appropriate figure legend. Tumor volume in mm 3 was determined using the formula (length x width 2 )/2, where length was the longest axis and width was the perpendicular measurement. Data are expressed as mean tumor volume + SE for each treatment group. All data was analyzed for significance by one-sided Student's t test where p ⁇ 0.05 was considered statistically significant. This research project was approved by the Animal Ethics Committee of the Austin Hospital.
  • ch806 in tumor and liver was calculated by calculation of % injected dose (ID) of 1 H In-ch806 from whole body gamma camera images obtained over one week following injection of 5-7mCi (200-280MBq) l u In-ch806. Liver and tumor dosimetry calculations were performed based on regions of interest in each individual patient u l In-ch806 infusion image dataset, corrected for background and attenuation, allowing calculation of cumulated activity. Dosimetry calculation was performed to derive the concentration of 1 ' 'ln-ch806 in tumor and liver over a one week period post injection.
  • ID % injected dose
  • Intact mAb's (50 mg) were digested in PBS with activated papain for 2-3 h at 37 0 C at a ratio of 1 :20 and the papain was inactivated with iodoacetamide. The digestion was then passed over a column of Protein- A sepharose (Amersham) in 2OmM sodium phosphate buffer pH 8.0, with the flow-through further purified by cation exchange using on a Mono-S column (Amersham). Protein was then concentrated using a 10,000 MWCO centrifugal concentrator (Millipore).
  • Fab-peptide complexes a molar excess of lyophilised peptide was added directly to the Fab and incubated for 2 hours at 4°C before setting up crystallisation trials.
  • Mapping of mAb 175 using EGFR fragments expressed in mammalian cells [0164] The day prior to transfection with these fragments, human 293T embryonic kidney fibroblasts were seeded at 8x10 5 per well in 6-well tissue culture plates containing 2 ml of media. Cells were transfected with 3-4 ⁇ g of plasmid DNA complexed with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.
  • cell cultures were aspirated and cell monolayers lysed in 250 ⁇ l of lysis buffer (1% Triton X-100, 10% glycerol, 150 mM NaCl, 50 mM HEPES pH 7.4, 1 mM EGTA and Complete Protease Inhibitor mix (Roche). Aliquots of cell lysate (10-15 ⁇ l) were mixed with SDS sample buffer containing 1.5% ⁇ -mercaptoethanol, denatured by heating for 5 min at 100 0 C and electrophoresed on 10% NuPAGE Bis-Tris polyacrylamide gels (Invitrogen).
  • lysis buffer 1% Triton X-100, 10% glycerol, 150 mM NaCl, 50 mM HEPES pH 7.4, 1 mM EGTA and Complete Protease Inhibitor mix (Roche). Aliquots of cell lysate (10-15 ⁇ l) were mixed with SDS sample buffer containing 1.5% ⁇ -mercaptoethanol, denatured by heating for 5 min
  • nitrocellulose membranes were then electro-transferred to nitrocellulose membranes that were rinsed in TBST buffer (1OmM Tris-HCI, pH 8.0, 10OmM NaCl and 0.1% Tween-20) and blocked in TBST containing 2.5% skim milk for 30 min at room temperature. Membranes were incubated overnight at 4 0 C with 0.5 ⁇ g/ml of mAb 175 in blocking buffer. Parallel membranes were probed overnight with mAb 9Bl 1 (1 :5000, Cell Signaling Technology, Danvers, Massachussets) to detect the c-myc epitope.
  • Membranes were washed in TBST, and incubated in blocking buffer containing horseradish peroxidase-conjugated rabbit anti-mouse IgG (Biorad) at a 1:5000 dilution for 2 h at room temperature. Blots were then washed in TBST, and developed using autoradiographic film following incubation with Western Pico Chemiluminescent Substrate (Pierce, Rockford, Illinois). Mapping of mAb 175 using EGFR fragments expressed in mammalian cells and yeast
  • yeast cells were then induced for protein display by transferring to minimal media containing galactose, and incubated with shaking at 30°C for 24 h. Cultures were then stored at 4°C until analysis.
