GB2479536A

GB2479536A - Blood filtration device for removing tumour cells

Info

Publication number: GB2479536A
Application number: GB1006060A
Authority: GB
Inventors: Jon Lacy-Colson
Original assignee: Individual
Current assignee: Individual
Priority date: 2010-04-12
Filing date: 2010-04-12
Publication date: 2011-10-19
Also published as: GB201006060D0

Abstract

An apheresis/extra corporeal filtration device for selective capture of circulating tumour cells from a patient's blood comprises an inlet 4 to receive the patient's blood and a capture agent 1. The capture agent 1 binds to an extracellular portion of a protein expressed by the circulating tumour cell. A preferred protein is E-cadherin. The device also includes means for separating captured circulating tumour cells from the blood 8 and an outlet 9 for return of the remainder of the blood, depleted of circulating tumour cells, to the patient. The capture agent 1 may be immobilized on a solid support. The capture agent 1 may comprise a peptide comprising the amino acid sequence LFSHAVSSNG.

Description

APHERESIS DEVICE AND RELATED METHODS

FIELD OF THE INVENTION

The invention relates to apheresis devices and components thereof. The devices are useful for selective capture of circulating tumour cells. The devices incorporate a suitable capture agent which binds to an extracellular portion of a protein expressed by the circulating tumour cell. Preferred proteins are cadherins.

BACKGROUND OF THE INVENTION

Cancer Cancer is a worldwide problem affecting all communities and the lives of tens of millions of people annually. The total global cancer burden in the year 2002 comprised 10.1 million new cases, 6.2 million deaths and 22.4 million persons living with cancer (GLOBECAN 2002). The vast majority of cancers are of epithelial cell origin.

Interestingly the primary tumour itself is rarely directly responsible for the patient's death, instead this arises more frequently as a result of distant metastatic disease. Metastasis is developed as a result of blood borne spread of tumour cells. Taking colorectal cancer as an example, over 60% of patients are free of metastasis at presentation, but -50% of patients can be expected to develop these lesions during the course of their disease.

Hence a proportion of patients at a relatively early stage without detectable local or distant metastases at presentation and time of initial surgical treatment, later go on to develop metastatic disease and ultimately succumb to this.

Current chemotherapy regimes offer up to a 5% survival advantage in colorectal cancer.

Because of the numbers involved any treatment which showed even a small overall percentage effect on outcome might potentially have a desirable effect on many thousands of people on a yearly basis, and hence be a very attractive proposition.

Cadherins and E-cadherin The cadherins are a family of calcium-dependent membrane glycoproteins, most of which act as cell adhesion molecules (Munro and Blaschuk 1996). Epithelial (E)-Cadherin was the first member of the classical or type I sub-family of Cadherins to be discovered (Munro and Blaschuk 1996). Other members of this group are neural (N)-, retinal (R)-and placental (P)-Cadherin, with varying tissue distributions as their names suggest (Geiger and Ayalon 1992).

The classical Cadherins are transmembrane glycoproteins which mediate intercellular adhesion. Loss of this adhesive function seems to be an important factor in tumour pathogenesis and is associated with loss of cell-cell contact inhibition of proliferation, enabling cells to escape from growth control as well as promoting tumour-cell detachment from the primary site and facilitating spread to distant sites (Wijnhoven BPL, Pignatelli M 1999).

There are at least two areas of the classical Cadherin glycoproteins, which are highly conserved not only in all the classical Cadherins but also across species (Blaschuk and Reynolds 2002). First the His-Ala-Val (HAV).amino acid sequence in the EC1 domain (Blaschuk et al 1990) which is the designated classical Cadherin cell adhesion recognition (CAR) sequence. Furthermore the amino acids immediately flanking the HAV sequence are specific between different sub-types and within that sub-type are also conserved between species. Second the Trp residue located at the amino terminus of the EC1 domain is also highly conserved as are the surrounding residues also highly conserved both across sub groups and species (Shapiro et al 1995).

