JP2007510625A - LIM2 inhibitor of LMO2 - Google Patents

Abstract

The present invention relates to inhibitors of protein function. In particular, the present invention relates to molecular inhibitors that play a pivotal role in angiogenesis, hematopoiesis and embryogenesis. The use of such inhibitors in the regulation of angiogenesis and hematopoiesis is described.

Description

  The present invention relates to inhibitors of protein function. In particular, the present invention relates to molecular inhibitors that play a pivotal role in angiogenesis, hematopoiesis and embryogenesis. The use of such inhibitors in the regulation of angiogenesis, hematopoiesis and embryogenesis has been described.

  LM02 is a master regulator of several major pathways in embryogenesis, hematopoiesis and angiogenesis and performs its function in the above situation by protein-protein interactions involving the LIM domain. The most appropriate function of LMO2 from a therapeutic point of view is a role in angiogenesis, for example LMO2 is a blood vessel that constitutes a remodeling process that is essential and essential for the expansion of primary solid tumor growth and metastatic accumulation It plays a role in the regulation of endothelial division and migration. The development of drugs that can interfere with LMO2 function in protein interactions would be very useful therapeutically to prevent abnormal angiogenesis, such as in tumors.

Numerous different translocations have been studied by molecular cloning of chromosomal translocation binding and cDNA copies of genes affected by chromosomal translocation (reviewed in ¹ ). Further studies of chromosomal translocations in leukemia are performed when the T cell receptor (TCR) β chain locus is mapped to chromosome 7, band q35, and chromosomal translocations in T cell tumors are the development of normal lymphocyte lineages. ² was suggested to be mediated by intrinsic chromosomal instability of the TCR gene undergoing rearrangement. This was confirmed by cloning ^5,7 to ¹⁰ HOX11 and LMO2 cloning ^3-6 and chromosomal translocations site by TCRβ of TCRδ and chromosomal translocations binding sites by TCR a LMO1 and LMO2.

Early analysis of gene structure after chromosomal translocation has shown that gene fusion or oncogene activation (forced expression by a new chromosomal environment) has occurred (reviewed in ¹ ) . Furthermore, although transcription factors are frequent targets of chromosomal translocations ^11,12 , a clear distinction appears to be possible during chromosomal translocations in chronic and acute leukemia ¹² , only the latter category being transcription factors It became clear in the early 1990s that it was involved. Thus, chromosomal translocation - master genes model occurs ^12, it in the model transcriptional regulators and generating modulator is a major target of chromosomal translocations, and the forced expression or gene fusions thereof transfer balance It was postulated to change and thereby affect the regulation of cell fate in diseased cells.

  The chromosomal translocation-gene of acute cancer is the upstream master gene and its product regulates the downstream pathway. Thus, the major part of the directionality of chromosomal translocation is due to the specificity of transcription factor interactions that occur after chromosomal translocation and the resulting influence on the transcription pathway.

The LMO2 gene of T cell acute leukemia (T-ALL) is a chromosomal translocation-master gene paradigm (reviewed in ¹³ ). LM02 belongs to four gene families encoding small LIM-only proteins, two of which are involved in independent chromosomal translocations that give rise to T-ALL. LMO2 is located on chromosome 11, band p13, and is specifically activated in T cells by chromosomal translocation by 14q11 or 7q35. These chromosomal translocation breakpoints occur upstream of the native LMO2 promoter, resulting in forced expression of the LMO2 protein in the translocated cell (the protein product itself is not affected thereby). The gene is transcribed and translated into a 156αα protein containing two zinc-binding LIM domains, each with two LIM fingers (each LIM finger coordinates to a zinc atom and a finger of about 16-20 residues Contains a zinc binding motif of cysteine or three cysteines plus one histidine or aspartic acid residue, FIG. Although the LIM domain is structurally related to the GATA Zn finger, the characteristic of the LIM domain is the separation of each LIM Zn finger by only two αα. Function of LIM domains, chromosomal translocation - protein is a constant feature of the master gene product - in protein interactions ^14-16. LMO2 can bind to the DNA binding complex TAL1 / SCL (another T-ALL translocation-related protein), LDB1 and GATA-1, found in red blood cells ¹⁷ . This complex can bind to a double DNA site containing an E box separated from the GATA site by about 12 base pairs. The present inventors have isolated a peptide aptamer set that can specifically bind to the second LIM domain of LMO2 and inhibit the cellular function of LMO2. These reagents are useful for inhibiting tumor angiogenesis, blocking LMO2-mediated T cell leukemia, and inhibiting LMO2 function in clinical conditions where angiogenesis is important, such as ischemia, inflammation and diabetic retinopathy It should be.

