WO2009023266A1

WO2009023266A1 - Generation of antibodies to cell-surface receptors and cancer-associated proteins including egfr family members

Info

Publication number: WO2009023266A1
Application number: PCT/US2008/009772
Authority: WO
Inventors: Antony Burgess; Thomas Garrett
Original assignee: Ludwig Institute For Cancer Research
Priority date: 2007-08-14
Filing date: 2008-08-14
Publication date: 2009-02-19

Abstract

The present invention relates generally to the generation of 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 generally 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). Mutant or modified such receptors or proteins, wherein the cysteine rich regions or cysteine loops are altered, or wherein the conformation of the receptor or protein is altered, serve as targets for isolating or screening modulators, including as epitopes for generating antibodies, particularly antibodies which prefer or are specific for a cancer-associated or tumorigenic form(s) of the protein or receptor. The invention also relates to the use of the altered cell-surface receptors or cancer-associated proteins in generating modulators including antibodies which have anti-tumor or anti-cancer activity or in stimulating an immunological response. The invention further relates to antibodies specifically directed against the altered cell-surface receptors or cancer-associated proteins, including EGFR and EGFR family members.

Description

GENERATION OF ANTIBODIES TO CELL-SURFACE RECEPTORS AND CANCER-ASSOCIATED PROTEINS INCLUDING EGFR FAMILY MEMBERS

FIELD OF THE INVENTION

[0001] 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). Mutant or modified such receptors or proteins, wherein the cysteine rich regions or cysteine loops are altered, or wherein the conformation of the receptor or protein is altered, serve as targets for isolating or screening modulators, including as epitopes for generating antibodies, particularly antibodies which prefer or are specific for a cancer-associated or tumorigenic form(s) of the protein or receptor.

BACKGROUND OF THE INVENTION

[0002] The treatment of proliferative disease, particularly cancer, by chemotherapeutic means often relies upon exploiting differences in target proliferating cells and other normal cells in the human or animal body. For example, 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. In addition, 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.

[0003] The extracellular domains of tumor or cancer-associated proteins are appropriate targets for modulators and antibody therapies. In particular, 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. In some instances, the stability of the cysteine loops is relevant or even critical for activity. Thus, for the 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).

[0004] Because of their cell surface expression 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. R., et al. (1997) Ann Oncol 8: 1197-206; den Eynde, B. and Scott, A. M. (1998) Tumor Antigens. In: P. J. Delves and I. M. Roitt (eds.), Encyclopedia of Immunology, Second Edition edition, pp. 2424-31. London: Academic Press). Moreover, expression of the EGFR is associated with poor prognosis in a number of tumor types including stomach, colon, urinary bladder, breast, prostate, endometrium, kidney and brain {e.g., glioma). Consequently, a number of EGFR antibodies have been reported in the literature with several undergoing clinical evaluation (Baselga, J., et al. (2000) J Clin Oncol. 18: 904; Faillot, T., et al. (1996) Neurosurgery 39: 478-83; Seymour, L. (1999) Cancer Treat Rev 25: 301-12). Results from studies using EGFR mAbs in patients with head and neck cancer, squamous cell lung cancer, brain gliomas and malignant astrocytomas have been encouraging. The anti-tumor activity of most EGFR antibodies is enhanced by their ability to block ligand binding (Sturgis, E. M., et al. (1994) Otolaryngol Head Neck Surg 111 : 633-43; Goldstein, N. I., et al. (1995) Clin Cancer Res 1: 1311-8). The use of these antibodies, however, may be limited by uptake in organs that have high endogenous levels of EGFR such as the liver and skin (Baselga, J., et al. (2000) J Clin Oncol. 18: 904; Faillot, T., et al. (1996) Neurosurgery 39: 478-83).

[0006] A significant proportion of tumors containing amplifications of the EGFR gene {i.e., multiple copies of the EGFR gene) also co-express a truncated version of the receptor (Wikstrand, C. J., et al. (1998) J Neurovirol 4: 148-58) known as de2-7 EGFR, ΔEGFR, or Δ2-7 (terms used interchangeably herein) (Olapade-Olaopa, E. O., et al. (2000) Br J Cancer 82: 186-94). 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. As 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. J., et al (1998) J Neurovirol 4: 148-58; Humphrey, P. A., et al (1990) Proc Natl Acad Sci USA 87: 4207-11 ; Okamoto, S., et al (1996) Br J Cancer 73: 1366-72; Hills, D., et al (1995) Int J Cancer 63: 537-43).

[0007] However, one potential shortcoming of de2-7 EGFR antibodies is that only a proportion of tumors exhibiting amplification of the EGFR gene also express the de 2-7 EGFR. Therefore, de2-7 EGFR specific antibodies would be expected to be useful in only a percentage of EGFR positive tumors. Thus, antibodies which do not target normal tissues and EGFR in the absence of amplification, overexpression, or mutation, would be 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, has been previously described in 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. In particular, 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.

[0008] The citation of references herein shall not be construed as an admission that such is prior art to the present invention.

SUMMARY OF THE INVENTION

[0009] Thus, 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. One appropriate such target is the cysteine-rich proteins such as EGFR and EGFR family members, including ErbB2, ErbB3, and ErbB4.

[0010] 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. Although the sequence homology of the EGFR mAb806 epitope, cysteine loop 287-302 epitope (CGADS YEMEEDGVRKC) of EGFR, is relatively low in EGF family members ErbB2, ErbB3 and ErbB4, the size and location of the cysteine loop is conserved. Furthermore, there are two amino acid residues completely conserved (E293 and G298) and a further two where charge is conserved (E295 and R300). Finally, the overall structure of ErbB3 (and probably ErbB4), is very similar to that of the EGFR in that it adopts a tethered conformation that presumably untethers during activation. Thus, 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).

[0011] Also, other 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.

[0012] 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. In one such aspect, the tumor associated protein is a receptor with a cysteine loop or cysteine rich domain. In a preferred such aspect, the tumor associated protein is selected from a member of the EGFR family, and a member of the insulin receptor family. In an aspect, the EGFR family member is ErbB2, ErbB3 or ErbB4. In a particular aspect the insulin receptor family member is insulin receptor (ISNR) or insulin- like growth factor receptor (IGFlR).

[0013] 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.

[0014] 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. In particular, 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.

[0015] The invention includes cysteine mutated tumor-associated proteins or mutant cysteine loop peptides. Thus, 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.

[0016] 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.

[0017] Other objects and advantages will become apparent to those skilled in the art from a review of the ensuing detailed description, which proceeds with reference to the following illustrative drawings, and the attendant claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1: Alignment of amino acid sequences for CDR' s from mAb806 and mAbl75. Sequence differences between the two antibodies are bolded.

[0019] Figure 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.Δ2-7 cells (express the Δ2-7 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.

[0020] 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.

[0021] Figure 4: Antitumor effects of mAbl75 on brain and prostate cancer xenografts. A: Mice (n=5) bearing U87MG.Δ2-7 xenografts were injected i.p. with PBS, 1 mg of mAb 175 or mAb806 (positive control), three times weekly for two weeks on days 6, 8, 10, 13, 15 and 17 when the starting tumor volume was 100 mm³. Data are expressed as mean tumor volume ± SE. 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. D: Mice (n=5) bearing DU 145 xenografts were injected i.p. with PBS, 1 mg of mAbl75 or mAb806, daily on days 18- 22, 25-29 and 39-43 when the starting tumor volume was 85 mm³. Data are expressed as mean tumor volume ± SE.

[0022] Figure 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. [0023] 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. EC₅₀S 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). D: BaF3 cells expressing the wt {left panel) or the C271A/C283A {right panel) EGFR were stimulated with increasing concentrations of EGF in the presence of no antibody (open symbols) , mAb 528 (grey circles) or mAb806 (black triangles), both at lOμg/ml. Data are expressed as mean and sd of triplicate measurements.

[0024] Figure 7: A) Whole body gamma camera image of the biodistribution of ¹¹¹In- 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. B) Single Photon Computed Tomography (SPECT) image of the neck of this patient, showing uptake of ^{n 1}In-ChSOo in viable tumor (arrow), with reduced central uptake indicating necrosis. C) Corresponding CT scan of the neck demonstrating a large right neck tumour mass (arrow) with central necrosis.

[0025] Figure 8: A stereo model of the structure of the untethered EGFRl -621. The receptor backbone is traced in blue and the ligand TGF-α 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).

[0026] 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).

[0027] Figure 10 depicts the ErbB2 amino acid sequence (SEQ ID NO: 6) with the C277/C289 806 homologous region noted.

[0028] Figure 11 depicts the ErbB3 protein structure and domains.

[0029] 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.

[0030] Figure 13 depicts the ErbB4 protein structure and domains.

[0031] Figure 14 shows the ErbB4 amino acid sequence (SEQ ID NO: 9) and the homologous region to the 806 epitope is highlighted.

[0032] Figure 15 A and 15B depict the generation of plasmid constructs for soluble ErbB protein expression.

[0033] Figure 16 provides the insulin-like growth factor receptor (IGFlR) amino acid sequence (SEQ ID NO: 10), with the cysteines 282 and 303 underlined. [0034] Figure 17 provides the insulin receptor (INSR) amino acid sequence (SEQ ID NO: 11), with the cysteines 286 and 311 underlined.

DETAILED DESCRIPTION

[0035] In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook et al, "Molecular Cloning: A Laboratory Manual" (1989); "Current Protocols in Molecular Biology" Volumes I-III [Ausubel, R. M., ed. (1994)]; "Cell Biology: A Laboratory Handbook" Volumes I-III [J. E. Celis, ed. (1994))]; "Current Protocols in Immunology" Volumes I-III [Coligan, J. E., ed. (1994)]; "Oligonucleotide Synthesis" (MJ. Gait ed. 1984); "Nucleic Acid Hybridization" [B.D. Hames & SJ. Higgins eds. (1985)]; "Transcription And Translation" [B.D. Hames & SJ. Higgins, eds. (1984)]; "Animal Cell Culture" [R.I. Freshney, ed. (1986)]; "Immobilized Cells And Enzymes" [IRL Press, (1986)]; B. Perbal, "A Practical Guide To Molecular Cloning" (1984).

