WO2008068481A1 - New compounds, methods and uses - Google Patents

Abstract

The present invention provides the use of a compound comprising a binding moiety capable of binding selectively to integrin alpha 11 subunit or heterodimer thereof in the preparation of (a) a medicament for treating malignant tumour tissue, b) diagnostic or prognostic agent for malignant tumours and (c) an agent for detecting and/or imaging malignant tumour cells, such as NSCLC cells. The invention further provides methods for treating, diagnosing and imaging cells of malignant tumours.

Description

NEW COMPOUNDS, METHODS AND USES

TECB-NICAL FILED

The present invention relates to the use of compounds with a binding moiety capable of binding to an integrin αl 1 subunit, or heterodimer comprising the same (for example, an αl lβl heterodimer), in the treatment, diagnosis or detection of malignant tumour tissue. More specifically, the invention provides compounds in the treatment of malignant tumour tissue, e.g. non-small cell lung carcinoma, and methods of using the same.

BACKGROUND OF THE INVENTION

Tumour - stoma cell interactions

There is abundant evidence that tumour-stromal cell interaction plays critical role in tumour growth, invasion, metastases, angiogenesis, and chemoresistance. Factors derived from the "carcinoma-associated fibroblast (CAF)" or "activated fibroblasts" contribute to the transformation of immortalized epithelial cells and enhance the tumourigenicity of cancer cells. Irradiated fibroblasts increase the incidence of breast cancer compared to the non- irradiated fibroblasts when they are co-transplanted with untransformed mammary epithelia into mammary fat pad. These studies demonstrated that changes in stromal fibroblasts might contribute to epithelial transformation and tumourigenicity possibly through paracrine secretion of growth factors. However, the precise mechanisms underlying communication between stromal fibroblasts, tumour cells and the environment remains incompletely understood.

Integrins regulate diverse events by cell-cell and cell-matrix interactions

Integrins were originally identified as intermediary cell surface structures that linked the internal cytoskeleton with the immediate environment or extracellular cell matrix, and were considered functionally "dead" molecules. This reasoning was partially based on the observation that most integrins contain only a very small cytoplasmic tail lacking any signaling motifs. Today, integrins are known as highly complex structures that via interaction with other cell surface receptors and recruitment of intracellular adapter proteins participate in cell signaling from the inside and out (Phillips et al, 1988, Blood 71:831-43.), from outside and in (Law et al, 1999, Nature 401:808-81 L), and have been shown to transduce signals laterally across the cell membrane (Hynes, 2002, Cell 110:673-87; for review see Miranti & Brugge, 2002, Nat Cell Biol 4:ES3-90.).

Blocking of some of the best studied integrins using monoclonal antibodies or small molecule inhibitors has been shown to abrogate cell-cell and cell-matrix contacts resulting in intervention of diverse biological processes including development, tissue repair, angiogenesis, inflammation and haemostasis. Some of these antibodies are the subject of clinical phase trials (Shimaoka & Springer, 2003, Nat Rev Drug Discov 2:703-16).

The integrins are a large family of transmembrane receptors that mediate physical interactions between cells and extracellular matrix protein collagens. There are at least 24 different integrins formed as heterodimers of 1 S alpha and 8 beta (β) subunits, each having distinct ligand binding and signalling properties . They play crucial roles in diverse cellular and developmental processes including cell growth, differentiation and survival as well as in carcinogenesis, cancer cell invasion and metastases. The alphal 1 integrin subunit was first identified as a major integrin in cultured skeletal muscle cells . It contains a trans-membrane domain and a 24 amino acid cytoplasmic tail. The alphal 1 integrin chain can dimerize with betal to form one of the 4 collagen receptors. La human embryos, alphal 1 expression is localized to the mesenchyme.

Non-small cell lung carcinoma The prognosis for patients with lung cancer remains poor. Although improved surgical techniques have led to an increase in one-year survival for lung cancer from 34% in 1975 to 42% in 1998, the five-year survival rate for all stages combined is only 15%. This poor prognosis is primarily due to the fact that only a small portion of cases are diagnosed at an early stage and at the later stage when the cases are diagnosed it is difficult to treat. Lung cancers can be grossly divided into small cell lung cancer (SCLC) which accounts for approximately 25% of lung cancer cases and non- small cell lung carcinoma (NSCLC). NSCLC can be further subdivided into adenocarcinoma, lung cell carcinoma and squamus cell carcinoma, each of which account for about 25% of lung cancer cases.

Non-small cell lung carcinoma (NSCLC) is the predominant form of lung cancer, accounting for about 80% of all cases. NSCLC is treated by surgical resection, as well as by chemotherapeutic agents such as cyclophosphamide, methotrexate, ifosfamid and cis-platin. When localized, NSCLC can be treated surgically or in some cases with combined radiation and chemotherapy. However, about 50% of surgically resectable cases and about 80% of locally advanced cases will relapse. Therapy of locally advanced (Stage IIIB) NSCLC or NSCLC with distant metastases (Stage IV) is not curative. For NSCLC patients, only 25% survive for 5 years after diagnosis.

Several mouse monoclonal antibodies produced against antigens on small cell and non-small cell human lung cancer has been used in immunohistochemical assays to study tumour biology, lung cancer immuno localization, and to give clues to rumour ancestry. The antigens recognized by these antibodies are expressed on a variety of tumours as well as normal fetal tissue. As summarized in the proceedings of the First International Workshop on Antigens of Small Cell Lung Cancer, Sou hami et al, Lancet 2(8554): 325-6,1987, there are nearly 100 monoclonal antibodies being investigated to study small cell and non-small cell cancer of the lung. This workshop supported central registry coding of antibodies followed by blinded staining of a variety of normal and neoplastic tissues. One problem is that none of the antigens studied were specific for small cell lung cancer. Another problem is that none of the antigens studied were universally present on all small cell lung cancer specimens studied. Furthermore, rather than being strict tumour markers, these antigenic determinants seems to be markers of differentiation.

LT.S. Patent No. 4,569,788, disclose monoclonal antibodies which can be used to detect human non-small cell lung carcinoma and distinguish this type of cancer from all other types of lung cancer and normal tissue cells. These two antibodies may be utilized to distinguish non-small cell lung carcinoma form other forms of lung cancer by testing the tumour tissue.

WO 86102735 discloses monoclonal antibodies which bind to antigens associated with human non-small cell lung carcinomas. The antibodies are useful in detecting malignant cells associated with non-small cell lung carcinomas.

EP-A-O 203 552 similarly relates to monoclonal antibodies which bind to antigens associated with non-small cell lung carcinomas.

It is thus highly desirable in the light of aforementioned problems to detect tumour tissue e.g. carcinoma cells and its tumour stroma, particularly NSCLC (non-small cell lung carcinoma) tissue, at an early stage as well as to achieve more effective therapeutic approaches for said carcinoma. Thus, new methods and uses that inhibit tumour tissue growth and metastasis, particularly for NSCLC, are needed, which can be used alone or in concert with other agents to treat cancer, especially NSCLC, which typically involve metastases. In this respect, the present invention addresses this needs and interest. SUMMARY OF THE INVENTION

In view of the foregoing disadvantages known in the art of detection, diagnosis and treatment of malignant tumour tissue, particularly NSCLC (non-small cell lung carcinoma) the present invention provides uses and methods of compounds comprising binding a moiety capable of binding selectively to an integrin alphal 1 or a heterodimer thereof.

The present invention provides that alphal 1 plays important role in the ability of fibroblasts to promote the growth of A549 lung adenocarcinoma cells in vivo, and such activity was partially mediated by its ability to regulate the expression of insulin-like growth factor-2 (IGF2).

Thus, the present invention provides the use of a compound comprising a binding moiety capable of binding selectively to an integrin alpha-11 subunit or a heterodimer thereof in the preparation of a medicament for treating a malignant tumour tissue. The invention further provides use of a compound comprising a binding moiety capable of binding selectively to an integrin alpha-11 subunit or a heterodimer thereof in the preparation of a diagnostic or prognostic agent for a malignant rumour tissue.

Even further, the invention provides use of a compound comprising a binding moiety capable of binding selectively to an integrin alpha-11 subunit or a heterodimer thereof in the preparation of an agent for detecting and/or imaging malignant tumour tissue. hi further embodiments, the uses according to the invention are wherein the intergrin alpha-11 subunit or heterodimer is expressed on the stroma cells associated to the malignant tumour tissue.

In even further embodiments, the stroma cells are fibroblast cells associated to the malignant tumour tissue.

In further embodiments, the use according to the invention are wherein the integrin alpha-11 subunit or heterodimer is expressed on the tumour cells associated to the malignant tumour tissue.

Li still even further embodiments, the tumour tissue is selected from the group consisting of tumour tissue of the breast, bile duct, brain, colon, stomach, reproductive organs, lung and airways, skin, gallbladder, liver, nasopharynx, nerve cells, kidney, prostate, lymph glands and gastrointestinal tract.

Even further embodiments are wherein the tumour tissue is a non-small cell lung carcinoma. In still even further embodiments, the binding moiety selectively binds to integrin alpha- 11 subunit.

In still even further embodiments, the malignant tumour tissue is metastatic. Also provided in the present invention is several methods e.g. a method of imaging a malignant tumour tissue in the body of an individual, the method comprising administering to the individual an effective amount of a compound comprising a binding moiety capable of binding selectively to integrin alpha- 11 subunit or a heterodimer thereof; a method of diagnosing or prognosing malignant tumour tissue in an individual, the method comprising administering to the individual an effective amount of a compound comprising a binding moiety capable of binding selectively to integrin alpha- 11 subunit or a heterodimer thereof; a method of treating an individual with a malignant tumour tissue, the method comprising administering to the individual an effective amount of a compound comprising a binding moiety capable of binding selectively to integrin alpha-11 subunit or a heterodimer thereof.

Even further, a method for monitoring the progression of a malignant tumour tissue in an individual is provided, the method comprising the steps of: (a) providing a sample of a malignant tumour tissue collected from the individual at a first time point and measuring the amount of integrin alpha-11 subunit protein therein; (b) providing a sample of a malignant rumour tissue collected from the individual at a second time point and measuring the amount of integrin alpha-11 subunit protein therein; and (c) comparing the amount of integrin alpha- 11 subunit protein measured in steps (a) and (b), wherein an increased amount of integrin alpha-11 subunit protein measured in step (b) compared to step (a) is indicative of a progression in the malignant tumour tissue.

Still even further the invention provides a method of identifying cells associated with a malignant tumour tissue, the method comprising measuring the amount of integrin alpha-11 subunit protein in a sample of cells to be tested and comparing it to the amount of integrin alpha-11 subunit protein in a sample of cells from a known malignant tumour tissue.

Also provided is a method of distinguishing between different types or stages of a malignant tumour tissue, the method comprising measuring the amount of integrin alpha-11 subunit protein in a sample of cells to be tested and comparing it to the amount of integrin alpha-11 subunit protein in a sample of cells from a malignant tumour tissue of a known type or stage.

The present invention further provides a method of screening for candidate compounds with efficacy in the treatment of a malignant tumour tissue, the method comprising the steps of: (a) contacting a molecule to be tested with an integrin alpha-11 subunit (or a fragment or binding sequence thereof); and (b) detecting the presence of a complex containing the integrin alpha- 11 subunit (or fragment thereof) and the molecule to be tested and wherein the molecule to be tested being identified as a candidate compound if the complex is detected in step(b).

SHORT DESCRIPTION OF DRAWINGS

Figure 1 A shows that alpha 11 is overexpressed in both lung adenocarcinoma and squamous cell carcinoma at protein level by Western blot on three of four unselected paired primary NSCLC and corresponding non-neoplastic lung tissue. (N: corresponding non- neoplastic lung tissue; T: tumour samples; loading control: Ponceau staining). The loading control is shown in supplemental.

Figure IB shows qPCR assay of alphal 1 mRNA expression in microdissected stromal (S) compared to tumour cells in two unselected primary NSCLC tissues. The figure shows that expression was significantly higher in the stromal tissue. Figure 1C and D shows immunofluorescent images of normal lung tissue (C) and an adenocarcinoma (D) that were double stained with antibodies to alphal 1 (red) and epithelial cell marker cytokeratin (green). The αl 1 (alphal 1) staining was negligible in nonneoplastic lung tissue, but was mainly confined to the stroma in the tumour sample. Figure C shows that the alphal 1 protein was mainly localized in the stroma bordering the invasive tumour cell nests, and it was not detectable in control normal lung sections using immunofluorescence microscopy. Figure D shows that alphal 1 was differentially overexpressed 2-fold or higher in four of 6 paired-samples of primary NSCLC and their corresponding non-neoplastic lung tissue.

Figure IE shows RT-qPCR analysis of alphal 1 mRNA expression in primary lung tumours and gum metastases. A 4-5-fold higher expression in the metastatic timors compared to primary tumours in the lungs was seen. mRNA expression of alphal 1 (black bar) and IGF2 (empty bar) in carcinoma-associated fibroblasts (CAFs) and their corresponding normal lung fibroblasts (CP). Four of five CAFs (520, 305, 619 and 836) showed higher alphal 1 and IGF2 expression. All expression levels were relative to that of CP for sample 520. Figure IF shows expression of rat ITGAl 1 (alphal 1) and IGF2 mRNA in the primary (Orthl-3) and gum metastatic (Metl-2) tumours of the NCI-H460 rat orthotopic model. All expression levels of rITGAl 1 and rIGF2 were relative to those of Orth-1 tumour. Figure 2A-D shows the effect of ITGAl 1 (alphal 1) expression in immortalized mouse embryonic fibroblasts (MEF) on the tumourigenicity of A549 lung adenocarcinoma cells.

Figure 2A shows tumour growth of A549 lung adenocarcinoma cells when co- implanted with immortalized wild type (WT) and αl 1 deficient (KO) MEFs in a 1 :1 ratio in the subcutaneous tissue of SCID mice. A549, KO, WT and KI were tumour formation by respective cell lines alone, while A549+WT/KO/KI represent tumour formation by A549 cells co-implanted with alphal 1 expressing MEF (WT), alphal 1 deficient MEF (KO)₅ or KO MEF that have re-expressed hITGAl 1 (KI). The A549+WT group showed markedly greater tumour growth than A549+KO group (p=0.024).

Figure 2B shows protein expression of mouse integrin subunits betal, alphal, alρha2, and alphal 1 in WT, KO and KI MEFs.

Figure 2C shows mouse Itgal 1 (black bar) and human ITGAl 1 (empty bar) rnRNA expressions in xenograft tumours formed by WT, KO and KI cells alone or when co- implanted with A549 cells. WT tumour expressed mouse alphal 1 within normal range. However, when WT was co-implanted with A549, there was a ~10-fold increase in mouse alphal 1 expression level. KI tumours only expressed human alphal 1. The low level of mouse alphal 1 detected in KO tumours was putatively of endogenous stromal cell origin. Note that no detectable human alphal 1 in WT and KO and trace level of mouse alphal 1 in KI and A549+KI exemplified the specificity of primers. AU expression levels were arbitrarily referenced to the mean.of A549+KO tumours.

Figure 2D shows that CD31 rnRNA expression levels in xenograft tumours as specified in (C). Error bar represents standard error.

Figure 3A-E shows regulation of IGF2 expression by ITGAl 1 in MEF and the effect on A549 cell tumour formation when co-implanted with these MEFs.

Figure 3A shows mouse (m) IGF2 mRNA expression levels in tumours formed by ITGAI l /alphal 1 expressing (WT), alphal 1 deficient (KO) and h-alphall re-expressing KO MEF (ICI), and the corresponding tumours formed when these MEFs were co-implanted with A549 lung adenocarcinoma cells. Note the lack of mIGF2 expression when alphal 1 was not expressed. The results is of a RT-qPCR that showed ~1 OO and >200 fold higher expression levels of the gene IGF2 in WT vs. KO tumours (p=0.001) and A549+WT vs. A549+KO tumours (p=0.001), respectively. Also, figure 3 A shows that KI cells restored the IGF2 expression in xenographs of KI alone (J)=O.002) or with A549 (p=0.006). Figure 3B shows the corresponding expression of mIGFl rnRNA in the tumours specified in (A). mIGFl mRNA expression was only elevated in tumours formed by KI or A549+KI cells, and note the greater magnitude of expression changes for mIGF2 than mIGFl. Figure 3C shows that mIGF2 mRNA in cell lines that stably express the shRNA constructs; A 70% mIGF2 knock down efficiency (vs. shLuc) was noted in WT and KI cells, while the level did not change in KO cells with already very low mIGF2 expression.

Figure 3D shows tumour growth rates of A549 cells co-implanted with WT_ShiG_F2 and WTshUic (control) MEFs. Figure 3E shows mIGF2 expression of xenograft tumours formed by A549+WT_ShLu_c and A549+WT_shi_GF2 MEFs showing a 70% stable downregulation of mIGF2 in the latter tumours.

Figure 4A-D shows mRNA expressions of other collagen receptor integrins in mouse xenograft tumours. Figure 4 A shows that alphal expression was slightly increased with alpha 1 1 down- regulation, but this was noted in vivo.

Figure 4B shows that alpha2 expression was up-regulated significantly consequent to the loss of alphal 1 , and that this was noted both in vitro and in vivo.

Figure 4C shows that alphal 0 mRNA expression was up-regulated by approximately 2-fold consequent to loss of alphal 1 expression.

Figure 4D shows that betal expression was either unchanged or inconsistently changed in relation to alphal 1 expression changes. In all instances, expression levels in various tumours were expressed relative to the means of A549+KO tumours.

DETAILED DESCRIPTION OF THE INVENTION

The present invention shows that stromal expression of alphal 1 is important for the tumourigenicity of malignant tumours such as NSCLC.

Integrin is one of the most important mediators of cell-extracellular matrix protein interaction. Integrins are localized mainly at focal adhesions. They transmit both biochemical and mechanical signals from the matrix proteins to cytoskeletal machinery of cells. They also activate signal transduction cascades that are important in cellular proliferation, movement and survival. Integrin may also affect these cellular factions through interactions with other transmembrane proteins including growth factor receptors. Studies on the role of integrins in cancer development and biology have mainly focused on their expression on tumour or endothelial cells. The patterns of expression changes in various tumour types are complex and often involved cell type or disease specific effects. While most such effects result from direct interaction of integrins on tumour cells and the stromal matrix proteins, our study suggests that altered integrin expression in tumour stromal fibroblasts may also play important role in the growth of carcinoma cells. Among the collagen receptor integrins, overexpression of alphal and alpha2 have been reported in squamous cell carcinoma and correlated with increased invasion, but the loss of alpha2 expression has also been associated with tumour progression in breast and prostate carcinoma cells. In lung cancer, the precise role of these two collagen receptor integrins remains ambiguous and often contradictory (Gogali, A. et al. (2004) Exp Oncol 26, 106-10).

With alphal 1 being expressed largely by stromal fibroblasts, the present invention shows that alphal 1 provides interstitial collagen to influence the growth of rumour cells. The present invention also provides that this mechanism is mediated by its ability to regulate thightly the fibroblast expression of IGF2, which is a potent growth stimulator of epithelial tumour cells.

