US20100056762A1 - Specific binding proteins and uses thereof - Google Patents

Specific binding proteins and uses thereof Download PDF

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Publication number
US20100056762A1
US20100056762A1 US12/388,504 US38850409A US2010056762A1 US 20100056762 A1 US20100056762 A1 US 20100056762A1 US 38850409 A US38850409 A US 38850409A US 2010056762 A1 US2010056762 A1 US 2010056762A1
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United States
Prior art keywords
amino acid
egfr
antibody
canceled
acid sequence
Prior art date
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Abandoned
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US12/388,504
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English (en)
Inventor
Lloyd J. Old
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ludwig Institute for Cancer Research Ltd
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Ludwig Institute for Cancer Research Ltd
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Publication date
Family has litigation
First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=42077108&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=US20100056762(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Priority claimed from US10/145,598 external-priority patent/US7589180B2/en
Application filed by Ludwig Institute for Cancer Research Ltd filed Critical Ludwig Institute for Cancer Research Ltd
Priority to US12/388,504 priority Critical patent/US20100056762A1/en
Assigned to LUDWIG INSTITUTE FOR CANCER RESEARCH reassignment LUDWIG INSTITUTE FOR CANCER RESEARCH ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: RENNER, CHRISTOPH, PANOUSIS, CON, SCOTT, ANDREW MARK, COLLINS, PETER, JOHNS, TERRANCE GRANT, JUNGBLUTH, ACHIM, STOCKERT, ELISABETH, HUANG, HUEI-JEN SU, BURGESS, ANTONY WILKS, CAVENEE, WEBSTER K., OLD, LLOYD J., RITTER, GERD, NICE, EDOUARD COLLINS
Priority to RU2011138154/10A priority patent/RU2549678C2/ru
Priority to ES10704485.1T priority patent/ES2540802T3/es
Priority to MX2011008767A priority patent/MX2011008767A/es
Priority to BRPI1012340-7A priority patent/BRPI1012340B1/pt
Priority to SG10201801945TA priority patent/SG10201801945TA/en
Priority to CN201510419051.2A priority patent/CN105399829A/zh
Priority to PL15160629T priority patent/PL2913344T3/pl
Priority to DK10704485T priority patent/DK2398828T3/en
Priority to PE2015002701A priority patent/PE20160535A1/es
Priority to NZ716762A priority patent/NZ716762A/en
Priority to CN201510004733.7A priority patent/CN104650232B/zh
Priority to SG2011058476A priority patent/SG173688A1/en
Priority to KR1020117021501A priority patent/KR101579769B1/ko
Priority to US13/201,061 priority patent/US9072798B2/en
Priority to HUE15160629A priority patent/HUE037061T2/hu
Priority to CN201080017279.4A priority patent/CN102405235B/zh
Priority to EP17184273.5A priority patent/EP3301117A3/de
Priority to MYPI2016001104A priority patent/MY185858A/en
Priority to PCT/US2010/024407 priority patent/WO2010096434A2/en
Priority to HUE10704485A priority patent/HUE025435T2/en
Priority to KR1020227004795A priority patent/KR20220025913A/ko
Priority to PL10704485T priority patent/PL2398828T3/pl
Priority to KR1020187032867A priority patent/KR20180125044A/ko
Priority to EP15160629.0A priority patent/EP2913344B1/de
Priority to KR1020177018394A priority patent/KR101921046B1/ko
Priority to SI201031541T priority patent/SI2913344T1/sl
Priority to KR1020217006120A priority patent/KR20210028271A/ko
Priority to PT151606290T priority patent/PT2913344T/pt
Priority to SG10201407281UA priority patent/SG10201407281UA/en
Priority to ES15160629.0T priority patent/ES2645663T3/es
Priority to PE2020000069A priority patent/PE20200609A1/es
Priority to EP10704485.1A priority patent/EP2398828B1/de
Priority to CA2752584A priority patent/CA2752584C/en
Priority to PT107044851T priority patent/PT2398828E/pt
Priority to LTEP15160629.0T priority patent/LT2913344T/lt
Priority to NZ627103A priority patent/NZ627103A/en
Priority to NO15160629A priority patent/NO2913344T3/no
Priority to PE2011001515A priority patent/PE20120569A1/es
Priority to UAA201407733A priority patent/UA117807C2/uk
Priority to NZ595224A priority patent/NZ595224A/xx
Priority to MYPI2011003853A priority patent/MY165583A/en
Priority to NZ608773A priority patent/NZ608773A/en
Priority to KR1020157020913A priority patent/KR101809441B1/ko
Priority to DK15160629.0T priority patent/DK2913344T3/da
Priority to RU2014120536A priority patent/RU2673724C2/ru
Priority to UAA201111099A priority patent/UA106607C2/uk
Priority to SI201030953T priority patent/SI2398828T1/sl
Priority to AU2010216168A priority patent/AU2010216168B2/en
Priority to MX2014000475A priority patent/MX361876B/es
Priority to JP2011551178A priority patent/JP5859314B2/ja
Priority to KR1020197018017A priority patent/KR20190076069A/ko
Publication of US20100056762A1 publication Critical patent/US20100056762A1/en
Priority to US13/078,764 priority patent/US20110313230A1/en
Priority to IL214643A priority patent/IL214643A/en
Priority to GT201100223A priority patent/GT201100223A/es
Priority to CL2011002003A priority patent/CL2011002003A1/es
Priority to MX2018015915A priority patent/MX2018015915A/es
Priority to CO11116706A priority patent/CO6420350A2/es
Priority to EC2011011335A priority patent/ECSP11011335A/es
Priority to US14/226,430 priority patent/US20140322130A1/en
Priority to JP2014206544A priority patent/JP6199269B2/ja
Priority to PH12014502419A priority patent/PH12014502419A1/en
Priority to CY20151100459T priority patent/CY1124849T1/el
Priority to US14/737,381 priority patent/US20160046726A1/en
Priority to HRP20150656TT priority patent/HRP20150656T1/hr
Priority to HK15107189.9A priority patent/HK1206752A1/xx
Priority to CL2015003090A priority patent/CL2015003090A1/es
Priority to AU2015255246A priority patent/AU2015255246B2/en
Priority to HK16102177.3A priority patent/HK1214278A1/zh
Priority to JP2016088333A priority patent/JP6317780B2/ja
Priority to HK16108847.0A priority patent/HK1220706A1/zh
Priority to US15/661,864 priority patent/US20180057606A1/en
Priority to CY20171101103T priority patent/CY1119591T1/el
Priority to HRP20171636TT priority patent/HRP20171636T1/hr
Priority to AU2017276184A priority patent/AU2017276184A1/en
Priority to JP2018063673A priority patent/JP2018148887A/ja
Priority to RU2018130075A priority patent/RU2018130075A/ru
Priority to DO2020000032A priority patent/DOP2020000032A/es
Priority to US16/818,062 priority patent/US20210032366A1/en
Priority to JP2020076499A priority patent/JP2020195368A/ja
Priority to AU2021201724A priority patent/AU2021201724A1/en
Priority to JP2022092047A priority patent/JP2022137018A/ja
Priority to US18/307,566 priority patent/US20240117073A1/en
Abandoned legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
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    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
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    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
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    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
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    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to specific binding members, particularly antibodies and fragments thereof, which bind to amplified epidermal growth factor receptor (EGFR) and to the in-frame deletion of exons 2 to 7 of EGFR, resulting in a truncated EGFR receptor missing 267 amino acids from the extracellular domain (de2-7 EGFR).
  • EGFR epidermal growth factor receptor
  • the epitope recognized by the specific binding members, particularly antibodies and fragments thereof is enhanced or evident upon aberrant post-translational modification.
  • the binding members of the present invention may also be used in therapy in combination with chemotherapeutics or anti-cancer agents and/or with other antibodies or fragments thereof.
  • chemotherapeutic means often relies upon exploiting differences in target proliferating cells and other normal cells in the human or animal body.
  • many chemical agents are designed to be taken up by rapidly replicating DNA so that the process of DNA replication and cell division is disrupted.
  • Another approach is to identify antigens on the surface of tumor cells or other abnormal cells which are not normally expressed in developed human tissue, such as tumor antigens or embryonic antigens.
  • antigens can be targeted with binding proteins such as antibodies which can block or neutralize the antigen.
  • the binding proteins, including antibodies and fragments thereof may deliver a toxic agent or other substance which is capable of directly or indirectly activating a toxic agent at the site of a tumor.
  • EGFR epidermal growth factor receptor
  • Such antibodies may mediate their efficacy through both modulation of cellular proliferation and antibody dependent immune functions (e.g. complement activation).
  • the use of these antibodies may be limited by uptake in organs that have high endogenous levels of EGFR such as the liver and skin (Baselga et al., 2000; Faillot et al., 1996).
  • a significant proportion of tumors containing amplifications of the EGFR gene also co-express a truncated version of the receptor (Wikstrand et al. (1998)
  • the de2-7 EGFR has been reported in a number of tumor types including glioma, breast, lung, ovarian and prostate (Wikstrand et al. (1997) Cell surface localization and density of the tumor-associated variant of the epidermal growth factor receptor, EGFRvIII. Cancer Res. 57, 4130-40; Olapade-Olaopa et al. (2000) Evidence for the differential expression of a variant EGF receptor protein in human prostate cancer. Br. J. Cancer. 82, 186-94; Wikstrand, et al. (1995) Monoclonal antibodies against EGFRvIII in are tumor specific and react with breast and lung carcinomas and malignant gliomas. Cancer Res. 55, 3140-8; Garcia de Palazzo et al.
  • de2-7 EGFR antibodies are that only a proportion of tumors exhibiting amplification of the EGFR gene also express the de2-7EGFR (Ekstrand et al. (1992) Amplified and rearranged epidermal growth factor receptor genes in human glioblastomas reveal deletions of sequences encoding portions of the N-and/or C-terminal tails. Proc. Natl. Acad. Sci. U.S.A. 89, 4309-13). The exact percentage of tumors containing the de2-7 EGFR is not completely established, because the use of different techniques (i.e. PCR versus immunohistochemistry) and various antibodies, has produced a wide range of reported values for the frequency of its presence.
  • gliomas express de2-7 EGFR with expression being lowest in anaplastic astrocytomas and highest in glioblastoma multiforme (Wong et al. (1992); Wikstrand et al. (1998) The class III variant of the epidermal growth factor receptor (EGFR): characterization and utilization as an immunotherapeutic target. J. Neurovirol. 4, 148-58; Moscatello et al. (1995) Frequent expression of a mutant epidermal growth factor receptor in multiple human tumors. Cancer Res. 55, 5536-9). The proportion of positive cells within de2-7 EGFR expressing gliomas has been reported to range from 37-86% (Wikstrand et al. (1997)).
  • de2-7 EGFR 27% of breast carcinomas and 17% of lung cancers were found to be positive for the de2-7 EGFR (Wikstrand et al. (1997); Wikstrand et al. (1995); Wikstrand et al.(1998); and Hills et al., 1995).
  • de2-7 EGFR specific antibodies would be expected to be useful in only a percentage of EGFR positive tumors.
  • the present invention provides isolated specific binding members, particularly antibodies or fragment thereof, which recognizes an EGFR epitope which does not demonstrate any amino acid sequence alterations or substitutions from wild-type EGFR and which is found in tumorigenic, hyperproliferative or abnormal cells and is not generally detectable in normal or wild type cells
  • wild-type cell as used herein contemplates a cell that expresses endogenous EGFR but not the de 2-7EGFR and the term specifically excludes a cell that over-expresses the EGFR gene;
  • wild-type refers to a genotype or phenotype or other characteristic present in a normal cell rather than in an abnormal or tumorigenic cell).
  • the present invention provides specific binding members, particularly antibodies or fragments thereof, which recognizes an EGFR epitope which is found in tumorigenic, hyperproliferative or abnormal cells and is not generally detectable in normal or wild type cells, wherein the epitope is enhanced or evident upon aberrant post translational modification or aberrant expression.
  • the EGFR epitope is enhanced or evident wherein post-translational modification is not complete or full to the extent seen with normal expression of EGFR in wild type cells.
  • the EGFR epitope is enhanced or evident upon initial or simple carbohydrate modification or early glycosylation, particularly high mannose modification, and is reduced or not evident in the presence of complex carbohydrate modification.
  • the specific binding members which may be antibodies or fragments thereof, such as immunogenic fragments thereof, do not substantially bind to or recognize normal or wild type cells containing normal or wild type EGFR epitope in the absence of aberrant expression and in the presence of normal EGFR post-translational modification.
  • the specific binding member of the invention may be antibodies or fragments thereof, which recognizes an EGFR epitope which is present in cells overexpressing EGFR (e.g., EGFR gene is amplified) or expressing the de2-7 EGFR, particularly in the presence of aberrant post-translational modification, and that is not generally detectable in cells expressing EGFR under normal conditions, particularly in the presence of normal post-translational modification.
  • the present inventors have discovered novel monoclonal antibodies, exemplified herein by the antibodies designated mAb806, ch806, hu806, mAb175, mAb124, and mAb1133, which specifically recognize aberrantly expressed EGFR.
  • the antibodies of the present invention recognize an EGFR epitope which is found in tumorigenic, hyperproliferative or abnormal cells and is not generally detectable in normal or wild type cells, wherein the epitope is enhanced or evident upon aberrant post-translational modification.
  • the novel antibodies of the invention also recognize amplified wild type EGFR and the de2-7 EGFR, yet bind to an epitope distinct from the unique junctional peptide of the de2-7 EGFR mutation.
  • the antibodies of the present invention specifically recognize aberrantly expressed EGFR, including amplified EGFR and mutant EGFR (exemplified herein by the de2-7 mutation), particularly upon aberrant post-translational modification. Additionally, while these antibodies do not recognize the EGFR when expressed on the cell surface of a glioma cell line expressing normal amounts of EGFR, they do bind to the extracellular domain of the EGFR (sEGFR) immobilized on the surface of ELISA plates, indicating the recognition of a conformational epitope. These antibodies bind to the surface of A431 cells, which have an amplification of the EGFR gene but do not express the de2-7 EGFR. Importantly, these antibodies did not bind significantly to normal tissues such as liver and skin, which express levels of endogenous, wild type (wt) EGFR that are higher than in most other normal tissues, but wherein EGFR is not aberrantly expressed or amplified.
  • wt wild type
  • the antibodies of the present invention can specifically categorize the nature of EGFR tumors or tumorigenic cells, by staining or otherwise recognizing those tumors or cells wherein aberrant EGFR expression, including EGFR amplification and/or EGFR mutation, particularly de2-7EGFR, is present. Further, the antibodies of the present invention demonstrate significant in vivo anti-tumor activity against tumors containing amplified EGFR and against de2-7 EGFR positive xenografts.
  • the unique specificity of these antibodies to bind to the de2-7 EGFR and amplified EGFR, but not to the normal, wild type EGFR, provides diagnostic and therapeutic uses to identify, characterize and target a number of tumor types, for example, head and neck, breast, or prostate tumors and glioma, without the problems associated with normal tissue uptake that may be seen with previously known EGFR antibodies.
  • the invention provides specific binding proteins, such as antibodies, which bind to the de2-7 EGFR at an epitope which is distinct from the junctional peptide but which do not substantially bind to EGFR on normal cells in the absence of amplification of the EGFR gene.
  • amplification it is meant to include that the cell comprises multiple copies of the EGFR gene.
  • the epitope recognized by the inventive antibodies is located within the region comprising residues 273-501 of the mature normal or wild type EGFR sequence, and preferably comprises residues 287-302 of the mature normal or wild type EGFR sequence. Therefore, also provided are specific binding proteins, such as antibodies, which bind to the de2-7 EGFR at an epitope located within the region comprising residues 273-501 and/or 287-302 of the EGFR sequence.
  • the epitope may be determined by any conventional epitope mapping techniques known to the person skilled in the art. Alternatively, the DNA sequence encoding residues 273-501 and/or 287-302 could be digested, and the resultant fragments expressed in a suitable host. Antibody binding could be determined as mentioned above.
  • the antibodies are ones which have the characteristics of the antibodies which the inventors have identified and characterized, in particular recognizing aberrantly expressed EGFR, as found in amplified EGFR and de2-7EGFR.
  • the invention provides antibodies capable of competing with the inventive antibodies, under conditions in which at least 10% of an antibody having the VH and VL sequences of the inventive antibodies are blocked from binding to de2-7EGFR by competition with such an antibody in an ELISA assay.
  • anti-idiotype antibodies are contemplated and are exemplified herein.
  • the anti-idiotype antibodies LMH-11, LMH-12 and LMH-13 are provided herein.
  • CDRs complementarity-determining regions
  • binding proteins such as antibodies which are based on the CDRs of the inventive antibodies identified, particularly the CDR3 regions, will be useful for targeting tumors with amplified EGFR regardless of their de2-7 EGFR status.
  • inventive antibodies do not bind significantly to normal, wild type receptor, there would be no significant uptake in normal tissue, a limitation of EGFR antibodies currently being developed.
  • an isolated antibody capable of binding EGFR on tumors containing amplifications of the EGFR gene, wherein cells of the tumors contain multiple copies of the EGFR gene, and on tumors that express the truncated version of the EGFR receptor de2-7, wherein the antibody does not bind to the de2-7 EGFR junctional peptide consisting of the amino acid sequence of SEQ ID NO:13, wherein the antibody binds to an epitope within the sequence of residues 287-302 of human wild-type EGFR, and wherein the antibody does not comprise a heavy chain variable region sequence having the amino acid sequence set forth in SEQ ID NO:2 and does not comprise a light chain variable region sequence having the amino acid sequence set forth in SEQ ID NO:4.
  • an isolated antibody wherein the antibody comprises a heavy chain and a light chain, the heavy chain having the amino acid sequence set forth in SEQ ID NO:42, and the light chain having the amino acid sequence set forth in SEQ ID NO:47.
  • an isolated antibody wherein the antibody comprises a heavy chain and a light chain, the heavy chain having the amino acid sequence set forth in SEQ ID NO:129, and the light chain having the amino acid sequence set forth in SEQ ID NO:134.
  • an isolated antibody wherein the antibody comprises a heavy chain and a light chain, the heavy chain having the amino acid sequence set forth in SEQ ID NO:22, and the light chain having the amino acid sequence set forth in SEQ ID NO:27.
  • an isolated antibody wherein the antibody comprises a heavy chain and a light chain, the heavy chain having the amino acid sequence set forth in SEQ ID NO:32, and the light chain having the amino acid sequence set forth in SEQ ID NO:37.
  • an isolated antibody wherein the antibody comprises a heavy chain and a light chain, wherein the variable region of the heavy chain comprises polypeptide binding domain regions having amino acid sequences highly homologous to the amino acid sequences set forth in SEQ ID NOS:44, 45, and 46.
  • an isolated antibody wherein the antibody comprises a heavy chain and a light chain, wherein the variable region of the light chain comprises polypeptide binding domain regions having amino acid sequences highly homologous to the amino acid sequences set forth in SEQ ID NOS:49, 50, and 51.
  • an isolated antibody wherein the antibody comprises a heavy chain and a light chain, wherein the variable region of the heavy chain comprises polypeptide binding domain regions having amino acid sequences highly homologous to the amino acid sequences set forth in SEQ ID NOS:130, 131, and 132.
  • an isolated antibody wherein the antibody comprises a heavy chain and a light chain, wherein the variable region of the light chain comprises polypeptide binding domain regions having amino acid sequences highly homologous to the amino acid sequences set forth in SEQ ID NOS:135, 136, and 137.
  • an isolated antibody wherein the antibody comprises a heavy chain and a light chain, wherein the variable region of the heavy chain comprises polypeptide binding domain regions having amino acid sequences highly homologous to the amino acid sequences set forth in SEQ ID NOS:23, 24, and 25.
  • an isolated antibody wherein the antibody comprises a heavy chain and a light chain, wherein the variable region of the light chain comprises polypeptide binding domain regions having amino acid sequences highly homologous to the amino acid sequences set forth in SEQ ID NOS:28, 29, and 30.
  • an isolated antibody wherein the antibody comprises a heavy chain and a light chain, wherein the variable region of the heavy chain comprises polypeptide binding domain regions having amino acid sequences highly homologous to the amino acid sequences set forth in SEQ ID NOS:33, 34, and 35.
  • an isolated antibody wherein the antibody comprises a heavy chain and a light chain, wherein the variable region of the light chain comprises polypeptide binding domain regions having amino acid sequences highly homologous to the amino acid sequences set forth in SEQ ID NOS:38, 39, and 40.
  • an isolated antibody wherein the isolated antibody is the form of an antibody F(ab′)2, scFv fragment, diabody, triabody or tetrabody.
  • an isolated antibody further comprising a detectable or functional label.
  • the detectable or functional label is a covalently attached drug.
  • the label is a radiolabel.
  • an isolated antibody wherein the isolated antibody is peglyated.
  • an isolated nucleic acid which comprises a sequence encoding an isolated antibody recited herein.
  • a method of preparing an isolated antibody comprising expressing a nucleic acid as recited above and herein under conditions to bring about expression of the antibody, and recovering the antibody.
  • a method of treatment of a tumor in a human patient which comprises administering to the patient an effective amount of an isolated antibody recited herein.
  • kits for the diagnosis of a tumor in which EGFR is aberrantly expressed or in which EGFR is expressed in the form of a truncated protein comprising an isolated antibody recited herein.
  • the kit further comprises reagents and/or instructions for use.
  • composition comprising an isolated antibody as recited herein.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable vehicle, carrier or diluent.
  • the pharmaceutical composition further comprises an anti-cancer agent selected from the group consisting of chemotherapeutic agents, anti-EGFR antibodies, radioimmunotherapeutic agents, and combinations thereof.
  • the chemotherapeutic agents are selected from the group consisting of tyrosine kinase inhibitors, phosphorylation cascade inhibitors, post-translational modulators, cell growth or division inhibitors (e.g. anti-mitotics), signal transduction inhibitors, and combinations thereof.
  • the tyrosine kinase inhibitors are selected from the group consisting of AG1478, ZD1839,ST1571, OSI-774, SU-6668, and combinations thereof.
  • the anti-EGFR antibodies are selected from the group consisting of the anti-EGFR antibodies 528,225, SC-03,DR8.3, L8A4, Y10, ICR62, ABX-EGF, and combinations thereof.
  • a method of preventing and/or treating cancer in mammals comprising administering to a mammal a therapeutically effective amount of a pharmaceutical composition as recited herein.
  • a method for the treatment of brain-resident cancers that produce aberrantly expressed EGFR in mammals comprising administering to a mammal a therapeutically effective amount of a pharmaceutical composition as recited herein.
  • the brain-resident cancers are selected from the group consisting of glioblastomas, medulloblastomas, meningiomas, neoplastic astrocytomas and neoplastic arteriovenous malformations.
  • a unicellular host transformed with a recombinant DNA molecule which encodes an isolated antibody recited herein.
  • the unicellular host is selected from the group consisting of E. coli, Pseudomonas, Bacillus, Streptomyces, yeasts, CHO, YB/20, NSO, SP2/0, R1.1, B-W, L-M, COS 1, COS 7, BSC1, BSC40, and BMT10 cells, plant cells, insect cells, and human cells in tissue culture.
  • a method for detecting the presence of amplified EGFR, de2-7EGFR or EGFR with high mannose glycosylation wherein the EGFR is measured by: (a) contacting a biological sample from a mammal in which the presence of amplified EGFR, de2-7EGFR or EGFR with high mannose glycosylation is suspected with an isolated antibody of claim 1 under conditions that allow binding of the EGFR to the isolated antibody to occur; and (b) detecting whether binding has occurred between the EGFR from the sample and the isolated antibody; wherein the detection of binding indicates that presence or activity of the EGFR in the sample.
  • the detection of the presence of the EGFR indicates the existence of a tumor or cancer in the mammal.
  • an isolated antibody capable of binding EGFR on tumors containing amplifications of the EGFR gene, wherein cells of the tumors contain multiple copies of the EGFR gene, and on tumors that express the truncated version of the EGFR receptor de2-7, wherein the antibody comprises a heavy chain and a light chain, the heavy chain having an amino acid sequence that is substantially homologous to the amino acid sequence set forth in SEQ ID NO:42, and the light chain having an amino acid sequence that is substantially homologous to the amino acid sequence set forth in SEQ ID NO:47.
  • the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:42, and wherein the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:47.
  • an isolated antibody capable of binding EGFR on tumors containing amplifications of the EGFR gene, wherein cells of the tumors contain multiple copies of the EGFR gene, and on tumors that express the truncated version of the EGFR receptor de2-7, wherein the antibody comprises a heavy chain and a light chain, wherein the variable region of the heavy chain comprises polypeptide binding domain regions having amino acid sequences highly homologous to the amino acid sequences set forth in SEQ ID NOS:44, 45, and 46, and wherein the variable region of the light chain comprises polypeptide binding domain regions having amino acid sequences highly homologous to the amino acid sequences set forth in SEQID NOS:49, 50, and 51.
  • an isolated antibody capable of binding EGFR on tumors containing amplifications of the EGFR gene, wherein cells of the tumors contain multiple copies of the EGFR gene, and on tumors that express the truncated version of the EGFR receptor de2-7, wherein the antibody comprises a heavy chain and a light chain, the heavy chain having an amino acid sequence that is substantially homologous to the amino acid sequence set forth in SEQ ID NO:129, and the light chain having an amino acid sequence that is substantially homologous to the amino acid sequence set forth in SEQ ID NO:134.
  • the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:129, and wherein the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:134.
  • an isolated antibody capable of binding EGFR on tumors containing amplifications of the EGFR gene, wherein cells of the tumors contain multiple copies of the EGFR gene, and on tumors that express the truncated version of the EGFR receptor de2-7, wherein the antibody comprises a heavy chain and a light chain, wherein the variable region of the heavy chain comprises polypeptide binding domain regions having amino acid sequences highly homologous to the amino acid sequences set forth in SEQ ID NOS:130, 131, and 132, and wherein the variable region of the light chain comprises polypeptide binding domain regions having amino acid sequences highly homologous to the amino acid sequences set forth in SEQ ID NOS:135, 136, and 137.
  • an isolated antibody capable of binding EGFR on tumors containing amplifications of the EGFR gene, wherein cells of the tumors contain multiple copies of the EGFR gene, and on tumors that express the truncated version of the EGFR receptor de2-7, wherein the antibody comprises a heavy chain and a light chain, the heavy chain having an amino acid sequence that is substantially homologous to the amino acid sequence set forth in SEQ ID NO:22, and the light chain having an amino acid sequence that is substantially homologous to the amino acid sequence set forth in SEQ ID NO:27.
  • the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:22, and wherein the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:27.
  • an isolated antibody capable of binding EGFR on tumors containing amplifications of the EGFR gene, wherein cells of the tumors contain multiple copies of the EGFR gene, and on tumors that express the truncated version of the EGFR receptor de2-7, wherein the antibody comprises a heavy chain and a light chain, wherein the variable region of the heavy chain comprises polypeptide binding domain regions having amino acid sequences highly homologous to the amino acid sequences set forth in SEQ ID NOS:23, 24, and 25, and wherein the variable region of the light chain comprises polypeptide binding domain regions having amino acid sequences highly homologous to the amino acid sequences set forth in SEQ ID NOS:28, 29, and 30.
  • an isolated antibody capable of binding EGFR on tumors containing amplifications of the EGFR gene, wherein cells of the tumors contain multiple copies of the EGFR gene, and on tumors that express the truncated version of the EGFR receptor de2-7, wherein the antibody comprises a heavy chain and a light chain, the heavy chain having an amino acid sequence that is substantially homologous to the amino acid sequence set forth in SEQ ID NO:32, and the light chain having an amino acid sequence that is substantially homologous to the amino acid sequence set forth in SEQ ID NO:37.
  • the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:32, and wherein the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:37.
  • an isolated antibody capable of binding EGFR on tumors containing amplifications of the EGFR gene, wherein cells of the tumors contain multiple copies of the EGFR gene, and on tumors that express the truncated version of the EGFR receptor de2-7, wherein the antibody comprises a heavy chain and a light chain, wherein the variable region of the heavy chain comprises polypeptide binding domain regions having amino acid sequences highly homologous to the amino acid sequences set forth in SEQ ID NOS:33, 34, and 35, and wherein the variable region of the light chain comprises polypeptide binding domain regions having amino acid sequences highly homologous to the amino acid sequences set forth in SEQ ID NOS:38, 39, and 40.
  • an isolated antibody capable of binding EGFR on tumors containing amplifications of the EGFR gene, wherein cells of the tumors contain multiple copies of the EGFR gene, and on tumors that express the truncated version of the EGFR receptor de2-7, wherein the antibody does not bind to the de2-7 EGFR junctional peptide consisting of the amino acid sequence of SEQ ID NO:13, wherein the antibody binds to an epitope within the sequence of residues 287-302 of human wild-type EGFR,
  • the antibody comprising a light chain and a heavy chain, wherein the variable region of the light chain comprises a first polypeptide binding domain region having an amino acid sequence corresponding to the amino acid sequence set forth in Formula I:
  • X aa1 is an amino acid residue having an uncharged polar R group
  • X aa2 is an amino acid residue having a charged polar R group
  • X aa3 is selected from the group consisting of A, G, and an amino acid residue which is conservatively substituted for A or G;
  • variable region of the heavy chain comprises a first polypeptide binding domain region having an amino acid sequence corresponding to the amino acid sequence set forth in Formula IV:
  • X aa4 is selected from the group consisting of F, Y, and an amino acid residue which is conservatively substituted for F or Y;
  • X aa5 is an amino acid residue having an uncharged polar R group
  • X aa6 is selected from the group consisting of G, A, and an amino acid residue which is conservatively substituted for G or A,
  • X aa7 is a basic amino acid residue
  • X aa8 is selected from the group consisting of V, A, and an amino acid residue which is conservatively substituted for V or A,
  • the antibody does not comprise a heavy chain variable region sequence having the amino acid sequence set forth in SEQ ID NO:2 and does not comprise a light chain variable region sequence having the amino acid sequence set forth in SEQ ID NO:4.
