WO2005039613A1

WO2005039613A1 - Lim2 inhibitor of lm02

Info

Publication number: WO2005039613A1
Application number: PCT/GB2004/004299
Authority: WO
Inventors: H. Rabbitts Terence; Alex Appert
Original assignee: Medical Research Council
Priority date: 2003-10-16
Filing date: 2004-10-08
Publication date: 2005-05-06
Also published as: JP2007510625A; GB0324265D0; EP1673101A1

Abstract

The present invention relates to inhibitors of protein fanction. In particular the invention relates to inhibitors of a molecule which plays pivotal roles in angiogenesis, haernatopoesis and embryogenesis. Uses of such inhibitors in the modulation of angiogenesis and haematopoeisis are described.

Description

LIM2 INHIBITORS OF LM02

The present invention relates to inhibitors of protein function. In particular the invention relates to inhibitors of a molecule which plays pivotal roles in angiogenesis, haernatopoesis and embryogenesis. Uses of such inhibitors in the modulation of angiogenesis, haematopoeisis and embryogenesis are described.

L O2 is a master regulator of several key pathways in embryogenesis, haematopoiesis and vascular formation and carries out its function in those settings via protein-protein interactions involving the LEvl domains. The most pertinent function of LMO2 from a therapeutic standpoint is its role in angiogenesis where LMO2 plays a part in control of vascular endothelial division and migration which constitutes the remodeling process which is, for instance, inherent in and essential for primary solid tumour growth and expansion of metastatic deposits. Development of reagents which can impede the function of LMO2 in protein interactions would be of great therapeutic value for preventing pathogenic angiogenesis such as in tumours.

The molecular cloning of chromosomal translocation junctions and of cDNA copies of genes affected by the chromosomal translocations has resulted in a plethora of different translocations being studied (reviewed in ). The study of chromosomal translocations in leukaemia was taken further when the T cell receptor (TCR) β chain locus was mapped to chromosome 7, band q35, suggesting that chromosomal translocations in T cell tumours are mediated by the intrinsic chromosomal instability of TCR genes, which undergo rearrangement in normal lymphoid lineage development ². This was confirmed by cloning of LMOl and LM02 at chromosomal translocation junctions with TCRδ and TCRa ^3'6 and of HOX11 and LM02 at chromosomal translocations with TCRβ ⁵' ^1A °.

Early analysis of gene structure after chromosomal translocations had occurred showed that either gene fusion or oncogene activation (enforced expression due to the new chromosomal environment) had resulted (reviewed in '). Further, it became clear in the early 1990s that transcription factors were a frequent target of chromosomal translocations "' but that a clear distinction could be drawn between chromosomal translocations in chronic and acute leukaemias ¹² and only the latter category involved transcription factors. This led to the chromosomal translocation-master gene model , in which it was postulated that transcription regulators and developmental regulators were the major target of chromosomal translocations, and that their enforced expression or gene fusion, altered the balance of transcription thereby affecting the control of cell fate in the afflicted cell.

The chromosomal translocation-genes in acute cancers are upstream master genes whose products control downstream pathways. Therefore, a major part of the tropism of chromosomal translocations is due to the specificity of transcription factor interactions which occur after the chromosomal translocation and the consequent effect on transcription pathways.

The LM02 gene in T cell acute leukaemias (T-ALL) is a paradigm of a chromosomal translocation-master gene (reviewed in ¹³). LM02 belongs to a family of four genes encoding small LIM-only proteins, two of which are involved in independent chromosomal translocations causing T-ALL. LM02 is located on chromosome 11, band pl3 and is activated by chromosomal translocations with 14ql l or 7q35, specifically in T cells. The breakpoints of these chromosomal translocations occur upstream of the natural LM02 promoter(s) causing enforced expression of LMO2 protein in cells with the translocation (the protein product per se is not affected by this). The gene is transcribed and translated into a 156 αα protein, comprising two zinc-binding LLM domains, each with two LLM fingers (each LIM finger comprises a zinc-binding motif of either four cysteines or three cysteines plus a histidine or aspartate residue co-ordinating the zinc atom and a finger of approximately 16-20 residues, Fig. 1). While the LLM domain is structurally related to the GATA Zn- finger, the characteristic of a LIM domain is the separation of each LLM Zn- finger by two αα only. The function of the LLM domain is in protein-protein interactions ^14*16 which is a consistent feature of the chromosomal translocation-master gene products. LMO2 can bind to TALl/SCL (another T-ALL translocation associated protein), LDB1 and GATA-1 in a DNA-binding complex found in erythroid cells ^1V. This complex can bind to a bipartite DNA site, comprising an E box separated from a GATA site by about 12 base pairs. We have isolated a set of peptide aptamers which bind specifically to the second LEV! domain of LMO2 and which can inhibit the cellular function of LMO2. These reagents should be useful for inhibition of tumour angiogenesis, for inhibition LMO2 -mediated T cell leukaemia and for inhibition of LMO2 function in clinical conditions where neo-vascularisation is important such as ischaemia, inflammation and diabetic retinopathy.

Summary of the invention

The present inventors have screened a library of peptide aptamers, based on an external loop of the bacterial protein thioredoxin (Trx), and have identified a set of peptides (known herein as 'anti-LMO2 reagents/LIM2 inhibitors') which bind specifically to the second LIM domain (LLM2) of the LMO2 protein. These peptide aptamers have surprisingly been found to inhibit the function of LMO2 in embryogenesis and angioenesis which is dependent upon the transcription factor LMO2.

The inventors therefore consider that these LLM2 inhibitors/anti-LMO2 reagents are of great therapeutic value in preventing LMO2 function in pathogenic circumstances such as tumour angiogenesis, for the inhibition LMO2-mediated T cell leukaemia and for inhibition of LMO2 function in clinical conditions where neo-vascularisation is important such as ischaemia, inflammation and diabetic retinopathy.

Thus in a first aspect the present invention provides an agent which is capable of binding to the LEVI2 domain of LMO2 and inhibiting the functional activity of LMO2 (a LLM2 inhibitor/anti-LMO2 reagent).

According to the present invention the term 'inhibiting' (the functional activity of LMO2) includes within its scope the significant inhibition of the functional activity of

LMO2 as compared with a suitable control. Advantageously, the term 'inhibiting' (the functional activity of LMO2) refers to a 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% inhibition of the functional activity of LMO2 as compared with a suitable control. Most advantageously, the term 'inhibiting' (the functional activity of LMO2) refers to a 95, 96, 97, 98, 99 or 100% inhibition of the functional activity of LMO2 as compared with a suitable control. Suitable controls will be familiar to those skilled in the art and are described in the detailed description of the invention and also the Examples.

The present inventors have surprisingly found that all LLM2 inhibitor peptides (peptides which substantially inhibit the functional activity of LMO2) share a consensus sequence which is described below. The consensus sequence confers upon the peptide certain structural features which the inventors believe are essential to inhibit the functional activity of LMO2. Thus, in a preferred embodiment of the above aspect of the invention, the LIM2 inhibitor is a peptide and comprises the consensus sequence:

His/CXXC C is a zinc binding Cys His represents histidine X is any amino acid.

According to the above aspect of the invention, advantageously, the LIM2 inhibitor is a peptide and comprises the consensus sequence:

HhHis/CXX'Ch

Where h represents a hydrophobic amino acid. His represents histidine, C represents cysteine,

X represents any amino acid and X represents a charged amino acid. Advantageously, the peptide LLM2 inhibitor according to the invention is an aptamer and comprises the consensus sequence shown above.

More advantageously a LLM2 inhibitor according to the invention comprises any one or more of the ten peptide sequences depicted in figure 2c as clone 207, clone 209, clone 85, clone 81, clone 63, clone 72, clone 247, clone 90, clone 69 and clone 202 and designated SEQ No 2 to SEQ No 11 respectively.

More advantageously still a LIM2 inhibitor according to the invention is any one or more of the ten peptide sequences depicted in figure 2 as clone 207, clone 209, clone 85, clone 81, clone 63, clone 72, clone 247, clone 90, clone 69 and clone 202 and designated SEQ No 2 to SEQ ID No 11 respectively.

Most advantageously, a LEVI2 inhibitor according to the invention is any one or more of those peptide sequences depicted in figure 2 as clone 207, clone 209 and clone 85 and designated SEQ ID NO 2, 3 and 4 respectively.

Peptide inhibitors according to the present invention may be used in unconstrained form or constrained at the C-terminal and/or N-terminal within a scaffold. Suitable scaffolds will be familiar to those skilled in the art and may be naturally occurring or synthetic. Suitable naturally occurring scaffolds are described in the detailed description of the invention. According to the present invention, advantageously, the scaffold is a protein scaffold and the thioredoxin protein.

According to the present invention, inhibitors according to the invention may comprise one or more intracellular targeting agents. Advantageously, an intracellular targeting agent according to the present invention is a nuclear localisation signal. Suitable nuclear localisation signals will be familiar to those skilled in the art and are described in the detailed description of the invention. In a further aspect the present invention provides a composition comprising one or more LIM2 inhibitors/anti-LMO2 reagents according to the invention and a pharmaceutically acceptable carrier, diluent or exipient.

In a preferred embodiment of the above aspect of the invention, the LIM2 inhibitor is a peptide and comprises the consensus sequence:

His/CXXC C is a zinc binding Cys His represents histidine X is any amino acid.

According to the above aspect of the invention, advantageously, the LEVI2 inhibitor is a peptide and comprises the consensus sequence:

HhHis/CXX'Ch

X represents any amino acid and X¹ represents a charged amino acid.

Advantageously, the peptide LLM2 inhibitor according to the invention is an aptamer and comprises the consensus sequence shown above.

Advantageously, the peptide LDVI2 inhibitor according to the invention is an aptamer and possesses the consensus sequence shown above. More advantageously a LIM2 inhibitor according to the invention comprises any one or more of the ten peptide sequences depicted in figure 2c as clone 207, clone 209, clone 85, clone 81, clone 63, clone 72, clone 247, clone 90, clone 69 and clone 202 and designated SEQ No 2 to SEQ No 11 respectively. Most advantageously a LIM2 inhibitor according to the invention is any one or more of the ten peptide sequences depicted in figure 2 as clone 207, clone 209, clone 85, clone 81, clone 63, clone 72, clone 247, clone 90, clone 69 and clone 202 and designated SEQ No 2 to SEQ No 11 respectively.

In an alternative embodiment of the above aspect of the invention, the LIM2 inhibitor/anti-LMO2 reagent is an antibody as herein defined.