The integrin alphal 1 subunit is commonly overexpressed in non-small cell lung carcinoma (NSCLC) (>80% of the cells), and immunolocalization study showed that the protein expression is mainly in rumour stroma. Alphal 1 is also commonly over-expressed in cancer-associated fibroblasts.

A first aspect of the invention provides the use of a compound comprising a binding moiety capable of binding selectively to an integrin alpha- 11 subunit or a heterodimer thereof in the preparation of a medicament for treating a malignant tumour tissue. It will be appreciated by persons skilled in the art that the medicament may be used for proplrylactic and/or therapeutic purposes, i.e. the medicament may be administered to a subject in need thereof in an amount sufficient to give prophylactic and/or therapeutic effect. By "tumour" we include an abnormal mass of tissue that results from excessive cell division that is uncontrolled and progressive, also called a neoplasm, comprising mainly tumour cells.

By "tumour stroma" we include the stroma surrounding the tumour and/or the tumour cells.

By "tumour tissue" we include tumour cells as such and the surrounding rumour stroma.

By "malignant" we include the meaning of a tumour having the properties of anaplasia, invasion and/or metastasis.

By "turnourigenicity" we include capable of causing or producing tumours.

A second aspect of the invention provides the use of a compound comprising a binding moiety capable of binding selectively to an integrin alpha- 11 subunit or a heterodimer thereof in the preparation of a diagnostic or prognostic agent for a malignant tumour tissue. ^•

A third aspect of the invention provides a use of a compound comprising a binding moiety capable of binding selectively to an integrin alpha- 11 subunit or a heterodimer thereof in the preparation of an agent for detecting and/or imaging malignant tumour tissue in a body of an individual, either in vivo or in vitro.

Persons skilled in the art will appreciate that the above aspects of the invention may be used for any tumour tissue found to over-express an integrin alpha- 11 subunit. Examples of tumour tissue are tumour tissue selected from the group consisting of tumour tissue of the breast, bile duct, brain, colon, stomach, reproductive organs, lung and airways, skin, gallbladder, liver, nasopharynx, nerve cells, Iddney, prostate, lymph glands and gastrointestinal tract. In one embodiment the tumour tissue is a non-small cell lung carcinoma and its surrounding tumour stroma.

In one embodiment, the integrin alpha- 11 subunit or heterodimer is expressed on the stroma cells associated to the malignant tumour cells in said tumour tissue. In still a further embodiment, the stroma cells are fibroblast cells associated to the malignant tumour tissue. Other stroma cells that may be associated to the malignant tumour tissue are stem cells, such as mesenchumal stem cells, or dendritic cells.

Yet another aspect of the invention provides for a use of a compound comprising a binding moiety capable of binding selectively to integrin alpha- 11 subunit or a heterodimer thereof in the preparation of an agent for inhibiting metastases or metastatic spread. The agent is administered to a subject in need thereof in an amount sufficient to inhibit metastases, and/or metastatic spread.

Thus, in one aspect of the invention, the present invention can be used to inhibit, prevent or slow down the progression of malignancies. Another aspect of the invention is to inhibit, prevent or slow down metastases or metastatic progression.

The present invention can also be used to inhibit, prevent or slow down the invasion of healthy tissue by malignant tumour tissue.

It is yet a further aspect of the invention that the tumour tissue treated is a solid tumour. Solid tissue tumours contemplated for treatment according to the invention include but are not limited to tumour tissue of carcimomas (e.g. non-small cell lung carcinoma, ), prostate cancers, and metastatic lesions of other primary tumours. Preferably, the tumour and its stroma is a solid tumour, e.g. a non-small cell lung carcinoma, or a metastatic lesion of other primary tumours.

By "carcinoma" we include a malignant new growth that arises from epithelium, found in skin or, more commonly, in the lining of body organs, for example, breast, prostate, lung, stomache or bowel. Carcinomas tend to infiltrate into adjacent tissue and spread (metastasize) to distant organs, for example to bone, liver, lung or the brain.

In further embodiments the tumour tissue is selected from the group consisting of tumour tissue of the breast, bile duct, brain, colon, stomach, reproductive organs, lung and airways, skin, gallbladder, liver, nasopharynx, nerve cells, kidney, prostate, lymph glands and gastrointestinal tract. In one particular embodiment, the tumour tissue is of non- small cell lung carcinoma type. In a iurther aspect of the invention, the agent can be administered alone or in combination with other cancer treatment therapies in a multi-treatment format. Examples are where the subject is further treated with a chemotherapy, an immunotherapy, surgery, radiation therapy, hyperthermia, or a drug to ameliorate the adverse effects of a cancer therapy. For example, if the tumour tissue is a carcinoma, the subject can be administered the agent after having had the carcinoma tumour tissue surgically removed. In non-small cell lung cancer (NSCLC), results of standard treatment are poor except for the most localized cancers. AU newly diagnosed patients with NSCLC are potential candidates for studies evaluating new forms of treatment. Surgery is the most potentially curative therapeutic option for this disease; radiation therapy can produce a cure in a small number of patients and can provide palliation in most patients. Adjuvant chemotherapy may provide an additional benefit to patients with resected NSCLC. In advanced-stage disease, chemotherapy offers modest improvements in median survival, though overall survival is poor. Chemotherap}' has produced short-term improvement in disease-related symptoms. Several clinical trials have attempted to assess the impact of chemotherapy on tumour-related symptoms and quality of life. In total, these studies suggest that tumour-related symptoms may be controlled by chemotherapy without adversely affecting overall quality of life. Current areas under evaluation include combining local treatment (surgery), regional treatment (radiation therapy), and systemic treatments (chemotherapy, immunotherapy, and targeted agents) and developing more effective systemic therapy. The chemotherapeutic agent can be any one or more of the following: cisplatin, carboplatin, paclitaxel (Taxol), docetaxel (Taxotere), topotecan, irinotecan, vinorelbine, and gemcitabine.

Yet another aspect of the invention provides for a use of a compound comprising a binding moiety capable of binding selectively to integrin alpha-11 subunit or a heterodimer thereof in the preparation of an agent for treating metastases to the brain, lung, liver, or bone.

Another aspect of the invention provides for a combination therapy wherein a binding moiety capable of binding selectively to integrin alpha-11 subunit or a heterodimer thereof in the preparation of an agent are used in combination with other tumour treatment therapies as known in the art.

A neoplasm, or tumour, is an abnormal, unregulated, and disorganised proliferation of cell growth. A neoplasm is malignant, or cancerous, if it has properties of destructive growth, invasiveness and metastasis. Invasiveness refers to the local spread of a neoplasm by infiltration or destruction of surrounding tissue, typically breaking through the basal laininas that define the boundaries of the tissues, thereby often entering the body's circulatory system. Metastasis typically refers to the dissemination of tumour cells by lymphatics or blood vessels. Metastasis also refers to the migration of tumour cells by direct extension through serous cavities, or subarachnoid or other spaces.

Through the process of metastasis, tumour cell migration to other areas of the body establishes neoplasms in areas away from the site of initial appearance. There are essential steps in the formation of metastasis in all tumour tissue. The steps include the following:

1. After neoplastic transformation, progressive proliferation of neoplastic cells supported by the organ/tissue environment in which the neoplasm is located.

2. Neovascularisation or angiogenesis of the tumour for further growth beyond 1 to 2 mm in diameter.

3. Down-regulation of expression of cohesive molecules wherein the cells have increased motility or ability to detach from the primary lesion.

4. Detachment and embolisation of single tumour cells or cell aggregates, with the vast majority of these cells being rapidly destroyed. 5. Once tumour cells survive the detachment and embolisation step, they must go on to proliferate within the lumen of the blood vessel. The cells will then go on to extravasate into the organ parenchyma by mechanism similar to those operative during invasion. 6. Tumour cells with the appropriate cell surface receptors can respond to paracrine growth factors and hence proliferate in the organ parenchyma. 7. Tumour cell evasion of host defences (both specific and nonspecific immune responses). 8. For a metastasis to proliferate beyond 1 to 2 mm in diameter, the metastases must develop a vascular network.

Thus, if a primary tumour tissue is given enough time to go through these steps, it will form metastatic lesions at a site or sites distant to the primary tumour tissue. The present invention disclosed herein may inhibit, slow down or prevent one or more one or more of these steps in the metastatic process. For additional details on the mechanism and pathology of tumour metastasis, see Isaiah J. Fidler, "Molecular Biology of Cancer: Invasion and Metastasis," in Cancer: Principles & Practice of Oncology pp 135-152 (Vincent T. DeVita et al., editors, 5^th ed., 1997).

Tumour cell invasion is believed to occur by a three-step process:

1 ) tumour cell attachment to extracellular matrix;

2) proteolytic dissolution of the matrix; and

3) movement of the cells through the dissolved barrier.

This process can occur repeatedly and can result in metastases, i.e. secondary tumours, at sites distant from the original, primary, tumour tissue.

By the term "subject" or "patient" as used herein is meant to include a mammal. The mammal can be a canine, feline, primate, bovine, ovine, porcine, camelid, caprine, rodent, or equine. Preferably the mammal is human.

By the term "primary tumour" is meant the original neoplasm and not a metastatic lesion located in another tissue or organ in the patient's body.

By the terms "metastatic disease", "metastases" and "metastatic lesion" are meant a group of cells which have migrated to a site distant relative to the primary tumour.

As used herein, the singular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an antibody" includes a plurality of such antibodies and reference to "the dosage" includes reference to one or more dosages and equivalents thereof known to those skilled in the art, and so forth.

By "capable of binding selectively" we include binding moieties which bind at least 10-fold more strongly to integrin alpha- 11 subunit or a heterodimer thereof than to another proteins (in particular other integrins, such as alphalO, alphal and apha2 having most identity with alphal 1); preferably at least 50-fold more strongly and more preferably at least 100-fold more strongly. Advantageously, the binding moiety is capable of binding selectively to integrin alpha- 11 subunit or a heterodimer thereof under physiological conditions, e.g. in vivo. Suitable methods for measuring relative binding strengths include immunoassays, for example where the binding moiety is an antibody (see Harlow & Lane, "Antibodies: A Laboratory", Cold Spring Habor Laboratory Press, New York). Alternatively, binding may be assessed using competitive assays or using Biacore^® analysis (Biacore International AB, Sweden).

Further embodiments are wherein the binding moiety binds exclusively to an integrin alpha- 11 subunit or a heterodimer thereof.

In one embodiment, the binding moiety selectively binds to an integrin alpha- 11 subunit. Conveniently, the binding moiety selectively binds to a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO 1 or natural variants thereof.

1 MDLPRGLWAWALSLWPGFTDTFNMDTRKPRVIPGSRTAFFGYTVQQHDISGNKWLVVGA 61 PLETNGYQKTGDVYKCPVIHGNCTKLNLGRVTLSNVSERKDNMRLGLSLATNPKDNSFLA 12ICSPLWSHECGSSYYTTGMCSRVNSNFRFSKTVAPALQRCQTYMDIVIVLDGSNSIYPWVE 18IVQHFLINILKKFYIGPGQIQVGVVQYGEDWHEFHLNDYRSVKDWEAASHIEQRGGTET 24IRTAFGIEFARSEAFQKGGRKGAKKVMIVITDGESHDSPDLEKVIQQSERDNVTRYAVAVL 30IGYYNRRGINPETFLNEIKYIASDPDDKHFFNVTDEAALKDIVDALGDRIFSLEGTNKNET 36ISFGLEMSQTGFSSHWEDGVLLGAVGAYDWNGAVLKETSAGKVIPLRESYLKEFPEELKN 42IHGAYLGYTVTSWSSRQGRVYVAGAPRFNHTGKVILFTMHNNRSLTIHQAMRGQQIGSYF 48IGSEITSVDIDGDGVTDVLLVGAPMYFNEGRERGKVYVYELRQNRFVYNGTLKDSHSYQNA 54IRFGSSIASVRDLNQDSYNDVWGAPLEDNHAGAIYIFHGFRGSILKTPKQRITASELATG 60ILQYFGCSIHGQLDLNEDGLIDLAVGALGNAVILWSRPWQINASLHFEPSKINIFHRDCK 66IRSGRDATCLAAFLCFTPIFLAPHFQTTTVGIRYNATMDERRYTPRAHLDEGGDRFTNRAV 72ILLSSGQELCERINFHVLDTADYVKPVTFSVEYSLEDPDHGPMLDDGWPTTLRVSVPFWNG 78ICNEDEHCVPDLVLDARSDLPTAMEYCQRVLRKPAQDCSAYTLSFDTTVFIIESTRQRVAV 84IEATLENRGENAYSTVLNISQSANLQFASLIQKEDSDGSIECVNEERRLQKQVCNVSYPFF 90IRAKAKVAFRLDFEFSKSIFLHHLEIELAAGSDSNERDSTKEDNVAPLRFHLKYEADVLFT 96IRSSSLSHYEVKLNSSLERYDGIGPPFSCIFRIQNLGLFPIHGIMMKITIPIATRSGNRLL 02IKLRDFLTDEVANTSCNIWGNSTEYRPTPVEEDLRRAPQLNHSNSDWSINCNIRLVPNQE 081INFHLLGNLWLRSLKALKYKSMKIMVNAALQRQFHSPFIFREEDPSRQIVFEISKQEDWQ 14IVPIWIIVGSTLGGLLLLALLVLALWKLGFFRSARRRREPGLDPTPKVLE

[SEQ ID NO 1]

By "natural variants" we include, for example, allelic variants. Typically, these will vary from the given sequence by only one or two or three, and typically no more than 10 or 20 amino acid residues. Typically, the variants have conservative substitutions.

Also included are fragments of integrin alpha 11, for example as described in WO 00/75187 (see pg 9 therein). Examples of fragments of the integrin subunit all are peptides of the cytoplasmic domain, especially a peptide comprising essentially the amino acid sequence ALWICLGFFRSARRRREPGLDPTPVLE [SEQ ID NO:2] ; a peptide of the I-domain, especially a peptide comprising essentially the amino acid sequence from about amino acid No. 156 to about amino acid No. 355 of SEQ ID No. 1, and especially a peptide comprising essentially the amino acid sequence QT\⁷MDIVIVLDGSNSIYPWVEVQHFLINILKKFYIGPGQIQVGWQYGEDWHEFHLN DYRSVKD WE AASHIEQRGGTETRTAFGIEF ARSEAFQKGGRKGAKKVMIVITDGES HDSPDLEKVIQQSERDNVTRYA VA VLGYYNRRGINPETFLNEIKYIASDPDDKHFFNV TDEAALKDIVDALGDRIFSLEGT [SEQ ID NO:3]; and the extracellular extension region, especially a peptide comprising esentially the amino acid sequence from about amino acid No. 804 to about amino acid No. 826 of SEQ ID No. 1 , especially a peptide comprising essentially the amino acid sequence EYCQRVLRKPAQDCSAYTLSFDT [SEQ ID NO:4]. A further fragment of the alphal 1 molecule is the extracellular domain of the alphal 1 molecule, and especially a peptide comprising essentially the amino acid sequence from about amino acid No. 24 to amino acid No. 1143, especially a peptide comprising essentially the amino acid sequence

NMDTRKPRVIPGSRTAFFGYTVQQHDISGNKWLVVGAPLETNGYQKTGDVYKCPVI HGNCTKLNLGRVTLSNVSERKDNMRLGLSLATNPKDNSFLACSPLWSHECGSSYYTT GMCSRVNSNFRFSKTVAP ALQRCQTYMDrVrVLDGSNSIYP WVEVQHFLINILKKF YI GPGQIQVGWQYGED WHEFHLND YRSVKD WE AASHIEQRGGTETRTAFGIEFARS EAFQKGGRKGAKKVMIVITDGESHDSPDLEKVIQQSERDNVTRY AVAVLGYYNRRGI NPETFLNEIKYIASDPDDKHFFNVTDEAALKDIVDALGDRIFSLEGTNKNETSFGLEMS QTGFSSHWEDGVLLGAVGAYDWNGAVLKETSAGKVIPLRESYLKEFPEELKNHGA YLGYWTSWSSRQGRVYVAGAPRFrøTGKVTLFTMHNNRSLTmQAMRGQQIGSYF GSEITSVDIDGDGVTD VLLVGAPMYFNEGRERGKVYVYELRQNRFVYNGTLKDSHS YQNARFGSSIASVRDLNQDSYNDVVVGAPLEDNHAGATYIFHGFRGSILKTPKQRITA SELATGLQYFGCSIHGQLDLNEDGLIDLAVGALGNAVILWSRPWQINASLHFEPSKI NIFHRDCKRSGRDATCLAAFLCFTPIFLAPHFQTTTVGIRYNATMDERRYTPRAHLDE GGDRFTNRAVLLSSGQELCERTNFFA^LDTADYVKPVTFSVEYSLEDPDHGPMLDDG ^' WPTTLRVSVPFWNGOSIEDEHCVPDLVLDARSDLPTAMEYCQRVLRKPAQDCSAYTL SFDTTVFIIESTRQRVAVEATLENRGENAYSTVLNISQSANLQFASLIQKEDSDGSIECV NEERRLQKQVCNVSYPFFRAKAKVAFRLDFEFSKSIFLHHLEIELAAGSDSNERDSTK EDM^APLRFHLKYEADVLFTRSSSLSHYEVKLNSSLERYDGIGPPFSCIFRIQNLGLFPI HGiMMKiTiPiATRSGNRLLKLRDFLTDEVANTscNiWGNSTEYRPTP VΈEDLRRAPQL NHSNSDWSINCNIRLVPNQEINFHLLGNLWLRSLKALKYKSMKIMVN AALQRQFHS PFIFREEDPSRQIWEISKQEDWQWI [SEQ ID NO:5]

Variants, such as natural variants, of the above polypeptide sequence include polypeptides comprising a sequence with at least 60% identity to the amino acid sequence of SEQ ID NO: I₅ preferably at least 70% or 80% or 85% or 90% identity to said sequences, and more preferably at least 95%, 96%, 91%, 98% or 99% identity to said amino acid sequences.

^' Percent identity can be determined by methods well known in the art, for example using the LALIGN program (Huang and Miller, Adv. Appl. Math. (1991) 12:337-357) at the Expasy facility site fh1ty://www.ch.embnet.org/software/L ALIGN_form.html) using as parameters the global alignment option, scoring matrix BLOSUM62, opening gap penalty -14, extending gap penalty -4.

Alternatively, the percent sequence identity between two polypeptides may be determined using suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group and it will be appreciated that percent identity is calculated in relation to polypeptides whose sequence has been aligned optimally.

In an alternative preferred embodiment, the binding moiety selectively binds to a heterodimer comprising an integrin alpha- 11 subunit, such as an alpha- 11 /beta- 1 heterodimer.

In one embodiment, the binding moiety is a nucleic acid molecule capable of inhibiting expression of an integrin alpha- 11 subunit.

The compound and/or binding moiety therein may be a polypeptide.

In a particularly embodiment of the invention, the binding moiety is an antibody or antigen- binding fragment or derivative thereof.