It has been shown (Noe et al 1999) that the synthetically produced peptide LFSHAVSSNG (an exact copy of the highly conserved and highly specific CAR amino acid sequence found in the E-cadherin EC-1 domain) can specifically inhibit aggregation of human E-cadherin bearing cells, presumably via competitive inhibition of the normal E-cadherin mediated cell-cell adhesion complex. Altering the amino acids either side of the HAV sequence abolishes this effect (Noe et al 1999, Williams et al 2000).

Role of E-cadherin expression in cancer metastasis Studies in breast cancer have shown that although reduced expression of E-cadherin in primary tumours is related to increased metastases, a similar pattern of loss of expression is not seen in the metastases, which very rarely show loss of expression (Bukholm 1K et al 2000) of E-cadherin.

In a recent paper (Whe&er 2005) JMD Wheeler observes that a high proportion of CRC's exhibited hypermethylation of the E-cadherin promoter and that this correlated with reduced E-cadherin expression. Wheeler goes on to say that this hypermethylation may be reversed restoring gene function and partially undoing the cancer phenotype. As Ilyas points out in an earlier paper (Ilyas 2000) "tumour cells in a metastatic deposit are in a different environment and are faced with a different set of selection pressures driving tumour evolution in a different direction" and as Buckham et al have shown, whereas primary breast tumours show reduced expression of E-cadherin the metastases from these have a significantly increased re-expression of E-cadherin.

DESCRIPTION OF THE INVENTION

It has yet to be determined whether this re-expression of E-cadherin in metastatic deposits is due to the selection of a sub set of metastatic cells which have never lost E-cadherin expression, or whether individual cells loose and then re-acquire E-cadherin expression. This does however raise the possibility that what Wheeler viewed as an undoing of the cancer phenotype may in reality be aiding the metastatic process.

It is hypothesised that even patients with relatively early stage cancers already have a systemic burden of malignant cells within their circulation, and that this "soup" of malignant cells circulating within their blood stream can fuel the later development of metastases. It has been shown (AIIen-Mersh et al 2007) that even careful surgical manipulation of tumours can lead to an increased tumour cell burden in the blood, and with the high numbers of malignant cells which can be demonstrated in the circulation it might reasonably be said that it is surprising that even more patients do not develop metastases. The absence of metastases in some patients suggests that the development of blood borne metastases is an inefficient process and hence this stage in the disease process might therefore pose an attractive therapeutic target for their prevention.

As E-cadherin plays such an important role in epithelial cell-cell adhesion it is an attractive hypothesis that decreased expression of this protein in epithelial tumours might lead to an increased susceptibility to metastasis via facilitating the shedding of malignant cells from the main tumour mass into the circulation. This is a hypothesis that has been tested in a number of epithelial cancers including colorectal.

Moreover, the data relating to the specificity of the amino acid sequence of the cadherin domains in terms of permitting cell adhesion suggest that this interaction is a highly specific way of targeting cells bearing the E-cadherin EC-1 domain.

In light of these observations, the inventor has determined that capture of tumour cells from the peripheral circulation may represent a useful therapy for lowering the burden of tumour cells and helping to prevent metastases.

The invention accordingly provides an apheresis or extra corporeal filtration device for selective capture of circulating tumour cells from a patient's blood comprising: a) An inlet to receive the patient's blood b) a capture agent for capture of circulating tumour cells from the patient's blood, which capture agent binds to an extracellular portion of a protein expressed by the circulating tumour cell C) means for separating captured circulating tumour cells from the remainder of the blood; and d) an outlet for return of the remainder of the blood, depleted of circulating tumour cells, to the patient.

Similarly, the invention provides (in a second aspect) an apheresis or extra corporeal filtration device for selective capture of circulating tumour cells from a patient's blood comprising a capture agent for capture of circulating tumour cells from the patient's blood immobilized on a solid support, wherein the capture agent binds to an extracellular portion of a protein expressed by the circulating tumour cell.

The circulating tumour cells may be cells of epithelial origin. They may be derived from any tumour type. One specific example is colorectal cancer.

Apheresis relates specifically to passing the blood of a subject through an apparatus that separates out one or more specific constituents of the blood and permits return of the remainder to the subject. It is thus an extracorporeal technique and hence the devices of the invention may also be referred to as extra corporeal filtration devices.