  We screened a peptide aptamer library based on the outer loop of the bacterial protein thioredoxin (Trx) and set a peptide that specifically binds to the second LIM domain (LIM2) of the LMO2 protein (“anti-LM02 reagent”). / LIM2 inhibitor ", known herein). These peptide aptamers were surprisingly found to inhibit LMO2 function in embryogenesis and angiogenesis that depend on the transcription factor LMO2.

  Thus, these LIM2 inhibitors / anti-LM02 reagents prevent LMO2 function in pathological situations such as tumor angiogenesis, block LMO2-mediated T cell leukemia, and such as ischemia, inflammation and diabetic retinopathy It is believed to be quite useful therapeutically in inhibiting LMO2 function in clinical situations where angiogenesis is important.

  Therefore, in the first aspect, the present invention provides an agent (LIM2 inhibitor / anti-LM02 reagent) capable of binding to the LIM domain of LMO2 and inhibiting the functional activity of LMO2.

  According to the present invention, the term “inhibits” (functional activity of LM02) includes within the scope of the present invention significant inhibition of the functional activity of LM02 as compared to a suitable control. Advantageously, the term “inhibits” (functional activity of LMO2) is 20%, 30%, 40%, 50%, 60%, 70%, 80% of the functional activity when compared to a suitable control. , 90% inhibition. Most advantageously, “inhibit” (functional activity of LM02) refers to a 95, 96, 97, 98, 99 or 100% inhibition of the functional activity of LM02 when compared to a suitable control. Suitable controls are well known to those skilled in the art and are also described in the detailed description and examples of the present invention.

  The inventors have surprisingly found that all LIM2 inhibitor peptides (peptides that substantially inhibit the functional activity of LM02) have the consensus sequence described below. The consensus sequence provides the peptide with certain structural features that we 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 the consensus sequence:
His / CXXXC
(Wherein C is a zinc bond Cys;
His stands for histidine,
X is any amino acid)
including.

According to the above aspect of the invention, advantageously the LIM2 inhibitor is a peptide and the consensus sequence HhHis / CXX ¹ Ch
(Wherein h represents a hydrophobic amino acid,
His stands for histidine,
C represents cysteine,
X represents any amino acid, and X ¹ represents a charged amino acid)
including

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

  More advantageously, LIM2 inhibitors according to the invention are illustrated in FIG. 2c as clone 207, clone 209, clone 85, clone 81, clone 63, clone 72, clone 247, clone 90, clone 69 and clone 202, It includes any one or more of the 10 peptide sequences designated respectively in numbers 2-11.

  More advantageously, further LIM2 inhibitors according to the invention are illustrated in FIG. 2 as clone 207, clone 209, clone 85, clone 81, clone 63, clone 72, clone 247, clone 90, clone 69 and clone 202. And any one or more of the 10 peptide sequences specified in SEQ ID NOs: 2 to 11, respectively.

  Most advantageously, the LIM2 inhibitor according to the present invention is represented by any one or more of the peptide sequences depicted in FIG. 2 as clone 207, clone 209, clone 85 and designated respectively in SEQ ID NOs: 2, 3 and 4. is there.

  The peptide inhibitors according to the invention can be used in an unconstrained form or in a form in which the C-terminal and / or N-terminal is constrained in a scaffold. Suitable scaffolds are well known to those skilled in the art and may be natural or synthesized. Suitable natural scaffolds are described in the detailed description of the invention. According to the invention, advantageously the scaffold is a protein scaffold and a thioredoxin protein.

  According to the present invention, an inhibitor according to the present invention can include one or more intracellular targeting agents. Advantageously, the intracellular targeting agent according to the present invention is a nuclear localization signal. Suitable nuclear localization signals are well known to those skilled in the art and are described in the detailed description of the invention.

