NZ576119A - Combination therapy involving egf inhibitors - Google Patents

Abstract

Provided is the use of an inhibitor of HB-EGF and an inhibitor of AREG in the preparation of a medicament formulated for simultaneous or sequential use in the combination treatment of neoplastic disease; wherein, in said combination treatment, said inhibitor of HBEGF and said inhibitor of AREG act synergistically; wherein said inhibitor of HB-EGF is an antibody molecule which binds said HB-EGF or a nucleic acid molecule which inhibits expression of HB-EGF; and wherein said inhibitor of AREG is an antibody molecule which binds AREG or a nucleic acid molecule which inhibits expression of AREG. Further provided are compositions comprising the two inhibitors and specific amino acid sequences of the antibodies therein.

Description

1 2 3 4 6 7 8 9 11 12 13 14 16 17 18 19 21 22 23 24 26 27 28 29 31 32 1 Received at IPONZ on 01.06.2011 Combination Therapy Comprising an Inhibitor of HB-EGF and an Inhibitor of AREG Field of the Invention The present invention relates to cancer treatment. In particular it relates to methods of determining susceptibility to resistance to anti-cancer drugs, methods for overcoming such resistance and combination therapies for the treatment of cancer.

Background to the Invention Cancer is the leading cause of mortality in the Western countries . A large number of chertiotherapeutic agents have been developed over the last 50 years to treat cancers. The majority of chemotherapeutic agents can be divided into: alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and antitumour agents. All of these drugs affect cell division, or DNA synthesis and function in some way.

The effectiveness of particular chemotherapeutic agents varies between cancers, between patients and over time in Individual patients. Cancerous cells exposed to a chemotherapeutic agent may develop resistance to such an agent, and quite often cross-resistance to several other antineoplastic agents as well. Moreover, the narrow therapeutic index of many chemo therapeut ic agents further limits their use. Accordingly, it is often necessary to change treatments of patients with cancer if the first or second line therapy is not sufficiently effective or ceases to be sufficiently effective. In many cases 2 1 combinations of particular treatments have been . 2 found to be particularly effective. 3 4 For example, colorectal cancer is one of the most currently diagnosed cancers in Europe and one with 6 the poorest 5 year survival rates. For more than 40 7 years, inhibitors of thymidylate synthase, for 8 example 5-Fluorouracil (5-Fu), have been the 9 treatment of choice for this cancer. Thymidylate synthase inhibitors act by causing DNA damage due to 11 misincorporation of FUTP into RNA and DNA (Longley 12 et al Nat Rev Cancer, 3:330-338, 2003; Backus et al 13 Oncol research 2000;12 (5) :231-9) . More recently new 14 chemotherapeutic agents have been introduced to the clinic, for example the topoisomerase I inhibitors 16 (e.g. irinotecan: CPT-11) and DNA damaging agents 17 (e.g. oxaliplatin: alkylating agents). 18 ■ 19 These chemotherapeutics agents, 5-Fu included, can be used alone but it is common that clinical regimes 21 incorporate a combination. Indeed combined 22 chemotherapy has shown promising results by 23 improving the response rates in patients by acting 24 on the tumors through different pathways.

Nevertheless many patients still cannot be treated 26 through these regimes because of drug resistance 27 either acquired or inherent. In vitro and in vivo 28 studies have demonstrated that increased TS 29 expression correlates with increased resistance to 5-FU (Johnston et al, Cancer Res., 52: 4306-4312, 31 1992). Other upstream determinants of 5-FU 3 1 chemosensitivity include the 5-FU-degrading enzyme 2 dihydropyrimidine dehydrogenase and 5-FU-anabolic 3 enzymes such as orotate phosphoribosyl transferase 4 (Longley et al Nat Rev Cancer, 3:330-338, 2003). 6 The use of antimetabolites e.g. tomudex (TDX) and 7 platinum containing compounds e.g. oxaliplatin is 8 similarly limited by resistance. 9 Further, the choice of chemotherapy is further 11 complicated by cancer type and, for example, whether 12 or not the cancer is associated with a p53 mutation. 13 For example, as described in W02005/053739, whereas 14 the combination of platinum based chemotherapeutics with antiFas antibodies was shown to have a 16 synergistic cytotoxic effect in tumours with wild 17 type p53, such synergy was not seen in p53 mutant 18 cells. 19 5-Fu, CPT-11 and oxaliplatin remain front line 21 therapies, but the development of non responsive 22 tumours or chemotherapy resistant cancer remains a 23 major obstacle to successful chemotherapy. Due to 24 the importance of early treatment of cancers, there is a clear need for tools which enable prediction of 26 whether a particular therapy, either single or 27 combination, will be effective against particular 28 tumours in individual patients. Moreover, there 29 remains the need for new treatment regimes to increase the repertoire of treatments available to 31 the physician. 4 1 Summary of the Invention 2 3 The present inventors have investigated proteins 4 upregulated in response to treatment with different classes of chemotherapy and have surprisingly shown 6 that a variety of genes encoding peptide growth 7 factors of the Epidermal Growth Factor (EGF) family 8 are overexpressed in a number of different tumour 9 cell line models of cancer, from a number of different types of cancer, following in vivo 11 challenge with different physiologically relevant 12 doses of different classes of chemotherapy. 13 14 Further investigation by the inventors has surprisingly shown that combinations of inhibitors 16 of different EGFs results in a surprisingly dramatic 17 reduction in tumour cell growth and proliferation 18 compared to the reduction when inhibitors of a 19 single EGF were tested. 21 Accordingly, in a first aspect of the present 22 invention, there is provided a method of treating 23 • neoplastic disease in a subject, said method 24 comprising the simultaneous, sequential or separate, administration to said subject of an effective 26 amount of (i) an inhibitor of a first EGF and 27 (ii) an inhibitor of a second EGF, wherein said 28 first and second EGF are different EGFs. 29 In a second aspect of the invention, the invention 31 provides a pharmaceutical composition comprising (i) 32 an inhibitor of a first EGF and 1 (ii) an inhibitor of a second EGF, wherein said 2 first and second EGFs are different EGFs. 3 4 . A third aspect of the invention provides kit comprising, in combination for simultaneous, 6 separate, or sequential use in the treatment of 7 neoplastic disease, 8 (i) an inhibitor of a first EGF and 9 (ii) an inhibitor of a second EGF, wherein said ■ first and second EGF are different EGFs. 11 12 Any EGF may be used in the first, second or third 13 aspects of the invention. Thus the first and second 14 EGFs may each be independently selected from the group consiting of HB-EGF, AREG, TGF, EREG, BTC, NRG 16 1, NRG2, NRG3, and NRG4. 17 18 In.one embodiment, the first EGF is HB-EGF and said 19 second EGF,is selected from the group consisting of AREG, TGF, EREG, BTC, and NRG3. 21 22 In a particular embodiment, the first EGF is HB-EGF 23 and said second EGF is AREG. 24 Any inhibitors of an EGF may be used. EGF inhibitors 26 which may be used in the present invention include 27 any molecule which reduces expression of the gene 28 encoding the EGF or antagonizes the EGF protein. 29 Such molecules may include, but are not limited to, antibodies, antibody fragments, immunoconjugates, 31 small molecule inhibitors, peptide inhibitors, 32 specific binding members, non-peptide small organic 6 1 molecules, nucleic acid molecules which inhibit EGF 2 expression, such as siRNA, antisense molecules or 3 oligonucleotide decoys. 4 ' In one embodiment, the inhibitors of said first and 6 second EGFs are different. In a particular 7 embodiment, the inhibitor of the first EGF is not an 8 inhibitor of the second EGF and vice versa. 9 In one embodiment of the invention, the inhibitor of 11 each EGF is a specific inhibitor of that EGF, i.e. 12 not an inhibitor of another EGF. 13 14 In one embodiment, said inhibitor of said first EGF is an antibody which binds said first EGF or a 16 nucleic acid molecule which inhibits EGF expression. 17 18 As described above, in one embodiment, said first 19 EGF is HB-EGF. 21 In one such embodiment the inhibitor of the first 22 EGF is an siRNA having sense and antisense sequences 23 shown as Sequence ID Nos 1 and 2 respectively: 24 .25 Sequence ID No: 1: 2 6 GAAAAUCGCUUAUAUACCUUU 27 Sequence ID No: 2: 2 8 AGGUAUAUAAGCGAUUUUCUU 29 In another such embodiment the inhibitor of the HB- 31 EGF is an siRNA having sense and antisense sequences 32 shown as Sequence ID Nos 3 and 4 respectively: 33 34 Sequence ID No: 3: 7 1 UGAAGUUGGGCAUGACUAAUU 2 Sequence ID No: 4: 3 UUAGUCAUGCCCAACUUCAUU 4 In another such embodiment the inhibitor of the HB- 6 EGF is an siRNA having sense and antisense sequences 7 shown as Sequence ID Nos 5 and 6 respectively: . 8 ' 9 Sequence ID No: 5: GGACCCAUGUCUUCGGAAAUU 11 Sequence ID No: 6: 12 UUUCCGAAGACAUGGGUCCUU 13 14 In another such embodiment the inhibitor of the HB- EGF is an siRNA having sense and antisense sequences 16 shown as Sequence ID Nos 7 and 8 respectively: 17 18 Sequence ID No: 7: 19 GGAGAAU G CAAAUAU GUAUU Sequence ID No: 8: 21 UCACAUAUUUGCAUUCUCCUU 22 23 In one embodiment, the inhibitor of HB -EGF 24 comprises a pool of two, three or four of the siRNA molecules (wherein each molecule comprises the sense 26 and complementary antisense molecule) shown above 27 i.e. two, three or four of the sense/antisense pairs 28 selected from the group consisting of Sequence ID 29 No: 1/Sequence ID No: 2, Sequence ID No: 3/Sequence ID No: 4, Sequence ID No: 5/Sequence ID No: 6, and 31 Sequence ID No: 7/Sequence ID No: 8. 32 33 34 In one embodiment, said inhibitor of said second EGF is an antibody which binds said second EGF or a 36 nucleic acid molecule which inhibits EGF expression. 37 8. 1 In one embodiment, said second EGF is AREG. In such 2 an embodiment, an antibody which may be used as the 3 inhibitor of AREG is the anti-AREG antibody 6E11 1E9 4 1C6. The VH and VL sequences of the 6E11 1E9 1C6 antibody have been determined by the inventors and 6 are described infra. 1 8 In one embodiment of the invention, siRNA molecules 9 which may be used in the invention as an inhibitor of AREG is an siRNA having sense and antisense 11 sequences shown as Sequence ID Nos 9 and 10 12 respectively: 13 Sequence ID No: 9 14 UGAUAACGAACCACAAAUAUU Sequence ID No: 10 16 UAUUUGUGGUUCGUUAUCAUU 18 In another such embodiment the inhibitor of AREG is 19 an siRNA' having sense and antisense sequences shown as Sequence ID Nos 11 and 12 respectively: 21 22. Sequence ID No: 11 2 3 UGAGUGAAAUGCCUUCUAGUU 24 Sequence ID No: 12 . CUAGAAGGCAUUUCACUCAUU 26 27 In another such embodiment the inhibitor of AREG is 28 an siRNA having sense and antisense sequences shown 29 as Sequence ID Nos 13 and 14 respectively: 31 Sequence ID No: 13 32 GUUAUUACAGUCCAGCUUAUU 33 Sequence ID No: 14 34 UAAGCUGGACUGUAAUAACUU 36 In another such embodiment the inhibitor of AREG is 37 an siRNA having sense and" antisense sequences shown 38 as Sequence ID Nos 15 and 16 respectively: 9 1 2 Sequence ID No: 15 3 GAAAGAAACUUCGACAAGAUU 4 Sequence ID No: 16 ■ UCUUGUCGAAGUUUCUUUCUU 6 7 In one embodiment, the inhibitor of AREG comprises a 8 pool of two, three or four of the siRNA molecules 9 (wherein each molecule comprises the sense and complementary antisense molecule) shown above i.e. 11 two, three or four of the sense/antisense pairs 12 selected from the group consisting of Sequence ID 13 No: 9/Sequence ID No: 10, Sequence ID No: 14 11/Sequence- ID No: 12, Sequence ID No: 13/Sequence ID No: 14, and Sequence ID No: 15/Sequence ID No: 16 16. 17 18 As described in the Examples, the inventors have 19 developed the antibodies with specificity for AREG, which may be used in the invention. The antibodies 21 have found to be particularly efficacious. 22 23 Accordingly, in a fourth aspect of the invention, 24 there is provided an antibody molecule having binding specificity for AREG, wherein the antibody 26 molecule is the 6E11 1E9 1C6 antibody, or a fragment 27 thereof. 28 29 The VH and VL domain sequences of the 6E11 1E9 1C6 antibody has been determined by the inventor and are 31 as follows. 32 33 6E11 1E9 1C6 VH sequence (Sequence ID No: 27) : 1 MECNWILPFILSVTSGVYSQVQLQQSGAELARPGASVKLSCKASGYTFTRYW 2 MQWIKQRPGQGLEWIGAIYPGNGDIRYTQKFKGKATLTADKSSSTAYMQLSS 3 LASE D SAVYYCARGTTP S SYWGQGTLVTVSAAKTTAP SVYP LAPVCGDTTGS 4 SVTLGCLVKGYF 6 6E11 1E9 1C6 VL sequence (Sequence ID No: 28) 7 8 MMSPAQFLFLLVLWIRETSGDWMTQTPLTLSVSIGQPASISCKSSQSLLDS 9 DGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVE AEDLGVYYCWQGTHFPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGAS 11 WCFLNNFYPK' 12 13 Thus, in one embodiment of the invention the 14 antibody molecule having binding specificity for AREG of arid for use in the invention is an antibody 16 molecule comprising at least one of the CDRs of the 17 6E11 1E9 1C6 VH region and/or at least one of the 18 CDRs of the 6E11 1E9 1C6 VL region. In one 19 embodiment, the antibody molecule comprises all three of the CDRS of the 6E11 1E9 1C6 VH region 21 and/or all three of the CDRS of the 6E11 1E9 1C6 VL 22 region. 23 24 In one embodiment, the specific binding member comprises an antibody variable domain (which may be 26 VH or VL) having the VH domain sequence shown above, 27 or an antibody variable domain (which may be VL or 28 VH) having the antibody VL domain sequence shown 29 above, or both. 11 1 The antibody molecule may be a whole antibody. In 2 one alternative embodiment, the antibody molecule 3 may be an antibody fragment such as an scFv. 4 The provision of the antibody molecules of the 6 present invention enables the development of related 7 antibodies which also inhibit tumour cell growth and 8 which optionally have similar or greater binding 9 specificity. 11 Accordingly, further encompassed within the scope of 12 this aspect of the present invention are antibody 13 molecules comprising an antibody variable domain (VH 14 or VL) having the 6E11 1E9 1C6 VH sequence shown above in which 5 or less, for example 4, 3, 2, or 1 16 amino acid substitutions have been made in at least 17 one CDR and wherein the specific binding member 18 retains the ability to inhibit the tumour cell 19 growth. Also encompassed by the invention are antibody molecules comprising an antibody variable 21 domain (VL or VH) having the 6E11 1E9 1C6 VL 22 sequence shown above in which 5 or less, for example 23 4, 3, 2, or 1 amino acid substitutions have been 24 made in at least one CDR and wherein the specific binding member retains the ability to inhibit the 2 6 tumour cell growth. 27 28 The method of any one of the first to third aspects 29 of the invention may further comprise the simultaneous, sequential or separate, administration 31 to said subject of an effective amount of (iii) a 12 1 chemotherapeutic agent. 2 3 In one embodiment, the chemotherapeutic agent is 4 selected from the group consisting of antimetabolites, topoisomerase inhibitors, 6 alkylating agents, anthracyclines, and plant 7 alkaloids. 8 9 The inventors have further shown that particular combinations of EGF inhibitors with topoisomerase 11 inhibitors attenuate tumour cell growth to an extent 12 greater than could be predicted from the effects of 13 each inhibitor alone. 14 Accordingly, in a sixth aspect of the invention, 16 there is provided a method of treating neoplastic 17 disease in a subject, said method comprising the 18 simultaneous, sequential or separate, administration 19 to said subject of an effective amount of (i) an inhibitor of an EGF, wherein said inhibitor is a 21 nucleic acid molecule which inhibits EGF expression 22 or an anti EGF antibody, and wherein said EGF is HB- 23 EGF or AREG, and (ii) a topoisomerase inhibitor. 24 In a seventh aspect of the invention, there is 26 provided a pharmaceutical composition for the 27 treatment of cancer, said composition comprising an 28 effective amount of (i) an inhibitor of an EGF , 29 wherein said inhibitor is a nucleic acid molecule which inhibits EGF expression or an anti EGF 31 antibody, and wherein said EGF is HB-EGF or AREG, 32 and (ii) a topoisomerase inhibitor. 2 3 4 6 7 8 9 11 12 13 14 16 17 18 19 21 22 23 24 26 27 28 29 31 32 Received at IPONZ on 29.02.2012 13 Ail eighth aspect of the invention provides comprising, in combination for simultaneous, separate, or sequential use in the treatment of neoplastic disec itive amount of (i) an inhibitor of an EGF, wherein said inhibitor is a nucleic acid molecule w hibits EGF expression or an anti EGF antibody, and wherein said EGF is HB-EGF or AREG, and (ii) a topoisomerase inhibitor.