  • Raw ascites fluid containing the c-myc monoclonal antibody 9E10 was obtained from Covance (Richmond, CA).
  • yeast cells 1 x 10 6 yeast cells were washed with ice-cold FACS buffer (PBS containing 1 mg/ml BSA) and incubated with either anti-c-myc ascites (1:50 dilution), or human EGFR monoclonal antibody (10 ⁇ g/ml) in a final volume of 50 ⁇ l, for 1 hr at 4°C. The cells were then washed with ice cold FACS buffer and incubated with phycoerythrin-labelled anti-mouse IgG (1 :25 dilution), in a final volume of 50 ⁇ l for 1 h at 4 0 C, protected from light.
  • FACS buffer PBS containing 1 mg/ml BSA
  • human EGFR monoclonal antibody 10 ⁇ g/ml
  • Stable cell lines expressing the mutant EGFR were obtained by selection in neomycin-containing medium. After final selection, mRNA was isolated from each cell line, reverse transcribed and the EGFR sequence amplified by PCR. All mutations in the expressed EGFR were confirmed by sequencing the PCR products. The level of EGFR expression was determined by FACS analysis on a FACStar (Becton and Dickinson, Franklin Lakes, NJ) using the anti-EGFR antibody mAb528(9;70) at 10 ⁇ g/ml in PBS, 5% FCS, 5 mM EDTA followed by Alexa 488-labeled anti-mouse Ig (1 :400 final dilution). Background fluorescence was determined by incubating the cells with an irrelevant, class-matched primary antibody. All cells were routinely passaged in RPMI, 10% FCS, 10% WEHI3B conditioned medium and 1.5 mg/ml G418.
  • Cells expressing the wtEGFR or C271A/C283 A-EGFR were washed and incubated for 3 hr in medium without serum or IL-3. Cells were collected by centrifugation and resuspended in medium containing EGF (100 ng/ml) or an equivalent volume of PBS. Cells were harvested after 15min, pelleted and lysed directly in SDS/PAGE sample buffer containing ⁇ -mercaptoethanol. Samples were separated on NuPAGE 4-12% gradient gels, transferred to Immobilon PVDF membrane and probed with anti-phosphotyrosine (4G10, Upstate Biotechnologies) or anti-EGFR antibodies (mAb806, produced at the LICR). Reactive bands were detected using chemiluminescence.
  • the median fluorescence values were chosen as most representative of peak shape and fluorescence intensity and were used to derive the ratio of mAb 806 to mAb 528 binding. Crystal structure determinations of 175, and 806 Fab, Fab-peptide complexes and the NMR structure of the 806 peptide epitope in solution
  • Crystals of native 806 Fab were grown by hanging drop vapour diffusion using 10mg/ml Fab and a reservoir containing 0.1 M Sodium acetate buffer pH 4.6, 6-8% PEG6000 and 15-20% (Isopropanol.
  • crystals were transfered to a cryoprotectant solution containing 0.1 M Sodium acetate buffer pH 4.6, 10% PEG6000, 15-20% Isopropanol and 10% glycerol. Crystals were then mounted in a nylon loop and flash frozen directly into liquid nitrogen.
  • Crystals of 806 Fab-peptide complex were grown by hanging drop vapour diffusion using lOmg/ml Fab-peptide complex and a reservoir containing 0.2M ammonium acetate 16-18% PEG 5,000 monomethylether, crystals quality was then improved through seeding techniques.
  • crystals were transfered to a cryoprotectant solution consisting of reservoir supplemented with 25% glycerol. Crystals were then mounted in a nylon loop and flash frozen directly into liquid nitrogen.