According to the above embodiment of the invention, advantageously the antibody is an scFv. In an alternative embodiment of the above aspect of the invention, the antibody is an dAb as defined herein. More advantageously, antibody LIM2 inhibitors according to the present invention are intracellular binding antibodies as herein defined. Most advantageously they are intracellularly binding scFv molecules, idAb (intracellularly binding single domain antibodies) which may be heavy chain domain idAbs or light chain domain idAbs. In a preferred embodiment of this aspect of the invention, an antibody LLM2 inhibitor is an intracellularly binding antibody comprising at least one CDR having the amino acid sequences depicted in Fig 8 and designated SEQ LD No 12(a), 12(b) and 12(c). In a further preferred embodiment of this aspect of the invention, the antibody LIM2 inhibitor is an intracellularly binding idAb or scFv comprising at least one CDR having the amino acid sequences depicted in Fig 8 and designated SEQ LD No 12(a), 12(b) and 12(c). In a still further preferred embodiment of this aspect of the invention, the antibody L1M2 inhibitor is an intracellularly binding idAb or scFv comprising at least two CDRs having the amino acid sequences depicted in Fig 8 and designated SEQ ID No 12(a), 12(b) and 12(c). In a yet further preferred embodiment of this aspect of the invention, the antibody LLM2 inhibitor is an intracellularly binding idAb or scFv comprising all three CDRs having the amino acid sequences depicted in Fig 8 and designated SEQ LD No 12(a), 12(b) and 12(c). In a preferred embodiment of this aspect of the invention, the antibody LEV12 inhibitor is an intracellularly binding scFv comprising the amino acid sequence depicted in Fig 8 and designated SEQ ID No 12, other than the Ser-Gly linker. In a most preferred embodiment of this aspect of the invention, the antibody LIM2 inhibitor is an intracellularly binding scFv consisting of the amino acid sequence depicted in Fig 8 and designated SEQ LD No 12. Thus in a further aspect the present invention provides an anti-LMO2 antibody (LLM2 inhibitor) which comprises at least one CDR having the amino acid sequences depicted in Fig 8 and designated SEQ LD No 12(a), 12(b) and 12(c).

Preferably, an anti-LMO2 antibody according to the invention is fused or conjugated to a domain or sequence from a protein responsible for translocational activity. Details of suitable translocation peptides are provided in the detailed description of the invention.

Advantageously, the present invention provides an anti-LMO2 antibody (LLM2 inhibitor) which comprises three CDRs having the amino acid sequences depicted in Fig 8 and designated SEQ LD No 12(a), 12(b) and 12(c) respectively and which together form an antigen binding site.

In a further aspect still the present invention provides nucleic acid encoding an anti- LMO2 antibody (LIM2 inhibitor) which comprises at least one CDR having the amino acid sequences depicted in Fig 8 and designated SEQ LD No 12(a), 12(b) and 12(c).

Advantageously, the present invention provides nucleic acid encoding an anti-LMO2 antibody (LLM2 inhibitor) which comprises three CDRs having the amino acid sequences depicted in Fig 8 and designated SEQ ID No 12(a), 12(b) and 12(c) respectively and which together form an antigen binding site.

In a preferred embodiment of this aspect of the invention, the antibody LLM2 inhibitor is an intracellularly binding scFv comprising the amino acid sequence depicted in Fig 8 and designated SEQ LD No 12, other than the ser-gly linker.

In a further aspect the invention provides the use of an LLM2 inhibitor according to the invention or a composition comprising it in inhibiting the functional activity of LMO2.

In a further aspect the present invention provides the use of a LLM2 inhibitor according to the invention in the preparation of a medicament for inhibiting the functional activity of LMO2. The present invention have shown that LMO2 has many and varied roles within a mammal. Thus as herein defined the term the 'functional activity' of LMO2 refers to one or more, several, or all of the roles performed by LMO2 within a mammal. Moreover, the term the 'inhibition of the functional activity of LMO2' refers to the substantial inhibition as described herein of one or more, several, or all of the functions performed by LMO2 within a mammal.

The present inventors have shown that LMO2 plays a role in the conditions in the group consisting of the following: angiogenesis (pathogenic angiogenesis and non- pathogenic angiogenesis), embryogenesis and haernatopoesis including leukaemogenesis.

As referred to herein the term 'angiogenesis' refers to the process of primary capillary remodelling which results in the formation of a mature vascular system. In contrast the term 'vasculogenesis' describes the primary process of vasculature formation in which the primary capillary network is formed. Importantly, the present inventors have shown that LMO2 plays a critical role in the process of angiogenesis but not vasculogenesis.

As referred to herein the term 'pathogenic angiogenesis' refers to any disease condition/state in which angiogenesis plays a role. The present inventors have shown that 'pathogenic angiogenesis' contributes to the conditions selected from the group consisting of the following: tumour angiogenesis, tumour metastasis, ischemia, inflammation and diabetic retinopathy. Thus as herein defined the term 'pathogenic angiogenesis' includes within its scope all of the conditions referred to above. Advantageously, according to the present invention, the pathogenic angiogenesis is tumour angiogenesis and/or tumour metastasis.

In addition to the roles of LMO2 in 'pathogenic angiogenesis' LMO2 also plays a role in 'non-pathogenic angiogenesis' during certain events such as wound healing and menstruation. Thus according to the present invention the term 'non-pathogenic angiogenesis' includes within its scope wound healing and menstruation.

According to the above aspects of the invention, the use may be in vitro or in vivo. Advantageously the use is in vivo.

As described above, the present inventors have shown that LMO2 plays a role in processes such as angiogenesis, embryogenesis and haematopoeisis. Accordingly, the inventors consider that LIM2 inhibitors according to the invention will be of great therapeutic value in the prophylaxis and/or treatment of conditions including: tumour formation, tumour metastasis, LMO2 mediated T-cell leukemia, inflammation, ischemia, diabetic retinopathy, the modulation of wound healing and the modulation of menstruation.

Thus, in yet a further aspect of the invention, there is provided an LEVI2 inhibitor according to the invention or a composition comprising it for use in medicine.

In yet a further aspect the invention provides the use of one or more LIM2 inhibitors according to the invention in the preparation of a medicament for the prophylaxis and/or treatment of one or more conditions selected from the group consisting of: tumour formation, tumour metastasis, LMO2 mediated T-cell leukemia, inflammation, ischemia, diabetic retinopathy and wound healing.

In yet a further aspect the invention provides a method for the prophylaxis and/or treatment of any one or more conditions selected from the group consisting: tumour formation, tumour metastasis, LMO2 mediated T-cell leukemia, inflammation, ischemia, diabetic retinopathy and wound healing comprising the step of administering to an individual in need of such treatment one or more LIM2 inhibitors according to the invention.

According to the above aspects of the invention advantageously, the condition is tumour formation and/or tumour metastasis. In a preferred embodiment of the above aspect of the invention, the LLM2 inhibitor is a peptide and comprises the consensus sequence: His/CXXC

C represents a zinc binding Cys His represents histidine X is any amino acid.

According to the above aspect of the invention, advantageously, the LLM2 inhibitor is a peptide and comprises the consensus sequence:

HhHis/CXX'Ch

Where h represents a hydrophobic amino acid. His represents histidine, C represents cysteine, X represents any amino acid and X¹ represents a charged amino acid.

Advantageously, the peptide LIM2 inhibitor according to the invention is an aptamer and comprises the consensus sequence shown above.

Advantageously, the peptide LLM2 inhibitor according to the invention is an aptamer and possesses the consensus sequence shown above. More advantageously a LIM2 inhibitor according to the invention comprises any one or more of the ten peptide sequences depicted in figure 2c as clone 207, clone 209, clone 85, clone 81, clone 63, clone 72, clone 247, clone 90, clone 69 and clone 202 and designated SEQ No 2 to SEQ No 11 respectively. Most advantageously a LEM2 inhibitor according to the invention is any one or more of the ten peptide sequences depicted in figure 2c as clone 207, clone 209, clone 85, clone 81, clone 63, clone 72, clone 247, clone 90, clone 69 and clone 202 and designated SEQ No 2 to SEQ No 11 respectively. In an alternative embodiment of the above aspect of the invention, the antibody is an scFv. More advantageously, antibody LLM2 inhibitors according to the present invention are intracellular binding antibodies as herein defined. Most advantageously they are intracellularly binding scFv molecules, idAb (intracellularly binding single domain antibodies) which may be heavy chain domain idAbs or light chain domain idAbs. In a preferred embodiment of this aspect of the invention, an antibody LIM2 inhibitor is an intracellularly binding antibody comprising at least one CDR having the amino acid sequences depicted in Fig 8 and designated SEQ LD No 12(a), 12(b) and 12(c). In a further preferred embodiment of this aspect of the invention, the antibody LLM2 inhibitor is an intracellularly binding idAb or scFv comprising at least one CDR having the amino acid sequences depicted in Fig 8 and designated SEQ ID No 12(a), 12(b) and 12(c). In a still further preferred embodiment of this aspect of the invention, the antibody LLM2 inhibitor is an intracellularly binding idAb or scFv comprising at least two CDRs having the amino acid sequences depicted in Fig 8 and designated SEQ LD No 12(a), 12(b) and 12(c). In a yet further preferred embodiment of this aspect of the invention, the antibody LLM2 inhibitor is an intracellularly binding idAb or scFv comprising at three CDRs having the amino acid sequences depicted in Fig 8 and designated SEQ LD No 12(a), 12(b) and 12(c). In a most preferred embodiment of this aspect of the invention, the antibody LLM2 inhibitor is an intracellularly binding scFv consisting of the amino acid sequence depicted in Fig 8 and designated SEQ LD No 12.

The uses and methods of the present invention are suitable for the treatment of mammalian individuals including but not limited to those selected from the group consisting of the following: human, mouse, rat, guinea-pig, hamster, rabbit, dog, cat, goat, monkey, horse. Advantageously, the uses and methods of the present invention are suitable for the treatment of human individuals.

The invention will now be described by the following Examples which should in no way be considered limiting of the invention.

Definitions. 'LMO2' is a transcription factor. Significantly, it is a master regulator of several key pathways in embryogenesis, haematopoiesis and vascular formation and carries out its function via protein-protein interactions involving LLM domains.

The term a 'peptide' refers to 10 or less, 15 or less, 16 or less, 17 or less, 18 or less, 19 or less, 20 or less, 25 or less, 50 or less amino acid joined together at the N-terminus of each amino acid via an amide bond. According to the present invention, advantageously, the term 'peptide' according to the present invention refers to 25 or less amino acids joined to one another via amide bonds. Most advantageously, the term 'peptide' refers to 20 or less amino acids joined together at their N-terminus. Peptides according to the present invention may be branched or linear. Advantageously peptides according to the invention are linear.

The term 'protein' refers to a collection of one or more polypeptide chains. As herein defined 'a polypeptide chain' describes more than 50 amino-acids joined together via amide bonds at the N-terminus of each amino acid to form a chain. Polypeptide chains according to the present invention may be branched or linear. One or more polypeptide chains comprise a protein molecule which possesses secondary and tertiary structural elements. These structural elements determine the structure and function of the resultant protein.

An 'anti-LMO2 reagent/LIM2 inhibitor' refers to an agent which binds to the LLM- 2 domain of LMO2 and substantially inhibits the functional activity of LMO2. Such agents may be naturally occurring or synthetic. Suitable synthetic molecules include small molecules. Suitable naturally occurring molecules include proteins in particular antibodies which may be monoclonal or polyclonal, peptides and/or nucleic acid molecules.

