FIELD OF THE INVENTION

The present invention relates to molecules which are useful for delivering functional entities to Transmembrane Endothelial Marker 8 (TEM8) expressing cells or tissues.

BACKGROUND TO THE INVENTION

The de novo generation of blood vessels (neovascularization) and the growth of new blood capillaries from existing vessels (angiogenesis) are important processes for embryonic development, tissue regeneration and remodelling. However, these processes are also associated with a number of pathologies including inflammation, tumour growth and metastasis. Unlike highly controlled physiologic angiogenesis, disease-associated angiogenesis, such as occurs during tumour formation and development, results in chaotic, inefficient, and permeable vessels that are distinct from the normal vasculature. Because such vessels are accessible to the systemic vasculature and are unique vital components of the tumour's growth strategy, they have been targeted for both therapeutic and imaging purposes.

Methods for imaging sites of angiogenesis and neovascularization can be generally defined as either functional imaging or molecular targeting imaging techniques. Functional imaging techniques include ultrasound, CT perfusion, dynamic contrast MRI and functional PET scans. However, these techniques are relatively nonspecific for tumour angiogenesis and are only semi-quantitative.

Molecular targeting imaging techniques involve the binding of labelled molecules to highly expressed markers on the endothelium of tumour vasculature. Among the molecules that are known targets for imaging 'vasular endothelial growth factor' (VEGF) and its receptors and matrix metalloproteinases (MMPs).

Therapeutic targeting of angiogenesis has generally focused on anti-VEGF therapies. Known anti-VEGF molecules include monoclonal antibodies such as bevacizumab (Avastin), antibody derivatives such as ranibizumab (Lucentis) and orally-available small molecules that inhibit the tyrosine kinases stimulated by VEGF. However, a number of studies into the consequences of VEGF inhibitor use have shown that, although they can reduce the growth of primary tumours, VEGF inhibitors can concomitantly promote invasiveness and metastasis of tumours. As such VEGF inhibitors have proved to be less effective for the treatment of diseases associated with angiogenesis than expected.

Products which enable regions of angiogenesis and neovascularisation to be targeted, and which are not associated with the disadvantages described above, are therefore required for both therapeutic and imaging applications.

DESCRIPTION OF THE FIGURES

Figure 1 - SDS-PAGE of mPAd4-His, mPAd4-lg-His and mPAd4-AP-His peptides produced in an E.coli cytoplasm expression system and purified by Ni ²⁺ column purification.

Figure 2 - Alkaline phosphatase enzyme activity of the mPAd4-AP peptide isolated in elutions EL1-4. Figure 3 - Quantification of sprouts in a rat aortic rings treated with (i) media + 1% FCS, (ii) VEGF (10ng/ml) or (iii) VEGF (10ng/ml) + mPAd4-Fc (50Mg/ml).

Figure 4 - Images of rat aortic rings treated with (i) media + 1 % FCS, (ii) VEGF (10ng/ml) or (iii) VEGF (10ng/ml) + mPAd4-Fc (50pg/ml).

SUMMARY OF ASPECTS OF THE INVENTION

The present inventors have developed a series of targeting molecules comprising a Tumour Endothelial Marker 8 (TEM8) binding entity. The molecules can be used to deliver functional entities to cells and tissues which express TEM8, such as sites of neovascularization and angiogenesis.

In a first aspect the present invention provides a molecule having the general formula [A]-[B]-[C]; wherein,

[A] is a targeting entity which binds Tumour Endothelial Marker 8 (TEM8), [B] is cleavage site recognised and cleaved by a tumour-specific protease; and,

[C] is a functional payload. The functional payload may, itself, be capable of being internalised through a cell membrane.

Alternatively, the functional payload may comprise an internalisation sequence which causes the functional payload as a whole to be internalised through a cell membrane, once it has been cleaved from the targeting portion of the molecule [A].

The internalisation sequence may be or comprise a basic (positively charged) amino acid sequence. The internalisation sequence may be or comprise a series of basic amino acids, such as arginine. The functional payload may comprise a string of arginine residues, for example between 3 and 15 arginine residues. The functional payload may comprise the amino acid sequence (Arg) ₉ . The targeting molecule may also comprise a neutralising moiety which counteracts the charge of, for example, the internalisation sequence. The neutralising moiety may comprise an acidic (negatively charged) amino acid sequence separated from the basic amino acid sequence by the amino acid sequence which is recognised and cleaved by the tumour-specific protease.

The neutralising moiety may comprise a series of basic amino acid residues such as glutamic acid. The neutralising moiety may be or comprise the sequence (Glu) ₆ .

The targeting molecule may also comprise a flexible linker sequence between the entity which binds TEM8 and the amino acid sequence which is recognised and cleaved by a tumour-specific protease.

The flexible linker sequence may be or comprise the sequence (Gly) _n -Ser, n being an integer between 1 and 8. The flexible linker sequence may be or comprise the sequence Gly-Gly-Gly-Gly-Ser. The flexible linker may have a length equivalent to that of Gly-Gly-Gly-Gly-Ser such that is causes [A] and [B] to be separated by an equivalent distance to this amino acid sequence. The amino acid sequence [B] may be recognised and cleaved by a matrix- metalloproteinase (MMP) or ADAM-17. The MMP may be MMP2 and/or MMP9. The cleavage site may be or comprise an amino acid sequence which is recognised and cleaved by a tumour-specific protease. It may, for example, be or comprise the sequence Pro-Leu-Gly-Leu-Ala-Gly.

The TE 8 binding entity may be a Fab, scFv, V _HH domain.

The TEM8 binding entity may be derived from domain 4 of anthrax protective antigen (pAd4). For example it may be or comprise the sequence shown as SEQ ID NO 2.