In one embodiment of any one of the sixth, seventh or eighth aspects of the invention, the EGF is HB-EGF . In another embodiment, the EGF is AREG.

In another aspect of the invention there is provided use of an inhibitor of HB-EGF and an inhibitor of AREG in the preparation of a medicament foririu 1 for simultaneous or sequ.ent.ial use in. the combination treatment of neoplastic disease; wherein, in said combin i treatment, said Lbitor of HB-EGF and said, inhibitor of AREG act synerg ally; wherein said inhibitor of HB-EGF is an antibody molecule which binds said HB-EGF or a nu : acid molecule which inhibits expression of HB-EGF; and wherein said inhibitor of AREG is an antibody molecule which binds AREG or a nucleic acid molecule which inhibits express! i.

In another aspect of the invention there is provided use of an inhibitor of HB-EGF and an inh AREG in the preparation of a rrw ment for the combination treatment of neoplastic disease, wherein 1 2 3 4 6 7 8 9 11 12 13 14 16 17 18 19 21 22 23 24 26 27 28 29 31 32 Received at IPONZ on 29.02.2012 13a said medicament is formulated for administration according to a dosage regime comprising administering said inhibitor of HB-EGF to a patient, where said inhibitor of AREG was administered within the previous 72 hours, or said inhibitor of AREG is administered simultaneously or within the subsequent 72 hours; wherein, with said dosage regime, said inhibitor of HB-EGF and said inhibitor of AREG act syne wherein said inhibitor of HB-EGF is an antibody molecule which binds said HB-EGF or a nu acid molecule which inhibits expression of HB-EGF; and wherein j inhibitor of AREG is an antibody molecule which binds AREG or a nucleic acid molecule w libits expression of AREG.

In another aspect of the invention there is provided combination treatment products comprising an inhibitor of HB-EGF and an inhibitor of AREG as a combined preparation for simultaneous or sequential use in the combination treatment of neoplastic disease; wherein, in said combination treatment, said inhibitor of HB-EGF and said inhibitor of AREG act synergistically; wherein said inhibitor of HB-EGF is an antibody molecule which binds said HB-EGF or a nu acid molecule which inhibits expression of HB-EGF; and wherein said inhibitor of AREG is an antibody molecule which binds AREG or a nucleic acid molecule which inhibits expression of AREG. 1 2 3 4 6 7 8 9 11 12 13 14 16 17 18 19 Received at IPONZ on 02.02.2012 13b In these aspects of the invention, any t- merase inhibitor may be used. In a particular embodiment, the topoisomerase inhibitor is CPT-11. In another embodiment, the topoisomerase inhibitor is an active metabolite of CPT-ll, for example SN-38.

In one embodiment, wherein the EGF is AREG, the EGF inhibitor is the anti-AREG antibody 6E11 1E9 1C6.

The methods of the invention may be used to treat any neoplastic disease. In a particular embodiment, the neoplastic disease is cancer. For example, neoplastic diseases which may be tr< using the compositions and methods of the invention include, but are not limited to, colorectal cancer, breast cancer, lung cancer, prostate cancer, hepatocellular cancer, lymphoma, leukaemia, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, renal cancer, thyroid cancer, 14 1 melanoma, carcinoma, head and neck cancer, and skin 2 cancer. 3 4 In one particular embodiment, the neoplastic disease is colorectal cancer. 6 7 In another embodiment, the neoplastic disease is 8 breast cancer. 9 In another another embodiment, the neoplastic 11 disease is lung cancer. 12 13 As described in the Examples, the inventors have 14 shown that certain EGFs are upregulated by chemotherapies in p53 mutant tumour cells as well as 16 in p53 wild type tumours. This is particularly 17 surprising given that resistance to chemotherapy has 18 previously been shown to be largely dependent on p53 19 status. 21 In a particular embodiment, the neoplastic disease 22 is a cancer comprising a p53 mutation. 23 24 Further provided by the invention in a ninth aspect is a method of inducing and/or enhancing expression 26 of a gene encoding an EGF protein in a cell or 27 tissue; said method comprising administration of a 28 topoisomerase inhibitor to said cell or tissue, 29 wherein said EGF is selected from the group consisting of AREG, TGF, EREG, BTC, and NRG3. 1 The demonstration by the present inventors that 2 expression of EGFs are upregulated in response to 3 treatment with diverse topoisomerase inhibitors 4 suggests that the therapeutic effect of treatment with these chemotherapies may, in certain patients, 6 be compromised by the upregulation of EGFs. 7 8 Thus, the invention may be used in assays to 9 determine whether or not treatment with a topoisomerase inhibitor e.g. CPT-11 or an analogue 11 thereof may be effective in a particular patient. 12 13 Thus, in a tenth aspect of the present invention, 14 there is provided an in vitro method for evaluating the response of tumour cells from a subject to the 16 presence of a topoisomerase inhibitor to predict 17 response of the tumour cells in vivo to treatment 18 with the topoisomerase inhibitor, which method 19 comprises: (a) providing a sample of tumour cells from a 21 subject; 22 (b) exposing a portion of said sample of tumour . 23 cells to said topoisomerase inhibitor; 24 (c) comparing expression of one or more genes encoding one or more EGFs wherein said EGF is 26 selected from the group consisting of AREG, TGF, 27 EREG, BTC, and NRG3 in said portion of the sample of 28 tumour cells exposed to said topoisomerase inhibitor 29 with expression of said gene(s) in a control portion of said sample which has not been exposed to said 31 topoisomerase inhibitor; wherein enhanced expression 32 in the portion of sample exposed to said 16 1 topoisomerase inhibitor is indicative of decreased 2 sensitivity to said topoisomerase inhibitor. 3 4 The invention further represents a tool for prognosis and diagnosis of a subject afflicted with 6 a tumour. For the purpose of prognosis, determining 7 the expression level of a gene before and after 8 chemotherapeutic treatment would identify if the 9 subject will respond to a combinatory treatment approach. For the purpose of diagnosis the 11 expression profile of a tumours genetic response to 12 chemotherapy would identify which combination 13 therapy would be most effective for that tumour. 3.4 Thus, an eleventh aspect of the invention provides a 16 method of prognosis for evaluating the response of a 17 patient to combination therapy comprising a 18 topoisomerase inhibitor and an inhibitor of an EGF, 19 said method comprising (a) determining expression of a gene encoding an EGF in an in vitro sample 21 containing tumour cells obtained from a subject 22 prior to treatment with said chemotherapeutic 23 treatment 24 (b) determining expression of said gene encoding said EGF, wherein said EGF is selected, from the 26 group consisting of AREG, TGF, EREG, BTC, and NRG3, 27 in an in vitro sample containing tumour cells 28 obtained from a subject after treatment with said 29 chemotherapeutic treatment; (c) comparing expression in (b) with expression in 31 (a), wherein enhanced expression in (b) compared to 32 (a) is indicative that the patient may benefit from 17 1 combination therapy comprising a topoisomerase 2 inhibitor and an inhibitor of said EGF. 3 4 In the tenth or eleventh aspects of the invention the expression of gene(s) encoding one or more EGFs 6 may be determined. For example, the expression of 7 genes encoding at least two, for example three, four 8 or five of AREG, TGF, EREG, BTC, and NRG3 may be 9 determined. -11 In another embodiment of the tenth or eleventh 12 aspects of the invention the expression of genes 13 encoding at least two, for example three, or four of 14 TGF, EREG, BTC, and NRG3 may be determined. 16 In aspects of the invention involving the 17 determination of expression of a gene encoding an 18 EGF, the expression of any gene encoding said EGF in 19 the subject may be determined. 21 For example, in an embodiment in which the EGF is 22 AREG, the gene may be NM_001657.' In an embodiment, 23 in which the EGF is HB-EGF, the gene may be 24 NM_0 0194 5. 26 In an embodiment of the invention, expression of 27 said gene in the sample exposed to said 28 chemotherapeutic agent is considered to be enhanced 29 if the expression is at least 1.5-fold, preferably •at least 2-fold, more preferably at least 5-fold, 31 that of the one or more genes in the control portion 18 1 of said sample which has not been exposed to said 2 chemotherapeutic agent. 3 4 In the present application, unless the context demands otherwise, where reference is made to a' 6 chemotherapeutic agent and an EGF modulator, the 7 chemotherapeutic agent and the EGF modulator are 8 different agents. Generally, the chemotherapeutic 9 agent will have a' different mode of action from the EGF'modulator. In one embodiment, the 11 chemotherapeutic agent will not inhibit the EGF. 12 13 In a further aspect of the invention, there is 14 provided the use of an inhibitor of a first EGF in the preparation of a medicament for simultaneous, 16 separate or sequential use with an inhibitor of a 17 second EGF for the treatment of neoplastic disease; 18 wherein said first and second EGFs are different 19 EGFs. 21 Another aspect of the invention provides the use of 22 an inhibitor of a second EGF in the preparation of a 23 medicament for simultaneous, separate or sequential 24 use with an inhibitor of a first EGF for the treatment of neoplastic disease; wherein said first 26 and second EGFs are different EGFs. 27 28 Another aspect which is provided is the use of an 29 inhibitor of an EGF, wherein said'inhibitor is a nucleic acid molecule 31 which inhibits EGF expression or an anti EGF 32 antibody, and wherein said EGF is HB-EGF or AREG, 19 1 in the preparation of a medicament for the 2 simultaneous, separate or sequential use with a 3 topoisomerase inhibitor in the treatment of a 4 neoplastic disease. 6 Further provided is the use of a topoisomerase 7 inhibitor in the preparation of a medicament for 8 simultaneous, separate or sequential use with an 9 inhibitor of an EGF in the treatment of a neoplastic disease, 11 wherein said inhibitor of an EGF is a nucleic acid 12 molecule which inhibits EGF expression or^an anti 13 EGF antibody, and wherein said EGF is HB-EGF or 14 AREG. 16 Preferred and alternative features of each aspect of 17 the invention are as for each of the other aspects 18 mutatis mutandis unless the context demands 19 otherwise. 2 0 21 Detailed Description 22 23 As described above and in the Examples, the present 24 invention is based on the demonstration that expression of various EGF genes and proteins are 26 upregulated in tumour cells in the presence of 27 certain chemotherapies and that particular 28 combinations of EGF inhibitors and chemotherapeutic 29 agents as well as particular combinations of two or more EGF inhibitors demonstrate superadditive 31 effects in the attenuation of tumour cell growth. 1 Assays 2 3 As described above, in one embodiment, the present 4 invention relates to methods of screening samples comprising tumour cells for expression of EGF genes 6 in order to determine suitability for treatment 7 using particular chemotherapeutic agents. 8 9 The methods of the invention may involve the determination of expression of any gene encoding an 11 EGF. The EGF-family of peptide growth factors' is 12 made up of 10 members which have the ability to 13 selectively bind the ErrB receptors (ErrBl or EGF 14 receptor, ErrB2 or Her2, ErrB3 and ErrB4). 16 In one embodiment of the invention, the EGF is a 17 ligand of ErbB-1, for example, amphiregulin (AREG), 18 TGF, Epiregulin (EREG) or BTC. 19 In another embodiment, the EGF is a ligand of ErbB- 21 4, for example NRG3 22 23 Accession details are provided for each of these 2'4 genes below.

