Use of anti-EGFR antibodies in treating diseases mediated by EGFR mutants

Abstract

The present invention relates to the use of anti-EGFR antibodies in treating diseases mediated by EGFR mutants, and to treating EGFR-mediated diseases resistant to tyrosine kinase inhibitor therapy, particularly cancer. Methods are provided for treating cancer and reducing tumor growth in individuals resistant to standard therapy who have secondary EGFR mutations, particularly tyrosine kinase domain mutations. The present invention provides methods for treating tyrosine kinase inhibitor-resistant cancers using anti-EGFR antibodies. Methods of treating recurrent lung cancer, including non-small cell lung cancer, resistant to tyrosine kinase inhibitors using the antibody anti-EGFR mAb806 are described.

Description

[0001] This application is a divisional application of an invention patent application filed on January 24, 2008, with application number 200880006008.1 and invention name “Use of anti-EGFR antibodies in treating diseases mediated by EGFR mutants”. Field of the Invention

[0002] The present invention relates to the treatment of EGFR-mediated diseases, particularly cancer, that are resistant to tyrosine kinase inhibitor therapy. Methods are provided for treating cancer and reducing tumor growth in individuals with secondary EGFR mutations, particularly tyrosine kinase domain mutations, that are resistant to standard therapy. Background of the Invention

[0003] Targeted cancer therapies are designed to disrupt the function of specific molecules required for carcinogenesis and tumor growth, thereby killing or preventing the growth of cancer cells (Ji H et al. (2006) Cell Cycle 5(18):2072-2076 Epub 2006 Sep15). In contrast to conventional cytotoxic chemotherapy, such targeted cancer therapies are more effective and less harmful to normal cells. The main effort in the field of targeted cancer therapy has been to develop agents targeting epidermal growth factor receptor (EGFR). EGFR is a member of the ErbB family of closely related receptors (including EGFR (ErbB-1), Her2 / neu (ErbB-2), Her3 (ErbB-3) and Her4 (ErbB-4)). Activation of EGFR leads to activation of receptor tyrosine kinases and a series of downstream signaling events (the signaling events mediate cell proliferation, motility, adhesion, invasion and resistance to chemotherapy and inhibition of apoptosis (2-4)), processes that are crucial for the continued proliferation and survival of cancer cells.

[0004] To date, two main types of anti-EGFR agents have entered the clinical setting: anti-EGFR antibodies and small molecule EGFR tyrosine kinase inhibitors (TKIs) (5, 6). Anti-EGFR antibodies, such as cetuximab, are designed to bind to the extracellular domain of EGFR and block activation of EGFR downstream signaling (7). Cetuximab (also known as antibody 225, U.S. Patent 4,943,533) was generated against A431 cells, which express high levels of wild-type EGFR. In contrast, small molecule TKIs, such as gefitinib (compound ZD 1839, Iressa) or erlotinib (compound OSI-774, Taserva), compete with ATP for binding to the extracellular catalytic domain of the EGFR tyrosine kinase, thereby preventing EGFR autophosphorylation and downstream signaling (4).

[0005] Both anti-EGFR drug types have shown some clinical efficacy in subsets of patients with a variety of different types of cancer. Treatment with gefitinib or erlotinib in patients with lung cancer harboring EGFR kinase domain mutations generally produces significant clinical responses (5,8). However, the efficacy of gefitinib or erlotinib in lung adenocarcinoma or other histological subtypes such as squamous cell carcinoma with wild-type EGFR is limited (9,10). In addition, gefitinib or erlotinib has been shown in preclinical and clinical trials to be largely ineffective in inhibiting the function of EGFRvIII mutants (11), a different activating EGFR mutation in which there is a frame-matched deletion of exons II to VII (also known as EGFR de2-7). EGFRvIII is commonly found in glioblastoma and has recently been found in subsets of human lung squamous cell carcinoma (12) and in most head and neck cancers (13).

[0006] Cetuximab has been shown to be effective in patients with non-small cell lung cancer (NSCLC) and a small subset of patients with head and neck cancer and colorectal cancer. However, the response to cetuximab does not appear to be related to the level of EGFR expression. Therefore, it is unclear why these patients respond to cetuximab treatment while other cancer patients whose tumors have high EGFR expression do not respond to cetuximab treatment (14).

[0007] Since the expression of the EGFR vIII mutant receptor is restricted to tumor cells, it represents a highly specific target for antibody therapy. Accordingly, polyclonal and monoclonal antibodies have been generated that are specific for a unique peptide of de2-7 EGFR. A series of mouse mAbs isolated after immunization with a unique de2-7 peptide all showed selectivity and specificity for truncated receptors and targeted de2-7 EGFR-positive xenografts grown in nude mice (Wikstrand CJ et al. (1995) Cancer Res 55:3140-3148; Okamoto, S et al. (1996) Br J Cancer 73:1366-1372; Hills D et al. (1995) Int J Cancer 63:537-543; Reist CJ et al. (1997) Cancer Res 57:1510-1515; Reist CJ et al. (1995) Cancer Res 55:4375-4382; U.S. Patent 5,401,828). Examples of anti-EGFR vIII antibodies include ABX-EGF (panitumumab), DH8.3, L8A.4, and Y10.

[0008] MAb806 is a novel murine antibody that was initially generated using intact cells expressing the EGFR vIII mutant as an immunogen to recognize a unique truncated mutant EGFRvIII (15-17). Importantly, the epitope recognized by mAb806 is inaccessible in inactive wild-type (wt) EGFR but is exposed in transitional forms of wt EGFR in cells overexpressing EGFR and expressing EGFRvIII (18). Epitope studies were supported by immunohistochemical studies demonstrating that mAb806 binds to an epitope present in gliomas and a wide range of epithelial cancers but not in normal human tissues (16,19). These and other preclinical data suggest that mAb806 may have a different spectrum of clinical activity and side effect profile than cetuximab and other anti-EGFR antibodies. In xenograft models, mAb806 has demonstrated potent antitumor activity without targeting normal tissue. Thus, the unique targeting capabilities of mAb806 represent a new paradigm for cancer-specific molecular targeted therapy.

[0009] When overexpressed or activated by mutations, tyrosine kinases (including EGFR) contribute to the development of cancer, and these mutated tyrosine kinases (TKs) often provide targets or sensitivities for selective and specific cancer therapies. Somatic mutations in the tyrosine kinase domain of the EGFR gene are associated with sensitivity of lung cancer to certain tyrosine kinase inhibitors (TKIs), including gefitinib and erlotinib. An in-frame EGFR deletion in exon 19 (del L747-S752) and a common point mutation in codon 858 (exon 21) (L858R) have been identified in non-small cell lung cancer and adenocarcinoma and are associated with sensitivity to the TKIs gefitinib and erlotinib (Lynch TJ et al. (2004) N Engl J Med 350:2129-2139; Paez JG et al. (2004) Science 304:1497-1500; Pao W et al. (2004) PNAS 101(36):13306-13311). Recent studies have shown that 10-30% of NSCLC patients have EGFR kinase domain mutations, while 5% of lung squamous cell carcinoma (SCC) patients have extracellular domain EGFRvIII mutations (12, 20). Methods for determining the responsiveness of cancer to EGFR-targeted therapies based on the evaluation of mutations in EGFR, particularly in the kinase domain, and predicted inhibitor sensitivity in patients are described by Bell et al. (WO 2005 / 094357 and US20060147959).

[0010] Resistance to chemotherapy or targeted cancer therapy, mediated by secondary resistance or compensatory mutations, is a challenge. Despite continued treatment with TKIs, tumors that are sensitive to TKIs (including gefitinib or erlotinib) ultimately continue to develop. A secondary mutation (T790M) on site 790 of EGFR has been identified in tumor biopsies of recurrent and resistant patients (Kobayashi S et al. (2005) N Engl J Med 352(8):786-792). This mutation is predicted to result in steric hindrance of the binding of inhibitors in the ATP-kinase-binding pocket.

[0011] Given the existence and prevalence of acquired resistance to TKIs in EGFR-mediated diseases and the significant cancer recurrence rate, there is a clinical need for a wider range of effective treatment options using EGFR-targeted agents that effectively address, target or circumvent acquired resistance in EGFR mutants and EGFR-mediated diseases.

[0012] Citation of a reference herein should not be construed as an admission that such reference is prior art with respect to the present invention. SUMMARY OF THE INVENTION

[0013] Mutations that activate epidermal growth factor receptor (EGFR) have been identified in many EGFR-mediated cancers (including lung cancer). EGFR mutations have been identified in human non-small cell lung cancer (NSCLC), with 5% of human lung squamous cell carcinomas having EGFRvIII mutations and 10-30% of lung adenocarcinomas having EGFR kinase domain mutations. EGFR-targeted monoclonal antibody mAb806 recognizes conformational epitopes of wild-type (wt) EGFR and truncated EGFRvIII mutants. In order to further characterize the application of mAb806 to EGFR-mediated cancer therapy, mAb806 was used to treat genetically engineered mice with lung tumors driven by mutations in EGFRvIII or EGFR kinase domains. The present invention demonstrates that anti-EGFR vIII antibodies, particularly mAb806, are very effective in blocking EGFRvIII signaling and inducing tumor cell apoptosis, thereby leading to significant tumor regression in EGFRvIII-driven murine lung cancer. A different EGFR-targeting antibody, cetuximab, generated against cells expressing high levels of wild-type EGFR, failed to show activity in these genetically defined lung tumors. Furthermore, treatment with mAb806 induced significant tumor regression in mouse lung tumors driven by a well-recognized and clinically relevant EGFR kinase domain mutation (L858R). This kinase domain mutation has been shown to be sensitive to TKI therapy, specifically gefitinib or erlotinib.

[0014] Acquired resistance to TKIs (including gefitinib or erlotinib) is an ongoing challenge, and despite continued therapy, tumors that are sensitive to TKIs eventually continue to develop. This acquired resistance can be mediated by secondary resistance or compensatory mutations, particularly including a secondary mutation at position 790 of EGFR (T790M). Researchers now demonstrate that anti-EGFR antibodies, particularly mAb806, are effective against the T790M mutation, resulting in significant tumor regression in EGFR T790M / L858R-driven murine lung cancer. Overall, these data demonstrate that anti-EGFR antibodies, particularly mAb806, provide an effective alternative or adjunct in the treatment of patients (including cancer patients, particularly lung cancer patients) with EGFR kinase domain mutations.

[0015] The present invention provides a method for treating a tyrosine kinase inhibitor-resistant EGFR-mediated disease in a mammal, wherein the resistant EGFR-mediated disease is the result of a secondary mutation in EGFR that produces a mutated EGFR and wherein the mutation is different from an EGFR vIII mutation, the method comprising administering to the mammal an effective amount of an anti-EGFR antibody that can bind to and inhibit the mutated EGFR. In a specific aspect, the secondary EGFR mutation is an EGFR tyrosine kinase domain mutation. In another aspect, the tyrosine kinase domain mutation is T790M.