The TEM8 binding entity may be or comprise a variant of the sequence shown as SEQ ID No. 2 which comprises one or more amino acid mutations, (i.e. additions, deletions of substitutions) in order to enhance its capacity to bind to TEM8. The variant sequence may have at least 80, 90, 95 or 98% sequence identity with the amino acid sequence shown as SEQ ID No. 2. The targeting moiety may, for example have a mutation of arginine to serine at position 659 and/or methionine to arginine at position 662.

The TEM8 binding entity may comprise the sequence shown as SEQ ID NO 3. The functional payload may comprise a detectable agent which may be or comprise a fluorescent peptide, for example an infra red fluorescent peptide.

The functional payload may be or comprise red fluorescent protein (mRFP1). The functional payload may be or comprise a therapeutic entity.

The functional payload may be or comprise a cytotoxic drug, for example it may comprise the catalytic domain of Pseudomonas Exotoxin. The functional payload may be or comprise a toxin. In a second aspect the present invention provides a nucleotide sequence which encodes a molecule according to the first aspect or the invention.

In a third aspect the present invention provides a construct comprising a nucleotide sequence according to the second aspect of the invention.

In a fourth aspect the present invention provides a vector or host cell comprising a nucleotide sequence according to the second aspect of the invention or a construct according to the third aspect of the invention.

In a fifth aspect the present invention provides a molecule according to the first aspect of the invention for use in treating a disease involving neovascularisation and/or angiogenesis. In a sixth aspect the present invention provides a molecule according to the first aspect of the invention for use in diagnosing a disease involving neovascularisation and/or angiogenesis.

In a seventh aspect the present invention provides the use of a molecule according to the first aspect of the invention in the manufacture of a medicament for treating a disease involving neovascularisation and/or angiogenesis in a subject.

In an eighth aspect the present invention provides the use of a molecule according to the first aspect of the invention in the manufacture of a medicament for diagnosing a disease involving neovascularisation and/or angiogenesis in a subject.

In a ninth aspect the present invention provides a method for treating a disease involving neovascularisation in a subject which comprises the step of administering a molecule according to the first aspect of the invention to the subject.

In a tenth aspect the present invention provides a method for diagnosing a disease involving neovascularisation and/or angiogenesis in a subject which comprises the step of administering a molecule according to the first aspect to the subject. The disease involving neovascularisation and/or angiogenesis may be cancer. In a eleventh aspect the present invention involves a method for delivering a detectable agent, a cytotoxic agent and/or a therapeutic entity to a cell expressing TEM8, comprising contacting the cell with a molecule according to the first aspect of the invention.

The targeting moiety targets the molecule of the present invention to cells and tissues which express TEM8, such as sites of neovascularization and angiogenesis. If in the presence of a tumour nearby, a tumour specific protease cleaves the molecule of the invention at the cleavage site [B], so that the functional payload is released. The targeting moiety may remain bound to TEM8.

The functional payload is then internalised and accumulates in cells, where it performs its diagnostic or therapeutic function. The present invention therefore provides specific products and methods for targeting tumour sites which involve neovascularization and/or angiogenesis for imaging and therapeutic purposes which have improved specificity when compared to existing products. DETAILED DESCRIPTION

MOLECULE

In a first aspect, the present invention provides a molecule. The molecule comprises a targeting entity which is able to bind Tumour Endothelial Marker 8 (TEM8).

TUMOUR ENDOTHELIAL MARKER 8 (TEM8)

TEM8 is a type I transmembrane protein with three domains: an N-terminal, extracellular von Willebrand factor type A domain (vWA domain), a single transmembrane spanning domain, and a C-terminal cytosolic domain.

The extracellular domain is -200 amino acids and is closely related to a-integrin inserted (I) domains. It is the extracellular domain which is responsible for binding TEM8 ligands. TEM8 is highly conserved across diverse species. Its expression is documented as restricted to angiogenic vessels in vivo, yet no functional significance has been assigned to it. TEM8 is reported to be weakly detected in normal adult tissues {e.g., lung, brain, kidney, and muscle) but abundant in tumour endothelial cells and the vasculature of developing embryos. The receptor is expressed at sites of neovascularisation; however its physiological ligand and cellular functions have not been determined. It has been reported that TEM8 is expressed by human umbilical vein endothelial cells (HUVEC) and is up-regulated during the process of endothelial cell differentiation and tube formation. Increased TEM8 expression enhances the motility of endothelial cells and antagonism of TEM8 indicates it to be a component of the migratory response.

TEM8 appears to promote endothelial cell migration through enhancing cell-matrix interactions on collagen and may play a role in angiogenesis, converting a normal quiescent cell state to one that is reactive and highly mobile. TEM8 has been shown to be a docking protein or receptor for Bacillus anthracis toxin, the causative agent of the disease anthrax. The binding of the protective antigen (PA) component of the tripartite anthrax toxin to this receptor mediates delivery of toxin components to the cytosol of cells. Once inside the cell, the other two components of anthrax toxin, edema factor (EF) and lethal factor (LF) disrupt normal cellular processes.