  • X aa5 is N or Q.
  • X aa1 is N or S.
  • X aa2 is D or E.
  • X aa3 is A or G.
  • X aa4 is F or Y.
  • X aa5 is N or Q.
  • X aa6 is G or A
  • X aa7 is independently K or R.
  • X aa8 is V or A.
  • an isolated antibody capable of binding EGFR on tumors containing amplifications of the EGFR gene, wherein cells of the tumors contain multiple copies of the EGFR gene, and on tumors that express the truncated version of the EGFR receptor de2-7, wherein the antibody does not bind to the de2-7 EGFR junctional peptide consisting of the amino acid sequence of SEQ ID NO:13, wherein the antibody binds to an epitope within the sequence of residues 273-501 of human wild-type EGFR,
  • variable region of the light chain comprises a first polypeptide binding domain region having the amino acid sequence HSSQDINSNIG (SEQ ID NO:18); a second polypeptide binding domain region having the amino acid sequence HGTNLDD (SEQ ID NO:19); and a third polypeptide binding domain region having the amino acid sequence VQYAQFPWT (SEQ ID NO:20),
  • variable region of the heavy chain comprises a first polypeptide binding domain region having the amino acid sequence SDFAWN (SEQ ID NO:15); a second polypeptide binding domain region having an amino acid sequence corresponding to the amino acid sequence set forth in Formula VIII:
  • X aa9 is an amino acid residue having an uncharged polar R group
  • VTAGRGFPY (SEQ ID NO: 17)
  • the antibody binds to an epitope within the sequence of residues 287-302 of human wild-type EGFR.
  • X aa9 is N or Q.
  • binding domain regions are carried by a human antibody framework.
  • the human antibody framework is a human IgG1 antibody framework.
  • an isolated antibody capable of binding EGFR on tumors containing amplifications of the EGFR gene, wherein cells of the tumors contain multiple copies of the EGFR gene, and on tumors that express the truncated version of the EGFR receptor de2-7, wherein the antibody comprises a heavy chain and a light chain, the heavy chain having an amino acid sequence that is substantially homologous to the amino acid sequence set forth in SEQ ID NO:2, and the light chain having an amino acid sequence that is substantially homologous to the amino acid sequence set forth in SEQ ID NO:4.
  • the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:2, and wherein the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:4.
  • an isolated antibody capable of binding EGFR on tumors containing amplifications of the EGFR gene, wherein cells of the tumors contain multiple copies of the EGFR gene, and on tumors that express the truncated version of the EGFR receptor de2-7, wherein the antibody comprises a heavy chain and a light chain, wherein the variable region of the heavy chain comprises polypeptide binding domain regions having amino acid sequences highly homologous to the amino acid sequences set forth in SEQ ID NOS:15, 16, and 17, and wherein the variable region of the light chain comprises polypeptide binding domain regions having amino acid sequences highly homologous to the amino acid sequences set forth in SEQ ID NOS:18, 19, and 20.
  • FIG. 1 presents the results of flow cytometric analysis of glioma cell lines.
  • U87MG light gray histograms
  • U87MG. ⁇ 2-7 dark gray histograms
  • cells were stained with either an irrelevant IgG2b antibody (open histograms), DH8.3 (specific for de2-7 EGFR), mAb806 or 528 (binds both wild type and de2-7 EGFR) as indicated.
  • FIGS. 2A-D present the results of ELISA of mAb806, mAbDH8.3 and mAb528.
  • A binding of increasing concentrations of mAb806 ( ⁇ ) DH8.3 ( ⁇ ) or 528 ( ⁇ ) antibody to sEGFR coated ELISA plates.
  • B inhibition of mAb806 and mAb528 binding to sEGFR coated ELISA plates by increasing concentrations of soluble EGFR (sEGFR) in solution.
  • sEGFR soluble EGFR
  • binding of increasing concentrations of DH8.3 to the de2-7 junctional peptide illustrates binding curves for mAb806 and mAb528 to immobilized wild-type sEGFR (D).
  • FIGS. 2E and 2F graphically present the results of BIAcore binding studies using C-terminal biotinylated peptide and including a monoclonal antibody of the invention, along with other known antibodies, among them the L8A4 antibody which recognizes the junction peptide of the de2-7 EGFR mutant, and controls.
  • FIG. 3 depicts the internalization of mAb806 and the DH8.3 antibody.
  • U87MG. ⁇ 2-7 cells were pre-incubated with mAb806 ( ⁇ ) or DH8.3 ( ⁇ ) at 4° C., transferred to 37° C. and internalization determined by FACS. Data represents mean internalization at each time point ⁇ SE of 3 (DH8.3) or 4 (mAb806) separate experiments.
  • FIGS. 6A-C illustrate flow cytometric analysis of cell lines containing amplification of the EGFR gene.
  • A431 cells were stained with either mAb806, DH8.3 or 528 (black histograms) and compared to an irrelevant IgG2b antibody (open histogram).
  • FIGS. 7A and 7B illustrate biodistribution (% ID/g tumor tissue) of radiolabeled (a) 125 I-mAb806 and (b) 131 I-528 in nude mice bearing U87MG. ⁇ 2-7 and A431 xenografts.
  • FIGS. 8A-D illustrate biodistribution of radiolabeled 125 I-mAb806 (open bar) and 131 I-528 (filled bar) and antibodies expressed as (A, B) tumor:blood or (C, D) tumor:liver ratios in nude mice bearing (A, C) U87MG. ⁇ 2-7 and (B, D) A431 xenografts.
  • FIGS. 9A and 9B illustrate anti-tumor effect of mAb806 on (A) U87MG and (B) U87MG. ⁇ 2-7 xenograft growth rates in a preventative model.
  • Mice were injected i.p. with either 1 mg of mAb806 ( ⁇ ); 0.1 mg of mAb806 ( ⁇ ); or vehicle ( ⁇ ) starting one day prior to tumor cell inoculation. Injections were given three times per week for two weeks as indicated by the arrows. Data are expressed as mean tumor volume ⁇ S.E.
  • FIG. 10 illustrates the anti-tumor effect of mAb806 on (A) U87MG, (B) U87MG. ⁇ 2-7 and (C) U87MG.wtEGFR xenografts in an established model.
  • Mice were injected i.p. with either 1 mg doses of mAb806 ( ⁇ ); 0.1 mg doses of mAb806 ( ⁇ ); or vehicle ( ⁇ ) starting when tumors had reached a mean tumor volume of 65-80 mm 3 . Injections were given three times per week for two weeks as indicated by the arrows. Data are expressed as mean tumor volume ⁇ S.E.
  • FIGS. 11A and 11B illustrate anti-tumor effect of mAb806 on A431 xenografts in (A) preventative and (B) established models.
  • FIG. 12 illustrates the anti-tumor effect of treatment with mAb806 combined with treatment with AG1478 on A431 xenografts in a preventative model. Data are expressed as mean tumor volume ⁇ S.E.
  • FIG. 13 depicts mAb806 binding to A431 cells in the presence of increasing concentrations of AG1478 (0.5 ⁇ M and 5 ⁇ M).
  • FIG. 14 illustrates the (A) nucleic acid sequence and the (B) amino acid translation thereof of the 806 VH gene (SEQ ID NO:1 and SEQ ID NO:2, respectively).
  • FIG. 15 illustrates the (A) nucleic acid sequence and the (B) amino acid translation thereof of the 806 VL gene (SEQ ID NO:3 and SEQ ID NO:4, respectively).
  • FIG. 16 shows the VH sequence numbered according to Kabat, with the CDRs boxed. Key residues of the VH are 24, 37, 48, 67 and 78.
  • FIG. 17 shows the VL sequence numbered according to Kabat, with the CDRs boxed. Key residues of the VL are 36, 46, 57 and 71.
  • FIGS. 18A-18D show the results of in vivo studies designed to determine the therapeutic effect of combination antibody therapy, particularly mAb806 and the 528 antibody. Mice received inoculations of U87MG.D2-7 (A and B), U87MG.DK (C), or A431 (D) cells.
  • FIGS. 19A-D show analysis of internalization by electron microscopy.
  • U87MG. ⁇ 2-7 cells were pre-incubated with mAb806 or DH8.3 followed by gold conjugated anti-mouse IgG at 4° C., transferred to 37° C. and internalization examined at various time points by electron microscopy.
  • B internalization of mAb806 by macropinocytosis (arrow) after 2 min;
  • C localization of DH8.3 to lysosomes (arrow) after 20 min;
  • D localization of mAb806 to lysosomes (arrow) after 30 min.
  • Original magnification for all images is X30,000.
  • FIG. 20 shows autoradiography of a U87MG. ⁇ 2-7 xenograft section collected 8 hr after injection of 125 I-mAb806.
  • FIG. 21 shows flow cytometric analysis of cell lines containing amplification of the EGFR gene.
  • HN5 and MDA-468 cells were stained with an irrelevant IgG2b antibody (open histogram with dashed line), mAb806 (black histogram) or 528 (open histogram with closed lines).
  • the DH8.3 antibody was completely negative on both cell lines (data not shown).
  • FIG. 22 shows immunoprecipitation of EGFR from cell lines.
  • the EGFR was immunoprecipitated from 35 S-labeled U87MG. ⁇ 2-7 or A431 cells with mAb806, sc-03 antibody or a IgG2b isotype control. Arrows at the side indicate the position of the de2-7 and wt EGFR. Identical banding patterns were obtained in 3 independent experiments.
  • FIG. 23 shows autoradiography of an A431 xenograft section collected 24 hr after injection of 125 I-mAb806, areas of localization to viable tissue are indicated (arrows).
  • FIGS. 24A and 24B show extended survival of nude mice bearing intracranial U87MG. ⁇ EGFR (A) and LN-Z308.
  • ⁇ EGFR (B) xenografts with systemic mAb806 treatment.
  • ⁇ EGFR cells (5 ⁇ 10 5 ) were implanted into nude mice brains, and the animals were treated with either mAb806, PBS, or isotype IgG from post-implantation days 0 through 14.
  • FIGS. 24C and 24D show growth inhibition of intracranial tumors by mAb806 treatment.
  • Nude mice five per group, treated with either mAb806 or the isotype IgG control, were euthanized on day 9 for U87MG.EGFR (C) and on day 15 for LN-Z308. ⁇ EGFR (D), and their brains were harvested, fixed, and sectioned. Data were calculated by taking the tumor volume of control as 100%. Values are mean ⁇ SD. ***, P ⁇ 0.001; control versus mAb806. Arrowheads, tumor tissue.
  • FIG. 24E shows extended survival of nude mice bearing intracranial U87MG. ⁇ EGFR xenografts with intratumoral mAb806 treatment.
  • U87MG. ⁇ EGFR cells were implanted as described.
  • 10 mg of mAb806 or isotype IgG control in a volume of 5 ⁇ l were injected at the tumor-injection site every other day starting at day 1 for five times.
  • FIG. 25 shows mAb806 extends survival of mice with U87MG.wtEGFR brain tumors but not with U87MG.DK. or U87MG brain tumors.
  • U87MG (A), U87MG.DK (B), or U87MG.wtEGFR (C) cells (5 ⁇ 10 5 ) were implanted into nude mice brains, and the animals were treated with mAb806 from post-implantation days 0 through 14 followed by observation after discontinuation of therapy.
  • FIG. 26A shows FACS analysis of mAb806 reactivity with U87MG cell lines.
  • U87MG, U87MG. ⁇ EGFR, U87MG.DK, and U87MG.wtEGFR cells were stained with anti-EGFR mAbs 528, EGFR.1, and anti- ⁇ EGFR antibody, mAb806.
  • Monoclonal EGFR. 1 antibody recognized wtEGFR exclusively and monoclonal 528 antibody reacted with both wtEGFR and ⁇ EGFR.
  • mAb806 reacted intensively with U87MG. ⁇ EGFR and U87MG.DK and weakly with U87MG.wtEGFR. Bars on the abscissa, maximum staining of cells in the absence of primary antibody. Results were reproduced in three independent experiments.
  • FIG. 26B shows mAb806 immunoprecipitation of EGFR forms. Mutant and wtEGFR were immunoisolated with anti-EGFR antibodies, 528, EGFR. 1, or anti- ⁇ EGFR antibody, mAb806, from (Lane 1) U87MG, (Lane 2) U87 ⁇ .EGFR, (Lane 3) U87MG.DK, and (Lane 4) U87MG.wtEGFR cells, and were then detected by Western blotting with anti-pan EGFR antibody, C13.
  • FIG. 27 shows systemic treatment with mAb806 decreases the phosphorylation of ⁇ EGFR and Bel-XL expression in U87MG. ⁇ EGFR brain tumors.
  • U87MG. ⁇ EGFR tumors were resected at day 9 of mAb806 treatment, immediately frozen in liquid nitrogen and stored at ⁇ 80° C. before tumor lysate preparation.
  • FIG. 28 shows mAb806 treatment leads to a decrease in growth and vasculogenesis and to increases in apoptosis and accumulating macrophages in U87MG. ⁇ EGFR tumors.
  • Tumor sections were stained for Ki-67.
  • Cell proliferative index was assessed by the percentage of total cells that were Ki-67 positive from four randomly selected high power fields ( ⁇ 400) in intracranial tumors from four mice of each group. Data are the mean ⁇ SE.
  • Apoptotic cells were detected by TUNEL assay.
  • Apoptotic index was assessed by the ratio of TUNEL-positive cells: total number of cells from four randomly selected high-power fields ( ⁇ 400) in intracranial tumors from four mice of each group. Data are the mean ⁇ SE.
  • Tumor sections were immunostained with anti-CD31 antibody. MVAs were analyzed by computerized image analysis from four randomly selected fields ( ⁇ 200) from intracranial tumors from four mice of each group. Peritumoral infiltrates of macrophages in mAb806-treated U87MG. ⁇ EGFR tumors. Tumor sections were stained with anti-F4/80 antibody.
  • FIG. 29 shows flow cytometric analysis of parental and transfected U87MG glioma cell lines.
  • Cells were stained with either an irrelevant IgG2b antibody (open histograms) or the 528 antibody or mAb806 (filled histograms) as indicated.
  • FIG. 30 shows immunoprecipitation of EGFR from cell lines.
  • the EGFR was immunoprecipitated from 35 S-labeled U87MG.wtEGFR, U87MG. ⁇ 2-7, and A431 cells with mAb806 (806), sc-03 antibody (c-term), or a IgG2b isotype control (con). Arrows, position of the de2-7 and wt EGFR.
  • FIG. 31 shows representative H&E-stained paraffin sections of U87MG. ⁇ 2-7 and U87MG.wtEGFR xenografts.
  • U87MG. ⁇ 2-7 collected 24 days after tumor inoculation
  • U87MG.wtEGFR collected 42 days after tumor inoculation
  • FIG. 32 shows immunohistochemical analysis of EGFR expression in frozen sections derived from U87MG, U87MG. ⁇ 2-7, and U87MG.wtEGFR xenografts. Sections were collected at the time points described in FIG. 31 above. Xenograft sections were immunostained with the 528 antibody (left panel) and mAb806 (right panel). No decreased immunoreactivity to either wtEGFR, amplified EGFR, or de2-7 EGFR was observed in xenografts treated with mAb806. Consistent with the in vitro data, parental U87MG xenografts were positive for 528 antibody but were negative for mAb806 staining.
  • FIG. 33 shows a schematic representation of generated bicistronic expression constructs. Transcription of the chimeric antibody chains is initiated by Elongation Factor-1 promoter and terminated by a strong artificial termination sequence. IRES sequences were introduced between coding regions of light chain and NeoR and heavy chain and dhfr gene.
  • FIG. 34 shows a biodistribution analysis of the ch806 radiolabeled with either (A) 125 I or (B) 111 In was performed in BALB/c nude mice bearing U87MG-de2-7 xenograft tumors. Mice were injected with 5 ⁇ g of radiolabeled antibody and in groups of 4 mice per time point, sacrificed at either 8, 28, 48 or 74 hours. Organs were collected, weighed and radioactivity measured in a gamma counter.
  • FIG. 35 depicts (A) the % ID gram tumor tissue and (B) the tumor to blood ratio.
  • Indium-111 antibody shows approximately 30% ID/gram tissue and a tumor to blood ratio of 4.0.
  • FIG. 36 depicts the therapeutic efficacy of chimeric antibody ch806 in an established tumor model.
  • 3 ⁇ 10 6 U87MG. ⁇ 2-7 cells in 100 ⁇ l of PBS were inoculated s.c. into both flanks of 4-6 week old female nude mice.
  • mAb806 was included as a positive control.
  • Treatment was started when tumors had reached a mean volume of 50 mm 3 and consisted of 1 mg of ch806 or mAb806 given i.p. for a total of 5 injections on the days indicated. Data was expressed as mean tumor volume ⁇ S.E. for each treatment group.
  • FIG. 37 shows CDC Activity on Target (A) U87MG.de2-7 and (B) A431 cells for anti-EGFR chimeric IgG1 antibodies ch806 and control cG250. Mean (bars; ⁇ SD) percent cytotoxicity of triplicate determinations are presented.
  • FIG. 38 shows ADCC on target (A) U87MG.de2-7 and (B) A431 cells at Effector:Target cell ratio of 50:1 mediated by ch806 and isotype control cG250 (0-10 ⁇ g/ml). Results are expressed as mean (bars; ⁇ SD) percent cytotoxicity of triplicate determinations.
  • FIG. 39 shows ADCC mediated by 1 ⁇ g/ml parental mAb806 and ch806 on target U87MG.de2-7 cells over a range of Effector:Target ratios. Mean (bars; ⁇ SD) of triplicate determinations are presented.
  • FIG. 40 shows twenty-five hybridomas producing antibodies that bound ch806 but not huIgG were initially selected.
  • Four of these anti-ch806 hybridomas with high affinity binding (clones 3E3, 5B8, 9D6 and 4D8) were subsequently pursued for clonal expansion from single cells by limiting dilution and designated Ludwig Institute for Cancer Research Melbourne Hybridoma (LMH) -11, -12, -13 and -14, respectively.
  • LMH Ludwig Institute for Cancer Research Melbourne Hybridoma
  • two hybridomas that produced mAbs specific for huIgG were also cloned and characterized further: clones 2C10 (LMH-15) and 2B8 (LMH-16).
  • FIG. 41 shows that after clonal expansion, the hybridoma culture supernatants were examined in triplicate by ELISA for the ability to neutralize ch806 or mAb806 antigen binding activity with sEGFR621.
  • Mean (+SD) results demonstrated the antagonist activity of anti-idiotype mAbs LMH-11, -12, -13 and -14 with the blocking in solution of both ch806 and murine mAb806 binding to plates coated with sEGFR (LMH-14 not shown).
  • FIG. 42 shows microtitre plates that were coated with 10 ⁇ g/ml purified (A) LMH-11, (B) LMH-12 and (C) LMH-13.
  • the three purified clones were compared for their ability to capture ch806 or mAb806 in sera or 1% FCS/Media and then detect bound ch806 or mAb806.
  • Isotype control antibodies hu3S193 and m3S193 in serum and 1% FCS/Media were included in addition to controls for secondary conjugate avidin-HRP and ABTS substrate.
  • Results are presented as mean ( ⁇ SD) of triplicate samples using biotinylated-LMH-12 (10 ⁇ g/ml) for detection and indicate LMH-12 used for capture and detection had the highest sensitivity for ch806 in serum (3 ng/ml) with negligible background binding.
  • FIG. 43 shows validation of the optimal pharmacokinetic ELISA conditions using 1 ⁇ g/ml anti-idiotype LMH-12 and 1 ⁇ g/ml biotinylated LMH-12 for capture and detection, respectively.
  • Three separate ELISAs were performed in quadruplicate to measure ch806 in donor serum ( ⁇ ) from three healthy donors or 1% BSA/media ( ⁇ ) with isotype control hu3S193 in serum ( ⁇ ) or 1% BSA/media ( ⁇ ). Controls for secondary conjugate avidin-HRP ( ⁇ ) and ABTS substrate (hexagon) alone were also included with each ELISA.
  • FIG. 44 depicts an immunoblot of recombinant sEGFR expressed in CHO cells, blotted with mAb806.
  • Recombinant sEGFR was treated with PNGaseF to remove N-linked glycosylation (deglycosylated), or untreated (untreated), the protein was run on SDS-PAGE, transferred to membrane and immunoblotted with mAb806.
  • FIG. 45 depicts immunoprecipitation of EGFR from 35 S-labelled cell lines (U87MG. ⁇ 2-7, U87MG-wtEGFR, and A431) with different antibodies (SC-03, 806 and 528 antibodies).
  • FIG. 46 depicts immunoprecipitation of EGFR from different cells (A431 and U87MG. ⁇ 2-7) at different time points (time 0 to 240 minutes) after pulse-labeling with 35 S methionine/cysteine.
  • Antibodies 528 and 806 are used for immunoprecipitation.
  • FIG. 47 depicts immunoprecipitation of EGFR from various cell lines (U87MG ⁇ 2-7, U87MG-wtEGFR and A431) with various antibodies (SC-03, 806 and 528) in the absence of ( ⁇ ) and after Endo H digestion (+) to remove high mannose type carbohydrates.
  • FIG. 48 depicts cell surface iodination of the A431 and U87MG. ⁇ 2-7 cell lines followed by immunoprecipitation with the 806 antibody, and with or without Endo H digestion, confirming that the EGFR bound by mAb806 on the cell surface of A431 cells is an EndoH sensitive form.
  • FIG. 49 shows the pREN ch806 LC Neo Vector.
  • FIG. 50 shows the pREN ch806 HC DHFR Vector.
  • FIGS. 51A-D shows the mAb124 VH and VL chain nucleic acid and amino acid sequences.
  • FIGS. 52A-D shows the mAb1133 VH and VL chain nucleic acid and amino acid sequences.
  • FIG. 53 shows a DNA plasmid graphic of the combined, double gene Lonza plasmid including pEE12.4 containing the hu806H (VH+CH) expression cartridge, and pEE6.4 containing the hu806L (VL+CL) expression cartridge.
  • FIG. 54 shows the DNA sequence of the combined Lonza plasmid described in FIG. 53 . This sequence also shows all translations relevant to the hu806 antibody.
  • the plasmid has been sequence-verified, and the coding sequence and translation checked. Sections of the sequence have been shaded to identify regions of interest; the shaded regions correspond to actual splice junctions.
  • the color code is as follows:
  • FIGS. 55A and 55B show the hu806 translated amino acid sequences, and give the Kabat numbers for the VH and VL chains, with CDRs underlined.
  • FIGS. 56A and 55B show the initial step in veneering design, the grading of amino acid residues in the mAb806 sequence for surface exposure. Grades are given in the number of asterisks (*) above each residue, with the most exposed residues having three asterisks. These figures also include a design indicating how the initial oligonucleotides overlapped to form the first veneered product (VH and VL).
  • FIG. 57 shows a map of surface-exposed residues for veneering of mAb806 (variable light chain).
  • FIG. 58 shows a map of codon optimized huIgG1 heavy chain DNA sequence and amino acid translation.
  • FIG. 59 shows the protein alignment comparing the hu806 VH+CH amino acid sequence (8C65AAG hu806 VH+CH) to the original reference file for the mAb806 VH. Highlighted regions indicate conserved amino acid sequences in the VH. The CDRs are underlined. Asterisks reflect changes that were planned and carried out in the initial veneering process. The numbered sites are references to later modifications.
  • FIG. 60 shows the corresponding alignment for the hu806 VL+CL amino acid sequence (8C65AAG hu806 signal+VL+CL). It contains an additional file (r2vk1 hu806 signal+VL+CL), a precursor construct, which was included to illustrate the change made at modification #7.
  • FIG. 61 shows a nucleotide and amino acid alignment of the hu806 signal+VL and CL sequences (8C65AAG hu806 Vl+Cl) with the corresponding ch806 sequences (PREN ch806 LC Neo; LICR). It has been modified and annotated as described in FIG. 62 .
  • FIG. 61 shows the nucleotide alignment of the hu806 signal+VH sequence (8C65AAG hu806 VH) with the corresponding mAb806 sequence [mAb806 VH before codon change (cc) and veneering (ven)].
  • the nucleotide changes behind the amino acid changes of FIGS. 59 and 60 are illustrated, as well as showing conservative nucleic acid changes that led to no change in amino acid.
  • the intron between the signal and the VH in hu806 has been removed for easier viewing.
  • the signal sequence and CDRs are underlined.
  • the corresponding amino acid sequence has been superimposed on the alignment.
  • FIG. 63 shows binding of purified hu806 antibody obtained from transient transfectant 293 cells to recombinant EGFR-ECD as determined by Biacore. No binding to the EGFR-ECD was observed with purified control human IgG1 antibody.
  • FIG. 64 shows the GenBank formatted text document of the sequence and annotations of plasmid 8C65AAG encoding the IgG1 hu806.
  • FIG. 65 shows the alignment of amino acid sequences for CDRs from mAb806 and mAb175. Sequence differences between the two antibodies are bolded.
  • FIG. 66 shows immunohistochemical staining of cell lines and normal human liver with mAb175.
  • Biotinylated mAb175 was used to stain sections prepared from blocks containing A431 cells (over-express the wtEGFR), U87MG. ⁇ 2-7 cells (express the ⁇ 2-7EGFR) and U87MG cells (express the wtEGFR at modest levels).
  • B Staining of normal human liver (400 ⁇ ) with mAb175 (left panel), isotype control (centre panel) and secondary antibody control (right panel). No specific sinusoidal or hepatocyte staining was observed.
  • FIG. 67 shows reactivity of mAb806 and mAb175 with fragments of the EGFR displayed on yeast.
  • A Representative flow cytometry histograms depicting the mean fluorescence signal of mAb175 and mAb806-labeling of yeast-displayed EGFR fragments. With yeast display a percentage of cells do not express protein on their surface resulting in 2 histogram peaks. The 9E10 antibody is used as a positive control as all fragments contain a linear C-terminal c-myc tag.
  • B Summary of antibody binding to various EGFR fragments.
  • C The EGFR fragments were denatured by heating yeast pellets to 800° C. for 30 min. The c-myc tag was still recognized by the 9E10 anti-myc antibody in all cases, demonstrating that heat treatment does not compromise the yeast surface displayed protein.
  • the conformation sensitive EGFR antibody mAb225 was used to confirm denaturation.
  • FIG. 68 shows the antitumor effects of mAb175 on brain and prostate cancer xenografts.
  • B Cells were stained with two irrelevant antibodies (blue, solid and green, hollow), mAb 528 for total EGFR (pink, solid), mAb806 (light blue, hollow) and mAb175 (orange, hollow) and then analyzed by FACS.
  • DU145 cells were lysed, subjected to IP with mAb 528, mAb806, mAb175 or two independent irrelevant antibodies and then immunoblotted for EGFR.
  • FIG. 69 shows crystal structures of EGFR peptide 287-302 bound to the Fab fragments
  • A Cartoon of Fab 806, with the light chain, red; heavy chain, blue; bound peptide, yellow; and the superposed EGFR 287-302 from EGFR, purple.
  • B Cartoon of Fab 175 with the light chain, yellow; heavy chain, green; bound peptide, lilac; and EGFR 287-302 from EGFR(DI-3), purple.
  • C Detail from (B) showing the similarity of EGFR 287-302 in the receptor to the peptide bound to FAb 175. Peptides backbones are shown as C ⁇ traces and the interacting side chains as sticks.
  • O atoms are colored red; N, blue; S, orange and C, as for the main chain.
  • D Superposition of EGFR with the Fab175:peptide complex showing spacial overlap. Coloring as in (C) with the surface of EGFR187-286 colored turquoise.
  • E Orthogonal view to (D) with EGFR187-286 shown in opaque blue and the surface of the light (orange) and heavy (green) chains transparent.
  • F Detailed stereoview of 175 Fab complex looking into the antigen-binding site. Coloring as in (C) and side chain hydrogen bonds dotted in black. Water molecules buried upon complex formation are shown as red spheres.
  • FIG. 70 shows the influence of the 271-283 cysteine bond on mAb806 binding to the EGFR.
  • A Cells transfected with wtEGFR, EGFR-C271A, EGFR-C283A or the C271A/C283A mutant were stained with mAb528 (solid pink histogram), mAb806 (blue line) or only the secondary antibody (purple) and then analyzed by FACS. The gain was set up using a class-matched irrelevant antibody.
  • B BaF3 cells expressing the EGFR-C271A or C271/283A EGFR were examined for their response to EGF in an MTT assay as described. EC 50S were derived using the Bolzman fit of the data points.