[0016] In certain embodiments of the method, the anti-EGFR antibody is mAb806 antibody or an active fragment thereof. MAb806 includes murine antibodies, recombinant antibodies, or humanized antibodies.

[0017] Additional anti-EGFR antibodies, including those targeting the EGFRvIII mutant, can be used in the therapeutic methods of the present invention.Exemplary and known anti-EGFR antibodies can be selected from ABX-EGF (panitumumab), DH8.3, L8A4 and / or active fragments thereof.

[0018] Diseases mediated by EGFR for treatment in the methods include cancers. EGFR-mediated cancers include glioblastoma, head and neck cancer, pancreatic cancer, lung cancer, cancer of the nervous system, gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, kidney cancer, retinal cancer, skin cancer, liver cancer, reproductive-urinary cancer, and bladder cancer. In specific aspects, the EGFR-mediated cancer is lung adenocarcinoma, lung squamous cell carcinoma, or non-small cell lung cancer.

[0019] The present invention provides a method for reducing EGFR-mediated tumor growth in a cancer patient, wherein the cancer patient has been previously treated with one or more tyrosine kinase inhibitors and has developed recurrent disease and tumor growth, the method comprising administering to the patient an effective amount of an anti-EGFR antibody such that recurrent disease and tumor growth are inhibited and reduced.

[0020] In certain embodiments of the present method for reducing tumor growth, the anti-EGFR antibody is mAb806 antibody or an active fragment thereof. MAb806 includes murine antibodies, recombinant antibodies, or humanized antibodies.

[0021] Additional anti-EGFR antibodies may be used, including those targeting the EGFRvIII mutant.Exemplary and known anti-EGFR antibodies may be selected from ABX-EGF (panitumumab), DH8.3, L8A4 and / or active fragments thereof.

[0022] In a specific clinical aspect, recurrent disease and tumor growth in cancer patients are the result of secondary EGFR mutations, which are EGFR tyrosine kinase domain mutations. A specific secondary EGFR mutation is the tyrosine kinase domain mutation T790M.

[0023] The present invention also provides a method for treating EGFR-mediated cancer in a mammal, comprising administering to the mammal a tyrosine kinase inhibitor and an anti-EGFR antibody, wherein the anti-EGFR antibody is administered after treatment with the tyrosine kinase inhibitor as a second-line therapy to inhibit potential secondary mutated EGFR that is resistant to the tyrosine kinase inhibitor.

[0024] The EGFR-mediated cancer can be selected from glioblastoma, head and neck cancer, pancreatic cancer, lung cancer, cancer of the nervous system, gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, kidney cancer, retinal cancer, skin cancer, liver cancer, reproductive-urinary cancer and bladder cancer. In specific aspects, the cancer is lung adenocarcinoma, lung squamous cell carcinoma or non-small cell lung cancer.

[0025] In one aspect of the present method, the tyrosine kinase inhibitor is a reversible tyrosine kinase inhibitor. The reversible tyrosine kinase inhibitor can be an aniliniquinazoline compound and be selected from gefitinib, erlotinib, AG1478, ST1571 and SU-6668.

[0026] In another aspect of the method, the tyrosine kinase inhibitor is an irreversible tyrosine kinase inhibitor.Exemplary irreversible tyrosine kinase inhibitors are known in the art and include, but are not limited to, EKB-569, EKI-569, HKI-272, HKI-357, and BIBW 2992.

[0027] Other objects and advantages will become apparent to those skilled in the art from the following description with reference to the accompanying drawings which are illustrated below. Summary of the Figures

[0028] Figure 1Describe the mouse lung cancer tumor driven by EGFRvIII expression to mAb806 and ch806 antibody treatment sensitive but anti-cetuximab treatment. Tet-op-EGFRvIII / CCSP-rtTA, Ink4A / Arf- / - mice were treated by IP injection of 0.5mg / dose of mAb806 or ch806 or 1mg / dose of cetuximab once daily. After the treatment of the first week, the antibody was given every two days with the same dosage. Serial MRI was carried out at the specified time point to show the corresponding sections of representative mice in each treatment group. The bar diagram (Bar diagram) illustrated as mean ± standard deviation (SD) illustrates the tumor regression measured by MRI, and uses Student's exact t test (Student's exact t test) to perform statistical analysis. All mice were made to continue diet doxycycline throughout the experiment. H: indicates the area of ​​the heart.

[0029] Figure 2 A and 2B depict the histopathological characteristics of lung adenocarcinoma in mice driven by EGFRvIII for mAb806 treatment.(A) lung adenocarcinoma (top frame) driven for more than 8 weeks was expressed by EGFRvIII. After 1 week of treatment with mAb806, tumor began to taper off and had the fibrosis (middle frame) of increase. When the 5th week ended mAb806 treatment, lung samples were generally normal (bottom frame). Arrows showed fibrotic nodules, which were composed of fibroblasts and macrophages. Tumor cells were not found in this specific fibrotic area. Left frame: 100X, in frame: 800X.(B) similar patterns and the intensity of immunohistological staining of total EGFR can be observed in control mice and in mice (left upper and left lower frame) treated with mAb806 for 1 week; when compared with untreated tumor (upper right frame), after 1 week of treatment, the intensity of phospho-EGFR staining of tumor cells decreased (lower right frame). Representative photos are taken at 200X magnification. (C) TUNEL staining shows that when compared with untreated tumor (upper left frame), the nucleus (red arrow) of apoptosis in the lung tumor (lower left frame) driven by EGFRvIII after treating 1 week with mAb806 increases. Representative photos are taken at 200X magnification. The bar graph examples expressed as mean ± SD illustrate the apoptosis index in lung tumors before and after the treatment of mAb806 at least 200 times high power fields (HPF). Statistical analysis is performed using Student's exact t test (right frame).

[0030] Figure 3Shown is a Western blot analysis of whole lung lysates from Tet-op-EGFRvIII / CCSP-rtTA, Ink4A / Arf- / - mice treated with mAb806. Whole lung lysates from tumors harvested from mice at various time points following mAb806 treatment were analyzed. Inhibition of EGFR phosphorylation was observed as early as one week of treatment, whereas total EGFR levels decreased only after five weeks of treatment. Throughout mAb806 administration, Erk1,2 phosphorylation was inhibited by the antibody, but AKT phosphorylation remained at levels comparable to untreated controls at both treatment time points. β-actin was used as a loading control.

[0031] Figure 4 A and 4B. Mouse lung adenocarcinoma driven by the EGFR kinase domain mutation L858R responds to ch806 treatment. (A) Tet-op-EGFR L858R-IRES-luciferase / CCSP-rtTA mice were treated with ch806 at 0.5 mg / dose IP injection once daily for 4 weeks. MRI showed a reduction in tumor volume after 2 and 4 weeks of treatment. Bar graphs, expressed as mean ± SD, illustrate tumor regression measured by MRI, with statistical analysis performed using Student's exact t-test (right panel). H: Heart region indicated. (B) Histopathological analysis showed that tumors in Tet-op-EGFR L858R-IRES-luciferase / CCSP-rtTA mice (two panels on the right) shrank and had significant macrophage infiltration when compared to untreated controls (two panels on the left). Arrows indicate foci of residual tumor. Photos at 100X and 800X magnification are shown as indicated by the footnotes in the figure.

[0032] Figure 5 A and 5B depict the results of treating EGFR T790M-L858R lung tumors using mAb806 versus cetuximab. Mice that continued a doxycycline diet for more than 8 weeks were subjected to MRI to record tumor burden. mAb806 was delivered to mice with lung tumors by IP injection of 0.5 mg dose once daily for 4 weeks. Cetuximab was administered to mice by IP injection of 1 mg / dose once daily for 4 weeks. Littermate mice were used as controls (untreated) for all treatment studies. (A) Mice were imaged with MRI at week 0, 2, and 4 or 5 to determine the reduction in tumor volume. (B) After treatment and MRI imaging were completed, mice were killed for further histological and biochemical studies. Detailed Description of the Invention

[0033] According to the present invention, conventional molecular biology, microbiology and recombinant DNA techniques within the capabilities of those skilled in the art may be employed. Such techniques are fully described in the literature. See, for example, Sambrook et al., "Molecular Cloning: A Laboratory Manual" (1989); "Current Protocols in Molecular Biology" Vol. I-III [Ausubel, RM, ed. (1994)]; "Cell Biology: A Laboratory Handbook" Vol. I-III [JECelis, ed. (1994))]; "Immunology" Vol. I-III [Coligan, JE, ed. (1994)]; "Oligonucleotide Synthesis" (MJ Gaited. 1984); "Nucleic Acid Hybridization" [BD Hames & S. J. Higgins eds. (1985)]; "Transcription And Translation" [BD Hames & S. J. Higgins, eds. (1984)]; "Animal CellCulture" [RI Freshney, ed. (1986)]; "Immobilized Cells And Enzymes" [IRL Press, (1986)]; B. Perbal, "A Practical Guide To Molecular Cloning" (1984).

[0034] Accordingly, if and when appearing herein, the following terms shall have the definitions set forth below.

[0035] The term "antibody" describes an immunoglobulin, whether natural or partially or completely synthetically produced. Antibodies include any immunoglobulin that binds to a specific epitope, including antibodies and fragments thereof. The term includes polyclonal, monoclonal, recombinant, humanized, and chimeric antibodies. The term also encompasses any polypeptide or protein having a binding domain that is or is homologous to an antibody binding domain. The term also includes CDR-grafted antibodies.

[0036] Since antibodies can be modified in many ways, the term "antibody" should be interpreted as encompassing any specific binding member or substance having a binding domain that requires specificity. Thus, the term encompasses antibody fragments, derivatives, functional equivalents, and homologs of antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or fully or partially synthesized. Thus, immune chimeric molecules comprising an immunoglobulin binding domain or equivalent fused to another polypeptide are included. The cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023, as well as U.S. Patents 4,816,397 and 4,816,567.

[0037] It has been shown that fragments of intact antibodies can function to bind antigens. Examples of binding fragments are (i) a Fab fragment consisting of the VL, VH, CL, and CH1 domains; (ii) an Fd fragment consisting of the VH and CH1 domains; (iii) an Fv fragment consisting of the VL and VH domains of a single antibody; (iv) a dAb fragment consisting of the VH domain (Ward, ES et al., Nature 341, 544-546 (1989)); (v) isolated CDR regions; (vi) a F(ab')2 fragment, a bivalent fragment comprising two linked Fab fragments; (vii) a single-chain Fv molecule (scFv), in which the VH and VL domains are linked by a peptide linker that 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 (WO 94 / 13804; P. Holliger et al. Proc. Natl. Acad. Sci. USA 90 6444-6448, (1993)).