The term 'inhibiting' (the functional activity of LMO2) refers to the significant inhibition of the functional activity of LMO2 as compared with a suitable control. Advantageously, the term 'inhibiting' (the functional activity of LMO2) refers to a 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% inhibition of the functional activity of LMO2 as compared with a suitable control. Most advantageously, the term 'inhibiting' (the functional activity of LMO2) refers to a 95, 96, 97, 98, 99 or 100% inhibition of the functional activity of LMO2 as compared with a suitable control. Suitable controls will be familiar to those skilled in the art and are described in the detailed description of the invention and also the Examples. LMO2 has many and varied roles within a mammal. Thus as herein defined the term the 'functional activity' of LMO2 refers to one or more, several, or all of the roles performed by LMO2 within the cell. Moreover, the term the 'inhibition of the functional activity of LMO2' refers to the substantial inhibition as described herein or one or more, several, or all of the functions performed by LMO2 within a mammal. The present inventors have shown that LMO2 plays a role in the conditions in the group consisting of the following: angiogenesis (including pathogenic angiogenesis and non-pathogenic angiogenesis), certain forms of T-cell leukemia, inflammation, wound-healing, ischemia and diabetic retinopathy.

Antibodies as used herein, refers to complete antibodies or antibody fragments capable of binding to a selected target, and including Fv, ScFv, Fab' and F(ab')₂, monoclonal and polyclonal antibodies, engineered antibodies including chimeric, CDR-grafted and humanised antibodies, and artificially selected antibodies produced using phage display or alternative techniques. Small fragments, such as Fv and ScFv, possess advantageous properties for diagnostic and therapeutic applications on account of their small size and consequent superior tissue distribution. Preferably, the LIM2 inhibitor antibody according to the present invention is a single heavy domain antibody (heavy domain dAb), a single light chain domain antibody (light domain dAb) or an scFv molecule. More preferably the LLM2 inhibitor antibody according to the present invention is an intracellularly binding single domain antibody molecule (idAb) as defined herein. As herein defined the term 'antibody' also includes within its scope molecules which comprise an antigen binding moiety comprising at least one heavy chain variable domain and at least one antibody constant region domain.

CDR (complementarity determining region) of an immunoglobulin molecule heavy and light chain variable domain describes those amino acid residues which are not framework region residues and which are contained within the hypervariable loops of the variable regions. These hypervariable loops are directly involved with the interaction of the immunoglobulin with the ligand. Residues within these loops tend to show less degree of conservation than those in the framework region.

Intracellular means inside a cell. The cell may be any cell, prokaryotic or eukaryotic, and is preferably selected from the group consisting of a bacterial cell, a yeast cell and a higher eukaryote cell. Most preferred are yeast cells and mammalian cells. As used herein, therefore, "intracellular" antibodies and targets or ligands are antibodies and targets/ligands which are present within a cell (including the cytoplasm and the nucleus). In addition the term 'Intracellular' refers to environments which resemble or mimic an intracellular environment. Thus, "intracellular" may refer to an environment which is not within the cell, but is in vitro. For example, the method of the invention may be performed in an in vitro transcription and/or translation system, which may be obtained commercially, or derived from natural systems. Accordingly, an 'intracellularly binding antibody' according to the present invention is an antibody as herein defined which is capable of specific binding to one or more antigens within an intraceuular enviroment as defined herein.

The term 'angiogenesis' refers to the process of primary capillary remodelling which results in the formation of a mature vascular system. In contrast the term 'vasculogenesis' describes the primary process of vasculature formation in which the primary capillary network is formed. Importantly, the present inventors have shown that LMO2 plays a critical role in the process of angiogenesis but not vasculogenesis.

The term 'pathogenic angiogenesis' refers to any disease condition/state in which angiogenesis plays a role. The present inventors have shown that 'pathogenic angiogenesis' contributes to the conditions selected from the group consisting of the following: tumour angiogenesis, tumour metastasis, ischemia, inflammation and diabetic retinopathy. Thus as herein defined the term 'pathogenic angiogenesis' includes within its scope all of the conditions referred to above. Advantageously, according to the present invention, the pathogenic angiogenesis is tumour angiogenesis and/or tumour metastasis.

LMO2 also plays a role in 'non-pathogenic angiogenesis' during certain events such as wound healing and menstruation. Thus according to the present invention the term 'non-pathogenic angiogenesis' includes within its scope wound healing and menstruation.

Brief Description of the Figures.

Figure 1. LIM domains: structure and function

The intriguing aspect of LMO2 function is that this small molecule (156 amino acids) could have such an key role. This is partly explained by its structure, which comprises two LIM domains (B) ⁵, each itself being comprised of two zinc-binding LIM fingers, in which a single zinc atom is co-ordinated between cysteine (cys), histidine (his) or aspartate (asp) residues (A) ^42_44. LIM domains are protein interaction modules ¹⁴' ¹⁵ and LMO2 seems to be a versatile protein partner playing a bridging role in DNA-binding complexes of differing composition in normal cells or in T cell leukaemia cells ^{15"18, 45}.

Figure 2: Sequence of peptides binding to the LMO2 protein

A peptide library comprising 20mer peptides fused into the Trx protein (used as a scaffold) was screened against a clone expressing an LMO2 bait. Clones encoding LMO2-interacting Trx-peptides were isolated and the sequence of the peptides obtained. The isolated peptide sequences are shown. From 3 x 10⁶ transformants, we isolated 15 plasmids that express peptide aptamers that interact with LMO2.

A. Sequences of the fifteen peptides clones interacting with LMO2 in the yeast two- hybrid assay. Conserved cys (C) and his (H) residues are in bold and underlined.

B. Sequences of the ten peptides interacting with LMO2 in yeast 2-hybrid assay and which were also active in a mammalian cells 2-hybrid assays using CHO cells. Conserved cysteine (C) and histidine (H) residues are in bold and underlined, conserved hydrophobic residues are underlined and residues identical with the motif from LMO2 LLM2 are indicated by •. The fold stimulation of luciferase activity generated in a mammalian two-hybrid assay with each Trx-peptide is shown on the LHS of the figure (+/- standard deviation).

C. Sequences of the ten peptides interacting with LMO2. Conserved cysteine (C) and histidine (H) residues are in bold and underlined. The short conserved aptamer motif is shown at the top of the diagram and conserved hydrophobic residues are underlined. C refers to a conserved charged residue in the aptamer motif.

Figure 3: Mammalian interaction assay of anti-LMO2 peptide aptamers

Clones expressing GAL4 DBD-Trx-peptide (pM vectors) were used as baits and their interaction tested against various clones expressing prey proteins (NP16 fusion vectors) which were co-transfected into CHO cells together with a luciferase reporter clone.

Luciferase activity was measured after 24 hours. Values shown are fold stimulation compared with the empty pM bait vector alone. The NP16 prey clones used were vector only (pVP16+), LMO2, the individual LMI domains of LMO2 (LIMl or LLM2), LMO4, LMOl, the protein kinase TTK (zinc finger type C₂H₂), the oestrogen receptor α (zinc finger type C₄) (NP16-ER), Ldbl or Gatal. (ΝB: The Gal4-207 has not been tested against TTK, ERα, Ldbl and Gatal..)

A. Mammalian two-hybrid data with Trx207

B. Mammalian two-hybrid data with 207 peptide not in a scaffold C. Mammalian two-hybrid data with Trx209

D. Mammalian two-hybrid data with Trx85

Figure 4: Erythroid differentiation of ES cell-derived embryoid bodies

Embryonic stem cells (ES) were cultured in vitro to produce erythroid differentiation and the numbers of erythroid cells measured by expression of the erythroid marker Terl 19. ES clones were wild type (CCB); null LMO2 cells (H16); heterozygous LMO2 (with a lacZ gene knocked-in LM02, clone KZ26); ES cells with a knock-in of Trx207 into one allele of LM02 (AF6) or the same clone stably transfected with a CMV-promoter driven Trx207 (6B23); ES cells with a knock-in of Trx into one allele of LM02 (BB4); heterozygous LM02 (KZ26) stably transfected with a EFα-promoter driven Trx207 (GI) or heterozygous LM02 (KZ26) stably transfected with a EFα-promoter driven Trx only (A5). A. Percentage of cells expressing Terl 19 in dissociated 11 day-old embryoid bodies.

B. Percentage inhibition of Terl 19 expressing cells in dissociated 11 day-old embryoid bodies.

Figure 5: In vitro differentiation of ES into the erythroid lineage. Each ES cells were grown in 1MDM containing 40% of methylcellulose, 20% of FBS, lOOU/ml of penicillin, lOOμg/ml of streptomycin, lOOμM of MTG, lOμg/ml of Human Insulin, 2mM of L-glutamine, lOng/ml of recombinant mouse NEGF, lU/ml of recombinant mouse Epo, 25ng/ml of recombinant human FGF and 5ng/ml of recombinant mouse IL-6 for 11 days. Embryoid bodies were dissociated with collagenase A to single ES cells for FACS analysis. FACS analysis was performed with biotinylated anti-mouse erythroid cells mAb (Ter-119) and streptavidin-phycoerythrin conjugate. Each assay was carried out in duplicate (shown by closed or open boxes).

Figure 6: de novo development of vascular endothelial cells (vasculogenesis) in the process of ES cell differentiation. Each ES cells were grown as described in Figure 5. Embryoid bodies were dissociated with collagenase A and FACS analysis was performed with biotinylated anti- mouse CD31mAb and streptavidin-phycoerythrin conjugate. Each assay was carried out in duplicate (shown by closed or open boxes).

Figure 7: beta-galactoside expression in ES-cell derived endothelial cells during angiogenesis. ES cells were injected to the inner cell mass of C57BL/6 blastocysts and implanted into recipient females. Embryos were isolated at day 10.5, stained with Xgal as described (Yamada et al., 1998) and whole mount photographs obtained.

Figure 8: Amino acid sequence of scFv which binds to the L1M2 domain of LMO2 and inhibits the functional activity of LMO2 (an anti-LMO2 antibody). V_H CDRs are underlined and also shaded and designated (a), (b) and (c). V_L CDRS are shaded and designated (a), (b), (c). The sequence of the Gly-Ser linker is underlined. The sequence is of the scFv is designated SEQ ID No: 12. Detailed description of the invention

General Techniques.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridisation techniques and biochemistry). Standard techniques are used for molecular, genetic and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (1999) 4^th Ed, John Wiley & Sons, Inc. which are incorporated herein by reference) and chemical methods. In addition Harlow & Lane., A Laboratory Manual Cold Spring Harbor, N.Y, is referred to for standard Immunological Techniques.

(A) Characteristics of LIM2 inhibitors according to the invention.

In a first aspect the present invention provides an agent which is capable of binding to the LLM2 domain of LMO2 and inhibiting its functional activity.