By "antibody" we include substantially intact antibody molecules, as well as chimaeric antibodies, humanised antibodies, human antibodies (wherein at least one amino acid is mutated relative to the naturally occurring human antibodies), single chain antibodies, bispecific antibodies, antibody heavy chains, antibody light chains, homodimers and heterodimers of antibody heavy and/or light chains, and antigen binding fragments and derivatives of the same.

By "antigen-binding fragment" we include a functional fragment of an antibody that is capable of binding to the integrin alpha- 11 subunit or a heterodimer thereof.

Preferably, the antigen-binding fragment is selected from the group consisting of Fv fragments (e.g. single chain Fv and disulphide-bonded Fv), Fab-like fragments (e.g. Fab fragments, Fab' fragments and F(ab)₂ fragments), single variable domains (e.g. VH and V_L domains), domain antibodies (dAbs, including single and dual formats [i.e. dAb-linker-dAb]) and nanobodies (for example, see Revets et al., 2005, Expert Opin Biol Tlier. 5(1): 111-24).

The advantages of using antibody fragments, rather than whole antibodies, are several-fold. The smaller size of the fragments may lead to improved pharmacological properties, such as better penetration of solid tissue. Moreover, antigen-binding fragments such as Fab, Fv, ScFv and dAb antibody fragments can be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of the said fragments.

Also included within the scope of the invention are modified versions of antibodies and an antigen-binding fragment thereof, e.g. modified by the covalent attachment of polyethylene glycol or other suitable polymer.

Although the antibody may be a polyclonal antibody, it is preferred if it is a monoclonal antibody. In some circumstances, particularly if the antibody is going to be administered repeatedly to a human patient, it is preferred if the monoclonal antibody is a human monoclonal antibody or a humanised monoclonal antibody.

Methods of generating antibodies and antibody fragments are well known in the art. For example, antibodies may be generated via any one of several methods which employ induction of in vivo production of antibody molecules, screening of immunoglobulin libraries (Orlandi. et al, 1989. Proc. Natl. Acad. Sd. U.S.A. 86:3833-3837; Winter et al, 1991, Nature 349:293-299) or generation of monoclonal antibody molecules by cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the Epstein-Barr virus (EBV)-hybridoma technique (Kohler et al, 1975. Nature 256:4950497; Kozbor et al., 19S5. J. Immunol. Methods 81:31-42; Cote et al, 1983. Proc. Natl. Acad. ScL USA 80:2026-2030; Cole et al, 1984. MoI. Cell. Biol. 62:109-120).

Suitable monoclonal antibodies to selected antigens may be prepared by known techniques, for example those disclosed in "Monoclonal Antibodies: A manual of techniques ", H Zola (CRC Press, 1988) and in "Monoclonal Hybridoma Antibodies: Techniques and Applications ", J G R Hurrell (CRC Press, 1982).

Antibody fragments can be obtained using methods well known in the art (see, for example, Harlow & Lane, 1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory, New York). For example, antibody fragments according to the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment. Alternatively, antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.

It will be appreciated by persons skilled in the art that for human therapy or diagnostics, human or humanised antibodies are preferably used. Humanised forms of non-human {e.g. murine) antibodies are genetically engineered chimaeric antibodies or antibody fragments having preferably minimal-portions derived from non-human antibodies. Humanised antibodies include antibodies in which complementary determining regions of a human antibody (recipient antibody) are replaced by residues from a complementary deterrnining region of a non human species (donor antibody) such as mouse, rat of rabbit having the desired functionality. In some instances, Fv framework residues of the human antibody are replaced by corresponding non-human residues. Humanised antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported complementarity determining region or framework sequences. In general, the humanised antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the complementarity determining regions correspond to those of a non human antibody and all, or substantially all, of the framework regions correspond to those of a relevant human consensus sequence. Humanised antibodies optimally also include at least a portion of an antibody constant region, such as an Fc region, typically derived from a human antibody (see, for example, Jones et al, 1986. Nature 321:522-525; Riechmann et al, 1988, Nature 332:323-329; Presta, 1992, Curr. Op. Struct. Biol. 2:593-596).

Methods for humanising non-human antibodies are well known in the art. Generally, the humanised antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues, often referred to as imported residues, are typically taken from an imported variable domain. Humanisation can be essentially performed as described (see, for example, Jones et al, 1986, Nature 321:522-525; Reichmann et al, 1988. Nature 332:323-327; Verhoeyen et al, 1988, Science 239:1534- 1536; US 4,816,567) by substituting human complementarity determining regions with corresponding rodent complementarity detenriining regions. Accordingly, such humanised antibodies are chimaeric antibodies, wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanised antibodies may be typically human antibodies in which some complementarity determining region residues and possibly some framework residues are substituted by residues from analogous sites in rodent antibodies.

Human antibodies can also be identified using various techniques known in the art, including phage display libraries (see, for example, Hoogenboom & Winter, 1991, J MoI Biol 227:381; Marks et al, 1991, J. MoI. Biol. 222:581; Cole et al, 1985, In: Monoclonal antibodies and Cancer Therapy, Alan R. Liss, pp. 77; Boerner et al, 1991. J. Immunol. 147:86-95).

Antibodies λvith binding affinity for the integrin alpha- 11 subunit are described in

International Patent Application No. PCT/SE2000/01135 (Publication No. WO 00/75187). Examples of antibodies are a polyclonal antiserum (all cyt) was produced against the peptide CRREPGLDPTPKVLE [SEQ ID NO:6] from the integrin all cytoplasmic domain. Peptide synthesis and conjugation to Keyhole limpet hemocyanin, immunization of rabbits and affinity purification was performed at Innovagen AB (Lund, Sweden).

Further, monoclonal antibodies may be generated by immunisation of e.g. mice, using hybridoma technology, as referred to above. In a further alternative embodiment, the binding moiety is a polypeptide capable of binding selectively to the integrin alpha- 11 subunit or a heterodimer thereof. Polypeptide binding moieties can be identified by means of a screen. A suitable method or screen for identifying peptides or other molecules which selectively bind a target protein or polypeptide may comprise contacting the target protein or polypeptide with a test peptide or other molecule under conditions where binding can occur, and then determining if the test molecule or peptide has bound the target protein or peptide. Methods of detecting binding between two moieties are well known in the art of biochemistry. Preferably, the known technique of phage display is used to identify peptides or other ligand molecules suitable for use as binding moieties. An alternative method includes the yeast two hybrid system.

Polypeptide binding moieties and compounds for use in the invention may be made by methods well known to persons skilled in the art (for example, see Sambrook & Russell, 2000, Molecular Cloning, A Laboratory Manual, Third Edition, Cold Spring Harbor, New York).

In brief, expression vectors may be constructed comprising a nucleic acid molecule which is capable, in an appropriate host, of expressing the pol)φeptide binding moiety or compound encoded by the nucleic acid molecule.

A variety of methods have been developed to operably link nucleic acid molecules, especially DNA, to vectors, for example, via complementary cohesive termini. For instance, complementary homopolymer tracts can be added to the DNA segment to be inserted into the vector DNA. The vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide an alternative method of joining the DNA segment to vectors. The DNA segment, e.g. generated by endonuclease restriction digestion, is treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, enzymes that remove protruding, 3 '-single-stranded termini with their 3 '-5'- exonucleolytic activities, and fill in recessed 3 '-ends with their polymerising activities. The combination of these activities therefore generates blunt-ended DNA segments. The blunt-ended segments are then incubated with a larger molar excess of linker molecules in the presence of an enzyme that is able to catalyse the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase. Thus, the products of the reaction are DNA segments carrying polymeric linker sequences at their ends. These DNA segments are then cleaved with the appropriate restriction enzyme and ligated to an expression vector that has been cleaved with an enzyme that produces termini compatible with those of the DNA segment.

Synthetic linkers containing a variety of restriction endonuclease site are commercially available from a number of sources including International Biotechnologies Inc., New Haven, CN, USA.

A desirable way to modify the DNA encoding the polypeptide of the invention is to use PCR. This method may be used for introducing the DNA into a suitable vector, for example by engineering in suitable restriction sites, or it may be used to modify the DNA in other useful ways as is known in the art.

In this method the DNA to be enzymatically amplified is flanked by two specific primers which themselves become incorporated into the amplified DNA. The said specific primers may contain restriction endonuclease recognition sites which can be used for cloning into expression vectors using methods known in the art.

The DNA (or in the case of retroviral vectors, RNA) is then expressed in a suitable host to produce a polypeptide comprising the compound of the invention or binding moiety thereof. Thus, the DNA encoding the polypeptide may be used in accordance with known techniques, appropriately modified in view of the teachings contained herein, to construct an expression vector, which is then used to transform an appropriate host cell for the expression and production of the compound of the invention or binding moiety thereof. Such techniques include those disclosed in US Patent Nos. 4,440,859 issued 3 April 1984 to Rutter et al, 4,530,901 issued 23 July 1985 to Weissman, 4,582,800 issued 15 April 1986 to Crowl, 4,677,063 issued 30 June 1987 to Mark et αl, 4,678,751 issued 7 July 1987 to GoeddeL 4,704,362 issued 3 November 19S7 to Itakura et αl, 4,710,463 issued 1 December 1987 to Murray, 4,757,006 issued 12 July 1988 to Toole, Jr. et αl, 4,766,075 issued 23 August 1988 to Goeddel et al and 4,810,648 issued 7 March 1989 to Stalker, all of which are incorporated herein by reference.

The DNA (or in the case or retroviral vectors, RNA) encoding the polypeptide constituting the compound of the invention or binding moiety thereof may be joined to a wide variety of other DNA sequences for introduction into an appropriate host. The companion DNA will depend upon the nature of the host, the manner of the introduction of the DNA into the host, and whether episomal maintenance or integration is desired.

Generally, the DNA is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression. If necessary, the DNA may be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognised by the desired host, although such controls are generally available in the expression vector. The vector is then introduced into the host through standard techniques. Generally, not all of the hosts will be transformed by the vector. Therefore, it will be necessary to select for transformed host cells. One selection technique involves incorporating into the expression vector a DNA sequence, with any necessary control elements, that codes for a selectable trait in the transformed cell, such as antibiotic resistance. Alternatively, the gene for such selectable trait can be on another vector, which is used to co-transform the desired host cell.

Host cells that have been transformed by the expression vector of the invention are then cultured for a sufficient time and under appropriate conditions known to those skilled in the art in view of the teachings disclosed herein to permit the expression of the polypeptide, which can then be recovered.

Many expression systems are known, including bacteria (for example, E. coli and Bacillus subtilis), j'easts (for example Saccharomyces cerevisiae), filamentous fungi (for example Aspergillus), plant cells, animal cells and insect cells.

The vectors typically include a prokaryotic replicon, such as the CoIEl ori, for propagation in a prokaryote, even if the vector is to be used for expression in other, non-prokaryotic, cell types. The vectors can also include an appropriate promoter such as a prokaryotic promoter capable of directing the expression (transcription and translation) of the genes in a bacterial host cell, such as E. coli, transformed therewith.

A promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur. Promoter sequences compatible with exemplary bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention.

Typical^' prokaryotic vector plasmids are pUCIS, pUC19, pBR322 and pBR329 available from Biorad Laboratories, (Richmond, CA, USA) and pTrc99A and pKK223-3 available from Pharmacia, Piscataway, NJ, USA.

A typical mammalian cell vector plasmid is pSVL available from Pharmacia, Piscataway, NJ, USA. This vector uses the SV40 late promoter to drive expression of cloned genes, the highest level of expression being found in T antigen-producing cells, such as COS-I cells.

An example of an inducible mammalian expression vector is pMSG, also available from Pharmacia. This vector uses the glucocorticoid-inducible promoter of the mouse mammary tumour virus long terminal repeat to drive expression of the cloned gene.

Useful yeast plasmid vectors are pRS403-406 and pRS413-416 and are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA. Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (Yips) and incorporate the yeast selectable markers HIS3, TRPJ, LEU2 and URA3. Plasmids ρRS413-416 are Yeast_ Centromere plasmids (Ycps).

Other vectors and expression systems are well known in the art for use with a variety of host cells.

The host cell can be either prokaryotic or eukaryotic. Bacterial cells are preferred prokaryotic host cells and typically are a strain of E. coli such as, for example, the E. coli strains DH5 available from Bethesda Research Laboratories Inc., Bethesda, MD, USA, and RRl available from the American Type Culture Collection (ATCC) of Rockville, MD, USA (No. ATCC 31343). Preferred eukaryotic host cells include yeast, insect and mammalian cells, preferably vertebrate cells such as those from a mouse, rat, monkey or human fibroblastic and kidney cell lines. Yeast host cells include YPH499, YPH500 and YPH501 which are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA. Preferred mammalian host cells include Chinese hamster ovary (CHO) cells available from the ATCC as CRL 1658 and 293 cells which are human embryonic kidney cells. Preferred insect cells are SfP cells which can be transfected with baculovirus expression vectors.

Transformation of appropriate cell hosts with a DNA construct of the present invention is accomplished by well known methods that typically depend on the type of vector used. With regard to transformation of prokaryotic host cells, see, for example, Cohen et al (1972) Proc. Natl. Acad. ScL USA 69, 2110 and Sambrook et al (1989) Molecular Cloning, A Laboratory) Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Transformation of yeast cells is described in Sherman et al (1986) Methods In Yeast Genetics, A Laboratory Manual, Cold Spring Harbor, NY. The method of Beggs (197S) Nature 275, 104-109 is also useful. With regard to vertebrate cells, reagents useful in transfecting such cells, for example calcium phosphate and DEAE-dextran or liposome formulations, are available from Stratagene Cloning Systems, or Life Technologies Inc., Gaithersburg, MD 20877, USA.

Electroporation is also useful for transforming and/or transfecting cells and is well known in the art for transforming yeast cells, bacterial cells, insect cells and vertebrate cells.

For example, many bacterial species may be transformed by the methods described in Luchansky et al (1988) MoI. Microbiol. 2, 637-646 incorporated herein by reference. The greatest number of transformants is consistently recovered following electroporation of the DNA-cell mixture suspended in 2.5 PEB using 6250V per cm at 25 μFD.

Methods for transformation of yeast by electroporation are disclosed in Becker & Guarente (1990) Methods Enzymol 194, 182.

Successfully transformed cells, i.e. cells that contain a DNA construct of the present invention, can be identified by well-known techniques. For example, cells resulting from the introduction of an expression construct of the present invention can be grown to produce the polypeptide of the invention. Cells can be harvested and lysed and their DNA content examined for the presence of the DNA using a method such as that described by Southern (1975) J. MoI. Biol. 98, 503 or Berent et al (1985) Biotech. 3, 208. Alternatively, the presence of the protein in the supernatant can be detected using antibodies as described below.

In addition to directly assaying for the presence of recombinant DNA, successful transformation can be confirmed by well known immunological methods when the recombinant DNA is capable of directing the expression of the protein. For example, cells successfully transformed with an expression vector produce proteins displaying appropriate antigenicity.

Samples of cells suspected of being transformed are harvested and assayed for the protein using suitable antibodies.

The host cell may be a host cell within a non-human animal body. Thus, transgenic non- human animals which express a compound according to the first aspect of the invention (or a binding moiety thereof) by virtue of the presence of the transgene are included. Preferably, the transgenic non-human animal is a rodent such as a mouse. Transgenic non-human animals can be made using methods well known in the art.

Methods of cultivating host cells and isolating recombinant proteins are well known in the art. It will be appreciated that, depending on the host cell, the compounds of the invention (or binding moieties thereof) produced may differ. For example, certain host cells, such as yeast or bacterial cells, either do not have, or have different, post-translational modification systems which may result in the production of forms of compounds of the invention (or binding moieties thereof) which may be post-translationally modified in a different way.

It is preferred that compounds of the invention (or binding moieties thereof) are produced in a eukaryotic system, such as a mammalian cell.

According to a less preferred embodiment, the compounds of the invention (or binding moieties thereof) can be produced in vitro using a commercially available in vitro translation system, such as rabbit reticulocyte lysate or wheatgerm lysate (available from Promega). Preferably, the translation system is rabbit reticulocyte lysate. Conveniently, the translation system may be coupled to a transcription system, such as the TNT transcription-translation system (Promega). This system has the advantage of producing suitable mRNA transcript from an encoding DNA polynucleotide in the same reaction as the translation.

It will be appreciated by persons skilled in the art that compounds for use in the medicament embodiment of the invention preferably should inhibit one or more biological activities of the integrin alpha- 11 subunit or heterodimer thereof. Such inhibition of the biological activity of the integrin alpha- 11 subunit or heterodimer thereof by a compound ma}' be in whole or in part. For example, the compound may inhibit the biological activity of the integrin alpha- 11 subunit or heterodimer thereof by at least 10%, preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, and most preferably by 100% compared to the biological activity of the integrin alpha- 11 subunit or heterodimer thereof in a carcinoma, such as the carcinoma cell or the stroma associated cell such e.g. a fibroblast cell, which have not been exposed to the compound. In a preferred embodiment, the compound is capable of inhibiting the biological activity of the integrin alpha- 11 subunit or heterodimer thereof by 50% or more compared to the biological activity of the integrin alpha- 11 subunit or heterodimer thereof in carcinoma cells, such as tumour cells or fibroblast cells, which have not been exposed to the compound. As used herein, "biological activity" refers to the effect of integrin alphal 1 or its heterodimer upon a living organism, tissue or cell. Included herein, but not limited to, is binding to its natural ligand(s), as well as down-stream events therefrom, causing direct or indirect effects on a living organism. Examples of the invention are inhibiting, slowing down or preventing metastases, tumourigenicity, tumour progression. Tumour progression may be e.g. proliferation or differentiation of the tumour cell.

hi a particular embodiment the compound inhibits one or more biological activities of the integrin alpha- 11 subunit selectively. By 'selectively' we mean that the compound inhibits the biological activity of the integrin alpha- 11 subunit or heterodimer thereof to a greater extent than it modulates the activity of other proteins in the carcinoma, such as the tumour cells or associated cells, e.g. stroma cells exemplified as fibroblast cells. In one embodiment the compound inhibits only the biological activity of the integrin alpha- 11 subunit or heterodimer thereof, although it will be appreciated that the expression and activity of other proteins within the carcinoma cells or associated stroma cells, such as fibroblast cells, may change as a downstream consequence of a selective inhibition of the integrin alpha- 11 subunit or heterodimer thereof. Thus, we exclude agents which have a non-specific effect on gene expression, tumourigenicity, and/or tumour cell or tumour tissue progression e.g. cancer cell growth and differentiation.

Advantageously, the compound is also selective in the sense that it acts preferentially on the biological activity of the integrin alpha- 11 subunit in malignant tumour tissue, such as carcinoma tumour tissue, and of cells being malignant cells and its stroma associated cells such as fibroblast cells (i.e. cell-specific inhibition). In one embodiment the compound inhibits the biological activity of the integrin alpha- 11 subunit in rumour stroma cells. In another embodiment, the compound inhibits the biological activity of the intergin alpha-11 subunit in tumour cells.

Thus, in one embodiment, the carcinoma tumour tissue is metastatic.

In a further embodiment, the medicament is for inhibiting, slowing down or prevention of migration of malignant tumour tissue cells.