[0036] Therefore, if appearing herein, the following terms shall have the definitions set out below.

A. TERMINOLOGY

[0037] 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.

[0038] It is important to appreciate that the term "aberrant expression" has been specifically chosen herein to encompass the state where abnormal (usually increased) quantities/levels of the protein are present, irrespective of the efficient cause of that abnormal quantity or level. Thus, 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.

[0039] In this latter connection, certain of the work of the inventors that is presented herein to illustrate the invention includes the analysis of samples certain of which exhibit abnormal protein levels resulting from amplification of EFGR. This therefore accounts for the presentation herein of experimental findings where reference is made to amplification and for the use of the terms "amplification/amplified" and the like in describing abnormal levels of EFGR. However, it is the observation of abnormal quantities or levels of the protein that defines the environment or circumstance where clinical intervention as by resort to the binding members of the invention is contemplated, and for this reason, the present specification considers that the term "aberrant expression" more broadly captures the causal environment that yields the corresponding abnormality in EFGR levels.

[0040] Accordingly, while the terms "overexpression" and "amplification" in their various grammatical forms are understood to have distinct technical meanings, they are to be considered equivalent to each other, insofar as they represent the state where abnormal EFGR protein levels are present in the context of the present invention. Consequently, the term "aberrant expression" has been chosen as it is believed to subsume the terms "overexpression" and "amplification" within its scope for the purposes herein, so that all terms may be considered equivalent to each other as used herein. [0041] The term "antibody "describes an immunoglobulin whether natural or partly or wholly synthetically produced. The term also covers any polypeptide or protein having a binding domain which is, or is homologous to, an antibody binding domain. CDR grafted antibodies are also contemplated by this term.

[0042] As antibodies can be modified in a number of ways, the term "antibody" should be construed as covering any specific binding member or substance having a binding domain with the required specificity. Thus, 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.

[0043] It has been shown that fragments of a whole antibody can perform the function of binding antigens. Examples of 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. et al., Nature 341, 544-546 (1989)) which consists of a VH domain; (v) isolated CDR regions; (vi) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al, Science, 242, 423-426, 1988; Huston et al, PNAS USA, 85, 5879-5883, 1988); (viii) multivalent antibody fragments (scFv dimers, trimers and/or tetramers (Power and Hudson, J Immunol. Methods 242: 193-204 9 (2000))(ix) bispecific single chain Fv dimers (PCT/US92/09965) and (x) "diabodies", multivalent or multispecific fragments constructed by gene fusion (WO94/13804; P. Holliger et al Proc. Natl. Acad. Sci. USA 90 6444-6448, (1993)). [0044] 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.

[0045] The phrase "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.

[0046] 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, Fab¹, F(ab')₂ and F(v), which portions are preferred for use in the therapeutic methods described herein.

[0047] 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. et al (1995) Int. J. Cancer 63(4):537-543), antibody L8A4 and YlO (Reist, CJ et al (1995) Cancer Res. 55(19):4375-4382; Foulon CF et al. (2000) Cancer Res. 60(16):4453-4460), ICR62 (Modjtahedi H et al (1993) Cell Biophys. Jan-Jun;22(l -3): 129-46; Modjtahedi et al (2002) P.A.A.C.R. 55(14):3140-3148, or the antibody of Wikstrand et al (Wikstrand C. et al (1995) Cancer Res. 55(14):3140-3148). 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. In the bispecific antibodies of the present invention 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.

[0048] 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.

[0049] 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.

[0050] The term "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). [0051] "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.

[0052] 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.

[0053] The term "comprise" is generally used in the sense of include, that is to say permitting the presence of one or more features or components.

[0054] 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. In the case of the peptide of the invention referred to above, those of skill in the art will appreciate that 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.

[0055] The term "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.

[0056] Also, as used herein, the terms "glycosylation" and "glycosylated" includes 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. In the biosynthesis of N-linked 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. The protein then moves to the Golgi where further modification and chain elongation occurs. O-linked modifications can occur with the simple addition of the OGIcNAc monosaccharide alone at those Ser or Thr sites which can also under different conditions be phosphorylated rather than glycosylated. [0057] As used herein, "pg" means picogram, "ng" means nanogram, "ug" or "μg" mean microgram, "mg" means milligram, "ul" or "μl" mean microliter, "ml" means milliliter, "1" means liter.

[0058] The amino acid residues described herein are preferred to be in the "L" isomeric form. However, 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. NH₂ 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. In keeping with standard polypeptide nomenclature, J. Biol. Chem., 243:3552-59 (1969), abbreviations for amino acid residues are shown in the following Table of Correspondence:

[0059] TABLE OF CORRESPONDENCE

SYMBOL AMINO ACID

1 -Letter 3 -Letter

Y Tyr tyrosine

G GIy glycine

F Phe phenylalanine

M Met methionine

A Ala alanine

S Ser serine

I He isoleucine

L Leu leucine

T Thr threonine

V VaI valine

P Pro proline

K Lys lysine

H His histidine

Q GIn glutamine E GIu glutamic acid

W Trp tryptophan

R Arg arginine

D Asp aspartic acid

N Asn asparagine

C Cys cysteine

[0060] It should be noted that all 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.

[0061] 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.

[0062] 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.

[0063] 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. In discussing the structure of particular double-stranded DNA molecules, 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). [0064] An "origin of replication" refers to those DNA sequences that participate in DNA synthesis.

[0065] 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 5¹ (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.

[0066] 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.

[0067] 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. For purposes of defining the present invention, 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. Within the promoter sequence will be found 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.

[0068] 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.

[0069] 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.

[0070] The term "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. The term "primer" as used herein 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. For example, for diagnostic applications, depending on the complexity of the target sequence, 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. For example, 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. Alternatively, 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.

[0071] As used herein, the terms "restriction endonucleases" and "restriction enzymes" refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.

[0072] 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. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, 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.

[0073] 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. [0074] It should be appreciated that also within the scope of the present invention are 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-. By "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:

Phenylalanine (Phe or F) UUU or UUC Leucine (Leu or L) UUA or UUG or CUU or CUC or CUA or CUG Isoleucine (He or I) AUU or AUC or AUA Methionine (Met or M) AUG Valine (VaI or V) GUU or GUC ofGUA or GUG Serine (Ser or S) UCU or UCC or UCA or UCG or AGU or AGC Proline (Pro or P) CCU or CCC or CCA or CCG Threonine (Thr or T) ACU or ACC or ACA or ACG Alanine (Ala or A) GCU or GCG or GCA or GCG Tyrosine (Tyr or Y) UAU or UAC Histidine (His or H) CAU or CAC Glutamine (GIn or Q) CAA or CAG Asparagine (Asn or N) AAU or AAC Lysine (Lys or K) AAA or AAG Aspartic Acid (Asp or D) GAU or GAC Glutamic Acid (GIu or E) GAA or GAG Cysteine (Cys or C) UGU or UGC Arginine (Arg or R) CGU or CGC or CGA or CGG or AGA or AGG Glycine (GIy or G) GGU or GGC or GGA or GGG Tryptophan (Trp or W) UGG Termination codon UAA (ochre) or UAG (amber) or UGA (opal)

[0075] It should be understood that the codons specified above are for RNA sequences. The corresponding codons for DNA have a T substituted for U.

[0076] 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.

[0077] The following is one example of various groupings of amino acids:

Amino acids with nonpolar R groups

Alanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine, Tryptophan, Methionine

Amino acids with uncharged polar R groups

Glycine, Serine, Threonine, Cysteine, Tyrosine, Asparagine, Glutamine

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)

[0078] Another grouping may be those amino acids with phenyl groups: Phenylalanine, Tryptophan, Tyrosine

[0079] Another grouping may be according to molecular weight (i.e., size of R groups):

Glycine 75

Alanine 89

Serine 105

Proline 115

Valine 117

Threonine 119

Cysteine 121

Leucine 131

Isoleucine 131

Asparagine 132

Aspartic acid 133

Glutamine 146

Lysine 146

Glutamic acid 147

Methionine 149

Histidine (at pH 6.0) 155

Phenylalanine 165

Arginine 174

Tyrosine 181

Tryptophan 204

[0080] Particularly preferred substitutions are:

- Ly s for Arg and vice versa such that a positive charge may be maintained;

- GIu for Asp and vice versa such that a negative charge may be maintained; - Ser for Thr such that a free -OH can be maintained; and

- GIn for Asn such that a free NH₂ can be maintained.

[0081] Amino acid substitutions may also be introduced to substitute an amino acid with a particularly preferable property. For example, a Cys may be introduced as a potential site for disulfide bridges with another Cys. In disrupting a cysteine loop or the potential for disulfide bridge(s) 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, β-turns in the protein's structure.

[0082] 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.

[0083] 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. Thus, 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.

[0084] The phrase "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.

[0085] The phrase "therapeutically 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. For example, 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.

[0086] 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.

[0087] The term "standard hybridization conditions" refers to salt and temperature conditions substantially equivalent to 5 x SSC and 65⁰C for both hybridization and wash. However, one skilled in the art will appreciate that such "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. Such standard hybridization conditions are easily determined by one skilled in the art according to well known formulae, wherein hybridization is typically 10-20⁰C below the predicted or determined T_m with washes of higher stringency, if desired.

[0088] 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.

[0089] As 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, ED₅₀ (the dose therapeutically effective in 50% of the population) and LD₅₀ (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, LD₅₀/ED₅₀. 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.

[0090] For any compound, 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.

[0091] The pharmaceutical 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.

[0092] 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. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.