The devices of the invention may incorporate a suitable inlet for blood taken from a patient. Similarly the devices may include an outlet for return of blood to the patient.

The inlet and outlet may comprise one or more filters to prevent entry of non-blood components into the device, The inlet and outlet effectively connect the device to the extracorporeal circuit. This extracorporeal circuit allows the blood to circulate from the patient through the device of the invention and back to the patient. The circuit may be formed of suitable sterile tubing in certain embodiments. The circuit may be connected at the inlet and outlet via any suitable connection or coupling. The inlet and outlet may form an integral part of the outer shell or housing of the device.

The devices of the invention incorporate a capture agent for capture of circulating tumour cells from the patients blood. The capture agent binds to an extracellular portion of a protein expressed by the circulating tumour cell, In binding to the protein expressed on the outside of tumour cells, the tumour cells are effectively captured and are not returned to the patient. Any suitable protein expressed in tumour cells and including a portion accessible to a capture agent of the invention may be employed. The protein may be one expressed exclusively or at increased levels by (circulating) tumour cells, although this is not essential. In certain embodiments, the protein expressed by the circulating tumour cell is a cell-cell adhesion molecule. The cell-cell adhesion molecule may comprise a cadherin or a non-cadherin calcium independent cell-cell adhesion molecule.

Any suitable cadherin molecule may be employed -a large number are known in the art.

In specific embodiments, the cadherin comprises, consists essentially of or consists of E-cadherin. Multiple different capture agents may be employed in a single device in certain embodiments.

In further embodiments, the capture agent binds the EC1 domain of E-cadherin. The EC1 domain of E-cadherin is the extracellular portion of E-cadherin containing the highly conserved amino acid sequences, including the His-Ala-Val (HAV) amino acid sequence -the classical Cadherin cell adhesion recognition (CAR) sequence -and the Trp residue located at the amino terminus of the EC1 domain. In specific embodiments, the capture agent comprises a peptide comprising, consisting essentially of or consisting of the amino acid sequence LFSHAVSSNG (SEQ ID NO: 1). Suitable amino acid subsitutions may be made in this peptide sequence provided that the resultant peptide is still an effective capture agent according to the invention. Typically the substitutions will be outside of the strictly conserved HAV sequence. One, two, three, four or five etc. substitutions may be made in certain embodiments. Any substitutions will typically be conservative in nature. Thus any replacement amino acid may be size and/or charge matched with the amino acid that is being substituted, as would be understood by one skilled in the art. The peptide may comprise deletion of one or two amino acids from either end provided the peptide retains the ability to (specifically) bind to the EC1 domain of E-cadherin. Of course the peptide, with our without modification, may be provided in the context of a larger peptide or protein, with the proviso that the peptide retains the ability to (specifically) bind to the EC1 domain of E-cadherin. Modified peptides can readily be tested for their ability to bind the EC1 domain of E-cadherin. For example, a sample containing E-cadherin expressing cells may be passed over a column upon which the peptide is immobilized. The retention of E-cadherin cells can then be determined, for example by suitable labeling of the cells.

In alternative embodiments, the non-cadherin calcium independent cell-cell adhesion molecule comprises epithelial cell adhesion molecule (EpCAM). EpCAM is expressed in almost all carcinomas and thus represents a particularly useful general target protein in the devices and methods of the invention. Antibodies are known in the art which can bind to this molecule and thus can form the basis of a suitable capture agent.