[0038] The "antibody combining site" is the structural portion of an antibody molecule consisting of the light or heavy chain and the light chain variable and hypervariable regions, which specifically binds to an antigen.

[0039] The phrase "antibody molecule" as used herein in its various grammatical forms includes intact immunoglobulin molecules and immunologically active portions of immunoglobulin molecules.

[0040] Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules, and those portions of immunoglobulin molecules that contain the paratope, including those portions referred to in the art as Fab, Fab', F(ab')2, and F(v), which are preferably used in the therapeutic methods described herein.

[0041] The antibodies may also be bispecific, where 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. recruiting effector function etc. The bispecific antibodies of the present invention include those in which one binding domain of the antibody is a specific binding member of the present invention (including fragments thereof) and the other binding domain is a different antibody or fragment thereof, including different anti-EGFR antibodies such as antibody 528 (U.S. Patent No. 4,943,533), chimeric and humanized 225 antibodies (U.S. Patent No. 4,943,533 and WO / 9640210), anti-de2-7 antibodies such as DH8.3 (Hills, D. et al. (1995) Int. J. Cancer 63(4):537-543), antibodies L8A4 and Y10 (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) PAACR55(14):3140-3148 or the antibodies 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 specific cell type, such as in a neural or glial cell specific antibody. In the bispecific antibodies of the present invention, one binding domain of an antibody of the present invention may be combined with other binding domains or molecules that recognize specific cell receptors and / or regulate cells in a specific manner, such as as an immunomodulator (e.g., an interleukin), a growth regulator or a cytokine (e.g., tumor necrosis factor (TNF), in particular, the TNF bispecific modality described in USSN 60 / 355,838 filed February 13, 2002 (incorporated herein by reference in its entirety)) or a toxin (e.g., ricin) or an anti-mitotic or apoptotic agent or factor.

[0042] The Fab and F(ab')2 portions of the antibody molecule can be prepared by proteolytic reactions of substantially intact antibody molecules by papain and pepsin, respectively, using well-known methods. See, for example, U.S. Patent No. 4,342,566 to Theofilopolous et al. The Fab' antibody molecule portion is also well known and is produced from the F(ab')2 portion, which is then reduced with mercaptoethanol to form the disulfide bond connecting the two heavy chain portions, and then alkylated with a reagent such as iodoacetamide to form the resulting protein thiol. Antibodies comprising intact antibody molecules are preferred herein.

[0043] The phrase "monoclonal antibody" in its various grammatical forms refers to an antibody that has only one antibody combining site capable of immunoreacting with a specific antigen. Thus, a monoclonal antibody typically displays a single binding affinity for any antigen with which it immunoreacts. Monoclonal antibodies can also encompass antibody molecules having multiple antibody combining sites, each of which is immunospecific for a different antigen; for example, bispecific (chimeric) monoclonal antibodies.

[0044] The term "antigen binding domain" describes the portion of an antibody that comprises a region that specifically binds to part or all of an antigen and is complementary to part or all of the antigen. When the antigen is large, the antibody may only bind to a specific portion of the antigen, which is called an epitope. The antigen binding domain can be provided by one or more antibody variable domains. Preferably, the antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[0045] The terms "mAb806," "806 antibody," "monoclonal antibody 806," "ch806," "humanized 806," and any variants not specifically listed are used interchangeably herein and as used throughout this application and claims. Thus, antibodies are likewise encompassed, including recombinant, chimeric, genetically modified, or alternative antibodies. These modifications may be intentional, such as those obtained by site-directed mutagenesis, or may be accidental, such as those obtained by mutations in a host that is a producer of the antibody or fragment thereof. Likewise, the terms "mAb806," "806 antibody," "monoclonal antibody 806," "ch806," and "humanized 806" are intended to include within their scope the publicly disclosed proteins and immunoglobulins explicitly referenced herein and known to those skilled in the art, as well as all substantially homologous analogs and allelic variations. The mAb806 antibody, including its production, specific activities, amino acid and nucleic acid sequences, antigen binding domains, and variable region sequences, are disclosed and known to those skilled in the art, including those provided in WO 02 / 092771; Luwor RB et al. (2001) Cancer Res 61:5355-5361; Mishima K et al. (2001) Cancer Res 61:5349-5354; Johns TG et al. (2002) Int J Cancer 98:398-408; Jungbluth AA et al. (2003) Proc Natl Acad Sci 100(2):639-644 (each of which is incorporated herein by reference in its entirety).

[0046] The amino acid residues described herein are preferably present in the "L" isomeric form. However, any L amino acid residue may be substituted with a residue present in the "D" isomeric form, provided that the polypeptide retains the desired immunoglobulin-binding functional properties. NH2 refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxyl group present at the carboxyl terminus of a polypeptide. In accordance with standard polypeptide nomenclature, J. Biol. Chem., 243:3552-59 (1969), the abbreviations for amino acid residues are shown in the following table of correspondence:

[0047] Correspondence table

[0048]

[0049]

[0050] It should be noted that all amino acid residue sequences are represented herein by the molecular formula (formulae) arranged in the conventional direction from amino terminal to carboxyl terminal in the direction from left to right. In addition, it should be noted that the dash (dash) at the start or end of the amino acid residue sequence represents the peptide bond connected to the other sequence of one or more amino acid residues. The above table is provided so that the three-letter and one-letter symbols that alternate in this article are corresponding.

[0051] It should be understood that also within the scope of the compositions used in the methods of the present invention are DNA sequences that encode and / or express effective anti-EGFR antibodies, particularly including mAb806 and ch806, which encode anti-EGFR antibodies, antigen-binding domains thereof, or active fragments thereof having the same amino acid sequence as the publicly disclosed mAb806 antibody known to those skilled in the art, but are degenerate from the known mAb806 sequence. "Degenerate from..." means that different three-letter codons are used to specify specific amino acids. It is well known in the art that the following codons can be used interchangeably to encode each specific amino acid:

[0052] Phenylalanine (Phe or F) UUU or UUC

[0053] Leucine (Leu or L) UUA or UUG or CUU or CUC or CUA or CUG

[0054] Isoleucine (Ile or I) AUU or AUC or AUA

[0055] Methionine (Met or M) AUG

[0056] Valine (Val or V) GUU or GUC or GUA or GUG

[0057] Serine (Ser or S) UCU or UCC or UCA or UCG or AGU or AGC

[0058] Proline (Pro or P) CCU or CCC or CCA or CCG

[0059] Threonine (Thr or T) ACU or ACC or ACA or ACG

[0060] Alanine (Ala or A) GCU or GCG or GCA or GCG

[0061] Tyrosine (Tyr or Y) UAU or UAC

[0062] Histidine (His or H) CAU or CAC

[0063] Glutamine (Gln or Q) CAA or CAG

[0064] Asparagine (Asn or N) AAU or AAC

[0065] Lysine (Lys or K) AAA or AAG

[0066] Aspartic acid (Asp or D) GAU or GAC

[0067] Glutamate (Glu or E) GAA or GAG

[0068] Cysteine ​​(Cys or C) UGU or UGC

[0069] Arginine (Arg or R) CGU or CGC or CGA or CGG or AGA or AGG

[0070] Glycine (Gly or G) GGU or GGC or GGA or GGG

[0071] Tryptophan (Trp or W) UGG

[0072] Stop codon UAA (ochre) or UAG (amber) or UGA (opal)

[0073] It will be understood that the codons specified above are for RNA sequences. The corresponding codons for DNA have T substituted for U.

[0074] Can be included in anti-EGFR antibody sequence, carry out mutation so that specific codon is changed into the codon of encoding different amino acids in mAb806 antibody sequence.Such mutation is usually produced by minimum nucleotide change as far as possible.Can be in non-conservative mode (that is, by codon from the amino acid belonging to the amino acid type with specific size or feature is changed into the amino acid belonging to another type) or in conservative mode (that is, by codon from the amino acid belonging to the amino acid type with specific size or feature is changed into the amino acid belonging to the same type) carry out such substitution mutation to change the amino acid in the obtained protein.Such conservative change usually causes less change in the structure and function of the obtained protein.Non-conservative change is more likely to change the structure, activity or function of the obtained protein.It should be understood that the present invention includes sequences that comprise conservative changes that do not significantly change the activity or binding characteristics of the obtained immunoglobulin and antibody.

[0075] Similarly, it is expected that certain EGFR mutations (which may even significantly affect or change the activity of EGFR), such as the EGFR kinase domain mutations described and utilized herein, may not affect the recognition, binding or inhibition of EGFR by anti-EGFR antibodies (particularly including anti-EGFR vIII mutant antibodies, particularly including mAb806 antibodies). Therefore, it is expected that mAb806 may be similarly effective against other hitherto unrecognized or hitherto unknown EGFR mutations, particularly secondary mutations generated in anticancer therapies. These mutations may be generated as a result of TKI inhibitory therapy or as a result of other therapies for resistant EGFR-mediated diseases, which may target the kinases or other activities of EGFR.

[0076] Amino acids can be classified as similar or different, conservative or non-conservative. Amino acids can be classified based on their R groups (e.g., nonpolar, uncharged polar, charged polar, amino acids with a phenyl group), based on their molecular weight or the size of their R groups, and based on molecular weight. Particularly preferred substitutions are: Lys for Arg and vice versa, so that the positive charge is maintained; Glu for Asp and vice versa, so that the negative charge is maintained; Ser for Thr, so that the free -OH is maintained; and Gln for Asn, so that the free NH2 is maintained.

[0077] Amino acid substitutions can also be introduced to replace amino acids with particularly preferred properties. For example, a Cys can be introduced into a potential position for forming a disulfide bond with another Cys. His can be introduced as a unique "catalytic" site (i.e., His can function as an acid or a base and is the most common amino acid in biochemical catalysis). Pro can be introduced due to its unique planar structure (which induces a beta turn in the structure of the protein).

[0078] 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.

[0079] The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce allergic or similar adverse reactions such as gastric upset, dizziness, and the like when administered to a human.

[0080] The phrase "therapeutically effective amount" is used herein to refer to an amount sufficient to prevent and preferably reduce by at least about 20%, more preferably at least 30%, more preferably at least 50%, more preferably at least 70%, more preferably at least 90% a clinically significant change in the S phase activity of a target cell mass or a significant change in the size or dimensions of a target cell mass or tumor, or other pathological features that may accompany their presence and activity.