  In yet another aspect, the present invention provides a composition comprising one or more LIM2 inhibitors / anti-LM02 reagents according to the present invention and a pharmaceutically acceptable carrier, diluent or excipient.

In a preferred embodiment of the above aspect of the invention, the LIM2 inhibitor is a peptide and the consensus sequence:
His / CXXXC
(Wherein C is a zinc bond Cys;
His stands for histidine,
X is any amino acid)
including.

According to the above aspect of the invention, advantageously the LIM2 inhibitor is a peptide and the consensus sequence:
HhHis / CXX ¹ Ch
(Wherein h represents a hydrophobic amino acid,
His stands for histidine,
C represents cysteine,
X represents any amino acid, and X ¹ represents a charged amino acid)
including.

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

  Advantageously, the peptide LIM2 inhibitor according to the invention is an aptamer and has the above consensus sequence. More advantageously, LIM2 inhibitors according to the invention are illustrated in FIG. 2c as clone 207, clone 209, clone 85, clone 81, clone 63, clone 72, clone 247, clone 90, clone 69 and clone 202, It includes any one or more of the 10 peptide sequences designated respectively in numbers 2-11. Most advantageously, further LIM2 inhibitors according to the present invention are illustrated in FIG. 2 as clone 207, clone 209, clone 85, clone 81, clone 63, clone 72, clone 247, clone 90, clone 69 and clone 202. And any one or more of the 10 peptide sequences specified in SEQ ID NOs: 2 to 11, respectively.

  In another embodiment of the above aspect of the invention, the LIM2 inhibitor / anti-LM02 reagent is an antibody as defined herein.

  According to the above embodiment of the invention, advantageously the antibody is an scFv. In another embodiment of the above aspect of the invention, the antibody is a dAb as defined herein. More advantageously, the antibody LIM2 inhibitor according to the invention is an intracellular binding antibody as defined herein. Most advantageously they are intracellular binding scFv molecules, idAbs (intracellular 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, the antibody LIM2 inhibitor is at least 1 having the amino acid sequence depicted in FIG. Intracellularly bound antibody containing one CDR. In yet another preferred embodiment of this aspect of the invention, the antibody LIM2 inhibitor has the amino acid sequence depicted in FIG. 8 and designated in SEQ ID NOs: 12 (a), 12 (b) and 12 (c). An intracellular binding idAb or scFv comprising at least one CDR having. In yet another preferred embodiment of this aspect of the invention, the antibody LIM2 inhibitor has the amino acid sequence depicted in FIG. 8 and designated in SEQ ID NOs: 12 (a), 12 (b) and 12 (c). Intracellularly bound idAb or scFv containing at least two CDRs. In yet another preferred embodiment of this aspect of the invention, the antibody LIM2 inhibitor has the amino acid sequence depicted in FIG. 8 and designated in SEQ ID NOs: 12 (a), 12 (b) and 12 (c). Intracellularly bound idAb or scFv containing all three CDRs. In a preferred embodiment of this aspect of the invention, the antibody LIM2 inhibitor is an intracellular binding scFv comprising the amino acid sequence depicted in FIG. 8 and designated SEQ ID NO: 12, in addition to the Ser-Gly linker. In the most preferred embodiment of this aspect of the invention, the antibody LIM2 inhibitor is an intracellular binding scFv consisting of the amino acid sequence depicted in FIG. 8 and designated SEQ ID NO: 12.

  Thus, in yet another aspect, the invention provides an anti-antibody comprising at least one CDR having the amino acid sequence depicted in FIG. 8 and designated SEQ ID NOs: 12 (a), 12 (b) and 12 (c). LMO2 antibodies (LIM2 inhibitors) are provided.

  Preferably, the anti-LM02 antibody according to the invention is fused or bound to the domain or sequence of the protein responsible for translocation activity. Details of suitable translocation peptides are provided in the detailed description of the invention.

  Advantageously, the present invention comprises three CDRs having the amino acid sequences illustrated in FIG. 8 and specified respectively in SEQ ID NOs: 12 (a), 12 (b) and 12 (c), and antigen binding as a whole An anti-LM02 antibody (LIM2 inhibitor) that forms a site is provided.