The capture agent of the invention may be any agent suitable for specific capture of circulating tumour cells through binding to an extracellular portion of a protein expressed by the circulating tumour cell. In certain embodiments, the capture agent comprises, consists essentially of or consists of an antibody or a portion or derivative thereof which retains the ability to bind the extracellular portion of the protein expressed by the circulating tumour cell. Thus, in specific embodiments, the capture agent comprises an antibody which binds specifically to a cell-cell adhesion molecule. The cell-cell adhesion molecule may comprise a cadherin or a non-cadherin calcium independent cell-cell adhesion molecule, as discussed herein. In certain embodiments, the antibody binds specifically to E-cadherin, such as to the EC1 domain of E cadherin. In such embodiments, the CAR sequence, or the peptide comprising, consisting essentially of or consisting of the amino acid sequence LFSHAVSSNG (SEQ ID NO: 1), as defined herein, may be used as an antigen to raise specific antibodies. The term "antibody" thus incorporates all derivatives and variants thereof which retain specific antigen binding capabilities. Both monoclonal and polyclonal antibodies may be utilised. Derivatised versions, which may be humanized versions of non-human antibodies for example, are also contemplated. Derivatives include, but are not limited to, heavy chain antibodies, single domain antibodies, nanobodies, Fab fragments, scFv etc. As indicated, the devices of the invention may incorporate means for separating captured circulating tumour cells from the blood. Thus, blood entering the device comes into contact with the capture agent, which leads to effective capture of the tumour cells in the blood through binding to an extracellular portion of a protein expressed by the circulating tumour cell. The blood which is returned to the patient must be separated from the captured tumour cells which are bound to the capture agent. This may be achieved through any suitable means. In certain embodiments, the means for separating captured circulating tumour cells from the blood comprises a membrane, such as a semi-permeable membrane which is impermeable to captured circulating tumour cells but is permeable to the remainder of blood components. Permeability may be achieved on a size-exclusion basis. Suitable pore diameters can readily be derived by one skilled in the art based upon the dimensions of the known components of blood and the size of the tumour cells in combination with the bound capture agent. In order to facilitate this separation process, the capture agent may be immobilized on a suitable material which increases the size significantly. As an example, beads such as sepharose beads may be conjugated to the capture agent to produce a larger molecular structure. Alternatively, the capture agent may form part of a larger macromolecule, such as a peptide as portion of a larger protein, which may be a fusion protein in some embodiments.

In other embodiments, the device incorporates a surface, such as a solid support, upon which the capture agent is immobilized and over which the patient's blood is passed.

Thus, once the circulating tumour cells become bound to the capture agent they are effectively removed from the blood (via immobilization) which can then be circulated back to the patient. The apheresis devices of the invention may be column devices in certain embodiments. The column devices may incorporate a solid support of the invention upon which is immobilized a capture agent of the invention. The device may thus comprise a solid support on which the capture agent is immobilized and may be in the form of a column (containing the solid support) through or over which the blood passes. The invention thus also relates to a solid support upon which a suitable capture agent is immobilized, as discussed herein. The surface upon which the capture agent is immobilized and over which the patient's blood is passed may thus be comprised of a suitable affinity matrix. The affinity matrix may be contained in a suitable column. The affinity matrix may be comprised of any suitable material. Typically, the material is of high molecular weight and may be of suitable dimensions to ensure that the material will not enter the blood that exits the device (and is returned to the patient). The material is inert such that it does not react or degrade upon coming into contact with the blood. An example of a suitable carbohydrate matrix material is agarose, which may be provided in the form of beads. Beads may be of any suitable size or shape.

It is noted that all materials used in the devices of the invention may be composed of materials which do not activate coagulation or may be treated, for example with heparin, to achieve this property.

Any suitable method of immobilization may be employed, as would be readily appreciated by one skilled in the art. Immobilization may be direct or indirect. Direct immobilization may be achieved through a range of chemical conjugation techniques.

This may rely upon various functional groups found in the capture agents or introduced therein, together with a suitably derivatized solid support material.

One specific method of immobilization of the capture agent is via the interaction between biotin and avidin (such as streptavidin). For example, the capture agent may be biotinylated and the surface of the support may carry avidin. Suitable spacer regions may be incorporated into the capture agent as required in order to prevent the biotin from affecting binding of the capture agent to the target extracellular portion of the protein expressed by the tumour cells. Alternatively, immobilization may rely upon antigen-antibody interactions in certain embodiments. A fusion of the capture agent and an antigen may be utilized, in particular a fusion protein if the capture agent is protein based, and the corresponding antibody specific to the antigen included in the support to act as a means to immobilize the capture agent via binding to the antigen.