[0081] Antibodies or active fragments can be formulated into therapeutic compositions in the form of neutralized pharmaceutically acceptable salts. Pharmaceutically acceptable salts include acid addition salts formed with inorganic acids such as hydrochloric acid or phosphoric acid or with organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. (formed with free amino groups of polypeptides or antibody molecules). Salts formed from free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, aluminum hydroxide, calcium hydroxide or ferric hydroxide and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0082] Compositions comprising therapeutic antibodies or active fragments are conventionally administered intravenously, for example, by injection in unit doses. The term "unit dose," when used in relation to the therapeutic compositions of the present invention, refers to physically discrete units suitable as unitary dosages for human use, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with the required diluent (i.e., carrier or vehicle).

[0083] In a manner compatible with the dosage form and in a therapeutically effective amount, the composition is administered. The amount to be administered depends on the ability of the subject to be treated, the subject's immune system to utilize the active ingredient and the degree of inhibition expected or the size of the targeted tumor mass. The precise amount of the active ingredient that needs to be administered depends on the doctor's judgment and is unique for each individual. However, suitable dosage can be in the range of approximately 0.1 to 20, preferably approximately 0.5 to approximately 10, and more preferably 1 to several milligrams of active ingredient / kg individual body weight / day, and depends on the approach used. The appropriate regimen for initially administering and boosting injections is also variable, but usually after initial administration, by subsequent injection or other routes of administration with 1 hour or several hours of interval repeat administration. Alternatively, comprise using a continuous intravenous infusion that is enough to keep 10 nanomoles to 10 micromolar concentrations in blood.

[0084] As used herein, "pg" means picogram, "ng" means nanogram, "ug" or "μg" means microgram, "mg" means milligram, "ul" or "μl" means microliter, "ml" means milliliter, and "l" means liter.

[0085] In order to further characterize the application of mAb806 in EGFR-mediated cancer therapy, the present invention describes the purposes of mAb806 for the treatment of genetically engineered mice with lung tumors driven by EGFRvIII or EGFR kinase domain mutations. Each of these mutations is clinically relevant and significant for EGFR-mediated diseases, particularly cancer (including lung cancer, pancreatic cancer, colorectal cancer, head and neck cancer and glioblastoma). The present invention verifies that anti-EGFR vIII antibodies, particularly mAb806, are very effective in blocking EGFRvIII signal conduction and inducing tumor cell apoptosis, thereby causing significant tumor regression in the mouse lung cancer driven by EGFRvIII. Cetuximab, an antibody targeting EGFR uniquely produced by cells expressing high levels of wild-type EGFR, can not show activity in these genetically determined lung tumors. In addition, mAb806 was used to induce significant tumor regression in the treatment of mouse lung tumors driven by recognized and clinically relevant EGFR kinase domain mutations (L858R). It has been shown that this kinase domain mutation is sensitive to TKI therapy, particularly gefitinib or erlotinib. Acquired resistance to TKIs, including gefitinib or erlotinib, is an ongoing challenge, and despite continued treatment, tumors that are sensitive to TKIs eventually continue to develop. This acquired resistance can be mediated by secondary resistance or compensatory mutations, particularly the secondary mutation (T790M) at site 790 of EGFR. Researchers now show that anti-EGFR antibodies, particularly mAb806, are effective against the T790M mutation, resulting in significant tumor regression in EGFR T790M / L858R-driven murine lung cancer. Overall, these data demonstrate that anti-EGFR antibodies, particularly mAb806, provide an effective alternative or adjuvant in the treatment of patients with EGFR kinase domain mutations, including cancer patients, particularly lung cancer patients.

[0086] Thus, therapeutic and diagnostic applications and methods are provided and generated by demonstrating the anti-tumor activity of anti-EGFR antibodies, particularly mAb806. As initially suggested and further elaborated herein, the present invention includes pharmaceutical interventions in cascades and signaling pathways involving EGFR, thereby modulating the tumorigenic capacity associated with EGFR mutations, including kinase domain mutations (primary and secondary resistance mutations).

[0087] The data provided herein demonstrate the activity of mAb806 against EGFR kinase domain mutations including L858R and T790M against TKIs. It is expected that additional kinase domain mutations or EGFR secondary mutations may exist or develop with continued and advanced guided anti-EGFR therapy. HKI272, an irreversible inhibitor of EGFR binding at cysteine ​​T797 (corresponding to cysteine ​​530 in the EGFR vIII deletion mutant), is being investigated in preclinical studies. If not anticipated, resistance secondary mutations with substitutions on cysteine ​​are also possible. These additional EGFR secondary mutants will be candidates for anti-EGFR antibody therapy.

[0088] Therefore, the present invention provides a method for treating a tyrosine kinase inhibitor-resistant EGFR-mediated disease in a mammal, wherein the resistant EGFR-mediated disease is the result of a secondary mutation in EGFR to produce a mutated EGFR, and wherein the mutation is different from the EGFR vIII mutation, and the method comprises administering an effective amount of an anti-EGFR antibody that can bind to and inhibit the mutated EGFR to the mammal. In a specific aspect, the secondary EGFR mutation is an EGFR tyrosine kinase domain mutation. In another aspect, the tyrosine kinase domain mutation is T790M. In a specific embodiment of the method, the anti-EGFR antibody is mAb806 antibody or its active fragment. MAb806 includes mouse antibodies, recombinant antibodies or humanized antibodies. Other anti-EGFR antibodies, including antibodies targeting EGFRvIII mutants, can be used for the methods of the present invention. Exemplary and known anti-EGFR antibodies can be selected from ABX-EGF (panitumumab), DH8.3, L8A4 and its active fragment.

[0089] The EGFR-mediated disease for treatment is particularly cancer and can be selected from glioblastoma, head and neck cancer, pancreatic cancer, lung cancer, cancer of the nervous system, gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, kidney cancer, retinal cancer, skin cancer, liver cancer, reproductive-urinary cancer and bladder cancer. In specific aspects, the EGFR-mediated cancer is lung adenocarcinoma, lung squamous cell carcinoma or non-small cell lung cancer.

[0090] The present invention includes the method for reducing the tumor growth of EGFR mediation in cancer patients, wherein said cancer patients have been treated with one or more tyrosine kinase inhibitors before and recurrent disease and tumor growth have occurred, and the method comprises that said patient is used the anti-EGFR antibody of significant dose so that recurrent disease and tumor growth are suppressed and reduced.In the specific embodiment of this method for reducing tumor growth, anti-EGFR antibody is mAb806 antibody or its active fragment.MAb806 comprises mouse antibody, recombinant antibody or humanized antibody.Other anti-EGFR antibody can be used, including those antibodies of targeting EGFRvIII mutant.Exemplary and known anti-EGFR antibody can be selected from ABX-EGF (panitumumab), DH8.3, L8A4 and / or its active fragment.

[0091] In a specific clinical aspect, recurrent disease and tumor growth in cancer patients are the result of secondary EGFR mutations, which are EGFR tyrosine kinase domain mutations. A specific secondary EGFR mutation is the tyrosine kinase domain mutation T790M.

[0092] The present invention also provides a method for treating EGFR-mediated cancer in a mammal, comprising administering to the mammal a tyrosine kinase inhibitor and an anti-EGFR antibody. In one aspect, the tyrosine kinase inhibitor and the anti-EGFR antibody are administered simultaneously. In one aspect, the tyrosine kinase inhibitor and the anti-EGFR antibody are administered simultaneously, sequentially, and repeatedly, before or after conventional chemotherapy.

[0093] The present invention also provides a method for treating EGFR-mediated cancer in a mammal, comprising administering to the mammal a tyrosine kinase inhibitor and an anti-EGFR antibody, wherein the anti-EGFR antibody is administered after treatment with the tyrosine kinase inhibitor as a second-line therapy to inhibit potential secondary mutated EGFR that is resistant to the tyrosine kinase inhibitor.

[0094] The tyrosine kinase inhibitor can be a reversible tyrosine kinase inhibitor or an irreversible tyrosine kinase inhibitor. The inhibitory tyrosine kinase inhibitor can be an aniline quinazoline compound and is selected from gefitinib, erlotinib, AG 1478, ST1571 and SU-6668. Exemplary irreversible tyrosine kinase inhibitors are known in the art and include, but are not limited to, EKB-569, EKI-569, HKI-272, HKI-357 and BIBW 2992 (Kwak EL et al. (2005) Proc Natl Acad Sci USA 102(21):7665-70).

[0095] The EGFR-mediated cancer can be selected from glioblastoma, head and neck cancer, pancreatic cancer, lung cancer, cancer of the nervous system, gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, kidney cancer, retinal cancer, skin cancer, liver cancer, reproductive-urinary cancer and bladder cancer. In particular, the cancer is lung adenocarcinoma, lung squamous cell carcinoma or non-small cell lung cancer.

[0096] Anti-EGFR antibodies, particularly mAb806, can be administered alone or in combination with other anti-EGFR antibodies in the methods. Thus, Mab806 can be administered continuously or in combination with cetuximab. mAb806 can also be administered continuously or in combination with other anti-EGFRvIII antibodies including ABX-EGF (panitumumab), DH8.3, L8A4, and / or active fragments thereof.

[0097] The anti-EGFR antibodies can be formulated into pharmaceutical compositions with appropriate carriers and at strengths effective for administration to patients by various methods. A variety of administration techniques can be used, including parenteral techniques such as subcutaneous, intravenous, and intraperitoneal injections, catheterization, and the like. The amount of the antibody or its active fragment can vary and should, in particular, be based on the recommendations and prescriptions of a qualified physician or veterinarian, including consideration of the results and data provided herein.

[0098] Anti-EGFR antibodies for use in the present invention, including mAb806, may provide useful diagnostic applications, including imaging applications or diagnostic biopsy applications, for diagnosing and / or monitoring cancer patients (including after or upon conclusion of TKI treatment).

[0099] Labels commonly used in such studies are radioactive elements, enzymes, chemicals and other substances that fluoresce when exposed to ultraviolet light. Many fluorescent materials are known and can be used as labels. These include, for example, fluorescein, rhodamine, auramine, Texas Red, AMCA Blue and Lucifer Yellow.

[0100] The antibodies of the present invention may be labeled with a detectable or functional marker. Detectable markers include, but are not limited to, radioactive markers such as isotopes. 3 H. 14 C. 32 P. 35 S. 36 Cl, 51 Cr, 57 Co、 58 Co、 59 Fe, 90 Y. 121 I. 124 I. 125 I. 131 I. 111In, 211 At 198 Au, 67 Cu, 225 Ac, 213 Bi, 99 Tc and 186 Re can be linked to the antibodies of the present invention using conventional chemistry known in the art of antibody imaging. Labels also include fluorescent labels and labels routinely used in the art for MRI-CT imaging. They also include enzyme labels, such as horseradish peroxidase. Labels also include chemical moieties such as biotin, which can be detected by binding to a specific cognate detectable moiety, such as labeled avidin.