  In yet another aspect, the invention relates to an anti-LM02 antibody comprising at least one CDR having the amino acid sequence depicted in FIG. 8 and designated in SEQ ID NOs: 12 (a), 12 (b) and 12 (c) Nucleic acids encoding (LIM2 inhibitors) are provided.

  Advantageously, the present invention comprises three CDRs having the amino acid sequences illustrated in FIG. 8 and specified respectively in SEQ ID NOs: 12 (a), 12 (b) and 12 (c), and antigen binding as a whole Nucleic acids encoding anti-LM02 antibodies (LIM2 inhibitors) that form sites are provided.

  In a preferred embodiment of this aspect of the invention, the antibody LIM2 inhibitor is an intracellular binding scFv comprising the amino acid sequence depicted in FIG. 8 and designated SEQ ID NO: 12, in addition to the ser-gly linker. In yet another aspect, the present invention provides the use of a LIM2 inhibitor according to the present invention or a composition comprising it in inhibiting the functional activity of LMO2.

  In yet another aspect, the present invention provides the use of a LIM2 inhibitor according to the present invention in the manufacture of a medicament for inhibiting the functional activity of LMO2.

  The present invention has shown that LMO2 has many different roles in mammals. Accordingly, the term “functional activity” of LMO2 as defined herein refers to one or more, several or all of the actions performed by LMO2 in mammals. Furthermore, the term “inhibition of the functional activity of LM02” refers to substantial inhibition as described herein, one or more, several or all of the functions performed by LM02 in a mammal.

  The inventors have shown that LMO2 has some effect in the group of conditions consisting of angiogenesis (pathological and non-pathological angiogenesis), embryogenesis and leukemogenesis.

  As used herein, the term “angiogenesis” refers to the process of primary capillary remodeling that forms the mature vasculature. On the other hand, the term “angiogenesis” refers to the primary process of angiogenesis in which a primary capillary network is formed. Importantly, we have shown that LM02 plays an important role in the angiogenesis process but not the angiogenesis process.

  As used herein, the term “pathological angiogenesis” refers to any disease state / situation in which angiogenesis performs some action. The inventors have shown that “pathological angiogenesis” contributes to a condition selected from the group consisting of tumor angiogenesis, tumor metastasis, ischemia, inflammation and diabetic retinopathy. Accordingly, the term “pathological angiogenesis” as defined herein includes within the scope of the present invention all of the above mentioned conditions. Advantageously, according to the invention, the pathological angiogenesis is tumor angiogenesis and / or tumor metastasis.

  In addition to the action of LMO2 in “pathological angiogenesis”, LMO2 also acts in “non-pathological angiogenesis” in certain events such as wound healing and menstruation. According to the present invention, the term “non-pathological angiogenesis” includes within the scope of the present invention wound healing and menstruation.

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

  As mentioned above, the present invention has shown that LMO2 plays a role in processes such as angiogenesis, embryogenesis and hematopoiesis. Accordingly, LIM2 inhibitors according to the present invention prevent and / or treat conditions including tumorigenesis, tumor metastasis, LMO2-mediated T cell leukemia, inflammation, ischemia, diabetic retinopathy, modulation of wound healing and regulation of menstruation The present inventors believe that it will be quite useful therapeutically.

  Accordingly, in yet another aspect of the present invention, there is provided a LIM2 inhibitor according to the present invention or a composition comprising the same for use in medicine.

  In yet another aspect, the present invention prevents and prevents one or more conditions selected from the group consisting of tumorigenesis, tumor metastasis, LMO2-mediated T cell leukemia, inflammation, ischemia, diabetic retinopathy and wound healing. There is provided the use of one or more LIM2 inhibitors according to the present invention in the manufacture of a medicament for treatment.

  In yet another aspect, the present invention provides tumor formation, tumor metastasis, LMO2-mediated T cell leukemia comprising administering one or more LIM2 inhibitors according to the present invention to a patient in need of such treatment. A method for preventing and / or treating any one or more conditions selected from the group consisting of inflammation, ischemia, diabetic retinopathy and wound healing is provided.

  According to the above aspect of the invention, advantageously the condition is tumorigenesis and / or tumor metastasis.