  • Crystals of 175 Fab-peptide complex were initially grown by free interface diffusion using a Topaz crystallisation system (Fluidigm, San Francisco). Microcrystals were grown by hanging drop vapour diffusion using 7mg/ml Fab with similar conditions 0.1 M Bis-tris propane buffer, 0.2M ammonium acetate and 18% PEG 10,000. Microcrystals were then improved by streak seeding into 0.15m Sodium formate and 15% PEG 1500 to yield small plate shaped crystals. For data collection crystals were transfered to a cryoprotectant solution consisting of reservoir supplemented with 25% glycerol. Crystals were then mounted in a nylon loop and flash frozen directly into liquid nitrogen.
  • l5 N-labelled peptide was produced recombinantly as a fusion to the SH2 domain of SHP2 using the method previously described by Fairlie et a ⁇ .(18), except that the E. coli were grown in Neidhardt's minimal medium supplemented with 15 NH 4 Cl(ZP).
  • the peptide was cleaved from the fusion partner using CNBr, purified by reversed-phase HPLC and its identity confirmed by MALDI-TOF mass spectrometry and N-terminal sequencing.
  • the methionine residue within the 806 antibody-binding sequence was mutated to leucine to enable cleavage from the fusion partner, but not within the peptide itself.
  • ErbB3 - 80% of Tumours of the gastro-intestinal tract
  • the monoclonal antibobdy mAb806 reacts with the truncated form of the EGFR known as the EGFR de-2-7 receptor as well as over expressed/amplified wtEGFR. This antibody binds to a transitional form of the wt EGFR as it does not bind to non amplified wtEGFR.
  • Mab806 recognizes the epitope (CGADSYEMEEDGVRKC) in EGFR which is a loop at amino acids 287-302 of EGFR. This epitope is otherwise hidden under normal conditions, and is exposed for antibody binding and recognition upon amplification, overexpression, and in the de 2-7 EGFR mutant.
  • Wild-type and mutant ErbB constructs were transfected into the interleukin-3-dependent murine hemopoietic cell line BaF/3 by electroporation. Transfected cells were selected in G418. Viable cells were screened for ErbB expression by FACS analysis on a Guava Flow Cytometer (Guava-USA) Using the relevant Antibodies for the ErbB Receptors (refer below) diluted in 1% HS A/PBS, followed by Alexa 488-labeled anti-mouse Ig (1 :400 final dilution). Background fluorescence was determined by incubating the cells with an irrelevant, class-matched primary antibody. Positive pools were sorted for the appropriate level of ErbBR expression on a Mo-Flow (Cytamation) [0084] All cells were routinely passaged in RPMI, 10% FCS, Penicillin/streptomycin,
  • ErbB2 Ab-3 (Anti-c-cErbB2) mAb (Extracellular domain) (Calbiochem)
  • ErbB3 Ab-4 (Anti-c-ErbB3) mAb (Extracellular domain) (Calbiochem)
  • ErbB4 MABl 1311 mAb (Extracellular domain) (R&D systems)
  • ErbB Antigen ErbB double mutant soluble form (extracellular domain) [0086]
  • the immunization regime will comprise of the above schedule on a monthly basis up to 4 months.
  • Antibodies will be screened by ELISA, flow analysis, and hemadsorption assay
  • ErbB3 and ErbB4 clones are obtained from Origene Technologies, Inc (Rockville, MD) as follows:
  • PrecisionShuttle pCMV-entry vector It is C-terminal Myc and Flag tagged. It has a
  • Kanamycin resistance gene for cloning into E.coli and a Neomycin resistance gene for cloning into mammalian systems Kanamycin resistance gene for cloning into E.coli and a Neomycin resistance gene for cloning into mammalian systems.
  • RO630S the isoschizomer of Sgfl
  • MIu I Catalogue number: RO 198s have been ordered from NEB.
  • the destination vector is a pCMV6-AC-Myc-HIS vector.
  • TGT Cysteine
  • GCT Alanine
  • TGT Cysteine
  • GCT Alanine
  • TGT Cysteine
  • GCT Alanine
  • Cysteine (TGT) at position 279 is mutated to an Alanine (GCT) by substituting the Thymine and Guanine to Guanine and Cytosine at positions 835 and 836 (signal sequence subtracted).