In still a further embodiment, the medicament is for inhibiting, slowing down or prevention of tumour tissue progression of malignant tumour tissue cells. In a further embodiment, the tumour tissue progression is tumour growth. In still a further embodiment, the tumour tissue progression is tumour tissue cell differentiation.

In still a further embodiment, the medicament is for altering tumour tissue associated stroma cell expression of alphal l. By "altering expression of alpha 11" we include inhibiting and blocking - fully or partially - the expression of alpha 11 thereby altering the expression. Said blocking or inhibiting may be via a binding moiety according to the invention. Said binding moiety may block or bind either at protein level, by e.g. an antibody or similar means or at nucleic acid level, e.g. by anti-sense technology known in the art or similar means.

In one embodiment, the binding moiety is an antibody.

Further embodiments are wherein the medicament is for inhibiting, slowing down or prevention of tumourigenicity of malignant tumour tissue cells. Optionally, the compounds used in the present the invention also comprise a further moiety. Such further moiety may be any further moiety which confers on the compound a useful property with respect to the treatment or imaging or diagnosis of carcinomas, e.g. target cell specific portions, cytotoxic moieties and/or detectable moieties.

Thus, in one embodiment, the compound comprises a target cell specific portion with binding affinity for malignant tumour tissue cells or cells associated with the malignant cells such as tumour stroma cells e.^•ag.⁴ fibroblast cells.

By "target cell specific" portion we mean a portion of the compound which comprises one or more binding sites which recognise and bind to entities on the target tumour tissue including cells associated with the malignant cells such as tumour stroma cells e.g. fibroblast cells. Upon contact with the target cell, the target cell specific portion may be internalised along with the alpha- 11 binding portion.

The entities recognised by the target cell-specific portion are expressed predominantly, and preferably exclusively, on the target cell. The target cell specific portion may contain one or more binding sites for different entities expressed on the same target cell type, or one or more binding sites for different entities expressed on two or more different target cell types.

In one particular embodiment the target cell-specific portion recognises the target cell with high avidity.

By "high avidity" we mean that the target cell-specific portion recognises the target cell with a binding constant of at least K_d = 10^"6 M, preferably at least Kd = 10^"9 M, suitably Kd = 10^{" 10}M, more suitably K_d = 10^"11 M₅ yet more suitably still K_d = 10^"12M, and more preferably IQ = 10^"15M or even K_d = 10^"IS M.

The entity which is recognised may be an}' suitable entity which is expressed by rumour cells, including its associated cells such as stroma cells e.g. . Often, the entity which is recognised will be an antigen.

Examples of antigens include those listed in Table 1. Table 1

Tumour Tissue Associated Antigens

Antigen Antibody Existing Uses

Carcino-embryonic C46 (Amersham) Imaging & Therapy of Antigen 85A12 (Unipath) colon/rectum tumours.

Placental Alkaline H17E2 (ICRP, Imaging & Therapy of Phosphatase Travers & Bodmer) testicular and ovarian cancers.

Pan Carcinoma NR-LU-10 (NeoRx Imaging & Therapy of Corporation) various carcinomas incl. small cell lung cancer.

Polymorphic Epithelial HMFGl (Taylor- Imaging & Therapy of

Mucin (Human milk fat Papadimitriou, ICRF) ovarian cancer, pleural globule (Antisoma pic) effusions, breast, lung

& other common epithelial cancers.

Human milk mucin SM-S(IgGl)¹ Diagnosis, Imaging core protein & Therapy of breast cancer

β-human Chorionic W14 Targeting of enzyme Gonadotropin (CPG2) to human xenograft choriocarcinoma in nude mice. (Searle et at (198I) Br. J. Cancer 44, 137-144) A Carbohydrate on L6 (IgG2a)² Targeting of alkaline Human Carcinomas phosphatase. (Senter eϊ al (198S) -PrOC. Natl. Acad. ScL USA 85, 4842-4846

CD20 Antigen on B 1 F5 (IgG2a)³ Targeting of alkaline

Lymphoma (normal and phosphatase. (Sente^'r et neoplastic) al (1988) Proc. Natl. Acad. ScL USA 85, 4842-4846

¹ Burchell et al (1987) Cancer Res. 47, 5476-5482

² Hellstrόm et al (1986) Cancer Res. 46, 3917-3923

³ Clarke et al (1985) Proc. Natl. Acad. Sd. USA 82, 1766-1770

Other antigens include alphafoetoprotein, Ca- 125, prostate specific antigen and members of the epidermal growth factor receptor family, namely EGFR, erb B3 and erb B4.

Preferably, the target cell specific portion is an antibody or antigen-binding fragment or derivative thereof. Thus, in one embodiment, the compound may comprise a binding moiety capable of binding selectively to integrin alpha- 11 subunit or a heterodimer thereof and a further binding moiety capable of further binding to the target tumour cells (e.g. non-small cell lung cancer cell or associated stroma cells e.g. fibroblast cells), for example a bi-specific antibody or bi-specific fragment or variant thereof.

In an additional embodiment, the further moiety is one which is useful in killing or imaging cells associated with malignant carcinoma. Preferably, the further moiety is one which is able to kill the cells to which the compound is able to bind.

Advantageously, the binding moiety and further moiety are covalently attached. In a preferred embodiment of the invention the further moiety is directly or indirectly cytotoxic. In particular the further moiety is preferably directly or indirectly toxic to malignant tumour tissue cells, such as NSCLC cells.

By "directly cytotoxic" we include the meaning that the moiety is one which on its own is cytotoxic. By "indirectly cytotoxic" we include the meaning that the moiety is one which, although is not itself cytotoxic, can induce cytotoxicity, for example by its action on a further molecule or by further action on it.

For example, the cytotoxic moiety is a cytotoxic chemotherapeutic agent. Suitable cytotoxic chemotherapeutic agents are well known in the art.

Cytotoxic chemotherapeutic agents, such as anticancer agents, include: alkylating agents including nitrogen mustards such as mechlorethamine (HN₂), cyclophosphamide, ifosfamide, melphalan (L-sarcolysin) and chlorambucil; ethyl enirnines and memylmelarnines such as hexamethyknelamine, thiotepa; alkyl sulphonates such as busulfan; nitrosoureas such as carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU) and streptozocin (streptozotocin); and triazenes such as decarbazine (DTIC; dimethyltriazenoirnidazole- carboxamide); Antimetabolites including folic acid analogues such as methotrexate (arnethopterin); pyrimidine analogues such as fiuorouracil (5-fluorouracil; 5-FU), fioxuridine (fluorodeoxyuridine; FUdR) and cytarabine (cytosine arabinoside); and purine analogues and related inhibitors such as mercaptopurine (6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; TG) and pentostatin (2'-deoxycoformycin). Natural Products including vinca alkaloids such as vinblastine (VLB) and vincristine; epipodophyllotoxins such as etoposide and teniposide; antibiotics such as dactinomyciα (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin) and mitomycin (mitomycin C); enzymes such as L-asparaginase; and biological response modifiers such as interferon alphenomes. Miscellaneous agents including platinum coordination complexes such as cisplatin (c/s-DDP) and carboplatin; anthracenedione such as mitoxantrone and anthracycline; substituted urea such as hydroxyurea; methyl hydrazine derivative such as procarbazine (N-methylhydrazine, MIH); and adrenocortical suppressant such as mitotane (cψ'-DDD) and aπmoglutethimide; t&xol and analogues/derivatives; and hormone agonists/antagonists such as flutamide and tamoxifen. Several of these agents have previously been attached to antibodies and other target site- delivery agents, and so compounds of the invention comprising these agents may readily be made by the person skilled in the art. For example, carbodiimide conjugation (Bauminger & Wilchek (1980) Methods Enzymol 70, 151-159; incorporated herein by reference) may be used to conjugate a variety of agents, including doxorubicin, to antibodies or peptides.

Carbodiimides comprise a group of compounds that have the general formula R₁-N=C=N-R₂, where Ri and R₂ can be aliphatic or aromatic, and are used for synthesis of peptide bonds. The preparative procedure is simple, relatively fast, and is carried out under mild conditions. Carbodiimide compounds attack carboxylic groups to change them into reactive sites for free amino groups.

The water soluble carbodiimide, l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) is particularly useful for conjugating a functional moiety to a binding moiety and may be used to conjugate doxorubicin to tumour homing peptides. The conjugation of doxorubicin and a binding moiety requires the presence of an amino group, which is provided by doxorubicin, and a carboxyl group, which is provided by the binding moiety such as an antibody or peptide.

In addition to using carbodiimides for the direct formation of peptide bonds, EDC also can be used to prepare active esters such as N-hydroxysuccinimide (NHS) ester. The NHS ester, which binds only to amino groups, then can be used to induce the formation of an amide bond with the single amino group of the doxorubicin. The use of EDC and NHS in combination is commonly used for conjugation in order to increase yield of conjugate formation (Bauminger & Wilchek, supra, 1980).

Other methods for conjugating a functional moiety to a binding moiety also can be used. For example, sodium periodate oxidation followed by reductive alkylation of appropriate reactants can be used, as can glutaraldehyde cross-linking. However, it is recognised that, regardless of which method of producing a conjugate of the invention is selected, a determination must be made that the binding moiety maintains its targeting ability and that the functional moiety maintains its relevant function.

In a further embodiment of the invention, the cytotoxic moiety is a cytotoxic peptide or polypeptide moiety by which we include any moiety which leads to cell death. Cj'totoxic peptide and polypeptide moieties are well known in the art and include, for example, ricin, abrin, Pseudomonas exotoxin, tissue factor and the like. Methods for linking them to targeting moieties such as antibodies are also known in the art. The use of ricin as a cytotoxic agent is described in Burrows & Thorpe (1993) Proc. Natl. Acad. Sci. USA 90, 8996-9000, incorporated herein by reference, and the use of tissue factor, which leads to localised blood clotting and infarction of a tumour, has been described by Ran et al (199S) Cancel- Res. 58, 4646-4653 and Huang et al (1997) Science 275, 547-550. Tsai et al (1995) Dis. Colon Rectum 38, 1067-1074 describes the abrin A chain conjugated to a monoclonal antibody and is incorporated herein by reference. Other ribosome inactivating proteins are described as cytotoxic agents in WO 96/06641. Pseudomonas exotoxin may also be used as the cytotoxic polypeptide moiety (see, for example, Aiello et al (1995) Proc. Natl. Acad. Sci USA 92, 10457-10461 ; incorporated herein by reference).

Certain cytokines, such as TNFα and IL-2, may also be useful as cytotoxic agents.

Likewise, certain radioactive atoms may also be cytotoxic if delivered in sufficient doses. Thus, the cytotoxic moiety may comprise a radioactive atom which, in use, delivers a sufficient quantity of radioactivity to the target site so as to be cytotoxic. Suitable radioactive atoms include phosphorus-32, iodine-125, iodine-131, indium-Ill, rhenium-186, rhenium- 1 SS or yttrium-90, or any other isotope which emits enough energy to destroy neighbouring cells, organelles or nucleic acid. Preferably, the isotopes and density of radioactive atoms in the compound of the invention are such that a dose of more than 4000 cGy (preferably at least 6000, 8000 or 10000 cGy) is delivered to the target site and, preferably, to the cells at the target site and their organelles, particularly the nucleus. The radioactive atom may be attached to the binding moiety in known ways. For example EDTA or another chelating agent may be attached to the binding moiety and used to attach 1¹¹In or ⁹⁰Y. Tyrosine residues may be directly labelled with ¹²⁵I or ¹³¹I.

The cytotoxic moiety may be a suitable indirectly cytotoxic polypeptide. In a particularly preferred embodiment, the indirectly cytotoxic polypeptide is a polypeptide which has enzymatic activity and can convert a relatively non-toxic prodrug into a cytotoxic drug. When the binding moiety is an antibody this type of system is often referred to as ADEPT (Antibody-Directed Enzyme Prodrug Therapy). The system requires that the binding moiety locates the enzymatic portion to the desired site in the body of the patient (i.e. the malignant tumour cells, e.g. NSCLC cells) and after allowing time for the enzyme to localise at the site, administering a prodrug which is a substrate for the enzyme, the end product of the catalysis being a cytotoxic compound. The object of the approach is to maximise the concentration of drug at the desired site and to minimise the concentration of drug in normal tissues (see Senter, P. D. et al (1988) "Anti-tumour effects of antibody- alkaline phosphatase conjugates in combination with etoposide phosphate" Proc. Natl. Acad. Sci. USA 85, 4842-4846; Bagshawe (1987) Br. J. Cancer 56, 531-2; and Bagshawe, K.D. et al (1988) "A cytotoxic agent can be generated selectively at cancer sites" Br. J. Cancer. 58, 700-703.)

Clearly, any binding moiety with specificity the integrin alpha- 11 subunit or a heterodimer thereof may be used in place of an antibody in this type of directed enzyme prodrug therapy system.

The enzyme and prodrug of the system using a targeted enzyme as described herein may be any of those previously proposed. The cytotoxic substance may be any existing anti-cancer drug such as an alkylating agent; an agent which intercalates in DNA; an agent which inhibits any key enzymes such as dihydrofolate reductase, thymidine synthetase, ribonucleotide reductase, nucleoside kinases or topoisomerase; or an agent which effects cell death by interacting with any other cellular constituent. Etoposide is an example of a topoisomerase inhibitor. Reported prodrug systems include: a phenol mustard prodrug activated by an E. coli β- glucuronidase (Wang et al, 1992 and Roffler el al, 1991); a doxorubicin prodrug activated by a human β-glucuronidase (Bosslet et al, 1994); further doxorubicin prodrugs activated by coffee bean α-galactosidase (Azoulay et al, 1995); daunorubicin prodrugs, activated by coffee bean α-D-galactosidase (Gesson et al, 1994); a 5-fluorouridine prodrug activated by an E. coli β-D-galactosidase (Abraham et al, 1994); and methotrexate prodrugs (e.g. methotrexate- alanine) activated by carboxypeptidase A (Kuefher et al, 1990, Vitols et al, 1992 and Vitols et al, 1995). These and others are included in the Table 2 below.

Table 2

Enzyme Prodrug

Derivatives of L-glutamic acid and benzoic acid

Carboxypeptidase G2 mustards, aniline mustards, phenol mustards and phenylenediamine mustards; fluorinated derivatives of these

Alkaline phosphatase Etoposide phosphate

Mitomycin phosphate

Beta-glucuronidase £>-Hydroxyaniline mustard-glucuronide

Epirubicin-glucuronide

Penicillin- V-amidase Adriamycin-N phenoxyacetyl Penicillin-G-amidase N-(4'-hydroxyphenyl acetyl) palytoxin

Doxorubicin and melphalan

Beta-lactamase Nitrogen mustard-cephalosporin jP-phenylenediamine; doxorubicin derivatives; vinbl astine derivati ve-cephalo sporin, cephalosporin mustard; a taxol derivative

Beta-glucosidase Cyanophenylmefhyl-beta-D-gluco- pyranosiduronic acid

Nitroreductase 5-(Azaridin- 1 -yl-)-2,4-dinitrobenzamide Cytosine deaminase 5-Fluorocytosine Carboxypeptidase A Methotrexate- alanine The above table is adapted from Bagshawe (1995) DrugDev. Res. 34, 220-230, from which fαll references for these various systems may be obtained; the taxol derivative is described in Rodrigues, MX. et al (1995) Chemistiγ & Biology 2, 223.

Suitable enzymes for forming part of the enzymatic portion a compound of the invention include: exopeptidases, such as carboxypeptidases G, Gl and G2 (for glutamylated mustard prodrugs), carboxypeptidases A and B (for MTX-based prodrugs) and aminopeptidases (for 2- α-aminocyl MTC prodrugs); endopeptidases, such as e.g. thrombolysin (for thrombin prodrugs); hydrolases, such as phosphatases (e.g. alkaline' phosphatase) or sulphatases (e.g. aryl sulphatases) (for phosphylated or sulphated prodrugs); amidases, such as penicillin amidases and arylacyl amidase; lactamases, such as β-lactamases; glycosidases, such as β- glucuronidase (for β-glucuronomide anthracyclines), α-galactosidase (for amygdalin) and β- galactosidase (for β-galactose anthracycline); deaminases, such as cytosine deaminase (for 5FC); kinases, such as urokinase and thymidine kinase (for gancyclovir); reductases, such as nitroreductase (for CB 1954 and analogues), azoreductase (for azobenzene mustards) and DT- diaphorase (for CB 1954); oxidases, such as glucose oxidase (for glucose), xanthine oxidase (for xanthine) and lactoperoxidase; DL-racemases, catalytic antibodies and cyclodextrins.

Preferably, the prodrug is relatively non-toxic compared to the cytotoxic drug. Typically, it has less than 10% of the toxicity, preferably less than 1% of the toxicity as measured in a suitable in vitro cytotoxicity test.

It is likely that the moiety which is able to convert a prodrug to a cytotoxic drug will be active in isolation from the rest of the compound but it is necessary only for it to be active when (a) it is in combination with the rest of the compound and (b) the compound is attached to, adjacent to or internalised in target cells.

When each moiety of the compound is a polypeptide, the two portions may be linked together by any of the conventional ways of cross-linking polypeptides, such as those generally described in O'Sullivan et al (1979) Anal. Biochem. 100, 100-108. For example, the binding moiety may be enriched with thiol groups and the further moiety reacted with a bifunctional agent capable of reacting with those thiol groups, for example the N-hydroxysuccinimide ester of iodoacetic acid (NHIA) or N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP). Amide and thioether bonds, for example achieved with m-maleimidobenzoyl-N- hydroxysuccinimide ester, are generally more stable in vivo than disulphide bonds.

Alternatively, the compound may be produced as a fusion compound by recombinant DNA techniques whereby a length of DNA comprises respective regions encoding the two moieties of the compound of the invention either adjacent one another or separated by a region encoding a linker peptide which does not destroy the desired properties of the compound. Conceivably, the two portions of the compound may overlap wholly or partly.

In a further preferred embodiment, the cytotoxic moiety may be a radiosensitizer.

Radiosensitizers include fluoropyrimidines, thymidine analogues, hydroxyurea, gemcitabine, fludarabine, nicotinamide, halogenated pyrimidines, 3-aminobenzamide, 3- aminobenzodiamide, etanixadole, pimonidazole and misonidazole (see, for example, McGinn et al (1996) J. Natl. Cancer Inst. 88, 1193-11203; Shewach & Lawrence (1996) Invest. New Drugs 14, 257-263; Horsman (1995) Acta Oncol. 34, 571-587; Shenoy & Singh (1992) Clin. Invest. 10, 533-551 ; Mitchell et al (1989) Int. J. Radial Biol. 56, 827-836; Iliakis & Kurtzman (1989) Int. J. Radial Oncol. Biol. Phys. 16, 1235-1241; Brown (1989) Int. J. Radial Oncol. Biol. Phys. 16, 987-993; Brown (1985) Cancer 55, 2222-2228).

Also, delivery of genes into cells can radiosensitise them, for example delivery of the p53 gene or cyclin D (Lang et al (1998) J. Neurosurg. 89, 125-132; Coco Martin et al (1999) Cancer Res. 59, 1134-1140).