B. DETAILED DISCLOSURE.

[0093] 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). We have determined the 3D-structures of the EGFR.₂₈₇-₃₀₂ peptide epitope bound to Fabs of antibodies mAb806 and mAbl75. In the presence of the antibody, the peptide epitope adopts a conformation very similar to that found in both forms of the receptor. However, binding the mAb806 or mAbl75 antibodies to the wtEGFR structure would be prohibited by significant steric clashes of the Fab with the CRl domain in both the tethered and untethered conformations. Examination of the 3D conformation of the CRl domain suggested that breaking of a disulfide bond just before the epitope should allow the CRl domain to open up sufficiently to allow binding of either antibodies. The cystine mutant EGFRc_{27 IA/}C_{283 A} 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). Whereas 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/C2_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

[0094] 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.

[0095] This was confirmed by the construction of EGFR with the two cystines preceding the epitope being replaced by alanine. Not only did the antibodies recognise 100 % of the mutant EGFR (as opposed to less than 10 % for wtEGFR) but this mutant was ligand- activatable, ie it was not 'broken' but just weakened to allow antibody binding.

[0096] This work demonstrated a number of principles which can not only be applied to the generation of similar antibodies which bind other members of the EGFR family but to other cell-surface receptor classes:

[0097] 1) The epitope in a cryptic epitope. That is, it 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.

[0098] 2) It appears possible that the antibodies could bind to EGFR with the correct post-translational modifications. However, as the epitope lies at the geographic centre of the ectodomain, access of an Fab to the epitope is severely sterically hindered by the bulky domains surrounding the epitope. As before, the conformation would still have to open up to some extent to provide access to the epitope.

[0099] It should be noted that in rapidly dividing cancer cells there is a high level of protein synthesis and receptors may not always have the correct post-translation modifications. Furthermore, the transformation to a malignant cell may include upregulation of a particular receptor, such as EGFR, to promote cell proliferation. Therefore, the receptor driving this cancer may be especially susceptible to having partially processed or misfolded forms on the cell surface. [0100] Thus, 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. One good target would be cysteine-rich proteins such as EGFR and EGFR family members, including ErbB2, ErbB3, and ErbB4.

[0101] Although the sequence homology of the EGFR mAb806 loop 287-302 epitope (CGADSYEMEEDGVRKC) (SEQ ID NO: 1) is relatively low in EGF family members ErbB3 and ErbB4, the size and location of the cysteine loop is conserved. Furthermore, there are two amino acid residues completely conserved (E293 and G298) and a further two where charge is conserved (E295 and R300). Finally, the overall structure of ErbB3 (and probably ErbB4), is very similar to that of the EGFR in that it adopts a tethered conformation that presumably untethers during activation. Thus, 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).

[0102] TABLE 1 below 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.

TABLE 1

Positions with conserved physicochemical properties of amino acids all boxed [0103] Also, other 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.

[0104] 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. In one such aspect, the tumor associated protein is a receptor with a cysteine loop or cysteine rich domain. In a preferred such aspect, the tumor associated protein is selected from a member of the EGFR family, and a member of the insulin receptor family. In an aspect, the EGFR family member is ErbB2, ErbB3 or ErbB4. In a articular aspect the insulin receptor family member is insulin receptor (ISNR) or insulin- like growth factor receptor (IGFlR).

[0105] 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.

[0106] 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. In particular, 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.

[0107] The invention includes cysteine mutated tumor-associated proteins or mutant cysteine loop peptides. Thus, 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.

[0108] 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.

EXAMPLES

[0109] The invention may be better understood by reference to the following non-limiting Examples, which are provided as exemplary of the invention. The following examples are presented in order to more fully illustrate the preferred embodiments of the invention and should in no way be construed, however, as limiting the broad scope of the invention.

EXAMPLE 1

SUMMARY

[0110] 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. We have determined the 3D-structures of the EGFR_287-302 peptide epitope bound to Fabs of antibodies mAb806 and mAbl75. In the presence of the antibody, the peptide epitope adopts a conformation very similar to that found in both forms of the receptor. However, binding the mAb806 or mAbl75 antibodies to the wtEGFR structure would be prohibited by significant steric clashes of the Fab with the CRl domain in both the tethered and untethered conformations. Examination of the 3D conformation of the CRl domain suggested that breaking of a disulfide bond just before the epitope should allow the CRl domain to open up sufficiently to allow binding of either antibodies. 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). Whereas 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_/C2₈₃A- 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 SIGNIFICANCE

[0111] 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.

INTRODUCTION

[0112] Understanding the activation of the EGFR by its family of ligands has been challenging but elegant genetic(7-5), biophysical(4-#) and more recently, crystallographic(P-i 7) studies have revealed many of the complex series of conformational changes and aggregation events required to activate the EGFR intracellular tyrosine kinase domain(7#). Amidst these complexities it is apparent that in solution the EGFR extracellular domain adopts at least two fundamental conformations: an inactive tethered conformation and an active untethered or extended, ligand-bound "back-to-back" dimer. The EGFR was the first growth factor receptor to be associated with cancer (19;20). 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.

[0113] Two major classes of agents have been developed to target the EGFR: tyrosine kinase inhibitors (TKI's) and monoclonal antibodies (mAb's). TKJ's such as gefitinib (ZDl 839) and erlotinib (OSI-774) competitively bind to the ATP pocket of EGFR to inhibit its activation. In contrast, antibodies against EGFR, such as cetuximab (C225) and panitumumab (ABX-EGFR) competitively inhibit ligand binding and thereby prevent receptor activation. 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 Δ2- 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.

[0114] Many 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 Δ2-7 EGFR form of the EGΕR(35;36) mentioned above. The Δ2-7 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. Several monoclonal antibodies directed to this junctional peptide have been described (34) and therefore represent potential therapeutics specific for the Δ2-7 EGFR. We generated a panel of Δ2-7 EGFR specific antibodies using NR6 cells (as variant of 3T3 devoid of endogenous EGFR family member) over-expressing this truncated EGFR. While showing robust binding to the Δ2-7 EGFR, some of these antibodies also bind wtEGFR when over-expressed but not when it was expressed at physiological levels. The best described of these antibodies MAb806 (35; 37; 38), appears not to bind cells expressing less than 1 x 10⁵ EGFR on their surface, but only where higher expression levels lead to a distinct population of mAb806 reactive EGFR (5-10% of the total receptor population) (35,37,38). [0115] Subsequent epitope mapping studies have shown that mAb806 binds to a short cysteine loop between amino acids 287-302 on the extracellular domain that is only exposed transiently as the EGFR moves from the tethered to the extended conformation (23,28). Thus, mAb806 reactivity is found only in cells with favorable conditions for receptor untethering, such as the presence of mutations (e.g. Δ2-7 EGFR), over- expression or activation of the receptor. In the case of EGFR over-expression, there appears to be increased untethering as a result of both ligand-independent EGFR activation and changes in glycosylation(JP). These conditions are common in tumour cells but are rare in normal tissues, thereby allowing mAb806 to preferentially target tumour cells over normal tissues, such as the liver. Indeed, the results from our recently completed Phase I clinical trial with a chimeric version of mAb806 demonstrates that the epitope targeted by this antibody is not exposed on normal tissue but is accessible on a range of EGFR positive tumors(28; 40). In xenografts mAb806 has shown robust antitumor activity against U87MG glioma cells expressing the Δ2-7 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

[0116] The EGFR amino acid sequence between cysteine residues 287 and 302 is sufficient for the binding mAb806. However, while the truncation found in the Δ2-7 EGFR clearly exposes this cysteine loop for binding by mAb806, the mechanism of mAb806-wtEGFR binding has only been partially resolved. The crystal structure of the EGFR has been solved for both the full length extracellular domain and EGFR-ECD i_-5Oi fragment bound to ligand. Analysis of these structures make it evident that 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_-50I (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. Under these conditions a larger percentage of the EGFR should form ligated back-to-back dimers, thus preventing mAb806 binding; an observation that was clearly confirmed(¥5). However, the effect of ligand on mAb806 binding in a steady state, such as might occur in cells with a robust EGFR/ligand autocrine loop, is unknown. Interestingly, while binding of mAb806 to cell surface wtEGFR is dependant on the conformation of the receptor, in the immunological sense, the epitope is not conformational as mAb806 is an excellent probe for EGFR in Western blots, i.e. it is capable of recognizing the denatured receptor. Clearly, accessibility to the epitope as determined by EGFR conformation, is the most critical factor with respect to mAb806 binding, not the conformation of the epitope itself. MAb806 also binds to EGFR immobilized on plastic and surface plasmon resonance chips(57).

[0117] In this report we also describe the biological activity, specificity and epitope of other antibodies, raised in the same manner as mAb806. In order to understand the unique specificity of these antibodies we determined the 3D structures for the mAb806 peptide epitope (EGFR287_-3o₂) bound to the Fab fragment of mAb806 and mAbl75 and the free Fab fragments. The orientation of EGFR_287-3Oa on the receptor and the conformation of this peptide bound to antibody confirmed that mAb806 must bind a specific form of the EGFR and that this form must be folded differently to the wtEGFR observed in either the tethered or extended conformation. Using point mutations we examine the influence of an adjacent cysteine loop (amino acids 271-283) on EGFR structure and mAb806/l 75 reactivity as this loop appears to severely restrict binding of these antibodies. We report the efficacy of mAb806 and 175 against DU 145 xenografts, a prostate cell line that possesses a robust TGF-α/EGFR autocrine stimulation loop, and the binding of radiolabeled-mAb806 to a head and neck cancer patient being treated in a Phase I setting(4¥). RESULTS mAbl75 specificity

[0118] Preliminary binding studies suggested that mAbl75 displayed similar specificity for EGFR as mAb806. In the CDR regions of mAb806 (IgG2b) and maB175 (IgGl), the amino acid sequences are almost identical, with only one amino acid difference in each

(Figure 1). All these differences preserve the charge and size of the side-chains. Clearly, these antibodies have arisen independently.