According to the present invention, a 'LEVI2 inhibitor/anti-LMO2 reagent' refers to an agent which binds to the LEVI-2 domain of LMO2 and substantially inhibits the functional activity of LMO2. Such agents may be naturally occurring or synthetic. Suitable synthetic molecules include small molecules. Suitable naturally occurring molecules include proteins (in particular antibodies which may be monoclonal or polyclonal) peptides and/or nucleic acid molecules. Advantageously, anti-LMO2 antibodies according to the present invention are scFv molecules or dAb molecules as herein defined. More advantageously, antibody LEVI2 inhibitors according to the present invention are intracellular binding antibodies (intrabodies) as herein defined. Most advantageously they are intracellularly binding scFv molecules, idAb (intracellularly binding single domain antibodies) which may be heavy chain domain idAbs or light chain domain idAbs. In a preferred embodiment of this aspect of the invention, an antibody LLM2 inhibitor is an intracellularly binding antibody comprising at least one CDR having the amino acid sequences depicted in Fig 8 and designated SEQ LD No 12(a), 12(b) and 12(c). In a further preferred embodiment of this aspect of the invention, the antibody LLM2 inhibitor is an intracellularly binding idAb or scFv comprising at least one CDR having the amino acid sequences depicted in Fig 8 and designated SEQ LD No 12(a), 12(b) and 12(c). In a still further preferred embodiment of this aspect of the invention, the antibody LLM2 inhibitor is an intracellularly binding idAb or scFv comprising at least two CDRs having the amino acid sequences depicted in Fig 8 and designated SEQ LD No 12(a), 12(b) and 12(c). In a yet further preferred embodiment of this aspect of the invention, the antibody LIM2 inhibitor is an intracellularly binding idAb or scFv comprising all three CDRs having the amino acid sequences depicted in Fig 8 and designated SEQ LD No 12(a), 12(b) and 12(c). In a still further preferred embodiment of this aspect of the invention, the antibody LLM2 inhibitor is an intracellularly binding scFv comprising, preferably consisting of the amino acid sequence depicted in Fig 8 and designated SEQ LD No 12, other than the Ser-Gly linker. In a most preferred embodiment of this aspect of the invention, the antibody LLM2 inhibitor is an intracellularly binding scFv comprising, preferably consisting of the amino acid sequence depicted in Fig 8 and designated SEQ LD No 12.

(i) Antibodies.

Antibody preparation

Antibodies raised against the L1M2 domain of LMO2 may be prepared using standard laboratory techniques. Either recombinant proteins or those derived from natural sources can be used to generate antibodies. For example, the protein (or "immunogen") is administered to challenge a mammal such as a monkey, goat, rabbit or mouse. The resulting antibodies can be collected as polyclonal sera, or antibody- producing cells from the challenged animal can be immortalized (e.g. by fusion with an immortalizing fusion partner to produce a hybridoma), which cells then produce monoclonal antibodies. lai. Polyclonal antibodies The antigen protein is either used alone or conjugated to a conventional carrier in order to increases its immunogenicity, and an antiserum to the peptide-carrier conjugate is raised in an animal, as described above. Coupling of a peptide to a carrier protein and immunizations may be performed as described (Dymecki et al. (1992) J. Biol. Chem., 267: 4815). The serum is titered against protein antigen by ELISA or alternatively by dot or spot blotting (Boersma and Nan Leeuwen (1994) J. Νeurosci. Methods, 51: 317). The serum is shown to react strongly with the appropriate peptides by ELISA, for example, following the procedures of Green et al. (1982) Cell, 28: 477.

laii. Monoclonal antibodies Techniques for preparing monoclonal antibodies are well known, and monoclonal antibodies may be prepared using any candidate antigen, preferably bound to a carrier, as described by Arnheiter et al. (1981) Nature, 294, 278. Monoclonal antibodies are typically obtained from hybridoma tissue cultures or from ascites fluid obtained from animals into which the hybridoma tissue was introduced. Nevertheless, monoclonal antibodies may be described as being "raised against" or "induced by" a protein.

After being raised, monoclonal antibodies are tested for function and specificity by any of a number of means. Similar procedures can also be used to test recombinant antibodies produced by phage display or other in vitro selection technologies. Monoclonal antibody-producing hybridomas (or polyclonal sera) can be screened for antibody binding to the immunogen, as well. Particularly preferred immunological tests include enzyme-linked immunoassays (ELISA), immunoblotting and immunoprecipitation (see Voller, (1978) Diagnostic Horizons, 2: 1, Microbiological Associates Quarterly Publication, Walkersville, MD; Voller et al. (1978) J. Clin. Pathol., 31: 507; U.S. Reissue Pat. No. 31,006; UK Patent 2,019,408; Butler (1981) Methods Enzymol., 73: 482; Maggio, E. (ed.), (1980) Enzyme Immunoassay, CRC Press, Boca Raton, FL) or radioimmunoassays (RIA) (Weintraub, B., Principles of radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March 1986, pp. 1-5, 46-49 and 68-78), all to detect binding of the antibody to the immunogen against which it was immunogen must be labeled to facilitate such detection. Techniques for labelling antibody molecules are well known to those skilled in the art (see Harrow and Lane (1989) Antibodies, Cold Spring Harbor Laboratory, pp. 1-726.).

(lb) Western (antibody) blotting

Western blotting to test for the specificity of antibody binding may be performed using methods familiar to those skilled in the art and detailed in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (1999).

Intrabodies.

Intrabody (intracellularly binding antibodies) LLM2 inhibitors are particularly useful in inhibiting the functional activity of LMO2. The structure and characteristics of these antibodies which permits their function within an intracellular environment are described in PCT/GB2002/003512, PCT/GB2003/001077, GB 0226728.4 and GB0026727.6. These applications are herein incorporated by reference. Advantageously, an intrabody (intracellularly binding antibody) according to the present invention is an scFv comprising, preferably consisting of the amino acid sequence shown in figure 8 and depicted as SEQ LD No 12.

(it) Peptides as LIM2 inhibitors.

According to the present invention, the term a 'peptide' refers to 10 or less, 15 or less, 16 or less, 17 or less, 18 or less, 19 or less, 20 or less, 25 or less, 50 or less amino acid joined together at the N-terminus of each amino acid via an amide bond. According to the present invention, advantageously, the term 'peptide' according to the present invention refers to 25 or less amino acids joined to one another via amide bonds. Most advantageously, the term 'peptide' refers to 20 or less amino acids joined together at their N-terminus. Peptides according to the present invention may be branched or linear. Advantageously peptides according to the invention are linear.

In contrast the term 'protein' refers to a collection of one or more polypeptide chains. As herein defined 'a polypeptide chain' describes more than 50 amino-acids joined together via amide bonds at the N-terminus of each amino acid to form a chain. Polypeptide chains according to the present invention may be branched or linear. One or more polypeptide chains comprise a protein molecule which possesses secondary and tertiary structural elements. These structural elements determine the structure and function of the resultant protein.

In a preferred embodiment of the invention, the LLM2 inhibitor is a peptide and comprises the consensus sequence: His/CXXC

C is a zinc binding Cys His represents histidine X is any amino acid.

Advantageously, the LLM2 inhibitor is a peptide and comprises the consensus sequence:

HnHis/CXX'Ch

Advantageously, the peptide LLM2 inhibitor according to the invention is an peptide aptamer (20 mer) and comprises the consensus sequence shown above. It has previously been shown that the LLM domain provides an interface for protein interactions ^14"16 and the LEVI-only protein LMO2 binds to several partners through its LLM domains.

The present inventors have shown that by modifying the LLM-domain protein-protein interactions of LMO2, it is possible to control the critical specific step of endothelial remodelling such as in tumour neo-vascularisation.

The anti-LMO2 peptide aptamers according to the present invention are typified by the 207 peptide which has the conserved cys-x-x-cys motif embedded in the 20 amino acids which comprise the peptide domain within the Trx protein scaffold. The anti-

LMO2 peptide aptamers described here bind specifically to the LMO2 LLM domain and do not bind efficiently to any of the LMO-only proteins. Analysis of the peptide aptamers indicates that LMO2 binding is dependent on the cys/his-x-x-cys motif and that the conservation of flanking hydrophobic residues on either side and a charged residue between. The 207 peptide is effective in both specific binding (Fig. 3) and in the inhibition of LMO2 function as exemplified by an erythropoiesis assay (Fig. 4).

The peptide aptamers bind to the LLM2 domain of LMO2 and examination of the peptide sequences does not reveal any obvious reason for this nor for the specificity for LMO2 over other members of the LMO family.

Amino acid types.

Charged, polar uncharged and hydrophobic (non-polar) amino acids as described herein are categorised in the table below. In the case of the conservative replacement of amino acids, amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:

AROMATIC H F W Y

(iia) Protein scaffolds for use with peptide inhibitors according to the invention.

Peptide LMO2 inhibitors according to the invention may be used in unconstrained form or alternatively constrained at the N and/or C-terminus of peptide via one or more attachments with a scaffold. Suitable scaffolds may be naturally occurring or synthetic. Advantageously, according to the present invention, the scaffold is a protein scaffold.

Suitable scaffolds will be familiar to those skilled in the art and include those selected from the group consisting of the following: scaffolds based on immunoglobulin domains, scaffolds based on bacterial receptors such as SpA, those based on fibronectin, lipcallin, CTLA4 Other suitable scaffolds include those based on fibronectin and affibodies. Other suitable scaffolds include lipocallin and CTLA4, as described in van den Beuken et al., J. Mol. Biol. (2001) 310, 591-601, and scaffolds such as those described in WO0069907 (Medical Research Council), which are based for example on the ring structure of bacterial GroEL or other chaperone polypeptides.

Advantageously, according to the present invention, the scaffold is the Trx protein (bacterial protein thioredoxin).

liii. Nucleic acid anti-LMO2 reagents/LLM2 inhibitors

According to the present invention, LIM2 inhibitors/anti-LOM2 reagents may comprise nucleic acid molecules. Advantageously, such a LLM2 inhibitor is anti-sense RNA. Methods for the preparation of such RNA and their modes for use will be familiar to those skilled in the art.

(C) Inhibition of the functional activity ofLM02 using LIM2 inhibitors/anti-LM02 reagents according to the invention. In a further aspect the invention provides the use of an LLM2 inhibitor according to the invention or a composition comprising it in inhibiting the functional activity of LMO2.

The present invention have shown that LMO2 has many and varied roles within a mammal. Thus as herein defined the term the 'functional activity' of LMO2 refers to one or more, several, or all of the roles performed by LMO2 within a mammal and which are described herein. Moreover, the term the 'inhibition of the functional activity of LMO2' refers to the substantial inhibition as described herein or one or more, several, or all of the functions performed by LMO2 within a mammal.

According to the present invention the term 'inhibiting' (the functional activity of LMO2) includes within its scope the significant inhibition of the functional activity of LMO2 as compared with a suitable control. Advantageously, the term 'inhibiting' (the functional activity of LMO2) refers to a 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% inhibition of the functional activity of LMO2 as compared with a suitable control. Most advantageously, the term 'inhibiting' (the functional activity of LMO2) refers to a 95, 96, 97, 98, 99 or 100% inhibition of the functional activity of LMO2 as compared with a suitable control. Suitable controls will be familiar to those skilled in the art and are described in the detailed description of the invention and also the Examples.

The present inventors have shown that LMO2 plays a role in the conditions in the group consisting of the following: angiogenesis (pathogenic angiogenesis and non- pathogenic angiogenesis), embryogenesis and haematopoesis including leukaemogenesis.