In a preferred embodiment of the above aspect of the invention, the LIM2 inhibitor is a peptide and the consensus sequence:
His / CXXXC
(Wherein C is a zinc bond Cys;
His stands for histidine,
X is any amino acid)
including.

According to the above aspect of the invention, advantageously the LIM2 inhibitor is a peptide and the consensus sequence:
HhHis / CXX ¹ Ch
(Wherein h represents a hydrophobic amino acid,
His stands for histidine,
C represents cysteine,
X represents any amino acid, and X ¹ represents a charged amino acid)
including.

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

  Advantageously, the peptide LIM2 inhibitor according to the invention is an aptamer and has the consensus sequence shown above. More advantageously, LIM2 inhibitors according to the invention are illustrated in FIG. 2c as clone 207, clone 209, clone 85, clone 81, clone 63, clone 72, clone 247, clone 90, clone 69 and clone 202, It includes any one or more of the 10 peptide sequences designated respectively in numbers 2-11. Most advantageously, LIM2 inhibitors according to the present invention are illustrated in FIG. 2c as clone 207, clone 209, clone 85, clone 81, clone 63, clone 72, clone 247, clone 90, clone 69 and clone 202, Any one or more of the 10 peptide sequences respectively designated by numbers 2-11.

  In another embodiment of the above aspect of the invention, the antibody is a scFv. Even more advantageously, the antibody LIM2 inhibitor according to the invention is an intracellular binding antibody as defined herein. Most advantageously they are intracellular binding scFv molecules, idAbs (intracellular 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, the antibody LIM2 inhibitor comprises at least a CDR having the amino acid sequence depicted in FIG. 8 and specified in SEQ ID NOs: 12 (a), 12 (b) and 12 (c). Intracellularly bound antibody comprising one. In yet another preferred embodiment of this aspect of the invention, the antibody LIM2 inhibitor has the amino acid sequence depicted in FIG. 8 and designated in SEQ ID NOs: 12 (a), 12 (b) and 12 (c). Intracellularly bound idAb or scFv comprising at least one CDR having. In yet another preferred embodiment of this aspect of the invention, the antibody LIM2 inhibitor has the amino acid sequence depicted in FIG. 8 and designated in SEQ ID NOs: 12 (a), 12 (b) and 12 (c). Intracellularly bound idAb or scFv comprising at least two CDRs having. In yet another preferred embodiment of this aspect of the invention, the antibody LIM2 inhibitor has the amino acid sequence depicted in FIG. 8 and designated in SEQ ID NOs: 12 (a), 12 (b) and 12 (c). Intracellularly bound idAb or scFv containing all three CDRs (at). In the most preferred embodiment of this aspect of the invention, the antibody LIM2 inhibitor is an intracellular binding scFv consisting of the amino acid sequence depicted in FIG. 8 and designated SEQ ID NO: 12.

  Uses and methods of the invention include mammals including, but not limited to, those selected from the group consisting of humans, mice, rats, guinea pigs, hamsters, rabbits, dogs, cats, goats, monkeys, horses. Suitable for treating an individual. Advantageously, the uses and methods of the present invention are useful for treating human individuals.

  The present invention is described by the following examples which should not be construed as limiting the invention in any way.

Definitions “LM02” is a transcription factor. Importantly, LM02 is a master regulator of several major pathways in embryogenesis, hematopoiesis and angiogenesis and performs its function through protein-protein interactions involving the LIM domain.

  The term “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 acids that are joined together by amide bonds at the N-terminus of each amino acid Say. According to the present invention, advantageously, the term “peptide” according to the present invention refers to 25 or fewer amino acids linked to each other by amide bonds. Most advantageously, the term “peptide” refers to no more than 20 amino acids linked together at the N-terminus. The peptides according to the invention may be branched or linear. Advantageously, the peptides according to the invention are linear.

  The term “protein” refers to a collection of one or more polypeptide chains. The “polypeptide chain” defined in the present specification refers to 51 or more amino acids that are joined together by an amide bond at the N-terminus of each amino acid to form a chain. The polypeptide chain of the present invention may be branched or linear. One or more polypeptide chains include protein molecules having secondary and tertiary structural elements. These structural elements determine the structure and function of the resulting protein.