The further moiety may be one which becomes cytotoxic, or releases a cytotoxic moiety, upon irradiation. For example, the boron-10 isotope, when appropriately irradiated, releases alpha (α) particles which are cytotoxic (for example, see LIS 4, 348, 376 to Goldenberg; Primus et al (1996) Bioconjug. Chan. 7, 532-535).

Similarly, the cytotoxic moiety may be one which is useful in photodynamic therapy such as photofiϊn (see, for example, Dougherty et al (1998) J. Natl. Cancer Inst. 90, 889-905).

The further moiety may comprise a nucleic acid molecule which is directly or indirectly cytotoxic. For example, the nucleic acid molecule may be an antisense oligonucleotide which, upon localisation at the target site is able to enter cells and lead to their death. The oligonucleotide, therefore, may be one which prevents expression of an essential gene, or one which leads to a change in gene expression which causes apoptosis.

Examples of suitable oligonucleotides include those directed at bcl-2 (Ziegler et al (1997) J. Natl. Cancer Inst. 89, 1027-1036), and DNA polymerase α and topoisomerase Ilα (Lee et al (1996) Anticancer Res. 16, 1805-1811.

Peptide nucleic acids may be useful in place of conventional nucleic acids (see Knudsen & Nielsen (1997) Anticancer Drugs 8, 113-118).

In a further embodiment, the binding moiety may be comprised in a delivery vehicle for delivering nucleic acid to the target. The delivery vehicle may be any suitable delivery vehicle. It may, for example, be a liposome containing nucleic acid, or it may be a virus or virus-like particle which is able to deliver nucleic acid. In these cases, the binding moiety is typically present on the surface of the delivery vehicle. For example, the binding moiety, such as a suitable antibody fragment, may be present in the outer surface of a liposome and the nucleic acid to be delivered may be present in the interior of the liposome. As another example, a viral vector, such as a retroviral or adenoviral vector, is engineered so that the binding moiety is attached to or located in the surface of the viral particle thus enabling the viral particle to be targeted to the desired site. Targeted delivery systems are also known such as the modified adenovirus system described in WO 94/10323 wherein, typically, the DNA is carried within the adenovirus, or adenovirus-like, particle. Michael et al (1995) Gene Therapy 2, 660-668 describes modification of adenovirus to add a cell-selective moiety into a fibre protein. Targeted retroviruses are also available for use in the invention; for example, sequences conferring specific binding affinities may be engineered into pre-existing viral env genes (see Miller & Vile (1995) Faseb J. 9, 190-199 for a review of this and other targeted vectors for gene therapy).

Immunoliposomes (antibody-directed liposomes) may be used in which the binding moiety is an antibody. For the preparation of immuno-liposomes MPB-PE (N-[4-(p-maleimidophenyl)- butyryl]-phosphatidylethanolamine) is synthesised according to the method of Martin & Papahadjopoulos (1982) J. Biol. Chem. 257, 286-288. MPB-PE is incorporated into the liposomal bilayers to allow a covalent coupling of the antibody, or fragment thereof, to the liposomal surface. The liposome is conveniently loaded with the DNA or other genetic construct for delivery to the target cells, for example, by forming the said liposomes in a solution of the DNA or other genetic construct, followed by sequential extrusion through polycarbonate membrane filters with 0.6 μm and 0.2 μm pore size under nitrogen pressures up to 0.8 MPa. After extrusion, entrapped DNA construct is separated from free DNA construct by ultracentrifugation at 80 000 x g for 45 min. Freshly prepared MPB-PE-liposomes in deoxygenated buffer are mixed with freshly prepared antibody (or fragment thereof) and the coupling reactions are carried out in a nitrogen atmosphere at 4^°C under constant end over end rotation overnight. The immunoliposomes are separated from unconjugated antibodies by ultracentrifugation at 80 000 x g for 45 min. Immunoliposomes may be injected intraperitoneally or directly into the tumour tissue.

The nucleic acid delivered to the target site (z^'.e. malignant tumour tissue cells) may be any suitable DNA which leads, directly or indirectly, to cytotoxicity. For example, the nucleic acid may encode a ribozyme which is cytotoxic to the cell, or it may encode an enzyme which is able to convert a substantially non-toxic prodrug into a cytotoxic drug (this latter system is sometime called GDEPT: Gene Directed Enzyme Prodrug Therapy).

Ribozymes which may be encoded in the nucleic acid to be delivered to the target are described in Cech and Herschlag "Site-specific cleavage of single stranded DNA" US 5,180,818; Altaian et al "Cleavage of targeted RNA by RNAse P" US 5,168,053, Cantin et al "Ribozyme cleavage of HIV-I RNA" US 5,149,796; Cech et al "RNA ribozyme restriction endoribonucleases and methods", US 5,116,742; Been et al "RNA ribozyme polymerases, dephosphorylases, restriction endonucl eases and methods", US 5,093,246; and Been et al "RNA ribozyme polymerases, dephosphorylases, restriction endoribonucleases and methods; cleaves single-stranded RNA at specific site by transesterification", US 4,987,071, all incorporated herein by reference. Suitable targets for ribozymes include transcription factors such as c-fos and c-myc, and bcl-2. Durai et al (1997) Anticancer Res. 17, 3307-3312 describes a hammerhead ribozyme against bcl-2.

EP 0 415 731 describes the GDEPT system. Similar considerations concerning the choice of enzyme and prodrug apply to the GDEPT system as to the ADEPT system described above.

The nucleic acid delivered to the target site may encode a directly cytotoxic polypeptide. Alternatively, the further moiety may comprise a polypeptide or a polynucleotide encoding a polypeptide which is not either directly or indirectly cytotoxic but is of therapeutic benefit. Examples of such polypeptides include antiproliferative or anti-inflammatory cytokines, and antiproliferative, immunomodulatory or factors influencing blood clotting which may be of benefit in treating malignant tumours.

The further moiety may usefully be an inhibitor of angiogenesis such as the peptides angiostatin or endostatin. The further moiety may also usefully be an enzyme which converts a precursor polypeptide to angiostatin or endostatin. Human matrix metallo-proteases such as macrophage elastase, gelatinase and stromolysin convert plasminogen to angiostatin (Cornelius et al (1998) J. Immunol. 161, 6845-6S52). Plasminogen is a precursor of angiostatin.

In a further embodiment of the invention, the further moiety comprised in the compound of the invention is a readily detectable moiety.

By a "readily detectable moiety" we include the meaning that the moiety is one which, when located at the target site following administration of the compound of the invention into a patient, may be detected, typically non-invasively from outside the body and the site of the target located. Thus, the compounds of this embodiment of the invention are useful in imaging and diagnosis.

Typically, the readily detectable moiety is or comprises a radioactive atom which is useful in imaging. Suitable radioactive atoms include ^99mTc and ¹²³I for scintigraphic studies. Other readily detectable moieties include, for example, spin labels for magnetic resonance imaging (MRI) such as ¹²³I again, ¹³¹1, ¹¹¹In, ¹⁹F, ¹³C, ¹⁵N, ¹⁷O, gadolinium, manganese or iron. Clearly, the compound for use in the invention must have sufficient of the appropriate atomic isotopes in order for the molecule to be readily detectable.

The radio- or other labels may be incorporated in the compound of the invention in known ways. For example, if the binding moiety is a polypeptide it may be biosynthesised or may be synthesised by chemical amino acid synthesis using suitable amino acid precursors involving, for example, fluorine- 19 in place of hydrogen. Labels such as ^99mTc, ¹²³1, ¹⁸⁶Rh, ¹⁸⁸Rh and ¹¹¹In can, for example, be attached via cysteine residues in the binding moiety. Yttrium-90 can be attached via a lysine residue. The IODOGEN method (Fraker et al (1978) Biochem. Biophys. Res. Comm. 80, 49-57) can be used to incorporate ¹²³I. Reference ("Monoclonal Antibodies in Immunoscintigraphy", J-F Chatal, CRC Press, 1989) describes other methods in detail.

In a further preferred embodiment of the invention the further moiety is able to bind selectively to a directly or indirectly cytotoxic moiety or to a readily detectable moiety. Thus, in this embodiment, the further moiety may be any moiety which binds to a further compound or component which is cytotoxic or readily detectable.

The further moiety may, therefore be an antibody which selectively binds to the further compound or component, or it may be some other binding moiety such as streptavidin or biotin or the like. The following examples illustrate the types of molecules that are included in the invention; other such molecules are readily apparent from the teachings herein.

For example, the compound may comprise or consist of a bispecific antibody wherein one binding site comprises the binding moiety (which selectively binds to the integrin alpha- 11 subunit or a heterodimer thereof) and the second binding site comprises a moiety which binds to, for example, an enzyme which is able to convert a substantially non-toxic prodrug to a cytotoxic drug.

Alternatively, the compound may comprise an antibody which selectively binds to the integrin alpha- 11 subunit or a heterodimer thereof, to which is bound biotin. Avidin or streptavidin which has been labelled with a readily detectable label may be used in conjunction with the biotin labelled antibody in a two-phase imaging system wherein the biotin labelled antibody is first localised to the target site in the patient, and then the labelled avidin or streptavidin is administered to the patient. Bispecific antibodies and biotin/streptavidin (avidin) systems are reviewed by Rosebrough (1996) O JNucl. Med. 40, 234-251.

In one embodiment of the invention, the binding moiety and the further moiety are polypeptides which are fused. The uses of the above aspects of the invention provide agents and medicaments for treating, detecting, imaging and/or diagnosing malignant tumour tissue, e.g. carcinomas, as described in more detail below. In a one particular embodiment, the malignant tumour tissue is metastatic.

Thus, the invention provides medicaments for inhibiting the migration of tumour tissue cells (for example, towards a chemoattractant).

It will be appreciated by persons skilled in the art that compounds used in the above-described aspects of the invention are preferably provided in the form of a pharmaceutical composition comprising the compound and a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" is included that the formulation is sterile and pyrogen free. Suitable pharmaceutical carriers are well known in the art of pharmacy. The carrier(s) must be "acceptable" in the sense of being compatible with the compound of the invention and not deleterious to the recipients thereof. Typically, the carriers will be water or saline which will be sterile and pyrogen free; however, other acceptable carriers may be used. Thus, "pharmaceutically acceptable carrier" and "pharmaceutically acceptable excipient" includes any compound(s) used in forming a part of the formulation that is intended to act merely as a carrier, i.e., not intended to have biological activity itself. The pharmaceutically acceptable carrier or excipient is generally safe, non-toxic, and neither biologically nor othenvise undesirable. A pharmaceutically acceptable carrier or excipient as used herein includes both one and more than one such carrier or excipient.

The terms "treating", and "treatment", and the like are used herein to generally mean obtaining a desired pharmacological and physiological effect. Further, it refers to any process, action, application, therapy, or the like, wherein a mammal, including a human being, is subject to medical aid with the object of improving the mammal's condition, directly or indirectly. More specifically, it may be one or more of the following inhibiting, preventing, alleviating non-small cell lung carcinoma, reduction, slowing, inhibition of non-small cell lung carcinoma cell migration, loss of metastatic lesions in any solid tumour tissue, inhibited or reduced development of new metastatic lesions in any solid tumour tissue after treatment has started.

The term "inhibition" in the context of neoplasia, tumour tissue growth, metastases, invasiveness, etc., maybe assessed by delayed appearance of primary or secondary tumours and tumour tissue, slowed development of primary or secondary tumours and tumour tissue, decreased occurrence of primary or secondary tumours and rumour tissue, slowed or decreased severity of secondary effects of disease, arrested rumour tissue growth and regression of tumour tissue, among others. In the extreme, complete inhibition, is referred to herein as prevention.

The term "prevention" includes either preventing the onset of clinically evident neoplasia, tumour tissue growth, metastases, invasiveness, preventing onset of primary or secondary tumours and tumour tissue, etc., altogether or preventing the onset of a preclinically evident stage of neoplasia, rumour tissue growth, metastases, invasiveness, onset of primary or secondary tumour tissue in individuals at risk. Also intended to be encompassed by this definition is the prevention of initiation for malignant cells or to arrest or reverse the progression of premalignant cells to malignant cells. This includes prophylactic treatment of those at risk of developing the neoplasia, tumour tissue growth, metastases, invasive tumours, and onset of primary or secondary tumours and its surrounding tumour stroma.

The compounds for use in the invention can be formulated at various concentrations, depending on the efficacy/to xicity of the compound being used. Preferably, the formulation comprises the agent of the invention at a concentration of between 0.1 μM and 1 mM, more preferably between 1 μM and 100 μM, between 5 μM and 50 μM, between 10 μM and 50 μM, between 20 μM and 40 μM and most preferably about 30 μM. For in vitro applications, formulations may comprise a lower concentration of a compound of the invention, for example between 0.0025 μM and 1 μM.

It will be appreciated by persons skilled in the art that the medicaments and agents will generally be administered in admixture with a suitable pharmaceutical excipient diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice (for example, see Remington: TJie Science and Practice of Pharmacy, 19^th edition, 1995, Ed. Alfonso Gennaro, Mack Publishing Company, Pennsylvania, USA).

For example, the medicaments and agents can be administered orally, buccally or sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which ma}' contain flavouring or colouring agents, for immediate-, delayed- or controlled-release applications. The medicaments and agents may also be administered via intracavernosal injection.

Such tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch

(preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxy-propylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the compounds of the invention may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.

The medicaments and agents of the invention can also be administered parenterally, for example, intravenously, intra-articularly, intra-arterially, intraperitoneally, intra-thecally, intraventricular!}', intrasternally, intracranially, intra-muscularly or subcutaneously, or they may be administered by infusion techniques. They are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile poλvders, granules and tablets of the kind previously described.

For oral and parenteral administration to human patients, the daily dosage level of the medicaments and agents will usually be from 1 to 1000 mg per adult (i.e. from about 0.015 to 15 mg/kg), administered in single or divided doses.

The medicaments and agents can also be administered intranasally or by inhalation and are conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurised container, pump, spray or nebuliser with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoro-methane, dichlorotetrafluoro-ethane, a hydrofiuoroalkane such as 1,1,1,2-tetrafluoroethane (HFA 134A3 or 1,1,1 ,2,3,3,3- heptafluoropropane (HFA 227EA3), carbon dioxide or other suitable gas. In the case of a pressurised aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurised container, pump, spray or nebuliser may contain a solution or suspension of the active compound, e.g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g. sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of a compound of the invention and a suitable powder base such as lactose or starch.

Aerosol or dry powder formulations are preferably arranged so that each metered dose or 'puff contains at least 1 mg of a compound of the invention for delivery to the patient. It will be appreciated that the overall daily dose with an aerosol will vary from patient to patient, and may be administered in a single dose or, more usually, in divided doses throughout the day.

Alternatively, the medicaments and agents can be administered in the form of a suppository or pessary, or they may be applied topically in the form of a lotion, solution, cream, ointment or dusting powder. The compounds of the invention may also be transdermally administered, for example, by the use of a skin patch. They may also be administered b}' the ocular route. For application topically to the skin, the medicaments and agents can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, they can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2- octyldodecanol, benzyl alcohol and water.

Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier.

Where the medicament or agent is a polypeptide, it may be preferable to use a sustained- release drug delivery system, such as a microsphere. These are designed specifically to reduce the frequency of injections. An example of such a system is Nutropin Depot which encapsulates recombinant human growth hormone (rhGH) in biodegradable microspheres that, once injected, release rhGH slowly over a sustained period.

Sustained-release immunoglobulin compositions also include liposomally entrapped immunoglobulin. Liposomes containing the immunoglobulin are prepared by methods known per se. See, for example Epstein et ai, Proc. Natl. Acad. ScL USA 82: 36S8-92 (1985); Hwang et ah, Proc. Natl. Acad. ScI USA 77: 4030-4 (1980); U.S. Patent Nos. 4,485,045; 4,544, 545; 6,139,869; and 6,027,726. Ordinarily, the liposomes are of the small (about 200 to about 800 Angstroms), unilamellar type in which the lipid content is greater than about 30 mole percent (mol. %) cholesterol; the selected proportion being adjusted for the optimal immunoglobulin therapy.

Alternatively, polypeptide medicaments and agents can be administered b3' a surgically implanted device that releases the drug directly to the required site. Electroporation therapy (EPT) systems can also be employed for the administration of proteins and polypeptides. A device which delivers a pulsed electric field to cells increases the permeability of the cell membranes to the drag, resulting in a significant enhancement of intracellular drag delivery.

Proteins and polypeptides can also be delivered by electroincorporation (EI). EI occurs when small particles of up to 30 microns in diameter on the surface of the skin experience electrical pulses identical or similar to those used in electroporation. In EI, these particles are driven through the stratum corneum and into deeper layers of the skin. The particles can be loaded or coated with drags or genes or can simply act as "bullets" that generate pores in the skin through which the drags can enter.

An alternative method of protein and polypeptide deliver}' is the thermo-sensitive ReGeI injectable. Below body temperature, ReGeI is an injectable liquid while at body temperature it immediately forms a gel reservoir that slowly erodes and dissolves into known, safe, biodegradable polymers. The active drag is delivered over time as the biopolymers dissolve.

Protein and polypeptide pharmaceuticals can also be delivered orally. One such system employs a natural process for oral uptake of vitamin B 12 in the body to co-deliver proteins and polypeptides. By riding the vitamin B 12 uptake system, the protein or polypeptide can move through the intestinal wall. Complexes are produced between vitamin B 12 analogues and the drag that retain both significant affinity for intrinsic factor (IF) in the vitamin B12 portion of the complex and significant bioactivity of the drag portion of the complex.

A fourth aspect of the invention provides a method of imaging malignant tumour tissue in the body of an individual, the method comprising administering to the individual an effective amount of a compound as defined above.

In one embodiment of this aspect of the invention, the method comprises the further step of detecting the location of the compound in the individual. Detecting the compound or antibody can be achieved using methods well known in the art of clinical imaging and diagnostics. The specific method required will depend on the type of detectable label attached to the compound or antibodjr. For example, radioactive atoms may be detected using autoradiography or in some cases by magnetic resonance imaging (MRI) as described above. A fifth aspect of the invention provides a method of diagnosing or prognosing a malignant tumour tissue in an individual, the method comprising administering to the individual an effective amount of a compound as defined above. The method may be one which is an aid to diagnosis, i.e. additional tests may be required in order to reach a firm diagnosis.

In one embodiment of this aspect of the invention, the method of diagnosing, or aiding diagnosis of, a malignant tumour tissue in an individual comprises the further step of detecting the location of the compound in the individual.

A sixth aspect of the invention provides a method of treating an individual in need of treatment, the method comprising administering to the individual an effective amount of a compound as defined above. In one embodiment, the patient in need of treatment has a malignant tumour tissue, e.g. a non-small cell lung carcinoma.

Persons skilled in the art will further appreciate that the medicaments and agents described above have utility in both the medical and veterinary fields. Thus, the medicaments and agents may be used in the treatment of both human and non-human animals (such as horses, dogs and cats). In some embodiments the patient is human.