[0101] Functional markers include substances designed to be targeted to the site of a tumor to cause destruction of tumor tissue. Such functional markers include cytotoxic drugs such as 5-fluorouracil or ricin, and enzymes such as bacterial carboxypeptidases or nitroreductases that can convert prodrugs into active drugs at the site of the tumor.

[0102] Radiolabeled anti-EGFR antibodies and fragments thereof are used for in vitro diagnostic techniques and for in vivo radioimaging techniques and for radioimmunotherapy. In the case of in vivo imaging, the specific binding members of the present invention can be conjugated to imaging agents rather than radioisotopes, including magnetic resonance imaging enhancing agents, wherein, for example, antibody molecules are loaded with a large amount of paramagnetic ions via a chelating group. Examples of chelating groups include EDTA, porphyrins, polyamine crown ethers, and polyoximes. Examples of paramagnetic ions include gadolinium, iron, manganese, rhenium, europium, lanthanum, holmium, and ferbium. In another aspect of the invention, radiolabeled specific binding members, particularly antibodies and fragments thereof, particularly radioimmunoconjugates are used for radioimmunotherapy, particularly as radiolabeled antibodies for cancer treatment. In a further aspect, radiolabeled specific binding members, particularly antibodies and fragments thereof, are used in radioimmuno-guided surgery techniques, wherein they can identify and indicate the presence and / or location of cancerous, precancerous, tumorous, neoplastic and hyperproliferative cells before, during or after surgery to remove such cells.

[0103] The immunoconjugates or antibody fusion proteins of the invention, in which specific binding members of the invention, particularly antibodies and fragments thereof, are conjugated or linked to other molecules or agents, also include, but are not limited to, binding members conjugated to chemical ablation agents, toxins, immunomodulators, cytokines, cytotoxic agents, chemotherapeutic agents or drugs.

[0104] Radioimmunotherapy (RAIT) has entered the clinic and demonstrated efficacy using various antibody immunoconjugates. 131 I-labeled humanized anti-carcinoembryonic antigen (anti-CEA) antibody hMN-14 (Behr TM et al. (2002) Cancer 94(4 Suppl):1373-81) and has been evaluated in medullary thyroid carcinoma. 90 The same antibody is labeled with Y (Stein R et al. (2002) Cancer 94 (1): 51-61). Radioimmunotherapy using monoclonal antibodies has also been evaluated and reported for Hodgkin's lymphoma and pancreatic cancer (Goldenberg DM (2001) Crit Rev Oncol Hematol 39 (1-2): 195-201; Gold DV et al. (2001) Crit Rev Oncol Hematol 39 (1-2): 147-54). Radioimmunotherapy using specific antibodies is also described in U.S. Patents 6,306,393 and 6,331,175. Radioimmunoguided surgery (RIGS) has also entered the clinic and demonstrated efficacy and usefulness, including the use of anti-CEA antibodies and antibodies directed against tumor-associated antigens (Kim JC et al. (2002) Int J Cancer 97(4):542-7; Schneebaum S et al. (2001) World J Surg 25(12):1495-8; Avital S et al. (2000) Cancer 89(8):1692-8; McIntosh DG et al. (1997) Cancer Biother Radiopharm 12(4):287-94).

[0105] The antibodies of the invention can be administered to a patient in need of treatment by any appropriate route, typically by injection into the bloodstream or CSF or directly into the site of the tumor. The precise dose will depend on many factors, including whether the antibody is for diagnostic or therapeutic use, the size and location of the tumor, the precise nature of the antibody (whether it is a complete antibody, a fragment, or a bispecific antibody, etc.), and the nature of the detectable or functional label attached to the antibody. When a radionuclide is used for treatment, an appropriate maximum single dose is about 45 mCi / m 2Up to a maximum of approximately 250mCi / m 2 . The preferred dosage is in the range of 15 to 40 mCi, with a more preferred dosage range of 20 to 30 mCi or 10 to 30 mCi. Such treatment may require bone marrow or stem cell replacement. Common antibody dosages for tumor imaging or tumor treatment may be in the range of 0.5 to 40 mg, preferably 1 to 4 mg of F(ab')2 form of antibodies. Naked antibodies are preferably administered in a dosage of 20 to 1000 mg protein / dose, or 20 to 500 mg protein / dose, or 20 to 100 mg protein / dose. This is a dosage for single treatment of adult patients, which can be adjusted proportionally for children and infants, and can also be adjusted proportionally for other antibody forms according to molecular weight. Treatment can be repeated under the guidance of a doctor at intervals of once a day, twice a week, once a week or once a month.

[0106] The present invention may be better understood by reference to the following non-limiting examples which are provided as illustrations of the invention. The following examples are provided to illustrate preferred embodiments of the invention in more detail and should not, however, be construed as limiting the broad scope of the invention.

[0107] Example 1

[0108] The therapeutic anti-EGFR antibody 806 generates responses in mice with de novo EGFR mutant-dependent lung cancer.

[0109] Activating epidermal growth factor receptor (EGFR) mutations occur in human non-small cell lung cancer (NSCLC), with 5% of human lung squamous cell carcinomas having EGFRvIII mutations and 10-30% of lung adenocarcinomas having EGFR kinase domain mutations. The monoclonal antibody mAb806 targeting EGFR recognizes conformational epitopes of wild-type (wt) EGFR and truncated EGFRvIII mutants. In order to further explore the anti-cancer spectrum of this antibody for EGFR-targeted cancer therapy, mAb806 was used to treat mice genetically engineered to have lung tumors driven by EGFRvIII or EGFR kinase domain mutations. Our results demonstrate that mAb806 is very effective in blocking EGFRvIII signaling and inducing tumor cell apoptosis, thereby causing significant tumor regression in EGFRvIII-driven mouse lung cancer. Cetuximab, another antibody targeting EGFR, cannot show activity in these genetically determined lung tumors. Furthermore, treatment of mouse lung tumors driven by EGFR kinase domain mutations with mAb806 induced significant tumor regression, albeit to a lesser extent than that observed in EGFRvIII-driven tumors. Overall, these data support the hypothesis that mAb806 may provide significant activity in the treatment of NSCLC patients harboring these two types of EGFR mutations.

[0110] introduction

[0111] Targeted cancer therapies are designed to disrupt the function of specific molecules required for carcinogenesis and tumor growth, thereby killing or preventing the growth of cancer cells (1). In contrast to conventional chemotoxic chemotherapy, such targeted cancer therapies can be more effective and less harmful to normal cells. A major effort in the field of targeted cancer therapy has been to develop agents that target the epidermal growth factor receptor (EGFR). EGFR is a member of the ErbB family of closely related receptors, including EGFR (ErbB-1), Her2 / neu (ErbB-2), Her3 (ErbB-3), and Her4 (ErbB-4). Activation of EGFR leads to activation of receptor tyrosine kinases and a series of downstream signaling events that mediate cell proliferation, motility, adhesion, invasion, resistance to chemotherapy, and inhibition of apoptosis (2-4), processes that are crucial for the continued proliferation and survival of cancer cells.

[0112] To date, two main types of anti-EGFR agents have entered the clinical setting: anti-EGFR antibodies and small molecule EGFR tyrosine kinase inhibitors (TKIs) (5,6). Anti-EGFR antibodies such as cetuximab are designed to bind to the extracellular domain of EGFR and block activation of EGFR downstream signaling (7). In contrast, small molecule TKIs such as gefitinib or erlotinib compete with ATP for binding to the extracellular catalytic domain of EGFR tyrosine kinase, thereby preventing EGFR autophosphorylation and downstream signaling (4).

[0113] Both anti-EGFR drug classes have shown some clinical efficacy in subsets of patients with a variety of different types of cancer. Treatment with gefitinib or erlotinib in patients with lung cancer harboring mutations in the EGFR kinase domain generally produces significant clinical responses (5,8). However, the efficacy of gefitinib or erlotinib in lung adenocarcinoma or other histological subtypes such as squamous cell carcinoma with wild-type EGFR is limited (9,10). In addition, gefitinib or erlotinib has been shown in preclinical and clinical trials to be largely ineffective in inhibiting the function of EGFRvIII mutants (11), a different activating EGFR mutation in which there is an in-frame deletion of exons II to VII. EGFRvIII is commonly found in glioblastomas and has recently been found in a subtype of human squamous cell lung carcinoma (12) and in a large subset of head and neck cancers (13). Cetuximab has been shown to be effective in patients with non-small cell lung cancer (NSCLC) and in a small subset of patients with head and neck cancer, as well as colorectal cancer. However, the response to cetuximab does not appear to be related to the level of EGFR expression. Therefore, it is unclear why these patients responded to cetuximab treatment while other cancer patients whose tumors have high EGFR expression did not respond to cetuximab treatment (14).

[0114] MAb806 is a novel murine antibody that was originally generated to recognize a unique truncated mutant of EGFRvIII (15-17). Importantly, the epitope recognized by mAb806 is inaccessible in inactive wild-type (wt) EGFR but is exposed in a transitional form of wt EGFR in cells overexpressing EGFR and expressing EGFRvIII (18). Epitope studies were supported by immunohistochemistry studies demonstrating that the 806 antibody binds to an epitope present in gliomas and a wide range of epithelial cancers but not in normal human tissues (16,19). These and other preclinical data suggest that mAb806 may have a different spectrum of clinical activity and side effect profile than cetuximab and other anti-EGFR antibodies. In xenograft models, mAb806 has demonstrated potent antitumor activity without targeting normal tissue. Thus, the unique targeting capabilities of mAb806 represent a new paradigm for cancer-specific molecular targeted therapy.

[0115] Recent studies have shown that 10-30% of NSCLC patients harbor EGFR kinase domain mutations, while 5% of patients with squamous cell carcinoma (SCC) of the lung harbor EGFRvIII mutations in the extracellular domain (12,20). To investigate the clinical potential of mAb806 for cancer-specific targeted therapy in patients with NSCLC harboring EGFR mutations, we used two established mouse lung cancer models that rely on either EGFRvIII or EGFR kinase domain mutants. Our data demonstrate that mAb806 is highly effective in the treatment of murine NSCLC driven by expression of either EGFRvIII or EGFR kinase domain mutants and suggest that this antibody may have clinical activity in patients whose tumors harbor similar mutations.

[0116] result

[0117] Treatment with mAb806, but not cetuximab, induced tumor regression in mice bearing lung tumors harboring the EGFRvIII mutation.