More specifically, the inventors consider that LMO2 plays a role in any one or more of the conditions selected from the group consisting of: tumour formation, tumour metastasis, LMO2 mediated T-cell leukemia, inflammation, ischemia, diabetic retinopathy, wound healing and menstruation. (ci) Role of LMO2 within mammals:

(cia) Role of LMO2 in haematopoetic cell fate.

By studying T cell tumours arising in an Z O2-gain-of-function transgenic mouse model, we have been able to detect an analogous LMO2-associated complex, in this case which binds a bipartite sequence comprising tandem E boxes ¹⁸. These observations lead to two conclusions. Firstly, as LMO2 appears to be a bridging component of the protein complex detected in erythroid cells, it seems possible that the components of the LMO2-complex might vary through-out haematopoietic differentiation, controlling various, stage-specific functions by regulating sets of genes. A corollary is that LMO2 has distinct roles in control of haematopoietic cell fate. Secondly, the role of LMO2 in T-ALL is probably by enforced formation of protein complexes (the direct result of enforced LM02 expression after the chromosomal translocation). Thus gene regulation via LMO2-complexes may be affected; this may occur by direct DNA-binding complexes or by sequestration of proteins from their normal roles ¹³.

The role of LMO2 in controlling cell fate in normal haematopoiesis has been documented mainly by gene targeting experiments. Null mutation were made in

LM02 in embryonic stem cells (ES cells) by homologous recombination and heterozygous mice were generated. Breeding of these mice resulted in embryonic lethality at E9-10 due to a failure of yolk sac erythropoiesis ¹⁹, showing that LMO2 is necessary for primitive erythropoiesis in mouse embryogenesis. Studies on a possible role for LMO2 in adult (definitive) haematopoiesis was conducted with chimaeric mice ²⁰ and this revealed that no haematopoiesis occurs in the absence of the LM02 gene. Thus LMO2 functions in a cell autonomous way during the early stages of definitive haematopoiesis. This important function may be at the level of the pluripotent stem cell, at the level of the immediate multi-potential progeny or even perhaps before this, when mesoderm gives rise to these precursors.

(cib) Role of LMO2 in embryogenesis. Using a lacZ knock-in of the endogenous LM02 gene to establish a reporter system for 91

LM02 expression in embryogenesis , we found that from E8.5, LM02 has a more specific intra-embryonic expression pattern, in endothelial cells and blood progenitor cells. At E10.5, when definitive haematopoiesis is thought to begin in the aorta- gonad-mesonephros (AGM) region, LM02 is expressed in endothelial cells of the vascular system and multi-potent blood progenitor cells ^{21* 22}. Additional sites of expression were observed in the limb buds and hippocampus. The expression pattern of LM02 in yolk sac and blood progenitor cells is consistent with its function in primitive and definitive haematopoiesis. The first intra-embryonic haematopoietic stem cell activity is thought to appear in the AGM region at around El 0.5, and that haematopoietic stem cells are derived from endothelium of large arteries, such as dorsal aorta ²³' ²⁴. In mouse embryogenesis, haemangioblasts (putative common precursors of endothelium and blood cells) are proposed to arise from unspecified posterior mesoderm and thus the primary capillary network is made from these haemangioblasts. This first process of vasculature construction in which the capillary network is formed is called vasculogenesis. The more mature vascular system is made by the remodeling of primary capillary network, in the process called angiogenesis ²⁵. The earlier role of LMO2 before specification of haematopoietic stem cells, especially in construction of vascular system, was studied by following the fate of Z O2-null ES cells in chimaeric ²¹. LM02-nu\\ ES cells can contribute to the capillary network in chimaeric mice before E9. At about E10, however, a marked vascular disorganization is observed in chimaeric mice, due to failure of LMO2-null ES cell contribution in endothelium. After El l, there is no contribution of Z O2-null ES cells to the endothelium of large arteries. These results indicate that LMO2 is necessary for angiogenesis, but not for vasculogenesis. LM02 is expressed in both endothelial cells and blood progenitor cells, and has dual functions both in haematopoiesis and angiogenesis. Considering the close relationship between endothelium and blood cell specification, the role of LMO2 during this critical stage of mouse development is pivotal. This key role for the protein may in turn explain why null mutation of LM02 results in failure of adult haematopoiesis. (cic Role of LMO2 in tumour angiogenesis.

The present inventors have also shown that LMO2 protein is essential for tumour angiogenesis since ES cells lacking LMO2 cannot support vascular formation during tumour growth ²⁶ . There are a number of situations in adults where this remodeling process of angiogenesis is required, such as wound healing or menstruation and in pathological conditions, such as tumour angiogenesis, ischaemia, inflammation and diabetic retinopathy ²⁷. In cancer, angiogenesis is a key part of solid tumour growth and invasion (metastasis). Like all cells, cancers cells require oxygen and carbon dioxide exchange and therefore solid tumours must develop a blood supply to facilitate their growth ²⁸' ²⁹. This is achieved by subverting local vessel endothelium in the process of tumour angiogenesis ²⁵' ³⁰ which allows for sprouting of existing endothelium into the tumour deposits . This angiogenic switch occurs when there is a relative increase angiogenesis stimulating factors and in tumour growth, the cancer cells become angiogenic to stimulate the necessary remodeling state. Furthermore, the concept of mosaic blood vessels is strongly supported by data which shows that a significant percentage of the vessel wall can be made from tumour cells and these cells are continually shed into the vessel lumen, and therefore the blood circulation contributing to the metastatic population. In addition, so-called angioblast-like endothelial precursors may be recruited from the bone marrow providing another source of vessel wall cells. These features of angiogenesis have been proposed as a major target for patient treatment in cancer ²⁸' ²⁹. Several key molecules have been identified which control angiogenesis, such as vascular endothelial growth factor ³⁴ which stimulates the process of vessel sprouting, but little is known of transcription regulators of vessel remodeling. Some candidates have come from the study of chromosomal translocations in human leukaemias which has led to the identification of novel genes, many of which are transcription factors (reviewed in '). The LM02 gene is an example. The gene is normally expressed in endothelial cells of mouse embryos where it is required for angiogenesis after vascularisation has occurred ²¹. Augmented expression of LMO2 occurs in tumour neo-vasculature. Furthermore, in the absence of LMO2, the development of a mature vascular system in solid tumours is prevented in an ES-cell tumour model.

(cii) Assay to test the functional activity ofLM02.

Assays to assess the functional activity of LMO2 will be familiar to those skilled in the art. A suitable assay measures the in vitro differentiation ES cells into erythroid cells which is known to be dependent on the function of the LMO2 protein as null mutation of LMO2 inactivates the erythroid differentiation pathway ¹⁹' ²⁰. Null mutation of LMO2 also results in loss of angiogenesis .

The present inventors were able to observe inhibiting effects on erythropoiesis by expressing the Trx207 protein in ES cells. The levels of inhibition varied between ES clones with the maximal level reaching -70%. This incomplete inhibition is thoiught by the inventors is to due to variable levels of Trx207 protein expression in the selected ES clones. There may also be a problem emanating from expressing the Trx protein with a 20mer peptide (aptamer) added at the external loop (data not shown) and this may mean that different scaffolds may be more effective in presenting the peptide.

Thus it may be that anti-LMO2 peptide aptamers, such as 207 peptide, are more effective in different scaffolds. Indeed it may be that an unconstrained peptide aptamer is most effective. Evidence for this is provided in the form that a GAL4-207 fusion protein was able to interact with the LLM2 domain of LMO2 at comparable levels to the Trx207 protein (Fig. 3).

(ciii) Delivery of anti-LMO2 reagents/LLM2 inhibitors to the cells of an individual Generally the LLM2 inhibitors according to the invention will be delivered to the cell. Advantageously, it is delivered to the cell nucleus.

(Ciiia^') Expression within cells. In order to introduce such molecules into an intracellular environment, cells are advantageously transfected with nucleic acids which encode the one or more LLM2 inhibitors.

Nucleic acids encoding such inhibitors can be incorporated into vectors for expression. As used herein, vector (or plasmid) refers to discrete elements that are used to introduce heterologous DNA into cells for expression thereof. Selection and use of such vehicles are well within the skill of the artisan. Many vectors are available, and selection of appropriate vector will depend on the intended use of the vector, the size of the nucleic acid to be inserted into the vector, and the host cell to be transformed with the vector. Each vector contains various components depending on its function and the host cell for which it is compatible. The vector components generally include, but are not limited to, one or more of the following: an origin of replication, one or more marker genes, an enhancer element, a promoter, a transcription termination sequence and a signal sequence.

Moreover, nucleic acids encoding the LLM2 inhibitors according to the invention may be incorporated into cloning vectors, for general manipulation and nucleic acid amplification purposes.

Both expression and cloning vectors generally contain nucleic acid sequence that enable the vector to replicate in one or more selected host cells. Typically in cloning vectors, this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and includes origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeast and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2m plasmid origin is suitable for yeast, and various viral origins (e.g. SV 40, polyoma, adenovirus) are useful for cloning vectors in mammalian cells. Generally, the origin of replication component is not needed for mammalian expression vectors unless these are used in mammalian cells competent for high level DNA replication, such as COS cells. Most expression vectors are shuttle vectors, i.e. they are capable of replication in at least one class of organisms but can be transfected into another class of organisms for expression. For example, a vector is cloned in E. coli and then the same vector is transfected into yeast or mammalian cells even though it is not capable of replicating independently of the host cell chromosome. DNA may also be replicated by insertion into the host genome. However, the recovery of genomic DNA is more complex than that of exogenously replicated vector because restriction enzyme digestion is required to excise the nucleic acid. DNA can be amplified by PCR and be directly transfected into the host cells without any replication component.

Expression and cloning vectors usually contain a promoter that is recognised by the host organism and is operably linked to the desired nucleic acid. Such a promoter may be inducible or constitutive. The promoters are operably linked to the nucleic acid by removing the promoter from the source DNA and inserting the isolated promoter sequence into the vector. Both the native promoter sequence and many heterologous promoters may be used to direct amplification and/or expression of nucleic acid encoding the peptide. The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

Gene transcription from vectors in mammalian hosts may be controlled by promoters derived from the genomes of viruses such as polyoma virus, adenovirus, fowlpox virus, bovine papilloma virus, avian sarcoma virus, cytomegalovirus (CMV), a retrovirus and Simian Virus 40 (SV40), from heterologous mammalian promoters such as the actin promoter or a very strong promoter, e.g. a ribosomal protein promoter, and from promoters normally associated with immunoglobulin sequences.

Transcription of a nucleic acid by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are relatively orientation and position independent. Many enhancer sequences are known from mammalian genes (e.g. elastase and globin). However, typically one will employ an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270) and the CMV early promoter enhancer. The enhancer may be spliced into the vector at a position 5' or 3' to the desired nucleic acid, but is preferably located at a site 5' from the promoter.

Advantageously, a eukaryotic expression vector may comprise a locus control region (LCR). LCRs are capable of directing high-level integration site independent expression of transgenes integrated into host cell chromatin, which is of importance especially where the gene is to be expressed in the context of a permanently- transfected eukaryotic cell line in which chromosomal integration of the vector has occurred.