Gene Accession No BTC NM_0 0172 9 HB-EGF : NM_0 0194 5 AREG NM_0 01657 TGFA NM_0 032 3 6 EREG NM_0 014 32 NRG 3 NM_0 01010848 21 1 2 The expression of any gene encoding an EGF of 3 interest may be determined. 4 For example, where the EGF is AREG, the Areg gene 6 may be NM_001657. 7 8 In a particular embodiment of the invention, the 9 gene is Areg having accession no: NM_00.1657. In another particular embodiment of the invention, the 11 gene is the HB-EGF gene having accession no: 12 NM_0 0194 5. 13 14 The expression of each gene may be measured using any technique known in the art. Either mRNA or 16 protein can be measured as a means of determining 17 up-or down regulation of expression of a gene. 18 Quantitative techniques are preferred. However semi- 19 quantitative or qualitative techniques can also be used. Suitable techniques for measuring gene 21 products include, but are not limited to, SAGE 22 analysis, DNA microarray analysis, Northern blot, 23 Western blot, immunocytochemical analysis, and 24 ELISA. " 26 In the methods of the invention, RNA can be detected 27 using any of the known techniques in the art. 28 Preferably an amplification step is used as the 2 9 amount of RNA from the sample may be very small.

Suitable techniques may include RT-PCR, 31 hybridisation of copy mRNA (cRNA) to an array of 32 nucleic acid probes and Northern Blotting. 22 1 2 For example, when using mRNA detection, the method 3 may be carried out by converting the isolated mRNA 4 to cDNA according to standard methods; treating the converted cDNA with amplification reaction reagents 6 (such as cDNA PCR reaction reagents) in a container 7 along with an appropriate mixture of nucleic acid 8 primers; reacting the contents of the container to 9 produce amplification products; and analyzing the amplification products to detect the presence of 11 gene expression products of one or more genes 12 encoding Areg in the sample. Analysis may be 13 accomplished using Northern Blot analysis to detect 14 the presence of the gene products in the amplification product. Northern Blot analysis is 16 known in the art. The analysis step may be further 17 accomplished by quantitatively detecting the 18 presence of such gene products in the amplification 19 products, and comparing;the quantity of product detected against a panel of expected values for 21 known presence or absence in normal and malignant 22 tissue derived using similar primers. 23 24 Primers for use in methods of the invention will of course depend on the gene(s), expression of which is 26 being determined. In one embodiment of the 27 invention, one or more of the following primer sets 28 may be used: 29 Forward : TTTTTTGGATCCAATGACACCTACTCTGGGAAGCGT (SEQ 31 ID No:17) 23 1 Reverse : TTTTTTAAGCTTAATTTTTTCCATTTTTGCCTCCC(SEQ ID 2 No:18) 3 And Exon Spanning 4 Forward : TTTTTTGGATCCCTCGGCTCAGGCCATTATGCTGCT(SEQ ID No:19) 6 Reverse : TTTTTTAAGCTTTACCTGTTCAACTCTGACTG(SEQ ID 7 No:20) 8 9 Forward 5'-TTTCTGGCTGCAGTTCTCTCGGCACT-3'(SEQ ID No:21) 11 Reverse 5'-CCTCTCCTATGGTACCTAAACATGAGAAGCCCC-3'(SEQ 12 ID No:22) 13 14 In e.g. determining gene expression in carrying out methods of the invention, conventional molecular 16 biological, microbiological and recombinant DNA 17 techniques known in the art may be employed. 18 Details of such techniques are described in, for 19 example, Current Protocols in Molecular Biology/ 5th ed.,Ausubel et al. eds., John Wiley & Sons, 2005 21 and, Molecular Cloning: a Laboratory Manual: 3rd 22 edition Sambrook et al., Cold Spring Harbor 23 Laboratory Press, 2001. 24 The assays of the invention may be used to monitor 26 disease progression, for example using biopsy 27 samples at different times. In such embodiments, 28 instead of comparing the expression of EGF against a 29 control sample which has not been exposed to said chemotherapeutic agent, the expression of the EGF is 31 compared against a sample obtained from the same 32 tissue at an earlier time point, for example from 24 1 days, weeks or months earlier. 2 3 The methods of the invention may be used to 4 determine the suitability for treatment of any suitable cancer with a chemotherapeutic agent e.g. 6 CPT-11 or analogues thereof. For example the 7 methods of the invention may be used to determine 8 the sensitivity or resistance to treatment of 9 cancers including, but not limited to, gastrointestinal, such as colorectal, te, head and 11 neck cancers. 12 13 In a particular embodiment of the invention, the 14 methods of the invention may be used to determine the sensitivity or resistance to treatment of 16 colorectal cancer. 17 18 In another particular embodiment of the invention, 19 the methods of the invention may be used to 'determine the sensitivity or resistance to treatment 21 of lung cancer. 22 23 In another particular embodiment of the invention, 24 the methods of the invention may be used to determine the sensitivity or resistance to treatment 26 of breast cancer. 27 28 The nature of the tumour or cancer will determine 29 the nature of the sample which is to be used in the methods of the invention. The sample may be, for 31 example, a sample from a tumour tissue biopsy, bone 32 marrow biopsy or circulating tumour cells in e.g. 1 blood. Alternatively, e.g. where the tumour is a 2 gastrointestinal tumour, tumour cells may be 3 isolated from faeces samples. Other sources of 4 tumour cells may include plasma, serum, cerebrospinal fluid, urine, interstitial fluid, 6 ascites fluid etc. 7 8 For example, solid tumour samples collected in 9 complete tissue culture medium with antibiotics.

Cells may be manually teased from the tumour 11 specimen or, where necessary, are enzymatically 12 disaggregated by incubation with collagenase/DNAse 13 and suspended in appropriate media containing, for 14 example, human or animal sera. 16 In other embodiments, biopsy samples may be isolated 17 and frozen or fixed in fixatives such as formalin. 18 The samples may then be tested for expression levels 19 of genes at a later stage. 21 In determining treatment, it may e desirable to 22 determine p53 status of a cancer. For example, p53 23 status may be useful as it may dictate the type of 24 chemotherapy which should be used in combination with particular EGF proteins. p3 status may be 26' detemined using conventional methods. For example, 27 the use of immunohistochemistry may be used to 28 identify hotspot mutations while gene sequencing or 29 other DNA analysis methodologies may also be employed. This analysis may suitably be performed on 31 isolated tumour tissue. 26 1 Chemotherapeutic Agents 2 Chemotherapeutic agents may be used in certain 3 embodiments of the the present invention. For 4 example agents which may be used include antimetabolites, including thymidylate synthase 6 inhibitors, nucleoside analogs, platinum cytotoxic 7 agents, topoisomerase inhibitors or antimicrotubules 8 agents. Examples of thymidylate synthase inhibitors 9 which may be used in the invention include 5-FU, MTA and TDX. An example of an antimetabolite which may 11 be used is tomudex (TDX). Examples of platinum 12 cytotoxic agents which may be used include cisplatin 13 and oxaliplatin. 14 Chemotherapeutic agents which may be used in the 16 present invention in addition or instead of the 17 specific agents recited above, may include 18 alkylating agents; alkyl sulfonates; aziridines; 19 ethylenimines; methylamelamines; nitrogen mustards; nitrosureas; anti-metabolites; folic acid analogues; 21 purine analogs; pyrimidine analogs; androgens; anti- 22 adrenals; folic acid replenishers; aceglatone; 23 aldophosphamide glycoside; aminolevulinic acid; 24 amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; 26 elliptinium acetate; etoglucid; gallium nitrate; 27 hydroxyurea; lentinan; ionidamine; mitoguazone; 28 mitoxantrone 29 In particular embodiments of the invention, the 31 chemotherapeutic agent is a topoisomerase inhibitor. 27 1 Any suitable topoisomerase inhibitor may be used in 2 the present invention. In a particular embodiment, 3 the topoisomerase inhibitor is a topoisomerase I ■ 4 inhibitor, for example a camptothecin. A suitable topoisomerase I inhibitor, which may be used in the 6 present invention is irenotecan (CPT-11) or its 7 active' metabolite SN-38. CPT-11 specifically acts in 8 the S phase of the cell cycle by stabilizing a 9 reversible covalent reaction intermediate, referred to as a cleavage or cleavage complex and may also 11 induces G2-M cell cycle arrest. 12 13 In certain embodiments of the invention, the 14 chemotherapeutic agent is a fluoropyrimidine e.g. 5- FU. 16 17 Where reference is made to specific chemotherapeutic 18 agents,, it should be understood that analogues 19 including biologically active derivatives and substantial equivalents thereof, which retain the 21 antitumour activity of the specific agents, may be 22 used. 23 24 EGF Inhibitors As described above, the inventors have found that 26 ^combinations of two or more inhibitors of EGFS may 27 be used to obtain a dramatically enhanced tumour 28 cell growth attenuating effect. In certain 29 embodiments of the invention, any molecule' which reduces expression of an EGF gene or antagonizes the 31 'EGF protein may be used as the EGF inhibitor. In 28 1 particular embodiments, the EGF is HB-EGF, AREG, 2 TGF, EREG, BTC, or NRG3. 3 4 In one embodiment, inhibitors of HB-EGF and of AREG are used. 6 7 EGF inhibitors may include, but are not limited to,- 8 antibodies, antibody fragments, immunoconjugates, 9 small molecule inhibitors, peptide inhibitors, specific binding members, non-peptide small organic 11 molecules, antisense molecules, aptamers, or 12 oligonucleotide decoys. 13 14 Any Erbl or EGF receptor inhibitor should indirectly inhibit AREG activity. Suitable inhibitors include, 16 but are not limited to, PD169540 (a pan-ErbB 17 inhibitor) and IRESSA (an ErbBl-specific inhibitor). 18 19 Other suitable inhibitors may include CTyrphostin AG 1478 (a selective and potent inhibitor of EGF-R 21 kinase) which indirectly inhibits TGF-alpha; ZM 22 252868 is an Epidermal growth factor (EGF) receptor- 23 specific tyrosine kinase inhibitor which inhibits 24 TGF-alpha actions in ovarian cancer cells (Simpson et al, British Journal of Cancer, 7 9(7-8):1098-103, 26 1999). 27 28 A suitable inhibitor of HB-EGF may include CRM197 . 29 In one embodiment, an indirect inhibitor of the EGF- 31 receptor may be utilised. 32 29 1 In another embodiment, the inhibitor a direct 2 inhibitor of the EGF is used. In particular, 3 embodiments, a direct inhibitor is an antibody 4 molecule which binds EGF or a nucleic acid molecule which inhibits expression of said EGF. 6 7 In one embodiment, the inhibitor is an anti EGF 8 antibody. 9 The inventors have developed some novel antibodies J 11 for use in the present invention. In a particular, 12 embodiment, an antibody of or for use in the 13 invention is an antibody molecule having binding 14 specificity for AREG, wherein the antibody molecule is the 6E11 1E9 1C6 antibody, or a fragment thereof. 16 17 Antibody molecules of or for use in the invention 18 herein include antibody fragments and "chimeric" 19 antibodies in which a portion of the heavy and/or light chain is identical with or homologous to 21 corresponding sequences in antibodies derived from a 22 particular species or belonging to a particular 23 antibody class or subclass, while the remainder of 24 the chain (s) is identical with or homologous to corresponding sequences,in antibodies derived from 26 another species or belonging to another antibody 27 class or subclass, as well as fragments of such 28 antibodies, so long as they exhibit the desired 29 biological activity (see U. S. Patent No. 4, 816, 567 ; and Morrison et al., Proc. Natl. Acad. Sci. 31 USA, 81 : 6851-6855 (1984)). Chimeric antibodies of 32 interest herein include "primatized"antibodies 1 comprising variable domain antigen-binding sequences 2 derived from a non-human primate(e. g. Old World 3 Monkey, Ape etc), and human constant region 4 sequences. 6 An antibody molecule for use in the invention may be 7 a bispecific antibody or bispecific fragment. For 8 example, the antibody molecule or fragment may have 9 specificity for HB-EGF and for AREG. For example, In one embodiment, a bispecific antibody molecule for 11 use in the present invention may comprise a first . 12 heavy chain and a first light chain from the anti 13 6E11 1E9 1C6 and an additional antibody heavy chain 14 and light chain with binding specificity for HB-EGF.

A number of methods are known in the art for the 16 production of antibody bispecific antibodies and 17 fragments. For example, such methods include the 18 fusion of hybridomas or linking of Fab' fragments 19 (for example, see Songsivilai & Lachmann, Clin. Exp.