The full amino acid sequence of PA is shown as SEQ ID NO. 1. SEQ ID NO. 1

10 20 30 40 50 60

MKKRKVLIPL MALSTILVSS TGNLEVIQAE VKQENRLLNE SESSSQGLLG YYFSDLNFQA

70 80 90 100 110 120 PMVVTSSTTG DLSIPSSELE NIPSENQYFQ SAIWSGFIKV KKSDEYTFAT SADNHVTMWV

130 140 150 160 170 180

DDQEVINKAS NSNKIRLEKG RLYQIKIQYQ RENPTEKGLD FKLYWTDSQN KKEVISSDNL

190 200 210 220 230 240

QLPELKQKSS NSRKKRSTSA GPTVPDRDND GIPDSLEVEG YTVDVKNKRT FLSPWISNIH

250 260 270 280 290 300

EKKGLTKYKS SPEKWSTASD PYSDFEKVTG RIDKNVSPEA RHPLVAAYPI VHVDMENIIL 310 320 330 340 350 360

SKNEDQSTQN TDSQTRTISK NTSTSRTHTS EVHGNAEVHA SFFDIGGSVS AGFSNSNSST

370 380 390 400 410 420

VAIDHSLSLA GERTWAET G LNTADTARLN ANIRYVNTGT APIYNVLPTT SLVLGKNQTL

430 440 450 460 470 480

ATIKAKENQL SQILAPNNYY PSKNLAPIAL NAQDDFSSTP ITMNYNQFLE LEKTKQLRLD

490 500 510 520 530 540

TDQVYG IAT YNFENGRVRV DTGSNWSEVL PQIQETTARI IFNGKDLNLV ERRIAAVNPS

550 560 570 580 590 600

DPLETTKPDM TLKEALKIAF GFNEPNGNLQ YQGKDITEFD FNFDQQTSQN IKNQLAELNA

610 620 630 640 650 660

TNIYTVLDKI KLNAKMNILI RDKRFHYDRN NIAVGADESV VKEAHREVIN SSTEGLLLNI

670 680 690 700 710 720

DKDIRKILSG YIVEIEDTEG LKEVINDRYD MLNISSLRQD GKTFIDFKKY NDKLPLYISN

730 740 750 760

PNYKVNVYAV TKENTIINPS ENGDTSTNGI KKILIFSKKG YEIG

The binding of PA to TEM8 is believed to be mediated by domain 4 of PA (PAd4) and the use of anthrax PA to target the anthrax toxin receptor in mice has recently been shown to inhibit tumour progression. Engineering of PAd4 in the context of whole PA (83KDa) has resulted in a PA molecule that has selectively enhanced binding to TEM8.

An "entity which binds TEM8" as used herein refers to an entity which is able to bind to the extracellular domain of TEM8.

The entity may be a polypeptide or glycoprotein.

Techniques which can be used to identify entities that are able to bind to TEM8 are well known in the art and include, but are not limited to, ELISA, western blot, immunohistochemistry, flow cytometry, FRET, phage display libraries, yeast two- hybrid screens, co-immunoprecipitation, bimolecular fluorescence complementation and tandem affinity purification.

The present inventors have expressed the TEM8 specific 131 amino acids of the isolated mPAd4 (16.2kDa) in the context of C-terminal fusion proteins and have shown that PAd4 retains its ability to bind TEM8 in this form. The entity may therefore be a polypeptide sequence derived from PAd4 which retains the ability to bind to TE 8.

For example the entity which targets TEM8 may comprise SEQ ID NO. 2 or a variant thereof.

SEQ ID NO. 2

VGADESWKEAHREVINSSTEGLLLNIDKDIRKILSGYIVEIEDTEGLKEVINDRYDMLN ISSLRQDGKTFIDFKKYNDKLPLYISNPNYKVNVYAVTKENTIINPSENGDTSTNGIKKI LIFSKKGYEIGS

The TEM8 specific 131 amino acids of isolated mPAd4 may be modified to enhance binding to TE 8. The TEM8 binding entity may be or comprise a variant of the sequence shown as SEQ ID No. 2 which comprises one or more amino acid mutations, (i.e. additions, deletions of substitutions) in order to enhance its capacity to bind to TEM8. The variant sequence may have at least 80, 90, 95 or 98% sequence identity with the amino acid sequence shown as SEQ ID No. 2.

For example, the wild-type PAd4 may have a mutation of arginine to serine at position 659 and/or methionine to arginine at position 662.

For example, the entity which targets TEM8 may comprise SEQ ID NO. 3, wherein the residues mutated in comparison to the wild-type (SEQ ID NO. 2) are highlighted. SEQ ID NO. 3

VGADESWKEAHREVINSSTEGLLLNIDKDIRKILSGYIVEIEDTEGLKEVINDSYDRLN ISSLRQDGKTFIDFKKYNDKLPLYISNPNYKVNVYAVTKENTIINPSENGDTSTNGIK I LIFSKKGYEIG The targeting entity may be an antibody or a functional fragment thereof, or an antibody mimetic.

As used herein, "antibody" means a polypeptide having an antigen binding site which comprises at least one complementarity determining region CDR. The antibody may comprise 3 CDRs and have an antigen binding site which is equivalent to that of a domain antibody (dAb). The antibody may comprise 6 CDRs and have an antigen binding site which is equivalent to that of a classical antibody molecule. The remainder of the polypeptide may be any sequence which provides a suitable scaffold for the antigen binding site and displays it in an appropriate manner for it to bind the antigen. The antibody may be a whole immunoglobulin molecule or a part thereof such as a Fab, F(ab)' ₂ , Fv, single chain Fv (ScFv) fragment or Nanobody. The antibody may be a bifunctional antibody. The antibody may be non-human, chimeric, humanised or fully human.

Variable loops, three each on the variable light (V _L ) and variable heavy (V _H ) chains are responsible for the binding of an antibody to the antigen. These loops are referred to as the complementarity determining regions (CDRs). The CDRs (CDR1 , CDR2 and CDR3) of each of the V _H and V _L are arranged non-consecutively. Within the variable domain, CDR1 and CDR2 are found in the V region of the polypeptide chain, and CDR3 includes some of V, all of D (heavy chains only) and J regions. Since most sequence variation associated with immunoglobulins is found in the CDRs, these regions may be referred to as hypervariable regions. Among these, CDR3 shows the greatest variability as it is encoded by a recombination of the VJ in the case of a light chain region and VDJ in the case of heavy chain regions. Regions between CDRs in the variable domain of an immunoglobulin are known as framework regions. These are important for establishing the structure of the V _H and V _L domains. The variable domains of the H (V _H ) and L (V _L ) chains constitute an Fv unit and can interact closely to form a single chain Fv (ScFv) unit.