  • FIG. 71 shows: (A) Whole body gamma camera image of the biodistribution of 111 In ch806 in a patient with metastatic squamous cell carcinoma of the vocal cord, showing quantitative high uptake in tumor in the right neck (arrow). Blood pool activity, and minor catabolism of free 111 In in liver, is also seen. (B) Single Photon Computed Tomography (SPECT) image of the neck of this patient, showing uptake of 111 in-ch806 in viable tumor (arrow), with reduced central uptake indicating necrosis. (C) Corresponding CT scan of the neck demonstrating a large right neck tumor mass (arrow) with central necrosis.
  • SPECT Single Photon Computed Tomography
  • FIG. 72 shows a stereo model of the structure of the untethered EGFR1-621.
  • the receptor backbone is traced in blue and the ligand TGF- ⁇ in red.
  • the mAb806/175 epitope is drawn in turquoise and the disulfide bonds in yellow.
  • the atoms of the disulfide bond which ties the epitope back into the receptor are shown in space-filling format.
  • the model was constructed by docking the EGFR-ECD CR2 domain from the tethered conformation onto the structure of an untethered EGFR monomer in the presence of its ligand.
  • FIG. 73 shows the reactivity of mAb806 with fragments of the EGFR.
  • Lysates from 293T cells transfected with vectors expressing the soluble 1-501 EGFR fragment or GH/EGFR fragment fusion proteins (GH-274-501, GH-282-501, GH-290-501 and GH-298-501) were resolved by SDS-PAGE, transferred to membrane and immunoblotted with mAb806 (left panel) or the anti-myc antibody 9B11 (right panel).
  • FIGS. 74A and 74B show the mAb175 VH chain nucleic acid and amino acid sequences.
  • FIGS. 75A and 75B show the mAb175 VL chain nucleic acid and amino acid sequences.
  • FIG. 76 shows: (A) Volumetric product concentration and (B) viable cell concentration of GS-CHO (14D8, 15B2 and 40A10) and GS-NSO (36) hu806 transfectants in small scale (100 mL) shake flasks cultures. Product concentration was estimated by ELISA using the 806 anti-idiotype as coating antibody and ch806 Clinical Lot: J06024 as standard; (C) GS-CHO 40A10 transfectant cell growth and volumetric production in a 15L stirred tank bioreactor. Viable cell density ( ⁇ 10 5 cell/mL), cell viability ( ⁇ ) and production ( ⁇ mg/L).
  • FIG. 77 shows Size Exclusion Chromatography (Biosep SEC-S3000) Analysis of Protein-A purified hu806 antibody constructs produced by small scale culture and control ch806 and mAb 806. Chromatograms at A214 nm are presented in the upper panels and at A280 nm in the lower panel of each Figure.
  • FIG. 78 shows Size Exclusion Chromatography (Biosep SEC-S3000) Analysis of Protein-A purified hu806 antibody construct 40A10 following large scale production and Protein-A purification. Chromatogram at A214 nm is presented indicating 98.8% purity with 1.2% aggregate present.
  • FIG. 79 shows that precast 4-20% Tris/Glycine Gels from Novex, USA were used under standard SDS-PAGE conditions to analyze purified transfectant hu806 preparations (5 ⁇ g) GS CHO (14D8, 15B2 and 40A10) and GS-NSO (36) hu806 under reduced conditions. Proteins detected by Coomassie Blue Stain.
  • FIG. 80 shows that precast 4-20% Tris/Glycine Gels were used under standard SDS-PAGE conditions to analyze purified transfectant hu806 preparations (5 ⁇ g) GS CHO (14D8, 15B2 and 40A10) and GS-NSO (36) under non-reduced conditions. Proteins detected by Coomassie Blue Stain.
  • FIG. 81 shows that precast 4-20% Tris/Glycine Gels were used under standard SDS-PAGE conditions to analyze purified transfectant hu806 GS CHO 40A10 (5 ⁇ g) following large scale production. Proteins detected by Coomassie Blue Stain.
  • FIG. 82 shows Isoelectric Focusing gel analysis of purified transfectant hu806 GS CHO 40A10 (5 ⁇ g) following 15L production. Proteins detected by Coomassie Blue Stain. Lane 1, pI markers; Lane 2, hu806 (three isoforms, pI 8.66 to 8.82); Lane 3, pI markers.
  • FIG. 83 shows binding to A431 cells: Flow Cytometry analysis of Protein-A purified hu806 antibody preparations (20 ⁇ g/ml), and isotype control huA33 (20 ⁇ g/ml). Controls include secondary antibody alone (green) and ch806 (red). Hu806 constructs were produced by small scale culture.
  • FIG. 84 shows binding to A431 cells: Flow Cytometry analysis of purified mAb806, ch806 and hu806 40A10 antibody preparations (20 ⁇ g/ml) that bind ⁇ 10% of wild type EGFR on cell surface, 528 (binds both wild type and de2-7 EGFR) and irrelevant control antibody (20 ⁇ g/ml) as indicated.
  • FIG. 85 shows binding to U87MG.de2-7 glioma cells.
  • FIG. 86 shows specific binding of 125 I-radiolabelled 806 antibody constructs to: (A) U87MG.de2-7 glioma cells and (B) A431 carcinoma cells.
  • FIG. 87 shows Scatchard Analyses: 125 I-radiolabelled (A) ch806 and (B) hu806 antibody constructs binding to U87MG.de2-7 cells.
  • FIG. 88 shows Scatchard Analyses: 125 I-radiolabelled (A) ch806 and (B) hu806 antibody constructs binding to A431 cells.
  • FIG. 89 shows BIAcore analysis of binding to 287-302 EGFR 806 peptide epitope by (A) hu806 and (B) ch806 passing over the immobilized peptide in increasing concentrations of 50 nM, 100 nM, 150 nM, 200 nM, 250 nM and 300 nM.
  • FIG. 91 shows treatment of established A431 xenografts in BALB/c nude mice. Groups of 5 mice received 6 ⁇ 1 mg dose over 2 weeks antibody therapy as indicated (arrows). Mean ⁇ SEM tumor volume is presented until study termination.
  • FIG. 92 shows treatment of established U87MG.de2-7 xenografts in BALB/c nude mice. Groups of 5 mice received 6 ⁇ 1 mg dose over 2 weeks antibody therapy as indicated (arrows). Mean ⁇ SEM tumor volume is presented until study termination.
  • FIG. 93 shows deviations from random coil chemical shift values for the mAb806 peptide (A) N, (B) HN and (C) HA.
  • Peptide was prepared in H 2 O solution containing 5% 2 H 2 O, 70 mM NaCl and 50 mM NaPO 4 at pH 6.8. All spectra used for sequential assignments were acquired at 298K on a Bruker Avance500.
  • FIG. 94 shows whole body gamma camera images of Patient 7 A) Anterior, and B) Posterior, Day 5 post infusion of 111 In-ch806. High uptake of 111 In-ch806 in metastatic lesions in the lungs (arrows) is evident. C) and D) show metastatic lesions (arrows) on CT scan. E) 3D SPECT images of the chest, and F) co-registered transaxial images of SPECT and CT showing specific uptake of 111 In-ch806 in metastatic lesions.
  • FIG. 95 shows planar images of the head and neck of Patient 8 obtained A) Day 0, B) Day 3 and C) Day 7 post infusion of 111 In-ch806.
  • Initial blood pool activity is seen on Day 0, and uptake of 111 In-ch806 in an anaplastic astrocytoma in the right frontal lobe is evident by Day 3 (arrow), and increases by Day 7.
  • Specific uptake of 111 In-ch806 is confirmed in D) SPECT image of the brain (arrow), at the site of tumor (arrow) evident in E) 18 F-FDG PET, and F) MRI.
  • FIG. 96 shows similar uptake of 111 In-ch806 in tumor is evident in Patient 3 compared to Patient 4, despite differences in 806 antigen expression in screened tumor samples.
  • FIG. 97 shows pooled population pharmacokinetics of ch806 protein measured by ELISA. Observed and predicted ch806 (% ID/L) vs time post infusion (hrs).
  • specific binding member describes a member of a pair of molecules which have binding specificity for one another.
  • the members of a specific binding pair may be naturally derived or wholly or partially synthetically produced.
  • One member of the pair of molecules has an area on its surface, or a cavity, which specifically binds to and is therefore complementary to a particular spatial and polar organization of the other member of the pair of molecules.
  • the members of the pair have the property of binding specifically to each other.
  • types of specific binding pairs are antigen-antibody, biotin-avidin, hormone-hormone receptor, receptor-ligand, enzyme-substrate. This application is concerned with antigen-antibody type reactions.
  • allegement expression in its various grammatical forms may mean and include any heightened or altered expression or overexpression of a protein in a tissue, e.g. an increase in the amount of a protein, caused by any means including enhanced expression or translation, modulation of the promoter or a regulator of the protein, amplification of a gene for a protein, or enhanced half-life or stability, such that more of the protein exists or can be detected at any one time, in contrast to a nonoverexpressed state.
  • Aberrant expression includes and contemplates any scenario or alteration wherein the protein expression or post-translational modification machinery in a cell is taxed or otherwise disrupted due to enhanced expression or increased levels or amounts of a protein, including wherein an altered protein, as in mutated protein or variant due to sequence alteration, deletion or insertion, or altered folding is expressed.
  • abnormal quantities of protein may result from overexpression of the protein in the absence of gene amplification, which is the case e.g. in many cellular/tissue samples taken from the head and neck of subjects with cancer, while other samples exhibit abnormal protein levels attributable to gene amplification.
  • antibody describes an immunoglobulin whether natural or partly or wholly synthetically produced.
  • the term also covers any polypeptide or protein having a binding domain which is, or is homologous to, an antibody binding domain.
  • CDR grafted antibodies are also contemplated by this term.
  • antibody should be construed as covering any specific binding member or substance having a binding domain with the required specificity.
  • this term covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023 and U.S. Pat. Nos. 4,816,397 and 4,816,567.
  • binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fd fragment consisting of the VH and CH1 domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward, E. S. et al.
  • an “antibody combining site” is that structural portion of an antibody molecule comprised of light chain or heavy and light chain variable and hypervariable regions that specifically binds antigen.
  • antibody molecule in its various grammatical forms as used herein contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule.
  • Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contains the paratope, including those portions known in the art as Fab, Fab′, F (ab′) Z and F (v), which portions are preferred for use in the therapeutic methods described herein.
  • Antibodies may also be bispecific, wherein one binding domain of the antibody is a specific binding member of the invention, and the other binding domain has a different specificity, e.g. to recruit an effector function or the like.
  • Bispecific antibodies of the present invention include wherein one binding domain of the antibody is a specific binding member of the present invention, including a fragment thereof, and the other binding domain is a distinct antibody or fragment thereof, including that of a distinct anti-EGFR antibody, for instance antibody 528 (U.S. Pat. No. 4,943,533), the chimeric and humanized 225 antibody (U.S. Patent No. 4,943,533 and WO/9640210), an anti-de2-7 antibody such as DH8.3 (Hills, D.
  • the other binding domain may be an antibody that recognizes or targets a particular cell type, as in a neural or glial cell-specific antibody.
  • the one binding domain of the antibody of the invention may be combined with other binding domains or molecules which recognize particular cell receptors and/or modulate cells in a particular fashion, as for instance an immune modulator (e.g., interleukin(s)), a growth modulator or cytokine (e.g. tumor necrosis factor (TNF), and particularly, the TNF bispecific modality demonstrated in U.S. Ser. No. 60/355,838 filed Feb. 13, 2002, incorporated herein in its entirety) or a toxin (e.g., ricin) or anti-mitotic or apoptotic agent or factor.
  • an immune modulator e.g., interleukin(s)
  • a growth modulator or cytokine e.g. tumor necrosis factor (TNF)
  • TNF tumor necrosis factor
  • Fab and F(ab′) 2 portions of antibody molecules may be prepared by the proteolytic reaction of papain and pepsin, respectively, on substantially intact antibody molecules by methods that are well-known. See, for example, U.S. Pat. No. 4,342,566 to Theofilopolous et al.
  • Fab′ antibody molecule portions are also well-known and are produced from F (ab′) 2 portions followed by reduction of the disulfide bonds linking the two heavy chain portions as with mercaptoethanol, and followed by alkylation of the resulting protein mercaptan with a reagent such as iodoacetamide.
  • An antibody containing intact antibody molecules is preferred herein.
  • the phrase “monoclonal antibody” in its various grammatical forms refers to an antibody having only one species of antibody combining site capable of immunoreacting with a particular antigen.
  • a monoclonal antibody thus typically displays a single binding affinity for any antigen with which it immunoreacts.
  • a monoclonal antibody may also contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different antigen; e.g., a bispecific (chimeric) monoclonal antibody.
  • an antigen binding domain describes the part of an antibody which comprises the area which specifically binds to and is complementary to part or all of an antigen. Where an antigen is large, an antibody may bind to a particular part of the antigen only, which part is termed an epitope.
  • An antigen binding domain may be provided by one or more antibody variable domains.
  • an antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).
  • Post-translational modification may encompass any one of or combination of modification(s), including covalent modification, which a protein undergoes after translation is complete and after being released from the ribosome or on the nascent polypeptide co-translationally.
  • Post-translational modification includes but is not limited to phosphorylation, myristylation, ubiquitination, glycosylation, coenzyme attachment, methylation and acetylation.
  • Post-translational modification can modulate or influence the activity of a protein, its intracellular or extracellular destination, its stability or half-life, and/or its recognition by ligands, receptors or other proteins. Post-translational modification can occur in cell organelles, in the nucleus or cytoplasm or extracellularly.
  • the term “specific” may be used to refer to the situation in which one member of a specific binding pair will not show any significant binding to molecules other than its specific binding partner(s).
  • the term is also applicable where e.g. an antigen binding domain is specific for a particular epitope which is carried by a number of antigens, in which case the specific binding member carrying the antigen binding domain will be able to bind to the various antigens carrying the epitope.
  • the term “consisting essentially of” refers to a product, particularly a peptide sequence, of a defined number of residues which is not covalently attached to a larger product.
  • a product particularly a peptide sequence
  • minor modifications to the N-or C-terminal of the peptide may however be contemplated, such as the chemical modification of the terminal to add a protecting group or the like, e.g. the amidation of the C-terminus.
  • isolated refers to the state in which specific binding members of the invention, or nucleic acid encoding such binding members will be, in accordance with the present invention.
  • Members and nucleic acid will be free or substantially free of material with which they are naturally associated such as other polypeptides or nucleic acids with which they are found in their natural environment, or the environment in which they are prepared (e.g. cell culture) when such preparation is by recombinant DNA technology practiced in vitro or in vivo.
  • Members and nucleic acid may be formulated with diluents or adjuvants and still for practical purposes be isolated-for example the members will normally be mixed with gelatin or other carriers if used to coat microtitre plates for use in immunoassays, or will be mixed with pharmaceutically acceptable carriers or diluents when used in diagnosis or therapy.
  • Specific binding members may be glycosylated, either naturally or by systems of heterologous eukaryotic cells, or they may be (for example if produced by expression in a prokaryotic cell) unglycosylated.
  • glycoproteins include and encompasses the post-translational modification of proteins, termed glycoproteins, by addition of oligosaccarides. Oligosaccharides are added at glycosylation sites in glycoproteins, particularly including N-linked oligosaccharides and O-linked oligosaccharides. N-linked oligosaccharides are added to an Asn residue, particularly wherein the Asn residue is in the sequence N-X-S/T, where X cannot be Pro or Asp, and are the most common ones found in glycoproteins.
  • a high mannose type oligosaccharide (generally comprised of dolichol, N-Acetylglucosamine, mannose and glucose is first formed in the endoplasmic reticulum (ER). The high mannose type glycoproteins are then transported from the ER to the Golgi, where further processing and modification of the oligosaccharides occurs. O-linked oligosaccharides are added to the hydroxyl group of Ser or Thr residues. In O-linked oligosaccharides, N-Acetylglucosamine is first transferred to the Ser or Thr residue by N-Acetylglucosaminyltransferase in the ER.
  • O-linked modifications can occur with the simple addition of the OGlcNAc monosaccharide alone at those Ser or Thr sites which can also under different conditions be phosphorylated rather than glycosylated.
  • pg means picogram
  • ng means nanogram
  • ug means nanogram
  • ug means microgram
  • mg means milligram
  • ul or “ ⁇ l” mean microliter
  • ml means milliliter
  • 1 means liter.
  • 806 antibody refers to proteinaceous material including single or multiple proteins, and extends to those proteins having the amino acid sequence data described herein and presented in SEQ ID NO:2 and SEQ ID NO:4, and the chimeric antibody ch806 which is incorporated in and forms a part of SEQ ID NOS:7 and 8, and the profile of activities set forth herein and in the Claims. Accordingly, proteins displaying substantially equivalent or altered activity are likewise contemplated.
  • modifications may be deliberate, for example, such as modifications obtained through site-directed mutagenesis, or may be accidental, such as those obtained through mutations in hosts that are producers of the complex or its named subunits.
  • the terms “806 antibody”, “mAb806” and “ch806” are intended to include within their scope proteins specifically recited herein as well as all substantially homologous analogs and allelic variations.
  • humanized 806 antibody “hu806”, and “veneered 806 antibody” and any variants not specifically listed, may be used herein interchangeably, and as used throughout the present application and claims refer to proteinaceous material including single or multiple proteins, and extends to those proteins having the amino acid sequence data described herein and presented in SEQ ID NO:42 and SEQ ID NO:47, and the profile of activities set forth herein and in the Claims. Accordingly, proteins displaying substantially equivalent or altered activity are likewise contemplated. These modifications may be deliberate, for example, such as modifications obtained through site-directed mutagenesis, or may be accidental, such as those obtained through mutations in hosts that are producers of the complex or its named subunits. Also, the terms “humanized 806 antibody”, “hu806”, and “veneered 806 antibody” are intended to include within their scope proteins specifically recited herein as well as all substantially homologous analogs and allelic variations.
  • proteins displaying substantially equivalent or altered activity are likewise contemplated. These modifications may be deliberate, for example, such as modifications obtained through site-directed mutagenesis, or may be accidental, such as those obtained through mutations in hosts that are producers of the complex or its named subunits. Also, the terms “175 antibody” and “mAb175” are intended to include within their scope proteins specifically recited herein as well as all substantially homologous analogs and allelic variations.
  • 124 antibody and “mAb124”, and any variants not specifically listed, may be used herein interchangeably, and as used throughout the present application and claims refer to proteinaceous material including single or multiple proteins, and extends to those proteins having the amino acid sequence data described herein and presented in SEQ ID NO:22 and SEQ ID NO:27, and the profile of activities set forth herein and in the Claims. Accordingly, proteins displaying substantially equivalent or altered activity are likewise contemplated. These modifications may be deliberate, for example, such as modifications obtained through site-directed mutagenesis, or may be accidental, such as those obtained through mutations in hosts that are producers of the complex or its named subunits. Also, the terms “124 antibody” and “mAb124” are intended to include within their scope proteins specifically recited herein as well as all substantially homologous analogs and allelic variations.
  • 1133 antibody and “mAb1133”, and any variants not specifically listed, may be used herein interchangeably, and as used throughout the present application and claims refer to proteinaceous material including single or multiple proteins, and extends to those proteins having the amino acid sequence data described herein and presented in SEQ ID NO:32 and SEQ ID NO:37, and the profile of activities set forth herein and in the Claims. Accordingly, proteins displaying substantially equivalent or altered activity are likewise contemplated. These modifications may be deliberate, for example, such as modifications obtained through site-directed mutagenesis, or may be accidental, such as those obtained through mutations in hosts that are producers of the complex or its named subunits. Also, the terms “11133 antibody” and “mAb1133” are intended to include within their scope proteins specifically recited herein as well as all substantially homologous analogs and allelic variations.
  • amino acid residues described herein are preferred to be in the “L” isomeric form.
  • residues in the “D” isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property of immunoglobulin-binding is retained by the polypeptide.
  • NH 2 refers to the free amino group present at the amino terminus of a polypeptide.
  • COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide.
  • amino-acid residue sequences are represented herein by formulae whose left and right orientation is in the conventional direction of aminoterminus to carboxy-terminus. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino-acid residues.
  • the above Table is presented to correlate the three-letter and one-letter notations which may appear alternately herein.
  • a “replicon” is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control.
  • a “vector” is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.
  • a “DNA molecule” refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form, or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes.
  • linear DNA molecules e.g., restriction fragments
  • viruses e.g., plasmids, and chromosomes.
  • sequences may be described herein according to the normal convention of giving only the sequence in the 5′ to 3′ direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).
  • An “origin of replication” refers to those DNA sequences that participate in DNA synthesis.
  • a DNA “coding sequence” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxyl) terminus.
  • a coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences.
  • a polyadenylation signal and transcription termination sequence will usually be located 3′ to the coding sequence.
  • Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
  • a “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence.
  • the promoter sequence is bounded at its 3′terminus by the transcription initiation site and extends upstream (5′ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • a transcription initiation site (conveniently defined by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • Eukaryotic promoters will often, but not always, contain “TATA” boxes and “CAT” boxes.
  • Prokaryotic promoters contain Shine Dalgarno sequences in addition to the ⁇ 10 and ⁇ 35 consensus sequences.
  • An “expression control sequence” is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence.
  • a coding sequence is “under the control” of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.
  • a “signal sequence” can be included before the coding sequence. This sequence encodes a signal peptide, N-terminal to the polypeptide, that communicates to the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the media, and this signal peptide is clipped off by the host cell before the protein leaves the cell. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes.
  • oligonucleotide as used herein in referring to the probe of the present invention, is defined as a molecule comprised of two or more ribonucleotides, preferably more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of the oligonucleotide.
  • primer refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH.
  • the primer may be either single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent.
  • the exact length of the primer will depend upon many factors, including temperature, source of primer and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.
  • the primers herein are selected to be “substantially” complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5′end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the strand to hybridize therewith and thereby form the template for the synthesis of the extension product.
  • restriction endonucleases and “restriction enzymes” refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.
  • a cell has been “transformed” by exogenous or heterologous DNA when such DNA has been introduced inside the cell.
  • the transforming DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.
  • the transforming DNA may be maintained on an episomal element such as a plasmid.
  • a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA.
  • a “clone” is a population of cells derived from a single cell or common ancestor by mitosis.
  • a “cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.
  • Two DNA sequences are “substantially homologous” when at least about 75% (preferably at least about 80%, and most preferably at least about 90 or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II, supra; Nucleic Acid Hybridization, supra.
  • DNA sequences encoding specific binding members (antibodies) of the invention which code for antibodies having the disclosed sequences but which are degenerate to such sequences.
  • degenerate to is meant that a different three-letter codon is used to specify a particular amino acid. It is well known in the art that the following codons can be used interchangeably to code for each specific amino acid:
  • codons specified above are for RNA sequences.
  • the corresponding codons for DNA have a T substituted for U.
  • Mutations can be made in, for example, the disclosed sequences of antibodies of the present invention, such that a particular codon is changed to a codon which codes for a different amino acid.
  • Such a mutation is generally made by making the fewest nucleotide changes possible.
  • a substitution mutation of this sort can be made to change an amino acid in the resulting protein in a non-conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to another grouping) or in a conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to the same grouping).
  • Such a conservative change generally leads to less change in the structure and function of the resulting protein.
  • a non-conservative change is more likely to alter the structure, activity or function of the resulting protein.
  • the present invention should be considered to include sequences containing conservative changes which do not significantly alter the activity or binding characteristics of the resulting protein.
  • Another grouping may be those amino acids with phenyl groups:
  • Another grouping may be according to molecular weight (ie., size of R groups):
  • Amino acid substitutions may also be introduced to substitute an amino acid with a particularly preferable property.
  • a Cys may be introduced a potential site for disulfide bridges with another Cys.
  • a His may be introduced as a particularly “catalytic” site (i.e., His can act as an acid or base and is the most common amino acid in biochemical catalysis).
  • Pro may be introduced because of its particularly planar structure, which induces. (3-turns in the protein's structure.
  • Two amino acid sequences are “substantially homologous” when at least about 70% of the amino acid residues (preferably at least about 80%, and most preferably at least about 90 or 95%) are identical, or represent conservative substitutions.
  • a “heterologous” region of the DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature.
  • the gene when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism.
  • Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.
  • phrases “pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
  • terapéuticaally effective amount is used herein to mean an amount sufficient to prevent, and preferably reduce by at least about 30 percent, preferably by at least 50 percent, preferably by at least 70 percent, preferably by at least 80 percent, preferably by at least 90%, a clinically significant change in the growth or progression or mitotic activity of a target cellular mass, group of cancer cells or tumor, or other feature of pathology.
  • the degree of EGFR activation or activity or amount or number of EGFR positive cells, particularly of antibody or binding member reactive or positive cells may be reduced.
  • a DNA sequence is “operatively linked” to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that DNA sequence.
  • the term “operatively linked” includes having an appropriate start signal (e.g., ATG) in front of the DNA sequence to be expressed and maintaining the correct reading frame to permit expression of the DNA sequence under the control of the expression control sequence and production of the desired product encoded by the DNA sequence. If a gene that one desires to insert into a recombinant DNA molecule does not contain an appropriate start signal, such a start signal can be inserted in front of the gene.
  • standard hybridization conditions refers to salt and temperature conditions substantially equivalent to 5 ⁇ SSC and 65° C. for both hybridization and wash. However, one skilled in the art will appreciate that such “standard hybridization conditions” are dependent on particular conditions including the concentration of sodium and magnesium in the buffer, nucleotide sequence length and concentration, percent mismatch, percent formamide, and the like. Also important in the determination of “standard hybridization conditions” is whether the two sequences hybridizing are RNA-RNA, DNA-DNA or RNA-DNA. Such standard hybridization conditions are easily determined by one skilled in the art according to well known formulae, wherein hybridization is typically 10-20° C. below the predicted or determined Tm with washes of higher stringency, if desired.
  • the present invention provides a novel specific binding member, particularly an antibody or fragment thereof, including immunogenic fragments, which recognizes an EGFR epitope which is found in tumorigenic, hyperproliferative or abnormal cells wherein the epitope is enhanced or evident upon aberrant post-translational modification and not detectable in normal or wild-type cells.
  • the binding member such as the antibody, recognizes an EGFR epitope which is enhanced or evident upon simple carbohydrate modification or early glycosylation and is reduced or not evident in the presence of complex carbohydrate modification or glycosylation.
  • the specific binding member, such as the antibody or fragment thereof does not bind to or recognize normal or wild-type cells containing normal or wild-type EGFR epitope in the absence of overexpression and in the presence of normal EGFR post-translational modification.
  • the present invention further provides novel antibodies 806, 175, 124, 1133, ch806, and hu806 and fragment thereof, including immunogenic fragments, which recognizes an EGFR epitope, particularly the EGFR peptide ( 287 CGADSYEMEEDGVRKC 302 (SEQ ID NO:14)), which is exposed in tumorigenic, hyperproliferative or abnormal cells wherein the epitope is enhanced, revealed, or evident and not detectable in normal or wild-type cells.
  • the antibody recognizes an EGFR epitope which is enhanced or evident upon simple carbohydrate modification or early glycosylation and is reduced or not evident in the presence of complex carbohydrate modification or glycosylation.
  • the antibody or fragment thereof does not bind to or recognize normal or wild-type cells containing normal or wild-type EGFR epitope in the absence of overexpression, amplification, or a tumorigenic event.
  • the present inventors have discovered the novel monoclonal antibodies 806, 175, 124, 1133, ch806, and hu806 which specifically recognize amplified wild-type EGFR and the de2-7 EGFR, yet bind to an epitope distinct from the unique junctional peptide of the de2-7 EGFR mutation. Additionally, while mAb806, mAb175, mAb124, mAb1133, and hu806 do not recognize the normal, wild-type EGFR expressed on the cell surface of glioma cells, they do bind to the extracellular domain of the EGFR immobilized on the surface of ELISA plates, indicating a conformational epitope with a polypeptide aspect.
  • mAb806, mAb175, mAb124, mAb1133, ch806, and hu806 do not bind significantly to normal tissues such as liver and skin, which express levels of endogenous wtEGFR that are higher than in most other normal tissues, but wherein EGFR is not overexpressed or amplified.
  • mAb806, mAb175, mAb124, mAb1133, and hu806 demonstrate novel and useful specificity, recognizing de2-7 EGFR and amplified EGFR, while not recognizing normal, wild-type EGFR or the unique junctional peptide which is characteristic of de2-7 EGFR.
  • mAb806, mAb175, mAb124, mAb1133, and hu806 of the present invention comprises the VH and VL CDR domain amino acid sequences depicted in FIGS. 16 and 17 ; 74 B and 75 B; 51 B and 51 D; 52 B and 54 D; and 55 A and 55 B, respectively (SEQ ID NOS:2 and 4; 129 and 134; 22 and 27; 32 and 37; and 42 and 47, respectively).
  • the invention provides an antibody capable of competing with the 175 antibody, under conditions in which at least 10% of an antibody having the VB and VL sequences of the 175 antibody is blocked from binding to de2-7EGFR by competition with such an antibody in an ELISA assay.
  • anti-idiotype antibodies are contemplated herein.