A 'therapeutically effective amount', or 'effective amount', or 'therapeutically effective', as used herein, refers to that amount which provides a therapeutic effect in the treatment of carcinomas for a given administration regimen. This is a predetermined quantity of active material calculated to produce a desired therapeutic effect, e.g. reduced migration of tumour cells, and/or reduction of metastatic lesions as assessed, for example, by radiologic imaging, in association with the required additive and diluent, i.e. a carrier or administration vehicle. Further, it is intended to mean an amount sufficient to reduce and most preferably prevent, a clinically significant deficit in the activity, function and response of the host. Alternatively, a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in a host. As is appreciated by those skilled in the art, the amount of a compound may vary depending on its specific activity. Suitable dosage amounts may contain a predetermined quantity of active composition calculated to produce the desired therapeutic effect in association with the required diluent. In the methods and use for manufacture of compositions of the invention, a therapeutically effective amount of the active component is provided. A therapeutically effective amount can be determined by the ordinary skilled medical or veterinary worker based on patient characteristics, such as age, weight, sex, condition, complications, other diseases, etc., as is well known in the art.

In some embodiments, the tumour tissue is selected from the group consisting of tumour tissue of the breast, bile duct, brain, colon, stomach, reproductive organs, lung and airways, skin, gallbladder, liver, nasopharynx, nerve cells, kidney, prostate, lymph glands and gastrointestinal tract.

In one particular embodiment, the tumour tissue is a non-small cell lung carcinoma.

Thus, in one embodiment, the method comprises administering to the individual an amount of the compound sufficient to inhibit a biological activity of an integrin alpha- 11 subunit or heterodimer thereof in a carcinoma.

In one embodiment the compound inhibits migration of tumour tissue cells.

It will be appreciated by persons skilled in the art that such an effective amount of the compound or formulation thereof may be delivered as a single bolus dose (i.e. acute administration) or, more preferably, as a series of doses over time (i.e. chronic administration).

Depending on the particular compound used in imaging, diagnosis or treatment, the timing of administration may vary and the number of other components used in therapeutic systems disclosed herein may vary.

For example, in the case where the compound of the invention comprises a readily detectable moiety or a directly cytotoxic moiety, it may be that only the compound, in a suitable formulation, is administered to the patient. Of course, other agents such as immunosuppressive agents and the like may be administered. In the case of compounds which are detectably labelled, imaging takes place once the compound has localised at the target site.

However, if the compound is one which requires a further component in order to be useful for treatment, imaging or diagnosis, the compound of the invention may be administered and allowed to localise at the target site, and then the further component administered at a suitable time thereafter.

For example, in the case of the ADEPT and ADEPT-like systems above, the binding moiety- enzyme moiety compound is administered and localises to the target site. Once this is done, the prodrug is administered.

Similarly, for example, in respect of the compounds wherein the further moiety comprised in the compound is one which binds a further component, the compound may be administered first and allowed to localise at the target site, and subsequently the further component is administered.

Thus, in one embodiment a biotin-labelled antibody is administered to the patient and, after a suitable period of time, detectably labelled streptavidin is administered. Once the streptavidin has localised to the sites where the antibody has localised (i.e. the target sites) imaging takes place.

A seventh aspect of the invention provides a method for monitoring the progression of a malignant tumour tissue, such as a carcinoma, in an individual, the method comprising:

(a) providing a sample of malignant tumour tissue collected from the individual at a first time point and measuring the amount of integrin alpha- 11 subunit protein therein;

(b) providing a sample of malignant tumour tissue collected from the individual at a second time point and measuring the amount of integrin alpha- 11 subunit protein therein; and

(c) comparing the level of integrin alpha- 11 subunit protein measured in steps (a) and (b) wherein an increased amount of integrin alpha- 11 subunit protein measured in step (b) compared to step (a) is indicative of a progression in the malignant tumour tissue.

An eighth aspect of the invention provides a method of identifying cells associated with malignant tumour tissue, the method comprising measuring the amount of integrin alpha- 11 subunit protein in a sample of tumour tissue cells to be tested and comparing it to the amount of integrin alpha- 11 subunit protein in a sample of known malignant tumour tissue.

In one embodiment, the cells associating witn the malignant tumour tissue is a stroma cell, i.e. a tumour stroma cell. In one further embodiment, the tumour stroma cell is a fibroblast cell. Other tumour stroma cells may be stem cells, such as mesechymal stem cells, or dendritic cells.

In one further embodiment of the eighth aspect of the invention, the method further comprises comparing the amount of integrin alpha- 11 subunit protein in a sample of tissue to be tested with the amount of integrin alpha- 11 subunit protein in a control sample.

Conveniently, the control sample comprises corresponding healthy (i.e. non-tumour) tissue.

For example, when the tumour tissue is a non-small cell lung carcinoma, the control cells may be normal lung cells.

Advantageously, the cells to be tested are identified as malignant tumour tissue by the upregulation of integrin alpha-11 subunit protein levels compared to corresponding normal healthy cells. By "upregulated" we mean that the integrin alpha-11 subunit protein is increased by at least 10% compared to expression of the same gene in normal cells. Preferably, the level of the integrin alpha-11 subunit is protein increased by at least 20%,

30%, 40% or 50%. Most preferably amount of the integrin alpha-11 subunit is increased by at least 100%.

A further aspect of the invention provides a method of distinguishing between different types or stages of malignant tumour tissue, e.g. stages of a caarcinoma, the method comprising measuring the amount of integrin alpha-11 subunit protein in a sample of cells to be tested and comparing it to the amount of integrin alpha-11 subunit protein in a sample of malignant tumour tissue of a known type or stage. Preferably, the known malignant tumour tissue are characterised by the upregulation of integrin alpha- 11 subunit protein compared to normal non-tumour tissue.

The amount of integrin alpha- 11 subunit in a sample may be determined using methods well known in the art. One way for assaying integrin alpha- 11 protein levels in a biological sample are antibody-based techniques. For example, integrin alpha- 11 protein expression in tissues may be studied with classical immunohistological methods. In these, the specific recognition is provided by the primary antibody (polyclonal or monoclonal) but the secondary detection system can Utilize fluorescent, enzyme, or other conjugated secondary antibodies. As a result, an immunohistological staining of tissue section for pathological examination is obtained. Tissues can also be extracted, e.g. with urea and neutral detergent, for the liberation of integrin alpha-11 protein for western blot or dot/slot assay (Jalkanen et al., 1985, J. Cell. Biol. 101:976-985; Jalkanen et al, 1987, J. Cell. Biol. 105:3087-3096). In this technique, which is based on the use of cationic solid phases, quantitation of integrin alpha-11 protein can be accomplished using isolated integrin alpha-11 protein as a standard. This technique can also be applied to body fluids.

hi one embodiment, the tumour tissue is selected from the group consisting of tumour tissue of the breast, bile duct, brain, colon, stomach, reproductive organs, lung and airways, skin, gallbladder, liver, nasopharynx, nerve cells, kidney, prostate, lymph glands and gastrointestinal tract, hi a particular embodiment, the tumour tissue is a non-small lung cancer.

hi a further embodiment, the known malignant tumour tissue are characterised by the upregulation of the amount of integrin alpha-11 subunit protein compared to corresponding normal non-tumour tissue. In still a further embodiment, the known malignant tumour tissue is non-small cell lung carcinoma characterised by the upregulation of the amount of integrin alpha-11 subunit protein compared to normal lung tissue.

In a further embodiment, the method further comprises comparing the amount of integrin alpha-11 subunit protein in a sample of cells to be tested with the amount of integrin alpha-11 subunit protein in a control sample.

Ih a further embodiment, the binding moiety selectively binds to integrin alpha-11 subunit. In still a further embodiment, the binding moiety selectively binds to a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO 1 or natural variants thereof.

In still a further embodiment, the binding moiety selectively binds to a heterodimer comprising an integrin alpha- 11 subunit.

In still a further embodiment, the binding moiety is an antibody or antigen-binding fragment or derivative thereof.

A ninth aspect of the invention is a method of screening for (i.e. identifying) candidate compounds with efficacy in the treatment of malignant tumour tissue, the method comprising the steps of:

(a) contacting a molecule to be tested with an integrin alpha- 11 subunit (or a fragment or binding sequence thereof); and

(b) detecting the presence of a complex containing the integrin alpha- 11 subunit (or fragment thereof) and the molecule to be tested

the molecule to be tested being identified as a candidate compound if the complex is detected in step(b).

Persons skilled in the art will appreciate that the methods of the above aspects of the invention may be used for any tumour tissue found to over-express an integrin alpha- 11 subunit. Preferably, the tumour tissue is selected from the group consisting of tumour tissue of the breast, bile duct, brain, colon, stomach, reproductive organs, lung and airways, skin, gallbladder, liver, nasopharynx, nerve cells, kidney, prostate, lymph glands and gastrointestinal tract. In one embodiment, the tumour tissue is a carcinoma, such as e.g. non- small cell lung carcinoma.

In a particular embodiment, the test molecule is a polypeptide. Suitable peptide ligands that will bind to an integrin alpha- 11 subunit (or fragments or derivatives thereof) may be identified using methods known in the art.

One method, disclosed by Scott and Smith (1990) Science 249, 386-390 and Cwirla et al (1990) Pr oc. Natl, Acad. ScL USA 87, 6378-6382, involves the screening of a vast library of filamentous bacteriophages, such as Ml 3 or fd, each member of the library having a different peptide fused to a protein on the surface of the bacteriophage. Those members of the library that bind to integrin alpha-11 subunit (or a fragment thereof) or a heterodimer thereof are selected using an iterative binding protocol, and once the phages that bind most tightly have been purified, the sequence of the peptide ligands may be determined simply by sequencing the DNA encoding the surface protein fusion. Another method that can be used is the NovaTope (TM) system commercially available from Novagen, Inc., 597 Science Drive, Madison, WI 53711. The method is based on the creation of a library of bacterial clones, each of which stably expresses a small peptide derived from a candidate protein in which the ligand is believed to reside. The library is screened by standard lift methods using the antibody or other binding agent as a probe. Positive clones can be analysed directly by DNA sequencing to determine the precise amino acid sequence of the ligand.

Further methods using libraries of beads conjugated to individual species of peptides as disclosed by Lam et al (1991) Nature 354, 82-S4 or synthetic peptide combinatorial libraries as disclosed by Houghten et al (1991) Nature 354, 84-86 or matrices of individual synthetic peptide sequences on a solid support as disclosed by Pirrung et al in US 5143854 may also be used to identify peptide ligands.

It will be appreciated that screening assays which are capable of high throughput operation will be particularly preferred. Examples may include cell based assays and protein-protein binding assays. An SPA-based (Scintillation Proximity Assay; Amersham International) system may be used. For example, an assay for identifying a compound capable of modulating the activity of a protein kinase may be performed as follows. Beads comprising scintillant and a polypeptide that may be phosphorylated may be prepared. The beads may be mixed with a sample comprising the protein kinase and ³²P-ATP or P-ATP and with the test compound. Conveniently this is done in a 96-well format. The plate is then counted using a suitable scintillation counter, using known parameters for ³²P or ³³P SPA assays. Only ³²P or ³³P that is in proximity to the scintillant, i.e. only that bound to the polypeptide, is detected. Variants of such an assay, for example in which the polypeptide is immobilised on the scintillant beads via binding to an antibody, may also be used.

Other methods of detecting polypeptide/polypeptide interactions include ultrafiltration with ion spray mass spectroscopy/HPLC methods or other physical and analytical methods. Fluorescence Energy Resonance Transfer (FRET) methods, for example, well known to those skilled in the art, may be used, in which binding of two fluorescent labelled entities may be measured by measuring the interaction of the fluorescent labels when in close proximity to each other.

Alternative methods of detecting binding of a polypeptide to macromolecules, for example DNA, RNA, proteins and phospholipids, include a surface plasmon resonance assay, for example as described in Plant et al (1995) Analyt Biochem 226(2), 342-348. Methods may make use of a polypeptide that is labelled, for example with a radioactive or fluorescent label.

A further method of identifying a compound that is capable of binding to an integrin alpha-11 subunit is one where the polypeptide is exposed to the compound and any binding of the compound to the said polypeptide is detected and/or measured. The binding constant for the binding of the compound to the polypeptide may be determined. Suitable methods for detecting and/or measuring (quantifying) the binding of a compound to a polypeptide are well known to those skilled in the art and may be performed, for example, using a method capable of high throughput operation, for example a chip-based method. New technology, called VLSIP S™, has enabled the production of extremely small chips that contain hundreds of thousands or more of different molecular probes. These biological chips or arrays have probes arranged in arrays, each probe assigned a specific location. Biological chips have been produced in which each location has a scale of, for example, ten microns. The chips can be used to determine whether target molecules interact with any of the probes on the chip. After exposing the array to target molecules under selected test conditions, scanning devices can examine each location in the array and determine whether a target molecule has interacted with the probe at that location.

Biological chips or arrays are useful in a variety of screening techniques for obtaining information about either the probes or the target molecules. For example, a library of peptides can be used as probes to screen for drugs. The peptides can be exposed to a receptor, and those probes that bind to the receptor can be identified. See US Patent No. 5,874,219 issued 23 February 1999 to Rava et al.

It will be understood that it will be desirable to identify compounds that may modulate the activity of an integrin alpha- 11 subunit in vivo. Thus it will be understood that reagents and conditions used in the method may be chosen such that the interactions between the said and the interacting polypeptide are substantially the same as between a said naturally occurring polypeptide and a naturally occurring interacting polypeptide in vivo.

In further embodiments, the above method further comprises a step (c) of testing the selected candidate compound for efficacy in one or more additional models of non-small cell lung carcinoma.

It will be appreciated that in the method described herein, the ligand ma3' be a drug-like compound or lead compound for the development of a drug-like compound.

The teπn "drug-like compound" is well known to those skilled in the art, and may include the meaning of a compound that has characteristics that may make it suitable for use in medicine, for example as the active ingredient in a medicament. Thus, for example, a drug-like compound may be a molecule that may be synthesised by the techniques of organic chemistry, less preferably by techniques of molecular biology or biochemistry, and is preferably a small molecule, which may be of less than 5000 daltons and which may be water-soluble. A drug-like compound may additionally exhibit features of selective interaction with a particular protein or proteins and be bioavailable and/or able to penetrate target cellular membranes, but it will be appreciated that these features are not essential.

The term "lead compound" is similarly well known to those skilled in the art, and may include the meaning that the compound, whilst not itself suitable for use as a drug (for example because it is only weakly potent against its intended target, non-selective in its action, unstable, poorly soluble, difficult to S3τathesise or has poor bioavailability) may provide a starting-point for the design of other compounds that may have more desirable characteristics. Alternatively, the methods may be used as "library screening" methods, a term well known to those skilled in the art. Thus, for example, the method of the invention may be used to detect (and optionally identify) a polynucleotide capable of expressing a polypeptide activator of a protein listed in Table 1. Aliquots of an expression library in a suitable vector may be tested for the ability to give the required result.

It is appreciated that the compound according to the invention decreases the activity of the integrin alpha- 11 subunit. For example, the compound may bind substantially reversibly or substantially irreversibly to the active site of said protein. In a further example, the compound may bind to a portion of said protein that is not the active site so as to interfere with the binding of the said protein to its ligand. In a still further example, the compound may bind to a portion of said protein so as to decrease said protein's activity by an allosteric effect. This allosteric effect may be an allosteric effect that is involved in the natural regulation of the said protein's activity, for example in the activation of the said protein by an "upstream activator".

The invention further discloses a method of treating an individual with a malignant tumour tissue substantially as described herein, a method of diagnosing or prognosing a malignant tumour tissue in an individual substantially as described herein, and a method of imaging malignant tumour tissue substantially as described herein.

The following non-limiting examples below will further describe the invention. The results are given in short here below.

SV40 immortalized mouse embryonic fibroblasts established from the wild type (WT) and

Itgall deficient (KO) mice were tested for their tumourigenicity in immune deficient mice when implanted alone or co-implanted with the A549 human lung adenocarcinoma cells.

A549, WT and KO cells formed small tumours, but A549 co-implanted with the fibroblasts showed markedly increased tumour growth rate. Importantly, the growth was significantly greater for A549+WT compared to A549+KO tumours.

Re-expression of human integrin alphal 1 cDNA in KO cells resulted in the rescue of tumour growth rate to that comparable with the A549+WT tumours. Gene expression profiling indicated that IGF2 mRNA expression level was >200 folds lower in A549+KO compared to A549+WT tumours. Stable short hairpin shRNA downregulation of IGF2 in WT (WT_sh-IGF2) fibroblasts resulted in decreased growth rate of A549+WT_sh-i_GF2 compared to A549+WT tumours.

In the orthotopic NCI-H460 human lung cancer model, host stromal alphal 1 expression was significantly higher in metastatic compared to the primary tumours.

The results shows that alphal 1 is an important tumour stromal factor in NSCLC.

A further aspect of the invention provides a kit comprising a composition according to the invention. Thus, there may be provided a kit for use in the treatment of a malignant tumour tissue such as e.g. NSCLC.

Alternatively, the kit may comprise a detectable antibody or antigen-binding fragment or derivative thereof according to the invention, suitable for use in diagnosis, prognosis or imaging of cells. Such a diagnostic, prognostic or imaging kit may comprise, in an amount sufficient for at least one assay, the diagnostic, prognostic or imaging agent as a separately packaged reagent. Instructions for use of the packaged reagent are also typically included. Such instructions typically include a tangible expression describing reagent concentrations and/or at least one assay method parameter such as the relative amounts of reagent and sample to be mixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions and the like.

A further aspect of the invention is use of a marker comprising the integrin alphal 1 chain expressed on a cell surface of a malignant tumour tissue cell, such as a tumour cell or a tumour stroma cell, or intracellular in said cells, as a marker for malignant tumour tissue. Said use may be wherein the integrin alphal 1 chain is expressed as a heterodimer in combination with betal. Such use is made essentially as described herein. The use may be in vivo, as e.g. imaging, or in vitro, in e.g. tumour tissue samples essentially as described herein. Said use may in further aspect be a method for detection and/or identification if tumour tissue in a sample in vitro or in vivo essentially as described herein. The marker may also be used for modulating rumour cells and tumour stroma cells. Said modulation may be e.g. inhibiting, slowing down, or preventing migration of tumour cells, or even inhibiting, slowing down or preventing tumour progression of malignant tumour tissue cells, i.e. the tumour cells as such or the tumour stroma cells. Said tumour progression may be e.g. tumour growth or tumour differentiation.