The C-terminal domain of an antibody is called the constant region. In most H chains, a hinge region is found. This hinge region is flexible and allows the Fab binding regions to move freely relative to the rest of the molecule. The hinge region is also the place on the molecule most susceptible to the action of proteases which can split the antibody into the antigen binding site (Fab) and the effector (Fc) region.

The domain structure of the antibody molecule is favourable to protein engineering, facilitating the exchange between molecules of functional domains carrying antigen- binding activities (Fabs and Fvs) or effector functions (Fes).

Chimeric antibodies may be produced by transplanting antibody variable domains from one species (for example, a mouse) onto antibody constant domains from another species (for example a human). Fab, Fv and ScFv fragments with V _H and V _L joined by a polypeptide linker exhibit specificities and affinities for antigen similar to the original monoclonal antibodies. The ScFv fusion proteins can be produced with a non-antibody molecule attached to either the amino or the carboxy terminus. In these molecules, the Fv can be used for specific targeting of the attached molecule to a cell expressing the appropriate antigen. Bifunctional antibodies can also be created by engineering two different binding specificities into a single antibody chain. Bifunctional Fab, Fv and ScFv antibodies may comprise engineered domains such as CDR grafted or humanised domains.

Genes encoding immunoglobulins or immunoglobulin-like molecules can be expressed in a variety of heterologous expression systems. Large glycosylated proteins including immunoglobulins are efficiently secreted and assembled from eukaryotic cells, particularly mammalian cells. Small, non-glycosylated fragments such as Fab, Fv or scFv fragments can be produced in functional form in mammalian cells or bacterial cells.

The antigen-binding domain may be comprised of the heavy and light chains of an immunoglobulin, expressed from separate genes, or may use the light chain of an immunoglobulin and a truncated heavy chain to form a Fab and a F(ab)' ₂ fragment. Alternatively, truncated forms of both heavy and light chains may be used which assemble to form a Fv fragment. An engineered svFv fragment may also be used, in which case, only a single gene is required to encode the antigen-binding domain. The targeting entity may alternatively be a molecule which is not derived from or based on an immunoglobulin. A number of "antibody mimetic" designed repeat proteins (DRPs) have been developed to exploit the binding abilities of non-antibody polypeptides. Repeat proteins such as ankyrin or leucine-rich repeat proteins are ubiquitous binding molecules which occur, unlike antibodies, intra- and extracellularly. Their unique modular architecture features repeating structural units (repeats), which stack together to form elongated repeat domains displaying variable and modular target- binding surfaces. Based on this modularity, combinatorial libraries of polypeptides with highly diversified binding specificities can be generated. DARPins (Designed Ankyrin Repeat Proteins) are one example of an antibody mimetic based on this technology. For Anticalins, the binding specificity is derived from lipocalins, a family of proteins which perform a range of functions in vivo associated with physiological transport and storage of chemically sensitive or insoluble compounds. Lipocalins have a robust intrinsic structure comprising a highly conserved β-barrel which supports four loops at one terminus of the protein. These loops for the entrance to a binding pocket and conformational differences in this part of the molecule account for the variation in binding specificity between different lipocalins. Avimers are evolved from a large family of human extracellular receptor domainsby in vitro exon shuffling and phage display, generating multi-domain proteins with binding and inhibitory properties.

Versabodies are small proteins of 3-5kDa with >15% cysteines which form a high disulfide density scaffold, replacing the hydrophobic core present in most proteins. The replacement of a large number of hydrophobic amino acids, comprising the hydrophobic core, with a small number of disulphides results in a protein that is smaller, more hydrophilic, more resistant to proteases and heat and has a lower density of T-cell epitopes. All four of these properties result in a protein having considerably reduced immunogenicity. They may also be manufactured in E. coli, and are highly soluble and stable.

TUMOUR SPECIFIC PROTEASE

The targeting molecule according to the first aspect of the invention also includes a cleavage site. The cleavage site may be or comprise an amino acid sequence which is recognised and cleaved by a tumour-associated protease. In other words, the targeting molecule includes a recognition sequence for a tumour-associated protease.

The term 'tumour-associated protease' refers to a protease which is expressed by tumour cells.

The tumour-associated protease may be selectively expressed by tumour cells but not expressed by normal, healthy cells. The tumour-associated protease may be expressed at higher levels in tumour cells in comparison to healthy cells. For example, the tumour-specific protease may be expressed 2-fold, 5-fold, 10-fold, 50-fold or 100-fold higher in the tumour cell compared to a healthy, non-tumour cell of the same cell type and/or derived from the same tissue.

The tumour-associated protease is either secreted by the cell which expresses it or is localised to the cell membrane such that the protease activity is able to occur outside of the cell.

Co-localisation of the targeting molecule comprising a recognition sequence for a tumour-associated protease with that protease results in cleavage of the targeting molecule at the recognition sequence. Because the recognition sequence is positioned between the TEM8 targeting entity and the functional payload, the cleavage results in the liberation of the functional payload from the TEM8 targeting entity. This amino acid sequence that is recognised and cleaved by a tumour-associated protease provides an increased level of specificity for the targeting molecule of the present invention, as the functional payload must be cleaved from the TEM8 binding entity. This required cleavage can only occur at sites where a protease that is able to recognise and cleave the target amino acid sequence is expressed. The protease must also be localised such that it is able to access its recognition sequence within the targeting molecule.

A variety of protease enzymes are known to be expressed and/or released by cells. The human degradome, which makes up a complete list of proteases synthesized by human cells, is made up of over 500 proteases that are distributed into five broad classes: metalloproteinases, serine, cysteine, threonine, and aspartic proteases. Serine, cysteine and threonine proteases are involved in covalent catalysis and the nucleophile of the catalytic site is part of the specified amino acid. Metalloproteinases and aspartic proteases perform non-covalent catalysis and the nucleophile is an activated water molecule.