Eukaryotic expression vectors will also contain sequences necessary for the termination of transcription and for stabilising the mRNA. Such sequences are commonly available from the 5' and 3' untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding a LEM2 inhibitor according to the invention.

Particularly useful for practising the present invention are expression vectors that provide for the transient expression of nucleic acids in mammalian cells. Transient expression usually involves the use of an expression vector that is able to replicate efficiently in a host cell, such that the host cell accumulates many copies of the expression vector, and, in turn, synthesises high levels of the desired gene product.

(Ciiib) Direct delivery to cells.

LLM2 inhibitors may be directly introduced into cells by micro injection, or delivery using vesicles such as liposomes which are capable of fusing with the cell membrane. Viral fusogenic peptides are advantageously used to promote membrane fusion and delivery to the cytoplasm of the cell for subsequent translocation into the nucleus. (Ciiibl Nuclear localization signals

Nuclear localization signals are signals which are responsible for targeting an antigen to the nucleus. Such targeting sequences are reviewed generally in Baker et al., 1996, Biol Rev Camb Philos Soc 71, 637-702. Nuclear localisation sequences include the SV40 large T antigen consensus sequence PKKKRKV (reviewed in Dingwall, et al., 1991, Trends Biochem. Sci. 16, 478-481), or the bipartite nuclear localisation sequence as exemplified by nucleoplasmin protein (Dingwall, et al., 1987, EMBO J. 6, 69-74; Robbins, et al 1991, Cell 64, 615-623). Those skilled in the art will be aware of other suitable nuclear localization signals for use in targeting a peptide according to the invention to the cell nucleus..

Antibodies may be directly introduced to the cell by microinjection, or delivery using vesicles such as liposomes which are capable of fusing with the cell membrane. Viral fiisogenic peptides are advantageously used to promote membrane fusion and delivery to the cytoplasm of the cell.

Preferably, the anti-LMO2 antibody according to the invention is fused or conjugated to a domain or sequence from a protein responsible for cell membrane translocational activity. Preferred translocation domains and sequences include domains and sequences from the HLV-1 -trans-activating protein (Tat), Drosophila Antennapedia homeodomain protein and the herpes simplex- 1 virus VP22 protein. By this means, the anti-LMO2 antibody according to the invention is able to enter the cell or its nucleus when introduced in the vicinity of the cell.

Exogenously added HIV- 1 -trans-activating protein (Tat) can translocate through the plasma membrane and to reach the nucleus to transactivate the viral genome. Translocational activity has been identified in amino acids 37-72 (Fawell et al., 1994, Proc. Natl. Acad. Sci. U. S. A. 91, 664-668), 37-62 (Anderson et al., 1993, Biochem. Biophys. Res. Commun. 194, 876-884) and 49-58 (having the basic sequence RKKRRQRRR) of HIV-Tat. Vives et al. (1997), J Biol Chem 272, 16010-7 identified a sequence consisting of amino acids 48-60 (CGRKKRRQRRRPPQC), which appears to be important for translocation, nuclear localisation and trans-activation of cellular genes. Intraperitoneal injection of a fusion protein consisting of beta-galactosidase and a HIV-TAT protein transduction domain results in delivery of the biologically active fusion protein to all tissues in mice (Schwarze et al., 1999, Science 285, 1569-72)

The third helix of the Drosophila Antennapedia homeodomain protein has also been shown to possess similar properties (reviewed in Prochiantz, A., 1999, Ann N Y Acad Sci, 886, 172-9). The domain responsible for translocation in Antennapedia has been localised to a 16 amino acid long peptide rich in basic amino acids having the sequence RQLKIWFQNRRMKWKK (Derossi, et al., 1994, J Biol Chem, 269, 10444- 50). This peptide has been used to direct biologically active substances to the cytoplasm and nucleus of cells in culture (Theodore, et al., 1995, J. Neurosci 15, 7158- 7167). Cell internalisation of the third helix of the Antennapedia homeodomain appears to be receptor-independent, and it has been suggested that the translocation process involves direct interactions with membrane phospholipids (Derossi et al., 1996, J Biol Chem, 271, 18188-93).

The VP22 tegument protein of herpes simplex virus is capable of intercellular transport, in which VP22 protein expressed in a subpopulation of cells spreads to other cells in the population (Elliot and O'Hare, 1997, Cell 88, 223-33). Fusion proteins consisting of GFP (Elliott and O'Hare, 1999, Gene Ther 6, 149-51), thymidine kinase protein (Dilber et al., 1999, Gene Ther 6, 12-21) or p53 (Phelan et al., 1998, Nat Biotechnol 16, 440-3) with VP22 have been targeted to cells in this manner.

Particular domains or sequences from proteins capable of translocation through the nuclear and/or plasma membranes may be identified by mutagenesis or deletion studies. Alternatively, synthetic or expressed peptides having candidate sequences may be linked to reporters and translocation assayed. For example, synthetic peptides may be conjugated to fluoroscein and translocation monitored by fluorescence microscopy by methods described in Vives et al. (1997), J Biol Chem 272, 16010-7. Alternatively, green fluorescent protein may be used as a reporter (Phelan et al., 1998, Nat Biotechnol 16, 440-3). Any of the domains or sequences or as set out above or identified as having translocational activity may be used to direct the immunoglobulins into the cytoplasm or nucleus of a cell. The Antennapedia peptide described above, also known as penetratin, is preferred, as is HLV Tat. Translocation peptides may be fused N- terminal or C-terminal to single domain immunoglobulins according to the invention. N-terminal fusion is preferred.

Also of use for the delivery of antibodies to cells is the TLM peptide. The TLM peptide is derived from the Pre-S2 polypeptide of HBV. See Oess S, Hildt E Gene Ther 2000 May 7:750-8. Anti-DNA antibody technology is also of use. Anti-DNA antibody peptide technology is described in Alexandre Avrameas et al., PNAS val 95, pp 5601-5606, May 1998; Therese Ternynck et al., Journal of Autoimmunity (1998) 11, 511-521; and Bioconjugate Chemistry (1999), vol 10 Number 1, pp 87-93.

(D) Uses ofLIM2 inhibitors/anti-LM02 reagents according to the invention.

In yet a further aspect of the invention, there is provided an L1M2 inhibitor according to the invention or a composition comprising it for use in medicine.

In yet a further aspect still the invention provides the use of one or more LLM2 inhibitors according to the invention in the preparation of a medicament for the prophylaxis and/or treatment of one or more conditions selected from the group consisting of: tumour formation, tumour metastasis, LMO2 mediated T-cell leukemia, inflammation, ischemia, diabetic retinopathy and wound healing.

In yet a further aspect the invention provides a method for the prophylaxis and/or treatment of any one or more conditions selected from the group consisting: tumour formation, tumour metastasis, LMO2 mediated T-cell leukemia, inflammation, ischemia, diabetic retinopathy and wound healing comprising the step of administering to an individual in need of such treatment one or more LLM2 inhibitors according to the invention. Anti LMO2 reagents/LLM2 inhibitors according to the present invention may be employed in in vivo therapeutic and prophylactic applications, in vitro and in vivo diagnostic applications, in vitro assay and reagent applications, in functional genomics applications and the like.

LMO2 functions by protein interactions in haematopoiesis, angiogenesis and leukaemogenesis. Inhibitors of LMO2 are therefore research tools for studies of the former, for prevention and/or treatment of angiogenesis in tumour growth or other conditions in which vascular remodelling is important and in treatment of specific leukaemias. The anti-LMO2 peptide aptamers described here can be used in any of these various settings.

Therapeutic and prophylactic uses of anti-LMO2 reagents/LLM2 inhibitors and compositions according to the invention involve the administration of the above to a recipient mammal, such as a human. Preferably they involve the administration to the intracellular environment of a mammal.

Substantially pure peptides and/or scaffold molecules comprising them of at least 90 to 95% homogeneity are preferred for administration to a mammal, and 98 to 99% or more homogeneity is most preferred for pharmaceutical uses, especially when the mammal is a human. Once purified, partially or to homogeneity as desired, the immunoglobulin molecules may be used diagnostically or therapeutically (including extracorporeally) or in developing and performing assay procedures using methods known to those skilled in the art.

In the instant application, the term "prevention" involves administration of the protective composition prior to the induction of the disease. "Suppression" refers to administration of the composition after an inductive event, but prior to the clinical appearance of the disease.

The selected anti-LMO2/LLM2 inhibitors of the present invention can perturb protein function in vivo and thus will typically find use in preventing, suppressing or treating inflammatory states, tumour formation and/or metastasis, LMO2 mediated T-cell leukemia, ischemia and diabetic retinopathy. In addition the anti-LMO2 reagents/LIM2 inhibitors will find use in the modulation of wound-healing and/or menstruation.

The anti-LMO2 reagents/LEVI2 inhibitors, particularly anti-LMO2 peptides/LLM2 inhibitor peptides or anti-LMO2 intrabodies according to the invention (either in unconstrained form or constrained at their N and/or C-terminus by a scaffold molecule as referred to herein) advantageously further comprise a nuclear localisation signal. Such molecules are advantageously used as therapeutic agents in the treatment of one or more conditions referred to above.

Animal model systems which can be used to screen the effectiveness of the selected immunoglobulins of the present invention in protecting against or treating disease are available.

Generally, the selected anti-LMO2 reagents/LLM2 inhibitors of the present invention will be utilised in purified form together with pharmacologically appropriate carriers. Typically, these carriers include aqueous or alcoholic/aqueous solutions, emulsions or suspensions, any including saline and/or buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride and lactated Ringer's. Suitable physiologically-acceptable adjuvants, if necessary to keep a polypeptide complex in suspension, may be chosen from thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates.

intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as those based on Ringer's dextrose. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents and inert gases, may also be present (Mack (1982) Remington's Pharmaceutical Sciences, 16th Edition).

The selected inhibitors of the present invention may be used as separately administered compositions or in conjunction with other agents. These can include various immunotherapeutic drugs, such as cylcosporine, methotrexate, adriamycin or cisplatinum, and immunotoxins. Pharmaceutical compositions can include "cocktails" of various cytotoxic or other agents in conjunction with anti-LMO2 reagents/LLM2 inhibitors according to the present invention or even combinations of selected inhibitors according to the present invention.

The route of administration of pharmaceutical compositions according to the invention may be any of those commonly known to those of ordinary skill in the art. For therapy, the selected inhibitor compositions according to the invention can be administered to any patient in accordance with standard techniques. The administration can be by any appropriate mode, including parenterally, intravenously, intramuscularly, intraperitoneally, transdermally, via the pulmonary route, or also, appropriately, by direct infusion with a catheter. The dosage and frequency of administration will depend on the age, sex and condition of the patient, concurrent administration of other drugs, counter-indications and other parameters to be taken into account by the clinician.

The selected inhibitors of the present invention can be lyophilised for storage and reconstituted in a suitable carrier prior to use. Known lyophilisation and reconstitution techniques can be employed. It will be appreciated by those skilled in the art that lyophilisation and reconstitution can lead to varying degrees of functional activity loss and that use levels may have to be adjusted upward to compensate.