  • the present invention relates to specific binding members, particularly antibodies or fragments thereof, which recognizes an EGFR epitope which is present in cells expressing amplified EGFR or expressing the de2-7 EGFR and not detectable in cells expressing normal or wild-type EGFR, particularly in the presence of normal posttranslational modification.
  • an additional non-limiting observation or characteristic of the antibodies of the present invention is their recognition of their epitope in the presence of high mannose groups, which is a characteristic of early glycosylation or simple carbohydrate modification.
  • altered or aberrant glycosylation facilitates the presence and/or recognition of the antibody epitope or comprises a portion of the antibody epitope.
  • Glycosylation includes and encompasses the post-translational modification of proteins, termed glycoproteins, by addition of oligosaccarides.
  • Oligosaccharides are added at glycosylation sites in glycoproteins, particularly including N-linked oligosaccharides and O-linked oligosaccharides.
  • N-linked oligosaccharides are added to an Asn residue, particularly wherein the Asn residue is in the sequence N—X—S/T, where X cannot be Pro or Asp, and are the most common ones found in glycoproteins.
  • a high mannose type oligosaccharide (generally comprised of dolichol, N-Acetylglucosamine, mannose and glucose is first formed in the endoplasmic reticulum (ER). The high mannose type glycoproteins are then transported from the ER to the Golgi, where further processing and modification of the oligosaccharides normally occurs. O-linked oligosaccharides are added to the hydroxyl group of Ser or Thr residues. In O-linked oligosaccharides, N Acetylglucosamine is first transferred to the Ser or Thr residue by N Acetylglucosaminyltransferase in the ER. The protein then moves to the Golgi where further modification and chain elongation occurs.
  • the present inventors have discovered novel monoclonal antibodies, exemplified herein by the antibodies designated mAb806 (and its chimeric ch806), mAb175, mAb124, mAb1133, and hu806 which specifically recognize amplified wild-type EGFR and the de2-7 EGFR, yet bind to an epitope distinct from the unique junctional peptide of the de2-7 EGFR mutation.
  • the antibodies of the present invention specifically recognize overexpressed EGFR, including amplified EGFR and mutant EGFR (exemplified herein by the de2-7 mutation), particularly upon aberrant post-translational modification.
  • these antibodies do not recognize the normal, wild-type EGFR expressed on the cell surface of glioma cells, they do bind to the extracellular domain of the EGFR immobilized on the surface of ELISA plates, indicating a conformational epitope with a polypeptide aspect. Importantly, these antibodies do not bind significantly to normal tissues such as liver and skin, which express levels of endogenous wtEGFR that are higher than in most other normal tissues, but wherein EGFR is not overexpressed or amplified. Thus, these antibodies demonstrate novel and useful specificity, recognizing de2-7 EGFR and amplified EGFR, while not recognizing normal, wild-type EGFR or the unique junctional peptide which is characteristic of de2-7 EGFR.
  • the antibodies are ones which have the characteristics of the antibodies which the inventors have identified and characterized, in particular recognizing amplified EGFR and de2-7EGFR.
  • the antibodies are mAb806, mAb175, mAb124, mAb1133, and hu806 or active fragments thereof.
  • the antibody of the present invention comprises the VH and VL amino acid sequences depicted FIGS. 16 and 17 ; 74 B and 75 B; 51 B and 51 D; 52 B and 54 D; and 55 A and 55 B, respectively.
  • the epitope of the specific binding member or antibody is located within the region comprising residues 273-501 of the mature normal or wild-type EGFR sequence, and preferably the epitope comprises residues 287-302 of the mature normal or wild-type EGFR sequence. Therefore, also provided are specific binding proteins, such as antibodies, which bind to the de2-7 EGFR at an epitope located within the region comprising residues 273-501 of the EGFR sequence, and comprising residues 287-302 of the EGFR sequence.
  • the epitope may be determined by any conventional epitope mapping techniques known to the person skilled in the art. Alternatively, the DNA sequences encoding residues 273-501 and 287-302 could be digested, and the resultant fragments expressed in a suitable host. Antibody binding could be determined as mentioned above.
  • the member will bind to an epitope comprising residues 273-501, and more specifically comprising residues 287-302, of the mature normal or wild-type EGFR.
  • other antibodies which show the same or a substantially similar pattern of reactivity also form an aspect of the invention. This may be determined by comparing such members with an antibody comprising the VH and VL domains shown in SEQ ID NOS:2 and 4; 129 and 134; 22 and 27; 32 and 37; and 42 and 47, respectively. The comparison will typically be made using a Western blot in which binding members are bound to duplicate blots prepared from a nuclear preparation of cells so that the pattern of binding can be directly compared.
  • the invention provides an antibody capable of competing with mAb806 under conditions in which at least 10% of an antibody having the VH and VL sequences of one of such antibodies is blocked from binding to de2-7EGFR by competition with such an antibody in an ELISA assay.
  • anti-idiotype antibodies are contemplated and are illustrated herein.
  • the invention provides an antibody capable of competing with mAb175, mAb124, and/or mAb1133 under conditions in which at least 10% of an antibody having the VH and VL sequences of one of such antibodies is blocked from binding to de2-7EGFR by competition with such an antibody in an ELISA assay.
  • anti-idiotype antibodies are contemplated and are illustrated herein.
  • the invention provides an antibody capable of competing with mAb806, mAb175, mAb124, mAb1133 and/or hu806, under conditions in which at least 10% of an antibody having the VH and VL sequences of one of such antibodies is blocked from binding to de2-7EGFR by competition with such an antibody in an ELISA assay.
  • anti-idiotype antibodies are contemplated and are illustrated herein.
  • compositions of the peptide of the present invention include pharmaceutical composition and immunogenic compositions.
  • cells overexpressing EGFR e.g. by amplification or expression of a mutant or variant EGFR
  • those demonstrating aberrant post-translational modification may be recognized, isolated, characterized, targeted and treated or eliminated utilizing the binding member(s), particularly antibody(ies) or fragments thereof of the present invention.
  • a method of treatment of a tumor, a cancerous condition, a precancerous condition, and any condition related to or resulting from hyperproliferative cell growth comprising administration of mAb806, mAb175, mAb124, mAb1133, and/or hu806.
  • the antibodies of the present invention can thus specifically categorize the nature of EGFR tumors or tumorigenic cells, by staining or otherwise recognizing those tumors or cells wherein EGFR overexpression, particularly amplification and/or EGFR mutation, particularly de2-7EGFR, is present. Further, the antibodies of the present invention, as exemplified by mAb806 (and chimeric antibody ch806), mAb175, mAb124, mAb1133, and hu806, demonstrate significant in vivo anti-tumor activity against tumors containing amplified EGFR and against de2-7 EGFR positive xenografts.
  • the specific binding member of the invention recognizes tumor-associated forms of the EGFR (de2-7 EGFR and amplified EGFR) but not the normal, wild-type receptor when expressed in normal cells. It is believed that antibody recognition is dependent upon an aberrant posttranslational modification (e.g., a unique glycosylation, acetylation or phosphorylation variant) of the EGFR expressed in cells exhibiting overexpression of the EGFR gene.
  • an aberrant posttranslational modification e.g., a unique glycosylation, acetylation or phosphorylation variant
  • antibodies of the present invention have been used in therapeutic studies and shown to inhibit growth of overexpressing (e.g. amplified) EGFR xenografts and human de2-7 EGFR expressing xenografts of human tumors and to induce significant necrosis within such tumors.
  • the antibodies of the present invention inhibit the growth of intracranial tumors in a preventative model.
  • This model involves injecting glioma cells expressing de2-7 EGFR into nude mice and then injecting the antibody intracranially either on the same day or within 1 to 3 days, optionally with repeated doses.
  • the doses of antibody are suitably about 10 ⁇ g.
  • Mice injected with antibody are compared to controls, and it has been found that survival of the treated mice is significantly increased.
  • a method of treatment of a tumor, a cancerous condition, a precancerous condition, and any condition related to or resulting from hyperproliferative cell growth comprising administration of a specific binding member of the invention.
  • Antibodies of the present invention are designed to be used in methods of diagnosis and treatment of tumors in human or animal subjects, particularly epithelial tumors. These tumors may be primary or secondary solid tumors of any type including, but not limited to, glioma, breast, lung, prostate, head or neck tumors.
  • Immortal, antibody-producing cell lines can also be created by techniques other than fusion, such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See, e.g., M. Schreier et al., “Hybridoma Techniques” (1980); Hammering et al., “Monoclonal Antibodies And T cell Hybridomas” (1981); Kennett et al., “Monoclonal Antibodies” (1980); see also U.S. Pat. Nos. 4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,451,570; 4,466,917; 4,472,500; 4,491,632; and 4,493,890.
  • Panels of monoclonal antibodies produced against EFGR can be screened for various properties; i.e., isotype, epitope, affinity, etc.
  • monoclonal antibodies that mimic the activity of EFGR or its subunits can be readily identified in specific binding member activity assays.
  • High affinity antibodies are also useful when immunoaffinity purification of native or recombinant specific binding member is possible.
  • a myeloma or other self-perpetuating cell line is fused with lymphocytes obtained from the spleen of a mammal hyperimmunized with an appropriate EGFR.
  • Splenocytes are typically fused with myeloma cells using polyethylene glycol (PEG) 6000.
  • Fused hybrids are selected by their sensitivity to HAT.
  • Hybridomas producing a monoclonal antibody useful in practicing this invention are identified by their ability to immunoreact with the present antibody or binding member and their ability to inhibit specified tumorigenic or hyperproliferative activity in target cells.
  • a monoclonal antibody useful in practicing the present invention can be produced by initiating a monoclonal hybridoma culture comprising a nutrient medium containing a hybridoma that secretes antibody molecules of the appropriate antigen specificity.
  • the culture is maintained under conditions and for a time period sufficient for the hybridoma to secrete the antibody molecules into the medium.
  • the antibody-containing medium is then collected.
  • the antibody molecules can then be further isolated by well-known techniques.
  • DMEM Dulbecco's minimal essential medium
  • fetal calf serum An exemplary inbred mouse strain is the Balb/c.
  • EGFR or a peptide analog is used either alone or conjugated to an immunogenic carrier, as the immunogen in the before described procedure for producing anti-EGFR monoclonal antibodies.
  • the hybridomas are screened for the ability to produce an antibody that immunoreacts with the EGFR present in tumorigenic, abnormal or hyperproliferative cells.
  • Other anti-EGFR antibodies include but are not limited to the HuMAX-EGFr antibody from Genmab/Medarex, the 108 antibody (ATCC HB9764) and U.S. Pat. No. 6,217,866, and antibody 14E1 from Schering AG (U.S. Pat. No. 5,942,602).
  • the CDR1 regions comprising amino acid sequences substantially as set out as the CDR1 regions of SEQ ID NOS:2 and 4; 129 and 134; 22 and 27; 32 and 37; and 42 and 47, respectively, will be carried in a structure which allows for binding of the CDR1 regions to an tumor antigen.
  • this is preferably carried by the VL region of SEQ ID NO:4 (and similarly for the other recited sequences).
  • the CDR2 regions comprising amino acid sequences substantially as set out as the CDR2 regions of SEQ ID NOS:2 and 4; 129 and 134; 22 and 27; 32 and 37; and 42 and 47, respectively, will be carried in a structure which allows for binding of the CDR2 regions to an tumor antigen.
  • this is preferably carried by the VL region of SEQ ID NO:4 (and similarly for the other recited sequences).
  • the CDR3 regions comprising amino acid sequences substantially as set out as the CDR3 regions of SEQ ID NOS:2 and 4; 129 and 134; 22 and 27; 32 and 37; and 42 and 47, respectively, will be carried in a structure which allows for binding of the CDR3 regions to an tumor antigen.
  • this is preferably carried by the VL region of SEQ ID NO:4 (and similarly for the other recited sequences).
  • CDR regions for example CDR3 regions
  • CDR regions of the invention will be either identical or highly homologous to the specified regions of SEQ ID NOS:2 and 4; 129 and 134; 22 and 27; 32 and 37; and 42 and 47, respectively.
  • highly homologous it is contemplated that only a few substitutions, preferably from 1 to 8, preferably from 1 to 5, preferably from 1 to 4, or from 1 to 3 or 1 or 2 substitutions may be made in one or more of the CDRs.
  • the structure for carrying the CDRs of the invention will generally be of an antibody heavy or light chain sequence or substantial portion thereof in which the CDR regions are located at locations corresponding to the CDR region of naturally occurring VH and VL antibody variable domains encoded by rearranged immunoglobulin genes.
  • the structures and locations of immunoglobulin variable domains may be determined by reference to Kabat, E. A. et al, Sequences of Proteins of Immunological Interest. 4th Edition. US Department of Health and Human Services. 1987, and updates thereof, now available on the Internet (http://immuno.bme.nwu.edu)).
  • CDR determinations can be made in various ways. For example, Kabat, Chothia and combined domain determination analyses may be used. In this regard, see for example http://www.bioinf.org.uk/abs/#cdrid.
  • the amino acid sequences substantially as set out as the VH chain CDR residues in the inventive antibodies are in a human heavy chain variable domain or a substantial portion thereof, and the amino acid sequences substantially as set out as the VL chain CDR residues in the inventive antibodies are in a human light chain variable domain or a substantial portion thereof.
  • variable domains may be derived from any germline or rearranged human variable domain, or may be a synthetic variable domain based on consensus sequences of known human variable domains.
  • the CDR3-derived sequences of the invention for example, as defined in the preceding paragraph, may be introduced into a repertoire of variable domains lacking CDR3 regions, using recombinant DNA technology.
  • Marks et al ( Bio/Technology, 1992,10:779-783) describe methods of producing repertoires of antibody variable domains in which consensus primers directed at or adjacent to the 5′end of the variable domain area are used in conjunction with consensus primers to the third framework region of human VH genes to provide a repertoire of VH variable domains lacking a CDR3. Marks et al further describe how this repertoire may be combined with a CDR3 of a particular antibody.
  • the CDR3-derived sequences of the present invention may be shuffled with repertoires of VH or VL domains lacking a CDR3, and the shuffled complete VH or VL domains combined with a cognate VL or VH domain to provide specific binding members of the invention.
  • the repertoire may then be displayed in a suitable host system such as the phage display system of W092/01047 so that suitable specific binding members may be selected.
  • a repertoire may consist of from anything from 10 4 individual members upwards, for example from 10 6 to 10 8 or 10 10 members.
  • a further alternative is to generate novel VH or VL regions carrying the CDR3derived sequences of the invention using random mutagenesis of, for example, the mAb806 VH or VL genes to generate mutations within the entire variable domain.
  • random mutagenesis of, for example, the mAb806 VH or VL genes to generate mutations within the entire variable domain.
  • Another method which may be used is to direct mutagenesis to CDR regions of VH or VL genes.
  • Such techniques are disclosed by Barbas et al, (1994, Proc. Natl. Acad. Sci., USA, 91:3809-3813) and Schier et al (1996, J. Mol. Biol. 263:551-567).
  • a substantial portion of an immunoglobulin variable domain will comprise at least the three CDR regions, together with their intervening framework regions.
  • the portion will also include at least about 50% of either or both of the first and fourth framework regions, the 50% being the C-terminal 50% of the first framework region and the N-terminal 50% of the fourth framework region.
  • Additional residues at the N-terminal or C-terminal end of the substantial part of the variable domain may be those not normally associated with naturally occurring variable domain regions.
  • construction of specific binding members of the present invention made by recombinant DNA techniques may result in the introduction of N- or C-terminal residues encoded by linkers introduced to facilitate cloning or other manipulation steps.
  • Other manipulation steps include the introduction of linkers to join variable domains of the invention to further protein sequences including immunoglobulin heavy chains, other variable domains (for example in the production of diabodies) or protein labels as discussed in more detail below.
  • binding members comprising a pair of binding domains based on sequences substantially set out in SEQ ID NOS:2 and 4; 129 and 134; 22 and 27; 32 and 37; and 42 and 47, respectively, are preferred, single binding domains based on these sequences form further aspects of the invention.
  • binding domains based on the sequence substantially set out in VH chains such binding domains may be used as targeting agents for tumor antigens since it is known that immunoglobulin VH domains are capable of binding target antigens in a specific manner.
  • these domains may be used to screen for complementary domains capable of forming a two-domain specific binding member which has in vivo properties as good as or equal to the mAb806, ch806, mAb175, mAb124, mAb1133 and hu806 antibodies disclosed herein.
  • phage display screening methods using the so-called hierarchical dual combinatorial approach as disclosed in U.S. Pat. No. 5,969,108 in which an individual colony containing either an H or L chain clone is used to infect a complete library of clones encoding the other chain (L or H) and the resulting two-chain specific binding member is selected in accordance with phage display techniques such as those described in that reference. This technique is also disclosed in Marks et al, ibid.
  • Specific binding members of the present invention may further comprise antibody constant regions or parts thereof.
  • specific binding members based on VL chain sequences may be attached at their C-terminal end to antibody light chain constant domains including human Ck of C ⁇ chains, preferably C ⁇ chains.
  • specific binding members based on VH chain sequences may be attached at their C-terminal end to all or part of an immunoglobulin heavy chain derived from any antibody isotype, e.g. IgG, IgA, IgE, IgD and IgM and any of the isotype sub-classes, particularly IgG1, IgG2b, and IgG4. IgG1 is preferred.
  • mAb monoclonal antibody
  • the engineered mAbs have markedly reduced or absent immunogenicity, increased serum half-life and the human Fc portion of the mAb increases the potential to recruit the immune effectors of complement and cytotoxic cells (Clark 2000). Investigations into the biodistribution, pharmacokinetics and any induction of an immune response to clinically administered mAbs requires the development of analyses to discriminate between the pharmaceutical and endogenous proteins.
  • the antibodies, or any fragments thereof, may also be conjugated or recombinantly fused to any cellular toxin, bacterial or other, e.g. pseudomonas exotoxin, ricin, or diphtheria toxin.
  • the part of the toxin used can be the whole toxin, or any particular domain of the toxin.
  • Such antibody-toxin molecules have successfully been used for targeting and therapy of different kinds of cancers, see e.g. Pastan, Biochim Biophys Acta. 1997 Oct. 24; 1333 (2):C1-6; Kreitman et al., N. Engl. J. Med. 2001 Jul. 26; 345 (4):241-7; Schnell et al., Leukemia. 2000 January; 14 (1):129-35; Ghetie et al., Mol. Biotechnol. 2001 July; 18 (3):251-68.
  • Bi-and tri-specific multimers can be formed by association of different scFv molecules and have been designed as cross-linking reagents for T-cell recruitment into tumors (immunotherapy), viral retargeting (gene therapy) and as red blood cell agglutination reagents (immunodiagnostics), see e.g. Todorovska et al., J Immunol Methods. 2001 Feb. 1; 248 (1-2):47-66; Tomlinson et al., Methods Enzymol. 2000; 326:461-79; McCall et al., J. Immunol. 2001 May 15; 166 (10):6112-7.
  • Fully human antibodies can be prepared by immunizing transgenic mice carrying large portions of the human immunoglobulin heavy and light chains.
  • mice examples of such mice are the XenomouseTM (Abgenix, Inc.) (U.S. Pat. Nos. 6,075,181 and 6,150,584), the HuMAb-MouseTM (Medarex, Inc./GenPharm) (U.S. Pat. Nos. 5,545,806 and 5,569,825), the TransChromo Mouse (Kirin) and the KM Mouse (Medarex/Kirin), are well known within the art.
  • Antibodies can then be prepared by, e.g. standard hybridoma technique or by phage display. These antibodies will then contain only fully human amino acid sequences.
  • Fully human antibodies can also be generated using phage display from human libraries.
  • Phage display may be performed using methods well known to the skilled artisan, as in Hoogenboom et al. and Marks et al. (Hoogenboom H R and Winter G. (1992) J. Mol. Biol. 227 (2):381-8; Marks J D et al. (1991) J. Mol. Biol. 222 (3):581-97; and also U.S. Pat. Nos. 5,885,793 and 5,969,108).
  • the in vivo properties, particularly with regard to tumor:blood ratio and rate of clearance, of specific binding members of the invention will be at least comparable to mAb806.
  • a specific binding member Following administration to a human or animal subject such a specific binding member will show a peak tumor to blood ratio of >1:1.
  • the specific binding member will also have a tumor to organ ratio of greater than 1:1, preferably greater than 2:1, more preferably greater than 5:1.
  • the specific binding member will also have an organ to blood ratio of ⁇ 1:1 in organs away from the site of the tumor. These ratios exclude organs of catabolism and secretion of the administered specific binding member.
  • the binding members are secreted via the kidneys and there is greater presence here than other organs.
  • clearance will be at least in part, via the liver.
  • the peak localization ratio of the intact antibody will normally be achieved between 10 and 200 hours following administration of the specific binding member. More particularly, the ratio may be measured in a tumor xenograft of about 0.2-1.0 g formed subcutaneously in one flank of an athymic nude mouse.
  • Antibodies of the invention may be labelled with a detectable or functional label.
  • Detectable labels include, but are not limited to, radiolabels such as the isotopes 3 H, 14 C, 32 P, 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 F, 90 Y, 121 I, 124 I, 125 , 131 I, 111 In, 211 At, 198 Au, 67 CU, 225 Ac, 213 Bi, 99 Tc and 186 Re, which may be attached to antibodies of the invention using conventional chemistry known in the art of antibody imaging.
  • Labels also include fluorescent labels and labels used conventionally in the art for MRI-CT imagine. They also include enzyme labels such as horseradish peroxidase. Labels further include chemical moieties such as biotin which may be detected via binding to a specific cognate detectable moiety, e.g. labelled avidin.
  • Functional labels include substances which are designed to be targeted to the site of a tumor to cause destruction of tumor tissue.
  • Such functional labels include cytotoxic drugs such as 5-fluorouracil or ricin and enzymes such as bacterial carboxypeptidase or nitroreductase, which are capable of converting prodrugs into active drugs at the site of a tumor.
  • antibodies including both polyclonal and monoclonal antibodies, and drugs that modulate the production or activity of the specific binding members, antibodies and/or their subunits may possess certain diagnostic applications and may for example, be utilized for the purpose of detecting and/or measuring conditions such as cancer, precancerous lesions, conditions related to or resulting from hyperproliferative cell growth or the like.
  • the specific binding members, antibodies or their subunits may be used to produce both polyclonal and monoclonal antibodies to themselves in a variety of cellular media, by known techniques such as the hybridoma technique utilizing, for example, fused mouse spleen lymphocytes and myeloma cells.
  • small molecules that mimic or antagonize the activity(ies) of the specific binding members of the invention may be discovered or synthesized, and may be used in diagnostic and/or therapeutic protocols.
  • the radiolabeled specific binding members are useful in in vitro diagnostics techniques and in in vivo radioimaging techniques and in radioimmunotherapy.
  • the specific binding members of the present invention may be conjugated to an imaging agent rather than a radioisotope(s), including but not limited to a magnetic resonance image enhancing agent, wherein for instance an antibody molecule is loaded with a large number of paramagnetic ions through chelating groups.
  • chelating groups include EDTA, porphyrins, polyamines crown ethers and polyoximes.
  • paramagnetic ions examples include gadolinium, iron, manganese, rhenium, europium, lanthanium, holmium and erbium.
  • radiolabeled specific binding members particularly antibodies and fragments thereof, particularly radioimmunoconjugates, are useful in radioimmunotherapy, particularly as radiolabeled antibodies for cancer therapy.
  • the radiolabelled specific binding members, particularly antibodies and fragments thereof are useful in radioimmuno-guided surgery techniques, wherein they can identify and indicate the presence and/or location of cancer cells, precancerous cells, tumor cells, and hyperproliferative cells, prior to, during or following surgery to remove such cells.
  • Immunoconjugates or antibody fusion proteins of the present invention wherein the specific binding members, particularly antibodies and fragments thereof, of the present invention are conjugated or attached to other molecules or agents further include, but are not limited to binding members conjugated to a chemical ablation agent, toxin, immunomodulator, cytokine, cytotoxic agent, chemotherapeutic agent or drug.
  • Radioimmunotherapy has entered the clinic and demonstrated efficacy using various antibody immunoconjugates. 1311 labeled humanized anti-carcinoembryonic antigen (anti-CEA) antibody hMN-14 has been evaluated in colorectal cancer (Behr T M et al (2002) Cancer 94 (4Suppl): 1373-81) and the same antibody with 90Y label has been assessed in medullary thyroid carcinoma (Stein R et al (2002) Cancer 94 (1):51-61). Radioimmunotherapy using monoclonal antibodies has also been assessed and reported for non-Hodgkin's lymphoma and pancreatic cancer (Goldenberg D M (2001) Crit. Rev. Oncol. Hematol.
  • Radioimmunotherapy methods with particular antibodies are also described in U.S. Pat. Nos. 6,306,393 and 6,331,175.
  • Radioimmunoguided surgery (RIGS) has also entered the clinic and demonstrated efficacy and usefulness, including using anti-CEA antibodies and antibodies directed against tumor-associated antigens (Kim J C et al (2002) Jut. 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 D G et al (1997) Cancer Biother. Radiopharm. 12 (4):287-94).
  • Antibodies of the present invention may be administered to a patient in need of treatment via any suitable route, usually by injection into the bloodstream or CSF, or directly into the site of the tumor.
  • the precise dose will depend upon a number of factors, including whether the antibody is for diagnosis or for treatment, the size and location of the tumor, the precise nature of the antibody (whether whole antibody, fragment, diabody, etc), and she nature of the detectable or functional label attached to the antibody.
  • a radionuclia is used for therapy, a suitable maximum single dose is about 45 mCi/m 2 , to a maximum of about 250 mCi/m 2 .
  • Preferable dosage is in the range of 15 to 40 mCi, with a further preferred dosage range of 20 to 30 mCi, or 10 to 30 mCi.
  • Such therapy may require bone marrow or stem cell replacement.
  • a typical antibody dose for either tumor imaging or tumor treatment will be in the range of from 0.5 to 40 mg, preferably from 1 to 4 mg of antibody in F(ab′)2 form.
  • Naked antibodies are preferable administered in doses of 20 to 1000 mg protein per dose, or 20 to 500 mg protein per dose, or 20 to 100 mg protein per dose. This is a dose for a single treatment of an adult patient, which may be proportionally adjusted for children and infants, and also adjusted for other antibody formats in proportion to molecular weight. Treatments may be repeated at daily, twice-weekly, weekly or monthly intervals, at the discretion of the physician.
  • formulations may include a second binding protein, such as the EGPR binding proteins described supra.
  • this second binding protein is a monoclonal antibody such as 528 or 225, discussed infra.
  • Specific binding members of the present invention will usually be administered in the form of a pharmaceutical composition, which may comprise at least one component in addition to the specific binding member.
  • compositions according to the present invention may comprise, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • a pharmaceutically acceptable excipient such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. intravenous.
  • compositions for oral administration may be in tablet, capsule, powder or liquid form.
  • a tablet may comprise a solid carrier such as gelatin or an adjuvant.
  • Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
  • the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection.
  • Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included, as required.
  • compositions may be administered alone or in combination with other treatments, therapeutics or agents, either simultaneously or sequentially dependent upon the condition to be treated.
  • the present invention contemplates and includes compositions comprising the binding member, particularly antibody or fragment thereof, herein described and other agents or therapeutics such as anti-cancer agents or therapeutics, hormones, anti-EGFR agents or antibodies, or immune modulators. More generally these anti-cancer agents may be tyrosine kinase inhibitors or phosphorylation cascade inhibitors, post-translational modulators, cell growth or division inhibitors (e.g. anti-mitotics), or signal transduction inhibitors.
  • compositions can be administered in combination (either sequentially (i.e.
  • tyrosine kinase inhibitors including, but not limited to AG1478 and ZD1839, ST1571, OSI-774, SU-6668
  • doxorubicin including, but not limited to AG1478 and ZD1839, ST1571, OSI-774, SU-6668
  • doxorubicin including, but not limited to AG1478 and ZD1839, ST1571, OSI-774, SU-6668
  • doxorubicin including, but not limited to AG1478 and ZD1839, ST1571, OSI-774, SU-6668
  • doxorubicin including, but not limited to AG1478 and ZD1839, ST1571, OSI-774, SU-6668
  • doxorubicin including, but not limited to AG1478 and ZD1839, ST1571, OSI-774, SU-6668
  • doxorubicin including, but not limited to AG1478 and ZD1839, ST1571, OSI-774, SU-
  • these agents may be anti-EGFR specific agents, or tyrosine kinase inhibitors such as AG1478, ZD1839, ST1571, OSI-774, or SU-6668 or may be more general anti-cancer and anti-neoplastic agents such as doxorubicin, cisplatin, temozolomide, nitrosoureas, procarbazine, vincristine, hydroxyurea, 5-fluoruracil, cytosine arabinoside, cyclophosphamide, epipodophyllotoxin, carmustine, or lomustine.
  • doxorubicin cisplatin
  • temozolomide nitrosoureas
  • procarbazine vincristine
  • hydroxyurea 5-fluoruracil
  • cytosine arabinoside cyclophosphamide
  • epipodophyllotoxin carmustine, or lomustine.
  • composition may be administered with hormones such as dexamethasone, immune modulators, such as interleukins, tumor necrosis factor (TNF) or other growth factors or cytokines which stimulate the immune response and reduction or elimination of cancer cells or tumors.