A number of proteases have been identified as biomarkers for early diagnostic and prognostic markers, especially for cancer. Extracellular proteases, for example matrix-metalloproteases (MMPs) and serine proteases are strongly associated with cancer progression, specifically invasion and metastasis, primarily because of their ability to degrade extracellular matrices and proteins. Example proteases that are associated with tumour cells and cancer are provided in Table 1. The targeting molecule of the present invention may comprise a recognition sequence which is cleaved by one the proteases described therein.

Table 1 - Extracellular proteases associated with cancer

The targeting molecule may comprise a recognition sequence which is cleaved by Urokinase. Urokinase (urokinase-type plasminogen activator (uPA)) is linked to poor clinical prognosis of human carcinoma and has been implicated in cancer progression mechanisms such as angiogenesis, invasion and metastasis. uPA is a 411aa secreted protein, consisting of three domains: the serine protease domain, the kringle domain, and the growth factor domain. It is synthesized as a zymogen form (prourokinase or single-chain urokinase), and is activated by proteolytic cleavage between L158 and 1159. The two resulting chains are kept together by a disulphide bond.

The primary physiological substrate is plasminogen, which is an inactive form of the serine protease plasmin. Activation of plasmin triggers a proteolysis cascade that, depending on the physiological environment, participates in thrombolysis or extracellular matrix degradation. This mechanism of action links urokinase to vascular diseases and cancer.

Elevated expression levels of uPA and several other components of the plasminogen activation system are correlated with tumour malignancy. It is believed that the tissue degradation following plasminogen activation facilitates tissue invasion and, thus, contributes to metastasis.

The targeting molecule may include an amino acid sequence which is recognised and cleaved by a tumour-associated Matrix metalloproteinase (MMP). MMPs are zinc-dependent endopeptidases. Collectively, they are capable of degrading a wide range of extracellular matrix proteins and also process a number of bioactive molecules. They are known to be involved in the cleavage of cell surface receptors, the release of apoptotic ligands and chemokine and cytokine inactivation and activation. MMPs play a major role in cellular processes such as proliferation, migration, adhesion, differentiation, angiogenesis, apoptosis and host defence.

The targeting molecule may comprise an amino acid sequence which is recognised and cleaved by MMP-1 , -2, -8, -9, and/or -13.

The targeting molecule may comprise an amino acid sequence which is recognised and cleaved by MMP-2 and/or MMP-9.

The targeting molecule may comprise an amino acid sequence which is recognised and cleaved by MMP-2 and/or MMP-9, which sequence comprises Pro-Leu-Gly-Leu- Ala-Gly.

MMP-2 (gelatinase A) and MMP-9 (gelatinase B) are 72 and 92 kDa type IV collagenases, respectively. Their secretion is elevated in several types of human cancers and their elevated expression is associated with poor prognosis.

MMP-2 and MMP-9 play a key role in the degradation of type IV collagen and gelatin, the two main components of ECM. MMP-2 and MMP-9 are secreted in their latent zymogenic form, 72 and 92 kDa, respectively and are cleaved by other MMPs or proteases to yield the activated forms of 68, 58 and 54 kDa for MMP-2, and 84 kDa for MMP-9. Increased expression of MMP-2 and MMP-9 is reported in many human tumours, including ovarian, breast, colorectal, lung and prostate tumours and melanoma. The tumour-specific protease recognition sequence may be recognised by a protease localised to the membrane of the target cell. For example the protease may be an A Disintegrin and A Metalloproteinase (ADAM) protease, such as ADAM17.

FUNCTIONAL PAYLOAD

The molecule of the present invention comprises a functional payload. As described above, this payload is positioned on the opposite side of the protease recognition and cleavage sequence to the TEM8 targeting entity in the molecule (e.g. in the primary amino acid sequence of the molecule). Such an arrangement means that when the protease cleaves at its recognition sequence, the TEM8 targeting entity is separated from the functional payload (i.e. the TEM8 targeting entity and the functional payload become discrete entities).

The term 'functional payload' refers to an entity which is capable of providing an activity when it is delivered to the target cell. For example the entity may be able to alter or influence the functioning of the target cell or it may be detectable, such that the target cell in which it is localised is also detectable.

The functional payload may be a detectable entity, a cytotoxic drug or a therapeutic entity.

The detectable entity may be a fluorescent moiety, for example a fluorescent peptide. A "fluorescent peptide" refers to a polypeptide which, following excitation, emits light at a detectable wavelength. Examples of fluorescent proteins include, but are not limited to, green fluorescent protein (GFP), enhanced GFP, red fluorescent protein ( FP), blue fluorescent protein (BFP) and mCherry.

The detectable entity may be a radiolabel. The concept of radiolabelling is well known in the art and involves one or more of the atoms in a molecule of interest being substituted for an atom of the same chemical element, but of a different isotope (i.e. a radioactive isotope). Examples of radioactive isotopes used in biological research include tritium, carbon-14, sulphur-35 or phosphorus-33.

A "therapeutic entity" is any entity which may be useful for the treating of disease. Therapeutic agents include cytokines or haematopoietic factors including, but not limited to, IL-1 , IL-2, IL-4, IL-5, IL-13, IL-6, CSF-1 , M-CSF, GM-CSF, IFNa, IFNp, IFNy, IL-10, IL-12, VEGF, bone morphogenic proteins, FGFs, TNF and TGF .