  “Anti-LM02 reagent / LIM2 inhibitor” refers to an agent that binds to the LIM-2 domain of LMO2 and substantially inhibits the functional activity of LMO2. Such drugs may be natural or synthetic. Suitable synthetic molecules include small molecules. Suitable natural molecules include proteins, particularly antibodies, peptides and / or nucleic acid molecules, which may be monoclonal or polyclonal.

  The term “inhibit” (LMO2 functional activity) refers to significant inhibition of LMO2 functional activity compared to a suitable control. Advantageously, the term “inhibits” (the functional activity of LM02) is 20%, 30%, 40%, 50%, 60%, 70%, 80% of the functional activity of LM02 compared to a suitable control. %, 90% inhibition. Most advantageously, the term “inhibits” (functional activity of LM02) refers to a 95, 96, 97, 98, 99 or 100% inhibition of the functional activity of LM02 compared to a suitable control. Suitable controls are well known to those skilled in the art and are also described in the detailed description and examples of the present invention. LMO2 has a number of different roles in mammals. Accordingly, the term “functional activity” of LMO2 as defined herein refers to one or more, several or all of the actions performed by LMO2 in a cell. Furthermore, the term “inhibition of the functional activity of LM02” refers to substantial inhibition as described herein, one or more, several or all of the functions performed by LM02 in a mammal. We have LMO2 consisting of angiogenesis (including pathological and non-pathological angiogenesis), certain forms of T-cell leukemia, inflammation, wound healing, ischemia and diabetic retinopathy It was shown to have some effect on the group status.

As used herein, an antibody refers to an antibody fragment or a complete antibody capable of binding to a selected target, and Fv, ScFv, Fab ′ and F (ab ′) ₂ , monoclonal and polyclonal antibodies, chimeric Antibodies, engineered antibodies including CDR-grafted antibodies and humanized antibodies, and antibodies made using phage display or another technique and artificially selected. Small fragments such as Fv and ScFv have advantageous properties for diagnostic and therapeutic applications due to their small size and resulting excellent tissue distribution. Preferably, the LIM2 inhibitor antibody according to the invention is a single heavy chain domain antibody (heavy chain domain dAb), a single light chain domain antibody (light chain domain dAb) or an scFv molecule. More preferably, the LIM2 inhibitor antibody according to the present invention is an intracellular binding single domain antibody molecule (idAb) as defined herein. The term “antibody” as defined herein also includes, within the scope of the invention, a molecule comprising an antigen binding portion comprising at least one heavy chain variable domain and at least one antibody constant region domain.

  The CDRs (complementarity determining regions) of immunoglobulin molecule heavy and light chain variable domains refer to amino acid residues contained within the hypervariable loop of the variable region, not the framework region residues. These hypervariable loops are directly involved in immunoglobulin and ligand interactions. Residues in these loops tend to be less conserved than in framework regions.

  The inside of a cell means the inside of a cell. The cell may be any prokaryotic or eukaryotic cell and is preferably selected from the group consisting of bacterial cells, yeast cells and higher eukaryotic cells. Most preferred are yeast cells and mammalian cells. Thus, as used herein, “intracellular” antibodies and targets or ligands are antibodies and targets / ligands that are present in cells (including cytoplasm and nucleus). The term “intracellular” also refers to an environment that is similar to or mimics the intracellular environment. Thus, “intracellular” may refer to an environment that is in vitro rather than intracellular. For example, the methods of the invention can be carried out in an in vitro transcription and / or translation system, which can be obtained commercially or derived from natural systems. Accordingly, an “intracellularly bound antibody” according to the present invention is an antibody as defined herein that is capable of specifically binding to one or more antigens in an intracellular environment as defined herein.

  The term “angiogenesis” refers to the process of primary capillary remodeling that forms the mature vasculature. On the other hand, the term “angiogenesis” refers to the primary process of angiogenesis in which a primary capillary network is formed. Importantly, we have shown that LM02 plays an important role in the angiogenesis process but not the angiogenesis process.