  • hormones such as dexamethasone
  • immune modulators such as interleukins, tumor necrosis factor (TNF) or other growth factors or cytokines which stimulate the immune response and reduction or elimination of cancer cells or tumors.
  • TNF tumor necrosis factor
  • An immune modulator such as TNF may be combined together with a member of the invention in the form of a bispecific antibody recognizing the EGFR epitope recognized by the inventive antibodies, as well as binding to TNF receptors.
  • the composition may also be administered with, or may include combinations along with other anti-EGFR antibodies, including but not limited to the anti-EGFR antibodies 528, 225, SC-03, DR8.3, L8A4, Y10, ICR62 and ABX-EGF.
  • the present invention contemplates and includes therapeutic compositions for the use of the binding member in combination with conventional radiotherapy. It has been indicated that treatment with antibodies targeting EGF receptors can enhance the effects of conventional radiotherapy (Milas et al., Clin. Cancer Res. 2000 February:6 (2):701, Huang et al., Clin. Cancer Res. 2000 June:6 (6):2166).
  • combinations of the binding member of the present invention particularly an antibody or fragment thereof, preferably the mAb806, ch806, mAb175, mAb124, mAb1133 or hu806 or a fragment thereof, and anti-cancer therapeutics, particularly anti-EGFR therapeutics, including other anti-EGFR antibodies, demonstrate effective therapy, and particularly synergy, against xenografted tumors.
  • anti-cancer therapeutics particularly anti-EGFR therapeutics, including other anti-EGFR antibodies
  • AG 1478 (4-(3-chloroanilino)-6,7-dimethoxyquinazoline) is a potent and selective inhibitor of the EGF receptor kinase and is particularly described in U.S. Pat. No. 5,457,105, incorporated by reference herein in its entirety (see also, Liu, W. et al (1999) J. Cell Sci. 112:2409; Eguchi, S. et al. (1998) J. Biol. Chem. 273:8890; Levitsky, A. and Gazit, A. (1995) Science 267:1782).
  • the Specification Examples further demonstrate therapeutic synergy of antibodies of the present invention with other anti-EGFR antibodies, particularly with the 528 anti-EGFR antibody.
  • a subject therapeutic composition includes, in admixture, a pharmaceutically acceptable excipient (carrier) and one or more of a specific binding member, polypeptide analog thereof or fragment thereof, as described herein as an active ingredient.
  • the composition comprises an antigen capable of modulating the specific binding of the present binding member/antibody with a target cell.
  • compositions which contain polypeptides, analogs or active fragments as active ingredients are well understood in the art.
  • such compositions are prepared as injectables, either as liquid solutions or suspensions.
  • solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared.
  • the preparation can also be emulsified.
  • the active therapeutic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof
  • the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents which enhance the effectiveness of the active ingredient.
  • a polypeptide, analog or active fragment can be formulated into the therapeutic composition as neutralized pharmaceutically acceptable salt forms.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide or antibody molecule) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • the therapeutic polypeptide-, analog-or active fragment-containing compositions are conventionally administered intravenously, as by injection of a unit dose, for example.
  • unit dose when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for humans, 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.
  • compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount.
  • the quantity to be administered depends on the subject to be treated, capacity of the subject's immune system to utilize the active ingredient, and degree of EFGR binding capacity desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. However, suitable dosages may range from about 0.1 to 20, preferably about 0.5 to about 10, and more preferably one to several, milligrams of active ingredient per kilogram body weight of individual per day and depend on the route of administration. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration. Alternatively, continuous intravenous infusion sufficient to maintain concentrations of ten nanomolar to ten micromolar in the blood are contemplated.
  • compositions for oral administration may be in tablet, capsule, powder or liquid form.
  • a tablet may comprise a solid carrier such as gelatin or an adjuvant.
  • Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
  • the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection.
  • Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included, as required.
  • the present invention also relates to a variety of diagnostic applications, including methods for detecting the presence of stimuli such as aberrantly expressed EGFR, by reference to their ability to be recognized by the present specific binding member.
  • the EGFR can be used to produce antibodies to itself by a variety of known techniques, and such antibodies could then be isolated and utilized as in tests for the presence of particular EGFR activity in suspect target cells.
  • Diagnostic applications of the specific binding members of the present invention include in vitro and in vivo applications well known and standard to the skilled artisan and based on the present description. Diagnostic assays and kits for in vitro assessment and evaluation of EGFR status, particularly with regard to aberrant expression of EGFR, may be utilized to diagnose, evaluate and monitor patient samples including those known to have or suspected of having cancer, a precancerous condition, a condition related to hyperproliferative cell growth or from a tumor sample.
  • the assessment and evaluation of EGFR status is also useful in determining the suitability of a patient for a clinical trial of a drug or for the administration of a particular chemotherapeutic agent or specific binding member, particularly an antibody, of the present invention, including combinations thereof, versus a different agent or binding member.
  • This type of diagnostic monitoring and assessment is already in practice utilizing antibodies against the HER2 protein in breast cancer (Hercep Test, Dako Corporation), where the assay is also used to evaluate patients for antibody therapy using Herceptin.
  • In vivo applications include imaging of tumors or assessing cancer status of individuals, including radioimaging.
  • the diagnostic method of the present invention comprises examining a cellular sample or medium by means of an assay including an effective amount of an antagonist to an EFGR/protein, such as an anti-EFGR antibody, preferably an affinity-purified polyclonal antibody, and more preferably a mAb.
  • an EFGR/protein such as an anti-EFGR antibody, preferably an affinity-purified polyclonal antibody, and more preferably a mAb.
  • the anti-EFGR antibody molecules used herein be in the form of Fab, Fab′, F (ab′) 2 or F (v) portions or whole antibody molecules.
  • patients capable of benefiting from this method include those suffering from cancer, a pre-cancerous lesion, a viral infection, pathologies involving or resulting from hyperproliferative cell growth or other like pathological derangement.
  • Methods for isolating EFGR and inducing anti-EFGR antibodies and for determining and optimizing the ability of anti-EFGR antibodies to assist in the examination of the target cells are
  • the anti-EFGR antibody used in the diagnostic methods of this invention is an affinity purified polyclonal antibody. More preferably, the antibody is a monoclonal antibody (mAb).
  • the anti-EFGR antibody molecules used herein can be in the form of Fab, Fab′, F (ab′) 2 or F (v) portions of whole antibody molecules.
  • antibody(ies) to the EGFR can be produced and isolated by standard methods including the well known hybridoma techniques.
  • the antibody(ies) to the EGFR will be referred to herein as Ab 1 and antibody(ies) raised in another species as Ab 2 .
  • the EGFR forms complexes with one or more antibody(ies) or binding partners and one member of the complex is labeled with a detectable label.
  • a complex has formed and, if desired, the amount thereof, can be determined by known methods applicable to the detection of labels.
  • Ab 2 a characteristic property of Ab 2 is that it will react with Ab 1 .
  • Ab 1 raised in one mammalian species has been used in another species as an antigen to raise the antibody Ab 2 .
  • Ab 2 may be raised in goats using rabbit antibodies as antigens.
  • Ab 2 therefore would be anti-rabbit antibody raised in goats.
  • Ab 1 will be referred to as a primary or anti-EGFR antibody
  • Ab 2 will be referred to as a secondary or anti-Ab 1 antibody.
  • the labels most commonly employed for these studies are radioactive elements, enzymes, chemicals which fluoresce when exposed to ultraviolet light, and others.
  • fluorescent materials are known and can be utilized as labels. These include, for example, fluorescein, rhodamine, auramine, Texas Red, AMCA blue and Lucifer Yellow.
  • a particular detecting material is anti-rabbit antibody prepared in goats and conjugated with fluorescein through an isothiocyanate.
  • the EGFR or its binding partner(s) such as the present specific binding member can also be labeled with a radioactive element or with an enzyme.
  • the radioactive label can be detected by any of the currently available counting procedures.
  • the preferred isotope may be selected from 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, 111 In, 211 At, 198 Au, 67 Cu 225 Ac, 213 Bi, 99 Tc and 186 Re.
  • Enzyme labels are likewise useful, and can be detected by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques.
  • the enzyme is conjugated to the selected particle by reaction with bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde and the like. Many enzymes which can be used in these procedures are known and can be utilized. The preferred are peroxidase, ⁇ -glucuronidase, ⁇ -D-glucosidase, ⁇ -D-galactosidase, urease, glucose oxidase plus peroxidase and alkaline phosphatase.
  • U.S. Pat. Nos. 3,654,090; 3,850,752; and 4,016,043 are referred to by way of example for their disclosure of alternate labeling material and methods.
  • a particular assay system that may be advantageously utilized in accordance with the present invention, is known as a receptor assay.
  • the material to be assayed such as the specific binding member
  • the specific binding member is appropriately labeled and then certain cellular test colonies are inoculated with a quantity of both the labeled and unlabeled material after which binding studies are conducted to determine the extent to which the labeled material binds to the cell receptors. In this way, differences in affinity between materials can be ascertained.
  • a purified quantity of the specific binding member may be radiolabeled and combined, for example, with antibodies or other inhibitors thereto, after which binding studies would be carried out. Solutions would then be prepared that contain various quantities of labeled and unlabeled uncombined specific binding member, and cell samples would then be inoculated and thereafter incubated. The resulting cell monolayers are then washed, solubilized and then counted in a gamma counter for a length of time sufficient to yield a standard error of ⁇ 5%. These data are then subjected to Scatchard analysis after which observations and conclusions regarding material activity can be drawn. While the foregoing is exemplary, it illustrates the manner in which a receptor assay may be performed and utilized, in the instance where the cellular binding ability of the assayed material may serve as a distinguishing characteristic.
  • an assay useful and contemplated in accordance with the present invention is known as a “cis/trans” assay. Briefly, this assay employs two genetic constructs, one of which is typically a plasmid that continually expresses a particular receptor of interest when transfected into an appropriate cell line, and the second of which is a plasmid that expresses a reporter such as luciferase, under the control of a receptor/ligand complex.
  • one of the plasmids would be a construct that results in expression of the receptor in the chosen cell line, while the second plasmid would possess a promoter linked to the luciferase gene in which the response element to the particular receptor is inserted.
  • the compound under test is an agonist for the receptor
  • the ligand will complex with the receptor, and the resulting complex will bind the response element and initiate transcription of the luciferase gene.
  • the resulting chemiluminescence is then measured photometrically, and dose response curves are obtained and compared to those of known ligands.
  • the foregoing protocol is described in detail in U.S. Pat. No. 4,981,784 and PCT International Publication No. WO 88/03168, for which purpose the artisan is referred.
  • kits suitable for use by a medical specialist may be prepared to determine the presence or absence of aberrant expression of EGFR, including but not limited to amplified EGFR and/or an EGFR mutation, in suspected target cells.
  • one class of such kits will contain at least the labeled EGFR or its binding partner, for instance an antibody specific thereto, and directions, of course, depending upon the method selected, e.g., “competitive,” “sandwich,” “DASP” and the like.
  • the kits may also contain peripheral reagents such as buffers, stabilizers, etc.
  • test kit may be prepared for the demonstration of the presence or capability of cells for aberrant expression or post-translational modification of EGFR, comprising:
  • the diagnostic test kit may comprise:
  • test kit may be prepared and used for the purposes stated above, which operates according to a predetermined protocol (e.g., “competitive,” “sandwich,” “double antibody,” etc.), and comprises:
  • an assay system for screening potential drugs effective to modulate the activity of the EFGR, the aberrant expression or post-translational modification of the EGFR, and/or the activity or binding of the specific binding member may be prepared.
  • the receptor or the binding member may be introduced into a test system, and the prospective drug may also be introduced into the resulting cell culture, and the culture thereafter examined to observe any changes in the S-phase activity of the cells, due either to the addition of the prospective drug alone, or due to the effect of added quantities of the known agent(s).
  • the present invention further provides an isolated nucleic acid encoding a specific binding member of the present invention.
  • Nucleic acid includes DNA and RNA.
  • the present invention provides a nucleic acid which codes for a polypeptide of the invention as defined above, including a polypeptide as set out as the CDR residues of the VH and VL chains of the inventive antibodies.
  • the present invention also provides constructs in the form of plasmids, vectors, transcription or expression cassettes which comprise at least one polynucleotide as above.
  • the present invention also provides a recombinant host cell which comprises one or more constructs as above.
  • a nucleic acid encoding any specific binding member as provided itself forms an aspect of the present invention, as does a method of production of the specific binding member which method comprises expression from encoding nucleic acid therefor. Expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the nucleic acid. Following production by expression a specific binding member may be isolated and/or purified using any suitable technique, then used as appropriate.
  • nucleic acid molecules and vectors according to the present invention may be provided isolated and/or purified, e.g. from their natural environment, in substantially pure or homogeneous form, or, in the case of nucleic acid, free or substantially free of nucleic acid or genes origin other than the sequence encoding a polypeptide with the required function.
  • Nucleic acid according to the present invention may comprise DNA or RNA and may be wholly or partially synthetic.
  • Suitable host cells include bacteria, mammalian cells, yeast and baculovirus systems.
  • Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells and many others.
  • a common, preferred bacterial host is E. coli.
  • Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • Vectors may be plasmids, viral e.g. ‘phage, or phagemid, as appropriate.
  • phage plasmids
  • Many known techniques and protocols for manipulation of nucleic acid for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Short Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992. The disclosures of Sambrook et al. and Ausubel et al. are incorporated herein by reference.
  • a further aspect of the present invention provides a host cell containing nucleic acid as disclosed herein.
  • a still further aspect provides a method comprising introducing such nucleic acid into a host cell.
  • the introduction may employ any available technique.
  • suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. vaccinia or, for insect cells, baculovirus.
  • suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage.
  • the introduction may be followed by causing or allowing expression from the nucleic acid, e.g. by culturing host cells under conditions for expression of the gene.
  • the nucleic acid of the invention is integrated into the genome (e.g. chromosome) of the host cell. Integration may be promoted by inclusion of sequences which promote recombination with the genome, in accordance with standard techniques.
  • the present invention also provides a method which comprises using a construct as stated above in an expression system in order to express a specific binding member or polypeptide as above.
  • the present invention also relates to a recombinant DNA molecule or cloned gene, or a degenerate variant thereof, which encodes a specific binding member, particularly antibody or a fragment thereof, that possesses an amino acid sequence set forth in SEQ ID NOS:2 and 4; 129 and 134; 22 and 27; 32 and 37; and/or 42 and 47, preferably a nucleic acid molecule, in particular a recombinant DNA molecule or cloned gene, encoding the binding member or antibody has a nucleotide sequence or is complementary to a DNA sequence encoding one of such sequences.
  • DNA sequences disclosed herein may be expressed by operatively linking them to an expression control sequence in an appropriate expression vector and employing that expression vector to transform an appropriate unicellular host.
  • Such operative linking of a DNA sequence of this invention to an expression control sequence includes, if not already part of the DNA sequence, the provision of an initiation codon, ATG, in the correct reading frame upstream of the DNA sequence.
  • a wide variety of host/expression vector combinations may be employed in expressing the DNA sequences of this invention.
  • Useful expression vectors may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences.
  • Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g., E.
  • coli plasmids col E1, pCR1, pBR322, pMB9 and their derivatives, plasmids such as RP4; phage DNAs, e.g., the numerous derivatives of phage X, e.g., NM989, and other phage DNA, e.g., M13 and filamentous single stranded phage DNA; yeast plasmids such as the 2u plasmid or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like.
  • phage DNAs e.g., the numerous derivatives of phage X, e.g., NM989, and other phage DNA, e.g., M13 and filamentous single stranded phage
  • useful expression control sequences include, for example, the early or late promoters of SV40, CMV, vaccinia, polyoma or adenovirus, the lac system, the trp system, the TAC system, the TRC system, the LTR system, the major operator and promoter regions of phage ⁇ , the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase (e.g., Pho5), the promoters of the yeast-mating factors, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
  • a wide variety of unicellular host cells are also useful in expressing the DNA sequences of this invention.
  • These hosts may include well known eukaryotic and prokaryotic hosts, such as strains of E. coli, Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animal cells, such as CHO, YB/20, NSO, SP2/0, R1.1, B-W and L-M cells, African Green Monkey kidney cells (e.g., COS 1, COS 7, BSC1, BSC40, and BMT1O), insect cells (e.g., Sf9), and human cells and plant cells in tissue culture.
  • eukaryotic and prokaryotic hosts such as strains of E. coli, Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animal cells, such as CHO, YB/20, NSO, SP2/0, R1.1, B-W and L-M cells, African Green Monkey kidney
  • Suitable unicellular hosts will be selected by consideration of, e.g., their compatibility with the chosen vector, their secretion characteristics, their ability to fold proteins correctly, and their fermentation requirements, as well as the toxicity to the host of the product encoded by the DNA sequences to be expressed, and the ease of purification of the expression products.
  • specific binding member analogs may be prepared from nucleotide sequences of the protein complex/subunit derived within the scope of the present invention.
  • Analogs, such as fragments may be produced, for example, by pepsin digestion of specific binding member material.
  • Other analogs, such as muteins, can be produced by standard site-directed mutagenesis of specific binding member coding sequences.
  • Analogs exhibiting “specific binding member activity” such as small molecules, whether functioning as promoters or inhibitors, may be identified by known in vivo and/or in vitro assays.
  • a DNA sequence encoding a specific binding member can be prepared synthetically rather than cloned.
  • the DNA sequence can be designed with the appropriate codons for the specific binding member amino acid sequence. In general, one will select preferred codons for the intended host if the sequence will be used for expression.
  • the complete sequence is assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge, Nature, 292:756 (1981); Nambair et al., Science, 223:1299 (1984); Jay et al., J. Biol. Chem., 259:6311 (1984).
  • DNA sequences allow convenient construction of genes which will express specific binding member analogs or “muteins”.
  • DNA encoding muteins can be made by site-directed mutagenesis of native specific binding member genes or cDNAs, and muteins can be made directly using conventional polypeptide synthesis.
  • the present invention extends to the preparation of antisense oligonucleotides and ribozymes that may be used to interfere with the expression of the EGFR at the translational level.
  • This approach utilizes antisense nucleic acid and ribozymes to block translation of a specific mRNA, either by masking that mRNA with an antisense nucleic acid or cleaving it with a ribozyme.
  • Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule (See Weintraub, 1990; Marcus-Sekura, 1988.). In the cell, they hybridize to that mRNA, forming a double stranded molecule. The cell does not translate an mRNA in this double-stranded form. Therefore, antisense nucleic acids interfere with the expression of mRNA into protein. Oligomers of about fifteen nucleotides and molecules that hybridize to the AUG initiation codon will be particularly efficient, since they are easy to synthesize and are likely to pose fewer problems than larger molecules when introducing them into producing cells. Antisense methods have been used to inhibit the expression of many genes in vitro (Marcus-Sekura, 1988; Hambor et al., 1988).
  • Ribozymes are RNA molecules possessing the ability to specifically cleave other single stranded RNA molecules in a manner somewhat analogous to DNA restriction endonucleases. Ribozymes were discovered from the observation that certain mRNAs have the ability to excise their own introns. By modifying the nucleotide sequence of these RNAs, researchers have been able to engineer molecules that recognize specific nucleotide sequences in an RNA molecule and cleave it (Cech, 1988.). Because they are sequence-specific, only mRNAs with particular sequences are inactivated.
  • Tetrahymena-type ribozymes recognize four-base sequences, while “hammerhead”-type recognize eleven-to eighteen-base sequences. The longer the recognition sequence, the more likely it is to occur exclusively in the target mRNA species. Therefore, hammerhead-type ribozymes are preferable to Tetrahymena-type ribozymes for inactivating a specific mRNA species, and eighteen base recognition sequences are preferable to shorter recognition sequences.
  • DNA sequences described herein may thus be used to prepare antisense molecules against, and ribozymes that cleave mRNAs for EFGRs and their ligands.
  • NR6, NR6 ⁇ EGFR, , and NR6 wtEGFR cell lines were previously described (Batra et al. (1995) Epidermal Growth Factor Ligand-independent, Unregulated, Cell-Transforming Potential of a Naturally Occurring Human Mutant EGFRvIII Gene. Cell Growth Differ. 6(10): 1251-1259).
  • the NR6 cell line lacks normal endogenous EGFR. (Batra et al., 1995).
  • U87MG cell lines and transfections were described previously (Nishikawa et al. (1994) A mutant epidermal growth factor receptor common in human glioma confers enhanced tumorigenicity. Proc. Natl. Acad. Sci. U.S.A. 91, 7727-7731).
  • the U87MG astrocytoma cell line (Ponten, J. and Macintyre, E. H. (1968) Long term culture of normal and neoplastic human glia. Acta. Pathol. Microbiol. Scand. 74, 465-86) which endogenously expresses low levels of the wtEGFR, was infected with a retrovirus containing the de2-7 EGFR to produce the U87MG. ⁇ 2-7 cell line (Nishikawa et al., 1994). The transfected cell line U87MG.wtEGFR was produced as described in Nagane et al. (1996) Cancer Res. 56, 5079-5086.
  • U87MG cells express approximately 1 ⁇ 10 5 EGFR
  • U87MG.wtEGFR cells express approximately 1 ⁇ 10 6 EGFR, and thus mimic the situation seen with gene amplification.
  • the murine pro-B cell line BaF/3 which does not express any known EGFR related molecules, was also transfected with de2-7 EGFR. resulting in the BaF/3. ⁇ 2-7 cell line (Luwor et al. (2004) The tumor-specific de2-7 epidermal growth factor receptor (EGFR) promotes cells survival and heterodimerizes with the wild-type EGFR, Oncogene 23: 6095-6104).
  • Human squamous carcinoma A431 cells were obtained from ATCC (Rockville, Md.).
  • the epidermoid carcinoma cell line A431 has been described previously (Sato et al. (1987) Derivation and assay of biological effects of monoclonal antibodies to epidermal growth factor receptors. Methods Enzymol. 146, 63-81).
  • the de2-7 EGFR unique junctional peptide has the amino acid sequence: LEEKKGNYVVTDH (SEQ ID NO:13).
  • Biotinylated unique junctional peptides (Biotin-LEEKKGNYVVTDH (SEQ ID NO:5) and LEEKKGNYVVTDH-Biotin (SEQ ID NO:6)) from de2-7 EGFR were synthesized by standard Fmoc chemistry and purity (>96%) determined by reverse phase HPLC and mass spectral analysis (Auspep, Melbourne, Australia).
  • mAb528 to the wtEGFR (Sato et al. (1983) Mol. Biol. Med. 1(5), 511-529) and DH8.3, which was generated against a synthetic peptide spanning the junctional sequence of the ⁇ 2-7 EGFR deletion mutation.
  • the DH8.3 antibody (IgG1) which is specific for the de2-7 EGFR, has been described previously (Hills et al. (1995) Specific targeting of a mutant, activated EGF receptor found in glioblastoma using a monoclonal antibody. Int. J. Cancer. 63, 537-43,1995) and was obtained following immunization of mice with the unique junctional peptide found in de2-7 EGFR (Hills et al., 1995).
  • the 528 antibody which recognizes both de2-7 and wild-type EGFR, has been described previously (Masui et al. (1984) Growth inhibition of human tumor cells in athymic mice by anti-epidermal growth factor receptor monoclonal antibodies. Cancer Res. 44, 1002-7) and was produced in the Biological Production Facility, Ludwig Institute for Cancer Research (Melbourne, Australia) using a hybridoma (ATCC HB-8509) obtained from the American Type Culture Collection (Rockville, Md.).
  • the polyclonal antibody SC-03 is an affinity purified rabbit polyclonal antibody raised against a carboxy terminal peptide of the EGFR (Santa Cruz Biotechnology Inc.).
  • the murine fibroblast line NR6 ⁇ EGFR was used as immunogen.
  • Mouse hybridomas were generated by immunizing BALB/c mice five times subcutaneously at 2- to 3-week intervals, with 5 ⁇ 10 5 -2 ⁇ 10 6 cells in adjuvant. Complete Freund's adjuvant was used for the first injection. Thereafter, incomplete Freund's adjuvant (DifcoTM, Voigt Global Distribution, Lawrence, Kans.) was used. Spleen cells from immunized mice were fused with mouse myeloma cell line SP2/0 (Shulman et al. (1978) Nature 276:269-270).
  • these antibodies showed no reactivity (undiluted supernatant ⁇ 10%) with the native human glioblastoma cell line U87MG and U87MG wtEGFR , but were strongly reactive with U87MG ⁇ EGFR ; less reactivity was seen with A431.
  • 806 was unreactive with native U87MG and intensively stained U87MG ⁇ EGFR and to a lesser degree U87MG wtEGFR indicating binding of 806 to both, ⁇ EGFR and wtEGFR (see below).
  • mAb124, mAb806 and mAb1133 were then analyzed for reactivity with wtEGFR and ⁇ EGFR.
  • Detergent lysates were extracted from NR6 ⁇ EGFR , U87MG ⁇ EGFR as well as from A431. All three mAbs showed a similar reactivity pattern with cell lysates staining both the wtEGFR (170 kDa) and ⁇ EGFR protein (140 kDa).
  • mAbR.I known to be reactive with the wtEGFR (Waterfield et al. (1982) J. Cell Biochem.
  • mAb528, which is known to be non-reactive in western blot analysis was used instead of mAb528, which is known to be non-reactive in western blot analysis.
  • mAbR.I. showed reactivity with wild-type and ⁇ EGFR. All three newly generated clones showed reactivity with ⁇ EGFR and less intense with wtEGFR.
  • DH8.3 was solely positive in the lysate of U87MG ⁇ EGFR and NR6 ⁇ EGFR .
  • mAb124, mAb806, and mAb1133 revealed reactivity with mostly the basally located cells of the squamous cell carcinoma of A431 and did not react with the upper cell layers or the keratinizing component. DH8.3 was negative in A431 xenografts.
  • variable heavy (VH) and variable light (VL) chains of mAb806, mAb124 and mAb1133 were sequenced, and their complementarity determining regions (CDRs) identified, as follows:
  • mAb806 VH chain nucleic acid (SEQ ID NO:1) and amino acid (SEQ ID NO:2) sequences are shown in FIGS. 14A and 14B , respectively (signal peptide underlined in FIG. 14B ).
  • Complementarity determining regions CDR1, CDR2, and CDR3 are indicated by underlining in FIG. 16 .
  • mAb806 VL chain nucleic acid (SEQ ID NO:3) and amino acid (SEQ ID NO:4) sequences are shown in FIGS. 15A and 15B , respectively (signal peptide underlined in FIG. 15B ).
  • Complementarity determining regions CDR1, CDR2, and CDR3 are indicated by underlining in FIG. 17 .
  • mAb124 VH chain nucleic acid (SEQ ID NO:21) and amino acid (SEQ ID NO:22) sequences are shown in FIGS. 51A and 51B , respectively.
  • Complementarity determining regions CDR1, CDR2, and CDR3 are indicated by underlining.
  • mAb124 VL chain nucleic acid (SEQ ID NO:26) and amino acid (SEQ ID NO:27) sequences are shown in FIGS. 51C and 51D , respectively.
  • Complementarity determining regions CDR1, CDR2, and CDR3 are indicated by underlining.
  • mAb1113 VH chain nucleic acid (SEQ ID NO:31) and amino acid (SEQ ID NO:32) sequences are shown in FIGS. 52A and 52B , respectively.
  • Complementarity determining regions CDR1, CDR2, and CDR3 are indicated by underlining.
  • mAb1133 VL chain nucleic acid (SEQ ID NO:36) and amino acid (SEQ ID NO:37) sequences are shown in FIGS. 52C and 52D , respectively.
  • Complementarity determining regions CDR1, CDR2, and CDR3 are indicated by underlining.
  • mAb806 was initially selected for further characterization, as set forth herein and in the following Examples. mAb124 and mAb1133 were also selected for further characterization, as discussed in Example 26 below, and found to have properties corresponding to the unique properties of mAb806 discussed herein.
  • mAb806 stained U87MG. ⁇ 2-7 and U87MG.wtEGFR cells, indicating that mAb806 specifically recognizes the de2-7 EGFR and amplified EGFR ( FIG. 1 ).
  • DH8.3 antibody stained U87MG. ⁇ 2-7 cells, confirming that DH8.3 antibody specifically recognizes the de2-7 EGFR ( FIG. 1 ).
  • the 528 antibody stained both the U87MG. ⁇ 2-7 and U87MG.wtEGFR cell lines ( FIG. 1 ).
  • the 528 antibody stained U87MG. ⁇ 2-7 with a higher intensity than the parental cell as it binds both the de2-7 and wild-type receptors that are co-expressed in these cells ( FIG. 1 ).
  • sEGFR is the recombinant extracellular domain (amino acids 1-621) of the wild-type EGFR), and was produced as previously described (Domagala et al. (2000) Stoichiometry, kinetic and binding analysis of the interaction between Epidermal Growth Factor (EGF) and the Extracellular Domain of the EGF receptor. Growth Factors.
  • Antibodies were added to wells in triplicate at increasing concentration in 2% HSA in phosphate-buffered saline (PBS). Bound antibody was detected by horseradish peroxidase conjugated sheep anti-mouse IgG (Silenus, Melbourne, Australia) using ABTS (Sigma, Sydney, Australia) as a substrate and the absorbance measured at 405 nm.