[0118] Previous studies have identified an important role for EGFRvIII mutations in the maintenance of mutation-driven murine lung tumors. Blocking EGFRvIII activation resulted in significant tumor regression associated with apoptosis in a neonatal murine lung cancer model (12). Tet-op-EGFRvIII / CCSP-rtTA, Ink4A / Arf- / - mice developed lung adenocarcinomas with features of bronchioloalveolar carcinoma (BAC) after 8 to 10 weeks of doxycycline administration. Figure 1 , left frame; Figure 2 A, upper panel). After tumor-bearing mice were identified by MRI, 0.5 mg / dose of mAb806 was administered by intraperitoneal (IP) injection once daily for the first week and every 2 days thereafter for 4 weeks. Serial MRIs were performed at the end of 1, 3, and 5 weeks of treatment to determine changes in tumor volume and / or density. Significant tumor reduction was observed by MRI after 1 week of mAb806 treatment (mean reduction of 60% ± 5% in 6 mice, Figure 1 , upper panel). Tumor burden continued to decrease after 3 weeks of treatment (95% ± 8% mean reduction), and all 6 mice had complete tumor regression after 5 weeks of treatment. In contrast, treatment of mice with cetuximab failed to induce tumor regression in 4 Tet-op-EGFRvIII / CCSP-rtTA, Ink4A / Arf- / - mice even after 5 weeks of treatment using the same dosing regimen of 1 mg per mouse. We also observed that mice treated with cetuximab became increasingly frail, and some mice even died due to severe tumor burden during treatment (data not shown).

[0119] Pathological examination and MRI of the lungs from these mice revealed a decrease in tumor cellularity in adenocarcinomas after 1 week of treatment with mAb806 ( Figure 2 A, middle box). After 5 weeks, the lungs had focal fibrosis and scarring with sporadic mononuclear infiltrates; this likely represents areas of continued remodeling from regressed tumors ( Figure 2 A, lower frame). Although viable cancer cells can still be rarely observed in a few lesions of these fibrotic nodules, most of the fibrotic and scarred areas do not contain any tumor cells. In contrast, tumors from mice treated with cetuximab appeared unaffected, with no visible histological differences when compared to untreated tumors (data not shown). Thus, treatment with the mAb806 antibody resulted in rapid and significant tumor regression in the EGFRvIII-driven mouse lung cancer model, whereas cetuximab treatment was essentially ineffective.

[0120] MAb806 inhibits EGFRvIII phosphorylation and induces apoptosis of tumor cells in Tet-op-EGFRvIII / CCSP-rtTA, Ink4A / Arf- / - mice.

[0121] To determine whether intraperitoneally administered mAb806 recognizes its target in lung tumors, we performed immunohistochemical staining using antibodies against total EGFR and phospho-EGFR in lung tumors of mice treated with or without mAb806. As expected, mAb806 treatment had no effect on the expression of total EGFRvIII in tumor cells ( Figure 2 B). However, after 1 week of mAb806 treatment, the expression of phospho-EGFRvIII was reduced ( Figure 2 B). We then validated these findings by immunoblotting analysis using lung lysates collected at different time points during treatment with mAb806. After 1 week of mAb806 treatment, phospho-EGFRvIII levels were significantly reduced, whereas total EGFRvIII levels remained similar to those of untreated controls ( Figure 3 ), which shows that mAb806 has a strong inhibitory effect on EGFRvIII phosphorylation. Interestingly, total EGFRvIII levels are finally reduced after mAb806 is used for 5 weeks. One explanation for this phenomenon can be the significant reduction in the number of viable tumor cells. Consistent with this explanation, after mAb806 treatment for 1 week, TUNEL staining ( Figure 2C). In addition to changes in phospho-EGFR levels, one week of mAb806 treatment also reduced the expression of phospho-Akt and phospho-Erk1,2, downstream signaling molecules of EGFR that are functionally linked to anti-apoptotic and proliferative pathways. Surprisingly, a slight but reproducible increase in phospho-Akt levels was observed after 5 weeks of mAb806 treatment compared to the one-week treatment. This phosphorylation of Akt is unlikely to be initiated by EGFRvIII, as phospho-EGFRvIII was very low at this time point. It is likely that Akt can be activated by other signaling events involved in the lung remodeling process. These data suggest that mAb806 induces tumor regression in EGFRvIII mice by preventing EGFR activation and increasing apoptosis in tumor cells.

[0122] Ch806 treatment resulted in significant tumor regression in mouse lung tumors harboring the EGFRvIII mutation.

[0123] Ch806 is a humanized form of mAb806 (22). To determine whether the humanized antibody is as effective as murine mAb806 in the in vivo treatment of lung adenocarcinoma, we treated tumor-bearing Tet-op-EGFRvIII / CCSP-rtTA, Ink4A / Arf- / - mice with a dose of 0.5 mg of ch806 injected IP once daily for the first week and then once every two days for another 7 weeks. These mice underwent re-imaging at 1.5, 5, and 8 weeks of treatment and were then killed for histological analysis. We observed a significant decrease in tumor volume (43% ± 3%) by MRI scans starting at 1.5 weeks of treatment, with almost complete tumor regression (83% ± 7%) obtained at 8 weeks of treatment in each of the 4 mice treated with ch806 ( Figure 1 , lower panel). The histology of mice treated with ch806 (data not shown) was similar to the histology of tumors after mAb806 treatment and consistent with the MRI data.

[0124] Ch806 is effective in the treatment of murine lung tumors harboring the EGFR L858R mutation

[0125] To elucidate whether ch806 is effective against lung cancer driven by EGFR kinase domain mutations, EGFR L858R-IRES-luciferase / CCSP-rtTA mice were used. Ch806 was administered at 0.5 mg / mouse once daily for 4 weeks, and serial MRI scans were performed on all treated mice at the end of 1, 2, and 4 weeks of treatment. Tumor regression was observed after 2 weeks of ch806 treatment (21% ± 2%), and at 4 weeks of ch806 treatment, the tumor regression was 41% ± 2% ( Figure 4 A). Microscopic observation of the lungs of ch806-treated mice showed an increased diffuse cellular infiltration of macrophages, particularly in the areas surrounding the remaining viable tumor. Furthermore, macrophages were present in multiple areas of the tumor, suggesting that macrophage-mediated cytotoxicity may be responsible for the antibody-induced tumor regression ( Figure 4 B) It should also be noted that the presence of consolidation in the lungs due to the increased accumulation of macrophages associated with tumor cells can overestimate tumor volume measured by MR imaging.

[0126] discuss

[0127] EGFR mutations and activation events are common in human malignancies, including NSCLC. Activation of EGFR signaling can occur through receptor overexpression as well as through constitutive signaling due to gain-of-function mutant forms of EGFR. Approximately 10-30% of patients with NSCLC harbor EGFR kinase domain mutations in their lung tumors, and approximately 5% of patients with squamous cell lung cancer harbor specific EGFRvIII extracellular domain mutations (12, 20). Here, we show that mAb806 and its humanized form, ch806, are effective in treating murine lung cancers harboring both types of EGFR mutations. The significant tumor regression observed was associated with blockade of EGFRvIII signaling and subsequent increased apoptosis. In mice with lung tumors harboring EGFR kinase domain mutations, responses to ch806 were less pronounced than those reported for erlotinib and cetuximab, although they did have an objective response (41% ± 2%) radiographically and histologically (21, 23). On the contrary, after treatment with mAb806, in mice with lung tumors driven by EGFRvIII, almost complete tumor regression was obtained, but cetuximab was invalid. This latter result may not be surprising, because cetuximab is designed to interfere with the interaction between the ligand and the EGFR extracellular domain (24). It has been determined that the EGFRvIII mutation causes conformational changes and displays a constitutive kinase activity that is not dependent on ligand stimulation, which contributes to tumor formation (25). Although cetuximab has been approved by the FDA for cancer patients, there is no clear biomarker in order to predict the effect of the treatment of this antibody in individual patients, because response rate and total survival are not relevant to the expression of the EGFR protein measured by immunohistochemistry (14).

[0128] Although small molecule TKIs are effective in the treatment of many NSCLC patients with EGFR kinase domain mutations, all patients eventually develop resistance associated with the secondary mutation T790M (10, 26). Consistently, in vitro studies have shown that tumor cells with the T790M mutation are resistant to treatment with erlotinib (27, 28). Evidence from the crystal structure of the EGFR kinase domain with a secondary T790M mutation suggests that the effect of the T790M mutation on receptor function should be minimal. The T790M mutation may interfere with the binding of erlotinib to the ATPase pocket (27). However, the extracellular domain of the T790M mutant potentially provides a good target for antibody-based cancer therapies, including cetuximab and mAb806. This may mean that NSCLC tumors with secondary T790M point mutations that are resistant to small TKI treatment may respond to mAb806 treatment. To test this hypothesis, efforts are underway to generate mice with compound mutant EGFR alleles containing an activating kinase domain mutation and the T790M mutation.

[0129] The data released from Phase I clinical trials recently have shown that ch806 antibody, unlike cetuximab, selectively binds to the tumor cells of lung cancer including squamous cell lung cancer, but not to normal tissue (Scott, ASCO 2006). In this test, no significant toxicity of ch806 antibody was observed. Compared with other EGFR targeted cancer therapies including cetuximab and TKI treatment, ch806 seems to have much greater specificity by targeting the conformation-dependent epitope of the EGFR on the cancer cell but rarely targeting the wt EGFR on most (if not all) normal cells. Our results clearly show that mAb 806 has an effect on blocking EGFR signal transduction. Therefore, the unique targeting ability of ch806 represents a novel and exciting paradigm for cancer-specific molecular targeted therapy, which can benefit patients whose cancer depends on the uncontrolled EGFR signal transduction caused by overexpression or gain-of-function mutations (including EGFRvIII or EGFR kinase domain mutations).

[0130] method

[0131] Mouse cohorts

[0132] The generation of Tet-op-EGFRvIII / CCSP-rtTA, Ink4A / Arf- / - mice and Tet-op-EGFRL858R-IRES-luciferase / CCSP-rtTA mice has been described before (12,21). All mice were housed in a pathogen-free environment at Harvard School of Public Health and all mouse experiments performed were approved by the Institutional Animal Care and Use Committee (IACUC). Littermate mice were used as controls in all experiments. In order to induce EGFRvIII and EGFR L858R expression, mice were raised with a doxycycline diet (Research Diets, Inc.). The doxycycline withdrawal experiment in the previous study clearly identified that lung tumors from both alleles were completely dependent on doxycycline.

