Co-use of a clusterin inhibitor with an EGFR inhibitor to treat cancer

This patent application is a national stage filing under 35 U.S.C. §371 of international application No. PCT/CA2013/000167 filed on Feb. 22, 2013, which claimed priority to U.S. provisional application No. 61/601,786 filed Feb. 22, 2012. The entire contents of each of these priority applications are incorporated herein by reference.

In accordance with 37 C.F.R. §1,52(e)(5), a Sequence Listing in the form of a text file (entitled “Sequence Listing”, created on Jul. 28, 2014 and of 78 kilobytes) is incorporated herein by reference in its entirety.

The invention relates to combination of a clusterin inhibitor with an inhibitor of epidermal growth factor receptor (EGFR) for use in treatment of cancer. More particularly, the present invention encompasses the use of a clusterin inhibitor, such as an anti-clusterin antibody, to restore the sensitivity of cancer cells to an EGFR inhibitor or to potentiate the effect of the EGFR inhibitor.

Development of resistance to chemotherapy and invasion to other secondary sites are common features of solid tumor malignancies. It is well known that development of resistance to chemotherapeutic agents is caused by over-expression of proteins involved in drug and apoptosis resistance. The invasion process is also fairly well documented. Tumor invasion is caused by an increased motility of cancer cells and the expression of genes that cause degradation of the proteins in the extra-cellular matrix. There is growing evidence that resistance to chemotherapy and tumor invasion might share a common starting point through a biological process called the epithelial-to-mesenchymal transition (EMT). Recent studies have demonstrated that transforming growth factor beta (TGFβ) may be a critical mediator of EMT. Despite these advances, few therapeutic avenues are available to inhibit the development of chemo-resistance and the spread of cancer to other organs. It has also emerged in the recent literature that certain tumor cells undergoing EMT dedifferentiate and adopt stem cell-like properties (cancer stem cells or CSCs). As is the case for normal stem cells, CSCs are inherently refractory to chemotherapy and radiation therapy. Therefore, targeting a specific regulator of EMT and CSC maintenance represents a very promising therapeutic strategy to increase response to chemotherapeutic agents and to prevent recurrence of cancer.

Utilizing well characterized cell lines as models of EMT, proteins were identified that were up-regulated upon induction of EMT. One of these, a secreted protein termed clusterin (sCLU), was found to be stimulated during EMT and could, on its own, promote the EMT process (Lenferink et al., 2009). Several high-affinity antibodies were generated that interact with sCLU, which when tested in cell-based assays for their ability to block the EMT, those antibodies that neutralized EMT all bound to the same critical amino acid sequence in the sCLU protein. This discovery demonstrated that a specific region of sCLU was responsible for mediating its EMT-promoting activity. By blocking the EMT-epitope in sCLU, the antibodies, in particular an antibody designated 16B5 could block EMT as exemplified by the maintenance of the membrane expression of the epithelial cell marker, E-cadherin, when incubated with cancer cells. Furthermore, human xenograft animal studies using prostate cancer and pancreatic cancer tumors showed that blocking the activity of tumor-associated sCLU resulted in the increased response to standard chemotherapeutic drugs such as docetaxel and gemcitabine, as measured by a significant reduction in tumor growth. Taken together, these results demonstrated that blocking EMT with an antibody capable of interacting with a specific region in sCLU resulted in tumor growth inhibition and increased response to cytotoxic drugs (see international application No. PCT/CA2006/001505 published under No. WO2007/030930 and international application No. PCT/CA2010/0001882 published under No. WO2011/063523, the entire content of which is incorporated herein by reference

Lung cancer is one of the most common cancers and a leading cause of death worldwide, with over a million cases diagnosed yearly and non-small cell lung cancer (NSCLC) accounts for more than 80% of all lung cancers. Despite recent improvements in diagnostic and therapeutic approaches, the majority of patients are diagnosed with advanced NSCLC where the median survival remains poor (Adamo et al., 2009).

One of the most important targets in NSCLC is the epidermal growth factor receptor (EGFR), a member of the ErbB family of receptor tyrosine kinases, that is a cell membrane receptor that plays an important role in proliferation and survival of cancer cells. It is a large transmembrane glycoprotein that serves as a receptor for EGF and several additional endogenous ligands. It has three domains consisting of an extracellular region, a transmembrane domain and an intracellular tyrosine kinase (TK) domain. Functionally, ligand binding to EGFR induces receptor dimerization leading to a structural change that promotes autophosphorylation and activation of the intracellular TK domain. Consequently, EGFR activation influences multiple downstream signaling pathways, including Ras/Raf/mitogen-activated protein kinase (MAPK) and the phosphatidylinositol-3′-kinase (PI3K)/Akt pathway, which influence cell proliferation, invasiveness, motility, survival and apoptosis (Shigematsu et al., 2005).