Still a further aspect of the present invention is a method for identifying a tumour tissue and its cells, the method comprising the steps of a) providing a sample comprising a tumour tissue, b) detecting integrin alphal l chain expression on the cell surface or intracellular in the tumour tissue cells, c) scoring the integrin alpha 11 chain, d) identifying the tumour tissue and its cells according to the scoring in c) above. The sample may be a tumour tissue sample for in vitro analysis or a patient for in vivo analysis using e.g. imaging. Preferably, the patient is a human being, but mau also be any mammal, such as a mouse, horse, dog, cat, rat, cow, etc. The preferably, the detection is done by detecting protein expression. Protein expression may be detected using an immuno method using e.g. an antibody in immunohistochemical methods (IHC), FACS analysis, immunoprecipitaions and similar methods. Alternatively, protein expression is done using RT-PCR measuring rnRNA levels of the integrin alphal 1 chain. Examples of such RT-PCR and EHC-methods are given herein. The RT-PCR may even be a quantitative PCR (RT- QPCR) as exemplified herein. The scoring of protein levels expressed of the integrin alphal l subunit is done relative a control of normal tissue from the same type of tissue specimen. Thus, alphal l protein expression in NSCLC tumour tissue is scored relative normal lung tissue. The normal tissue sample may be collected from the same individual or a different individual as long as it is classified as a normal tissue sample. Preferably, the tissue sample is from the same individual. The detection and its scoring will ultimately lead to the identification of tumour tissue in said tumour tissue sample. Further details of said method is provided herein in the other methods and uses described and applies accordingly to the above method. Non-limiting examples which embody certain aspects of the invention will now be described, with reference to the figures herein.

EXAMPLES

Material and methods

Tissue samples

NSCLC and corresponding non-neoplastic lung parenchymal tissues were harvested and/or banked with informed consent from lung cancer resection specimens. The Research Ethics Board of the University Health Network has approved the use of these excess tissues for this project. Banked specimens were snap-frozen and stored in liquid nitrogen until use.

Mouse embryonic fibroblast cell lines SV40 immortalized mouse embryonic fibroblasts (MEF) cell lines were derived from wild type (WT) and alphal 1 -deficient (KO) E14.5 mouse embryos as previously reported (Popova et α/.(2004) Dev Biol 270, 427-42), cultured in Dulbecco's supplemented with 10% FBS. To re-express the integrin alphal 1 in KO fibroblasts, a full-length human integrin alphal 1 cDNA (3.5 kb) cloned into to the pBJ-1 vector (Tiger et α/.(2001) Dev Biol 237, 116-29) was co-transfected with a puromycin resistance vector and positive high expressing clones were selected. Clone 14 (KI) with confirmed stable expression of integrin alphal 1 was isolated and used in subsequent experiments.

Isolation of CAF (cancer-associated fibroblasts) Six independent cases of lung cancer resection specimens were used to establish primary cultures of CAFs from primary lung cancers and non-CAFs from the corresponding non-neoplastic lung parenchyma >5 cm distant from the tumour. Tissues were minced into small pieces and digested for 1 hr at 37⁰C in Dulbecco's Modified Eagle's Medium (DMEM) containing 10 % fetal bovine serum (FBS) and lmg/ml collagenase type IV (Boehringer Mannheim). The suspension was centrifuged at 1000 rpm for 5 min and the pellet was resuspended in the same serum containing DMEM medium.

Cell Culture and Xenograft Studies

A549 lung adenocarcinoma cell line was previously obtained from the American Type Culture Collection (ATCC, Manassas, VA USA) and routinely cultured in RPMI 1640 supplemented by 10% FBS. All cell lines were cultured at 37⁰C in a humidified 5%CO₂ atmosphere. The tumourigenicity of cell lines were tested in 6- week old male severe combined immunodeficiency (scid) mice. Fifty μl of medium containing 2x10 of A549 cells, WT, KO or ICI cells alone respectively, or 2x10⁶ A549 cells combined with 2x10⁶ respective MEF lines were co-implanted subcutaneously into the abdominal flanks of mice. Tumour growth was assessed by caliper measurement of tumour diameter in the longest dimension (L) and at right angles to that axis (T-F). Tumour volume was estimated using the formula π/6 x L x W^~. All mice were sacrificed after 29 days, fully autopsied to check for metastatic lesions. Tumour fragments were harvested and fixed in 10% buffered formalin for histological evaluation, and snap-frozen in liquid nitrogen for other studies. Difference in tumour growth rates of xenographs was tested using Mixed effects model estimation . The comparisons of mRNA expressions were using Wilcox two sample test. All statistical analyses were performed using SASv9.0 statistical software (SAS Institute, NC, USA).

Western blot

Western blot analysis was performed as previously described (Popova et al, (2004) Dev Biol 270, 427-442). Cells were prepared in protein lysis buffer [50mmol/L HEPES (pH 8.0), 10% glycerol, 1% Triton X-100, 150 mmol/L NaCl, lmmol/L EDTA, 1.5 mmol/L MgCl₂, lOOmmol/L NaF, and 10mmol/L Na₄P₂O₂H₂O supplemented with 5μg/L leupeptin, 5μg/ml aprotinin, and 100 μg/mL phenylmethylsulfonylfluoride]. Fifty μg of protein was separated by electrophoresis and transferred to nitrocellulose using a Trans-Blot cell (Bio- Rad). Membrane was then incubated with rabbit anti mouse al 1 antibody at 1:1000 dilution, followed by horse radish peroxidase (HRP)-coupled goat anti-rabbit IgG (Santa Cruz Biotechnology) and rmmunoreactivity was revealed by the ECL+ system (Amersham, UK).

Immunohistochemistiy

Frozen sections (5μm) of tissues were fixed in acetone at -2O⁰C for 10 min and air dried for an additional 20 min prior to staining. Following phosphate buffered saline (PBS) rinses, the slides were blocked with 10 % goat -serum and incubated with rabbit polyclonal alphal 1 antibody (Veiling et al (1999) J Biol Chem 274, 25735-42) (1 :500) and a cocktail of cytokeratin antibodies composed equal mixture of AE1/AE3 (1 :200), high molecular weight cytokeratin (1 :100), CK7 (1 :2000) and CK20 (1 :50) antibodies. All cytokeratin antibodies were obtained from Dako Canada (Burlington, ONT). After 1 hr incubation at room temperature in a moist chamber and subsequent washing in PBS, the sections were incubated for 30 min at room temperature with Cy 3 -conjugated goat anti-rabbit IgG (1 : 100 dilution, Jackson Immunology Laboratory, Bar Harbour, MA) and Cy2-conjugated goat anti-mouse IgG (1 :50 dilution, Jackson Immunology Laboratory, Bar Harbour, MA) secondary antibodies. The nuclei were counter-stained for 5 min with DAPI. The slides were then mounted with fluorescent mounting media (Vector Laboratories, Burlinghame, CA) and immunoreactivities were observed and recorded using a Zeiss 's Axioplan 2 fluorescent microscope.

RNA isolation and quantitative assay

The Arcturus's PixCell II Laser capture microdissection (LCM) system was used to microdissect independently tumour cells and their surrounding stroma from toulidine blue O stained frozen sections of 2 primary NSCLCs (Zhu et al, (2004) Clin Cancer Res 10, 1984- 91) . Total RNA from microdissected samples and xenograft tumour tissues was extracted using the Micro RNA Isolation Kit (Stratagene, La Jolla, CA) according to manufacturer's recommendations. The cDNA was S3'nthesized by using Superscript II RNase H^" Reverse Transcriptase kit (Invitrogen, Carlsbad, CA) and subjected to quantitative PCR analysis (qPCR) on HT SDS 7900 (Applied Biosystems Inc., Foster City, CA) according to User Bulletin #2 from Applied Biosystems Inc (Zhu et al, (2004) Clin Cancer Res 10, 19S4-91). - the primer sequences are provided in Table 3. All gene expression values were normalized using the house keeping genes (RPS 13 for human and GAPDH for mouse and rat) and calculated using the comparative ΔGr method (Zhu et al, (2004) Clin Cancer Res 10, 1984- 91).

Gene expression profiling of tumours

The expression profiles of human and mouse genes in tumours were profiled using the Affymetrix human U133A and MOE430A chips (Affymetrix, Santa Clara, CA). The cRNA synthesis, hybridization, washing, and scanning were performed by standard protocol provided by Affyrnetrix (Santa Clara, CA). The raw microarray data from both MOE430A and HGUl 33 A arrays was pre-processed using the RMA algorithm and the log scale expression levels of WT vs. KO and A549+WT vs. A549+KO were compared. shRNA downregulation ofIGF2 in MEF cells

Mouse IGF2 expression in WT fibroblast was silenced using the shRNA method. Hairpin sense

CCGGGACCGCGGCTTCTACTTCAGTTCAAGAGACTGAAGTAGAAGCCGCGGTCTT TTTG [SEQ DO NO: 7] and antisense

AATTCAAAAAGACCGCGGCTTCTACTTCAGTCTCTTGAACTGAAGTAGAAGCCGC GGTC [SEQ ID NO: 8] oligos against IGF2 (Sigma chemical, St. Louis, MO) were annealed and subcloned into pLKO.l YFP. This construct or the control shRNA against luciferase (Paddison, et al, (2002) GeneDev 16, 948-58) (lOug) was transfected into 293T cells together with 5ug of each of the packaging plasmids pMD.G, pMDLg/pRRE and pRsv-Rev (Barcyte-Lovejoy et al. (2006) Cancer Res 66, 5330-37) using calcium phosphate method. Forty eight hours later, the viral supernatant was collected and used to infect MEFs. YFP positive cells were sorted by Dako Moflo flowcytometeer (Dako North America, Inc., Carpinteria, CA).

Table 3

Primer sequences

gene product (bp) Forward primer Reverse primer

Mouse alpha11 112 GGCACCAACAAGMTGAGACC TCCAGTCATAGGCTCCCACAG [SEQ ID NO:9] [SEQ BD NO-.10] betai 112 TGCAATTGTCAAAGCCATGG CACAGTGCCTCCCAACACG [SEQ ID NO.-ll] [SEQ ID NO.-12] alphal 113 CAGAAGAGGTCCTGGTGGCA CCCCCGAGCTTCAGTGAAT

[SEQ ID NO: 13] [SEQ ID NO:14] alpha2 112 GCGGCAGAGATCGATACACA CCCCTGTCGGTACTTCTGCTT [SEQ ID NO:15] [SEQ ID NO:16]

Alphal 0 111 ACAAGCGCCCATGGAGTCT GTGAAGAGTCGTGGGTGGTGT [SEQ ID NO:17] [SEQ ID NO:18]

Igfϊ 111 CAGTTCGTGTGTGGACCGAG GCTCCGGAAGCAACACTCAT [SEQ ID NO:19] [SEQ ID NO:20]

Igf2 123 GGGAGCTTGTTGACACGCTT GCACTCTTCCACGATGCCA [SEQ ID NO:21] [SEQ ID NO:22] lgfir 111 TCCTGAAGGGCAATCTGCTT GGCGGATCTTCACGTAGCC [SEQ ID NO:23] [SEQ ID NO:24]

Igf2r 104 TCAGCCTCGGCGAGATTTA GCTGGTGATGGAGGAGAGCT [SEQ ID NO:25] [SEQ ID NO:26]

Cd31 98 GCCGCCATTACCTGACAAGTA CACAGACCAGAAGCCTGCTG [SEQ ID NO:27] [SEQ ID NO:28]

Gadph 101 AG GTTGTCTCCTGCGACTTCA CCAGGAAATGAGCTTGACAAAGTT [SEQ ID NO:29] [SEQ ID NO:30]

Human alphal 1 123 TTCCATGTCCTGGACACTGC ATTCCAGAAGGGCACCGAC [SEQ ID NO:31] [SEQ ID NO:32]

IGF1 112 CCCTGGGTTGCTGTAAGGGT GGAGCATTCAATTCACCAATCTC [SEQ ID NO:33] [SEQ ID NO:34]

IG F2 108 CTGTTCGGTTTGCGACACG AGAAGGTGAGAAGCACCAGCA [SEQ ID NO:35] [SEQ ID NO:36]

IGF1 R 111 TCTTCAAGGGCAATTTGCTCA GGCGGATCTTCACGTAGCC [SEQ ID NO:37] [SEQ ID NO:38]

IGF2R 93 AGGTGAAGCCCAACGATCAG ACACGACATCGAGATCGCC [SEQ ID NO:39] [SEQ ID NO:40]

RPS 13 100 GTTGCTGTTCGAAAGCATCTTG AATATCGAGCCAAACGGTGAA [SEQ ID NO:41] [SEQ ID NO:42]

Rat alphal 1 132 TCATCGAGATCCTGACAAAGTTCT GCGGCTTCCACCACATCTT [SEQ ID NO:43] [SEQ ID NO:44] igf2 Same as for mouse IGF2 Example 1 - Alpha 11 protein is overexpressed and found mainly in stroma of human NSCLC

Objective The objective of this example was to demonstrated that αl 1 mRNA overexpression in both lung adenocarcinoma and squamous cell carcinoma was confirmed at protein level by Western blot on primary NSCLC and corresponding non-neoplastic lung tissue

Materials and methods Materials and methods are given in the paragraphs above.

Results and conclusions

It is demonstrated that αl 1 is overexpressed in both lung adenocarcinoma and squamous cell carcinoma at protein level by Western blot on three of four unselected paired primary NSCLC and corresponding non-neoplastic lung tissue (Figure IA). Tumour cells separate from the stromal tissue by laser captured microdissection demonstrated that alphal 1 mRNA expression was 2-S folds higher in the latter (Fig. IB), despite low expression in the epithelial tumour cells. Immunofluorescence microscopy showed that the alphal 1 protein was mainly localized in the stroma (Figure 1 C) and was not appreciably detected in the control normal lung sections (Fig. ID).

Example 2 - alpha 11 overexpression in CAF and metastatic lesions

Objective The objective of this example is to demonstrate that αl 1 was differentially overexpressed in lung cancer stromal fibroblasts compared to non-neoplastic tissue.

Materials and methods

Material and methods are given in the paragraphs above. Results and conclusions

Alpha 11 expression in fibroblasts isolated from 6 pairs of primary NSCLC and their corresponding non-neoplastic lung tissue was compared. Alphal 1 was differentially overexpressed 2-fold or higher in four of these 6 paired-samples (Fig. IE). Further, alphal 1 expression was higher in metastatic compared to primary lung cancers. Since metastatic lung tumour samples are generally not available, we compared the primary and metastatic tumours in the previously well described rat orthotopic model of NCI-H460 cell lines (Liu, J. et al.^ Oncogene, 23:6316-24, 2004). In this model, the H460 cells may develop systemic metastases to the gum, rib bone, kidney and brain. RT-qPCR analysis of rat (host) alphal 1 mRNA expression in primary (ortho) tumours and gum metastases, one of the most frequent metastatic sites in this model, demonstrated 4-5-fold higher expression in the latter site (Fig. IF). These data strongly suggest that increased stromal alphal 1 mRNA expression was associated with tumour progression and metastasis.

Example 3 Alphal 1 expression in fibroblasts enhances tumourigenicity of NSCLC cells

Objective

The objective of this example is to see if alphal 1 in tumour stroma fibroblasts plays important role in the tumourigenicity of lung cancer cells.

Materials and methods

Material and methods are given in the paragraphs above.

Results and conclusions

To test the tumourigenicity, A549 lung adenocarcinoma cells when co-implanted with immortalized wild type (WT) and alphal 1 deficient (KO) MEFs in a 1 :1 ratio in the subcutaneous tissue of scid mice. Individual cell lines were also tested as controls. These cell lines formed small tumours; while the A549 cells formed poorly differentiated adenocarcinoma, the WT and KO MEFs forming fibrosarcomas. In contrast, A549 cells co- implanted with either WT or KO MEFs demonstrated enhanced tumour growth compared to individual cell line alone. The effect appeared synergistic rather than additive. More importantly, the A549+WT group showed markedly greater tumour growth than A549+KO group (p=0.024, Fig. 2A). To confirm the reduced tumourigenicity was due to the loss of alphal 1 expression in

KO fibroblasts, we re-express human al l gene into the KO cells (KI). KI cells re-expressed alphal 1 subunit, while all WT, KO and KI cells express alphal, alpha2 and betal integrin subunits (Fig. 2B and Figure 4). Importantly, the co-implanted A549+KI cells formed tumour at similar rate as the A549+WT mixture and at significantly greater rate than the A549+KO group (P=O.028). None of the experimental groups demonstrated metastasis. RT-qPCR analysis of tumours formed confirmed the high expression of alphal 1 in groups with WT fibroblasts and low expression in those with KO fibroblasts (Fig. 2C). It is worth noting that the co-implantation of A549 with WT fibroblasts induced a ~10-fold increase in mouse alphal 1 expression (figure 2C), suggesting that alphal 1 expression is regulated by factors derived from tumour cells.

The over-expression of integrins, including some of the collagen receptor integrins ^' such as αlβl and α2βl in tumour cells has been reported to play a role in angiogenesis. To explore whether the lower tumour growth rate in the absence of stromal alphal 1 was through angiogenesis, mouse CD31 mRNA expression level of the xenography was quantified by RT- qPCR. No significant differences were found (Fig. 2D), indicating that stromal alphal 1 mediated increase in tumourigenesis was unlikely by enhanced angiogenesis.

Example 4 - Regulation of the expression of IGF2 in fibroblasts

Objective

The objective of this example is to deliver insight in what genes active in mediating the alphal 1 effect on enhanced tumourigenicity.

Materials and Methods

Materials and methods are provided in the paragraphs above.

Results and conclusions

Microarray studies were conducted to derive insights into the genes that could potentially mediate alphal 1 effect on enhanced tumourigenicity in A549+WT compared to A549+KO tumours. With the mouse chip that would detect mainly gene expression of host derived mouse cells, alphal 1 expression or lack of it is associated with the differential expression of 2-fold or greater in 1630 probe sets. The highest differentially expressed gene was IGF2, which was 250-fold higher in alphal 1 WT compared to KO tumours. This was confirmed by RT-qPCR (Fig. 3A), which showed approximately 100 and >200 fold higher expression levels in WT compared to KO tumours (p=0.001), and in A549+WT compared to A549+KO tumours (p=0.001), respectively. In contrast, mouse IGFl expression was not affected (Fig. 3B), neither were human IGF2 nor hIGFl (data not shown). The results showed a specific regulation of IGF2 expression by alphal 1 in fibroblasts. This was further confirmed by the restoration of IGF2 expression in tumours of KI alone (p=0.002) or KI+A549 (p=0.006, Fig. 3A), and a good correlation between IGF2 and alphal 1 expression in CAFs and metastatic H460 tumours in nude rat orthotopic model (Fig. IE) and metastatic H460 tumours in the nude rat orthotopic model (Fig IF). In addition to the restoration of IGF2 expression by human alphal 1, a mild elevation of IGFl expression was noted in KI (p=0.006) and in A549+KI tumours (p=0.02, Fig. 3B). Compensatory effect within gene family is common in KO studies. We next asked whether the expression of other collagen receptor integrin subunit genes was also altered in KO tumours. RT-qPCR of xenographs showed a clear compensatory elevation of alpha2 expression in A549+KO (p=0.007 compared to A549+WT; p=0.014 compared to A549+KI). While the alphal 0 mRNA expression was high in KO as compared to WT (p=0.006), there was no significant difference between A549+KO and A549+WT. In addition, the levels of alphal and betal were inconsistently altered (Fig. 4).

Example 5 - IGF2 partially mediates the fibroblast alphal 1 effect on tumourigenicity of A549 cells.

Objective

The objective of this example is to further investigate the role of IGF2 in alphal 1 mediated enhanced tumourigenicity of A549 cells by fibroblasts.