The capture agents are thus specifically adapted for apheresis treatments. Accordingly, in a further aspect, the invention provides a capture agent capable of capturing a cell-cell adhesion molecule-expressing circulating tumour cell from the blood of a patient through binding to the cell-cell adhesion molecule for use in therapy. In a related aspect, the invention provides a capture agent capable of capturing a cell-cell adhesion molecule- expressing circulating tumour cell from the blood of a patient through binding to the cell-cell adhesion molecule for use in the removal of malignant cells from a patient's blood.

The discussion of the capture agents herein applies mutatis mutandis to these aspects of the invention.

The invention also provides for use of a device of the invention or a solid support of the invention in the selective removal of circulating tumour cells from the blood of a patient.

In a related aspect the invention provides a method of selectively removing circulating tumour cells from a patient's blood comprising; (a) contacting the blood taken from the patient with a capture agent to capture the circulating tumour cells from the patient's blood, wherein the capture agent binds to an extracellular portion of a protein expressed by the circulating tumour cell; and (b) separating the captured circulating tumour cells from the remainder of the blood.

Thus, the output of this method is blood from which (at least a proportion of) the tumour cells have been depleted. This blood can then be returned to the patient with the aim of improving the patient's prognosis.

In certain embodiments, the methods further comprise, prior to step (a), collecting the patient's blood. Similarly, the methods may further comprise, following step (b), infusing the remainder of the blood, depleted of circulating tumour cells, to the patient. Thus, the methods of the invention may in certain embodiments include the entire circuit from and to the patient.

The methods of the invention may be useful for reducing the tumour burden of the patient and/or preventing a metastasis or metastases developing. All aspects of the devices of the invention may be employed in the methods of the invention and so the discussion herein applies mutatis mutandis. Thus, the invention provides a method of selectively removing circulating tumour cells from a patient's blood comprising applying the patient's blood to a device of the invention. The patient's blood may be pre-isolated or may be isolated according to the method.

According to a further aspect, the invention provides a kit for producing a capture agent for use in the methods of the invention. The kit comprises: a) a peptide comprising the amino acid sequence HAV and in particular LFSHAVSSNG (SEQ ID NO: 1) as discussed herein; and b) means for immobilising the peptide to produce an immobilized capture agent.

As discussed herein, the peptide LFSHAVSSNG (SEQ ID NO: 1) includes the highly conserved amino acid sequences from E-cadherin that permit cell to cell interactions through binding of EC1 domains to one another. The peptide may be incorporated in a larger macromolecular structure. It may, for example, be produced as a fusion protein or as an EC1 domain peptide.

The means for immobilising the peptide permits the peptide to be attached to a solid surface or support. Suitable means for immobilization may include biotin molecules to which the peptide can be attached and avidin molecules incorporated into a suitable solid surface or support. Examples of a solid surface or support include beads or other particles for inclusion in a column, such as agarose beads.

The invention will now be described with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows schematically the structure of E-cadherin Figure 2 is a schematic representation of the cell-cell adhesion complex formed between epithelial cells, through interacting E-cadherin molecules.

Figure 3 represents a schematic diagram of an extracorporeal malignant cell filtration circuit, incorporating a device according to the invention. -11 -

Referring to figure 1, E-cadherin (epithelial) consists of 5 cadherin repeats (numbered 1 to 5 in the figure) in the extracellular domain, one transmembrane domain (6), and an intracellular domain (7) that binds p120-catenin (8) and beta-catenin (9). The intracellular domain (7) contains a highly-phosphorylated region which permits beta-catenin binding.

In epithelial cells, E-cadherin-containing cell-to-cell junctions are often adjacent to actin-containing filaments of the cytoskeleton.

The blown up portion of the figure shows domains ECI (1) and EC2 (2). Within the ECI domain, the conserved HAV and Trp residues, which facilitate cell-cell adhesion are shown schematically.

As shown in Figure 2 the first (1) and second (2) epithelial cell interact via the CAR sequence of the EC1 domains of E-cadherin expressed by the cells. The blown up portion of the figure shows how the conserved residues in the EC1 domains permit two cells to adhere to one another. It is noted that the context of these specific amino acids is critical to permit cell-cell adhesion to occur.