  • Soluble proteins will be made by subcloning the extracellular domain of each receptor into the Origene destination Vector containing both the C-terminal Myc and His tags.
  • Secreted HIS tagged protein can be purified on a Nickel column and used as part of an immunization strategy and hybridoma screening procedure.
  • Cell lysates can be prepared from these cells and used as part of an immunization strategy. These transfected cells will also be used for screening potential clones.
  • Stable cell lines will be generated under neomycin selection and positive clones will be selected according to their protein expression levels.
  • the protein will be purified using a Nickel -sepharose column and final concentration will be determined by spectrophotometric analysis.
  • Type 1 Insulin-like growth factor receptor as a mature polypeptide is composed of an alpha chain (residues 31-736) disulfide linked to a beta chain (residues 741-1367). On the cell surface receptors usually exist disulfide-linked dimers (2 alpha/2 beta chains) although heterodimers with the Insulin receptor can occur.
  • Type 1 Insulin-like growth factor receptor (IGFlR) sequence is known and is disclosed including in Genbank as Entrez Ace. No. P08069
  • the IGFlR has been implicated in cancer and a review of IGFlR as a target for cancer therapy can be found in Larsson et al (Larsson, O., Girnita, A. and Girnita L. (2005) British Journal of Cancer 92: 2097-2101).
  • the primary target disulfide bond to mutate in IGFlR is C282-C303 , wherein each of C282 and C303 is mutated to alanines.
  • the amino acid sequence of IGFlR, with Cys 282 and Cys 303 bolded is shown in Figure 16.
  • the Insulin receptor (INSR) sequence is known and publicly available including in Genbank at Entrez Ace. No. P06213.
  • the Insulin receptor mature polypeptide is composed of an alpha chain (residues 28-758) disulfide linked to a beta chain (residues 763-1382).
  • receptors usually exist as disulfide-linked dimers (2 alpha/2 beta chains) although heterodimers with the Typel IGF receptor can occur.
  • the insulin receptor is implicated in cancer (Sciacca, L., et al. (1999) Oncogene 18: 2471-2479; Vella, V., et al.
  • IR-A isoform (exon 11) denoted IR-A (Denley, A., et al. (2003). Horm Metab Res 35: 778- 785).
  • the target of a therapeutic antibody for cancer is IR-A or the Exon 11 form which is also missing residues 745-756, sequences KTSSGTGAEDPR.
  • the primary target disulfide bond to mutate in INSR is C286-C311 , wherein each of C286 and C311 is mutated to alanine.
  • the amino acid sequence of INSR, with Cys 286 and Cys 311 underlined is shown in Figure 17.

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Abstract

La présente invention concerne la production d'anticorps dirigés de façon générale contre des récepteurs de surface cellulaire et des protéines associées au cancer, et particulièrement contre des récepteurs ou des protéines présentant des régions riches en cystéine ou des boucles cystéine. Des récepteurs de surface cellulaire et des protéines associées au cancer présentant des régions riches en cystéine ou des boucles cystéine et qui sont appropriés dans la présente invention comprennent les récepteurs du facteur de croissance épidermique (EGFR) et des membres de la famille des EGFR, le récepteur de l'insuline, et des tyrosine kinases telles que IGFR (récepteur du facteur de croissance analogue à l'insuline), Ret et Ror. La présente invention propose un procédé permettant généralement de produire des modulateurs ou des liants, parmi lesquels des anticorps, dirigés contre des protéines associées au cancer, particulièrement contre des récepteurs de surface cellulaire, qui peuvent, particulièrement dans les cancers ou en cas d'amplification ou de surexpression ou autres états oncogènes, présenter des épitopes qui ne sont pas nécessairement disponibles dans des protéines correctement repliées et transformées (type sauvage ou situations normales). Lesdits récepteurs ou lesdites protéines mutant(e)s ou modifié(e)s, dans lesquel(le)s les régions riches en cystéine ou les boucles cystéine sont modifiées, ou dans lesquel(le)s la conformation du récepteur ou de la protéine est modifiée, servent de cibles pour isoler ou cribler des modulateurs, y compris d'épitopes pour produire des anticorps, particulièrement des anticorps qui préfèrent une ou des forme(s) oncogène(s) ou associée(s) au cancer de la protéine ou du récepteur ou qui sont spécifiques de celle(s)-ci. L'invention concerne également l'utilisation des récepteurs de surface cellulaire ou des protéines associées au cancer modifié(e) dans la production de modulateurs comprenant des anticorps qui présentent une activité anti-tumorale ou anti-cancéreuse ou dans la stimulation de la réponse immunologique. L'invention concerne en outre des anticorps spécifiquement dirigés contre les récepteurs de surface cellulaire ou les protéines associées au cancer modifié(e)s, parmi lesquel(le)s EGFR et des membres de la famille des EGFR.