Although EGFR is ubiquitously expressed, it is often modified in tumors cells. These modifications include gene amplification, overexpression of ligands and/or receptors and activating mutations. Overexpression or dysregulation of EGFR or its primary ligands is characteristic of many solid human tumors, including lung cancer. In NSCLC, between 43 and 83% of tumors overexpress EGFR (Adamo et al., 2009). Several agents against EGFR such as monoclonal antibodies that target the extracellular domain or small molecules are able to inhibit the TK activity.

The status of EGFR in metastatic NSCLC and the response to chemotherapy is the subject of much debate. Despite the high proportion of tumors with increased expression of EGFR, some clinical studies have shown that this was a poor predictor of response in first-line therapy (Barr et al., 2008). Furthermore, despite a mild but significant response in patients treated first-line with cisplatin, vinorelbine and cetuximab (EGFR monoclonal antibody) compared to chemotherapy alone, there was no correlation between cetuximab and EGFR over-expression (Mirshahidi and Hsueh, 2010). Overall, an overwhelming amount of clinical results with EGRF inhibitors in NSCLC showed that the status of the receptor was not important in first-line therapy until a recent study reported results showing that patients with high EGFR expression that were treated with cetuximab and chemotherapy exhibited an increase in overall survival compared to chemotherapy alone (Pirker et al., 2012). It is clear from these results that there are other mechanisms involving EGFR in tumors that influence the response of NSCLC patients to EGFR inhibitors.

Additional characteristics of EGFR that likely influence the response of inhibitors are those that permit the binding of small molecules to the TK activity of the receptor. A few have been developed and approved for cancer indications including gefitinib and erlotinib, two small molecules that mimic ATP-binding to this region thus preventing intracellular signaling. Neither of these inhibitors was found to be active in NSCLC in first-line therapy but significant clinical responses were achieved in second- and third line settings (Mirshahidi and Hsueh, 2010). Interestingly, EGFR overexpression had no influence on patient response but it was discovered that activating mutations in EGFR and certain other genes were critical. For example, activating mutations were found to lead to significant increase in progression free survival in patients treated with gefitinib (Costanzo et al., 2011). In addition, NSCLC patients treated with erlotinib who also had mutations in a gene called KRAS, showed no response (Herbst and Sandler, 2008). Thus, patient selection is critical for attempting to understand if they will be responders to EGFR TK inhibitors.

As described above, EMT can have a tremendous influence on the way tumors cells will respond to therapy and the ability of cancer cells to remain epithelial is critical for this response. In cell-based studies, cells that have increased expression of the epithelial cell marker, E-cadherin, are more sensitive to EGFR inhibitors (Barr et al., 2008). In agreement with these observations, there was a correlation between E-cadherin expression and sensitivity to erlotinib (Yauch et al., 2005). At the tumor level, it has been shown that restoration of E-cadherin expression increases sensitivity to EGFR inhibitors (Witta et al., 2006). To date, however, the link between EMT and EGFR status in clinical trials has not been clearly examined. However, given the lack of correlation between EGFR overexpression and response to EGFR inhibitors and the influence of activating EGFR mutations on their response, it is probable that additional influences such as EMT might be directly involved in increasing the efficacy of EGFR inhibitors in NSCLC patients.

This present application provides a method of treatment with an antibody that blocks EMT by inhibiting sCLU in tumors that express EGFR. The EGFR status in these tumors might include EGFR gene amplifications or amplification in EGFR ligands. Furthermore, the tumors cells might include increased autocrine signaling through EGFR and EGFR protein partners. The EGFR status might also include activating mutations in EGFR that cause the receptor to exhibit increased activity. In another embodiment, the tumors might be sensitive or resistant to EGFR inhibitors, including monoclonal antibodies against EGFR or small molecule inhibitors that abrogate the activity of EGFR. Furthermore, EGFR status might also include tumors that were a priori negative for EGFR expression that have reacquired EGFR.

When NSCLC cell lines that express sCLU are exposed to an anti-clusterin antibody, both the expression and the activation of EGFR are increased. In parallel, by virtue of EMT inhibition by the anti-clusterin antibody, the NSCLC cell lines also show increased E-cadherin expression. Taken together, NSCLC patients treated with a clusterin inhibitor in combination with EGFR inhibitors may show an increased response to the EGFR inhibitors.

The present invention generally relates to a combination of a clusterin inhibitor and EGFR inhibitors for cancer treatment.

Methods of the present invention encompass administration of a clusterin inhibitor capable of inhibiting epithelial-to-mesenchymal transition (EMT) and an EGFR inhibitor to an individual in need. An EGFR inhibitor may also be administered separately, concurrently or sequentially with the clusterin inhibitor.

The clusterin inhibitor may be administered especially when EGFR resistance is observed, detected or suspected.

Methods of the present invention also comprise administering a clusterin inhibitor to prevent EGFR resistance or to sensitize cancer cells to an EGFR inhibitor.