The compositions containing the present selected anti-LMO2 reagents/LLM2 inhibitors of the present invention or a cocktail thereof can be administered for prophylactic and/or therapeutic treatments. In certain therapeutic applications, an adequate amount to accomplish at least partial inhibition, suppression, modulation, killing, or some other measurable parameter, of a population of selected cells is defined as a "therapeutically-effective dose". Amounts needed to achieve this dosage will depend upon the severity of the disease and the general state of the patient's own immune system, but generally range from 0.005 to 5.0 mg of selected immunoglobulin per kilogram of body weight, with doses of 0.05 to 2.0 mg/kg/dose being more commonly used. For prophylactic applications, compositions containing the present selected inhibitor molecules or cocktails thereof may also be administered in similar or slightly lower dosages.

A composition containing one or more selected anti-LMO2 reagents/LLM2 inhibitor molecules according to the present invention may be utilised in prophylactic and therapeutic settings to aid in the alteration, inactivation, killing or removal of a select target cell population in a mammal. In addition, the selected repertoires of polypeptides described herein may be used extracorporeally or in vitro selectively to kill, deplete or otherwise effectively remove a target cell population from a heterogeneous collection of cells.

The invention will now be described by the following examples which are in way limiting of the invention.

Examples

Example 1 : Materials and Methods

Screening a peptide library with LMO2 using a yeast 2-hybrid assay

A truncated form of mouse LMO2 (aa 28-150) was fused to the LexA DNA binding domain in a modified version of pBTM116 vector in which the selectable marker for yeast TRP1 conferring resistance in a medium lacking tryptophan had been replaced by the LEU gene conferring resistance in a medium lacking leucine because the library vector was containing the TRPl gene as a selectable yeast marker. This LexA-LMO2 construct was used as a bait to screen 3 x 10⁶ transformants from a 20-mers peptide library ³⁵ (kindly provided by Dr. R. Brent) using the L40 yeast strain and following the protocol described by . In this library, which contain more than 10⁹ members, the active-site loop of E. coli thioredoxin (Trx) is used as a scaffold to display the 20-mer peptides. Mammalian two-hybrid assays

Yeast Trx-peptide sequences were subcloned into the mammalian VP16 vector pMVN (Clontech) as a fusion with the VP16 activation domain, whereas the LMO2 baits was fused to the GAL4 DNA binding domain in the pM vector ⁴⁰. The interaction between LMO2 and the peptides was tested in Chinese hamster ovary (CHO) cells. CHO cells were grown in alpha MEM with 10% FCS, penicillin and streptomycin. CHO cells were seeded onto 6 well-plates (3 x 10⁵ cells per well) 16-24h before transfection. Transfection was performed using lOμl of polyfect transfection reagent (Qiagen), 500 ng of each pM and pVP16 vectors, 500 ng of pG5 a luciferase reporter vector and 50 ng of pRL-CMV (Dual-Luciferase Reporter Assay System, Promega). Cells were harvested 24-36 hours after being transfected and luciferase activity assay was performed according to the manufacturer. The three best peptides were subcloned into the pM bait vector as a fusion with the GAL4 DNA binding domain and their interaction with different part of LMO2, other LLM-only proteins and other zinc finger proteins was tested.

Generation of ES cell lines expressing anti-LMO2 peptide aptamers

KZ26 and HI 6 cell lines have been previously described and are respectively LMO2 +/- (with a lacZ gene knock-in to one allele of LM02) or LM02 -/-. AF6 and BB4 cell lines had been made by knocking-in Trx207 or Trx respectively into one LMO2 allele of the wild type ES cell line CCB. 6B23 cell line is a subclone of AF6 which was stably transfected with a CMV-Trx207 expression clone, whereas GI and A5 cell lines were made from KZ26 by stably transfecting respectively EFα-Trx207 and EFα-Trx..

Differentiation of embryoid bodies and erythropoiesis assays

The in vitro differentiation of the different ES clones into embryoid bodies (EBs) was done as previously described ⁴¹ except that recombinant human vascular endothelial growth factor was used at 10 ng/ml, mouse erythropoietin at 1 unit/ml, human basic fibroblast growth factor 2 at 25 ng/ml and murine interleukin 6 at 5 ng/ml. EBs were formed and collected after 11 days of culture, dissociated 30 min in 1 ml of DMEM containing 1.5 mg of collagenase, and cells analysed by FACS for the expression the erythrocyte marker Terl 19. Example 2: Anti-LMO2 peptide aptamers are homologous to elements in LIM fingers

A yeast peptide aptamer library ³⁵' ³⁶ was screened in vivo with an LMO2 bait in which LMO2 was fused to the GAL4 DNA binding domain (DBD). The yeast peptide aptamer library consisted of a diverse set of clones expressing random peptides (20mers) incorporated into the bacterial thioredoxin (Trx) protein in an external loop suitable for interaction between peptide and target proteins. Two hundred and fifty clones were identified in the yeast screen and fifteen of these were designated to be specific binders after re-screening. The DNA was isolated from these plasmids and sequence data obtained (Fig. 2). The notable feature of these sequences is the presence of a motif cys-x-x-cys or his-x-x-cys in 13/15 clones (Fig. 2A). When these peptide aptamers were re-evaluated using a mammalian luciferase two-hybrid transcription activation assay (see below, shown in Fig. 3), it was found that the protein from the two clones lacking this motif did not interact with the LMO2 bait in this more stringent assay, whereas the others displayed variable efficiencies of activation in the two- hybrid assay. A comparison of these derived peptide aptamer sequences, each with the level of transcriptional activation of the luciferase reporter gene obtained in a mammalian 2-hybrid assay, is shown in Fig. 2B (the peptides are aligned according to the cys/his-x-x-cys motif). It is significant that the cys/his-x-x-cys motif in the anti- LMO2 peptides are highly homologous to part of the first LLM finger of the second LLM domain of LMO2 (i.e. his-leu-glu-cys) (Fig. 2B).

The homology of the anti-LMO2 peptide aptamers extends outside the cys/his- x-x-cys motif. The comparison of the various anti-LMO2 peptide aptamers (Fig. 2C) shows that the conserved region of the cys/his-x-x-cys motif extends a few residues on either of this region, with generally two hydrophobic residues on the N-terminal side and one hydrophobic residue on the C-terminal side of the core motif. Further, it is significant that often a charged amino acid occurs as the second of the 2 amino-acids in the centre of the cys/his-x-x-cys (Fig. 2C) and this is a feature of the equivalent area of the LMO2 LLM2 domain (Fig. 2B) . Finally the val-tyr dipeptide which flanks the LMO2 LLM2 cys/his-x-x-cys (the complete element of LMO2 LLM2 domain being val-tyr-his-leu-glu-cys-phe) is conserved in two of the peptide aptamers, but not in the two which show the greatest interaction with LMO2 (i.e. 207 and 209, Fig. 2B). Overall the conserved region of the anti-LMO2 peptide aptamers is hydrophobic- hydrophobic-cys-x-charged-cys-hydrophobic residue (Fig. 2C).

Example 3: Anti-LMO2 peptide aptamers bind specifically the second LIM domain of LMO2

The anti-LMO2 peptide aptamers were isolated from a library of random peptide expressed in yeast. The activity and specificity of the interaction with LMO2 was checked in a mammalian assay based on activation of a luciferase reported after interaction of anti-LMO2 peptide aptamer with an LMO2 protein bait expressed in CHO cells. The results of activation levels are summarised in Fig 2B, which shows the activity of each peptide aptamer expressed as fold increase in luciferase activity observed after co-transfection of clones expressing DBD-Trx aptamer fusion (termed the bait) and LMO2 fused to the VP16 transcriptional activation domain (termed the prey) . The four aptamers producing the highest levels of activation were made from clones Trx207, 209, 85 and 81. The specificity of binding by the anti-LMO2 peptide aptamers was assessed in the mammalian reporter assay in which various baits comprising GAL4 DBD-fused with either the Trx-anti-LMO2 peptide aptamer 207 (Fig. 3A), 209 (Fig.3 C) and 85 (Fig. 3D) or fused with the anti-LMO2 peptide aptamer 207 without the Trx scaffold, were co-expressed with various target proteins fused to the VP16 transcriptional transactivation domain. These 'preys' included LMO2, the individual LLM domains of LMO2 (i.e. LLM1 or LLM2), the related LLM-only proteins LMOl and LMO4, and the LMO-interacting proteins LDB1 and GATA1 (Fig. 3). We observed that the anti- LMO2 peptide aptamers bound only to LMO2 or the second LLM domain (LLM2) of LMO2, while Trx207 shows no significant binding to GATA1, Trx209 and 85 were able to interact with this protein to a small extent. In all three cases, binding to the isolated LLM2 domain of LMO2 was much better than binding to the intact LMO2. Furthermore, the anti-LMO2 peptide aptamers were specific for the LLM domain zinc- fingers as they showed no binding to the zinc finger domain found in oestrogen receptor α (zinc finger type C₄) or in the protein kinase TTK (zinc finger type C₂H₂). The data obtained with both yeast and CHO interaction assays used the peptide aptamers constrained at both N- and C-terminal ends within the thioredoxin protein scaffold. The ability of unconstrained peptide was difficult to assess at this stage of the analysis because we do not know the exact sequence requirements for binding with LMO2. As an intermediate test, a fusion protein bait was designed in which the 207 peptide was fused only at its N-teπninal end with the GAL4 DBD. This protein was used in the CHO reporter assay with an array of prey proteins comprising LLM proteins fused to VP16 (Fig. 3B). In this assay, we found that the unconstrained 207 peptide was equally effective in vivo in binding specifically to LMO2, and that the peptide binds to the LLM2 domain of LMO2 more efficiently than it binds to the complete LMO2 protein. These data imply that the 207 peptide has sequence specific binding to a region of the LLM2 domain which is independent of structural constraints within Trx.

Example 4: Anti-LMO2 peptide aptamers inhibit LMO2 protein function

LMO2 is a protein interaction module ¹⁵' ¹⁷ which functions by protein interaction in each of its functional settings, which includes haematopoiesis, angiogenesis and leukaemogenesis ³⁷. The possible use of the anti-LMO2 peptide aptamers to inhibit LMO2 function was assayed in an LMO2-dependent in vitro culture assay using the differentiation of embryonic stem cells (ES) cells into erythroid forms which is known to be dependent on LMO2 ¹⁹. When wild-type ES cells were differentiated (Fig. 4A, CCB), around 20-25% of cells differentiated into erythroid cells (measured by the expression of the erythroid surface protein Terl 19) and a similar number were obtained using LM02 +/- ES cells (Fig. 4A, ES clone KZ26 ^{20, 26}). On the other hand, null LM02 mutant ES cells did not undergo erythropoiesis (Fig. 4A, ES clone HI 6, 9ft

LM02 -/-) as previously described . When the Trx207 anti-LMO2 peptide aptamer was expressed in the differentiating ES cells either under the control of the endogenous LMO2 transcription promoter (as a knock-in of the LM02 gene, Fig. 4A, ES clone AF6) or as this knock-in of the LM02 gene plus an transgene expressing LM02 from the CMV promoter (Fig. 4 A, ES clone 6B23) or just a transgene expressing Trx207 from the EFα promoter (Fig. 4A, ES clone GI), a reduction in the number of erythroid cells occurred. No loss of erythroid differentiation occurred in control ES clones expressing Trx with no peptide insertion (Fig. 4A, ES clones BB4 and A5). Thus the ES clones expressing the Trx207 fusion showed an inhibition of formation of erythroid colonies to around 50-70% of the wild type levels (Fig. 4B) and this inhibition requires the presence of the 207 peptide aptamer. Thus the loss of erythropoiesis specifically in ES cells expressing the anti-LMO2 peptide aptamer 207 is indicative of an inhibition of LMO2 function and that this inhibition is mediated by the peptide sequence represented by the 207 peptide (Fig. 4).