Therapeutic agents also include chemotherapeutic agents, which may be a cytotoxic drug. A chemotherapeutic agent contemplated includes, without limitation, alkylating agents, nitrosoureas, ethylenimines/methylmelamine, alkyl sulfonates, antimetabolites, pyrimidine analogs, epipodophylotoxins, enzymes such as L- asparaginase; biological response modifiers such as IFNa, IL-2, G-CSF and GM- CSF; platinium coordination complexes such as cisplatin and carboplatin, anthracenediones, substituted urea such as hydroxyurea, methylhydrazine derivatives including N-methylhydrazine (MIH) and procarbazine, adrenocortical suppressants such as mitotane (ο,ρ'-DDD) and aminoglutethimide; hormones and antagonists including adrenocorticosteroid antagonists such as prednisone and equivalents, dexamethasone and aminoglutethimide; progestin such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogen such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogen such as tamoxifen; androgens including testosterone propionate and fluoxymesterone/equivalents; antiandrogens such as flutamide, gonadotropin- releasing hormone analogs and leuprolide; and non-steroidal antiandrogens such as flutamide.

The cytotoxic drug may be the catalytic domain of Pseudomonas Exotoxin.

The Pseudomonas exotoxin (PE), also known as exotoxin A, is an exotoxin produced by Pseudomonas aeruginosa.

PE is synthesized as a single 638-residue (69-kDa) polypeptide that is processed by the removal of a 25-residue N-terminal sequence before secretion as the 613-residue (66-kDa) native toxin.

PE comprises three major structural domains. The N-terminal domain I is divided into nonsequential but structurally adjacent domains la (residues 1 -252) and lb (365-404). The residues between domains la and lb comprise domain II (253-364), and the remaining C-terminal residues make up domain III (405-613).

Functionally, domain I of PE is the receptor-binding domain and targets the low density lipoprotein receptor related protein or the closely related variant LRP1 B for subsequent cellular internalization by receptor-mediated endocytosis.

Domain III is the catalytically active domain. It catalyzes the inactivation of elongation factor-2 (eEF2) by transferring an ADP-ribosyl group from NAD ⁺ to the diphthamide residue, a highly conserved, post-translationally modified histidine that is unique to eEF2. The functional catalytic activity may be provided by amino acids 405-613 or 395-613 of the native toxin.

PE-based recombinant immunotoxins (RITs) are known in the art. The most commonly employed cytotoxic fragment of PE in RITs is a 38-kDa version known as PE38, which contains a deletion of the majority of domain la (Δ1-250) and a portion of domain lb (Δ365-380) from native PE. Several RITs incorporating a 38-kDa fragment of PE are in preclinical evaluation or clinical trials. A "toxin" is a poison produced by a living cell or organism. Examples of toxins include, but are not limited to, cyanotoxins, heamotoxins, necrotoxins, neurotoxins, cytotoxins and myotoxins.

The functional payload may bind to an extracellular receptor. The target receptor for the functional payload may be present on the membrane of the cell which has bound the TEM8 targeting entity.

Alternatively the functional payload may bind to a target receptor on a cell which is in very close proximity to the cell which has bound the TEM8 targeting entity.

Upon binding to its target receptor, the functional payload activates the signalling pathway downstream of the receptor in order to bring about its functional effect.

The functional payload may comprise all or part of an antibody or antibody-like molecule (see above). The functional payload may comprise an effector domain from an antibody, such as all or part of an Fc domain. The functional payload may comprise all or part of a human Fc domain, the Fc domain may comprise the part of the sequence shown in bold in SEQ ID No. 5 (below) or a variant thereof having at least 85%, 90% or 95% sequence identity, which retains the effector function of the Fc domain.

INTERNALISATION SEQUENCE

The functional payload of the present invention may, itself, be capable of being internalised through a cell membrane once it has been cleaved from the targeting part of the molecule. Alternatively, the functional payload may comprise an intemalisation moiety, such as an intemalisation sequence which confers upon the functional payload the capacity of being internalised through a cell membrane once it has been cleaved from the targeting part of the molecule.

The functional payload may be internalised into a cell via, for example, receptor- mediated endocytosis, phagocytosis or pinocytosis.

The functional payload may be capable of being internalised through a cell membrane due to the presence of a protein transduction domain (PTD).

PTDs are short, basic peptide sequences present in many cellular and viral proteins that mediate translocation across cellular membranes. They are well known in the art and are widely used as tools for the delivery of high _r polypetides, polynucleotides, or nanoparticles to into cells.

Examples of PTD sequences and proteins which comprise PTDs and are able to translocate through cell membranes include, but are not limited to, Penetratin, polylysine, polyarginine, HIV Tat, HSV VP22, Kaposi FGF, Syn B1 , FGF-4, nuclear localization signal (NLS), anthrax toxin derivative 254-amino acids peptide segment, diphtheria toxin 'R' binding domain, MPG (HIV gp41/SV40 Tag NLS), pep-1 , WR peptide, and exotoxin A.

In all of these proteins, the activity of translocating across cellular membranes is confined to a short stretch of less than 30 amino acids highly enriched in basic residues.

For example the entity may comprise a polyarginine sequence of nine adjacent arginine residues (Arg ₉ ).

FLEXIBLE LINKER

The targeting molecule described herein may comprise a flexible linker sequence located between the TEM8 targeted entity and the protease recognition sequence. Such a linker sequence is included in the targeting molecule to minimise steric hindrance between the TEM8 targeting entity and the protease recognition sequence. Suitable flexible linker sequences are well known in the art.

The linker sequence may be, but is not limited to, a polyglycine or polyserine linker sequence. The linker sequence may, for example be (Gly ₄ Ser) ₂ , (Gly ₄ Ser) ₃ , (G ₄ S), (Gly ₃ Ser) ₄ , (G ₃ S), SerGly ₄ or SerGly ₄ SerGly ₄ .

The linker sequence may be Gly Ser or another linker of equivalent molecular length.

MOLECULE

The present invention provides molecule having the general formula [A]-[B]-[C];

wherein,

[A] is a targeting moiety which binds Tumour Endothelial Marker 8 (TEM8), [B] is an amino acid sequence recognised and cleaved by a tumour-specific protease; and,

[C] is a functional payload.