FIG. 1 . Lung cancer cell lines express clusterin. This figure shows RT-PCR analysis of clusterin mRNA expression in several cell lines derived from NSCLC (upper panel). As a control, the mRNA from actin was also amplified (lower panel) to ensure equal amounts a starting material in each reaction.

FIG. 2 . Lung cancer cell lines express clusterin (sCLU). This figure shows the quantification of sCLU that is secreted by lung cancer cell lines. The quantities were measured with a sCLU-specific ELISA and are expressed in ng/ml.

FIG. 3 . h16B5 increases E-cadherin expression in lung cancer cell lines. This figure demonstrates the increase in the epithelial character of A549 NSCLC cells when incubated with the sCLU inhibitor, h16B5. The left panel shows the cells incubated with a control IgG for 48 h whereas the right panel shows cells incubated with h16B5 under identical conditions.

FIG. 4 . Treatment of NSCLC cells with h16B5 results in increased EGFR expression. EGFR expression in the presence of control vehicle or h16B5 was measured using an EGFR-specific antibody in whole cell lysates prepared from either A549 or H226 cells.

FIG. 5 . Treatment of NSCLC cells with h16B5 results in increased EGFR phosphorylation. EGFR phosphorylation in the presence of control vehicle or h16B5 was measured using an phosphotyrosine-specific antibody in whole cell lysates prepared from either A549 or H226 cells.

FIG. 6 . Treatment of NSCLC cells with h16B5 results in increased EGFR phosphorylation. EGFR phosphorylation in the presence of control vehicle, h16B5 or a control IgG was measured using an phosphotyrosine-specific antibody in whole cell lysates prepared from either A549 or H226 cells.

FIGS. 7A and B. Combination of h16B5 with erlotinib result in an enhanced anti-tumor effect. Survival of H1299 and H460 cancer cells was determined in the presence of a combination of h16B5 with erlotinib in the absence (A) or presence of recombinant clusterin (B).

Epithelial growth factor receptor (EGFR) is overexpressed in several types of epithelial carcinomas. The existence of EGFR genetic alterations may render tumors more susceptible to EGFR inhibitors. However, patients with initial response to EGFR tyrosine kinase inibitors (TKIs) relapse within an average of one year. Although it has been shown that deletions in EGFR exon 9 or L858R mutations results in favorable clinical outcome, secondary molecular event such as mutations T790M, L747S and/or D761Y are associated with resistance and tumor relapse. Amplification of the MET receptor and activation of IGFR signaling that both activate the PI3/AKT pathway independently of EGFR have also been shown to drive secondary resistance.

The Applicant has come to the unexpected discovery that EGFR expression and phosphorylation is increased in cancer cells treated with a clusterin inhibitor. Treatment of cancer cells with the clusterin inhibitor is also accompanied with reappearance of an epithelial phenotype of the cancer cell, as illustrated by an increased E-cadherin expression at the cell surface.

Thus, not only may clusterin inhibitors induce reversal of the EMT phenotype, they may restore sensitivity of cancer cells to EGFR inhibitors.

The present invention generally relates to a combination of a clusterin inhibitor and EGFR inhibitors for use in cancer treatment.

The pharmaceutical combination may particularly comprise a clusterin inhibitor in association with a pharmaceutically acceptable carrier and an EGFR inhibitor in association with a pharmaceutically acceptable carrier.

Methods of the present invention encompass administration of a clusterin inhibitor and an EGFR inhibitor to an individual in need. The EGFR inhibitor may be administered separately, concurrently or sequentially with the clusterin inhibitor.

The clusterin inhibitor may be administered especially when EGFR resistance is observed, detected or suspected.

Methods of the present invention also comprise administering a clusterin inhibitor to prevent EGFR resistance or to sensitize cancer cells to an EGFR inhibitor.

In one aspect of the invention, the clusterin inhibitor may be administered prior to the EGFR inhibitor. For example, the clusterin inhibitor may be administered from a few hours to several days or months prior to administration of the EGFR inhibitor. In another aspect of the invention, the clusterin inhibitor may also be administered at the same time (e.g., same day) or between each treatment with the EGFR inhibitor.

In accordance with the present invention, the clusterin inhibitor may be administered in multiple doses prior to administration of the EGFR inhibitor, e.g., daily, every other day, once a week, twice a week, etc.

The method may also comprise testing for reversal of the epithelial-to-mesenchymal phenotype of the cancer cells before administration of the EGFR inhibitor. The EMT status may be determined, for example, by measuring expression levels of one or more genes/protein selected from E-cadherin, RAB25, integrin beta 6, vimentin, ZEBI and SIPI.

Clusterin inhibitors may be identified by their ability to impair clusterin expression, secretion or clusterin activity.

Exemplary embodiments of clusterin inhibitors include those identified, for example, by their ability to interfere with the EMT-promoting effect of secreted clusterin (sCLU) or of TGF-β. For example, carcinoma cells (e.g., 4T1: breast carcinoma cells, DU145: prostate cancer cells, etc.) may be treated with a putative clusterin inhibitor in the presence of TGF-β or sCLU and markers of EMT may be assessed as described below. A putative compound, which is capable of increasing the expression of epithelial markers and/or reducing the expression of mesenchymal markers, may be identified as a suitable clusterin inhibitor.