  • PBS phosphate-buffered saline
  • Both mAb806 and the 528 antibody displayed dose-dependent and saturating binding curves to immobilized wild-type sEGFR ( FIG. 2A ).
  • mAb806 must be binding to an epitope located within the wild-type EGFR sequence.
  • the binding of the 528 antibody was lower than that observed for mAb806, probably because it recognizes a conformational determinant.
  • the DH8.3 antibody did not bind the wild-type sEGFR even at concentrations up to 10 ⁇ g/ml ( FIG. 2A ).
  • sEGFR in solution was able to inhibit the binding of mAb806 to immobilized sEGFR ( FIG. 2C ), confirming that mAb806 can bind the wild-type EGFR under certain conditions.
  • the denatured sEGFR was unable to inhibit the binding of the 528 antibody ( FIG. 2C ), demonstrating that this antibody recognizes a conformational epitope.
  • the DH8.3 antibody exhibited dose-dependent and saturable binding to the unique de2-7 EGFR peptide ( FIG. 2D ). Neither mAb806 or the 528 antibody bound to the peptide, even at concentrations higher than those used to obtain saturation binding of DH8.3, further indicating mAb806 does not recognize an epitope determinant within this peptide.
  • biotinylated de2-7 specific peptide Biotin LEEKKGNYVVTDH (SEQ ID NO:5)
  • streptavidin Piereptavidin
  • Peptide C was immobilized on a Streptavidin microsensor chip at a surface density of 350RU ( ⁇ 30RU). Serial dilutions of mAbs were tested for reactivity with the peptide. Blocking experiments using non-biotinylated peptide were performed to assess specificity.
  • mAbL8A4 showed strong reactivity with Peptide C even at low antibody concentrations (6.25 nM) ( FIG. 2E ). mAb806 did not show detectable specific reactivity with Peptide C up to antibody concentrations of 100 nM (highest concentration tested) ( FIGS. 2E and 2F ). It was expected that mAbL8A4 would react with Peptide C because the peptide was used as the immunogen in the generation of mAbL8A4. Addition of the Junction Peptide (non-biotinylated, 50 ⁇ g/ml) completely blocks the reactivity of mAbL8A4 with Peptide C, confirming the antibody's specificity for the junction peptide epitope.
  • sEGFR was immobilized on a CM microsensor chip at a surface density of ⁇ 4000RU.
  • Serial dilutions of mAbs were tested for reactivity with sEGFR.
  • mAb806 was strongly reactive with denaturated sEGFR while mAbL8A4 did not react with denaturated sEGFR. Reactivity of mAb806 with denaturated sEGFR decreases with decreasing antibody concentrations. It was expected that mAbL8A4 does not react with sEGFR because mAbL8A4 was generated using the junction peptide as the immunogen and sEGFR does not contain the junction peptide.
  • mAb806 bound to the wtEGFR in cell lysates following immunoblotting (results not shown). This is different from the results obtained with DH8.3 antibody, which reacted with de2-7 EGFR but not wtEGFR. Thus, mAb806 can recognize the wtEGFR following denaturation but not when the receptor is in its natural state on the cell surface.
  • mAb806 and the DH8.3 antibody retained high immunoreactivity when iodinated and was typically greater than 90% for mAb806 and 45-50% for the DH8.3 antibody.
  • mAb806 had an affinity for the de2-7 EGFR receptor of 1.1 ⁇ 10 9 M ⁇ 1 whereas the affinity of DH8.3 was some 10-fold lower at 1.0 ⁇ 10 8 M ⁇ 1 .
  • mAb806 recognized an average of 2.4 ⁇ 10 5 binding sites per cell with the DH8.3 antibody binding an average of 5.2 ⁇ 10 5 sites.
  • U87MG. ⁇ 2-7 cells were incubated with either mAb806 or the DH8.3 antibody (10 ⁇ g/ml) for 1 h in DMEM at 4° C. After washing, cells were transferred to DMEM pre-warmed to 37° C. and aliquots taken at various time points following incubation at 37° C. Internalization was stopped by immediately washing aliquots in ice-cold wash buffer (1% HSA/PBS). At the completion of the time course cells were stained by FACS as described above.
  • This method was validated in one assay using an iodinated antibody (mAb806) to measure internalization as previously described (Huang et al. (1997) The enhanced tumorigenic activity of a mutant epidermal growth factor receptor common in human cancers is mediated by threshold levels of constitutive tyrosine phosphorylation and unattenuated signaling. J. Biol. Chem. 272, 2927-35).
  • U87MG. ⁇ 2-7 cells were grown on gelatin coated chamber slides (Nunc, Naperville, Ill.) to 80% confluence and then washed with ice cold DMEM. Cells were then incubated with mAb806 or the DH8.3 antibody in DMEM for 45 min at 4° C. After washing, cells were incubated for a further 30 min with gold-conjugated (20 nm particles) anti-mouse IgG (BBInternational, Cambridge, UK) at 4° C. Following a further wash, pre-warmed DMEM/10% PCS was added to the cells, which were incubated at 37° C. for various times from 1-60 min.
  • mAb806 appeared to be internalized by macropinocytosis ( FIG. 19 ). In fact, a detailed analysis of 32 coated pits formed in cells incubated with mAb806 revealed that none of them contained antibody. In contrast, around 20% of all coated-pits from cells incubated with DH8.3 were positive for antibody, with a number containing multiple gold grains. A statistical analysis of the total number of gold grains contained within coated-pits found that the difference was highly significant (p ⁇ 0.01). After 20-30 min both antibodies could be seen in structures that morphologically resemble lysosomes ( FIG. 19C ). The presence of cellular debris within these structures was also consistent with their lysosome nature.
  • mAb806 and the DH8.3 antibody were compared in nude mice containing U87MG xenografts on one side and U87MG. ⁇ 2-7 xenografts on the other.
  • a relatively short time period was chosen for this study as a previous report demonstrated that the DH8.3 antibody shows peak levels of tumor targeting between 4-24 h (Hills et al. (1995) Specific targeting of a mutant, activated EGF receptor found in glioblastoma using a monoclonal antibody. Int. J. Cancer. 63, 537-43).
  • Tumor xenografts were established in nude BALB/c mice by s.c. injection of 3 ⁇ 10 6 U87MG, U87MG. ⁇ 2-7 or A431 cells.
  • de2-7 EGFR expression in U87MG. ⁇ 2-7 xenografts remained stable throughout the period of biodistribution as measured by immunohistochemistry at various time points (data not shown).
  • A431 cells retained their mAb806 reactivity when grown as tumor xenografts as determined by immunohistochemistry.
  • U87MG or A431 cells were injected on one side 7-10 days before U87MG. ⁇ 2-7 cells were injected on the other side because of the faster growth rate observed for de2-7 EGFR expressing xenografts.
  • Antibodies were radiolabeled and assessed for immunoreactivity as described above and were injected into mice by the retro-orbital route when tumors were 100-200 mg in weight. Each mouse received two different antibodies (2 ⁇ g per antibody): 2 ⁇ Ci of 125 I-labeled mAb806 and 2 ⁇ Ci of 131 I labelled DH8.3 or 528. Unless indicated, groups of 5 mice were sacrificed at various time points post-injection and blood obtained by cardiac puncture. The tumors, liver, spleen, kidneys and lungs were obtained by dissection. All tissues were weighed and assayed for 125 I and 131 I activity using a dual-channel counting Window.
  • % ID/g tumor determined by comparison to injected dose standards or converted into tumor to blood/liver ratios (i.e. % ID/g tumor divided by % ID/g blood or liver). Differences between groups were analyzed by Student's t-test. After injection of radiolabeled mAb806, some tumors were fixed in formalin, embedded in paraffin, cut into 5, ⁇ m sections and then exposed to X-ray film (AGFA, Mortsel, Belgium) to determine antibody localization by autoradiography.
  • AGFA X-ray film
  • mAb806 reached its peak level in U87MG. ⁇ 2-7 xenografts of 18.6% m/g tumor at 8 h ( FIG. 4A ), considerably higher than any other tissue except blood. While DH8.3 also showed peak tumor levels at 8 h, the level was a statistically (p ⁇ 0.001) lower 8.8% m/g tumor compared to mAb806 ( FIG. 4B ). Levels of both antibodies slowly declined at 24 and 48 h. Autoradiography of U87MG. ⁇ 2-7 xenograft tissue sections collected 8 hr after injection with 125 I-labeled mAb806 alone, clearly illustrates localization of antibody to viable tumor ( FIG. 20 ).
  • mAb806 showed the highest ratio at 24 h for both blood (ratio of 1.3) and liver (ratio of 6.1) ( FIGS. 5A and 5B ).
  • the DH8.3 antibody had its highest ratio in blood at 8 h (ratio of 0.38) and at 24 h in liver (ratio of 1.5) ( FIGS. 5A and 5B ), both of which are considerably lower than the values obtained for mAb806.
  • % ID/g tumor seen with mAb806 was similar to that reported for other de2-7 EGFR specific antibodies when using standard iodination techniques (Hills et al., 1995; Huang et al., 1997; Reist et al. (1995) Tumor-specific anti-epidermal growth factor receptor variant III monoclonal antibodies: use of the tyramine-cellobiose radioiodination method enhances cellular retention and uptake in tumor xenografts. Cancer Res. 55, 4375-82).
  • the reason for the early peak is probably two-fold. Firstly, tumors expressing the de2-7 EGFR, including the transfected U87MG cells, grow extremely rapidly as tumor xenografts. Thus, even during the relatively short period of time used in these biodistribution studies, the tumor size increases to such an extent (5-10 fold increase in mass over 4 days) that the % ID/g tumor is reduced compared with slow growing tumors. Secondly, while internalization of mAb806 was relatively slow compared to DH8.3, it is still rapid with respect to many other tumor antibody/antigen systems. Internalized antibodies undergo rapid proteolysis with the degradation products being excreted from the cell (Press et al.
  • L8A4 monoclonal antibody directed to the unique junctional peptide found in the de2-7 EGFR behaves in a similar fashion to mAb806 (Reist et al. (1997) In vitro and in vivo behavior of radiolabeled chimeric anti-EGFRvIII monoclonal antibody: comparison with its murine parent. Nucl Med. Biol. 24, 639-47).
  • this antibody had a similar internalization rate (35% at 1 hour compared to 30% at 1 hour for mAb806) and displayed comparable in vivo targeting when using 3T3 fibroblasts transfected with de2-7 EGFR (peak of 24% ID/g tumor at 24 hours compared to 18% ID/g tumor at 8 hours for mAb806) (Reist et al. (1997) Improved targeting of an anti-epidermal growth factor receptor variant III monoclonal antibody in tumor xenografts after labeling using N-succinimidyl 5-iodo-3-pyridinecarboxylate. Cancer Res. 57, 1510-5).
  • A431 cells are human squamous carcinoma cells and express high levels of wtEGFR. Low, but highly reproducible, binding of mAb806 to A431 cells was observed by FACS analysis ( FIG. 6 ).
  • the DH8.3 antibody did not bind A431 cells, indicating that the binding of mAb806 was not the result of low level de2-7 EGFR expression ( FIG. 6 ).
  • the anti-EGFR 528 antibody showed strong staining of A431 cells ( FIG. 6 ).
  • binding of mAb806 to A431 was characterized by Scatchard analysis. While the binding of iodinated mAb806 was comparatively low, it was possible to get consistent data for Scatchard. The average of three such experiments gave a value for affinity of 9.5 ⁇ 10 7 M ⁇ 1 with 2.4 ⁇ 10 5 receptors per cell. Thus, the affinity for this receptor was some 10-fold lower than the affinity for the de2-7 EGFR. Furthermore, mAb806 appears to only recognize a small portion of EGFR found on the surface of A431 cells. The 528 antibody measured approximately 2 ⁇ 10 6 receptors per cell which is in agreement with numerous other studies (Santon et al. (1986) Effects of epidermal growth factor receptor concentration on tumorigenicity of A431 cells in nude mice. Cancer Res. 46, 4701-5).
  • mAb806 reactivity was examined in 2 other cells lines exhibiting amplification of the EGFR gene.
  • HN5 head and neck cell line Kerok T T and Sutherland R M (1991) Differences in EGF related radiosensitisation of human squamous carcinoma cells with high and low numbers of EGF receptors. Br. J. Cancer. 64, 251-4
  • MDA-468 breast cancer cell line Breastmus et al. (1985) MDA-468, a human breast cancer cell line with a high number of epidermal growth factor (EGF) receptors, has an amplified EGF receptor gene and is growth inhibited by EGF. Biochem. Biophys. Res.
  • a second biodistribution study was performed with mAb806 to determine if it could target A431 tumor xenografts.
  • the study was conducted over a longer time course in order obtain more information regarding the targeting of U87MG. ⁇ 2-7 xenografts by mAb806, which were included in all mice as a positive control.
  • the anti-EGFR 528 antibody was included as a positive control for the A431 xenografts, since a previous study demonstrated low but significant targeting of this antibody to A431 cells grown in nude mice (Masui et al. (1984) Growth inhibition of human tumor cells in athymic mice by anti-epidermal growth factor receptor monoclonal antibodies. Cancer Res. 44, 1002-7).
  • mAb806 displayed almost identical targeting properties as those observed in the initial experiments ( FIG. 7A compared with FIG. 4A ).
  • levels of mAb806 in U87MG. ⁇ 2-7 xenografts slowly declined after 24 h but always remained higher than levels detected in normal tissue.
  • Uptake in the A431 xenografts was comparatively low, however there was a small increase in % ID/g tumor during the first 24 h not observed in normal tissues such as liver, spleen, kidney and lung ( FIG. 7A ).
  • Uptake of the 528 antibody was very low in both xenografts when expressed as % ID/g tumor ( FIG.
  • the tumor to blood ratio for the 528 antibody showed a similar profile to mAb806 although higher levels were noted in the A431 xenografts ( FIGS. 8A , B).
  • mAb806 had a peak tumor to liver ratio in U87MG. ⁇ 2-7 xenografts of 7.6 at 72 h, clearly demonstrating preferential uptake in these tumors compared to normal tissue ( FIG. 8C ).
  • Other tumor to organ ratios for mAb806 were similar to those observed in the liver (data not shown).
  • the peak tumor to liver ratio for mAb806 in A431 xenografts was 2.0 at 100 h, again indicating a slight preferential uptake in tumor compared with normal tissue ( FIG. 8D ).
  • mAb806 The effects of mAb806 were assessed in two xenograft models of disease—a preventative model and an established tumor model.
  • Tumor cells (3 ⁇ 10 6 ) in 100 ml of PBS were inoculated subcutaneously into both flanks of 4-6 week old female nude mice (Animal Research Centre, Western Australia, Australia).
  • Therapeutic efficacy of mAb806 was investigated in both preventative and established tumor models.
  • 5 mice with two xenografts each were treated intraperitoneally with either 1 or 0.1 mg of mAb806 or vehicle (PBS) starting the day before tumor cell inoculation. Treatment was continued for a total of 6 doses, 3 times per week for 2 weeks.
  • Tumor volume in mm 3 was determined using the formula (length ⁇ width 2 )/2, where length was the longest axis and width the measurement at right angles to the length (Clark et al. (2000) Therapeutic efficacy of anti-Lewis (y) humanized 3S 193 radioimmunotherapy in a breast cancer model: enhanced activity when combined with Taxol chemotherapy. Clin. Cancer Res. 6, 3621-3628).
  • Xenografts were excised and bisected. One half was fixed in 10% formalin/PBS before being embedded in paraffin. Four micron sections were then cut and stained with haematoxylin and eosin (H&E) for routine histological examination. The other half was embedded in Tissue Tek® OCT compound (Sakura Finetek, Torrance, Calif.), frozen in liquid nitrogen and stored at ⁇ 80° C. Thin (5 micron) cryostat sections were cut and fixed in ice-cold acetone for 10 min followed by air drying for a further 10 min.
  • H&E haematoxylin and eosin
  • Sections were blocked in protein blocking reagent (Lipshaw Immunon, Pittsburgh U.S.A.) for 10 min and then incubated with biotinylated primary antibody (1 mg/ml), for 30 min at room temperature (RT). All antibodies were biotinylated using the ECL protein biotinylation module (Amersham, Baulkham Hills, Australia), as per the manufacturer's instructions. After rinsing with PBS, sections were incubated with a streptavidin horseradish peroxidase complex for a further 30 min (Silenus, Melbourne, Australia).
  • AEC 3-amino-9-ethylcarbozole
  • mAb806 was examined for efficacy against U87MG and U87MG. ⁇ 2-7 tumors in a preventative xenograft model. Antibody or vehicle were administered i.p. the day before tumor inoculation and was given 3 times per week for 2 weeks. mAb806 had no effect on the growth of parental U87MG xenografts, which express the wtEGFR, at a dose of 1 mg per injection ( FIG. 9A ). In contrast, mAb806 significantly inhibited the growth of U87MG. ⁇ 2-7 xenografts in a dose dependent manner ( FIG. 9B ).
  • the mean tumor volume was 1637 ⁇ 178.98 mm 3 for the control group, a statistically smaller 526 ⁇ 94.74 mm 3 for the 0.1 mg per injection group (p ⁇ 0.0001) and 197 ⁇ 42.06 mm 3 for the 1 mg injection group (p ⁇ 0.0001).
  • Treatment groups were sacrificed at day 24 at which time the mean tumor volumes was 1287 ⁇ 243.03 mm 3 for the 0.1 mg treated group and 492 ⁇ 100.8 mm 3 for the 1 mg group.
  • mAb806 Given the efficacy of mAb806 in the preventative xenograft model, its ability to inhibit the growth of established tumor xenografts was then examined. Antibody treatment was as described in the preventative model except that it commenced when tumors had reached a mean tumor volume of 65 ⁇ 6.42 mm 3 for the U87MG. ⁇ 2-7 xenografts and 84 ⁇ 9.07 mm 3 for the parental U87MG xenografts. Once again, mAb806 had no effect on the growth of parental U87MG xenografts at a dose of 1 mg per injection ( FIG. 10A ).
  • mAb806 significantly inhibited the growth of U87MG. ⁇ 2-7 xenografts in a dose dependent manner ( FIG. 10B ).
  • the mean tumor volume was 935 ⁇ 215.04 mm 3 for the control group, 386 ⁇ 57.51 mm 3 for the 0.1 mg per injection group (p ⁇ 0.01) and 217 ⁇ 58.17 mm 3 for the 1 mg injection group (p ⁇ 0.002).
  • Sections from mAb806 or control treated U87MG xenografts were also stained with H&E and revealed no differences in cell viability between the two groups, further supporting the hypothesis that mAb806 binding induces decreased cell viability/necrosis within tumor xenografts.
  • mice with A431 xenografts contain an amplified EGFR gene and express approximately 2 ⁇ 10 6 receptors per cell.
  • mAb806 binds about 10% of these EGFR and targets A431 xenografts.
  • the mean tumor volume was 1385 ⁇ 147.54 mm 3 in the control group and 260 ⁇ 60.33 mm 3 for the 1 mg injection treatment group (p ⁇ 0.0001).
  • a dose of 0.1 mg mAb also significantly inhibited the growth of A431 xenografts in a preventative model.
  • mAb806 in the preventative A431 xenograft model, its ability to inhibit the growth of established tumor xenografts was examined. Antibody treatment was as described in the preventative model except it was not started until tumors had reached a mean tumor volume of 201 ⁇ 19.09 mm 3 . mAb806 significantly inhibited the growth of established tumor xenografts ( FIG. 11B ). At day 13, when control animals were sacrificed, the mean tumor volume was 1142 ⁇ 120.06 mm 3 for the control group and 451 ⁇ 65.58 mm 3 for the 1 mg injection group (p ⁇ 0.0001).
  • mAb806 also mediates in vivo antitumor activity against cells containing amplification of the EGFR gene.
  • mAb806 inhibition of U87MG.wtEGFR xenografts appears to be less effective than that observed with U87MG. ⁇ 2-7 tumors. This probably reflects the fact that mAb806 has a lower affinity for the amplified EGFR and only binds a small proportion of receptors expressed on the cell surface.
  • mAb806 treatment produced large areas of necrosis within these xenografts.
  • AG1478 (4-(3-Chloroanilino)-6,7-dimethoxyquinazoline) is a potent and selective inhibitor of the EGFR kinase versus HER2-neu and platelet-derived growth factor receptor kinase (Calbiochem Cat. No. 658552). Three controls were included: treatment with vehicle only, vehicle+mAb806 only, and vehicle+AG1478 only. The results are illustrated in FIG. 12 . 0.1 mg mAb806 was administered at 1 day prior to xenograft and 1, 3, 6, 8 and 10 days post xenograft. 400 pg AG 1478 was administered at 0, 2, 4, 7, 9, and 11 days post xenograft.
  • mAb806 to EGFR of A431 cells was evaluated in the absence and presence of AG1478.
  • Cells were placed in serum free media overnight, then treated with AG1478 for 10 min at 37° C., washed twice in PBS, then lysed in 1% Triton and lysates prepared by centrifugation for 10 min at 12,000 g. Lysate was then assessed for 806 reactivity by an ELISA in a modified version of an assay described by Schooler and Wiley, Analytical Biochemistry 277, 135-142 (2000). Plates were coated with 10 ⁇ g/ml of mAb806 in PBS/EDTA overnight at room temperature and then washed twice.
  • Immunohistochemical analysis was performed using 5 mm sections of fresh frozen tissue applied to histology slides and fixed for 10 minutes in cold acetone. Bound primary antibody was detected with biotinylated horse anti-mouse antibody followed by an avidin-biotin-complex reaction. Diaminobenzidine tetra hydrochloride (DAB) was used as chromogen. The extent of the immunohistochemical reactivity in tissues was estimated by light microscopy and graded according to the number of immunoreactive cells in 25% increments as follows:
  • the 528 antibody showed intense reactivity in all tumors, while DH8.3 immunostaining was restricted to those tumors expressing the de2-7 EGFR (Table 2). Consistent with the previous observations in FACS and rosetting assays, mAb806 did not react with the glioblastomas expressing the wtEGFR transcript from nonamplified EGFR genes (Table 2). This pattern of reactivity for mAb806 is similar to that observed in the xenograft studies and again suggests that this antibody recognizes the de2-7 and amplified EGFR but not the wtEGFR when expressed on the cell surface.
  • de2-7 EGFR in other tumor types was examined using a panel of 12 different malignancies.
  • the 528 antibody showed often homogeneous staining in many tumors analyzed except melanoma and seminoma.
  • DH8.3 immunoreactivity was restricted to the occasional focal tumor cell indicating there is little if any de2-7 EGFR expression in tumors outside the brain using this detection system (Table 4).
  • the mAb806 showed positive staining in 64% of head and neck tumors and 50% of lung carcinomas (Table 4). There was little mAb806 reactivity elsewhere except in urinary tumors that were positive in 30% of cases.
  • the reactivity seen with the mAb in these tumors maybe associated with EGFR gene amplification.
  • Case #3 also revealed a mutation (designated A2 in Table 5), which included the sequences of the de2-7 mutation but this did not appear to be the classical de2-7 deletion with loss of the 801 bases (data not shown). This case was negative for DH8.3 reactivity but showed reactivity with 806, indicating that 806 may recognize an additional and possibly unique EGFR mutation.
  • N WT 26 ++++ ++++++ +++ A 5′ MUT 27 neg. neg. neg. N WT 28 +++ neg. neg. N WT 29 neg. neg. neg. N WT 30 ++++++ ++++++ neg. N WT 31 ++++ neg. neg. N nd par det 32 ++ +++ ++ N 5′ MUT 33 +++ ++++ ++++ A 5′ MUT 34 ++++ +++ ++++ N WT 35 ++++ neg. ++++ A 5′ MUT 36 +++ ++ +++ A 5′ MUT 37 ++ + + A 5′ MUT 38 ++++ neg. neg. N WT 39 ++ neg. neg. N 5′ MUT 40 ++++ ++++ + A WT 41 ++ neg. neg. neg.
  • N WT 42 ++++++ ++++ neg.
  • a WT 43 ++++ neg. neg. nd nd 44 ++++ neg. neg.
  • mAb806 we treated nude mice bearing intracranial ⁇ EGFR-overexpressing glioma xenografts with intraperitoneal injections of mAb806, the isotype control IgG or PBS.
  • U87MG. ⁇ EGFR cells (1 ⁇ 10 5 ) or 5 ⁇ 10 5 LN-Z308.
  • ⁇ EGFR, A1207. ⁇ EGFR, U87MG, U87MG.DK, and U87MG.wtEGFR cells in 5 ⁇ l of PBS were implanted into the right corpus stratum of nude mice brains as described previously (Mishima et al. (2000) A peptide derived from the non-receptor binding region of urokinase plasminogen activator inhibits glioblastoma growth and angiogenesis in vivo in combination with cisplatin. Proc. Natl. Acad. Sci. U.S.A. 97, 8484-8489).
  • Systemic therapy with mAb806, or the IgG2b isotype control was accomplished by i.p. injection of 1 ⁇ g of mAbs in a volume of 100 ⁇ l every other day from post-implantation day 0 through 14.
  • 10 ⁇ g of mAb806, or the IgG2b isotype control in a volume of 5 ⁇ l were injected at the tumor-injection site every other day starting at day 1 for 5 days.
  • mice treated with PBS or isotype control IgG had a median survival of 13 days, whereas mice treated with mAb806 had a 61.5% increase in median survival up to 21 days (P ⁇ 0.001; FIG. 24A ).
  • mice 3 days post-implantation, after tumor establishment also extended the median survival of the mAb806-treated animals by 46.1% (from 13 days to 19 days; P ⁇ 0.01) compared with that of the control groups (data not shown).
  • mice bearing U87MG. ⁇ EGFR and LN-Z308. ⁇ EGFR xenografts were euthanized at day 9 and day 15, respectively. Tumor sections were histopathologically analyzed and tumor volumes were determined. Consistent with the results observed for animal survival, mAb806 treatment significantly reduced the volumes by about 90% of U87MG. ⁇ EGFR. (P ⁇ 0.001; FIG. 24C ) and LN-Z308. ⁇ EGFR by more than 95% (P ⁇ 0.001; FIG. 24D ) xenografts in comparison to that of the control groups. Similar results were obtained for animals bearing A1207. ⁇ EGFR tumors (65% volume reduction, P ⁇ 0.01; data not shown).
  • mAb806 reactivity with various tumor cells by FACS analysis. Stained cells were analyzed with a FACS Calibur using Cell Quest software (Becton-Dickinson PharMingen). For the first antibody, the following mAbs were used: mAb806, anti EGFR mAb clone 528, and clone EGFR.1. Mouse IgG2a or IgG2b was used as an isotype control.
  • mAb806 specificity by immunoprecipitation.
  • EGFRs in various cell lines were immunoprecipitated with antibodies mAb806, anti-EGFR mAb clone 528 (Oncogene Research Products, Boston, Mass.), or clone EGFR.1 (Oncogene Research Products).
  • cells were lysed with lysis buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 2 mM EDTA, 0.1% SDS, 0.5% sodium deoxycholate, 10 mM sodium PPi, 1 mM phenylmethlsulfonyl fluoride, 2 mM Na3 V0 4 , 5 ⁇ g/ml leupeptin, and 5 ⁇ g/ml aprotinin.
  • Antibodies were incubated with cell lysates at 4° C. for 1 h before the addition of protein-A and-G Sepharose.
  • Immunoprecipitates were washed twice with lysis buffer and once with HNTG buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 0.1% Triton X-100, and 10% glycerol], electrophoresed, and transferred to nitrocellulose membranes.
  • HNTG buffer 50 mM HEPES (pH 7.5), 150 mM NaCl, 0.1% Triton X-100, and 10% glycerol
  • antibody 528 recognized wtEGFR and mutant receptors ( FIG. 26B-panel IP: 528), whereas antibody EGFR.1 reacted with wtEGFR but not with the mutant species ( FIG. 26B , panel IP:EGFR.1). Moreover, the levels of mutant receptors in U87MG. ⁇ EGFR and U87MG.DK cells are comparable with those of wtEGFR in the U87MG.wtEGFR cells ( FIG. 26B , panel IP: 528).
  • antibody mAb806 was able to precipitate only a small amount of the wtEGFR from the U87MG.wtEGFR cell lysates as compared with the larger amount of mutant receptor precipitated from U87MG. ⁇ EGFR and U87MG.DK cells, and an undetectable amount from the U87MG cells ( FIG. 26B , panel IP:mAb806).
  • mAb806 recognizes an epitope in ⁇ EGFR that also exists in a small fraction of wtEGFR only when it is overexpressed on the cell surface (see further discussion of and references to the mAb806 epitope below).
  • angiogenesis in tumors were fixed in a solution containing zinc chloride, paraffin embedded, sectioned, and immunostained using a monoclonal rat anti-mouse CD31 antibody (Becton-Dickinson PharMingen; 1:200).
  • Assessment of tumor cell proliferation was performed by Ki-67 immunohistochemistry on formalin-fixed paraffin-embedded tumor tissues. After deparaffinization and rehydration, the tissue sections were incubated with 3% hydrogen peroxide in methanol to quench endogenous peroxidase. The sections were blocked for 30 min with goat serum and incubated overnight with the primary antibody at 4° C.
  • the Ki-67 labeling index was determined as the ratio of labeled: total nuclei in high-power (3400) fields.