[0119] We conducted a set of immunohistochemistry experiments to analyze the specificity of mAbl75 binding. mAbl75 stains sections of A431 xenografts that over- express the EGFR (Figure 2A) and sections of U87MG.Δ2-7 glioma xenografts that express the Δ2-7EGFR (Figure 2A). In contrast, mAbl75 does not stain U87MG xenograft sections. The U87MG cell line only expresses modest levels of the wild type EGFR (Figure 2A) and has no detectable EGFR autocrine loop. Most importantly, mAbl75 does not bind to normal human liver sections (Figure 2B). Thus, mAbl75 appears to demonstrate the same specificity as mAb806: i.e. it detects over-expressed and truncated human EGFR, but not the wtEGFR expressed at modest levels.

Identification of the m AbI 75 epitope

[0120] Since mAbl75 also binds the Δ2-7EGFR, in which amino acids 6-273 are deleted, and EGFRi-₅₀₁, the mAbl75 epitope must be contained within residues 274-501. When determining the epitope of mAb806, we expressed a series of c-myc-tagged EGFR fragments fused to the carboxy terminus of human GH, all terminating at amino acid 501(45/ 46). The mAbl75 also reacted with both the 274-501 and 282-501 EGFR fragments in Western blots, but did not detect fragments commencing at amino acid 290 or 298 (Supplemental Figure 9). The presence of all GH-EGFR fusion proteins was confirmed using the c-myc antibody, 9E10 (Supplemental Figure 9). Therefore, a critical determinant of the mAbl75 epitope is located near amino acid 290. Finally, a 274-501 EGFR fragment with the mAb806 epitope deleted (Δ287-302) was also negative for mAbl75 binding (Supplemental Figure 9), suggesting that this region similarly determined most of the mAbl75 binding. [0121] We used a second approach to characterize the mAbl75 epitope further. Fragments encompassing extracellular domains of the EGFR were expressed on the surface of yeast and tested for mAbl75 binding by indirect immunofluorescence using flow cytometry. The mAbl75 recognized the yeast fragment 273-621, which corresponds to the extracellular domain of the Δ2-7 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.

[0122] Using surface plasmon resonance (BIAcore) we investigated the binding of mAb 175 to the EGFR peptide (₂₈₇CGADS YEMEEDGVRKC₃₀₂). The EGFR_287-302 was immobilized on the biosensor surface using amine, thiol-disulfide exchange or Pms-Ser coupling chemistries. The latter method immobilizes the peptide exclusively through the N-terminal cysteine(¥7). mAbl75 bound the EGFR_287-302 in all orientations (Table 1). The affinity of mAb 175 for EGFR_287-302 ranged from 35 nM for Pms-serine coupling to 154 nM for amine coupling. In all cases the binding affinity of mAb 175 for EGFR_287-302 was lower than that obtained for mAb806 (Table 1). We also determined the affinity of mAbl75 to two different extracellular fragments of the EGFR. mAbl75 bound the 1-501 fragment with an affinity similar to that obtained using the peptide (16 nM versus 35 nM) (Table 2). As expected, the affinity of mAb 175 against the 1-621 full length extracellular domain, which can form the tethered conformation, was much lower (188 nM). Although 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). Clearly, 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

[0124] The panel of mutants of the 273-621 EGFR fragment, expressed on the surface of yeast (45; 46), was used to characterize the fine structure of the mAbl75 epitope. mAbl75 and mAb806 displayed a near identical pattern of reactivity to the mutants (Table 2). Disruption of the 287-302 disulfide bond only had a moderate effect on the epitope reactivity as the antibody bound to all mutants at C287 and to some but not all mutants at C302 (Table 3). Amino acids critical for mAbl75 binding include E293, G298, V299, R300 and C302 (Table 3). 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.

[0125] Table 3: Display of EGFR Epitope 287-302 mutations on yeast and the binding scores for mAb806 and mAbl75

Efficacy of mAbl75 against tumor xenografts stimulated by Δ2-7EGFR or an EGFR autocrine loop.

[0126] We examined the in vivo anti-tumor activity of mAb806 and mAbl75 against U87MG.Δ2-7 glioma xenografts. Xenografts were allowed to establish for 6 days before antibody therapy (3 times a week for 2 weeks on days indicated) commenced. At this time the average tumor volume was 100 mm³ (Figure 4A). mAbl75 treatment resulted in a reduction in overall tumor growth rate compared to treatment with vehicle or mAb806 and was highly significant at day 19 post-inoculation (P < 0.0001 versus control and P < 0.002 versus ma 806), when the control group was sacrificed for ethical reasons. The average tumor volume at this time was 1530, 300 and 100 mm³ for the vehicle, mAb806 and mAbl75 treatment groups, respectively (Figure 4A), confirming mAbl75 is antitumor activity against xenografts expressing the Δ2-7 EGFR.

[0127] Even though U87MG cells express approximately I X lO⁵ EGFR per cell, mAb 806 is not able to recognize any of the surface EGFR, and not surprisingly, does not inhibit U87MG in vivo growth. Furthermore these cells do not co-express any EGFR ligand. To test whether the EGFR epitope is transiently exposed and hence able to be recognized by mAb806 and mAb 175 in cells containing an EGFR autocrine loop. 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-α gene and thus exhibit an EGFR/TGF-α 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). Both techniques showed greater binding of mAbl75, however, when compared to mAb 528, which binds to the L2 domain, mAb 175 and mAb806 only bind a subset of EGFR on the surface of these cells (Figure 4B and 4C). Similar observations were seen with a second prostate cell line (LnCap); (data not shown) and a colon line (LIM1215) both of which also contain EGFR autocrine \oops(22;48). Clearly, mAb806 and mAb 175 can recognize only a small proportion of the EGFR on cells in the presence of an autocrine stimulation loop. [0128] Since mAbl75 and mAb806 bind more effectively to the EGFR expressed in DU 145 cells than U87MG cells, we conducted a study to analyse the anti-tumor activity of these antibodies in DU 145 xenografts grown in nude mice. Xenografts were allowed to establish for 18 days before therapy commenced (3 times a week for 3 weeks on days indicated). At this time the average tumor volume was 90 mm³ (Figure 4D). Both mAbl75 and mAb806 inhibited the growth of DU 145 xenografts. The control group was sacrificed on day 61 and had a mean tumor volume of 1145 mm³ compared with 605 and 815 mm³ for the mAb806 and mAbl75 groups respectively (p < 0.007 and 0.02 respectively) (Figure 4D).

3D-Structure of EGFR₂₈7-₃₀₂ in contact with the Fab fragments of mAb806 and mAbl75

[0129] In order to understand the molecular details of how mAb806 and mAbl75 could recognise EGFR in some, but not all conformations, the crystal structures of Fab fragments for both antibodies were determined in complex with the oxidized EGFR_287-302 epitope (at 2.0 and 1.59 A resolution respectively, Figures 5A & 5B) and alone (at 2.3 A and 2.8 A resolution, respectively). In both cases, the free and complexed Fab structures were essentially the same and the conformations of the peptide and CDR loops of the antibodies were well defined (Figure 5). The epitope adopts a β-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.

[0130] Of the 20 antibody residues in contact with the epitope, there are only two substitutions between mAb806 and mAbl75 (Figure 1). 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_287-302 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 EGFR₂₈7-₃₀2 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.

[0131] The conformation of the epitope backbone between 293 and 302 was essentially identical in the Fab806 and Fab 175 crystals (rms deviation = 0.4 A, for Ca atoms in these residues). Although constrained by the disulfide bond, the N-terminus of the peptide (287-292) does not make significant contact in either antibody structure and conformations in this region differ. However, this segment in the Fab806 complex appears rather disordered. More interestingly, 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). For EGFR_287-302 from the Fabl75 complex, the rms deviations in Ca positions are 0.66 and 0.75 A, respectively (Figure 5).

[0132] To gain further insight into the recognition of EGFR by mAb806 and mAbl75, the conformation of ¹⁵N labelled oxidized peptide EGFR_287-302 was studied by NMR spectroscopy in solution, free and in the presence of 806 Fab (see Supplemental Data for details). For the free peptide, resonances were assigned and compared to those for random coil. Essentially, the free peptide adopted a random coil structure, not the beta ribbon as seen in the native EGFR(14). Upon addition of the Fab, resonance shifts were observed. However, due to the weak signal arising from significant line broadening upon addition of the Fab and successful crystallisation of the complexes, the solution structure of the Fab806-epitope complex was not pursued further. Clearly though, when the peptide binds to the Fab fragment of mAb806 (or mAbl75) it appears that the Fab selects or induces the conformation of the peptide which matches that peptide in the native receptor. [0133] Why do mAb806 and mAbl75 recognise only some conformations of EGFR? We docked the Fab fragment of mAbl75 onto an extra-cellular domain of EGFR (tethered and untethered monomers) by superimposing EGFR_287-302- For a Δ2-7-like fragment there were no significant steric clashes with the receptor. In the untethered form there was substantially more accessible surface area of the Fab buried (920 A² compared with 550 A² in the tethered form). Therefore, this antigen may make additional contacts with non-CDR regions of the antibody, as has been indicated by yeast expression mutants(45). Conversely, docking the whole EGFR ectodomain onto the Fab, there is substantial spatial overlap with the part of the CRl domain preceding the epitope (residues 187-286) and running through the centre of the Fab (Figure 5Z), E). Hence, as the CRl domain has essentially the same structure in tethered or untethered conformations, mAb806 or mAbl75 will be unable to bind to either form of EGFR. Clearly, there must be a difference between the orientation of the epitope with respect to the CRl domain in either known conformations of the wtEGFR and the orientation that permits epitope binding. Inspection of the CRl domain indicated that the disulfide bond (271-283) preceding EGFR_287-302 constrains the polypeptide which blocks access to the epitope; disruption of this disulfide, even though it is not involved in direct binding to the antibodies, would be expected to allow partial unfolding of the CRl domain so that mAbl75 or mAb806 could gain access to the epitope.