Alternatively, the motility of carcinoma cells in the presence of the putative clusterin inhibitor may be assessed. For example, carcinoma cells may be treated with a putative clusterin inhibitor in the presence of TGF-β or sCLU and a wound healing assay or a black ink motility assay may be carried out as described for example in PCT/CA2006/001505. A putative compound, which is capable of inhibiting or reducing the motility of carcinoma cells in these types of assays, may be identified as a suitable clusterin inhibitor. It is to be understood that other techniques may be used to identified suitable clusterin inhibitors.

Clusterin inhibitors particularly encompassed by the present invention include for example, anti-clusterin antibodies or antigen binding fragments thereof.

In accordance with the present invention, clusterin inhibitors include anti-clusterin antibodies or antigen binding fragment capable of inhibiting EMT (e.g., in carcinoma cells).

Commonly used molecular markers of EMT include, for example, a reduced expression of E-cadherin, cytokeratin and β-catenin (in the membrane) and/or an increased expression of Snail, Slug, Twist, ZEB1, ZEB2, N-cadherin, vimentin, a-smooth muscle actin, matrix metalloproteinases etc. (see for example, Kalluri and Weinberg, The Journal of Clinical Investigation, 119(6), p 1420-1428; 2009; Fassina et al., Modern Pathology, 25; p 86-99; 2012; Lee et al., JCB; 172; p 973-981; 2006). An EMT phenotype may also be distinguished by an increased capacity for migration, invasion of by resistance to anoikis/apoptosis. Cells that are undergoing epithelial-to-mesenchymal transition may thus be detected by a reduction of epithelial markers and apparition of mesenchymal markers or EMT phenotypes.

Expression of markers may generally be determined by comparing their level of cellular expression (at the genetic level or at the protein level (e.g., including cell surface expression) in one state in comparison with another state. For example, the level of expression of one or more specific markers may be determined in cancerous cells in comparison with non-cancerous cells. Alternatively, the level of expression of one or more specific markers may be determined in cancerous cells that are resistant to a EGFR inhibitor in comparison with cancerous cells that are sensitive to the EGFR inhibitor. Furthermore, level of expression of one or more specific markers may be evaluated over values that are statistically representative of controls.

Individuals who would benefit from such treatment include those having carcinoma (i.e., epithelial carcinoma) including, prostate cancer, breast cancer (e.g., triple negative or basal-like), endometrial carcinoma, ovarian carcinoma, hepatocellular carcinoma, colorectal carcinoma, head and neck carcinoma (e.g., head and neck squamous cell carcinoma), lung carcinoma (e.g., non-small cell lung cancer), pancreatic cancer, renal cell carcinoma, etc. (including advanced or metastatic forms of these cancers).

Exemplary embodiments of anti-clusterin antibodies that may be used to carry the present invention include those that are capable of binding to amino acids 421 and 443 of a C-terminal portion of a β-subunit of human clusterin. More particular embodiments of antibodies or antigen binding fragment encompassed by the present invention include those described in international application No. PCT/CA2006/001505 published under No. WO2007/030930 and international application No. PCT/CA2010/0001882 published under No. WO2011/063523.

The present invention especially encompasses antibodies and antigen binding fragment having at least one complementary determining region (CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and/or CDRL3) identical to those of the antibodies identified as 16B5, 21B12, 20E11, 16C11 and 11E2. More particularly, the present invention especially encompasses antibodies and antigen binding fragment having a light chain and/or heavy chain identical to those of identified as 16B5, 21B12, 20E11, 16C11 and 11E2 (see also PCT/CA2006/001505) or to those identified as humanized 16B5 (h16B5), humanized 21B12 (h21B12), h16B5 VL consensus 1, h16B5 VL consensus 2, h16B5 VL consensus 3, h16B5 VH consensus 1, h16B5 VH consensus 2, h16B5 VH consensus 3, h21B12 VL consensus 1, h21B12 VL consensus 2, h21B12 VL consensus 3, h21B12 VH consensus 1, h21B12 VH consensus 2 or h21B12 VH consensus 3 (see also PCT/CA2010/0001882).

The amino acid sequence of the light chain and/or heavy chain variable regions of the antibody identified as 20E11, 16C11 and 11E2 are presented in SEQ ID NOs.:62-67, where the predicted complementarity determining regions are shown in bold.

Other exemplary embodiments of antibodies and antigen binding fragments include those that can compete with the antibodies identified herein.

The invention encompasses monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies and human antibodies (isolated) as well as antigen binding fragments having the characteristics described herein. Antibodies or antigen binding fragments encompassing permutations of the light and/or heavy chains between a monoclonal, chimeric, humanized or human antibody are also encompassed herewith.