[0133] In vivo targeted therapy using mAb806 or ch806 or cetuximab

[0134] The mice that will continue to carry out doxycycline diet for more than 8 weeks experience MRI to record lung tumor burden.By IP injection, MAb806 or ch806 (produced by Ludwig Institute for Cancer Research, Melbourne, Australia) are delivered into mice with lung tumors at 0.5mg / agent once a day.After 1 week of treatment, antibody is administered once every two days with the same dosage, and the number of weeks of other appointment is carried out.Use the same dosage regimen to administer cetuximab (commercially available from BMS pharmaceuticals) to mice by IP injection with 1mg / agent.Use MRI to image mice at the specified time point to determine the reduction of tumor volume, then kill mice to carry out further histological and biochemical research after completing treatment.All mice are kept on doxycycline diet during the whole experiment.Mice born in the same litter are used as the control of all drug treatment studies.

[0135] Pathological evaluation of lung tumors

[0136] Mice were euthanized at the indicated times, their left lungs dissected, and then snap-frozen for biochemical analysis. Their right lungs were then inflated with neutrally buffered 10% formalin under pressure (25 cm) for 10 minutes and then fixed overnight. Hematoxylin and eosin (H&E) staining was performed on 5 μm thick sections from formalin-fixed, paraffin-embedded tumor samples at the Department of Pathology at Brigham and Women's Hospital.

[0137] Immunohistochemical analysis was performed on formalin-fixed paraffin sections. Sections were deparaffinized in xylene and serially rehydrated in ethanol. For antibodies requiring antigen retrieval, antigen-unmasking solution (Vector Laboratories) was used according to the manufacturer's instructions. Slides were quenched in hydrogen peroxide (0.3%-3%) to block endogenous peroxidase activity and then washed in automated buffer (Fisher Scientific). Slides were blocked in 5% normal serum for 1 hour at room temperature and then incubated overnight at 4°C with primary antibodies diluted in blocking buffer. Slides were counterstained with hematoxylin using the avidin-biotin peroxidase complex method (Vector). Slides were serially dehydrated in ethanol, washed in xylene, and then mounted with Permount (Fisher). Biotinylated DBA lectin (Vector) was used at 1:100. Antibodies used were total EGFR and phospho-EGFR Y1068 (1:50, Cell Signaling Technology). Apoptosis was measured using the TUNEL assay (ApopTag kit; Intergen, Inc.) by counting positive cells.

[0138] Western blot analysis.

[0139] Snap-frozen lung tissue samples were homogenized in RIPA buffer (Boston Bioproducts) containing Complete Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktail Set I and II (EMDBiosciences). Lung lysates were clarified by centrifugation and then boiled for 5 minutes in 1x final sodium dodecyl sulfate (SDS) sample buffer (50 mM Tris (pH 6.8), 10% glycerol, 0.715 M β-mercaptoethanol, 2% SDS, and 0.01% bromophenol blue). Lysates were then separated by SDS polyacrylamide gel electrophoresis (PAGE), transferred to nitrocellulose membranes, and detected by immunoblotting with antibodies using SuperSignal West PicoChemiluminescent Substrate (Pierce Biotechnology). The antibodies used in this study were directed against total EGFR, phospho-EGFR (pY1068), total Akt, phospho-AKT (pS473), total Erk1 / 2, and phospho-ERK 1 / 2 (pT202 / pY204) (all from Cell Signaling); and β-actin (Santa Cruz Biotechnology, Inc.). Antibodies were used according to the manufacturer's recommended conditions.

[0140] MRI and tumor volume measurement

[0141] 1.5-2% isoflurane in 100% oxygen was used via a nose cone. Animals were anesthetized at Abbot Laboratories. To eliminate motion artifacts, cardiac and respiratory gating was used for all MRI studies. Because MR signal acquisition is synchronized with the cardiac and respiratory cycles, MR signals are acquired during each cardiac phase and the end-expiratory phase, significantly minimizing motion artifacts.

[0142] An MRI protocol optimized for the assessment of lung parenchyma and vasculature in normal mice (29) was adapted for operation at 4.7 dsRa (Biospec 47 / 40, Bruker BioSpin, Karlsruhe, Germany). The system was equipped with a shielded gradient system with a maximum power gradient of 30 G / cm and a cardiac-respiratory triggering system (BioTrig, Bruker BioSpin, Karlsruhe, Germany). The animals were then placed in a prone position, and electrodes for cardiac gating (front and left posterior pads) and a respiratory sensor were placed on their bodies, with the head first entering the system, with the center of the thorax aligned with the center of the radiofrequency cage coil (inner diameter 3 cm). For reproducible localization of the imaging region, low-resolution multislice images of the entire lung in the transverse and coronal planes (used as a terminal expiratory phase localizer) were first acquired using a fast spin echo sequence (RARE: Rapid Acquisition with Relaxation Enhancement, TR / Effective TE = 1000 / 28 milliseconds, bandwidth = 50 kHz, field of view = 30 mm, matrix = 128×128, slice thickness = 1 mm, number of excitations = 1). In addition, two-dimensional (2D) multislice gradient echo imaging was performed in multislice transverse and coronal planes encompassing the entire lung using cardiorespiratory gating. A pulse repetition time (TR) shorter than the duration of one cardiac cycle (range 150 to 200 milliseconds, average 178 milliseconds) was selected, where one k-space line was filled for each image for each single pulse. A minimum echo time (TE: 1.8 milliseconds) was used to reduce susceptibility effects that can reduce the MR signal due to interference between air / bone and tissue. Other scanning parameters are: flip angle = 22°, matrix size = 256x256, field of view (FOV) = 2.56 cm 2 , slice thickness = 1mm and number of excitations (NEX) = 4, providing 100μm 2 The total scan time in each plane was approximately 6-7 minutes, depending on the heart / respiratory rate of the individual animal. On each MR image, the area indicating the lung tumor was manually segmented and then measured using ImageJ (ver. 1.33, National Institute of Health) to calculate tumor volume.

[0143] References

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[0149] 6.Herbst,R.S.2002.Targeted therapy in non-small-cell lungcancer.Oncology (Williston Park).16:19-24.

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[0156] 13.Sok,J.C.,et al.2006.Mutant epidermal growth factor receptor(EGFRvIII)contributes to head and neck cancer growth and resistance to EGFRtargeting.Clin.Cancer.Res.12:5064-5073.

[0157] 14.Italiano,A.2006.Targeting the epidermal growth factor receptor incolorectal cancer:advances and controversies.Oncology.70:161-167.

[0158] 15.Jungbluth,A.A.,et al.2003.A monoclonal antibody recognizing humancancers with amplification / overexpression of the human epidermal growthfactor receptor.Proc.Natl.Acad.Sci.U S A.100:639-644.

[0159] 16.Luwor,R.B.,et al.2001.Monoclonal antibody 806 inhibits the growthof tumor xenografts expressing either the de2-7 or amplified epidermalgrowth.factor receptor(EGFR)but not wild-type EGFR.Cancer Res.61:5355-5361.

[0160] 17.Mishima,K.,et al.2001.Growth suppression of intracranialxenografted glioblastomas overexpressing mutant epidermal growth factorreceptors by systemic administration of monoclonal antibody(mAb)806,a novelmonoclonal antibody directed to the receptor.Cancer Res.61:5349-5354.

[0161] 18.Johns,T.G.,et al.2004.Identification of the epitope for theepidermal growth factor receptor-specific monoclonal antibody 806 revealsthat it preferentially recognizes an untethered form of thereceptor.J.Biol.Chem.279:30375-30384.

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[0165] 22.Panousis,C.,et al.2005.Engineering and characterisation ofchimeric monoclonal antibody 806(ch806)for targeted immunotherapy of tumoursexpressing de2-7EGFR or amplified EGFR.Br.J.Cancer.92:1069-1077.

[0166] 23.Politi,K.,et al.2006.Lung adenocarcinomas induced in mice bymutant EGF receptors found in human lung cancers respond to a tyrosine kinaseinhibitor or to down-regulation of the receptors.Genes.Dev.20:1496-1510.

[0167] 24.Li,S.,etal.2005.Structural basis for inhibition of the epidermalgrowth factor receptor by cetuximab.Cancer Cell.7:301-311.

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[0173] Example 2

[0174] Antibody 806 causes tumor regression in lung tumors harboring the EGFR T790M mutation

[0175] Mice expressing the human EGFR secondary mutation T790M were generated. mAb806 was delivered to mice bearing lung tumors via IP injection at 0.5 mg / dose once daily for 4 weeks. Serial MRI scans were performed on the treated mice at the end of 2 and 4 weeks of treatment as described below.

[0176] Generation of Tet-op-hEGFR T790M-L858R / CCSP-rtTA mouse population

[0177] To generate mice with inducible expression of the human EGFR T790M-L858R mutant, we constructed a 4.7-kb DNA segment consisting of seven direct repeats of the tetracycline (tet)-operon, followed by EGFR T790M-L858R cDNA and β-globin poly A. The construct was injected into FVB / N blastocysts, and offspring were screened using a PCR strategy. Fifteen Tet-op-hEGFR T790M-L858R founders were identified and then crossed with CCSP-rtTA mice (which show allele-specific targeting of reverse tetracycline trans-activator protein (rtTA) expression in type II alveolar epithelial cells (Fisher GH et al. (2001) Genes Dev 15(24):3249-62) to generate an inducible double transgenic mouse population with both activator and responder transgenes (Fisher GH et al. (2001) Genes Dev 15(24):3249-62; Perl AK, Tichelaar JW, and Whitsett JA. (2002) Transgenic Res 11(1):21-9). Four tightly regulated hEGFR T790M-L858R founders were identified by RT-PCR analysis. T790M-L858R (#17, #19, #24 and #29) founders, copy numbers from individual founders were determined by quantitative real-time PCR (JiH et al. (2006) Cancer Cell 9(6):485-95).

[0178] Tightly regulated expression of EGFR T790M-L858R at the RNA level in lung tissue

[0179] The inducibility of EGFR mutant transgene expression in the lung compartment was assessed at the RNA level by RT-PCR using human EGFR-specific primers. Lungs from the Tet-op-hEGFR T790M-L858R / CCSP-rtTA cohort of double transgenic mice from each potential founder were collected before and after 8 weeks of doxycycline administration and 3 days after doxycycline withdrawal following 8 weeks of doxycycline administration. EGFR mutant transcripts were not detected in non-transgenic mice or double transgenic mice not treated with doxycycline, but became readily detectable after 8 weeks of doxycycline administration; 3 days of doxycycline withdrawal completely abolished mutant EGFR transcription in all strains. To further confirm that mutant EGFR transcripts are inducible and tightly regulated by doxycycline, RT-PCR and quantitative real-time PCR using the same primers described above were performed on lung samples from founder #19 collected at serial time points of doxycycline administration and withdrawal. EGFR expression was observed after 1 week of doxycycline administration and remained at comparable levels throughout the 8-week administration period; doxycycline withdrawal was sufficient to prevent mutant EGFR expression, and no transgene expression was observed after 12 weeks of doxycycline withdrawal.