Immunol. 79: 315-321 (1990), Kostelny et al., J. 21 Immunol. 148:1547-1553 (1992)). In another 22 embodiement, bispecific antibodies may be formed as 23 "diabodies". 24 Antibody molecules, such as antibodies and antibody 26 fragments, for use in the present invention may be 27 produced .in any suitable way, either naturally or 28 synthetically. Such methods may include, for 29 example, traditional hybridoma techniques (Kohler and Milstein (1975) Nature, 256 :495-499), 31 recombinant DNA techniques (see e.g. U. S. Patent 32 No. 4,816, 567), or phage display techniques using 31 1 antibody libraries (see e.g. Clackson et al. (1991) 2 Nature, 352: 624-628 and Marks et al. (1992) Bio/ 3 Technology, 10: 779-783). Other antibody production 4 techniques are described in Using Antibodies: A Laboratory Manual, eds. Harlow and Lane, Cold Spring 6 Harbor Laboratory, 1999. 7 8 Traditional hybridoma techniques typically involve 9 the immunisation of a mouse or other animal with an antigen in order to elicit production of lymphocytes 11 capable of binding the antigen. The lymphocytes are 12 isolated and fused with a a myeloma cell line to 13 form hybridoma cells which are then cultured in 14 conditions which inhibit the growth of the parental myeloma cells but allow growth of the antibody 16 producing cells. The hybridoma may be subject to 17 genetic mutation, which may or may not alter the 18 binding specificity of antibodies produced. 19 Synthetic antibodies can be made using techniques known in the art (see, for example, Knappik et al, 21 J. Mol. Biol. (2000) 296, 57-86 and Krebs et al, J. 22 Immunol. Meth. (2001) 2154 67-84. 23 24 Modifications may be made in the VH, VL or CDRs of the binding members, or indeed in the FRs using any 26 suitable technique known in the art. For example, 27 variable VH and/or VL domains may be produced by 28 introducing a CDR, e.g. CDR3 into a VH or VL domain 29 lacking such a CDR. Marks et al. (1992) Bio/ Technology, 10: 779-783 describe a shuffling 31 technique in which a repertoire of VH variable 32 1 domains lacking CDR3 is generated and is then 2 combined with a CDR3 of a particular antibody to 3 produce novel VH regions. Using analogous 4 techniques, novel VH and VL domains comprising CDR derived sequences of the present invention may be 6 .produced. 7 8 Accordingly, antibodies and antibody fragments for 9 use in the invention may be produced by a method comprising: (a) providing a starting repertoire of 11 nucleic acids encoding a variable domain, wherein 12 the variable domain includes a CDRl, CDR2 or CDR3 13 to be replaced or the nucleic acid lacks an encoding 14 region for such a CDR; (b) combining the repertoire with a donor nucleic acid encoding an amino acid 16 sequence such that the donor nucleic acid, is 17 inserted into the CDR region in the repertoire so as 18 to provide a product repertoire of nucleic acids 19 encoding a variable domain; (c) expressing the nucleic acids of the product repertoire; (d) 21 ' selecting a specific antigen-binding fragment 22 specific for said target; and (e) recovering the 23 specific antigen-binding fragment or nucleic acid 24 encoding it. The method may include an optional step of testing the specific binding member for ability 26 to inhibit the activity of said target. 27 28- Alternative techniques of producing antibodies for 29 use in the invention may involve random mutagenesis of gene(s) encoding the VH or VL domain using, for' 31 example, error prone PCR (see Gram et al, 1992, 33 1 P.N.A.S. 89 3576-3580. Additionally or 2 alternatively, CDRs may be targeted for mutagenesis '3 e.g. using the'molecular evolution approaches 4 described by Barbas et al 1991 PNAS 3809-3813 and Scier 1996 J Mol Biol 263 551-567. 6 7 An antibody for use in the invention may be a• 8 "naked" antibody (or fragment therof) i.e. an 9 antibody (or fragment thereof) which is not conjugated with an "active therapeutic agent". An 11 "active therapeutic agent" is a molecule or atom 12 which is conjugated to a antibody moiety (including 13 antibody fragments, CDRs etc) to produce a 14 conjugate. Examples of such "active therapeutic agents" include drugs, toxins, radioisotopes, 16 immunomodulators, chelators, boron compounds, dyes 17 etc. 18 19 An EGF inhibitor for use in the invention may be in the form of an immunoconjugate, comprising an 21 antibody fragment conjugated to an "active 22 therapeutic agent". The therapeutic agent may be a 23 chemotherapeutic agent or another molecule.

V 24 Methods of producing immunoconjugates are well known 26 in the art; for example, see U. S. patent No. 27 5,057,313, Shih et al., Int. J. Cancer 41: 832-839 28 (1988); Shih et al., Int. J.Cancer 46: 1101-1106 29 (1990), Wong, Chemistry Of Protein Conjugation And Cross-Linking (CRC Press 1991); Upeslacis et al., 34 1 "Modification of Antibodies by Chemical Methods,"in 2 Monoclonal Antibodies: Principles And Applications, 3 Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc. 4 1995); Price, "Production and Characterization of Synthetic Peptide-Derived Antibodies," in Monoclonal '6 Antibodies: Production, Engineering And Clinical 7 Application, Ritter et al.(eds.), pages 60-84 8 (Cambridge University Press 1995) . 9 The antibody molecules for use in the invention may 11 comprise further modifications. For example the 12 antibody molecules can be glycosylated, pegylated, 13 or linked to albumin or a nonproteinaceous polymer. 14 Antisense/siRNA 16 17 Inhibitors of EGF and inhibitors of HB-EGF for use 18 in the present invention may comprise nucleic acid 19 molecules capable of modulating gene expression, for example capable of down regulating expression of a 21 sequence encoding an EGF protein. Such nucleic acid 22 molecules may include, but are not limited to 23 antisense molecules, short interfering nucleic acid 24 (siNA), for example short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro RNA, short 26 hairpin RNA (shRNA), nucleic acid sensor molecules, 27 allozymes, enzymatic nucleic acid molecules, and 28 triplex oligonucleotides and any other nucleic acid 2 9 molecule which can be used in mediating RNA interference "RNAi" or gene silencing in a sequence- 31 specific manner (see for example Bass, 2001, Nature, 1 411, 428-429; Elbashir et al., 2001, Nature, 411, 2 494-498; WO 00/44895; WO 01/36646; WO 99/32619; WO 3 00/01846; WO 01/29058; WO 99/07409; and WO 00/44914; 4 Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, 6 Science, 297, 2215-2218; Hall et al., 2002, Science, 7 297, 2232-2237; Hutvagner and Zamore, 2002, Science, 8 297, 2056-60; McManus et al., 2002, RNA, 8, 842-850; 9 Reinhart et al., 2002, Gene & Dev., 16, 1616-1626; and Reinhart & Bartel, 2002, Science, 297, 1831). 11 12 An "antisense nucleic acid", is a non-enzymatic 13 nucleic acid molecule that binds to target RNA by 14 means of RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al., 1993 Nature 365, 566) 16 interactions and alters the activity of the target 17 RNA (for a review, see Stein and Cheng, 1993 Science 18 261, 1004 and Woolf et al., U.S. Pat. No. 19 5,849,902). The antisense molecule may be complementary to a target sequence along a single 21 contiguous sequence of the antisense molecule or may 22 be in certain embodiments, bind to a substrate such 23 that the substrate, the antisense molecule or both 24 can bind such that the antisense molecule forms a loop such that the antisense molecule can be 2 6 complementary to two or more non-contiguous 27 substrate sequences or two or more non-contiguous 28 sequence portions of an antisense molecule can be 29 complementary to a target sequence, or both.

Details of antisense methodology are known in the 31 art, for example see Schmajuk et al., 1999, J. Biol. 32 Chem., 274, 21783-21789, Delihas et al., 1997, 36 1 Nature, 15, 751-753, Stein et al., 1997, Antisense 2 N. A. Drug. Dev., 7, 151, Crooke, 2000, Methods 3 Enzymol., 313, 3-45; Crooke, 1998, Biotech. Genet. 4 Eng. Rev., 15, 121-157, Crooke, 1997, Ad.

Pharmacol., 4 0, l-'4 9. 6 7 A "triplex nucleic acid" or "triplex 8 oligonucleotide" is a polynucleotide or 9 oligonucleotide that can bind to a double-stranded DNA in a sequence-specific manner to form a triple- 11 strand helix. Formation of such triple helix 12 structure has been shown to modulate transcription 13 of the targeted gene (Duval-Valentin et al., 1992, 14 Proc. Natl. Acad. Sci. USA, 89, 504). 16 Aptamers 17 18 Aptamers are nucleic acid (DNA and RNA) 19 macromolecules that bind tightly to a specific molecular target. They can be produced rapidly 21 through repeated rounds of in vitro selection for 22 example by SELEX (systematic evolution of ligands 23 by exponential enrichment) to bind to various 24 molecular targets such as small molecules, proteins, nucleic acids etc ( see Ellington and Szostak, 26 Nature 346 (6287) :818-822 (1990), Tuerk and Gold, 27 Science 249 (4968) :505-510 (1990) U.S. Patent No. 28 6, 867, 289; U.S.. Patent No. 5, 567, 588, U.S. Patent 29 No. 6, 699, 843). 31 In addition to exhibiting remarkable specificity, 32 aptamers generally bind their targets with very high 37 1 affinity; the majority of anti-protein aptamers have 2 equilibrium dissociation constants (Kds) in the 3 picomolar (pM) to low nanomolar (nM) range. 4 Aptamers are readily produced by chemical synthesis, possess desirable storage properties, and elicit 6 little or no immunogenicity in therapeutic 7 applications. 8 9 Non-modified aptamers are cleared rapidly from the bloodstream, with a half-life of minutes to hours, 11 ■ mainly due to nuclease degradation and renal 12 clearance a result of the aptamer's inherently low 13 molecular weight. However, as is known in the art, 14 modifications, such as 2'-fluorine-substituted pyrimidines, polyethylene glycol (PEG) linkage, etc. 16 (can be used to adjust the half-life of the 17 molecules to days or weeks as required.. 18 19 Peptide aptamers are proteins that are designed to interfere with other protein interactions inside 21 cells. They consist of a variable peptide loop 22 attached at both ends to a protein scaffold. This 23 double structural constraint greatly increases the 24 binding affinity of the peptide aptamer to levels comparable to an antibody's (nanomolar range). The 26 variable loop length is typically comprised of 10 to 27 20 amino acids, and the scaffold may be any protein 28 which has good solubility and compacity properties. 29 Aptamers may comprise any deoxyribonucleotide or ribonucleotide or modifications of these bases, such 31 as deoxythiophosphosphate (or phosphorothioate), 32 which have sulfur in place of oxygen as one of the 38 1 non-bridging ligands bound to the phosphorus. 2 Monothiophosphates aS have one sulfur atom and are 3 thus chiral around the phosphorus center. 4 Dithiophosphates are substituted at both oxygens and are thus achiral. Phosphorothioate nucleotides are 6 commercially available or can be synthesized by 7 several different methods known in the art.). 8 9 Treatment 11 "Treatment" or "therapy" includes any regime that 12 can benefit a human or non-human animal. The 13 treatment may be in respect of an existing condition 14 or may be prophylactic (preventative treatment).

Treatment may include curative, alleviation or 16 prophylactic effects. 17 ■ 18 "Treatment of cancer" includes treatment of 19 conditions caused by cancerous growth and/or vascularisation and includes the treatment of 21 neoplastic growths or tumours. Examples of tumours 22 that can be treated using the invention are, for 23 instance, sarcomas, including osteogenic and soft 24 tissue sarcomas, carcinomas, e.g., breast-, lung-, bladder-, thyroid-, prostate-, colon-, rectum-, 26 pancreas-, stomach-, liver-, uterine-, prostate , 27 cervical and ovarian carcinoma, non-small cell lung 28 cancer, hepatocellular carcinoma, lymphomas, 29 including Hodgkin and non-Hodgkin lymphomas, neuroblastoma, melanoma, myeloma, Wilms tumor, and 31 leukemias, including acute lymphoblastic leukaemia 39 1 and acute myeloblastic leukaemia, astrocytomas, 2 gliomas and retinoblastomas. . 3 ( 4 The invention may be particularly useful in the treatment of existing cancer and in the prevention 6 of the recurrence of cancer after initial treatment 7 or surgery. 8 9 Pharmaceutical Compositions 11 Pharmaceutical compositions according to the present 12 invention, and for use in accordance with the 13 present invention may comprise, in addition to 14 active ingredients, e.g (i) a chemotherapeutic agent and/or an EGF inhibitor or (ii) an inhibitor of a 16 first EGF and an inhibitor of a second EGF, a 17 pharmaceutically acceptable excipient, a carrier, 18 buffer stabiliser or other materials well known to 19 those skilled in the art (see, for example, (Remington: the Science and Practice of Pharmacy, 21 21st edition, Gennaro AR, et al, eds., Lippincott 22 Williams & Wilkins, 2005.). Such materials may 23 include buffers such as acetate, Tris, phosphate, 24 citrate, and other organic acids ; antioxidants; preservatives; proteins, such as serum albumin, 26 gelatin, or immunoglobulins ; hydrophilic polymers 27 such aspolyvinylpyrrolidone ; amino acids such as 28 glycine, glutamine, asparagine, histidine, arginine, 29 or lysine ; carbohydrates; chelating agents; tonicifiers; and surfactants. 40 1 2 The pharmaceutical compositions may also contain one 3 or more further active compound selected as 4 necessary for the particular indication being treated, preferably with complementary activities 6 - - that do not adversely affect the activity of the 7 composition of the invention. For example, in the 8 'treatment of cancer, in addition to one or more EGF 9 inhibitors and/or a chemotherapeutic agent, the formulation or kit may comprise an additional 11 component, for example a second or further EGF 12 inhibitor, a second or further chemotherapeutic 13 agent, or an antibody to a target other than the EGF 14 to which the said inhibitors bind, for example to a growth factor which affects the growth of a 16 particular cancer. 17 18 The active ingredients (e.g. EGF inhibitors, for 19 example HB-EGF inhibitors, AREG inhibitors, and/or chemotherapeutic agents) may be administered via 21 microspheres, microcapsules liposomes, other 22 microparticulate delivery systems. For example, 23 active ingredients may be entrapped within . 24 microcapsules which may be prepared, for example, by coacervation techniques or by interfacial 26 polymerization, for example, hydroxymethylcellulose 27 or gelatinmicrocapsules and poly- 28 (methylmethacylate) microcapsules, respectively, in 29 colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, 31 - nano-particles and nanocapsules) or in 41 1 macroemulsions. For further details, see Remington: 2 the Science and Practice of Pharmacy, 21st edition, 3 Gennaro AR, et al, eds., Lippincott_Williams & 4 Wilkins, 2005. 6 Sustained-release preparations may be used for 7 delivery of active agents. Suitable examples of 8 sustained-release preparations include semi- 9 . permeable matrices of solid hydrophobic polymers " containing the antibody, which matrices are in the 11 form of shaped articles, e. g. films, suppositories 12 or microcapsules. Examples of sustained-release 13 matrices include polyesters, hydrogels (for example, 14 poly (2-hydroxyethyl-methacrylate) , or poly (vinylalcohol)), polylactides (U. S. Pat. No. 3, 16 773, 919), copolymers of L-glutamic acid andy ethyl- 17 Lglutamate,non-degradable ethylene-vinyl acetate, 18 degradable lactic acid-glycolic acid copolymers, and 19 poly-D- (-)-3-hydroxybutyric acid. 21 As described above nucleic acids may also be used in 22 methods of treatment. Nucleic acid for use in the 23 invention may be delivered to cells of interest 24 using any suitable technique known in the art.