The antibodies or antigen binding fragments of the present invention may thus comprise amino acids of a human constant region and/or framework amino acids of a human antibody.

The term “antibody” refers to intact antibody, monoclonal or polyclonal antibodies. The term “antibody” also encompasses multispecific antibodies such as bispecific antibodies.

Human antibodies are usually made of two light chains and two heavy chains each comprising variable regions and constant regions. The light chain variable region comprises 3 CDRs, identified herein as CDRL1, CDRL2 and CDRL3 flanked by framework regions. The heavy chain variable region comprises 3 CDRs, identified herein as CDRH1, CDRH2 and CDRH3 flanked by framework regions.

The term “antigen-binding fragment”, as used herein, refers to one or more fragments of an antibody that retain the ability to bind to an antigen (e.g., KAAG1, secreted form of KAAG1 or variants thereof). It has been shown that the antigen-binding function of an antibody can be performed by fragments of an intact antibody. Examples of binding fragments encompassed within the term “antigen-binding fragment” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the V L , V H , C L and C H1 domains; (ii) a F(ab′) 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V H and C H , domains; (iv) a Fv fragment consisting of the V L and V H domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a V H domain; and (vi) an isolated complementarity determining region (CDR), e.g., V H CDR3. Furthermore, although the two domains of the Fv fragment, V L and V H , are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single polypeptide chain in which the V L and V H regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding fragment” of an antibody. Furthermore, the antigen-binding fragments include binding-domain immunoglobulin fusion proteins comprising (i) a binding domain polypeptide (such as a heavy chain variable region, a light chain variable region, or a heavy chain variable region fused to a light chain variable region via a linker peptide) that is fused to an immunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge region, and (iii) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region. The hinge region may be modified by replacing one or more cysteine residues with serine residues so as to prevent dimerization. Such binding-domain immunoglobulin fusion proteins are further disclosed in US 2003/0118592 and US 2003/0133939. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

A typical antigen binding site is comprised of the variable regions formed by the pairing of a light chain immunoglobulin and a heavy chain immunoglobulin. The structure of the antibody variable regions is very consistent and exhibits very similar structures. These variable regions are typically comprised of relatively homologous framework regions (FR) interspaced with three hypervariable regions termed Complementarity Determining Regions (CDRs). The overall binding activity of the antigen binding fragment is often dictated by the sequence of the CDRs. The FRs often play a role in the proper positioning and alignment in three dimensions of the CDRs for optimal antigen binding.

Antibodies and/or antigen binding fragments of the present invention may originate, for example, from a mouse, a rat or any other mammal or from other sources such as through recombinant DNA technologies.

Other clusterin inhibitor may include for example and without limitation siRNAs (e.g., targeting clusterin RNA) and antisenses (e.g., targeting clusterin RNA).

Exemplary embodiments of antisense include for example, those described in U.S. Pat. No. 7,569,551 (the entire content of which is incorporated herein by reference) and especially include OGX-011 (a.k.a., custirsen sodium, OncoGenex, see also SEQ ID NO.:61).

Exemplary embodiment of EGFR inhibitors include tyrosine kinase inhibitors such as, for example, gefitinib, erlotinib, imatinib, lapatinib or semazinib. Other exemplary embodiments of EGFR inhibitors include for example, monoclonal antibodies such as cetuximab, panitumumab, nimotuzumab, or metuzumab.

Other individuals who would benefit from treatment with the pharmaceutical combinations of the present invention include those that have a tumor which is resistant to one or more EGFR inhibitors. Such individuals may be selected prior to administration of the pharmaceutical combination.

EGFR resistance may be determined by evaluating clinical parameters such as tumor relapse or by measuring molecular markers of resistance, e.g., mutations, amplifications in EGFR or in the EGFR pathway (RAS/MAPK, phospholipase C, phosphatidylinositol 3-kinase/AKT, SRC/FAK pathways, etc.) and/or EMT markers.

Testing for resistance to an EGFR inhibitor may thus includes determining the presence of mutation in EGFR (e.g., mutation in the tyrosine kinase domain, truncating mutations, insertions), determining EGFR amplification.

As used herein “pharmaceutically acceptable carrier” or “pharmaceutical carrier” are known in the art and include, but are not limited to, 0.01-0.1 M or 0.05 M phosphate buffer or 0.8% saline. Additionally, such pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's orfixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, collating agents, inert gases and the like.

The experiments described herein were carried out with a humanized form of 16B5 (h16B5).