  • apoptotic cells in tumor tissue were detected by using the TUNEL method as described previously (Mishima et al., 2000). TUNEL-positive cells were counted at ⁇ 400. The apoptotic index was calculated as a ratio of apoptotic cell number: total cell number in each field. Analysis of the apoptotic index through TUNEL staining demonstrated a significant increase in the number of apoptotic cells in mAb806 treated tumors as compared with the control tumors (P ⁇ 0.001; FIG. 28 ).
  • MVAs microvascular areas
  • mice Female nude mice, 4-6 weeks old, were used as the experimental animals. Mice received subcutaneous inoculations of 3 ⁇ 10 6 tumor cells in each of their flanks.
  • mice then received injections of one of (i) phosphate buffered saline, (ii) mAb806 (0.5 mg/injection), (iii) mAb528 (0.5 mg/injection), or (iv) a combination of both mAbs.
  • mAb806 0.5 mg/injection
  • mAb528 0.5 mg/injection
  • a combination of both mAbs a combination of both mAbs.
  • mice received either 0.5 mg/injection of each mAb, or 0.25 mg/injection of each mAb.
  • the treatment protocol began 9 days after inoculation, and continued 3 times per week for 2 weeks (i.e., the animals were inoculated 9, 11, 13, 16, 18 and 20 days after they were injected with the cells).
  • the average tumor diameter was 115 mm 3 .
  • Each group contained 50 mice, each with two tumors.
  • FIG. 18A shows the results graphically.
  • mice In a second group of mice, the injected materials were the same, except the combination therapy contained 0.25 mg of each antibody per injection. The injections were given 10, 12, 14, 17, 19 and 21 days after inoculation with the cells. At the start of the therapy the average tumor size was 114 mm 3 . Results are shown in FIG. 18B .
  • mice received inoculations of U87MG.DK.
  • Therapeutic injections started 18 days after inoculation with the cells, and continued on days 20, 22, 25, 27 and 29.
  • the average tumor size at the start of the treatment was 107 mm 3 .
  • FIG. 18C summarizes the results.
  • the therapeutic injections were the same as in the first group.
  • mice which had been inoculated with A431 cells, received injections as in groups I and III, at 8, 10, 12 and 14 days after inoculation.
  • the average tumor size was 71 mm 3 . Results are shown in FIG. 18D .
  • the combination therapy did not inhibit the growth of U87MG.DK ( FIG. 18C ), indicating that antibody immune function was not the cause for the decrease seen in FIGS. 18A and 18B .
  • mAb806 has been unexpectedly been found to inhibit the growth of tumor xenographs expressing either de2-7 or amplified EGFR, but not wild-type EGFR
  • mAb806 stained U87MG.D2-7 and U87MG.wtEGFR cells, indicating that mAb806 specifically recognized the de2-7 EGFR and a subset of the overexpressed EGFR ( FIG. 29 ).
  • the 528 antibody stained both the U87MG.D2-7 and U87MG.wtEGFR cell lines ( FIG. 29 ).
  • the intensity of 528 antibody staining on U87MG.wtEGFR cells was much higher than mAb806, suggesting that mAb806 only recognizes a portion of the overexpressed EGFR.
  • the mAb806 reactivity observed with U87MG.wtEGFR cells is similar to that obtained with A431 cells, another cell line that over expresses the wtEGFR.3
  • mAb806 had an affinity for the de2-7EGFR receptor of 1.1 ⁇ 10 9 M ⁇ 1 and recognized an average (three separate experiments) of 2.4 ⁇ 10 5 binding sites/cell, as noted in Example 4.
  • affinity of mAb806 for the wtEGFR on A431 cells was only 9.5 ⁇ 10 7 M ⁇ 1 , as noted in Example 8.
  • mAb806 recognized 2.3 ⁇ 10 5 binding sites on the surface of A431, which is some 10-fold lower than the reported number of EGFR found in these cells.
  • mAb806 reactivity was further characterized in the various cell lines by immunoprecipitation after 35 S-labeling using mAb806, sc-03 (a commercial polyclonal antibody specific for the COOH-terminal domain of the EGFR) and a IgG2b isotype control. Briefly, cells were labeled for 16 h with 100 mCi/ml of Tran 35 S-Label (ICN Biomedicals, Irvine, Calif.) in DMEM without methionine/cysteine supplemented with 5% dialyzed FCS.
  • lysis buffer 1% Triton X-100, 30 mM HEPES, 150 mM NaCl, 500 ⁇ M 4-(2-aminoethyl)benzenesulfonylfluoride (AEBSF), 150 nM aprotinin, 1 ⁇ M E-64 protease inhibitor, 0.5 mM EDTA, and 1 ⁇ M leupeptin, pH 7.4) for 1 h at 4° C. Lysates were clarified by centrifugation for 10 min at 12,000 g and then incubated with 5 ⁇ g of appropriate antibody for 30 min at 4° C. before the addition of Protein A-Sepharose. Immunoprecipitates were washed three times with lysis buffer, mixed with SDS sample buffer, separated by gel electrophoresis using a 4-20% Tris/glycine gel that was then dried, and exposed to X-ray film.
  • AEBSF 4-(2-aminoethyl)benzenesulfonylfluor
  • mAb806 also immunoprecipitated a single band corresponding to the wtEGFR from both U87MG.wtEGFR and A431 cells ( FIGS. 22 and 30 ). Consistent with the FACS and Scatchard data, the amount of EGFR immunoprecipitated by mAb806 was substantially less than the total EGFR present on the cell surface. Given that mAb806 and the sc-03 immunoprecipitated similar amounts of the de2-7 EGFR, this result supports the notion that the mAb806 antibody only recognizes a portion of the EGFR in cells overexpressing the receptor. Comparisons between mAb806 and the 528 antibody showed an identical pattern of reactivity (data not shown).
  • mAb806 was also examined for efficacy against U87MG and U87MG. ⁇ 2-7 tumors in a preventative xenograft model. Antibody or vehicle was administered i.p. the day before tumor inoculation and was given three times per week for 2 weeks. At a dose of 1 mg/injection, mAb806 had no effect on the growth of parental U87MG xenografts that express the wtEGFR ( FIG. 9A ). In contrast, mAb806 inhibited significantly the growth of U87MG. ⁇ 2-7 xenografts in a dose-dependent manner ( FIG. 9B ).
  • the mean tumor volume was 1600 ⁇ 180 mm 3 for the control group, a significantly smaller 500 ⁇ 95 mm 3 for the 0.1 mg/injection group (P ⁇ 0.0001) and 200 ⁇ 42 mm 3 for the 1 mg/injection group (P ⁇ 0.0001).
  • Treatment groups were sacrificed at day 24, at which time the mean tumor volumes were 1300 ⁇ 240 mm 3 for the 0.1 mg treated group and 500 ⁇ 100 mm 3 for the 1 mg group (P ⁇ 0.005).
  • mAb806 Given the efficacy of mAb806 in the preventative xenograft model, its ability to inhibit the growth of established tumor xenografts was examined. Antibody treatment was as described in the preventative model, except that it commenced when tumors had reached a mean tumor volume of 65 mm 3 (10 days after implantation) for the U87MG. ⁇ 2-7 xenografts and 84 mm 3 (19 days after implantation) for the parental U87MG xenografts (see Example 10). Once again, mAb806 had no effect on the growth of parental U87MG xenografts, even at a dose of 1 mg/injection ( FIG. 10A ).
  • mAb806 significantly inhibited the growth of U87MG. ⁇ 2-7 xenografts in a dose-dependent manner ( FIG. 10B ).
  • the mean tumor volume was 900 ⁇ 200 mm 3 for the control group, 400 ⁇ 60 mm 3 for the 0.1 mg/injection group (P ⁇ 0.01), and 220 ⁇ 60 mm 3 for the 1 mg/injection group (P ⁇ 0.002).
  • Treatment of U87MG. ⁇ 2-7 xenografts with an IgG2b isotype control had no effect on tumor growth (data not shown).
  • mice containing A431 xenografts contain an amplified EGFR gene and express approximately 2 ⁇ 10 6 receptors/cells.
  • mAb806 binds 10% of these EGFRs and targets A431 xenografts (Garcia et al. (1993) Expression of mutated epidermal growth factor receptor by non-small cell along carcinomas. Cancer Res. 53, 3217-3220).
  • mAb806 significantly inhibited the growth of A431 xenografts when examined in the preventative xenograft model described previously ( FIG. 11A ).
  • mAb806 in the preventative A431 xenograft model, its ability to inhibit the growth of established tumor xenografts was examined. Antibody treatment was as described in the preventative model, except it was not started until tumors had reached a mean tumor volume of 200 ⁇ 20 mm 3 . mAb806 significantly inhibited the growth of established A431 xenografts ( FIG. 11B ). At day 13, the day control animals were sacrificed, the mean tumor volume was 1100 ⁇ 100 mm 3 for the control group and 450 ⁇ 70 mm 3 for the 1 mg/injection group (P ⁇ 0.0001).
  • Chimeric antibodies are a class of molecules in which heavy and light chain variable regions of for instance, a mouse, rat or other species are joined onto human heavy and light chain regions, Chimeric antibodies are produced recombinantly.
  • One advantage of chimeric antibodies is that they can reduce xenoantigenic effects, the inherent immunogenicity of non-human antibodies (for instance, mouse, rat or other species).
  • recombinantly prepared chimeric antibodies can often be produced in large quantities, particularly when utilizing high level expression vectors.
  • dhfr-CHO cells are transfected with an expression vector containing a functional DHFR gene, together with a gene that encodes a desired protein.
  • the desired protein is recombinant antibody heavy chain and/or light chain.
  • the recombinant cells develop resistance by amplifying the dhfr gene.
  • the amplification unit employed is much larger than the size of the dhfr gene, and as a result the antibody heavy chain is co-amplified.
  • both the expression level, and the stability of the cells being employed, are critical.
  • recombinant CHO cell populations lose homogeneity with respect to their specific antibody productivity during amplification, even though they derive from a single, parental clone.
  • Bicistronic expression vectors were prepaid for use in recombinant expression of the chimeric antibodies. These bicistronic expression vectors, employ an “internal ribosomal entry site” or “IRES.” In these constructs for production of chimeric anti-EGFR, the immunoglobulin chains and selectable markers cDNAs are linked via an IRES. IRES are cis-acting elements that recruit the small ribosomal subunits to an internal initiator codon in the mRNA with the help of cellular trans-acting factors. IRES facilitate the expression of two or more proteins from a polycistronic transcription unit in eukaryotic cells.
  • IRES elements have been successfully incorporated into vectors for cellular transformation, production of transgenic animals, recombinant protein production, gene therapy, gene trapping, and gene targeting.
  • the chimeric 806 antibody was generated by cloning the VH and VL chains of the 806 antibody from the parental murine hybridoma using standard molecular biology techniques.
  • the VH and VL chains were then cloned into the pREN mammalian expression vectors, the construction of which are set forth in SEQ ID NO:7 and SEQ ID NO:8, and transfected into CHO (DHFR ⁇ / ⁇ ve) cells for amplification and expression. Briefly, following trypsinization 4 ⁇ 10 6 CHO cells were co-transferred with 10 ⁇ g of each of the LC and HC expression vectors using electroporation under standard conditions.
  • the cells were added to 15 ml medium (10% fetal calf serum, hypoxanthine/thymidine supplement with additives) and transferred to 15 ⁇ 10 cm cell culture petri dishes. The plates were then placed into the incubator under normal conditions for 2 days.
  • 15 ml medium (10% fetal calf serum, hypoxanthine/thymidine supplement with additives)
  • Clones growing at 100 nM MTX were then passed onto the Biological Production Facility, Ludwig Institute, Melbourne, Australia for measurement of production levels, weaning off serum, cell banking.
  • the cell line has been shown to stably produce ⁇ 10mg/litre in roller bottles.
  • the nucleic acid sequence of the pREN ch806 LC neo vector is provided in SEQ ID NO:7.
  • the nucleic acid sequence of the pREN ch806 HC DHFR vector is provided in SEQ ID NO:8.
  • FIG. 33 depicts the vectors pREN-HC and pREN-LC, which employ an IRES.
  • the pREN bicistronic vector system is described and disclosed in co-pending U.S. Patent Application No. 60/355,838 filed Feb. 13, 2002, which is incorporated herein by reference in its entirety.
  • ch806 was assessed by FACS analysis to demonstrate that the chimeric 806 displays identical binding specificity to that of the murine parental antibody. Analysis was performed using wild-type cells (U87MG parental cells), cells overexpressing the EGF receptor (A431 cells and UA87.wtEGFR cells) and UA87. ⁇ 2-7 cells (data not shown). Similar binding specificity of mAb806 and ch806 was obtained using cells overexpressing EGFR and cells expressing the de2-7 EGFR. No binding was observed in wild-type cells. Scatchard analysis revealed a binding affinity for radiolabeled ch806 of 6.4 ⁇ 10 9 M ⁇ 1 using U87MGde2-7 cells (data not shown).
  • the 111 In-labelled ch806 shows some nonspecific retention in the liver, spleen and kidneys. This is common for the use of this isotope and decreases with time, which supports that this binding is non-specific to ch806 and due to 111 In binding.
  • Chimeric antibody ch806 was assessed for therapeutic efficacy in an established tumor model. 3 ⁇ 10 6 U87MG. ⁇ 2-7 cells in 100 ⁇ l of PBS were inoculated s.c. into both flanks of 4-6 week old female nude mice (Animal Research Center, Western Australia, Australia). The mAb806 was included as a positive control. The results are depicted in FIG. 36 . Treatment was started when tumors had reached a mean volume of 50 mm 3 and consisted of 1 mg of ch806 or mAb806 given i.p. for a total of 5 injections on the days indicated.
  • Tumor volume in mm 3 was determined using the formula (length ⁇ width 2 )/2, where length was the longest axis and width the measurement at right angles to the length. Data was expressed as mean tumor volume ⁇ S.E. for each treatment group.
  • the ch806 and mAb806 displayed nearly identical anti-tumor activity against U87MG. ⁇ 2-7 xenografts.
  • Murine anti-de2-7 EGFR monoclonal mAb806, chimeric antibody ch806 (IgG 1 ) and control isotype matched chimeric anti-G250 monoclonal antibody cG250 were prepared by the Biological Production Facility, Ludwig Institute for Cancer Research, Melbourne, Australia. Both complement-dependant cytotoxicity (CDC) and antibody-dependent cellular-cytotoxicity (ADCC) assays utilized U87MG.de2-7 and A431 cells as target cells.
  • the previously described U87MG.de2-7 cell line is a human astrocytoma cell line infected with a retrovirus containing the de2-7EGFR (Nishikawa et al. (1994) Proc. Natl. Acad. Sci. U.S.A.
  • Human squamous carcinoma A431 cells were purchased from the American Type Culture Collection (Manassas, Va.). All cell lines were cultured in DMEM/F-12 with Glutamax (Life Technologies, Melbourne, Australia) supplemented with 10% heat-inactivated FCS (CSL, Melbourne, Australia), 100 units/ml penicillin and 100 ⁇ g/ml streptomycin. To maintain selection for retrovirally transfected U87MG.de2-7 cells, 400 ⁇ g/ml G418 was included in the media.
  • PBMCs were isolated from healthy volunteer donor blood. Heparinized whole blood was fractionated by density centrifugation on Ficoll-Hypaque (ICN Biomedical Inc., Ohio, USA). PBMC fractions was collected and washed three times with RPMI + 1640 supplemented with 100 U/ml penicillin and 100 ⁇ g/ml streptomycin, 2 mM L-glutamine, containing 5% heat-inactivated FCS.
  • the cells were then washed three time with PBS (0.05M, pH 7.4) and a fourth wash with culture medium. Aliquots (1 ⁇ 10 4 cells/50 ⁇ l) of the labeled cells were added to each well of 96-well microtitre plates (NUNC, Roskilde, Denmark).
  • 50 ⁇ l ch806 or isotype control antibody cG250 were added in triplicate over the concentration range 0.00315-10 ⁇ g/ml, and incubated on ice 5 min. Fifty ⁇ l of freshly prepared healthy donor complement (serum) was then added to yield a 1:3 final dilution of the serum. The microtitre plates were incubated for 4 hr at 37° C. Following centrifugation, the released 51 Cr in the supernatant was counted (Cobra II automated Gamma Counter, Canberra Packard, Melbourne, Australia). Percentage specific lysis was calculated from the experimental 51 Cr release, the total (50 ⁇ l target cells+100 ⁇ l 10% Tween 20) and spontaneous (50 ⁇ l target cells+100 ⁇ l medium) release.
  • ch806-mediated ADCC effected by healthy donor PBMCs was measured by two 4-hr 51 lCr release assays.
  • labelled target cells were plated with the effector cells in 96-well “U” bottom microplates (NUNC, Roskilde, Denmark) at effector/target (E:T) cell ratios of 50:1.
  • E:T effector/target
  • 0.00315-10 ⁇ g/ml (final concentration) test and control antibodies were added in triplicate to each well.
  • the ADCC activity of ch806 was compared with the parental murine mAb806 over a range of Effector: Target cell ratios with the test antibody concentration constant at 1 ⁇ g/ml.
  • the percent (%) cytotoxicity was plotted versus concentration of antibody ( ⁇ g/ml).
  • ch806 mediated ADCC on target U87MG.de2-7 and A431 cells at E:T ratio of 50:1 is presented in FIG. 38 .
  • Effective ch806 specific cytotoxicity was displayed against target U87MG.de2-7 cells, but minimal ADCC was mediated by ch806 on A431 cells.
  • the levels of cytotoxicity achieved reflect the number of ch806 binding sites on the two cell populations.
  • Target U87MG.de2-7 cells express ⁇ 1 ⁇ 10 6 de2-7EGFR which are specifically recognized by ch806, while only a subset of the 1 ⁇ 10 6 wild-type EGFR molecules expressed on A431 cells are recognized by ch806 (see above Examples).
  • mice monoclonal anti-idiotypic antibodies (anti-ids) were generated and characterized for suitability as ELISA reagents for measuring ch806 in patient sera samples and use as positive controls in human anti-chimeric antibody immune response analyses. These anti-idiotype antibodies may also be useful as therapeutic or prophylactic vaccines, generating a natural anti-EGFR antibody response in patients.
  • mouse monoclonal anti-idiotypic antibodies were generated as follows. Splenocytes from mice immunized with ch806 were fused with SP2/0-AG14 plasmacytoma cells and antibody producing hybridomas were selected through ELISA for specific binding to ch806 and competitive binding for antigen ( FIG. 40 ). Twenty-five hybridomas were initially selected and four, designated LMH-11,-12,-13, and-14, secreted antibodies that demonstrated specific binding to ch806, mAb806 and were able to neutralize ch806 or mAb806 antigen binding activity ( FIG. 41 ). The recognition of the ch806/mAb806 idiotope or CDR region was demonstrated by lack of cross-reactivity with purified polyclonal human IgG.
  • LMH Ludwig Institute for Cancer Research Melbourne Hybridoma
  • anti-ch806 antibodies to concurrently bind two ch806 antibodies is a desirable feature for their use as reagents in an ELISA for determining serum ch806 levels.
  • results demonstrated the antagonist activity of anti-idiotype mAbs LMH-11, -12, -13, and -14 with the blocking in solution of both ch806 and murine mAb806 binding to plates coated with sEGFR ( FIG. 41 for LMH-11, -12, -13).
  • LMH-11 through -14 antibodies were identified as isotype IgGl ⁇ by mouse monoclonal antibody isotyping kit.
  • the recombinant sEGFR expressed in CHO cells was treated with PNGase F to remove N-linked glycosylation.
  • the protein was run on SDS-PAGE, transferred to membrane and immunoblotted with mAb806 ( FIG. 44 ).
  • the deglycosylated sEGFR ran faster on SDS-PAGE, indicating that the carbohydrates had been successfully removed.
  • the mAb806 antibody clearly bound the deglycosylated material demonstrating the antibody epitope is peptide in nature and not solely a glycosylation epitope.
  • Lysates prepared from cell lines metabolically labelled with 35 S, were immunoprecipitated with different antibodies directed to the EGFR ( FIG. 45 ).
  • the 528 antibody immunoprecipitated three bands from U87MG. ⁇ 2-7 cells, an upper band corresponding to the wild-type (wt) EGFR and two lower bands corresponding to the de2-7 EGFR.
  • These two de2-7 EGFR bands have been reported previously and are assumed to represent differential glycosylation (Chu et al. (1997) Biochem. J. June 15; 324 (Pt 3): 885-861).
  • mAb806 only immunoprecipitated the two de2-7 EGFR bands, with the wild-type receptor being completely absent even after over-exposure (data not shown).
  • mAb806 showed increased relative reactivity with the lower de2-7 EGFR band but decreased reactivity with the upper band when compared to the 528 antibody.
  • the SC-03 antibody a commercial rabbit polyclonal antibody directed to C-terminal domain of the EGFR, immunoprecipitated the three EGFR bands as seen with the 528 antibody, although the total amount of receptor immunoprecipitated by this antibody was considerably less. No bands were observed when using an irrelevant IgG2b antibody as a control for mAb806 (see Example 18).
  • the 528 antibody immunoprecipitated a single band from U87MG.wtEGFR cells corresponding to the wild-type receptor ( FIG. 45 ).
  • mAb806 also immunoprecipitated a single band from these cells, however, this EGFR band clearly migrated faster than the 528 reactive receptor.
  • the SC-03 antibody immunoprecipitated both EGFR reactive bands from U87MG.wtEGFR cells, further confirming that the mAb806 and 528 recognize different forms of the EGFR in whole cell lysates from these cells.
  • the 528 antibody immunoprecipitated a single EGFR band from A431 cells ( FIG. 45 ).
  • the 528 reactive EGFR band is very broad on these low percentage gels (6%) and probably reflects the diversity of receptor glycosylation.
  • a single EGFR band was also seen following immunoprecipitation with mAb806. While this EGFR band did not migrate considerably faster than the 528 overall broad reactive band, it was located at the leading edge of the broad 528 band in a reproducible fashion.
  • A431 and U87MG. ⁇ 2-7 cells were pulsed for 5 min with 35 S methionine/cysteine, then incubated at 37° C. for various times before immunoprecipitation with mAb806 or 528 ( FIG. 46 ).
  • the immunoprecipitation pattern in A431 cells with the 528 antibody was typical for a conformational dependent antibody specific for the EGFR.
  • a small amount of receptor was immunoprecipitated at 0 min (i.e. after 5 min pulse) with the amount of labelled EGFR increasing at each time point. There was also a concurrent increase in the molecular weight of the receptor with time.
  • mAb806 reactive EGFR material was present at high levels at 0 min, peaked at 20 min and then reduced at each further time point. Thus, it appears that mAb806 preferentially recognizes a form of the EGFR found at an early stage of processing.
  • the lower de2-7 EGFR band was fully sensitive to Endo H digestion, migrating faster on SDS-PAGE after Endo H digestion, demonstrating that this band represents the high mannose form of the de2-7 EGFR.
  • the upper de2-7 EGFR band was essentially resistant to Endo H digestion, showing only a very slight difference in migration after Endo H digestion, indicating that the majority of the carbohydrate structures are of the complex type.
  • the small but reproducible decrease in the molecular weight of the upper band following enzyme digestion suggests that while the carbohydrates on the upper de2-7 EGFR band are predominantly of the complex type, it does possess some high mannose structures.
  • these cells also express low amounts of endogenous wtEGFR that is clearly visible following 528 immunoprecipitation. There was also a small but noticeable reduction in molecular weight of the wild-type receptor following Endo H digestion, indicating that it also contains high mannose structures.
  • Cell surface iodination of the A431 celline was performed with 125 I followed by immunoprecipitation with the 806 antibody.
  • the protocol for surface iodination was as follows: The cell lysis, immunoprecipitation, Endo H digestion, SDS PAGE and autoradiography are as described above herein.
  • For labeling cells were grown in media with 10% FCS, detached with EDTA, washed twice with PBS then resuspended in 400 ⁇ l of PBS (approx 2-3 ⁇ 10 6 cells).
  • 8C65AAG 11891 bp; SEQ ID NO:41
  • 8C65AAG 11891 bp; SEQ ID NO:41
  • VH and CH regions are shown in FIG. 55A , with the VH region CDR1, CDR2, and CDR3 (SEQ ID NOS:44, 45, and 46, respectively) indicated by underlining.
  • VL and CL regions are shown in FIG. 55B , with the VL region CDR1, CDR2, and CDR3 (SEQ ID NOS:49, 50, and 51, respectively) indicated by underlining.
  • VL and VH chain of the mouse monoclonal antibody (mAb) 806 have been re-engineered by gene-synthesis and overlapping PCR primer technology.
  • CL (kappa) chain was assembled in the same manner.
  • vVL and vVH were also expressed in a scFv format that demonstrated good binding to the synthetic peptide that comprises the 806 antigenic epitope by ELISA and to recombinant EGF Receptor (EGFR) extracellular domain (ECD) as measured by surface plasmon resonance (SPR) analysis.
  • EGFR EGF Receptor
  • ECD extracellular domain
  • the v806VL and v806VH have been engineered into a full length human IgG1 context using a codon-optimized kappa-LC and a newly designed codon-and splice-site optimized human IgG1 heavy chain constant region to achieve stable gene expression in NSO and CHO cell systems.
  • the expression system is based on the LONZA GS expression system using the pEE12.4 and pEE6.4 heavy and light chain expression vectors as provided by LONZA Biologics.
  • the hu806 antibody product ( FIG. 55 ) obtained by transient expression of the 8C65AAG vector was reactive with recombinant EGFR-ECD by SPR, and with the synthetic EGFR 806 peptide epitope by ELISA.
  • the 8C65AAG vector was transferred to LICR affiliate Christoph Renner (University of Zurich) for generation of stable GS-NSO hu806 cell lines and to LICR, Melbourne Centre, for the generation of GS-CHO hu806 cell lines.
  • Antibody veneering is a humanization strategy aimed at countering HAMA (human anti-mouse antibody) responses.
  • Mouse mAbs are considered “foreign” antigens by a patient's immune system and an immune response is induced, even upon a single administration, preventing further use of the reagent in those patients.
  • the amino acid sequences of the VL and VH chains in mAb806 were analyzed, and each amino acid residue in the mAb806 protein sequence was graded for surface exposure ( FIG. 56 and FIG. 57 ). Only those amino acids that resided on the outside of the antibody molecule were considered for possible modification, as these were the only ones that would be exposed to antibody recognition.
  • the mAb806 protein sequence was compared to three human antibody sequences (VH36germ, CAD26810, and AAA37941). Wherever a mAb806 surface residue did not match the consensus of the human antibody sequences, that residue was identified to be changed to the consensus sequence. Initially 12 amino acids in the VL were subjected to veneering; and 14 in the VH chain of ch806 ( FIG. 56 and FIG. 57 ).
  • Codon optimization is a means of improving the heterologous expression of antibodies or other proteins based on the codon bias of the system used to express these antibodies.
  • One of the goals in the creation of hu806 was to utilize codon optimization to improve expression levels for this antibody.
  • the expression system is based upon the LONZA GS expression system using the pEE12.4 and pEE6.4 HC and LC expression vectors as provided by LONZA Biologics and NSO and/or CHO cells as production cells. Thus, decisions about which codon to use for a given amino acid were made with consideration for whether or not that codon would be favored in the NSO/CHO expression systems.
  • VH variable heavy
  • VL variable light
  • oligonucleotides were designed as overlapping sense and antisense primers. These oligos would overlap each other in such a way as to cover the entire hu806 VH or VL sequence, including the signal sequence, coding sequences, introns, and include a HindIII site at the 5′ terminus and a 3′ BamHI site at the 3′ terminus.
  • the oligonucleotide maps are presented in FIGS. 56B and 57B , and the primer details are provided below.
  • the hu806 VH or VL was assembled by PCR as follows: Initially v806hc- or v8061c-oligos 1, 2, 3, 4, oligos 5, 6, and oligos 7, 8, 9, 10 were combined in three separate reactions. Aliquots (50 pmol) of each flanking oligo, and 5 pmol of each internal oligo were added to a 50 ⁇ l PCR reaction containing 25 ⁇ l of 2 ⁇ HotStar Taq Master Mix (Qiagen) and 48 ⁇ l of nuclease free water.
  • Qiagen HotStar Taq Master Mix
  • thermo cycle program was as follows: 95° C.; 15′′, [94° C.; 30′′, 58° C.; 30′′, 72° C.; 30′′] ⁇ 20 cycles, 72° C.; 10′′, 4° C.
  • the products of these three reactions were excised after separation by gel electrophoresis. They were then purified using a salt column (Qiagen-Qiaspin Minipreps), and combined. These products were further amplified by PCR using primers 1 and 10.
  • the product of this second reaction included restriction enzyme sites for HindIII and BamHI, enabling insertion into expression plasmids.