Nucleic acid (optionally contained in a vector) may 26 be delivered to a patient's cells using in vivo or 27 ex vivo techniques. For in vivo techniques, 28 transfection with viral vectors (such as adenovirus, 29 Herpes simplex I virus, or adeno-associated virus) and lipid-based systems (useful lipids for lipid- 31 mediated transfer of the gene are DOTMA, DOPE and 32 DC-Choi, for example) may be used (see for example, 42 1 Anderson et al., Science 256 : 808-813 (1992). See 2 also WO 93/25673 ). 3 4 In ex vivo techniques, the nucleic acid is introduced into isolated cells of the patient with 6 the modified cells being administered to the patient 7 either directly or, for example, encapsulated within 8 porous membranes which are implanted into the 9 patient (see, e. g. U. S. Patent Nos. 4, 892, 538 10 and 5, 283, 187). Techniques available for 11^ introducing nucleic acids into viable cells may 12 include the use of retroviral vectors, liposomes, 13 electroporation, microinjection, cell fusion, DEAE- 14 dextran, the calcium phosphate precipitation method, etc. 16 17 The EGF inhibitors and/or chemotherapeutic agent may 18 be administered in a localised manner to a tumour 19 site or other desired site or may be delivered in a manner in which it targets tumour or other cells. 21 Targeting therapies may be used to deliver the 22 active agents more specifically to certain types of 23 cell, by the use of targeting systems such as 24 antibody or cell specific ligands. Targeting may be desirable for a variety of reasons, for example if 26 the agent is unacceptably toxic, or if it would 27 otherwise require too high a dosage, or if it would 28 not otherwise be able to enter the target cells. 29 Administration 43 1 2 In embodiments of the invention, where an EGF 3. inhibitor and a chemotherapeutic agent are used in 4 treatment, the EGF inhibitor may be administered simultaneously, separately or sequentially with the 6 chemotherapeutic agent. Likewise, in embodiments of 7 the invention, where a first EGF inhibitor and a 8 second EGF inhibitor are used in treatment together, 9 the first EGF inhibitor may be administered simultaneously, separately or sequentially with the 11 second EGF inhibitor. Where administered separately 12 or sequentially, they may be administered within any 13 suitable time period e. g. within 1, 2, 3, 6, 12, 14 24, 48 or 72 hours of each other. In preferred embodiments, they are administered within 6, 16 preferably within 2, more preferably within 1, most 17 preferably within 20 minutes of each other. 18 19 Kits 21 The invention further extends to various kits for 22 the treatment of cancer or the killing of tumour 23 cells. The kits may optionally include instructions 24 for the administration of each component, e.g. EGF inhibitor and chemotherapeutic agent, or first EGF 26 inhibitor and second EGF inhibitor, separately, 27 sequentially or simultaneously. 28 2 9 Dose 44 1 The EGF inhibitors and/or chemotherapeutic agents of 2 and for use in the invention are suitably 3 administered to an individual in a "therapeutically 4 effective amount", this being sufficient to show benefit to the individual. The actual dosage 6 regimen will depend on a number of factors ' including 7 the condition being treated, its severity, the 8 patient being treated, the agents being used, and 9 will be at the discretion of the physician. 11 In one embodiment of the methods, compositions or 12 kits, in which an EGF inhibitor and a 13 chemotherapeutic agent is used, the EGF inhibitor 14 and chemotherapeutic agent are administered in doses which produce a potentiating ratio. Likewise, in 16 one embodiment of the methods, compositions or kits, 17 in which a first EGF inhibitor and a second EGF 18 inhibitor is used, the EGF inhibitor and 19 chemotherapeutic agent are administered in doses which produce a potentiating ratio. 21 22 The term "potentiating ratio" in the context of the 23 present invention is used to indicate that two 24 components, e.g. EGF inhibitors,chemotherapeutic agents etc. are present in a ratio such that the 26 cytotoxic activity of the combination is greater 27 than that of either component alone or of the 28 additive activity that would be predicted for the 29 combinations based on the activities of the individual components. 45 1 Thus in a potentiating ratio, the individual 2 components act synergistically. 3 4 Synergism may be defined using a number of methods. 6 In one method, synergism may be determined by 7 calculating the combination index (CI) according to 8 the method of Chou and Talalay. CI values of 1, < 1, 9 and > 1 indicate additive, synergistic and antagonistic effects respectively. 11 12 . In one embodiment of the invention, the EGF 13 inhibitor and the chemotherapeutic agent are present 14 in concentrations sufficient to produce a CI of less than 1, such as less than 0.85. Likewise, in another 16 embodiment of the invention, the first EGF inhibitor 17 and the second EGF inhibitor are present in 18 concentrations sufficient to produce a CI of less 19 than 1, such as less than 0.85. 21 Synergism is preferably defined as an RI of greater 22 than unity using the method of Kern as modified by 23 Romaneli (1998a, b). The RI may be calculated as the 24 ratio of expected cell survival (Sep, defined as the product of the survival observed with component A 26 alone and the survival observed with component B 27 alone) to the observed cell survival (Sobs) for the 28 combination of A and B(RI=Se/Sobs). Synergism may 29 then be defined as an RI of greater than unity. 31 In one embodiment of the invention, the EGF 32 inhibitor and the chemotherapeutic agent ( or the 46 1 first EGF inhibitor and the second EGF inhibitor) 2 are provided in concentrations sufficient to produce 3 an RI of greater than 1.5, such as greater than 2.0, 4 for example greater than 2.25. 6 Thus in one embodiment the combined medicament^ 7 produces a synergistic effect when used to treat 8 tumour cells. 9 The optimal dose can be determined by physicians 11 based on a number of parameters including, for 12 example, age, sex, weight, severity of the condition 13 being treated, the active ingredient being 14 administered and the route of administration. 16 The invention will now be described further in the 17 following non-limiting examples with reference made 18 to the accompanying drawings in which: 19 Figure 1A illustrates analysis of AREG and beta 21 actin RNA expression in RKO +/+ with / without 22 either a 48 hour CPT11 treatment or 5-Fu treatment. 23 RNA levels were analyzed following 35 cycle of PCR 24 to determine relative differences in expression between treated and untreated samples; 26 27 Figure IB illustrates analysis of AREG and beta 28 actin RNA expression in HCT116 +/+ with / without a 29 48 hour CPTll treatment. RNA levels were analyzed following 35 cycles of PCR to determine relative 47 1 differences in expression between treated and 2 untreated samples; 3 4 Figure 2 illustrates western blot analysis of AREG and gamma tubulin protein expression in HCT116 +/+ 6 and RKO +/+ with / without a 48 hour CPT11 or 5-Fu 7 treatment; 8 9 Figure 3 illustrates confocal microscopy image of AREG and Actin protein in HCT 116 +/+ with or 11 without CPT-11 treatment; 12 13 Figure 4 illustrates analysis of AREG protein 14 - expression in H630 p53 mutant colorectal cancer cell lysates following a 48 hour CPT11 treatment. Western 16 blots were probed using an anti-amphiregulin 17 antibody. Enhanced AREG expression was observed 18 following chemotherapy (A and B illustrate two 19 separate experiments). 21 22 Figure 5 illustrates analysis of AREG and beta actin 23 RNA expression in H460 lung cancer cells with / 24 without either a 48 hour CPT11 treatment or 5-Fu treatment. RNA levels were analyzed following 35 26 cycle of PCR to determine relative differences in 27 expression between treated and untreated samples. 28 48 1 Figure 6 illustrates AREG upregulation following 2 chemotherapeutic challenge in A) HT29, B) HCT116 and 3 C) MDA-MB231 cells. Cells were treated with/without 4 chemotherapy for 48 hours. RT-PCR was performed with 1 pg of total RNA using primer pairs specific for 6 the human AREG gene or GAPDH. The PCR products were 7 separated on 1.5% agarose gel electrophoresis and 8 visualized by ethidium bromide staining. 9 .

Figure 7a illustrates specific AREG silencing by 11 siRNA in HCT116. HCT116 cells were transfected with 12 AREG specific siRNA (lOnM), or a control siRNA 13 (lOnM). AREG and Beta-actin gene expression were 14 measured by RT-PCR RNA 72 hrs after transfection.

Figure 7b) illustrates specific HB-EGF silencing by 16 siRNA in HCT116. HCT116 cells were transfected with 17 HB-EGF specific siRNA (lOnM), or a control siRNA 18 (lOnM). HB-EGF and Beta-actin gene expression were 19 measured by RT-PCR RNA 72 hrs after transfection 21 Figure 8 illustrates HCT116 cell proliferation 22 following AREG/HB-EGF silencing by siRNA and/or 23 Chemotherapy treatment. Cells were transfected with 24 AREG siRNA (lOnM), HB-EGF siRNA (lOnM) or a control siRNA (lOnM). Transfected cells were treated with no 26 drug or 3.5pM CPT-11. Cell proliferation was 27 analysed by MTT assay 48hr after 28 transfection/chemotherapy 29 49 1 Figure 9 illustrates specific AREG silencing by 2 siRNA in HT29 colorectal cancer cells. Cells were 3 transfected with AREG specific siRNA (lOnM), or a 4 control siRNA (lOnM). AREG and GAPDH gene expression were measured by RT-PCR RNA 72 hrs after 6 transfection. 7 8 Figure 10 illustrates HT29 cell proliferation 9 following AREG silencing by siRNA and/or Chemotherapy treatment. Cells were transfected with 11 AREG siRNA (lOnM) or a control siRNA (lOnM). 12 Transfected cells were treated with no drug or 3.5yM 13 CPT-11. Cell proliferation was analysed by MTT assay 14 48hr after transfection/chemotherapy. 16 Figure 11 illustrates Specific AREG silencing by 17 siRNA in MDA-MB231 cells. Cells were transfected 18 with AREG specific siRNA (lOnM), or a control siRNA 19 (lOnM). AREG and GAPDH gene expression were measured by RT-PCR RNA 72 hrs after transfection. 21 22 Figure 12 illustrates MDA-MB2 31 cell proliferation 23 following AREG silencing by siRNA and/or 24 chemotherapy treatment. Cells were transfected with AREG siRNA (lOnM), or a control siRNA (lOnM) and 26 varying doses of 5-FU. Cell proliferation was 27 analysed by MTT assay 48hr after 28 transfection/chemotherapy. 29 50 1 Figure 13 illustrates MDA-MB231 cell proliferation 2 following AREG/HB-EGF silencing by siRNA. Cells were 3 transfected with AREG siRNA (lOnM), HB-EGF siRNA 4 (lOnM) or a control siRNA (lOnM). Cell proliferation was analysed by MTT assay 48hr after 6 transfection/chemotherapy. 7 8 Figure 14 illustrates amplification of amphiregulin 9 fragments from cDNA library. Amphiregulin was amplified from kidney cDNA and PCR reaction was 11 analysed on 1.5% agarose gel stained with ethidium 12 bromide 13 14 Figure 15 illustrates colony PCR of AREG fragments.