SEQ ID NO.: 1

16B5 CDRH1: GFNIKDIYMH

SEQ ID NO.: 2

16B5 CDRH2: RIDPAYGNTKYDPKFQG

SEQ ID NO.: 3

16B5 CDRH3: RYDTAMDY

SEQ ID NO.: 4

16B5 CDRL1: KSSQSLLNSRTRKNYLA

SEQ ID NO.: 5

16B5 CDRL2: WASTRES

SEQ ID NO.: 6

16B5 CDRL3: KQSYNLWT

SEQ ID NO.: 7

h16B5 Humanized heavy chain variable region

SEQ ID NO.: 8

h16B5 Humanized light chain variable region

SEQ ID NO.: 9

Complete heavy chain immunoglobulin sequences for h16B5

TKYDPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCArRYDTAMDYwgqgtlvtvsSAS

SEQ ID NO.: 10

Complete light chain immunoglobulin sequences for h16B5

SEQ ID NO.: 11

21B12 CDRH1: GYTFTNYGMH

SEQ ID NO.: 12

21B12 CDRH2: WINTYTGEPTYADDFKG

SEQ ID NO.: 13

21B12 CDRH3: DGFLYFFDY

SEQ ID NO.: 14

21B12 CDRL1: KSSQSLLYSSNQKNYLA

SEQ ID NO.: 15

21B12 CDRL2: WASTRES

SEQ ID NO.: 16

21B12 CDRL3: QQYYIYPRT

SEQ ID NO.: 17

h21B12 Humanized heavy chain variable region

SEQ ID NO.: 18

h21B12 Humanized light chain variable region

SEQ ID NO.: 19

Complete heavy chain immunoglobulin sequences for h21B12

SEQ ID NO.: 20

Complete light chain immunoglobulin sequences for h21B12

SEQ ID NO.: 21

Complete nucleotide sequence of the heavy chain of h16B5

SEQ ID NO. : 22

Complete nucleotide sequence of the heavy chain of h21B12

SEQ ID NO.: 23

Complete nucleotide sequence of the light chain of h16B5

SEQ ID NO.: 24

Complete nucleotide sequence of the light chain of h21B12

SEQ ID NO.: 25 (murine 16B5 VL)

SEQ ID NO.: 26 (h16B5 VL consensus1)

STRES GVPDRFXGSGSGTDFTLTISSXQAEDXAVYYC KQSYNLW TFGXGTKLEXK;

X is an amino acid substitution in comparison with a corresponding

amino acid in the polypeptide set forth in SEQ ID NO.: 25.

SEQ ID NO.: 27 (h16B5 VL consensus2)

DIVMX a1 QSPX a2 SLAVSX a3 GEX a4 X a5 TX a6 X a7 C KSSQSLLNSRTRKNYLA WYQQKPGQX a8

PKLLIY WASTRES GVPDRFX a9 GSGSGTDFTLTISSX a10 QAEDX a11 AVYYC KQSYNL -

WT FGX a12 GTKLEX a13 K

X a1  is a neutral hydrophilic amino acid such as for example, T or S;

X a2  is D or S;

X a3  is an hydrophobic amino acid such as for example, L or A;

X a4  is a basic amino acid such as for example R or K;

X a5  is an hydrophobic amino acid such as for example A or V;

X a6  is an hydrophobic amino acid as for example I or M;

X a7  is N or S;

X a8  is P or S;

X a9  is a neutral hydrophilic amino acid such as for example S or T;

X a10  is an hydrophobic amino acid such as for example L or V;

X a11  is an hydrophobic amino acid such as for example V or L;

X a12  is Q or G and;

X a13  is I or F.

SEQ ID NO.: 28 (h16B5 VL consensus3)

DIVMX a1 QSPX a2 SLAVSX a3 GEX a4 X a5 TX a6 X a7 C KSSQSLLNSRTRKNYLA WYQQKPGQX a8

PKLLIY WASTRES GVPDRFX a9 GSGSGTDFTLTISSX a10 QAEDX a11 AVYYC KQSYNL -

WT FGX a12 GTKLEX a13 K

X a1  is T or S; Xag is D or S; X a3  is L or A; X a4  is R or K; X a5  is A or

V; X a6  is I or M; X a7  is N or S; X a8  is P or S; X a9  is S or T; X a10  is L

or V; X a11  is V or L; X a12  is Q or G and; X a13  is I or F.

SEQ ID NO.: 29 (murine 16B5 VH)

SEQ ID NO.: 30 (h16B5 VH consensus1)

TKYDPKFQG XXTITADTSXXTAYXXLSSLXSEDTAVYYCAR RYDTAMDY WGQGTXVTVSS;

X is an amino acid substitution in comparison with a corresponding amino

acid in the polypeptide set forth in SEQ ID NO.: 29.