The invention takes advantage of the CAR sequence of the ECI domain of E-cadherin in order to capture and thus remove tumour cells from the peripheral blood of a patient.

Referring to figure 3, the capture agent (1) includes one or more synthetically produced peptides comprising the amino acid sequence LFSHAVSSNG (SEQ ID NO:1) which represents the highly conserved and highly specific CAR amino acid sequence found in the E-cadherin EC-1 domain. The peptide is incorporated into a much larger macro-molecule carrier (2) which permits the separation of E-Cadherin bearing circulating tumour cells from a solution containing these cells, by a process of filtration. Thus, in patients with epithelial cancers, the malignant cells with the potential to form blood borne metastases may be removed from the blood via an extracorporeal apheresis circuit.

In use, blood from a patient with an epithelial cancer enters the apheresis device (3) via inlet (4). As blood enters the filtration chamber (5) it comes into contact with the macro-molecule carrier (2) upon which is immobilized the CAR sequence (1). The E-cadherin expressing malignant cells (6) adhere to the CAR sequence (1). The remainder of the blood (7) (and its constituents including the red and white blood cells (RBC and WBC)) passes through a semi-permeable membrane (8). The E-cadherin expressing malignant -12 -cells are unable to pass across the membrane as they are attached to the CAR sequence (1), which in turn is immobilized on the macro-molecule carrier (2) which is prevented from doing so because of its size. The blood exits via the outlet (9) and is returned to the patient depleted of potentially metastatic malignant cells.

In alternative embodiments instead of the membrane the device comprises a column or other solid support on which the capture agent is immobilised. As the capture agent is immobilized within the device itself, no membrane is required to prevent the captured tumour cells from being circulated back to the patient. For example, the column may be packed with beads upon which the capture agent is immobilized. Blood passes through the column and over the beads. The tumour cells are bound by the capture agent and retained by the column. The remainder of the blood, depleted of the tumour cells, can then be returned to the patient.

References Allen-Mersh TG, McCullough 1K, Patel H, Wharton RQ, Glover C, Jonas SK. Role of circulating primary tumour cells in predicting recurrence after excision of primary colorectal carcinoma. (2007) BJS. 94:96-1 05.

Blaschuk OW. Sullivan R. David S. Pouliot Y. Identification of a cadherin cell adhesion recognition sequence. Developmental Biology. 139(1):227-9, 1990 May.

Bukholm 1K, Nesland JM, Borresen-Dale AL. Re-expression of E-cadherin, ci-catenin and -catenin, but not of y-catenin, in metastatic tissue from breast cancer patients. (2000) J Path, 190: 15-19.

Geiger B. Ayalon 0. Cadherins. Annual Review of Cell Biology. 8:307-32, 1992.

Ilyas M. Adhesion molecule expression in breast cancer: the phoenix in tumour metastasis Journal of Pathology. 190(1):3-5, 2000 Jan.

Munro SB. Blaschuk OW. A comprehensive survey of the cadherins expressed in the testes of fetal, immature, and adult mice utilizing the polymerase chain reaction. Biology of Reproduction. 55(4):822-7, 1996 Oct.

-13 -Noe V, Willems J, Vandekerckhove J, van Roy F, Bruyne E and Mareel M. (1999).

Inhibition of adhesion and induction of epithelial cell invasion by HAV-containing E-cadherin-specific peptides. J Cell Sci. 112:127-135.

Wheeler JM, Kim HC, Efstathiou JA, et al. Hypermethylation of promoter region of E-cadherin gene (CDHI) in sporadic and ulcerative colitis associated colorectal cancer, (2001) Gut. 48(3), 367-71 Wijnhoven BP, Pignatelli M. E-cadherin-catenin: more than a "sticky" molecular complex.

(1999) Lancet; 354(9176):356-7 Williams E, Williams 0, Gour B, Blaschuk OW and Doherty P. A novel family of cyclic peptide antagonists suggests that N-cadherin specificity is determined by amino acids that flank the HAV motif. (2000) J Biol Chem; 275:4007-4012.