PCR amplification was carried out colonies to 16 identify colonies that had the amphiregulin fragment 17 successful cloned into the expression vector. PCR 18 reaction was analysed on 1.5% agarose gel stained 19 with ethidium bromide. Any positive colonies were selected for sequence and expression analysis 21 22 Figure 16 Panel A) illustrates the elution profile 23 of the purification of AREG recombinant protein from 24 500ml culture volume. Pellet from culture was resuspended in 8M Urea and then purified by mature 26 of the 6xHistidine tag. The elution samples were 27 collected and analysed by SDS -PAGE (Panel B). The 28 gel was stained with comassie blue. 51 1 Figure 17 illustrates ELISA result of AREG 2 monoclonal antibodies produced. Monoclonal 3 antibodies were tested by ELISA against recombinant 4 AREG protein and a negative control produced in similar method. 6 \ 7 Figure 18 illustrates western blot analysis of 8 monoclonal test bleeds. The monoclonal test bleeds 9 were tested by western blot against the recombinant amphiregulin protein and a negative control protein. 11 Equal amounts of protein were loaded on SDS-PAGE 12 gel. 13 14 Figure 19 illustrates western blot analysis of AREG monoclonal antibodies against recombinant protein. 16 Recombinant AREG protein and a negative control 17 protein were run on SDS-PAGE gel. The gel was 18 transferred to nitrocellulose membrane and probed 19 with the AREG monoclonal antibodies. 21 Figure 20 illustrates western blot analysis of AREG 22 monoclonal antibodies against whole cell lysated 23 from colorectal cell lines HCT116 and HT29. Whole 24 cell lysates from HCT116 and HT29 cell lines were prepared and ran on SDS-PAGE. Blots were probed with 26 AREG monoclonal antibodies a) 6E11 1E9 2D8 and b) 27 6E11 1E9 1C6. ( NB 6E11 1E9 2D8 has been 28 subsequently shown to be the sanme antibody as 6E11 29 1E9 1C6) . 52 1 Figure 21 illustrates confocal microscopy image of 2 AREG and actin protein in HCT 116 +/+ stained with 3 6E11 1E9 1C6 Monoclonal antibody 4 Figure 22 illustrates confocal microscopy image of 6 AREG and actin protein in HCT 116 +/+ stained with 7 6E11 1E9 2D8 Monoclonal antibody 8 9 Figure 23 illustrates FACS analysis in HCT116 colorectal cancer cell line treated with or without 11 2 . 5pM irinotecan for 48 hours. 12 13 Figure 24 illustrates FACS analysis in HCT116 cells 14 treated with or without 2.5pM irinotecan for 48 hours. Following treatment cells were stained with 16 AREG monoclonal antibodies and analysed by FACS 17 18 Figure 25 illustrates FACS analysis in H460 lung 19 carcinoma cell line treated with or without 2.5pM irinotecan for 48 hours. Following treatment cells 21 were stained with AREG monoclonal antibodies and 22 analysed by FACS 23 24 Figure 26 illustrates MDA MB231 cell proliferation after treatment with AREG antibody. Cell 26 proliferation was analysed by MTT assay 48hr after 27 treatment 28 53 1 Figure 27 illustrates MDA MB231 cell proliferation 2 after treatment with AREG antibody.' Cell 3 .proliferation was analysed by MTT assay 48hr after 4 treatment. 6 Figure 28 illustrates HCT116 cell proliferation 7 after treatment with AREG antibody. Cell 8 proliferation was analysed by MTT assay 48hr after 9 treatment. 11 Figure 2 9 illustrates MDA MB231 cell proliferation 12 after treatment. with AREG antibody. Cell 13 proliferation was analysed by MTT assay 48hr after 14 treatment. 16 Figure 30 illustrates HCT116 cell proliferation 17 after treatment with AREG antibody. Cell 18 proliferation was analysed by MTT assay 48hr after 19 treatment. 21 Figure 31 illustrates HCT116 cell proliferation 22 after treatment with AREG antibody. Cell 23 proliferation was analysed by MTT assay 48hr after 24 treatment 26 Figure 32 illustrates H460 lung carcinoma cell 27 proliferation following treatment with different 28 concentrations of AREG (6E11 1E9 1C6) antibody or an 29 isotype control antibody. Cells were seeded 24 hours 54 1 before treatment with either 25nM, 50nM or lOOnM 2 antibody. Cell viability was assayed 48 hours after 3 treatment by MTT assay. 4 Figure 33 illustrates HCT116 cell proliferation 6 following HB-EGF silencing by siRNA and/or treatment 7 with Anti AREG antibodies (6E11 1E9 2D8 & 6E11 1E9 8 1C6). Cells were transfected with HB-EGF siRNA 9 (50nM) or a control siRNA (50nM) .- Cell proliferation 10' was analysed by MTT assay 72hr after transfection. 11 12 Materials and Methods 13 14 Cell lines and culture conditions 16 The HCT116 (p53 wild type) human colorectal 17 adenocarcinoma cell line was maintained in McCoys 18 • (Invitrogen, UK). The RKO (p53 wild type) colorectal 19 carcinoma cell line, the MDA-MB231 human breast carcinoma cell line and the HT2 9 human colorectal 21 carcinoma cell line were each maintained in 22 Dulbecco's Modified Eagle's Medium (DMEM, 23 Invitrogen, UK). 24 colorectal cell lines were maintained in Dulbecco's Modified Eagle's Medium (DMEM, Invitrogen, UK). The 26 HH630 (p53 mutant) colorectal cancer cell lines were 27 maintained in Dulbecco's Modified Eagle's,Medium 28 (DMEM, Invitrogen, UK). The H460 (p53 mutant) lung 29 cancer cell lines were maintained in RPMI media 55 1 (Sigma Aldrich, UK). All medium was supplemented 2 with 10% FCS (normal (Invitrogen, UK) or dialysed 3 (Autogene Bioclear, UK)), 1% pen / strep, 1% L- 4 ' Glutamine (All Invitrogen, UK). 6 Xenograft models 7 6-8 week old female SCID mice were implanted with 2 8 x 106 HCT116+/+ human colorectal adenocarcinoma 9 cells into each flank. HCT116 cells in a log phase of growth were harvested, washed in PBS and 11 resuspended in HBSS. They were mixed with equal 12 volumes of matrigel to give a final concentration of 13 5 x 106 cells / ml. Mice were randomly separated 14 into treatment groups on day 5 after implantation and treated with different doses of chemotherapy. 5- 16 Fu (70 mgs/kg), CPT11 (70 mgs/kg) or saline control 17 solution and the mice have been sacrificed at 18 different time points (24 and 48 hrs after 19 injections). All drugs were administered through a bolus injection. Animals were sacrificed at various 21 time points and tumours were removed for analysis 22 23 Microarray analysis 24 Approximately lOpg total RNA was isolated from tumour cells and was used as the starting material 26 for preparation of probes. The microarray analysis 27 was carried using an Affymetrix U133 plus 2.0 28 GeneChip®. Probes were prepared as per the 29 manufacturers recommendations. 56 1 After RNA extraction, samples were reverse 2 transcribed into cDNA which was then purified on a 3 column prior to labelling. The probes were then 4 amplified and labelled using Oligo(dT)-primed in vitro transcription generating high-yield, 6 biotinylated targets from the 3'-end. The cRNA was 7 fragmented to obtain optimal assay sensitivity and 8 then subjected to quality control to confirm that 9 fragment sizes range from 35-200 nucleotides. cRNA was quantified on a spectrophotometer and the 11 quality of fragmented cRNA checked on a bioanalyser. 12 For the next stage the fragmented cRNA was 13 hybridised to the array for 16 hours at 45°C. 14 Following this the array was washed and stained with streptavidin-phycoerythrin (SAPE) using a fluidics 16 station and scanned using a GeneChip® Scanner 3000. 17 Stained images were then analysed. 18 19 Initially the data was scaled using the Affymetrix® GCOS (Genechip® Operating System) software, to 21 assess quality metrics. The data was then normalized 22 against a control sample. After normalisation the 23 data was filtered removing all genes where the noise 24 level obscured signal and were fold change was greater than 2-fold. Finally a confidence filter 26 where the t-test p-value were used to filter the 27 genes to derive lists of statistically robust data. 28 29 Each treatment type and timepoint was carried out in triplicate and statistics and filtering were applied 31 to the whole data set from each condition. 57 1 2 Chemotherapy treatment 3 a- Cell culture 4 Cells in a log phase of growth were seeded into T75 flask at -20% confluence and incubated overnight to 6 allow adherence to the plate. Wells were treated 7 with CPT11 (Irinotecan) and 5-Fu (Flourouracil) at 8 7 . 5|iM concentration for 48 hours. Chemotherapy was 9 substituted with saline in control flasks. After different time exposure to chemotherapy the cells 11 were harvested, washed 3 times in PBS and total RNA 12 was isolated using the RNA TAT-60 reagent according 13 to the manufacturer instructions. 14 Reverse transcription was performed with 2.5 yg of RNA using a High' Capacity cDNA Archive kit (Applied 16 Biosystem) according to the instruction of the 17 manufacturer. 18 19 b- organs For in vivo toxicity study, mice were inoculated 21 with 2 x 106 HCT116+/+. human colorectal 22 adenocarcinoma cells into each flank. Mice were 23 randomly separated into treatment groups on day 5 24 after implantation and treated or not with 5Fu (15mg/kg daily or 70mg/kg twice weekly). All drugs 26 were administered through a bolus injection. Animals 27 were sacrificed at various time points and organs 28 and tumor were removed for analysis. 58 1 Semi quantitative RT-PCR 2 Semi quantitative RT-PCR was performed using a PTC 3 225 Gradient Cycler (MJ Research Incorporated. The 4 PCR mixture, in a final volume of 25 pL, contains 12.5pL of 2x Biomix (Bioline,UK), 2 pL of primers 6 (10 pmol/L), 1 pL of cDNA and 9.5 pL of dH20) . PCR 7 conditions were initial denaturation step of' 95C for 8 10 minutes, followed by 35 cycles of 95°C for 30 sec 9 for denaturation; as annealing step 55°C for 30 sec; and extension at 72°C for 90 sec, with a final 11 extension of 72°C for 10 minutes. 5 pi of amplified 12 product reactions was loaded onto a 1.5% agarose gel 13 (0.001% ethidium bromide) which was ran at 90V for 14 30 to 40 minutes prior to analysis on a UV box. A Beta-actin control PCR amplification was performed 16 for each cDNA to check the level of cDNA charged in 17 the PCR mix. 18 19 For Example 2, Total RNA was isolated from cells ' following the RNA STAT-60 manufacturer's protocol 21 (Biogenesis, Poole, U.K.). RT-PCR was performed with 22 1 pg of total cell RNA using a Promega ImProm-II™ 23 Reverse Transcription System (Promega, Southhampton, 24 UK). PCR was performed using primer pairs specific for human AREG ( Forward 5'- 26 TTTTTTGGATCCCTCGGCTCAGGCCATTATGCTGCT-3'(SEQ ID 27 No:19), Reverse 5'-TTTTTTAAGCTTTACCTGTTCAACTCTGACTG- 28 3' (SEQ ID No:20)), human HB-EGF (Forward 5'- 29 TTTCTGGCTGCAGTTCTCTCGGCACT-3' (SEQ ID No:21), Reverse 5'-CCTCTCCTATGGTACCTAAACATGAGAAGCCCC-3'(SEQ 31 ID No:22)), human GAPDH as a control (Forward 59 1 5'ACCACAGTCCATGCCATCAC-3' (SEQ ID No:23), Reverse 5' 2 TCCACCACCCTGTTGCTGTA-3'(SEQ ID No:24)) and human 3 Beta-actin as a control (Forward 5'- 4 ATCTGGCACCACACCTTTACAATGAGCTGCG-3' (SEQ ID No:25), Reverse 5'-CGTCATACTCCTGCTTGCTGATCCACATCTGC-3'(SEQ 6 ID No:2 6)) . 7 The PCR products were separated on 1.5% agarose gel 8 and visualized by ethidium bromide staining. 9 Western blotting 11 a- Cell lysis 12 HCT 116, RKO, HT 29 and H630 are human colorectal 13 carcinoma cell lines. After different time exposure 14 to chemotherapy the cells were harvested, and washed in 1 X PBS. The cell pellet was then lysed 16 in a' suitable amount of 1 X RIPA lysis buffer (150mM 17 NaCl, lOmM Tris at pH 7.2, 0.1% SDS, 1.0% triton X- 18 100 and 5mM.EDTA) supplemented with protease 19 inhibitors. The cell lysate was briefly vortexed, incubated on ice for 10 min and then centrifuged at 21 12,000rpm to remove cell debris. Following 22 centrifugation, the lysate supernatant was removed 23 to a fresh eppendorf tube. 24 b- Quantitation of Whole Cell Lysates (WCL) 26 Protein concentrations were assayed using the BCA 27 Protein Assay (Pierce) according to the 28 manufacturer's instructions. The absorbance of each 29 sample at 620nm was assayed using a microplate reader. A standard BSA curve was plotted for each 60 1 experiment and the protein concentration of each 2 sample calculated. 3 4 c- Preparation of Whole Cell Lysate (WCL) protein samples 6 To each sample an equal volume of 5 X Western sample 7 buffer and 10% .of the final volume of |3- 8 mercaptoethanol (Sigma) was added. The samples were 9 denatured at 95°C for 5 minutes and placed on ice prior to loading onto SDS polyacrylamide gel (SDS 11 PAGE). lOpl of pre-stained protein molecular weight 12 marker was loaded into one well of the gel. 13 14 d- Electro-transfer of proteins to Polyvinylidene Flouride (PVDF) membrane 16 After electrophoresis the gel was placed in 1 X 17 Western transfer buffer. A piece of PVDF (Millipore) 18 was soaked in 100% methanol for 30 seconds then 19 washed with deionised H2O and equilibrated in 1 X transfer buffer. 21 22 The above were then assembled into a Trans-blot SD 23 semi-dry transfer cell (Bio-Rad) as follows: One 24 piece of blotting paper soaked in transfer buffer, the PVDF membrane, followed by the gel, then one 26 piece of blotting paper soaked in transfer buffer. 27 The proteins were then electrophoresed onto the 28 membrane at 20V for 90min. 29 61 1 e- Immunoblotting (Western blotting) 2 Following transfer, the PDVF membrane was washed 3 three times for 10 minutes with 1 X PBS/0.1% Tween 4 before being blocked for 1 hour in 1 X PBS/5% milk.

Once blocked the membrane was incubated with the 6 appropriate primary antibody at the relevant 7 dilution, in 1 X PBS/0.1 Tween/5% milk for 1 hour. 8 Following incubation the membrane was washed three 9 times with 1 X PBS/0.1% Tween before being probed with the appropriate secondary antibody (Bio-Rad) at •11 a dilution 1:5000 in 1 X PBS/5% milk for 1 hour. The 12 membrane was subsequently washed three times with 1 13 X PBS/0.1% Tween for 10 minutes each before 14 visualisation using Super Signal detection method (Pierce), as described by the manufacturers. Protein 16 bands were detected by exposure to autoradiograph 17 which was subsequently developed. If detection of a 18 second protein was required from the same 19 immunoblot, the membrane was placed in western stripping buffer, incubated for 30 min in a 50°C 21 rocking incubator. Following membrane stripping it 22 was washed in 1 X PBS/0.1% Tween, 5 times, for 10 23 min periods. The membrane was reprobed, as before 24 with the appropriate antibodies. 2 6 RNA interference 27 AREG, HB-EGF and Control siRNAs and Dharmafect 4 28 transfection reagent were obtained from Dharmacon, 29 (Lafayette, CO,USA). 62 1 For HB-EGF, the siRNAs used had the following 2 sequences: 3 1 Sense sequence 4 GAAAAUCGCUUAUAUACCUUU (Sequence ID No: 1) lAnti-sense sequence 6 AGGUAUAUAAGCGAUUUUCUU(Sequence ID No: 2) 7 8 2 Sense Sequence 9 UGAAGUUGGGCAUGACUAAUU(Sequence ID No: 3) 2 Anti-sense sequence 11 UUAGUCAUGCCCAACUUCAUU(Sequence ID No: 4) 12 13 3 Sense sequence 14 GGACCCAUGUCUUCGGAAAUU(Sequence ID No: 5) 3 Antisense sequence 16 UUUCCGAAGACAUGGGUCCUU(Sequence ID No: 6) 17 18 4 Sense Sequence 19 GGAGAAUGCAAAUAUGUAUU(Sequence ID No: 7) 4 Anti-sense Sequence 21 UCACAUAUUUGCAUUCUCCUU (Sequence ID No: 8) 22 23 For AREG, the siRNA sequences used had the following 24 sequences: 1 Sense Sequence 26 UGAUAACGAACCACAAAUAUU(Sequence ID No: 9) 27 1 Anti-Sense Sequence 28 UAUUUGUGGUUCGUUAUCAUU(Sequence ID No: 10) 29 2 Sense Sequence 31 UGAGUGAAAUGCCUUCUAGUU(Sequence ID No: 11) 32 2 Anti-sense Sequence 33 CUAGAAGGCAUUUCACUCAUU(Sequence ID No: 12) 34 3 Sense Sequence 36 GUUAUUACAGUCCAGCUUAUU(Sequence ID No: 13) 37 3 Anti-Sense Sequence 38, UAAGCUGGACUGUAAUAACUU(Sequence ID No: 14) 39 40 4 Sense Sequence 41 GAAAGAAACUUCGACAAGAUU(Sequence ID No: 15) 42 4 Anti-sense Sequence 43 UCUUGUCGAAGUUUCUUUCUU(Sequence ID No: 16) 44 63 1 Cells were seeded at 5 000 cells per well in a 96 2 well plate or 5x 105 cells per well in a 6 well 3 plate. The cells were cultured for 24 hours before 4 transfection. The siRNA was made up to lOOnM in serum free DMEM and left for 5 minutes at room 6 temperature. The Dharmafect transfection reagent was 7 also made up in the serum free DMEM and incubated 8 for 5 minutes at room temperature. The transfection 9 reagent was added to the siRNA and incubated at room temperature for 20 minutes. The media was removed 11 from the plate wells and antibiotic free DMEM was 12 added to the wells. After 20 minutes the siRNA was 13 added dropwise to the wells. The plates were 14 incubated at 37DC for 48 hours. 16 MTT assay 17 Cell viability was assessed by 3-(4, 5- 18 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium 19 bromide(MTT, Sigma) assay (Mosmann, T. 1983. J.