SEQ ID NO.: 31 (h16B5 VH consensus2)

X b1 VQLX b2 QSGAEX b3 X b4 KPGAX b5 VX b6 X b7 SCX b8 X b9 S GFNIKDIYMH WVX b10 QX b11 PX b12 X b13

GLEWX b14 G RIDPAYGNTKYDPKFQG X b15 X b16 TITADTSX b17 X b18 TAYX b19 X b20 LSSLX b21 S

EDTAVYYCAR RYDTAMDY WGQGTX b22 VTVSS;

X b1  is Q or E;

X b2  is V or Q;

X b3  is an hydrophobic amino acid such as for example V or L;

X b4  is K or V;

X b5  is a neutral hydrophilic amino acid such as for example T or S;

X b6  is a basic amino acid such as for example K or R;

X b7  is an hydrophobic amino acid such as for example I or L;

X b8  is K or T;

X b9  is V or T;

X b10  is a basic amino acid such as for example Q or K;

X b11  is A or R;

X b12  is G or E;

X b13  is a basic amino acid such as for example K or Q;

X b14  is an hydrophobic amino acid such as for example M or I;

X b15  is a basic amino acid such as for example R or K;

X b16  is an hydrophobic amino acid such as for example V or A;

X b17  is a neutral hydrophilic amino acid such as for example T or S;

X b18  is for example D or N;

X b19  is an hydrophobic amino acid such as for example M or L;

X b20  is E or Q;

X b21  is R or T and;

X b22  is L or S.

SEQ ID NO.: 32 (h16B5 VH consensus3)

X b1 VQLX b2 QSGAEX b3 X b4 KPGAX b5 VX b6 X b7 SCX b8 X b9 S GFNIKDIYMH WVX b10 QX b11 PX b12 X b13

GLEWX b14 G RIDPAYGNTKYDPKFQG X b15 X b16 TITADTSX b17 X b18 TAYX b19 X b20 LSSLX b21 S

EDTAVYYCAR RYDTAMDY WGQGTX b22 VTVSS;

X b1  is Q or E; X b2  is V or Q; X b3  is V or L; X b4  is K or V; X b5  is T or S;

X b6  is K or R; X b7  is I or L; X b8  is K or T; X b9  is V or T; X b10  is Q or K;

X b11  is A or R; X b12  is G or E; X b13  is K or Q; X b14  is M or I; X b15  is R

or K; X b16  is V or A; <<X b17  is T or S; X b18  is D or N; X b19  is M or

L; X b20  is E or Q; X b21  is R or T and; X b22  is L or S.

SEQ ID NO.: 33 (murine 21B12 VL)

SEQ ID NO.: 34 (h21B12 VL consensus1)

X is an amino acid substitution in comparison with a corresponding amino

acid in the polypeptide set forth in SEQ ID NO.: 33.

SEQ ID NO.: 35 (h21B12 VL consensus2)

DIVMX c1 QSPX c2 SLAVSX c3 GEX c4 X c5 TX c6 X c7 C KSSQSLLYSSNQKNYLA WYQQX c8 PGQX c9

PKLLIY WASTRES GVPDRFX c10 GSGSGTDFTLTISSX c11 X c12 AEDX c13 AVYYC QQYYIYP

RT FGX c14 GTKLEIK;

X c1  is a neutral hydrophilic amino acid such as for example T or S;

X c2  is D or S;

X c3  is an hydrophobic amino acid such as for example L or V;

X c4  is a basic amino acid such as for example R or K;

X c5  is an hydrophobic amino acid such as for example A or V;

X c6  is an hydrophobic amino acid such as for example I or M;

X c7  is N or S;

X c8  is a basic amino acid such as for example K or R;

X c9  is P or S;

X c10  is a neutral hydrophilic amino acid such as for example S or T;

X c11  is an hydrophobic amino acid such as for example L or V;

X c12  is a basic amino acid such as for example Q or K;

X c13  is an hydrophobic amino acid such as for example V or L and;

X c14  is Q or G.

SEQ ID NO.: 36 (h21B12 VL consensus3)

DIVMX c1 QSPX c2 SLAVSX c3 GEX c4 X c5 TX c6 X c7 C KSSQSLLYSSNQKNYLA WYQQX c8 PGQX c9

PKLLIY WASTRES GVPDRFX c10 GSGSGTDFTLTISSX c11 X c12 AEDX c13 AVYYC QQYYIYP

RT FGX c14 GTKLEIK;

X c1  is T or S; X c2  is D or S; X c3  is L or V; X c4  is R or K; X c5  is A or V;

X c6  is I or M; X c7  is N or S; X c8  is K or R; X c9  is P or S; X c10  is S or T;

X c11  is L or V; X c12  is Q or K; X c13  is V or L and; X c14  is Q or G.

SEQ ID NO.: 37 (murine 21B12 VH)

SEQ ID NO.: 38 (h21B12 VH consensus1)

X is an amino acid substitution in comparison with a corresponding amino

acid in the polypeptide set forth in SEQ ID NO.: 37.