The molecule may have the formula:

[A]-[D]-[B]-[C]

wherein [D] is a flexible linker, such as G ₄ S.

The molecule may have the formula:

[A]-[E]-[B]-[C], [A]-[D]-[E]-[B]-[C] or [A]-[E]-[D]-[B]-[C]

wherein E is a neutralising moiety such as E ₈ .

The molecule may have the general structure:

PAd4-(G ₄ S)-(E ₈ )-PLGLAG-(R ₉ )-[payload] wherein PAd4 is the TEM8-specific 131 amino acids of PA or a variant thereof, and [payload] is a diagnostic or therapeutic entity.

DIAGNOSING OR TREATING A DISEASE

A molecule of the present invention may be used for the diagnosis of a disease. In this aspect, the targeting molecule may be administered to a subject who may have or is suspected of having a disease which is associated with the expression of TEM8. A molecule of the present invention may be used for the treatment of a disease through use as a therapeutic entity. In this aspect the targeting molecule may be administered to a subject having an existing disease or condition in order to lessen, reduce or improve at least one symptom associated with the disease and/or to slow down, reduce or block the progression of the disease.

The disease may be one which involves neovascularisation and/or angiogenesis. NEOVASCULARISATION & ANGIOGENESIS

Neovascularization is the formation of original, functional microvascular networks with red blood cell perfusion and is usually characterised by capillary ingrowth and endothelial proliferation in unusual sites. Angiogenesis is characterized by the protrusion and outgrowth of capillary buds and sprouts from pre-existing blood vessels.

Diseases associated with neovascularization and angiogenesis include, but are not limited to, tumour growth (i.e. cancer), diabetic retinopathy, hemangiomas, arthritis, psoriasis.

The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention.

EXAMPLES

Example 1 - Mutation of wild-type PAd4 to improve TEM8 binding and expression and purification of mutated PAd4 (mPAd4) fusion proteins

In the wildtype protective antigen protein sequence (NCBI ACCESSION P13423, SEQ ID N0.1), the 131 amino acid sequence corresponding to domain 4 was identified and modified to include two amino acid substitutions identified in J Biol. Chem. 2007, 282:9834-9845. PAD7 Arginine at 659->to Serine / Methionine at 662-Mo Arginine to enhance the binding to TEM8.

Both the wildtype (wt) and the modified (m) PAd4 amino acid sequences were reverse translated to DNA optimised for E.coli codon usage. A methionine and alanine were introduced at the amino terminal and 3x alanine introduced at the carboxyly terminal, encoded by the Ncol and NotI restrictions sites respectively for in-frame cloning.

The designed DNA inserts mPAd4 and wtPAd4 were synthesised as Ncol-NotI fragments and inserted into a modified pET16b vector for cytoplasmic expression with a hexahistidine tag. The DNA encoding human CH2-CH3 domains (Fc), was PCR amplified as a Notl-Xhol fragment and inserted between the PAd4 domain and the hexahistidine tag using the inframe restriction sites. Likewise additional fusion proteins mRFP and alkaline phosphatase were added as a Notl-Xhol fragments.

The TEM8 specific 131 amino acids of the isolated mPAd4 were expressed with a histidine tag (mPAd4-His, SEQ ID NO. 4), as an immunofusion with human lgG1 CH1-CH2 domains (mPAd4-Fc, SEQ ID NO. 5), and as a fusion with red fluorescent protein (PAd4-RFP) and as a fusion with alkaline phosphatase (mPAd4-AP), each fused at the C-terminus. The peptides were expressed in an E.coli cytoplasm expression system and purification of the constructs is shown by SDS-PAGE of the expression extraction (under native conditions) and Ni ²⁺ column purification (Figure 1 , (a), (b), (c) and (d) respectively).

SEQ ID NO. 4

mPAd4- \s

MA VGADES VVKEAHREVINSSTEGLLLNIDKDIRKILSG Yl VEIED TE GLKEVINDS YDR LNISSLRQDGKTFIDFKKYNDKLPLYISNPNYKVNVYA VTKENTIINPSEN GDTSTNGI KKILIFSKKG YE/GAAALE H H H H H H

SEQ ID NO. 5

mPAd4-¥c

MAVGADESVVKEAHREVINSSTEGLLLNIDKDIRKILSGYIVEIEDTEGLKEVINDS YDR LNISSLRQDGKTFIDFKKYNDKLPL Yl SNPNYKVNVYA VTKENTIINPSENGD TS TNG I K/L/FS KG V£/GAAAPELLGGPSVFLFPPKPKDTLMISRTPEVTC\AA/DVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGKLEHHHHHH

Example 2 - Determination of functional protein expression

The cytoplasmic expression system described in Example 1 permitted the correct folding and sulphide bond formation as indicated by the alkaline phosphatase fusion retaining enzyme activity (Figure 2). 20μΙ Elution EL1 -4 from (d) mPAd4-AP (Example 1 , Figure 1 ) was incubated with 10ΟμΙ paranitrophenyl-phosphate (1 mg/ml) and the OD of the sample was determined at 405nm after 30 minutes. The control was the PAd4-His elution fraction 2. The Kon and for the PAd4 constructs on TEM8 were determined and the K _d calculated (shown in Table 2). These data indicate that the purified proteins exhibited high affinity binding for TE 8.

Table 2 - BiaCore SPR Determination of PAd4 Binding Kinetics to rTEM8

Example 3 - An in vitro rat aortic ring assay of angiogenesis

Rat aortic rings were isolated and treated with (i) media + 1 % FCS, (ii) VEGF (1 Ong/ml) or (iii) VEGF (1 Ong/ml) + mPAd4-Fc (50pg/ml).