Breaking of the EGFR 271-283 disulfide bond increases mAb806 binding [0134] 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). As this was one mechanism by which 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. As previously described, 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. To confirm that mAb806 recognizes the C271A/C283A mutant more efficiently than the wtEGFR, we determined the ratio of mAb806 binding to the binding of mAb528. Since both the wt and C271A/C283A EGFR were N-terminally FLAG- tagged, we also determined the ratio of mAb806 and mAb528 binding to the M2 antibody. As reported previously, 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).

[0135] Table 4: mAb806 reactivity with cells expressing the wt or C271 A/C283A EGFR

* Average of four independent clones

[0136] Mutation of the two cysteines did not compromise EGF binding or receptor function. BaF3 cells expressing the C271A/C283A EGFR mutant proliferate in the presence of EGF (Figure 6B). We have reproducibly observed a left-shift in the dose response curve for EGF in cells expressing the C271A/C283A mutations, suggesting either higher affinity for the ligand, or enhanced signaling potential for the mutant receptor. Western blotting analysis confirmed that the C271 A/C283A mutant is expressed at similar levels to the wtEGFR and is tyrosine phosphorylated in response to EGF stimulation (Figure 6C). Consistent with previous studies in other cell lines, 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.

Phase I Imaging study in Head and Neck Cancer

[0137] Eight patients [1 female and 7 male; mean age of 61 years (range 44-75)] completed this phase 1 trial as reported (44). All patients fulfilled inclusion criteria and, except for Patient 8 (who had a primary brain tumor), all had metastatic disease at study entry. Ab uptake by the tumor was seen in all patients, and ^{i π}In-ch806, the chermerized version of mAb806, demonstrated prompt and high level uptake in tumor (Figure 7). The clearance of ^{π ι}In-ch806 from normal organs (liver, lungs, kidney and spleen) showed no difference between dose levels(4¥). In particular, liver clearance showed no difference between dose levels, indicating no saturable antigen compartment in the liver for ch806. 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).

[0138] The measured peak tumor uptake of ^{u l}In-ch806 occured 5-7 days post infusion. Calculation of quantitative tumor uptake in Patients 1 and 3 could not be accurately performed due to proximity of target lesion to cardiac blood pool and patient movement. Peak ch806 uptake in tumor ranged from 5.21 to 13.73 x 10^'3 %ID/gm tumor tissue. Calculation of actual ch806 concentration in tumor showed peak values of (mean ± SD) 0.85 ± 0 μg/gm (5mg/m²), 0.92 ± 0 μg/gm (lOmg/m²), 3.80 ± l.lOμg/gm (20mg/m²), and 7.05 ± 1.40μg/gm (40mg/m²). DISCUSSION

[0139] When the levels or activity of the EGFR or the related erbB2 are perturbed, antibodies such as cetuximab and herceptin, that target EGFR family members, are important options for treating cancer. Determining the binding sites for these antibodies, the 3D-structures of both the target receptors and more recently, the antibody:receptor complexes, has improved our understanding of how these antibodies interfere with receptor activation. These studies have also suggested that targeting other epitopes on this receptor family may produce a new opportunities for using combinations of antibodies to improve cancer treatment.

[0140] Unfortunately, all of the currently available therapeutic anti-EGFR antibodies recognize the wtEGFR, which is expressed in virtually all normal tissues. Not only do the EGFR expressed in normal tissues represent a large sink for the antibodies, they are likely to be critical in the dose limiting toxicity (such as skin rash) observed and make use of antibody/cytotoxic conjugates impossible. Despite these problems, it should be noted that most normal tissues appear to lack activated EGFR, thus neutralizing anti- EGFR antibodies appear not have a profound effect on vital homeostatic signaling. In contrast, many tumors contain activated EGFR, either through autocrine/paracrine mechanisms, truncation, mutation, gene amplification and/or over-expression. Importantly, activated EGFR seems to contribute to tumorgenicity by enhancing cell movement, proliferation, invasion, angiogenesis and survival of tumour cells. Consequently, the administration of anti-EGFR antibodies or EGFR kinase inhibitors can decrease the growth and survival of the tumor cells. Antibodies directed to the unique junctional peptide in the Δ2-7 EGFR have the potential to target several tumors(57) without the difficulties associated with normal tissue uptake. In glioma, the expression of the Δ2-7 EGFR is accompanied by over-expression of the wtEGFR which would not be inhibited by other Δ2-7 EGFR antibodies, but should be inhibited by mAb806 or mAbl75.

[0141] Previously, we described an antibody, mAb806, which was raised against cells expressing Δ2-7 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 Δ2-7 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 intriguing 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. Like mAb806 the mAbl75 stains tumor cells which over-express the EGFR or which express the Δ2-7 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.

[0142] Both mAb806 and mAbl75 possess anti-tumor activity against human glioma xenografts that express the Δ2-7 EGFR and both induce a significant delay in tumor growth, although mAbl75 appeared slightly more potent in this model. Interestingly, 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-α(52) in an autocrine fashion. As with cell lines which over-express the EGFR, both antibodies only bind a small proportion of the surface EGFR on DU 145 cells. However, both antibodies inhibit the growth of DU 145 xenografts in nude mice. Thus, it appears that the presence of ligand under physiological conditions increases the availability of the transitional form of the EGFR recognized by these antibodies and targeting this form is sufficient to downregulate EGFR driven cell growth. [0143] This class of anti-EGFR antibodies may well have even wider anti-tumor action than first envisaged. Furthermore, the synergistic activity of mAb806, when used in combination with other EGFR therapeutics(47), suggests an immediate therapeutic role for antibodies of this class. mAb806 also binds to tumor cells that contain cancer- associated mutations which activate the EGFR kinase. mAb806 and mAbl75 selectively bind cells that have an activated EGFR and may be useful reagents for identifying and/or monitoring patients likely to respond to currently approved EGFR therapeutics.

[0144] Our structural studies with the EGFR_287-302 epitope indicate that both mAb806 and mAbl75 recognized the same 3D structural motif. The peptide residues in contact with mAb806 and mAbl75 exhibited almost identical structures in both cases, suggesting that this is the conformation of these amino acid, found in Δ2-7 EGFR, the generating antigen. Indeed, the peptide backbone of EGFR_287-302 seen in the antibody/peptide structures closely matches that occurring in both known conformations of EGFR structure. However, the orientation of the epitope in these structures would prevent antibody access to the relevant amino acids: which is consistent with the experimental observation that antibody 806 does not bind wtEGFR. Detailed inspection of the EGFR structure raised another intriguing possibility. 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.

[0145] On the surface of cells over-expressing the wtEGFR, there is clearly a sub- population of receptors in which the EGFR_287-302 epitope is accessible for mAb806 or mAbl75 binding. While access most readily occurs during receptor activation, it is not yet clear whether this sub-population of receptors are those in conformational transition to the untethered form, those in transition from the untethered form to the ligated activated state, or whether there is incomplete oxidation in a sub-set of the EGFR in which the disulfide bond between 271 and 283 has been damaged (reduced). If a reduced form of EGFR does exist on the surface of cancer cells, our data clearly shows it is likely to be active and capable of initiating cell signaling. The ability of mAb806 to inhibit the growth of xenografts over-expressing the wtEGFR, despite only binding a small sub- population of receptors and not inhibiting signaling downstream of the EGFR, remains an enigma. For this reason the concept that mAb806 binds a unique sub-set of EGFR that has unusual signaling properties has always been appealing, especially given its tremendous synergy with other EGFR therapeutics. If it exists on the cell surface of cancer cells, an EGFR reduced at the 271-283 disulfide could represent this unique form of the EGFR. Finally, it should be remembered that while the deletion in the Δ2-7EGFR is very large, it does end at amino acid 273. The Δ2-7 EGFR lacks this disulfide bond and is known to have different signaling properties to the wtEGFR. On the other hand, activating kinase mutations, autocrine loops and under-glycosylation of the EGFR also enhance mAb806 reactivity by increasing activation of the receptor, presumably without the need of breaking the 271-283 disulfide. These observations support the concept that the CRl domain can kink to allow access to EGFR_287-302 at some point during EGFR activation, but is protected from kinking in the tethered and ligand-bound states. We are currently conducting on-going studies to determine if the EGFR recognized by mAb806 contains a reduced 271-283 disulfide bond.

[0146] The analysis of the results of our Phase I trial of chimerized 806 (ch806) confirmed that the epitope targeted by mAb806 is tumor specific. Quantitative biodistribution analysis clearly demonstrates the rapid and specific uptake of ch806 in tumor. These data are consistent with the highest quantitative targeting of antibodies to antigens expressed on cancer cells and markedly superior to values of wtEGFR antibodies at equivalent doses(44;50). The uptake of ch806 in all normal tissues (including liver) was low, indicating no evidence of binding to wtEGFR in normal tissue, and in liver represented only blood pool activity and minor catabolism of free " ¹In- chelate. This is in marked distinction to antibodies that target wtEGFR (eg 225; Cetuximab), which have been shown to have very high uptake (20-30% ID) in liver retained for over 72 hours post infusion, despite large protein doses being administered (up to 300mg)(50). In addition, antibodies to wtEGFR require large loading doses to saturate normal tissue before tumour uptake is evident(50), and also have dose limiting toxicity from antibody binding to wtEGFR in skin and gut(55). These results indicate that mAb806 does not target normal tissue in human, and quantitative analysis of biodistribution confirms the tumor specificity of the EGFR epitope targeted by mAb806 invivo.

[0147] Targeting the EGFR₂g_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.