Immunol. Methods 65:55-63). To assess chemotherapy/ 21 siRNA interactions 5000 cells were seeded per well 22 on 96 well plate. After 24 hours cells were 23 transfected with siRNA and treated with various 24 chemotherapeutic agents at different concentrations.

After 48 hours MTT (l.Omg/ml) was added to each well 26 and cells were incubated at 37°C for 2 hours. The 27 culture media was removed and formazan crystals were 28 reabsorbed in 200pl DMSO. Cell viability was 29 determined by reading the absorbance of each well at 570nm using a microplate reader (Tecan Sunrise, 31 Biorad, UK). 64 1 2 Cloning of Amphiregulin(AREG) 3 The DNA sequence encoding the amphiregulin protein 4 was amplified by PCR from a cDNA library using gene- specific primers. The AREG gene was cloned into the 6 bacterial expression vector pETlOO allowing the 7 incorporation of a hexahistidine tag onto the N- 8 terminus of the recombinant protein. This construct 9 was then used to transform competent TOPIOF' E.coli cells (Invitrogen). Positive transformants were 11 selected by colony PCR using vector-specific primers 12 flanking the multiple cloning site. 13 14 Expression of recombinant AREG protein The positive clones were propagated overnight at 37 16 °C in 5 mis of Luria-Bertani (LB) broth supplemented 17 with 50 |J,M ampicillin. A 300 |_ll aliquot of this 18 culture was retained for inoculation of secondary 19 cultures and the remainder of the sample was miniprepped using the Qiagen miniprep kit and the 21 sequence verified by DNA sequencing. 22 23 Three secondary cultures were inoculated to allow 24 visualisation of protein expression. The cultures were induced with IPTG (final concentration 1 mM) 26 when the cultures had an OD of 0.2, 0.5 and 1.0 27 (A55o) respectively and then left for 4 hrs at 37 °C. 28 The cells were then harvested by centrifugation at 29 4000 rpm for 15 mins and the pellet resuspended in 1 ml of PBS/0.1 % Igepal supplemented with 1 p.1 of 31 lysonase. Samples were then analysed by SDS-PAGE and 32 western blotting to confirm expression of the 65 1 protein. The SDS-PAGE gel was stained overnight in 2 coomassie blue and destained the following day. 3 4 The recombinant AREG protein was then expressed in 500 mis of LB broth supplemented with ampicillin, 6 using the secondary culture as an inoculant and 7 induced with IPTG once the culture had reached the 8 optimal optical density. The culture was centrifuged 9 at 5000 rpm for 15 mins and the pellet retained for protein purification. 11 12 Protein Purification 13 The induced recombinant protein was solubilised in 14 50 mis of 8 M urea buffer (480g Urea, 29g NaCl, 3.12g NaH2P04 (dihydrate), 0.34g Imidazole) 16 overnight.. The solution was centrifuged at 6000 rpm 17 for 1 hr, after which the supernatant was filtered 18 using 0.8 |am gyrodisc filters before purification. 19 The protein was purified by its N-terminal 21 hexahistidine tag and refolded using on-column 22 . refolding by immobilized metal affinity 23 chromatography. Chelating hi-trap columns (Amersham 24 Biosciences) were charged using 100 mM nickel sulphate before attachment to the Aktaprime. 26 Refolding takes place by the exchange of the 8 M 27 urea buffer with a 5 mM imidazole wash buffer (29g 28 NaCl, 3.12g NaH2P04 (dihydrate) 0.34g Imidazole, pH 29 8.0 ) and elution of the protein using a 500 mM imidazole elution buffer (29g NaCl, 3.12g NaH2P04 31 (dihydrate), 34g Imidazole). The elution profile of 66 1 the purified recombinant protein was recorded and 2 can be seen in figure 16. 3 4 The eluted fractions were subjected to SDS-PAGE analysis to confirm recombinant protein presence in 6 eluted fractions. The gels were stained with 7 coomassie blue overnight and subsequently destained 8 to determine the fractions containing the AREG 9 protein. 11 Antibody generation 12 The refolded protein was used as an immunogen to 13 generate monoclonal antibodies. Five BALB/C mice 14 were immunized at three weekly intervals with 150 |Llg of purified recombinant protein and the antibody 16 titre was analysed after boosts three and five. A 17 test bleed was taken from each animal and tested at 18 1:1000 dilutions in western blotting against 100 ng 19 of antigen. Blots were developed using 3,3'- diaminobenzidine (DAB). 21 22 After the fifth boost, the spleen was removed from 23 the mouse and the antibody producing B cells were 24 fused with SP2 myeloma cells following standard protocols. Eleven days after the hybridoma fusion, 26 the plates were examined for cell growth. Clones 27 were screened by ELISA against recombinant protein 28 and selected positive hybridomas were cloned twice 29 by limiting dilution. 67 1 ELISA 2 The monoclonal antibodies were screened by ELISA to 3 determine which clones should be expanded. Maxi Sorb 4 96 well plates were coated with recombinant antigen by adding 100 J0.1 of coating buffer (Buffer A: 0.42g 6 sodium bicarbonate/100|il H2O, Buffer B: 0.53g sodium 7 carbonate/100|i.l H2O, pH 9.5) containing the 8 screening antigen to each well (100 ng/well). A 9 control antigen was also used to eliminate non- specific clones. The plates were incubated at 37 °C 11 for 1 hr to allow the antigen to bind to the well 12 and then blocked for 1 hr at room temperature by 13 adding 200 |0.1 PBS/3 % BSA to each well. 14 The blocking solution was removed from the plates 16 . and 100 |Xl of hybridoma supernatant was added to a 17 positive antigen and a control antigen well. The 18 screening plates were incubated with supernatant for 19 1 hr on a rocker at room temperature. The plates were washed three times with PBS-T, after which 100 21 |J.l of goat anti-mouse HRP conjugated secondary 22 antibody (1:3000) was added to each well and 23 incubated for 1 hr at room temperature. The plates 24 were washed three times with PBS-T and 100 |Lll of 3, 3' ,5,5'-tetramethylbenzidine (TMB) was added to 26 each well and incubated for 5 mins at 37 °C. 27 Positive wells were indicated by a colour 28 development and the reaction was stopped by addition 29 of 50 |J,1 1M HCL. Plates were read by a spectrophotometer at 450 nm and samples displaying a 31 positive reading in the screening well (+) with a 68 1 negative reading in the control well (-) were chosen 2 for further work. The cells from the original wells 3 were transferred into a 24 well plate and grown up. 4 Western blotting 6 The supernatants from the hybridoma cell lines were 7 analysed by western blotting to determine the 8 ability of the monoclonal antibodies to detect both 9 recombinant AREG and endogenous native AREG protein in a range of cancer cell lines, which are 11 representative of AREG expression in cancer. 12 Aliquots of HCT116 and HT29 whole cell lysates (-30 13 (0,g/ml) or recombinant AREG protein were separated by 14 SDS-PAGE and transferred onto Hybond-C Extra nitrocellulose membrane (Amersham Biosciences). The 16 membrane was blocked by incubation in PBS / 5 % 17 marvel for 1 hr at room temperature, after which it 18 was rinsed briefly in PBS. The monoclonal antibodies 19 were used at a 1:500 or 1:250 dilutions in PBS and incubated on the membrane overnight at 4°C while 21 gently rocking. The blot were then rinsed three 22 times with PBS / 1 % marvel and 0.1% Tween-20 and 23 then incubated with the goat anti-mouse HRP 24 conjugated secondary antibody at a 1:3000 dilution for 1 hr at room temperature while shaking. The 26 i blots were then rinsed three times with the PBS / 1 27 % marvel and 0.1 % Tween-20 solution, followed by a 28 short rinse in PBS. The blots were incubated with 29 ECL plus substrate (Amersham Bioscicences) for 5 mins at room temperature prior to analysis on the 31 Kodak imager. 69 1 Flow Cytometry Analysis 2 HCT116 or H460 cells were treated for 48 hours with 3 or without 2.5pM irinotecan. After 48 hours cells 4 were washed in PBS and blocked for 20 minutes in Normal Goat Serum. 5x10s cells were incubated with 6 AREG antibodies or isotype control for 2 hours and 7 washed in PBS-T. The cells were incubated with a 8 FITC conjugated goat anti-mouse antibody for 1 hour 9 and washed in PBS-T before analysis on BD FACS canto. 11 12 Results 13 14 Example 1 A xenograft study was set up to examine the genetic 16 response to 2 different chemotherapeutic drugs 24 17 and 48 hours after treatment. Each mouse was 18 implanted with equal volumes of HCT116 cells and 19 each condition was performed in triplicate. 4 groups of three mice were administered of lOOul CPT-11 21 (70mg/kg), 5-FU (70mg/kg) or saline control. Tumours 22 were then resected after 24h (5-FU) & 48h (CPT-11, 23 5-FU) . Average mass of the tumours did not vary over 24 control and drug treated groups. 26 ' RNA isolated from tumours in each of the 1'2 mice was 27 subjected to microarray analysis to measure mRNA 28 expression levels. Fold change values for drug 29 treated against untreated control is presented.

After 48 hours, the fold change values for AREG mRNA 31 expression in 5FU treated against untreated controls 70 1 was 2.1 with the fold change values for AREG mRNA 2 expression in CPTll treated against untreated 3 controls being 2.2. The data was passed through 4 stringent statistical filters and is considered statistically robust. The amphiregulin RNA was 6 significantly upregulated greater than 2 fold 7 relative to control. 8 9 Five other ErbB cognate ligands have also been found to be up-regulated by our micro-array analysis. TGF 11 and HB-EGF protein showed up-regulation when treated 12 by 5-FU. EREG protein showed up-regulation after 48h 13 treatment with both CPT-11 and 5-FU. BTC protein 14 showed up-regulation in all 3 conditions. NRG3 was upregulated after 48h treatment with both CPT-11 and 16 5-FU. The results are summarised in Table 1. 17 18 The genes were selected for further study as a 19 potential target for antagonists. To validate the expression data observed in the microarray semi 21 quantitive RT-PCR was carried out for the gene. RT- 22 PCR was carried out on RNA extracted from 23 colorectal cell lines (including HCT116, RKO, HT29 & 24 H630) following exposure to a relevant range of chemotherapeutic treatments 26 27 Results for the selected target using Q-PCR 28 validated the results observed in the microarray 29 analysis. AREG upregulation was validated in RKO cell lines 48h after treatment with CPT-11 and 5-FU 71 1 and in HCT116 cells 48h after treatment with 5-FU 2 (FIG 1) . 3 4 To make a suitable target for an AREG inhibitor preferential upregulation should be observed in 6 tumour tissue when compared to other vital organs. 7 For this experiment the inventors have used mouse 8 homologues of the targets and examined regulation in 9 mice organs, for the gene. None of the targets analysed displayed upregulation in the mouse organs 11 examined which suggests the chemotherapeutic 12 treatment has a more acute affect on expression in 13 cancer cells than stable tissue. 14 To show that target upregulation observed at the 16 mRNA level was mirrored at the protein level western 17 blot analysis was performed. AREG protein expression 18 in RKO and HCT116 p53 wild type colorectal cancer 19 cell lysates was analysed following a 48 hour CPTll or 5FU treatment. Western blots were probed using an 21 anti-AREG antibody. Enhanced AREG expression was 22 observed, following CPTll treatment with both the 23 HCT116 and RKO cell lines (FIG 2) . 24 Confocal microscopy was used to analyse the in vitro effects of CPT-11 treatment (24h and 48h) of HCT116 26 cells on AREG expression levels. The inventors 27 observed increasing levels of expression at each 28 time point when compared to untreated controls (FIG 29 3) . 72 1 Figure 4 illustrates analysis of AREG protein 2 expression in H630 p53 mutant colorectal cancer cell 3 lysates following a 48 hour CPTll treatment. Western 4. blots were probed using an anti-AREG antibody.