PCT/US2008/009772 2007-08-14 2008-08-14 Production d'anticorps dirigés contre des récepteurs de surface cellulaire et des protéines associées au cancer comprenant des membres de la famille des récepteurs du facteur de croissance épidermique (egfr) WO2009023266A1 (fr)

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US10253313B2 (en) 2009-10-30 2019-04-09 Novartis Ag Universal fibronectin type III bottom-side binding domain libraries
US9139825B2 (en) 2009-10-30 2015-09-22 Novartis Ag Universal fibronectin type III bottom-side binding domain libraries
US9512199B2 (en) 2010-07-30 2016-12-06 Novartis Ag Fibronectin cradle molecules and libraries thereof
US9085622B2 (en) 2010-09-03 2015-07-21 Glaxosmithkline Intellectual Property Development Limited Antigen binding proteins
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ITRM20100577A1 (it) * 2010-11-02 2012-05-03 Takis Srl Immunoterapia contro il recettore erbb-3
US9688738B2 (en) 2010-11-02 2017-06-27 Takis S.R.L. Immunotherapy against ErbB-3 receptor
US10464995B2 (en) 2011-10-31 2019-11-05 Bristol-Myers Squibb Company Fibronectin binding domains with reduced immunogenicity
US9765132B2 (en) 2011-10-31 2017-09-19 Bristol-Myers Squibb Company Fibronectin binding domains with reduced immunogenicity
US9522951B2 (en) 2011-10-31 2016-12-20 Bristol-Myers Squibb Company Fibronectin binding domains with reduced immunogenicity
US10604556B2 (en) 2011-10-31 2020-03-31 Bristol-Myers Squibb Company Fibronectin binding domains with reduced immunogenicity
US9416170B2 (en) 2011-10-31 2016-08-16 Bristol-Myers Squibb Company Fibronectin binding domains with reduced immunogenicity
US11279751B2 (en) 2011-10-31 2022-03-22 Bristol-Myers Squibb Company Fibronectin binding domains with reduced immunogenicity
US11408093B2 (en) 2011-10-31 2022-08-09 Bristol-Myers Squibb Company Fibronectin binding domains with reduced immunogenicity
WO2020191434A1 (fr) * 2019-03-22 2020-10-01 Olivia Newton-John Cancer Research Institute Molécules de liaison anti-her2
CN113993902A (zh) * 2019-03-22 2022-01-28 奥利维亚·牛顿-约翰癌症研究所 抗her2结合分子
EP3941947A4 (fr) * 2019-03-22 2022-12-14 Olivia Newton-John Cancer Research Institute Molécules de liaison anti-her2
CN113993902B (zh) * 2019-03-22 2025-03-14 瑟缇斯治疗私人有限公司 抗her2结合分子
CN111647074A (zh) * 2020-06-01 2020-09-11 皖南医学院 一种her3二聚化界面抗原肽、重组抗原肽、编码基因及其应用
CN111647074B (zh) * 2020-06-01 2023-12-19 皖南医学院 一种her3二聚化界面抗原肽、重组抗原肽、编码基因及其应用

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