[0148] In understanding, at a molecular level, how an antibody can recognise aberrant and activated forms of a growth factor receptor but not inactive wild-type receptor, this work can be used to generate antibodies to other targets for cancer therapeutics, for instance other members of the EGFR family. One method could use the disulfide mutant EGFR-C227A/C283A which binds antibodies mAb806 and mAbl75 stoichiometrically. If conformational perturbations seen for EGFR also occur when erbB2, erbB3 or erbB4 are overexpressed or activated continuously, then homologous disulfide mutants of these receptors may act as immunogens for creating other EGFR family member targeting antibodies with selectivity for tumors. Furthermore, when tumor cells overexpress other receptors, particularly those with disulfide rich domains such as Trk, a proportion of these receptors may be partially misfolded due to underglycosylation or transiently broken disulfide bonds. It is conceivable that disulfide mutant or truncated receptors could be used similarly as immunogens to potentially generate antibodies which recognise other aberrantly expressed receptors. EXPERIMENTAL PROCEDURES

Cell Lines

[0149] The Δ2-7 EGFR transfected U87MG.Δ2-7(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.

Antibodies, Fabs and peptides

[0150] 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

Mapping of mAbl75 using EGFR fragments expressed in mammalian cells and yeast

[0151] The mapping was performed as described in the Supplemental Data.

Surface plasmon resonance (BIAcore)

[0152] 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 5μl/min(¥7). The mAb806 and mAbl75 were passed over the sensor surface at a flow rate of 5μl/min at 25°C. The surfaces were regenerated between runs by injecting 10 mM HCl at a flow rate of lOμl/min.

Immunoprecipitation and Western blotting

[0153] 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 μg/ml for 60 minutes and captured by Sepharose- A beads overnight. Samples were then eluted with 2X NuPAGE SDS Sample Buffer (Invitrogen), resolved on NuPAGE gels (either 3-8% or 4-12%), electro-transferred onto Immobilon-P transfer membrane (Millipore) then probed with the relevant antibodies before detection by chemoluminescence radiography.

Immunohistochemistry

[0154] Frozen sections were stained with 5 μg/ml mAbl75 or irrelevant isotype control for 60 min at room temperature. Bound antibody was detected using the Dako Envision+ HRP detection system as per manufacturer's instructions. Sections were finally rinsed with water, counterstained with hematoxylin and mounted.

Xenograft Models

[0155] U87MG.Δ2-7 cells ( (3xlO⁶) in 100 μL 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 mm³ was determined using the formula (length x width²)/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.

Generation and characterization of stable cell lines expressing EGFR mutant constructs

[0156] Mutations of the (wt) EGFR were generated using a site-directed mutagenesis kit

(Stratagene, La Jolla, CA). The template for each mutagenesis was the human EGFR cDNA (accession number x00588)(2). Automated nucleotide sequencing of each construct was performed to confirm the integrity of the EGFR mutations. Wild type and mutant (C173A/C281A) EGFR were transfected into BaF/3 cells by electroporation. [0157] Further details on the characterization of the cell lines are presented in the Supplemental Data.

Crystal structure determinations of Fab 175, and Fab 806, Fab-peptide complexes and the NMR structure of the 806 peptide epitope in solution

[0158] Crystallographic procedures for preparing and analyzing the Fab 806, Fab 175 and the individual Fab-peptide complexes and details on NMR studies of the ¹⁵N-labelled 806 epitope peptide in solution are described in the Supplemental Data. Structures were determined by molecular replacement and refinement converged with R=0.225/Rfree=0.289 for Fab806 and R=0.226/Rfree=0.279 for Fab806:peptide; R=0.210/Rfree=0.305 for Fab806 and R=0.203/Rfree=0.257 for Fab806:peptide.

Biodistribution of chAb 806 Tumor in Patients

[0159] To demonstrate the tumor specificity of mAb806 invivo, a chimeric version (ch806) was engineered and produced under cGMP conditions(56). A Phase I first-in- man trial was conducted to evaluate the safety, biodistribution and immune response of ch806 in patients with 806 positive tumors, and the results of safety, biodistribution and pharmacokinetics have been reported previously(44). To define the specificity of ch806 in tumor compared to normal tissue (ie liver) in patients, the quantitative uptake of ch806 in tumor and liver was performed 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.

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EXAMPLE 2 SUPPLEMENTAL DATA

EXPERIMENTAL PROCEDURES

Cell lines

[0161] All cell lines were maintained in DMEM (Life Technologies, Grand Island, NY) containing 10% FCS (CSL, Melbourne), 2 mM glutamine (Sigma Chemical Co, St. Louis), and penicillin/streptomycin (Life Technologies, Grand Island). In addition, the U87MG.Δ2-7 cell line was maintained in 400mg/ml of Geneticin (Life Technologies, Inc, Grand Island). BaF/3(7) and BaF/3 cell lines expressing different EGF receptors(2) were maintained routinely in RPMI 1640 (GIBCO BRL) supplemented with 10% foetal calf serum (GIBCO BRL) and 10% WEHI-3B conditioned medium(5) as a source of IL-3. All cell lines were grown at 37°C in an air/CO₂ (95%-5%) atmosphere.

Antibodies and peptides

[0162] Nucleotide sequencing of the cDNAs corresponding to mAb 806 and 175 variable antibody regions was performed as described in detail previously(^), with corrections where differences were observed in the crystal structure. The EGFR-derived peptide

(287CGADSYEMEEDGVRKC302), containing the mAb 806 epitope(5), was synthesized using standard Fmoc chemistry, purified by RP-HPLC and characterized by mass spectral analysis.

[0163] Intact mAb's (50 mg) were digested in PBS with activated papain for 2-3 h at 37⁰C 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). For 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 8x10⁵ per well in 6-well tissue culture plates containing 2 ml of media. Cells were transfected with 3-4 μg of plasmid DNA complexed with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. 24 to 48 h after transfection, cell cultures were aspirated and cell monolayers lysed in 250 μl 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 μl) were mixed with SDS sample buffer containing 1.5% β-mercaptoethanol, denatured by heating for 5 min at 100⁰C and electrophoresed on 10% NuPAGE Bis-Tris polyacrylamide gels (Invitrogen). Samples 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 4⁰C with 0.5 μg/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

[0165] A series of overlapping c-myc-tagged EGFR ectodomain fragments, starting at residues 274, 282, 290 and 298 and all terminating at amino acid 501 and fused to growth hormone have been described previously(ό).

[0166] Expression of EGFR proteins on the yeast cell surface was performed as previously described(7). Briefly, transformed colonies were grown at 30°C in minimal media containing yeast nitrogen base, casein hydrolysate, dextrose, and phosphate buffer pH 7.4, on a shaking platform for approximately one day until an OD₆₀₀ of 5-6 was reached. 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). 1 x 10⁶ 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 μg/ml) in a final volume of 50 μl, 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 μl for 1 h at 4 ⁰C, protected from light. After washing the yeast cells with ice-cold FACS buffer, fluorescence data was obtained with a Coulter Epics XL flow cytometer (Beckman-Coulter), and analyzed with WinMDI cytometry software (J. Trotter, Scripps University). For determination of linear versus conformational epitopes, yeast cells were heated at 80°C for 30 min, then chilled on ice 20 min prior to labelling with antibodies. The series of EGFR mutants listed in Table 2 have been described previously(#).

Generation of cell lines expressing EGFR mutants

[0167] 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 μg/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.

EGF-dependent activation of mutant EGFR

[0168] 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 β-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.

Effect of EGF and antibodies on cell proliferation

[0169] Cells growing in log phase were harvested and washed twice with PBS to remove residual IL-3. Cells were resuspended in RPMI 1640 plus 10% FCS and seeded into 96- well plates at 10⁵ cells/well with carrier only or with increasing concentrations of EGF. Where appropriate, a fixed concentration of mAb528 or mAb806 (2 μg/well) was also added to the cultures. Proliferation was determined using the MTT assay(7/).

Reactivity with Conformation-specific Antibodies

[0170] Cells were collected by centrifugation and stained with the control or test antibodies (all at 10 μg/ml in FACS buffer for 40 min on ice, washed in FACS buffer) followed by Alexa 488-labeled anti-mouse Ig (1 :400 final dilution, 20 min on ice). The cells were washed with ice-cold FACS buffer, collected by centrifugation, and analyzed on a FACScan; peak fluorescence channel and median fluorescence were determined for each sample using the statistical tool in CellQuest (Becton and Dickinson). Background (negative control) fluorescence was deducted from all measurements. 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

[0171] 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. For data collection 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.

[0172] 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. 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.

[0173] 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.

[0174] Diffraction data on 806 Fab and 175 Fab complex crystals were collected in-house using a R-AXIS IV detector on a Rigaku micromax-007 generator fitted with AXCO optics, these data were then processed using CrystalClear. 806 Fab-peptide complex data were collected on an ADSC quantum315 CCD detector at beamline X29, Brookhaven National Laboratory, these data were processed with HKL2000(72) (data collection statistics are shown in Table 5). Native 806 Fab was solved by molecular replacement using the program MOLREP(Zi) using the coordinates of the Fab structure 2E8 refinement of the structure was performed in REFMAC5(Z4) and model building in Coot(/5). Both 806-peptide and 175 Fab-peptide structures were solved by molecular replacement using the program MOLREP using the coordinates of the 806 Fab structure, refinement and rebuilding were again performed in REFMAC5, and COOT and O. Validation of the final structures were performed with PROCHECK(7<5) and WHATCHECK(Z 7).

NMR studies

[0175] For NMR studies, ^l5N-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 ¹⁵NH₄Cl(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.

[0176] Samples used for NMR studies were prepared in H₂O solution containing 5% ²H₂O, 70 mM NaCl and 50 mM NaPO₄ at pH 6.8. All spectra were acquired at 298K on a Bruker Avance500 spectrometer using a cryoprobe. Sequential assignments of the peptide in the absence of m806Fab were established using standard 2D TOCSY and NOESY as well as ¹⁵N-edited TOCSY and NOESY spectra. Interaction between the peptide and fAb806was examined by monitoring ¹⁵N HSQC spectra of the peptide in the absence and presence of fAb806. Spectral perturbation of ¹⁵N HSQC spectra of the peptide in the presence of fAb806clearly indicates the peptide was able to bind to the fAb806under the presence solution conditions. Detailed conformation of the peptide in the complex form was, however, not determined.