[0180] Overexpression of the EGFR T790M-L858R mutant drives the development of lung adenocarcinomas with Bronchioloalveolar features and papillary adenocarcinoma in the airways.

[0181] To determine whether overexpression of hEGFR mutants drives lung tumorigenesis, double transgenic hEGFR T790M-L858R / CCSP-rtTA mice that were continuously administered with doxycycline were subjected to a series of magnetic resonance imaging (MRI) and then killed at different time points for histological examination of the lungs. Tumors could only be observed by MRI 5 to 6 weeks after the administration of doxycycline, and the tumor volume determined by MRI increased after prolonged doxycycline treatment. In contrast to untreated mice, early lesions began to occur in the parenchyma of the lungs after 2 to 3 weeks of doxycycline treatment. After 4 to 5 weeks, typical BACs appeared.

[0182] After 7 to 9 weeks, invasive adenocarcinomas with bronchioloalveolar features appeared and became the predominant histological pattern after 12 weeks of doxycycline treatment. The lung parenchymal adenocarcinomas observed in our mouse model were histologically similar to those in the previously described EGFR L858R mouse model (Ji, H., et al. (2006) Cancer Cell 9:485-495; Politi, K., et al. (2006) Genes Dev. 20:1496-1510) and were also similar to those seen in a subset of NSCLC patients who initially responded to erlotinib.

[0183] In addition to parenchymal adenocarcinoma, hEGFR T790M-L858R / CCSP-rtTA mice also develop bronchial papillary adenocarcinoma. Early papillary neoplasia was observed in the bronchioles after 2 to 3 weeks of continuous doxycycline administration, which then developed into adenocarcinoma within another 6 to 8 weeks. All 4 founders showed similar morphological features and similar tumorigenesis latency. Bronchial tumors were found in all 4 founders of hEGFR T790M-L858R / CCSP-rtTA mice identified in this study, but they were not present in all our EGFR L858R mice. Occasionally, metastases of adenocarcinoma were observed in the lymph nodes of mice with EGFR T790M-L858R-driven lung tumors but not in mice with EGFR L858R-driven tumors. IHC staining of bronchial and parenchymal tumors using specific cell markers showed different differentiation patterns. Prosurfactant protein C (SPC) is a unique biomarker for type II pneumocytes in the alveoli, while Clara cell secretory protein (CCSP) is specific for Clara cells in the bronchiolar epithelium. Most parenchymal tumors showed strong SPC staining, suggesting a type II pneumocyte origin, as expected. In contrast, bronchial tumors were negative for SPC. Interestingly, only a small subpopulation of bronchial tumor cells was positive for CCSP. This may be explained by a Clara cell origin followed by poor differentiation that results in loss of CCSP expression markers.

[0184] Expression of the hEGFR T790M-L858R mutant is important for tumor maintenance in parenchymal and bronchial adenocarcinomas.

[0185] Bronchial and parenchymal lung adenocarcinomas from hEGFR T790M-L858R / CCSP-rtTA mice stained positively with antibodies against total EGFR and phospho-EGFR, indicating that the expressed EGFR mutant was functionally active. After 3 days of doxycycline withdrawal, no positive signals from either antibody were observed, indicating that both tumor types were driven by EGFR T790M-L858R and that their survival was dependent on EGFR T790M-L858R. Following doxycycline withdrawal, we also observed increased positive staining using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, indicating that apoptosis was triggered.

[0186] Consistent with the apoptosis demonstrated by TUNEL staining, MRI results demonstrated complete regression of EGFR T790M-L858R-driven lung tumors after 10 days of doxycycline withdrawal. Microscopic analysis of lungs from the same mice examined by MRI revealed grossly normal lung histology. No tumor lesions were found in the airways or parenchyma of other tumor-bearing mice after 12 weeks of doxycycline withdrawal.

[0187] To better quantify mutant EGFR expression in tumors at the protein level, we performed western blotting using whole lung lysates from double-transgenic mice at different times of doxycycline administration. Although individual variability existed, EGFR phosphorylation was tightly regulated by doxycycline and synchronized with tumor development, confirming the important role of mutant EGFR signaling in tumor maintenance, as observed by IHC staining and MRI. Therefore, EGFR remains an attractive therapeutic target in our novel mouse lung cancer model.

[0188] Treatment of EGFR T790M-L858R-driven lung tumors with mAb806

[0189] The results of treatment of EGFR T790M-L858R lung tumors with mAb806 versus cetuximab are described in Figure 5 In the experiment, mice that were on a doxycycline diet for more than 8 weeks were subjected to MRI to record tumor burden. mAab806 was delivered into mice with lung tumors at a dose of 0.5 mg once a day by IP injection for 4 weeks. Cetuximab was administered to mice at 1 mg / dose by IP injection once a day for 4 weeks. MRI was used to image mice at 0, 2, and 4 or 5 weeks to determine the reduction in tumor volume. By using the treatment of mAb806, tumor volume decreased (more than 20%) at week 2 and decreased more significantly (more than 30%) at week 4. Although tumor volume began to decrease when treated with cetuximab for 2 weeks, tumor volume increased significantly after 5 weeks of treatment with cetuximab (the tumor volume observed during 5 weeks of cetuximab treatment was larger than the initial volume at week 0). After treatment and MRI imaging were completed, mice were killed for further histological and biochemical studies. For all treatment studies, littermate mice were used as controls (untreated).

[0190] The present invention may be embodied in other forms or carried out in other ways without departing from its spirit and essential characteristics. Therefore, the present disclosure is considered in all aspects to be illustrative and not restrictive, the scope of the present invention is indicated by the appended claims, and all changes that appear within the meaning and scope of equivalence are intended to be included therein.

[0191] Throughout this specification, various references are cited, each of which is herein incorporated by reference in its entirety.

[0192] Preferred embodiment:

[0193] 1. A method for treating a tyrosine kinase inhibitor-resistant EGFR-mediated disease in a mammal, wherein the resistant EGFR-mediated disease is the result of a secondary mutation in EGFR resulting in a mutant EGFR, and wherein the mutation is different from an EGFR vIII mutation, the method comprising administering to the mammal an effective amount of an anti-EGFR antibody capable of binding to and inhibiting the mutant EGFR.

[0194] 2. The method of item 1, wherein the secondary EGFR mutation is an EGFR tyrosine kinase domain mutation.

[0195] 3. The method of claim 2, wherein the tyrosine kinase domain mutation is T790M.

[0196] 4. The method of item 1, wherein the anti-EGFR antibody is mAb806 antibody or an active fragment thereof.

[0197] 5. The method of claim 4, wherein mAb806 is a recombinant antibody or a humanized antibody.

[0198] 6. The method of item 1, wherein the anti-EGFR antibody is selected from ABX-EGF (panitumumab), DH8.3, L8A4 and / or active fragments thereof.

[0199] 7. The method of claim 1, wherein the EGFR-mediated disease is cancer and is selected from glioblastoma, head and neck cancer, pancreatic cancer, lung cancer, cancer of the nervous system, gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, kidney cancer, retinal cancer, skin cancer, liver cancer, reproductive-urinary cancer, and bladder cancer.

[0200] 8. The method of claim 7, wherein the cancer is lung adenocarcinoma, lung squamous cell carcinoma, or non-small cell lung cancer.

[0201] 9. A method for reducing EGFR-mediated tumor growth in a cancer patient, wherein the cancer patient has previously been treated with one or more tyrosine kinase inhibitors and has developed recurrent disease and tumor growth, the method comprising administering to the patient an effective amount of an anti-EGFR antibody such that recurrent disease and tumor growth are inhibited and reduced.

[0202] 10. The method of claim 9, wherein the anti-EGFR antibody is mAb806 antibody or an active fragment thereof.

[0203] 11. The method of claim 10, wherein mAb806 is a recombinant antibody or a humanized antibody.

[0204] 12. The method of claim 9, wherein the anti-EGFR antibody is selected from ABX-EGF (panitumumab), DH8.3, L8A4 and / or active fragments thereof.

[0205] 13. The method of claim 9, wherein the cancer patient has developed a secondary EGFR mutation that is a mutation in the EGFR tyrosine kinase domain.

[0206] 14. The method of claim 13, wherein the tyrosine kinase domain mutation is T790M.

[0207] 15. A method for treating EGFR-mediated cancer in a mammal, the method comprising administering to the mammal a tyrosine kinase inhibitor and an anti-EGFR antibody, wherein the anti-EGFR antibody is administered after treatment with the tyrosine kinase inhibitor as a second-line therapy to inhibit potential secondary mutant EGFR that is resistant to the tyrosine kinase inhibitor.

[0208] 16. The method of claim 15, wherein the EGFR-mediated cancer is selected from glioblastoma, head and neck cancer, pancreatic cancer, lung cancer, cancer of the nervous system, gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, kidney cancer, retinal cancer, skin cancer, liver cancer, genitourinary cancer, and bladder cancer.

[0209] 17. The method of claim 16, wherein the cancer is lung adenocarcinoma, lung squamous cell carcinoma, or non-small cell lung cancer.

[0210] 18. The method of claim 15, wherein the tyrosine kinase inhibitor is a reversible tyrosine kinase inhibitor.

[0211] 19. The method of claim 18, wherein the reversible tyrosine kinase inhibitor is an aniline quinazoline compound and is selected from the group consisting of gefitinib, erlotinib, AG1478, ST1571 and SU-6668.

[0212] 20. The method of claim 15, wherein the tyrosine kinase inhibitor is an irreversible tyrosine kinase inhibitor.

[0213] 21. The method of claim 20, wherein the irreversible tyrosine kinase inhibitor is selected from the group consisting of EKB-569, EKI-569, HKI-272, HKI-357, and BIBW 2992.

Claims

1. Use of an effective amount of an anti-EGFR antibody capable of binding to and inhibiting mutant EGFR in the preparation of a medicament for treating tyrosine kinase inhibitor-resistant EGFR-mediated lung adenocarcinoma in a mammal, wherein the resistant EGFR-mediated lung adenocarcinoma is characterized by secondary T790M and L858R mutations in EGFR, and The anti-EGFR antibody is mAb806 antibody.

2. The use of claim 1, wherein mAb806 is a recombinant antibody or a humanized antibody.

3. Use of an effective amount of an anti-EGFR antibody capable of binding to and inhibiting mutant EGFR in the preparation of a medicament for reducing tyrosine kinase inhibitor-resistant EGFR-mediated tumor growth in patients with lung adenocarcinoma, wherein the lung adenocarcinoma patient has been previously treated with one or more tyrosine kinase inhibitors and has experienced recurrent disease and tumor growth, wherein the lung adenocarcinoma patient has secondary T790M and L858R mutations in EGFR, and The anti-EGFR antibody is mAb806 antibody.

Images