Figures 3 and 4 show that the mPAd4-lgG bound to TEM8 and inhibited VEGF induced angiogenesis in the rat aortic ring assay. Methodology for Ex vivo Aortic Ring Assay

From 180-200g Wistar Rats the thoracic aortas were removed and transferred to Optimem media (Gibco 31985-047) containing penicillin/streptomycin. The extraneous fat and other tissue from the aortas were carefully removed and any residual blood flushed out gently using a fine needle (25G) and syringe (1 ml) with Optimem media. After cleaning the aortas were transferred to fresh Optimem (Pen/Strep free). The aortas were sliced transversely into rings, approximately 0.5- mm in width without letting the aortas dry out. All the aortic rings from an individual aorta were placed in petri dishes containing optimem. The petri dish was then placed in an incubator at 37°C/5% C0 ₂ and left overnight.

Next day sterile water was added to sterile 10x DMEM (Gibco, cat. no. 12800-017) and mixed well. The water/DMEM mix was chilled on ice for approx. 5 mins. Collagen was slowly added (Collagen type 1 rat tail Millipore) to the chilled water/E4 mix ensuring that the final mixture was homogeneous by pipetting up-and-down.

The collagen 1 should was at a final concentration of 1.1-1.2 mg/ml and the final concentration of the DMEM is 1x. For 10ml of collagen embedding medium use: 1. 1 ml 10x DMEM, 6.1 ml ddH ₂ 0 (Sigma) & 2.9 ml rat-tail collagen type 1, assuming collagen 1 stock is 4mg/ml. The pH was adjusted by the addition of 1 M NaOH until the mixture just turns permanently pink from the original yellow colour. Each ring was transferred to 150μΙ of unpolymerised rat-tail collagen + DMEM in a 48 well plate.

The plate was then placed in the incubator after 10-15 minutes standing in the hood and then allowed to polymerise for 1 hour at 37°C/5% C0 ₂ . The plate was removed from the incubator and allowed to come to room temperature (approx.5-10 mins) and 200μΙ of aortic ring media (Optimem + 1% FCS + P/S + either 10 ng/ml VEGF or 10ng VEGF + 50μg/ml Pad4-Fc) gently added to each well and the plate placed back in the incubator. On Days 2-4, 120μΙ of media from each well was replaced with 200 ul of new aortic ring media.

The media was then refreshed every two days after embedding in collagen. After 7-10 days, phase contrast images of the rings were taken and the number of sprouts were counted and quantified per condition.

Example 4 - PAd4-Fc assessment in a murine syngeneic ovarian cancer model

Mice with syngeneic ovarian tumours are dosed at ~8mg/kg and ~5.2mg/kg with PAd4-Fc or Fc respectively.

Example 5 - TEM8 targeted delivery of therapeutic or diagnostic entities

The genes encoding the following molecules are assembled RFP-(Arg) ₉ -Protease Cleavage Site-(Glu) ₈ -mPAd4-(His) ₆ or as (His) ₆ -PAd4-(Glu) ₈ -Protease Cleavage Site- (Arg)g-mPAd. The molecules are produced in E.coli and purified as described above. The molecule is administered iv, ip or subcutaneously (0.1-0.5mg/mouse) to mice carrying a solid tumour. Irrespective of route of administration, similar circulating levels are reached within a 24 hours. After 48-72 hr the tumours and associated vasculature excised, processed for fixation and sectioning and imaged using fluorescent microscopy.

Upon binding to TEM8 in the newly forming blood vessels associated with the tumours, the presence of elevated levels of protease (MMP, urokinase) that recognise and cleave the peptide release mRFP-(Arg) ₉ or (Arg)g-mRFP which then translocate across the nearest cell membrane and accumulate inside the cells.

Materials and Methods

Expression and purification of PAd4 fusion proteins

Protein growth

· 4 L in LB Rosetta gami(DE3)pLysS carrying pET plasmid grown for 4 hours at 37 °C with ampicillin (100 pg/mL) and chloramphenicol (34 pg/mL).

• Induced (1 mM IPTG) and cells grown overnight at 18 °C

Cells harvested (pelleted by centrifugation at 4,000 xg ,10 mins) resuspended in 100 ml buffer (0.01 M Tris pH 8.0, 0.5 M NaCI, 10 mM imidazole, 0.1 % Triton X-100)

Cells lysed by emulsiflex (2 passages at pressure of 20,000 psi) (lys) and cell debris (pel) removed by centrifugation at 33,000 xg for 1 hour.

Purification

· Soluble fraction (s/n) was filtered using 0.45 pm syringe filter and manually loaded using a syringe on 4 x 1 mL His Trap HP columns (GE Healthcare). 1 mL His Trap HP columns were washed with 5 mL of buffer (0.01 M Tris pH 8.0, 0.5 M NaCI, 10 mM imidazole) (wash). Elution

• 5 mL of elution buffer (0.01 M Tris pH 8.0, 0.5 M NaCI, 500 mM imidazole) was manually injected into the 1 mL His Trap HP Ni Sepharose columns and 1 mL fractions collected in microcentrifuge tubes.

Eluted protein fractions were pooled and concentrated down to 10 mL and 0.22 pm syringe filtered before loading on FPLC. FPLC

• Protein was applied to a HiLoad™ 16/60 Superdex™200 gel filtration column pre equilibrated in elution buffer (0.02 M Tris pH 7.5, 0.15 M NaCl, 10 % glycerol).

SDS-PAGE samples

lys, s/n, pel, f/t, wash = 40 μΙ sample buffer + 10 μΙ sample

Elution fractions = 20 μΙ sample + 20 μΙ sample buffer

• Samples boiled at 100 °C for 10 minutes. Load 10 μΙ of sample to 15 % SDS gel. Gel run at 200 V for 1 hour. Gel stained in coomassie stain.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in caner immunology, cell biology, molecular biology or related fields are intended to be within the scope of the following claims.