[0177] Supplemental Table 5. Data Collection and Refinement Statistics

Data Collection

806(native) 806(peptide) 175(native) 175(peptide)

Space Group P2,2,2 P2, P2.2.2, P2,2,2

Cell Dimensions (A) α 140.37 35.92 36.37 83.17 b 74.62 83.16 94.80 69.26

C 83.87 72.21 β=92.43 108.90 71.47

Source in-house BNL X29 in-house in-house

Wavelength (A) 1.542 1.1 1.542 1.542

Resolution range (A) 29.7-2.2 50-2.0 50-2.8 14.18-1.59

(2.27-2.20) (2.07-2.0) (2.87-2.80) (1.65-1.59)

Emerge (%) 6.4 (26.7) 6.6 (28.2) 8.6 (30.0)

I/σl 12.2 (3.2) 22 (3.15) 10.2 (2.2)

Completeness (%) 98.3 (91.3) 96.6 (79.2) 98.4(90.5) 78.8 (1 1.8)

98.1 at 1.89 A

Total Reflections 156497 98374 205401 Unique reflections 44905 27692 9171 43879

Numbers in parentheses ar for the highest resolution shell.

[0178] Refinement

Resolution range (A) 20-2.3 72.17-2.00 50-2.6 14.18-1.6

Reflections 37397 26284 9171 41611

°αyst 0.225 0.226 0.210 0.203

^free 0.289 0.279 0.305 0.257

Protein Atoms 6580 3294 3276 3390

Solvent Atoms 208 199 46 247 r.m.s.d bond length (A) 0.022 0.007 0.015 0.014 r.m.s.d bond angle (°) 1.70 1.12 1.77 1.48

Average B-factor (A²) 40.3 33.6 37.5 20.7

Overall anisotropic B-factors (A²)

BI l -1.52 2.42 0.20 1.13

B22 2.22 -0.26 -1.022 -0.38

B33 -0.70 -2.1 1 1.03 -0.74

References

1. Palacios, R., Henson, G., Steinmetz, M., and McKearn, J. P. (1984) Nature. 309, 126-131.

2. Walker, F., Orchard, S. G., Jorissen, R. N., Hall, N. E., Zhang, H. H., Hoyne, P. A., Adams, T. E., Johns, T. G., Ward, C, Garrett, T. P., Zhu, H. J., Nerrie, M., Scott, A. M., Nice, E. C, and Burgess, A. W. (2004) J Biol Chem. 279, 22387-22398.

3. Ymer, S., Tucker, W. Q., Sanderson, C. J., Hapel, A. J., Campbell, H. D., and Young, I. G. (1985) Nature. %19-25;317, 255-258.

4. Panousis, C, Rayzman, V. M., Johns, T. G., Renner, C, Liu, Z., Cartwright, G., Lee, F. T., Wang, D., Gan, H., Cao, D., Kypridis, A., Smyth, F. E., Brechbiel, M. W., Burgess, A. W., Old, L. J., and Scott, A. M. (2005) Br. J Cancer. 92, 1069- 1077.

5. Johns, T. G., Adams, T. E., Cochran, J. R., Hall, N. E., Hoyne, P. A., Olsen, M. J., Kim, Y. S., Rothacker, J., Nice, E. C, Walker, F., Ritter, G., Jungbluth, A. A., Old, L. J., Ward, C. W., Burgess, A. W., Wittrup, K. D., and Scott, A. M. (2004) J Biol Chem. 279, 30375-30384.

6. Johns, T. G., Adams, T. E., Cochran, J. R., Hall, N. E., Hoyne, P. A., Olsen, M. J., Kim, Y. S., Rothacker, J., Nice, E. C, Walker, F., Ritter, G., Jungbluth, A. A., Old, L. J., Ward, C. W., Burgess, A. W., Wittrup, K. D., and Scott, A. M. (2004) J Biol Chem. 279, 30375-30384. 7. Johns, T. G., Adams, T. E., Cochran, J. R., Hall, N. E., Hoyne, P. A., Olsen, M. J., Kim, Y. S., Rothacker, J., Nice, E. C, Walker, F., Ritter, G., Jungbluth, A. A., Old, L. J., Ward, C. W., Burgess, A. W., Wittrup, K. D., and Scott, A. M. (2004) J Biol Chem. 279, 30375-30384.

8. Johns, T. G., Adams, T. E., Cochran, J. R., Hall, N. E., Hoyne, P. A., Olsen, M. J., Kim, Y. S., Rothacker, J., Nice, E. C, Walker, F., Ritter, G., Jungbluth, A. A., Old, L. J., Ward, C. W., Burgess, A. W., Wittrup, K. D., and Scott, A. M. (2004) J Biol Chem. 279, 30375-30384.

9. Masui, H., Kawamoto, T., Sato, J. D., Wolf, B., Sato, G., and Mendelsohn, J. (1984) Cancer Res. 44, 1002-1007.

10. Gill, G. N., Kawamoto, T., Cochet, C, Le, A., Sato, J. D., Masui, H., McLeod, C, and Mendelsohn, J. (1984) J Biol Chem. 259, 7755-7760.

1 1. van de Loosdrecht, A. A., Beelen, R. H., Ossenkoppele, G. J., Broekhoven, M. G., and Langenhuijsen, M. M. (199 >4) J Immunol Methods. 174, 311-320.

12. Otwinowski, Z. and Minor, W. (1997) processing of X-ray diffraction data collected in oscillationn mode Academic Press (New York).

13. Vagin, A. and Teplyakov, A. (1997) J. Appl. Cryst 30, 1022-1025.

14. Murshudov, G. N., Vagin, A. A., and Dodson, E. J. (1997) Acta crystallographica 53, 240-255.

15. Emsley, P. and Cowtan, K. (2004) Acta crystallographica 60, 2126-2132.

16. Laskowski, R. A., MacArthur, M. W., Moss, D. S., and Thornton, J. M. (1993) J. Appl. Cryst 26, 283-291.

17. Hooft, R. W., Vriend, G., Sander, C, and Abola, E. E. (1996) Nature 381, 272.

18. Fairlie, W. D., Uboldi, A. D., De Souza, D. P., Hemmings, G. J., Nicola, N. A., and Baca, M. (2002) Protein expression and purification 26, 171-178.

19. Neidhardt, F. C, Bloch, P. L., and Smith, D. F. (1974) Journal of bacteriology 119, 736-747.

EXAMPLE 3 ErbB Family Members

[0179]Because of their cell surface expression the ErbB family receptors are a promising target for antibody-based cancer therapies.

Amplifϊcation/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. E.g.;

ErbB2:- 25% of breast cancer

ErbB3:- 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.

[0180] 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. Although the sequence homology of the EGFR (ErbB2) mAb806 loop 287-302 epitope (CGADSYEMEEDGVRKC) is relatively low in EGFR family members, including ErbB3 and ErbB4, the size and location of the cysteine loop is conserved (see Table 1 above). The extracellular region of each family member is made up of four subdomains, Ll, CRl, L2 and CR2, were "L" signifies a leucine-rich repeat domain and "CR" a cysteine-rich region.

[008I]We are generating mAb806 like antibodies which will recognize the transitional state of the other ErbB family members; ErbB2, ErbB3 and ErbB4 and not the wt forms. Antibodies are being raised in mice immunised with Baf/3 B cell lines transfected with the ErbB2, ErbB3 or ErbB4 cysteine mutants, whereby these mutations allow the conformational exposure of epitopes found in tumour but not normal tissues. The sequences of ErbB2, ErbB3 and ErbB4 with cysteine regions for mutation highlighted, are depicted in Figures 10, 12, and 14 respectively.

[0082]Mutant ErbB constructs were transfected into the interleukin-3-dependent murine hemopoietic cell line BaF/3 which contains no detectable levels of ErbB family members (numbers correspond to amino acid sequences with signal sequences subtracted): ErbB2:-

Baf/3-ErbB2-C²⁷⁷A

Baf/3-ErbB2-C²⁸⁹A

Baf/3-ErbB2-C²⁷⁷A/C²⁸⁹A "Baf/3-ErbB2-CACA" ErbB3:-

Baf/3-ErbB3-C²⁷¹A

Baf/3-ErbB3-C²⁸²A

Baf/3-ErbB3-C^27IA/C²⁸²A "Baf/3-ErbB3-CACA" ErbB4:-

Baf/3-ErbB4-C²⁶⁸A

Baf/3-ErbB4-C²⁷⁹A

Baf/3-ErbB4-C²⁶⁸A/C²⁷⁹A "Baf/3-ErbB4-CACA"

Transfection of EGFR Constructs and Generation of Stable Cell Lines [0083] 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,

2mM Glutamax, 10% WEHDB conditioned medium and 1.5 mg/ml G418.

Note: Primary Antibodies for Flow Analysis.

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)

[0085] Immunization Schedule:

1. 5 x 10⁶ Baf/3 cells transfected with ErbBR double mutant by ip /mouse with Complete Freund Adjuvant (1^st)

2. 5 x 10⁶ Baf/3 cells transfected with ErbBR double mutant by ip /mouse with Incomplete Freund Adjuvant (IFA) (2^nd)

3. 5 x 10⁶ Baf/3 cells transfected with ErbBR double mutant by ip /mouse with IFA

(3^rd)

4. 5 x 10⁶ Baf/3 cells transfected with ErbBR double mutant OR ErbBR antigen 40 μg with IFA ip (4^th injection)

5. Serum test

6. Booster with ErbBR antigen 40 μg with IFA ip/mouse

7. Fusion on day 4 after booster

8. ErbB Antigen: ErbBR double mutant soluble form (extracellular domain) [0086] The immunization regime will comprise of the above schedule on a monthly basis up to 4 months.