Example 5: An anti-LMO2 intrabody (designated anti-LMO2 #14) can block angiogenesis in developing mouse embryos. Results and Discussion

Anti-LMO2 intrabodies were isolated by the intracellular capture method (Tanaka et al., 2003; Tse et al., 2002a; Tse et al., 2002b; Visintin et al., 1999). One intrabody (anti-LMO2 #14) binds to LMO2 LLM domains specifically in mammalian cell reporter assays (CHN & THR, unpublished). We wished to determine if anti-LMO2 #14 could interfere with biological functions of LMO2. First, we used an ES cell assay in vitro, comprising differentiation of ES into erythroid or primary endothelial cells in culture. ES cells were transfected with a clone expressing anti-LMO2 #14 and these cells, and control untransfected ES cells, were subjected to growth factor dependent differentiation as described (Keller et al., 1993). After the requisite culture period, cells were examined for expression of the surface marker Terl 19 (an erythroid specific surface protein) or CD31 (Pecam, a pan-endothelial protein). The presence of Terl 19 indicates erythroid differentiation and of CD31 indicates de novo formation of vascular endothelial cells (vasculogenesis). Angiogenesis does not occur in ES cell cultures. Figure 5 shows the results of in vitro differentiation of ES cells into the erythroid lineage. Comparable levels of differentiation into the erythroid cells were found with normal ES cells (CCB) and subclone KZ26, which is an established ES line with the lacZ gene knocked-in to one allele of Lmo2, rendering that allele null for Lmo2 expression but allowing β-galactosidase expression under the control of the Lmo2 promoter (serving as a reporter of Lmo2 expression) (Yamada et al., 1998). On the other hand, an ES cell with homozygous null mutation of Lmo2 does not undergo erythropoiesis (clone H16) (Yamada et al., 1998) and an ES clone expressing an anti- LMO2 peptide aptamer has severly inhibited capacity for erythropoiesis (clone 6B23). The effect of expressing anti-LMO2 intrabody #14 in ES cells is to produce a similar effect to null mutation of the Lmo2 gene. ES clone 3 A expresses anti-LMO2 #14 under the control of the pEF-BOS promoter. When these cells were induced to differeniate in culture using growth factors, very low levels (-10% of controls) of Terl l9-erythroid colonies were found. We conclude that the anti-LMO2 #14 intrabody inhibits the Lmo2 function in erythropoiesis.

As a control for the specificity of anti-LMO2 #14 inhibition of Lmo2 function in the ES cell assay, we assessed the development of CD31 (Pecam) expressing cells in the ES differentiation assay. These cells form during a vasculogenesis process in ES cells which is largely unaffected by null mutation of Lmo2 (Yamada et al., 2000) (Fig. 6, ES clone H16). Similar levels of CD31+ cells were observed with ES clones KZ26 (heterozygous for the Lmo2 gene), 6B23 (expressing anti-Lmo2 aptamer) or 3 A which expresses the anti-LMO2 #14 intrabody. Thus the data in Figs. 5 and 6 show that the anti-LMO2 #14 intrabody can affect the function of Lmo2 in erythropoiesis but not de novo vasculogenesis (paralleling the results defining Lmo2 function in gene targeting studies (Warren et al., 1994; Yamada et al., 1998; Yamada et al., 2000).

Lmo2 has a role in remodelling of vascular endothelium from pre-existing blood vessels (angiogenesis) but not in de novo development of blood capillaries (vasculogenesis) (Yamada et al., 2000). This function of LMO2 is therefore a therapeutic target for clinical indications involving angiogenesis. To show that the anti-LMO2 #14 intrabody could block angiogenesis, an assay based on angiogenesis in mouse embryos was used (Yamada et al., 2000). ES cells injected into donor blastocysts and transferred to pseudo-pregnant recipients develop into chimaeric embryos in which a substantial proportion of cells are derived from the injected ES cells. When KZ26 ES cells (heterozygous for Lmo2-lacZ knock-in) were injected into blastocysts and embryos stained for β-galactosidase activity (giving a blue coloration specifically in Lmo2 expressing cells) we observed staining in the developing vasculature (Yamada et al., 2000). Fig. 7 shows a specimen chimaeric embryo at embryonic day El 0.5, in which endothelial cells of remodelling blood vessels stain blue due to Lmo2 gene promoter expression (left panel). A similar analysis of chimaeric embryos generated with ES cells carrying a null mutation of Lmo2, showed no blue vascular formation (Fig. 7, right panel) due to failure of angiogenesis (remodelling) of the capillary network in the absence of Lmo2. An identical finding was made when chimaeric embryos were generated with ES cells expressing the anti- Lmo2 intrabody (Fig. 7, central panel). As we have shown that this intrabody binds to the Lmo2 protein in vivo (CHN & THR, unpublished) and that it blocks LMO2 function in ES cell assays (Figs. 5 and 6), we conclude that the anti-Lmo2 intrabody prevents angiogenesis by binding to Lmo2 and preventing the protein from performing its function in blood vessel endothelial cells.

Our results show that an anti-LMO2 intrabody (comprising an scFv antibody fragment) can interfere with LMO2 function in the angiogenesis segment of vascular formation but has no effect on de novo vascular endothelial cell formation. The data shown uses an chimaeric embryo assay for Lmo2 function in angiogenesis and can be extrapolated to other in vivo settings where angiogenesis occurs, such as normal situations like wound healing or clinical indications such as cancer. Using this anti- LMO2 intrabody, derivatives thereof, or other agents (such as small chemical entities derived based on the knowledge of the anti-LMO2 intrabody binding site) which perform a similar function suggests an important approach to management of angiogenesis in human and animal diseases.

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 molecular biology or related fields are intended to be within the scope of the following claims.

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Claims

1. An LLM2 inhibitor which is capable of binding to the LLM2 domain of LMO2 and inhibiting the functional activity of LMO2.

2. A LLM2 inhibitor according to claim 1 which is a peptide and comprises the consensus sequence:

His/CXXC C is a zinc binding Cys His represents histidine X is any amino acid.

3. A LLM2 inhibitor according to claim 2 which is a peptide and comprises the consensus sequence:

HhHis/CXX'Ch

X represents any amino acid and X¹ represents a charged amino acid.

4. A LLM2 inhibitor according to claim 2 or claim 3 which is a peptide aptamer.

5. A LLM2 inhibitor according to any one of claims 3 to 4 wherein the peptide is constrained within a scaffold.

6. A LLM2 inhibitor according to any of claims 2 to 5 wherein the peptide is constrained within a scaffold at least at the N-terminus of the peptide.

7. A LLM2 inhibitor according to any of claims 2 to 5 wherein the peptide is constrained within a scaffold at least at the C-terminus of the peptide.

8. A LLM2 inhibitor according to any of claims 5 to 7 wherein the peptide is constrained within a scaffold of : Thioredoxin protein (Trx).

9. A LLM2 inhibitor according to any preceding claim which is selected from the group of peptide aptamers consisting of the following: clone 207, clone 209, clone 85, clone 81, clone 63, clone 72, clone 247, clone 90, clone 69 and clone 202 as depicted in figure 2 and designated SEQ No 2 to SEQ No 11 respectively.

10. A LLM2 inhibitor according to claim 9 which is selected from the group consisting of the peptide aptamers consisting of the following: clone 207, clone 209, clone 85 as depicted in figure 2 and designated SEQ NO 2, 3 and 4 respectively.

11. A LLM2 inhibitor according to claim 1 which is an antibody.

12. A LLM2 antibody inhibitor according to claim 11 which comprises at least one CDR having the amino acid sequences depicted in Fig 8 and designated SEQ JD No 12(a), 12(b) and 12(c).

13. An anti-LMO2 antibody according to claim 12 which comprises all three CDRs having the amino acid sequences depicted in Fig 8 and designated SEQ ID No 12(a), 12(b) and 12(c) respectively and which together form an antigen binding site.

14. An anti-LMO2 antibody (LLM2 inhibitor) according to any of claims 11 to 13 which is an scFv or a dAb.

15. An anti-LMO2 antibody (LLM2 inhibitor) according to claim 14 which is an scFv.

16. An anti-LMO2 antibody (LLM2 inhibitor) according to any of claims 11 to 15 which is an intrabody (intracellular binding antibody).

17. Nucleic acid encoding an anti-LMO2 antibody (LLM2 inhibitor) according to any of claims 11 to 16.

18. A vector comprising nucleic acid according to claim 17.

19. A host cell comprising a vector according to claim 18.

20. A LLM2 inhibitor according to any preceding claim which further comprises a nuclear localisation signal.

21. 1 A LLM2 inhibitor according to any preceding claim which is is fused or conjugated to a domain or sequence from a protein responsible for cell membrane translocational activity.

22. A LIM2 inhibitor according to claim 21, wherein the domain or sequence responsible for cell membrane translocation activity is one or more of those selected from the group consisting of the following: the HLV-1 -trans-activating protein (Tat), Drosophila Antennapedia homeodomain protein and the herpes simplex- 1 virus VP22 protein.

23. A composition comprising one or more LLM2 inhibitors according to any preceding claim and a pharmaceutically acceptable carrier, diluent or exipient.

24. The use of an LLM2 inhibitor according to any of claims 1 to 19 or a composition according to claim 20 for inhibiting the functional activity of LMO2.

25. The use of a LLM2 inhibitor according to any of clams 1 to 19 or a composition according to claim 20 in the preparation of a medicament for inhibiting the functional activity of LMO2.

26. An LLM2 inhibitor according to any of claims 1 to 16 or a composition according to claim 23 for use in medicine.

27. The use of one or more LLM2 inhibitors according to any of claims 1 to 16 or a composition according to claim 23 in the preparation of a medicament for the prophylaxis and/or treatment of one or more conditions selected from the group consisting of: tumour formation, tumour metastasis, inflammation, LMO2 mediated T- cell leukemia and diabetic retinopathy.

28. A method for the prophylaxis and/or treatment of any one or more conditions selected from the group consisting: tumour formation, tumour metastasis, LMO2 mediated T-cell leukemia, inflammation, ischemia, diabetic retinopathy comprising the step of administering to an individual in need of such treatment one or more LLM2 inhibitors according to any of claims 1 to 16.

29. The use according to claim 26 or a method according to claim 27 wherein the condition is tumour formation and/or tumour metastasis.

30. The use according to claim 26 or a method according to claim 27 wherein the condition is LMO2 T-cell mediated leukemia.