Enhanced AREG expression was observed following 6 chemotherapy (A and B illustrate two separate 7 experiments)when compared to controls. This data 8 demonstrates that CPT-11 (or indeed analogues or 9 metabolites thereof) in combination with an ErbB cognate ligand (as shown to be upregulated) can be 11 used for the treatment of p53 mutant cancers. 12 13 Figure 5 illustrates analysis of AREG and beta actin 14 RNA expression in H460 lung cancer cells with / without chemotherapy (either a 4 8 hour CPTll 16 treatment or 5-Fu treatment). RNA levels were 17 analyzed following 35 cycle of PCR to determine 18 relative differences in expression between treated 19 and untreated samples. This data shows an enhanced expression of AREG following both CPT-11 and 5-Fu 21 challenge. 22 23 Table 1 Result of microarray analysis performed on 24 5FU & CPT-11 treated HCT116+/+ cells (In Vivo) for the different members of the EGF-family protein 73 Gene 24h 5-FU 48h 5-FU 48h CPT-11 EGF -2 . 4 -3.8 -2.5 (Epidermal Growth factor) TGF alpha 1.3 2.2 -2.1 (Transforming 1.8 1.0 -1.2 Growth factor alpha) BTC 1.1 1.9 2.4 (Betacellulin) HB-EGF 1.6 1.6 1.0 (Heparin- 3.1 -1.1 -1.3 Binding EGF- like growth factor) EREG -1.5 ' 1.9 1.5 (Epiregulin) 1.0 2.1 1.5 NRG1 N 0 T NRG 2 -2 .1 -1.5 -1.5 -2 . 5 -4 . 7 1.5 NRG 3 -2.1 1.6 1.2 NRG 4 -1.1 -1.8 1.2 74 1 This chemotherapy induced up-regulation of AREG has 2 been observed at both the mRNA and protein level 3 using p53 wt and mutant colorectal cancer cell lines 4 (HCT116+/+; RK0+/+ and H630). AREG has also been shown to be up-regulated at the mRNA level in H460 6 lung cancer cells and MDA breast cancer cells which 7 indicates that this effect may be observed over a 8 range of AREG expressing cancers. 9 As seen from the molecular analysis, these proteins 11 are selectively expressed after chemotherapy 12 treatment in different carcinoma cell lines. This 13 indicates that cancer cells, as a response to a 14 chemotherapy challenge, over-express six different growth factors of the same family. This response 16 seems to be a way used by the cancer cells to 17 overcome chemotherapy insult. By selectively 18 targeting these proteins, their role in cancer cell 19 survival may be at least reduced and at best inhibited, which may lead to a reduction in tumour 21 growth. Furthermore, the simultaneous targeting of 22 two or more over expressed ligands (by an antagonist 23 molecule like an antibody) may provide a useful 24 therapeutic strategy. 2 6 Example 2 27 Chemotherapy induced AREG up-regulation in 2 8 colorectal and breast cancer cell lines. 29 AREG up-regulation was further validated in several 31 carcinoma cell lines. In human HT29 colorectal 75 1 cancer cells and human HCT116 colorectal cancer 2 cells AREG mRNA up-regulation was observed after 3 treatment with IC50 dose of CPT 11 (Figure 6A and 4 6B). Moreover, after treatment with IC50 dose of 5- FU in human MDA-MB231 breast carcinoma cell line up- 6 regulation of AREG mRNA was shown (Figure 6C) . 7 8 Silencing of AREG and HB-EGF in cancer cells 9 siRNA potently down-regulated expression of AREG 11 (Figure 7A) and HB-EGF (Figure 7B) in HCT116 12 . colorectal cell line in comparison to untreated 13 cells, mock transfection and control siRNA. In 14 Figure 9 and Figure 11 respectively, AREG knockdown is also shown in HT29 colorectal cancer cells and in 16 MDA-MB2 31. 17 18 Synergistic attenuation of cell growth after 19 treatment with siRNA and chemotherapy in colorectal cancer 21 22 Following confirmation of AREG and HB-EGF silencing 23 by siRNA, MTT assays were performed to investigate 24 the effect of down-regulation of these two genes on cell growth. 26 27 AREG siRNA alone, HB-EGF siRNA alone and monotherapy 28 of CPT-11 had no significant effect on cell 29 viability compared to untreated cells, mock 76 1 transfection and control siRNA. However, co- 2 treatment of HCT116 with AREG siRNA and CPT-11 3 resulted in synergistic decreases in cell viability. 4 The same effect was observed when AREG siRNA was replaced with HB-EGF siRNA (Figure 8). 6 In another colorectal cancer cell line, HT29 similar 7 results were obtained as those observed with HCT116. 8 AREG siRNA alone and control siRNA alone had no 9 significant effect on the growth of the cells. The combination of AREG siRNA and CPT-11 had a 11 synergistic effect on cell viability resulting in 12 the decrease of cell growth (Figure 10) . 13 Collectively these results indicate that down- 14 regulation of AREG/HB-EGF expression in combination with chemotherapy had a significant effect on the 16 attenuation of cell growth in colorectal cancer. 17 18 Synergy between silencing of AREG and treatment with 19 chemotherapy led to an attenuation of cell growth in breast cancer 21 22 Following transfection with control siRNA alone, 23 transfection reagent alone (mock) and chemotherapy 24 treatment, a 20% reduction in cell growth was observed. A further 23% decrease was observed when 26 cells were transfected with AREG siRNA alone. 27 Treatment with varying doses of 5-FU (2.5 - 6pM) 28 showed similar results to that of AREG siRNA alone. 29 However, treatment with 7.5jiM 5-FU in combination with AREG siRNA lead to a further 20% reduction in 77 1 growth (60% overall reduction in growth in 2 comparison to the untreated, Figure 12). 3 4 When siRNA experiments and MTT assays were performed using a combination of AREG and HB-EGF the inventors 6 surprisingly observed a marked reduction in cell 7 growth. Experiments were performed in the MDA-MB2 31 8 breast cancer cell line, to assess if knocking down 9 AREG and HB-EGF had any affect on cell viability in breast cancer cells. 11 12 Remarkably, co-silencing AREG and HB-EGF resulted in 13 a significant decrease on cell viability compared to 14 controls. A decline in cell growth of ~75% was observed when MDA-MB231 cells were co-treated with 16 target siRNA compared to controls figure 13. 17 18 Development of AREG specific monoclonal antibodies. 19 A panel of murine monoclonal antibodies were raised 21 against recombinant human amphiregulin (Figure 14- 22 16). They were characterised by ELISA, western 23 blotting (whole cell lysates from colorectal cell 24 lines HCT116 and HT29) and confocal microscopy analysis for demonstration of specific recognition 26 of AREG (Figure 17-22). 27 28 Up-regulation of AREG as shown by FACS analysis 2 9 using AREG monoclonal antibodies on colorectal 30 cancer and lung carcinoma cell lines. 78 1 Flow cytometry analysis of HCT116 cells and H460 2 cells shows cell surface recognition of AREG when 3 assessed using the Anti AREG monoclonal antibodies 4 (Figure 23 shows FACS analysis of HCT116 cells treated with AREG clones 4G5 and 6E11 and an isotype 6 control). Furthermore, cells treated with 2.5pM 7 irinotecan for 48 hours prior to analysis showed up 8 to a 40% increase in the cell surface expression of 9 AREG. The Anti-AREG clone 6E11 1E9 detected a 20% increase in AREG expression in HCT116 cells after 11 treatment with irinotecan (Figure 24). 12 FACS analysis has also been carried out on the lung 13 carcinoma cell line H460. Two clones namely 3H5 and 14 3F8 both detected AREG expression on the surface and up-regulation in expression levels after treatment 16 with 2.5yM irinotecan (Figure 25). 17 These results demonstrate that the inventors' panel 18 of AREG monoclonal antibodies recognise the AREG 19 protein on the surface of the HCT116 cells. 21 Attenuation of cell growth in cancer cells after 22 treatment with AREG monoclonal antibodies. 23 MTT assays were performed on cancer cell lines, MDA- 24 MB231 breast cancer cells (Figure 26 & Figure 27) and HCT116 colorectal cancer cells (Figure 28) to 26 ascertain the effect of AREG monoclonal antibodies 27 on cell growth. The activities of different clones 28 were screened on the MDA-MDB231 cell line with 29 several showing -40% reduction in cell growth when compared to untreated cells. In HCT116 cells a more 31 pronounced effect was observed with -60% reduction 32 in the cell viability observed with clones 4G5 and 79 1 6E11. Figure 29 shows similar effects on cell 2 growth in the MDA-MB231 breast cancer cells with 3 AREG clone 6E11 1E9 2D8 ( which has been shown to be 4 the same clone as 6E11 1E9 1C6) . Figure 30 and 31 shows the effect of AREG antibodies in the HCT116 6 cell line. A 65% decrease in cell viability is 7 observed in Figure 30 and Figure 31 shows a 50% 8 decrease in cell growth. Figure 32 shows the effect 9 of the AREG antibody 6E11 1E9 1C6 in the lung cancer H460 cell line, comparing the effect against a 11 isotypic control antibody and showing that the 12 antibody significantly attenuates cell viability of 13 the lung cancer cells. Together these results show 14 that the AREG monoclonal antibodies have a significant effect on the cell viability of AREG 16 expressing cancer cells including colorectal, lung 17 and breast carcinoma. 18 19 Synergistic attenuation of growth on colorectal 2 0 cancer cells after treatment with HBEGF siRNA and 21 AREG antibodies. 22 The effect of down-regulation of the HBEGF siRNA in 23 combination with an AREG antibody was investigated 24 by performing an MTT cell viability assay (Figure 33). The HBEGF siRNA alone had a slight reduction on 26 cell growth while the AREG antibody 6E11 1E9 2D8 had 27 -50% reduction in cell growth. When the HBEGF siRNA 28 and 6E11 1E9 2D8 was added in combination a further 29 reduction in cell viability was observed. To see if this effect was synergistic the RI values as 31 described by Kern 1988 and modified by Romanelli 32 1998 were calculated. The RI value is calculated as 80 1 the ratio of expected survival (Sexp defined as the 2 product of the survival observed with drug A alone 3 and the survival observed with drug B alone) to the 4 observed cell survival (S0bs) for the combination of A and B. (RI=Sexp/S0bs) • Synergism is defined as RI>1. 6 The RI value for 6E11 1E9 2D8 was approximately 2. 7 However, the RI value for 6E11 1E9 1C6 was 8 calculated as being above 5. Collectively these 9 results show that the combined targeting of both HBEGF and AREG in the treatment of cancers 11 associated with Erb ligands or EGF (including 12 colorectal, breast and lung) results in a 13 synergistic attenuation of cell growth. 14 The development of non-responsive tumours or 16 chemotherapy resistant cancer remains a major 17 obstacle to successful treatment. There is a clear 18 need for tools which enable prediction of whether a 19 particular therapy either single or combination will be effective against particular tumours. Moreover, 21 there remains the need for new treatment regimes to 22 increase the repertoire of treatments available. 23 Combined therapies have shown promising results by 24 improving the response rates in patients by acting on the tumours through different mechanisms, the 26 inventors' data suggests inhibitory molecules, for 27 example antagonist antibodies, specific to AREG and 28 HB-EGF used alone or in combination with 29 chemotherapy could potentially be used to treat a wide variety of aggressive cancers including 31 colorectal, lung and breast cancer. 81 1 2 All documents referred to in this specification are 3 herein incorporated by reference. Various 4 modifications and variations to the described embodiments of the inventions will be apparent to 6 those skilled in the art without departing from the 7 scope and spirit of the invention. Although the 8 invention has been described in connection with 9 specific preferred embodiments, it should be understood that the invention as claimed should not 11 be unduly limited to such specific embodiments. 12 Indeed, various modifications of the described modes 13 of carrying out the invention which are obvious to 14 those skilled in the art are intended to be covered by the present invention. 16 17 18

Claims (19)

2 3 4 5 6 ? 8 9 10 11 12 13 14 15 16 1? 18 19 20 21 22 23 24 25 26 27 28 29 30 31 Received at IPONZ on 29.02.2012 82 C 3

1. Use of an inhibitor of HB-EGF' and an. inhibitor of AREG in the preparation of a medicament formi d for siinultaiieousor sequential use in the combination treatment of neoplastic disease; wh i, in said combination treatment, said inhibitor of HBEGF and said inhibitor of AREG act syn wherein said in! IGF is an antibody molecule which binds said HB-EGF or a nucleic acid molecule w hibits expression of HB-EGF; and wherein said inhibitor of AREG is an antibody molecule which binds AREG or a nucleic acid molecule which inhibits expression of AREG,

2. Use of an inhibitor of HB-EGF and an inh r of AREG in the preparation of a medicament for the combination treatment of neoplastic disease, wherein said medicament is formu ministration according to a dosage regime comprising administering said inhibitor of HB-EGF to a patient, where said inhibitor of .AREG was administered within the previous 72 hours, or said inhibitor of A,REG is administered simultaneously or within the subsequent 72 hours; 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 13 20 21 22 23 24 25 26 27 28 29 30 31 Received at IPONZ on 29.02.2012 83 wherein, with said dosage regime, said inhibitor of HB-EGF and said irxl act synergistically; wherein said inhibitor of HB-EGF is an antibody molecule which binds said HB-EGF or a nucleic acid molecule which inhibits expression of HB-EGF; and wherein said inhibitor of AREG is an antibody molecule which binds AREG or a n* c acid molecule which inhibits expression 3.

3, The use according to claim 1 or claim. 2, wherein said inhibitor of HB-EGF is a first siRNA and /or said inhibitor of AREG is a second siRNA.

4, The use according to claim 1 or claim. 2, wherein the inhi G is an anti AREG antibody molecule,

5. The use according to claim. 4, wherein the inhibitor of AREG is an antibody molecule which comprises a variable region having the amino acid sequence of Sequence ID No: 27 and a variable region, having the amino acid, sequence of S< ce ID No: 28.

6. The use according to any one of claims 1 to 5, wherein the medicament is for simultaneous or sequential use in combination treatment with a 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Received at IPONZ on 29.02.2012 84 chemotherapeutic agent. 7, The use according to claim 6, wherein the chemotherapeutic agent is selected from the group consisting of antinw , topoisomerase inhibitors, alkylating agents, anthracyclines, and plant alka.; 8, The use according to claim 7, wherein the-; chemotherapeutic agent is CPT-11, 9, The use according to claim 8, wherein the chemotherap : agent is 5-FU. 10, T! ording to any one of the claims 1 to 9, wherein said neopt^r. t ise is selected frona the group cc of colorectal cancer, breast cancer and lung cancer. 11, The use according to any one of claims 1 to 10, wherein, the net dsease is a. cancer comp Lg a p53 mutation. 12, A pharmaceutical composition comprising (i) an inhibitor of HB-EGF and (ii) an inhibitor of AREG,- wherein said inhibitor of AREG is an dy molecule which binds said AREG or a nucleic acid r ale which inhibits expression of said AREG; and wherein said inh >r of HB-EGF is an -body molecule which binds said HB-EGF or 1 2 3 4 5 6

7

8

9

10

11

12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Received at IPONZ on 29.02.2012 85 a nucleic acid molecule which inhibits expression of HB-EGF.

13. The composition according to claim 12, wherein the inhi 6 is an anti AREG antibody molecule.

14. The composition according to claim 12, whei:ein the inhibitor of AREG is an antibody molecule which comprises a variable region having the ami ce of Sequence ID No: 27 and a variable region having the amino acid sequence of Sequence ID No; 28.

15. The composition according to claim 12, wherein s-ho bnibitor of HB-EGF is a first siRNA and/or said in! of AREG is a second siRNA..

16. The use according to claim 1, or the composition according to claim 12, wherein the inhibitor an antibody molecule comprising at least one of the CDRs of the VH region having the amino acid sequence shown as Sequence ID No: 27 and/or at least one of the CDRs of the VL region having the amino acid sequence shown, as Sequence ID No: 28, wherein the antibody molecule has binding sp G.

17. The use or composition according to claim, 18, wherein the antibody r ale comprises 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 86 all three of the CDRS of the VH region having the amino acid sequence shown as Sequence ID-No: 27 and/or all three of the CDRS of the VL region having the amino acid sequence shown as Sequence ID No: 28.

18. The use or composition according to claim 17, wherein the antibody molecule comprises a variable region having the amino acid sequence of Sequence ID No: 27.

19. The use or composition according to claim 17 or claim 18, wherein the antibody molecule comprises a variable region having the amino acid sequence of Sequence ID No: 28. Fusion Antibodies Limited eys for the Applicant Aliens Arthur Robinson Patent & Trade Marks Attorneys