SEQ ID NO.: 39 (h21B12 VH consensus2)

QX d1 QLVQSGX d2 ELKKPGX d3 X d4 VKX d5 SCKAS GYTFTNYGMH WVX d6 QAPGX d7 GLX d8 W

MG WINTYTGEPTYADDFKG RFX d9 FSLX d10 TSX d11 STAYLQIX d12 X c13 LKX d14 EDTAX d15 YX d16

CAR DGFLYFFDY WGQGTX d17 X d18 TVSS;

X d1  is an hydrophobic amino acid such as for example V or I;

X d2  is S or P;

X d3  is A or E;

X d4  is a neutral hydrophilic amino acid such as for example S or T;

X d5  is an hydrophobic amino acid such as for example V or I;

X d6  is a basic amino acid such as for example R or K;

X d7  is a basic amino acid such as for example Q or K;

X d8  is E or K;

X d9  is an hydrophobic amino acid such as for example V or A;

X d10  is an acidic amino acid such as for example D or E;

X d11  is an hydrophobic amino acid such as for example V or A;

X d12  is S or N;

X d13  is S or N;

X d14  is A or N;

X d15  is V or T;

X d16  is an aromatic amino acid such as for example Y or F;

X d17  is L or T and;

X d18  is hydrophobic amino acid such as for example V or L.

SEQ ID NO.: 40 (h21B12 VH consensus3)

QX d1 QLVQSGX d2 ELKKPGX d3 X d4 VKX d5 SCKAS GYTFTNYGMH WVX d6 QAPGX d7 GLX d8 W

MG WINTYTGEPTYADDFKG RFX d9 FSLX d10 TSX d11 STAYLQIX d12 X d13 LKX d14 EDTAX d15 YX d16

CAR DGFLYFFDY WGQGTX d17 X d18 TVSS;

X d1  is V or I; X d2  is S or P; X d3  is A or E; X d4  is S or T; X d5  is V or I;

X d6  is R or K; X d7  is Q or K; X d8  is E or K; X d9  is V or A; X d10  is D or E;

X d11  is V or A; X d12  is S or N; X d13  is S or N; X d14  is A or N; X d15  is V

or T; <<X d16  is Y or F; X d17  is L or T and; X d18  is V or L.

SEQ ID NO.: 41 (human model of 16B5VL)

SEQ ID NO.: 42 (human model of 16B5VH)

SEQ ID NO.: 43 (human model of 21B12VL)

SEQ ID NO.: 44 (human model of 21B12VH)

SEQ ID NO.: 45

16B5 CDRH1 (shorter version): GFNIKDIY

SEQ ID NO.: 46

16B5 CDRH2 (shorter version): IDPAYGNT

SEQ ID NO.: 47

16B5 CDRH3: ARRYDTAMDY

SEQ ID NO.: 48

16B5 CDRL1 (shorter version): QSLLNSRTRKNY

SEQ ID NO.: 49

16B5 CDRL2 (shorter version): WAS

SEQ ID NO.: 50

16B5 CDRL3: KQSYNLWT

SEQ ID NO.: 51

21B12 CDRH1: GYTFTNYG

SEQ ID NO.: 52

21B12 CDRH2: INTYTGEP

SEQ ID NO.: 53

21B12 CDRH3: X 3 X 4 DGFLYFFDY

X 3  is A;

X 4  is R;

or X 3  and X 4  are outside the CDRH3

SEQ ID NO.: 54

21B12 CDRL1: QSLLYSSNQKNY

SEQ ID NO.: 55

21B12 CDRL2: WAS

SEQ ID NO.: 56

21B12 CDRL3: QQYYIYPRT

SEQ ID NO: 57

SEQ ID NO: 58

SEQ ID NO: 59

SEQ ID NO: 60

SEQ ID NO.: 61

clusterin antisense: 5′-CAGCAGCAGAGTCTTCATCAT-3′

SEQ ID NO.: 62 (20E11 variable light chain)

SEQ ID NO.: 63 (20E11 variable heavy chain)

SEQ ID NO.: 64 (11E2 variable light chain Gr1)

SEQ ID NO.: 65 (11E2 variable light chain GR2)

SEQ ID NO.: 66 (11E2 variable heavy chain)

SEQ ID NO.: 67 (16C11 variable heavy chain)

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Shigematsu H., L. Lin L, et al., (2005) “Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers.” J Natl Cancer Inst 97:339.

Barr, S., S. Thomson et al., (2008) “Bypassing cellular EGF receptor dependence through epithelial-to-mesenchymal-like transitions.” Clin Exp Metastasis 25:685.

Mirshahidi, H. R., C. T., Hsueh (2010) “Updates in non-small cell lung cancer-insights from the 2009 45th annual meeting of the American Society of Clinical Oncology.” J Hemat & Oncol 3:18.

Pirker, R., J. R., Peirera et al., (2012) “EGFR expression as a predictor of survival for first-line chemotherapy plus cetuximab in patients with advanced non-small-cell lung cancer: analysis of data from the phase 3 FLEX study.” Lancet 13:33.

Costanzo, R., M. C., Piccirillo (2011) “Gefitinib in non small cell lung cancer.” J Biomed Biotechnol 2011: 815269.

Herbst, R. S. and A. Sandler (2008) “Bevacizumab and erlotinib: A promising new approach to the treatment of advanced NSCLC.” Oncologist 13:1166.

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