  • v806 VH v806hc-1: GAGAAGCTTGCCGCCACCATGGATTGGACCTGGCGCATTC 52 v806hc-2: CCCTTCCTCCTCACTGGGATTTGGCAGCCCCTTACCTGTGGCGGCTGCT 53 ACCAGAAAGAGAATGCGCCAGGTCCAATCC v806hc-3: CCCAGTGAGGAGGAAGGGATCGAAGGTCACCATCGAAGCCAGTCAAG 54 GGGGCTTCCATCCACTCCTGTGTCTTCTCTAC v806hc-4: GACTCGGCTTGACAAGCCCAGGTCCACTCTCTTGGAGCTGCACCTGGCT 55 GTGGACACCTGTAGAGAAGACACAGGAGTGG v806hc-5: GGGCTTGTCAAGCCGAGTCAAACTTTGTCCCTAACATGTACTGTGTCCG 56 GATACTCTATCTCATCAGATTTTGCGTGGAATTGG v806hc-6: CCCAGAGTATGATATGTA
  • a codon-optimized version of the constant kappa light chain (CL) was prepared in a manner similar to that used for the variable regions
  • the initial PCR step involved the creation of only two preliminary products using oligos VKlcons-1, 2, 3, 4; and 5, 6, 7, 8.
  • the flanking restriction sites for this product were BamHI and NotI prior to plasmid insertion.
  • VK1cons-1 GACGGATCCTTCTAAACTCTGAGGGGGTCGGATGACG 72
  • VK1cons-2 GGAGCTGCGACGGTTCCTGAGGAAAGAAGCAAACAGGATGGTGTTTAA 73
  • VK1cons-3 GGAACCGTCGCAGCTCCCTCCGTGTTCATCTTCCCCCCATCCGACGAGC 74
  • VK1cons-5 GTGGAAAGTGGACAACGCACTACAGAGCGGGAACTCTCAGGAAAGCG 76 TGACAGAGCAGGACTCAAAAGATTCAACATACAGCC
  • VK1cons-6 CTTCACAGGCATATACCTTGTGCTTTTCATAATCAGCTTTTGACAGTGTC 77 AGGGTAGA
  • a synthetic, humanized version of the IgG1 constant heavy chain (CH) gene (SEQ ID NO:80) was purchased from GeneArt, Regensburg, Germany. The gene was codon optimized for expression in CHO/NSO cells. Details of the gene sequence, restriction sites, etc, are shown in FIG. 58 .
  • hu806 VH and VL sequences prepared in the manner described above were ligated into expression vectors containing generic constant regions. These vectors, provided by LICR affiliate Christoph Renner (University of Zurich, Switzerland), were known as pEAK8 HC (which contained a generic CH), and a33-xm-lc (which contained a generic CL). Vectors were digested using BamHI and HindIII in the presence of CIP then hu806 VH and VL were ligated into the corresponding vectors. The resulting plasmids were used to transform Top10 chemically competent E. coli (Invitrogen) according to the manufacturer's directions. Transformed E.
  • Coli were plated on LB+Ampicillin plates, and resistant clones were screened by restriction digestion and PCR. In general, eight positive clones detected in this manner would be isolated and further amplified. DNA purified from these colonies were analyzed by automated DNA sequencing.
  • Codon-optimized versions of the constant regions were added to these constructs by restriction enzyme-digestion and ligation using BamHI and NotI. These transformants were selected, sequenced, and analyzed as stated above. Prior to the full-length antibody chains being ligated into the Lonza GS system the BamHI site between the variable and constant region sequences was destroyed, in one case, by digestion using BamHI, fill-in using DNA Polymerase, and blunt-end ligation.
  • Restriction fragments containing hu806 (VH+CH) or hu806 (VL+CL) were then digested with NotI followed by HindIII. These digestions were designed to create a blunt end at the NotI site, and thus were done in series in the following manner:
  • the plasmid was first digested with NotI. Fully digested (single-cut) plasmid was separated by electrophoresis using a 1% agarose gel. This product was then excised and purified on a salt column and filled-in using DNA Polymerase. The product of this reaction was salt-column purified and then digested with HindIII. This product ( ⁇ 1.3 Kb for hu806 (VH+CH), and ⁇ 0.8 Kb for hu806 (VL+CL) was then separated by gel electrophoresis, excised, and purified.
  • Vectors pEE12.4 and pEE6.4 were each digested on HindIII and PmII.
  • hu806 VH+CH
  • hu806 VL+CL
  • pEE6.4 pEE6.4-hu806L
  • a combined, double gene Lonza plasmid was created to contain both the hu806 heavy and light chain sequences.
  • the pEE12.4-hu806H and pEE6.4-hu806L vectors were digested with NotI and SalI restriction enzymes.
  • the resultant fragments which contained the GS transcription unit and hCMV-MIE promoter, followed by the hu806 Heavy or Light chain expression cassette, were isolated and ligated together.
  • the resulting “combined” Lonza plasmid (Designated 8C65AAG) was used for single-plasmid transient transfections in a HEK 293 system and stable transfections in NSO and CHO systems.
  • a plasmid map is shown in FIG. 53 .
  • the reference file (mAb806 LC) incorrectly indicates Histidine (H), not the correct Tyrosine (Y) at position 91; the subject of modification #1.
  • the original, uncorrected file sequence is included in FIG. 60 , to illustrate the necessary modification made to hu806 at position 91.
  • Restriction enzyme sites were added to the introns surrounding the hu806 VH and VL regions. These restriction sites (unique in the pREN vector system, LICR) were designed to ease the process of making modifications to the expression cassettes.
  • the hu806 VH sequence, not including the initial signal region, could be removed or inserted by single-digestion on DraIII.
  • FseI could be used, in concert with NotI (PREN system) or EcoRI (Lonza System) to cut out the constant region, fulfilling the function of BamHI from the original sequence.
  • the restriction sites added were RsrII, having the same function as DraIII in the heavy chain, and PacI, which matched the function of FseI.
  • the protein sequence for the parental mAb806 at VH amino acids 81-87 is SVTIEDT (SEQ ID NO:96).
  • SVTIEDT SEQ ID NO:96
  • isoleucine and glutamic acid at positions 84 and 85 were changed to alanine-proline to read SVTAPDT (SEQ ID NO:97; FIG. 56 ).
  • Site-directed mutagenesis was used to generate this secondary change (SVTAADT, SEQ ID NO:98) using the primers listed below.
  • Final DNA and translated protein sequences are presented in FIG. 62 .
  • the hu806 heavy chain variable region sequence underwent three further mutations following the initial veneering: T70S, S76N and Q81K.
  • the change at position 76 from serine to asparagine represented a correction back to the original sequence of mAb806 molecule.
  • the additional changes in the framework were included because they represent residues that are not found in mouse antibodies but are found in human antibodies. Accordingly, the protein sequence TRDTSKSQFFLQ (SEQ ID NO: 101) was veneered to SRDTSKNQFFLK (SEQ ID NO: 102).
  • Final DNA and translated protein sequences in comparison to mAb806 are presented in FIG. 62 .
  • N-Glycosylation follows the scheme: N X S/T, where X is any amino acid.
  • the amino acid sequence from position 60 was N P S, which follows this scheme.
  • proline as in our example
  • cysteine is found at the X position for N-glycosylation. It was of concern that inconsistent glycosylation could lead to variations in the reactivity of the antibody.
  • asparagine was removed, and replaced with its most closely related amino acid, glutamine, removing any potential for this site to be glycosylated ( FIG. 59 and FIG. 62 ).
  • Transient transfection of 293FT cells with the final plasmid 8C65AAG was performed to enable the preparation of small quantities of hu806 for initial antigen binding verification.
  • Culture supernatants from several small-scale replicate transient transfections were pooled, concentrated and hu806 antibody was collected using a protein-A chromatography step.
  • Approximately 1-2 ⁇ g of hu806 antibody was obtained as measured by a quantitative huIgG1 ELISA and the antibody was analyzed by Biacore for binding to recombinant EGFR-ECD ( FIG. 63 ).
  • Bovine immunoglobulin from the cell culture medium co-purified with hu806 and represented the major fraction of total IgG, limiting quantitative assessment of hu806 binding.
  • RenVecUPSTREAM Sense primer, begins sequencing upstream of variable region in peak8, and a33xm vectors.
  • RenVecDwnstrmLC Antisense primer, begins sequencing downstream of variable region on a33-xm-lc light-chain plasmid. Anneals within non-codon-optimized LC constant region.
  • Upstrm Lonza Sense primer, begins sequencing upstream of variable region in Lonza vectors pEE 12.4 and pEE 6.4. Cannot be used with combined Lonza because this is a duplicate region in the combined plasmid.
  • Dnstrm 6-4 Antisense primer, begins sequencing downstream of constant region in Lonza vector pEE 6.4
  • Dnstrm 12-4 Antisense primer, begins sequencing downstream of constant region in Lonza vector pEE12.4
  • Cod-Opt LC const E Sense primer, internal to the codon-optimized light-chain v-kappa constant region
  • Cod-Opt LC const F Antisense primer, internal to the codon-optimized light-chain v-kappa constant region (vk).
  • 806HCspec Sense primer, internal and unique to the veneered version of the 806 HC variable region.
  • 806LCspec Sense primer, internal and unique to the veneered version of the 806 LC variable region.
  • GenBank formatted text document of the sequence and annotations of plasmid 8C65AAG encoding the IgG1 hu806 is set forth in FIG. 64 .
  • the veneering of the 806 anti-EGF receptor antibody involved mutation of 14 amino acids in the VH ( FIG. 59 and FIG. 62 ), and 12 changes to the VL chain ( FIG. 60 and FIG. 61 ) with codon optimization as indicated for expression in mammalian CHO or NSO cells.
  • the final double gene vector, designated 8C65AAG has been sequence-verified, and the coding sequence and translation checked. Binding to recombinant EGFR extracellular domain was confirmed by Biacore analyses using transiently expressed hu806 product.
  • Stable single clones producing high levels of intact hu806 antibody have been selected in glutamine-free medium as recommended by LONZA. Stable clones have been gradually weaned off serum to obtain serum-free cultures.
  • FIG. 76 presents the cell viability and antibody productivity charts for the four transfectants during the culture.
  • Product concentration was estimated by ELISA using the 806 anti-idiotype antibody LMH-12 (Liu et al. (2003) Generation of anti-idiotype antibodies for application in clinical immunotherapy laboratory analyses. Hybrid Hybridomics. 22(4), 219-28) as coating antibody, and ch806 Clinical Lot: J06024 as standard. Material at harvest was centrifuged and supernatant was 0.2 ⁇ m filtered then the antibodies were affinity purified by Protein-A chromatography.
  • the CHO-K1SV transfectant cell line expressing hu806 candidate clone 40A10 was cultured in a 15 L stirred tank bioreactor with glucose shot feeding for 16 days using CD-CHO (Invitrogen)/25 ⁇ M L-Methionine sulfoximine (MSX; Sigma)/GS supplements (Sigma) as the base media.
  • FIG. 76C presents the cell growth and volumetric production in the 15 L stirred tank bioreactor. Final yield was 14.7 L at 58 mg/L by ELISA.
  • the hu806 products from the small and large scale cultures were quantified by OD A280 nm.
  • the antibody samples recovered from rProtein-A were assessed by Size Exclusion Chromatography (SEC) (small scale, FIG. 77 ; large scale, FIG. 78 ), 4-20% Tris-Glycine SDS-PAGE under reduced and non-reduced conditions ( FIGS. 79-81 ), and Isoelectric Focusing was performed with an Amersham Multiphor II Electrophoresis system on an Ampholine PAG plate (pH 3.5-9.5) according to the manufacturer's instructions ( FIG. 82 ).
  • SEC Size Exclusion Chromatography
  • the Protein-A affinity purified hu806 antibodies displayed symmetrical protein peaks and identical SEC elution profiles to the ch806 clinical reference material.
  • the SDS-PAGE gel profiles were consistent with an immunoglobulin.
  • the IEF pattern indicated three isoforms with pI ranging from 8.66 to 8.82 which was consistent with the calculated pI of 8.4 for the protein sequence.
  • hu806 40A10 sample produced by large scale culture was also assessed by FACS for binding A431 as well as U87MG.de2-7 glioma cells expressing the variant EGFRvIII receptor (Johns et al., 2002). Representative results of duplicate analyses are presented in FIG. 84 and FIG. 85 , respectively. Controls included an irrelevant IgG2b antibody (shaded histograms), ch806 or 528 (binds both wild-type and de2-7 EGFR) as indicated.
  • the ch806 and the hu806 antibody demonstrated similar staining of the A431 and U87MG.de2-7 cell lines supporting our previous observations that mAb806 specifically recognized the de2-7 EGFR and a subset of the over-expressed EGFR (Luwor et al. (2001) Monoclonal antibody 806 inhibits the growth of tumor xenografts expressing either the de2-7 or amplified epidermal growth factor receptor (EGFR) but not wild-type EGFR. Cancer Res. 61(14), 5355-61). As expected, the 528 antibody stained both the U87MG.de2-7 and A431 cell lines ( FIGS. 84 and 85 ).
  • the antigen binding capabilities of the radioimmunoconjugates were assessed by cell adsorption assays (Lindmo et al. (1984) Determination of the immunoreactive fraction of radiolabeled monoclonal antibodies by linear extrapolation to binding at infinite antigen excess. J. Immunol. Methods. 72(1), 77-89) using the U87MG.de2-7 glioma cell line and A431 epidermoid carcinoma cells expressing the amplified EGFR gene.
  • Immunoreactive fractions of hu806 and ch806 radioconjugates were determined by binding to antigen expressing cells in the presence of excess antigen.
  • Results for U87MG.de2-7 cell binding of 125 I-hu806 and 125 I-ch806 are presented in FIG. 86A over the cell concentration range 20 ⁇ 10 6 to 0.03 ⁇ 10 6 cells/sample.
  • Results for A431 cell binding of 125 I-hu806 and 125 I-ch806 are presented in FIG. 86B over the cell concentration range 200 ⁇ 10 6 to 0.39 ⁇ 10 6 cells/sample.
  • the binding affinity for 125 I-hu806 binding EGFRvIII on U87MG.de2-7 cells was determined to be 1.18 ⁇ 10 9 M ⁇ 1 .
  • the Ka for 125 I-ch806 was 1.06 ⁇ 10 9 M ⁇ 1 .
  • the scatchard analysis on A431 cells demonstrated high affinity binding by both 806 constructs to a minor population of EGFR on these cells.
  • Biosensor analyses were performed on a BIAcore 2000 biosensor using a carboxymethyldextran-coated sensor chip (CM5).
  • the chip was derivatized on channel 3 with the 806 epitope peptide (EGFR amino acids 287-302; see U.S. patent application Ser. No. 11/060,646, filed Feb. 17, 2005; U.S. Provisional Patent Application No. 60/546,602, filed Feb. 20, 2004; and U.S. Provisional Patent Application No. 60/584,623, filed Jul. 1, 2004, the disclosure of each is which is hereby incorporated in its entirety), using standard amine coupling chemistry.
  • Channel 2 was derivatized with a control antigen used for system suitability determination.
  • Channel 1 was derivatized with ethanolamine and used as a blank control channel for correction of refractive index effects.
  • Samples of hu806 were diluted in HBS buffer (10 mM HEPES, pH 7.4; 150 mM NaCl; 3.4 mM di-Na-EDTA; 0.005% Tween-20), and aliquots (120 ⁇ l) containing 50 nM, 100 nM, 150 nM, 200 nM, 250 nM and 300 nM were injected over the sensor chip surface at a flow rate of 30 ⁇ l/min. After the injection phase, dissociation was monitored by flowing HBS buffer over the chip surface for 600 s.
  • Bound antibody was eluted and the chip surface regenerated between samples by injection of 20 ⁇ l of 10 mM sodium hydroxide solution. Positive control, ch806, was included. The binding parameters were determined using the equilibrium binding model of the BIAevaluation software.
  • FIG. 89 present the sensorgrams generated.
  • hu806 consistently demonstrated superior ADCC activity to the chimeric ch806 IgG1.
  • hu806 at 1 ⁇ g/mL effected an ADCC of 30% cytotoxicity in contrast to ch806 5% cytoxicity.
  • mice were injected subcutaneously into the right and left inguinal mammary line with 1 ⁇ 10 6 A431 adenocarcinoma cells or 1 ⁇ 10 6 U87MG.de2-7 glioma cells in 100 ⁇ l of PBS.
  • Tumor volume (TV) was calculated by the formula [(length ⁇ width 2 )/2] where length was the longest axis and width the measurement at right angles to length.
  • the in vivo therapy assessments with hu806 showed a marked reduction in A431 xenograft growth compared with PBS vehicle control.
  • the A431 xenograft growth curve observed for hu806 was highly comparable to the ch806 treatment group.
  • the PBS control group was euthanized at day 20.
  • the hu806 therapy demonstrated significant reduction in tumor growth by day 20 compared to the PBS controls (P ⁇ 0.001), and continued tumor growth retardation after day 20 similar to the ch806 group.
  • the Protein-A affinity purified hu806 antibodies displayed identical SEC elution profiles to the ch806 clinical reference material, and SDS-PAGE gel profiles consistent with an immunoglobulin.
  • the IEF pattern was consistent with the anticipated pI of 8.4.
  • hu806 antibody demonstrated highly comparable binding curves and affinity parameters to the ch806 antibody.
  • the binding affinity of hu806 and ch806 to EGFRvIII and over expressed wild-type EGFR are similar and in the low nanomolar range.
  • Cell binding through FACS analyses supported these observations.
  • the hu806 demonstrates markedly improved ADCC over the ch806 construct on target antigen positive A431 cells.
  • hu806 therapy demonstrated significant reduction in tumor growth by day 20 compared to the PBS controls and continued tumor growth retardation after day 20 similar to the ch806 group.
  • clone 175 (IgG2a) was selected for further characterization.
  • the hormone-independent prostate cell line DU145 (Mickey et al. (1977) Cancer Res. 37, 4049-4058) was obtained from the ATCC (atcc.org).
  • mAb806 and mAb175 were generated at the Ludwig Institute for Cancer Research (LICR) New York Branch and were produced and purified in the Biological Production Facility (Ludwig Institute for Cancer Research, Melbourne).
  • the murine fibroblast line NR6 ⁇ EGFR was used as immunogen.
  • Mouse hybridomas were generated by immunizing BALB/c mice five times subcutaneously at 2- to 3-week intervals, with 5 ⁇ 10 5 -2 ⁇ 10 6 cells in adjuvant. Complete Freund's adjuvant was used for the first injection. Thereafter, incomplete Freund's adjuvant (Difco) was used. Spleen cells from immunized mice were fused with mouse myeloma cell line SP2/0.
  • Intact mAbs (50 mg) were digested in PBS with activated papain for 2-3 hours at 37° C. at a ratio of 1:20 and the papain was inactivated with iodoacetamide. The digestion was then passed over a column of Protein-A sepharose (Amersham) in 20 mM sodium phosphate buffer pH 8.0, with the flow-through further purified by cation exchange using on a Mono-S column (Amersham). Protein was then concentrated using a 10,000 MWCO centrifugal concentrator (Millipore). For Fab-peptide complexes a molar excess of lyophilized peptide was added directly to the Fab and incubated for 2 hours at 4° C. before setting up crystallization trials.
  • human 293T embryonic-kidney fibroblasts were seeded at 8 ⁇ 10 5 per well in 6-well tissue culture plates containing 2 ml of media.
  • Cells were transfected with 3-4 ⁇ g of plasmid DNA complexed with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.
  • Lipofectamine 2000 Invitrogen
  • 24 to 48 h after transfection cell cultures were aspirated and cell mono layers lysed in 250 ⁇ ; of lysis buffer (1% Triton X-100, 10% glycerol, 150 mM NaCl, 50 mM HEPES pH 7.4, 1 mM EGTA and Complete Protease Inhibitor mix (Roche).
  • mAb 9B11 (1:5000, Cell Signaling Technology, Danvers, Mass.) to detect the c-myc epitope.
  • Membranes were washed in TBST, and incubated in blocking buffer containing horseradish peroxidase-conjugated rabbit anti-mouse IgG (Biorad) at a 1:5000 dilution for 2 h at room temperature. Blots were then washed in TBST, and developed using autoradiographic film following incubation with Western Pico Chemiluminescent Substrate (Pierce, Rockford, Ill.).
  • transformed colonies were grown at 30° C. in minimal media containing yeast nitrogen base, casein hydrolysate, dextrose, and phosphate buffer pH 7.4, on a shaking platform for approximately one day until an OD 600 of 5-6 was reached.
  • Yeast cells were then induced for protein display by transferring to minimal media containing galactose, and incubated with shaking at 30° C. for 24 h. Cultures were then stored at 4° C. until analysis.
  • Raw ascites fluid containing the c-myc monoclonal antibody 9E10 was obtained from Covance (Richmond, Calif.).
  • yeast cells were washed with ice-cold FACS buffer (PBS containing 1 mg/ml BSA) and incubated with either anti-c-myc ascites (1:50 dilution), or human EGFR monoclonal antibody (10 ⁇ g/ml) in a final volume of 50 ⁇ l, for 1 hr at 4° C. The cells were then washed with ice cold FACS buffer and incubated with phycoerythrin-labelled anti-mouse IgG (1:25 dilution), in a final volume of 50 ⁇ l for 1 h at 4° C., protected from light.
  • FACS buffer PBS containing 1 mg/ml BSA
  • human EGFR monoclonal antibody 10 ⁇ g/ml
  • a BIAcore 3000 was used for all experiments.
  • the peptides containing the putative InAb806 epitope were immobilized on a CM5 sensor chip using amine, thiol or Pms coupling at a flow rate of 5 ⁇ l/min (Wade et al. (2006) Anal Biochem. 348, 315-317).
  • the mAb806 and mAb175 were passed over the sensor surface at at flow rate of 5 ⁇ l/min at 25° C.
  • the surfaces were regenerated between runs by injecting 10 mM HCI at a flow rate of 10 ⁇ l/min.
  • lysis buffer 1% Triton X-100, 30 mM HEPES, 150 mM NaCl, 500 mM 4-(2-aminoethyl)benzenesulfonylfluoride, 150 nM aprotinin, 1 mM E-64 protease inhibitor, 0.5 mM EDTA, and 1 mM leupeptin, pH 7.4
  • lysis buffer 1% Triton X-100, 30 mM HEPES, 150 mM NaCl, 500 mM 4-(2-aminoethyl)benzenesulfonylfluoride, 150 nM aprotinin, 1 mM E-64 protease inhibitor, 0.5 mM EDTA, and 1 mM leupeptin, pH 7.4
  • Frozen sections were stained with 5 ⁇ g/ml mAb175 or irrelevant isotype control for 60 min at room temperature. Bound antibody was detected using the Dako Envision+HRP detection system as per manufacturer's instructions. Sections were finally rinsed with water, counterstained with hematoxylin and mounted.
  • Mutations of the wtEGFR were generated using a site-directed Inutagenesis kit (Stratagene, La Jolla, Calif.). The template for each mutagenesis was the human EGFR cDNA (accession number x00588) (Ullrich et al. (1984) Nature. 309, 418-425). Automated nucleotide sequencing of each construct was performed to confirm the integrity of the EGFR mutations. Wild-type and mutant (C173A/C281A) EGFR were transfected into BaF/3 cells by electroporation.
  • Stable cell lines expressing the mutant EGFR were obtained by selection in neomycin-containing medium. After final selection, mRNA was isolated from each cell line, reverse transcribed and the EGFR sequence amplified by PCR. All mutations in the expressed EGFR were confirmed by sequencing the PCR products. The level of EGFR expression was determined by FACS analysis on a FACStar (Becton and Dickinson, Franklin Lakes, N.J.) using the anti-EGFR antibody mAb528 (Masui et al. (1984) Cancer Res. 44, 1002-1007; Gill et al. (1984) J. Biol. Chem.
  • Cells expressing the wtEGFR or C271A/C283A-EGFR were washed and incubated for 3 hr in medium without serum or IL-3. Cells were collected by centrifugation and resuspended in medium containing EGF (100 ng/ml) or an equivalent volume of PBS. Cells were harvested after 15 min, pelleted and lysed directly in SDS/PAGE sample buffer containing p-mercaptoethanol. Samples were separated on NuPAGE 4-12% gradient gels, transferred to Immobilon PVDF membrane and probed with anti-phosphotyrosine (4G10, Upstate Biotechnologies) or anti-EGFR antibodies (mAb806, produced at the LICR). Reactive bands were detected using chemiluminescence.
  • Crystals of native 806 Fab were grown by hanging drop vapor diffusion using 10 mg/ml Fab and a reservoir containing 0.1M Sodium acetate bufter pH 4.6,6-8% PEG6000 and 15-20% Isopropanol.
  • crystals were transferred to a cryoprotectant solution containing 0.1M Sodium acetate buffer pH 4.6, 10% PEG6000, 15-20% Isopropanol and 10% glycerol. Crystals were then mounted in a nylon loop and flash frozen directly into liquid nitrogen.
  • Crystals of 806 Fab-peptide complex were grown by hanging drop vapor diffusion using 10 mg/ml Fab-peptide complex and a reservoir containing 0.2M ammonium acetate 16-18% PEG 5,000 monomethylether, crystals quality was then improved through seeding techniques.
  • crystals were transferred to a cryoprotectant solution consisting of reservoir supplemented with 25% glycerol. Crystals were then mounted in a nylon loop and flash frozen directly into liquid nitrogen.
  • Crystals of 175 Fab-peptide complex were initially grown by free interface diffusion using a Topaz crystallization system (Fluidigm, San Francisco). Microcrystals were grown by hanging drop vapor diffusion using 7 mg/ml Fab with similar conditions 0.1M Bis-tris propane buffer, 0.2M ammonium acetate and 18% PEG 10,000. Microcrystals were then improved by streak seeding into 0.15 m Sodium formate and 15% PEG 1500 to yield small plate shaped crystals. For data collection crystals were transferred to a cryoprotectant solution consisting of reservoir supplemented with 25% glycerol. Crystals were then mounted in a nylon loop and flash frozen directly into liquid nitrogen.
  • N-labelled peptide was produced recombinantly as a fusion to the SH2 domain of SHP2 using the method previously described by Fairlie et al. (Fairlie et al. (2002) Protein expression and purification 26, 171-178) except that the E. coil were grown in Neidhardt's minimal medium supplemented with 15 H 4 Cl (Neidhardt et al. (1974) Journal of bacteriology 119, 736-747).
  • the peptide was cleaved from the fusion partner using CNBr, purified by reversed-phase HPLC and its identity confirmed by MALDI-TOF mass spectrometry and N-terminal sequencing.
  • the methionine residue within the 806 antibody-binding sequence was mutated to leucine to enable cleavage from the fusion partner, but not within the peptide itself.
  • ch806 a chimeric version was engineered and produced under cGMP conditions (Panousis et al. (2005) Br. J. Cancer. 92, 1069-1077)
  • ch806 in tumor and liver was calculated by calculation of % injected dose (ID) of 111 In-ch806 from whole body gamma camera images obtained over one week following injection of 5-7 mCi (200-280 MBq) 111 In-ch806. Liver and tumor dosimetry calculations were performed based on regions of interest in each individual patient. 111 In-ch806 infusion image dataset, corrected for background and attenuation, allowing calculation of cumulated activity. Dosimetry calculation was performed to derive the concentration of 111 In-ch806 in tumor and liver over a one week period post injection.
  • ID % injected dose
  • variable heavy (VH) and variable light (VL) chains of mAb175 were sequenced, and their complementarity determining regions (CDRs) identified, as follows:

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US12/388,504 US20100056762A1 (en) 2001-05-11 2009-02-18 Specific binding proteins and uses thereof
RU2011138154/10A RU2549678C2 (ru) 2009-02-18 2010-02-17 Белки специфического связывания и их применения
ES10704485.1T ES2540802T3 (es) 2009-02-18 2010-02-17 Proteínas de unión específica y uso de las mismas
MX2011008767A MX2011008767A (es) 2009-02-18 2010-02-17 Proteinas de enlace especifico y usos de las mismas.
BRPI1012340-7A BRPI1012340B1 (pt) 2009-02-18 2010-02-17 Anticorpo anti-receptor do fator de crescimento epidérmico (egfr) isolado e imunoconjugado compreendendo um agente citotóxico e o dito anticorpo
SG10201801945TA SG10201801945TA (en) 2009-02-18 2010-02-17 Specific binding proteins and uses thereof
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PE2015002701A PE20160535A1 (es) 2009-02-18 2010-02-17 Proteinas de union al receptor del factor de crecimiento epidermico (egfr)
NZ716762A NZ716762A (en) 2009-02-18 2010-02-17 Specific binding proteins and uses thereof
CN201510004733.7A CN104650232B (zh) 2009-02-18 2010-02-17 特异性结合蛋白及其用途
SG2011058476A SG173688A1 (en) 2009-02-18 2010-02-17 Specific binding proteins and uses thereof
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US13/201,061 US9072798B2 (en) 2009-02-18 2010-02-17 Specific binding proteins and uses thereof
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SI201031541T SI2913344T1 (sl) 2009-02-18 2010-02-17 Specifični vezavni proteini in njihove uporabe
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PE2020000069A PE20200609A1 (es) 2009-02-18 2010-02-17 Proteinas de union al receptor del factor de crecimiento epidermico (egfr)
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US13/078,764 US20110313230A1 (en) 2001-05-11 2011-04-01 Specific binding proteins and uses thereof
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DO2020000032A DOP2020000032A (es) 2009-02-18 2020-02-12 Proteínas de unión específicas y sus usos
US16/818,062 US20210032366A1 (en) 2009-02-18 2020-03-13 Methods of treating neoplastic astrocytoma
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