Screening of Antibodies:

[0087] Antibodies will be screened by ELISA, flow analysis, and hemadsorption assay

(rosetting), including as set out as follows:

1. ELISA

ELISAs will be done against the following ErbB2, ErbB3, and ErbB4 constructs: ErbB2

M2-ErbB2-MYC- HIS ++ M2-ErbB2-CACA-MYC-HIS++++ Anti-Flag (M2) -

Anti- Myc -

Ant-His -

Biotinylated epitope peptide ++++

ErbB3

ErbB3-MYC- HIS ++ ErbBS-CACA-MYC-HIS ++++ Anti- Myc - Ant-His -

Biotinylated epitope peptide ++++

ErbB4

ErbB3-MYC- HIS ++

ErbBS-CACA-MYC-HIS ++++

Anti- Myc -

Ant-His -

Biotinylated epitope peptide ++++

2. Flow Analysis

[0088] Flow analysis is then performed using these for the ErbB2, ErbB3 and ErbB4 antibodies being screened, respectively:

ErbB2 Baf/3 -

Baf/3-ErbB2-CACA +++ Baf/3-wtErbB2 + BT474 (Breast) ++

ErbB3 Baf/3 -

Baf/3-ErbB3-CACA +++ Baf/3-wtErbB3 + MCF-7 (Breast) ++

ErbB4 Baf/3 -

Baf/3-ErbB4-CACA +++ Baf/3-wtErbB4 + T47D (Breast) ++

3. Hemadsorption Assays

[0089] Hemadsorption asays are performed on Baf/3 transfected with double cysteine mutations (+++), Baf/3 transfected with wild-type ErbB receptors ( +), Baf/3 cells ( -), and Cell lines endogenously expressing the receptors (+).

Methods for Generating the Constructs and Mutants: I. CLONES AND VECTORS FROM ORIGENE

[0090] ErbB3 and ErbB4 clones are obtained from Origene Technologies, Inc (Rockville, MD) as follows:

1) TrueORF Human ORP clone for ErbB3, transcript variant 1 in a 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. ORF size: 4028. Accession Number: NM_001982 Catalogue No.: RC209954 2) TrueORF Human ORF clone for ErbB4, transcript variant JM-a/CVT-1 in a

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.

ORF size: 3926.

Accession Number: NM_005235.

Catalogue No: RC220449

[0091] *Both ErbB3 and ErbB4 ORFs were cloned into a PrecisonShuttle Destination vector using Sgf I (5 '-end) (or the isoschizomer AsiS I) and MIu I (3 '-end) restriction enzymes.

[0092] *AsiS I Catalogue number: RO630S (the isoschizomer of Sgfl) and MIu I (Catalogue number: RO 198s have been ordered from NEB.

[0093] *The PrecisonShuttle Destination vector was obtained from Origene.

[0094] The destination vector is a pCMV6-AC-Myc-HIS vector.

It has an Ampicillin resistance gene for selection in E.coli and a Neomycin resistance gene for cloning into mammalian systems.

Catalogue No: PSlOOOI l

II. SITE DIRECTED MUTAGENESIS

[0095] ErbB3 and ErbB4 clones were mutated whereby specific Cysteine residues flanking the disulphides holding the "806-like" epitope in place, are substituted for

Alanines.

Clones with 3 types of mutations were constructed for ErbB3 and ErbB4 using

QuikChange II Site Directed Mutagenesis Kit from Stratagene:

Numbers correspond to amino acid sequences with signal sequences subtracted. [0096] ErbB3:

The Cysteine (TGT) at position 271 is mutated to an Alanine (GCT) by substituting the Thymine and Guanine to Guanine and Cytosine at positions 811 and 812 (signal sequence subtracted).

1) Cysteine (TGT) at position 282 is mutated to an Alanine (GCT) by substituting the Thymine and Guanine to Guanine and Cytosine at positions 844 and 845 (signal sequence subtracted).

2) A double mutation where the cysteines at positions 271 and 282 are both mutated to Alanines as above (signal sequence subtracted).

3) Soluble protein containing the same mutation as (1) above. *

4) Soluble protein containing the same mutation as (2) above. *

5) Soluble protein containing the same two mutations as (3) above. *

[0097] ErbB4:

The Cysteine (TGT) at position 268 is mutated to an Alanine (GCT) by substituting the Thymine and Guanine to Guanine and Cytosine at positions 802 and 803 (signal sequence subtracted).

6) 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).

7) A double mutation where the cysteines at positions 268 and 279 are both mutated to Alanines as above (signal sequence subtracted).

8) Soluble protein containing the same mutation as (7) above. *

9) Soluble protein containing the same mutation as (8) above. *

10) Soluble protein containing the same two mutations as (9) above. *

[0098] * 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. III. STABLE TRANSFECTION OF CONSTRUCTS INTO MAMMALIAN

CELLS

CHO cells:

[0099] Constructs encoding soluble ErbB3 and ErbB4 proteins will be cloned into the Origene destination vector and subsequently transfected into mammalian expression system (CHO cells) to establish stable clones for culture and soluble product purification.

[0100] Secreted HIS tagged protein can be purified on a Nickel column and used as part of an immunization strategy and hybridoma screening procedure.

Baf/3 B cells:

[0101] Constructs encoding the full length ErbB3 and ErbB4 in the Origene Entry

Vector, have been stably tranfected into BaF/3 cells.

[0102] 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.

IV. GENERATION OF SOLUBLE ErbB PROTEINS

[0103] The vectors and scheme for generating soluble ErbB proteins are depicted in

Figure 15A and 15B. The following constructs will be made and transfected into CHO

(Chinese hamster ovarian) cells by electroporation. Numbers correspond to amino acid sequences with signal sequences subtracted.

PCMV6-AC-HIS/MYC -ErbB2-C²⁷⁷A/C²⁸⁹A

PCMV6-AC-HIS/MYC -ErbB2

PCMV6-AC-HIS/MYC -ErbB3-C²⁷¹A/C²⁸²A

PCMV6-AC-HIS/MYC -ErbB3

PCMV6-AC-HIS/MYC -ErbB4-C²⁶⁸A/C²⁷⁹A

PCMV6-AC-HIS/MYC -ErbB4

[0104] Stable cell lines will be generated under neomycin selection and positive clones will be selected according to their protein expression levels. [0105] The protein will be purified using a Nickel -sepharose column and final concentration will be determined by spectrophotometric analysis.

EXAMPLE 4 Insulin-Like Growth Factor Receptor (IGF-I)

[0106] The Type 1 Insulin-like growth factor receptor (IGFlR) 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

[0107] 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).

[0108] 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.

EXAMPLE 5 Insulin Receptor

[0109] 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). On the cell surface, receptors usually exist as disulfide-linked dimers (2 alpha/2 beta chains) although heterodimers with the Typel IGF receptor can occur. [0110] The insulin receptor is implicated in cancer (Sciacca, L., et al. (1999) Oncogene 18: 2471-2479; Vella, V., et al. (2002) J Clin Endocrinol Metab 87: 245-254), including an 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.

[OHIO] 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.

[0112] This invention may be embodied in other forms or carried out in other ways without departing from the spirit or essential characteristics thereof. The present disclosure is therefore to be considered as in all aspects illustrate and not restrictive, the scope of the invention being indicated by the appended Claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.

[0113] Various references are cited throughout this Specification, each of which is incorporated herein by reference in its entirety.

Claims

CLAIMS:

1. 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.

2. The method of claim 1 wherein the tumor associated protein is a receptor with a cysteine loop or cysteine rich domain.

3. The method of claim 1 wherein the tumor associated protein is selected from a member of the EGFR family, and a member of the insulin receptor family.

4. The method of claim 3 wherein the EGFR family member is selected from ErbBl, ErbB2, ErbB3 and ErbB4.

5. The method of claim 3 wherein the insulin receptor family member is selected from insulin receptor (INSR) or insulin-like growth factor receptor (IGFlR).

6. A method for screening modulators, including antibodies, 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, 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 available epitope for antibodies.

7. The method of claim 6 wherein the tumor associated protein is a receptor with a cysteine loop or cysteine rich domain.

8. The method of claim 6 wherein the tumor associated protein is selected from a member of the EGFR family, and a member of the insulin receptor family.

9. The method of claim 8 wherein the EGFR family member is selected from ErbBl, ErbB2, ErbB3 and ErbB4.

10. The method of claim 6 wherein the insulin receptor family member is selected from insulin receptor (INSR) or insulin-like growth factor receptor (IGFlR).

1 1. A method for identifying agents capable of modulating the expression or activity of proteins involved in the processes leading to cancer, cancer pathology, and tumors which target cryptic or hidden cysteine loop or cysteine domains in cancer-associated proteins, particularly cell-surface receptors, 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 or epitope for agents.

12. The method of claim 1 1 wherein the agent is an antibody.

13. The method of claim 12 wherein the antibody is a monoclonal antibody.

14. The method of claim 11 wherein the tumor associated protein is selected from a member of the EGFR family, and a member of the insulin receptor family.

15. The method of claim 14 wherein the EGFR family member is selected from ErbBl, ErbB2, ErbB3 and ErbB4.

16. The method of claim 14 wherein the insulin receptor family member is selected from insulin receptor (INSR) or insulin-like growth factor receptor (IGFlR).

17. The method of claim 11 wherein the cysteine mutated tumor-associated protein is expressed as a soluble polypeptide.

18. A cysteine mutated tumor-associated protein or mutant cysteine loop peptide selected from ErbB2, ErbB3, ErbB4, INSR, and IGFlR.

19. The cysteine mutated protein or peptide of claim 18 wherein one or more cysteine is replaced by alanine.

20. A kit for the in vitro assay of compounds, including antibodies, for binding to cancer- associated proteins using mutated cysteine modified polypeptide(s) of a cancer-associated protein, or fragments thereof wherein the. cysteine-mutated peptides, proteins or fragments are modified at one or more cysteine positions by replacement with a distinct amino acid such that the cysteine-mutated peptides, proteins or fragments expose epitopes or targets previously unavailable for binding.