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims. Moreover, all embodiments described herein are considered to be broadly applicable and combinable with any and all other consistent embodiments, as appropriate.

Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties. *

Claims

-14 -CLAIMS1. An apheresislextra corporeal filtration device for selective capture of circulating tumour cells from a patient's blood comprising: e) An inlet to receive the patient's blood f) a capture agent for capture of circulating tumour cells from the patient's blood, which capture agent binds to an extracellular portion of a protein expressed by the circulating tumour cell g) means for separating captured circulating tumour cells from the remainder of the blood; and h) an outlet for return of the remainder of the blood, depleted of circulating tumour cells, to the patient.
2. An apheresis/extra corporeal filtration device for selective capture of circulating tumour cells from a patient's blood comprising a capture agent for capture of circulating tumour cells from the patient's blood immobilized on a solid support, wherein the capture agent binds to an extracellular portion of a protein expressed by the circulating tumour cell.
3. The device of claim 1 or 2 wherein the protein expressed by the circulating tumour cell is a cell-cell adhesion molecule.
4. The device of claim 3 wherein the cell-cell adhesion molecule comprises a cadherin or a non-cadherin calcium independent cell-cell adhesion molecule.
5. The device of claim 4 wherein the cadherin comprises E-cadherin.
6. The device of claim 5 wherein the capture agent binds the EC1 domain of E-cadherin.
7. The device of any one of claims 1 to 6 wherein the capture agent comprises a peptide comprising the amino acid sequence LFSHAVSSNG.
8. The device of claim 4 wherein the non-cadherin calcium independent cell-cell adhesion molecule comprises epithelial cell adhesion molecule (EpCAM).
9. The device of any one of claims 1 to 8 wherein the capture agent comprises an antibody or a portion or derivative thereof which retains the ability to bind the extracellular portion of the protein expressed by the circulating tumour cell.
10. The device of any one of claims ito 9 wherein the means for separating captured circulating tumour cells from the blood comprises a semi-permeable membrane which is impermeable to captured circulating tumour cells but is permeable to the remainder of blood components; and/or a surface upon which the capture agent is immobilized and over which the patient's blood is passed.
11. A solid support as defined in claim 2 and in any of claims 3 to 10 when dependent from claim 2.
12. A capture agent capable of capturing a cell-cell adhesion molecule-expressing circulating tumour cell from the blood of a patient through binding to the cell-cell adhesion molecule for use in therapy.
13. A capture agent capable of capturing a cell-cell adhesion molecule-expressing circulating tumour cell from the blood of a patient through binding to the cell-cell adhesion molecule for use in the removal of malignant cells from a patients blood.
14. The capture agent of claim 12 or 13 wherein the capture agent is as defined in any one of claims 1 to 10.
15. Use of a device according to any of claims 1 to 10 or a solid support as claimed in claim 11 in the selective removal of circulating tumour cells from the blood of a patient.
16. A method of selectively removing circulating tumour cells from a patient's blood comprising; -16- (a) contacting the blood taken from the patient with a capture agent to capture the circulating tumour cells from the patient's blood, wherein the capture agent binds to an extracellular portion of a protein expressed by the circulating tumour cell; and (b) separating the captured circulating tumour cells from the remainder of the blood.
17. The method of claim 16 further comprising, prior to step (a), collecting the patient's blood.
18. The method of claim 16 or 17 further comprising, following step (b), infusing the remainder of the blood, depleted to circulating tumour cells, to the patient.
19. The method of any of claims 16 to 18 wherein the method is for reducing the tumour burden of the patient and/or preventing a metastasis or metastases developing.
20. The method of any of claims 16 to 19 wherein the capture agent is as defined in any one of claims ito 8.
21. A method of selectively removing circulating tumour cells from a patient's blood comprising applying the patient's blood to a device of any one of claims 1 to 10.
22. A kit for producing a capture agent for use in the method of any one of claims 16 to 21 comprising: c) a peptide comprising the amino acid sequence LFSHAVSSNG; and d) means for immobilising the peptide to produce an immobilized capture agent.