Materials and methods

Materials and methods are given in the paragraphs above.

Results and conclusions

To investigate further the role of IGF2 in alphal 1 mediated enhanced tumourigenicity of A549 cells by fibroblasts, the IGF2 expression in WT fibroblasts was stably down regulated using short hairpin (sh) RNA. Compared with control luciferase-transduced WT cells (WTL_UO) and the parental WT cells, the WT cells that stably express the IGF2 shRNA (WT_Sh-io_F2) showed approximately 70% downregulation of IGF2 mRNA expression (Fig. 3C). When WT_Sh-i_GF2 cells were co-implanted with A549 cells in scid mice, significant growth inhibition compared to A549+WTL_UC tumour was noted (p=0.001, Fig. 3D). The knock down of mouse IGF2 was verified in xenographs (p=0.004, Fig. 3E). These results are consistent with IGF2 playing important role in fϊbroblast-induced enhancement of lung cancer tumour formation.

Example 6 and 7

Materials and methods for Example 6-7

Patient tissue samples were obtained from the University Health Network (UHN) Tissue Bank, following approval of this project by UHN Research Ethics Board. All tissues were collected within 30 min of resection and snap-frozen in liquid nitrogen; their qualities have been verified by histology. Tissues used in this analysis included 68 primary colorectal adenocarcinoma, 36 metastatic colorectal adenocarcinoma cancer in the liver, and 36 normal colorectal mucosa corresponding to a subset of the above mentioned primary colorectal cancers. RNA were isolated from the snap-frozen samples by the guanidine isothiocyanate phenol-chloroform method (Tsao MS, Liu N, Chen JR, Pappas J, Ho J, To C et al. Lung Cancer 199S;20:l— 16.), and then purified by Qiagen Rneasy kit (Qiagen Inc., Ontario, Canada). Four mg of total cellular RNA was reversed transcribed using Superscript II reverse transcriptase (Invitrogen Inc., Ontario, Canada). Five nanogram equivalent of cDNA was used for each quantitative PCR assay, which is performed in duplicate using the ABI PRISM 7700 Sequence Detection System, using SYBR green 2x master mix (Applied Biosystems, Foster City, CA). The cycle threshold (CT) represented the relative abundance of a transcript and the relative RNA expression level was estimated using the ΔΔCx method, as recommended by the manufacturer (Applied Biosystems). The expression level of each gene was normalized using the geometric means of 3 house keeping genes: β-actin (BACT), β2-microglobulin (B2M) and histocompatibility complex 1C (HLC). The primer sequences are as follows:

HLA-C: forward: GAGTATTGGGACCGGGAGACA [SEQ ID NO:45];

Reverse: CCGTCCTCGCTCTGGTTGTA [SEQ ID NO:46] B2M: forward: GAGTGCTGTCTCCATGTTTGATGT [SEQ ID NO:47]; Reverse: AAGTTGCCAGCCCTCCTAGAG[SEQ ID NO:48];

BACT: forward: GCATGGGTCAGAAGGATTCCTA [SEQ ID NO:49];

Reverse: TCCATGTCGTCCCAGTTGGT [SEQ ID NO:50] Al l: in Table 3 above; sequences for human specimens

IGF2: in Table 3 above; sequences for human specimens

Example 6 - RT-QPCR on colorectal carcinoma

Objective

The objective of the present example is to analyse alphal 1 expression by quantitative RT- PCR (RT-QPCR) in normal and tumour (primary and metastatic colorectal carcinomas)

Materials and methods

Materials and methods are given in the paragraphs above. Primers for alphal 1

(ITGAl 1) are given in table 2 above.

Results and conclusions

Figure 5 shows mRNA expression levels of ITGAl 1 (5a) in normal colorectal mucosa, primary carcinomas and metastases. All values expressed as ratio of fold change with respect to mean normal mucosa. Average of normal = 1. The housekeeping genes analysed are β2M, βAct, HLA-2. The line indicates median. The conclusion is that alphal 1 is significantly overexpressed in primary and metastatic colorectal cancers compared to normal colonic mucosa.

Example 7 - correlation between IGF2 and ITGAl 1

Objective

The objective of the present experiment is to investigate the correlation between IGF2 and

ITGAl 1 (alphal 1) expression in colorectal adenocarcinoma using RT-QPCR.

Materials and methods

Materials and methods are given in the paragraphs above. Results and conclusions

Figure 6 shows the correlation between transcript levels of IGF2 and ITGAl 1 mRNA expression identified by RT-QPCR. Statistics: Spearman R = .4314 (95% CI = .2820-.5604). * Average of normal expression set at 1 p value (two tailed O.0001). The results show significant correlation between IGF2 and ITGAl 1 mRNA expression in human colorectal cancer specimens.

Example 8 - Distribution of integrin αl 1 β 1' expression in human tumour tissue.

Objective

The objective of the present example is to analyse alphal 1 expression in human tumour tissue and corresponding normal samples.

Materials and methods

Tissue samples

Tissue arrays #T6235700-5 were from BioChain (CA, US) were used. In brief, the arrays include sections from 14 tumour tissues and corresponding normal tissue, 5-8μm frozen sections, acetone fixed, mounted on one glass slide. Tumours include carcinomas from breast, colon, kidney, liver, lung, pancreas, prostate, small intestine, stomach, ovary, and uterus.

Immunohistochemistry

The following staining protocol for analysis using light microscopy was used:

Warm slides at RT. Wash PBS (Gibco) 2 x 5'.

Block with Avidine-Biotin (DAKO, DK) blocking solution 10' + 10' (according to the manufactor's instructions).

Wash PBS 2 x 5'.

Block 5% Human Serum (Sigma-Aldrich, SE) 30' RT. Wash PBS 2 x 5'.

Block 4% Goat Serum (Jackson Laboratories) 30' RT.

Wash PBS 2 x 5'.

Incubate with primary Ab's 1 hour RT. Wash PBS 2 x 5'.

Incubate with secondary Ab's 45' RT. Wash PBS 2 x 5'.

Incubate 45' RT with Vectastain ABC Elite kit (preincubated for 45'). Wash PBS 2 x 5'.

Develop with DAB (Vector) 4 min or VIP (Vector) 3 min - stop in tap water 5'. Counterstain slides with Hematoxylin (Histolab Products AB, SE) 2 sec. or Methyl Green (Sigma-Aldrich, SE) 5 min.

Dehydrate (EtOH) (Kemetyl, SE) 95%, 100%, 100% - Tissue Clear Replacement (Histolab Products AB, SE).

Mount in Faramount (DAKO, DK).

Antibodies

Primary and secondary antibodies used to analyse the expression of αl 1 integrin were the following: • Polyclonal rabbit anti human αl 1 integrin (made against the cytoplasmic tail)

Innovagen (described in WO00/75187). Used at 7,5 ug/ml.

• Polyclonal rabbit anti human CD3 (T-cells) DAKO # A0452 Used at 7,5 ug/ml. Used as an isotype control.

• Secondary antibody: Goat anti rabbit biotinylated Vector # BA-1000

IHC Evaluation

The IHC staining was analysed with light microscopy in a Nikon Eclipse TE2000-S microscope. Photos were taken using Nikon Digital Sight DSU-I camera and stored in the database of the Image Analysis Program Visiopharm.

Staining was graded as follows:

0 = no staining

1 = few cells stained 2 = lot of cells stained

3 = majority of the cells in the tissues slide stained

The staining is further given + for strong staining and - for weak staining. Results and conclusions The results are shown in table 4. In this study, we have identified αl lβl integrin expression in a numbers of different tumours. The staining is mostly located to the stromal tissue of the tumour, and to, as judged by morphology, fibroblast-like cells. In the stroma, the staining is on the majority of the cells.

In most tumour tissues, the tumour cells did not stain positive for αl 1. However, in the tumour s of brain, breast and skeletal muscle, αl 1 staining was identified on large cells with plurimorph cell nuclei that are most likely tumour cells.

Table 4 al l staining of human carcinomas and their corresponding healthy tissue

General discussion for the examples

The present invention demonstrates that integrin alpha 11 expressing stromal fibroblasts have greater paracrine stimulating effect on the tumour formation of A549 lung adenocarcinoma cells than fibroblasts deficient in alphal 1 expression. This finding implicates alphal 1 integrin as a stromal factor that may modulate the growth of carcinoma cells during rumour formation. Furthermore, it is shown that this stromal-tumour cell interaction is mediated uniquely by the ability of alphal 1 to regulate the expression of IGF2 in fibroblasts. Considering that greated than 80% of NSCLC overexpress alphal I₅ stromal alpha 11 is a promising diagnostic and therapeutic target in NSCLC which is shown in the present invention.

Since betalc is not found in mouse cells (Svineng, G and Johansson, S. (1999) J Cell Sd 111 (pt 24), 4751-61) and the predominant variant betal_A does not affect IGF2 level (Goel et a (2006) Cancer Res 66 331-42), the present invention suggest that the regulation of IGF2 expression by alphal 1 in fibroblasts could be mediated by the cytoplasmatic domain of the alpha chain, e.g. via She-dependent signalling in an alpha-chain specific manner (Barberis et al (2000) J Biol Chem 275, 36532-40, and Wary, K.K., et α/.(1996) Cell 87, 733-43).

IGF2 is commonly overexpressed in human cancer including breast, colorectal, liver, esophageal, intestinal, and prostate cancer. The most commonly posited mechanism for its overexpression is the Loss of imprinting (LOI). However, other regulatory mechanisms of IGF2 expression have also been reported, including its induction by prolactin in breast carcinoma and PTEN in hepatoma cells. The concurrent elevated levels of alphal 1 and IGF2 in CAF suggest that in primary NSCLC, their expression is closely associated. This and the finding of alphal 1-regulated IGF2 expression in fibroblasts suggest that alphal 1 is also an important stromal factor to mediate the tumour growth enhancing activity of CAF in NSCLC. In summary, the present invention clearly indicates that stromal alphal 1 may promote the tumourigeriicity of A549 lung adenocarcinoma cells, and this is could partially be explained by the alphal 1 -induced IGF2 expression by expression in the stromal fibroblasts.

Therefore, alphal 1 represents a novel target for the development of diagnostic and therapeutic modalities in NSCLC.

Claims

1. Use of a compound comprising a binding moiety capable of binding selectively to an integrin alpha- 11 subunit or a heterodimer thereof in the preparation of a medicament for treating a malignant tumour tissue.

2. Use of a compound comprising a binding moiety capable of binding selectively to an integrin alpha- 11 subunit or a heterodimer thereof in the preparation of a diagnostic or prognostic agent for a malignant tumour tissue.

3. Use of a compound comprising a binding moiety capable of binding selectively to an integrin alpha- 11 subunit or a heterodimer thereof in the preparation of an agent for detecting and/or imaging malignant tumour tissue.

4. The use according to any of claims 1-3, wherein the intergrin alpha- 11 subunit or heterodimer is expressed on the stroma cells associated to the malignant tumour tissue.

5. The use according to claim 4, wherein the stroma cells are fibroblast cells associated to the malignant tumour tisseue.

6. The use according to any one of Claims 1 to 5 wherein the tumour tissue is selected from the group consisting of tumour tissue of the breast, bile duct, brain, colon, stomach, reproductive organs, lung and airways, skin, gallbladder, liver, nasopharynx, nerve cells, kidney, prostate, lymph glands and gastrointestinal tract.

7. The use according to any one of Claims 1 to 6 wherein the tumour tissue is a non- small cell lung carcinoma.

8. The use according to any one of Claims 1 to 7 wherein the binding moiety selectively binds to integrin alpha- 11 subunit.

9. The use according to any one of Claims 1 to S wherein the binding moiety selectively binds to a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 1, fragments or natural variants thereof.

10. The use according to any one of Claims 1 to 9 wherein the binding moiety selectively binds to a heterodimer comprising an integrin alpha- 11 subunit.

11. The use according to any one of the preceding claims wherein the binding moiety is an antibody or antigen-binding fragment or derivative thereof.

12. The use according to any one of the preceding claims wherein the malignant tumour tissue is metastatic.

13. The use according to any of the Claims 1 and 4-12 wherein the medicament is for inhibiting, slowing down or prevention of migration of malignant tumour cells of said tumour tissue.

14. The use according to any of Claims 1 and 4-12 wherein the medicament is for inhibiting, slowing down or prevention of tumour progression of malignant tumour cells of said tumour tissue.

15. The use according to claim 14, wherein the tumour progression is tumour growth.

16. The use according to claim 14, wherein the tumour progression is tumour cell differentiation.

17. The use according to claims 1 and 4-16, wherein the medicament is for altering tumour stroma cell expression of alphal 1.

18. The use according to Claims 1 and 4-12 wherein the medicament is for inhibiting, slowing down or prevention of tumourigeniciry of malignant tumour cells within the tumour tissue.

19. A method of detecting and/or imaging malignant tumour tissue in the body of an individual, the method comprising administering to the individual an amount of a compound comprising a binding moiety capable of binding selectively to integrin alpha- 11 subunit or a heterodimer thereof.

20. A method of diagnosing or prognosing malignant tumour tissue in an individual, the method comprising administering to the individual an amount of a compound comprising a binding moiety capable of binding selectively to integrin alpha- 11 subunit or a heterodimer thereof.

21. A method according to Claim 19 or 20 further comprising the step of detecting the location of the compound in the individual.

22. A method of treating an individual with a malignant tumour tissue, the method comprising administering to the individual an effective amount of a compound comprising a binding moiety capable of binding selectively to integrin alpha- 11 subunit or a heterodimer thereof.

23. A method according to any one of Claims 19 to 22 wherein the tumour tissue is selected from the group consisting of tumour tissue of the breast, bile duct, brain, colon, stomach, reproductive organs, lung and airways, skin, gallbladder, liver, nasopharynx, nerve cells, kidney, prostate, lymph glands and gastrointestinal tract.

24. A method according to any one of Claims 19 to 23 wherein the tumour tissue is a non- small cell lung carcinoma tumour tisue.

25. A method according to any one of Claims 19 to 24 wherein the compound inhibits migration of tumour cells of said tumour tissue.

26. A method for monitoring the progression of a malignant tumour tissue in an individual, the method comprising:

(a) providing a sample of a malignant tumour tissue collected from the individual at a first time point and measuring the amount of integrin alpha- 11 subunit protein therein;

(b) providing a sample of a malignant tumour tissue collected from the individual at a second time point and measuring the amount of integrin alpha- 11 subunit protein therein; and (c) comparing the amount of integrin alpha- 11 subunit protein measured in steps (a) and (b)

wherein an increased amount of integrin alpha- 11 subunit protein measured in step (b) compared to step (a) is indicative of a progression of the malignant tumour tissue.

27. A method of identifying cells associated with a malignant tumour tissue, the method comprising measuring the amount of integrin alpha- 11 subunit protein in a sample of cells to be tested and comparing it to the amount of integrin alpha- 11 subunit protein in a sample of cells from a known malignant tumour tissue.

28. The method according to claim 27, wherein the cells associating with the malignant tumour tissue is a stroma cell.

29. The method according to claim 28, Avherein the stroma cell is a fibroblast cell.

30. A method of distinguishing between different types or stages of a malignant tumour tissue, the method comprising measuring the amount of integrin alpha- 11 subunit protein in a sample of tissue to be tested and comparing it to the amount of integrin alpha- 11 subunit protein in a sample of tissue from a malignant tumour of a known type or stage.

31. A method according to any one of Claims 19 to 30 wherein the tumour tissue is selected from the group consisting of tumours of the breast, bile duct, brain, colon, stomach, reproductive organs, lung and airways, skin, gallbladder, liver, nasopharynx, nerve cells, kidney, prostate, lymph glands and gastrointestinal tract.

32. A method according to any one of Claims 19 to 31 wherein the tumour tissue is a non- small lung cancer.

33. A method according to any one of Claims 19 to 32 wherein the known malignant tumour tissue is characterised by the upregulation of the amount of integrin alpha- 11 subunit protein compared to corresponding normal non-tumour tissue.

34. A method according to Claim 33 wherein the known malignant tumour tissue is non- small cell lung carcinoma characterised by the upregulation of the amount of integrin alpha- 11 subunit protein compared to normal lung tissue.

35. A method according to any one of Claims 19 to 34 further comprising comparing the amount of integrin alpha- 11 subunit protein in a sample of tissue to be tested with the amount of integrin alpha- 11 subunit protein in a control tissue sample.

36. A method according to any one of Claims 19 to 34 wherein the tumour tissue to be tested is identified or distinguished as a malignant tumour tissue if the amount of integrin alpha- 11 subunit protein is upregulated compared to corresponding a normal non-tumour tissue.

37. A method according to any one of Claims 19 to 36 wherein the binding moiety selectively binds to integrin alpha- 11 subunit.

38. A method according to any one of Claims 19 to 36 wherein the binding moiety selectively binds to a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO 1, fragments or natural variants thereof.

39. A method according to any one of Claims 19 to 36 wherein the binding moiety selectively binds to a heterodimer comprising an integrin alpha- 11 subunit.

40. A method according to any one of Claims 19 to 36 wherein the binding moiety is an antibody or antigen-binding fragment or derivative thereof.

41. A method of screening for candidate compounds with efficacy in the treatment of a malignant tumour tissue, the method comprising the steps of:

(a) contacting a molecule to be tested with an integrin alpha- 11 subunit (or a fragment or binding sequence thereof); and

Sl (b) detecting the presence of a complex containing the integrin alpha- 11 subunit (or fragment thereof) and the molecule to be tested

and wherein the molecule to be tested being identified as a candidate compound if the complex is detected in step(b).

42. A method according to Claim 41 wherein the tumour tissue is selected from the group consisting of tumour tissue of the breast, bile duct, brain, colon, stomach, reproductive organs, lung and airways, skin, gallbladder, liver, nasopharynx, nerve cells, kidney, prostate, lymph glands and gastrointestinal tract.

43. A method according to Claim 41 or 42 wherein the tumour tissue is a non-small cell lung carcinoma.

44. A method according to any one of Claims 41 to 43 further comprising step (c) of testing the selected candidate compound for efficacy in one or more additional models of non-small cell lung carcinoma.

45. Use of a marker comprising the integrin alphal 1 chain expressed on a cell surface of a malignant tumour tissue cell, such as a tumour cell or a tumour stroma cell, or intracellular in said cells, as a marker for malignant tumour tissue.

46. The use according to claim 45, wherein the integrin alphal 1 chain is expressed as a heterodimer in combination with betal .

47. A method for identifying a tumour tissue and its cells in vivo or in vitro,the method comprising the steps of e) providing a sample comprising a tumour tissue, f) detecting integrin alphal 1 chain expression on the cell surface or intracellular in the tumour tissue cells, g) scoring the integrin alphal 1 chain, h) identifying the tumour tissue and its cells according to the scoring in c) above.

48. A method of treating an individual with a malignant tumour tissue substantially as described herein.

49. A method of diagnosing or prognosing a malignant tumour tissue in an individual substantially as described herein.

50. A method of detecting malignant tumour tissue substantially as described herein.

51. A method of imaging malignant tumour tissue substantially as described herein.