  The term “pathological angiogenesis” refers to any disease state / situation in which angiogenesis performs some action. The inventors have shown that “pathological angiogenesis” contributes to a condition selected from the group consisting of tumor angiogenesis, tumor metastasis, ischemia, inflammation and diabetic retinopathy. Accordingly, the term “pathological angiogenesis” as defined herein includes within the scope of the present invention all of the above mentioned conditions. Advantageously, according to the invention, the pathological angiogenesis is tumor angiogenesis and / or tumor metastasis.

  LMO2 also has some effect on “non-pathological angiogenesis” in certain events such as wound healing and menstruation. Thus, according to the present invention, the term “non-pathological angiogenesis” includes within the scope of the present invention wound healing and menstruation.

DETAILED DESCRIPTION OF THE INVENTION General Techniques Unless defined otherwise, all technical and scientific terms used herein are (for example, in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry). ) Has the same meaning as commonly understood by those skilled in the art. Molecular, genetic and biochemical methods (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbour Harb., Spring Spring Harb., Incorporated herein by reference). And Ausubel et al., Short Protocols in Molecular Biology (1999), 4th Edition, John Wiley & Sons, Inc.) and standard techniques of chemical methods are used. See also Harlow & Lane. , A Laboratory Manual Cold Spring Harbor, N.A. Y. To mention.

(A) Features of LIM2 inhibitors according to the present invention In a first aspect, the present invention provides an agent capable of binding to the LIM2 domain of LMO2 and inhibiting its functional activity.

  According to the present invention, “LIM2 inhibitor / anti-LM02 reagent” refers to an agent that binds to the LIM-2 domain of LMO2 and substantially inhibits the functional activity of LMO2. Such agents may be natural or synthesized. Suitable synthetic molecules include small molecules. Suitable natural molecules include proteins (especially antibodies that may be monoclonal or polyclonal), peptides and / or nucleic acid molecules. Advantageously, the anti-LM02 antibody according to the invention is a scFv molecule or a dAb molecule as defined herein. More advantageously, the antibody LIM2 inhibitor according to the invention is an intracellular binding antibody (intracellular antibody) as defined herein. Most advantageously they are intracellular binding scFv molecules, idAbs (intracellular 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, the antibody LIM2 inhibitor is at least 1 having the amino acid sequence depicted in FIG. 8 and specified in SEQ ID NOs: 12 (a), 12 (b) and 12 (c). Intracellularly bound antibody containing one CDR. In yet another preferred embodiment of this aspect of the invention, the antibody LIM2 inhibitor has the amino acid sequence depicted in FIG. 8 and designated in SEQ ID NOs: 12 (a), 12 (b) and 12 (c). An intracellular binding idAb or scFv comprising at least one CDR having. In yet another preferred embodiment of this aspect of the invention, the antibody LIM2 inhibitor has the amino acid sequence depicted in FIG. 8 and designated in SEQ ID NOs: 12 (a), 12 (b) and 12 (c). Intracellularly bound idAb or scFv containing at least two CDRs. In yet another preferred embodiment of this aspect of the invention, the antibody LIM2 inhibitor has the amino acid sequence depicted in FIG. 8 and designated in SEQ ID NOs: 12 (a), 12 (b) and 12 (c). Intracellularly bound idAb or scFv containing all three CDRs. In yet another preferred embodiment of this aspect of the invention, the antibody LIM2 inhibitor comprises, in addition to the Ser-Gly linker, the amino acid sequence depicted in FIG. 8 and designated SEQ ID NO: 12, advantageously then Is an intracellularly bound scFv. In the most preferred embodiment of this aspect of the invention, the antibody LIM2 inhibitor is an intracellularly bound scFv comprising, advantageously consisting of, the amino acid sequence depicted in FIG. 8 and designated SEQ ID NO: 12.

(I) Antibody
Antibody Generation Antibodies formed in the LIM2 domain of LMO2 can be generated using standard laboratory techniques. Antibodies can be made using recombinant proteins or those of natural origin. For example, a protein (ie, “immunogen”) is administered to challenge a mammal such as a monkey, goat, rabbit or mouse. The resulting antibody may be collected as a polyclonal serum, or the antibody-producing cells of the challenge animal are immortalized (eg, by creating a hybridoma by fusing with an immortal fusion partner) and then producing monoclonal antibodies in the cells You may let them.

1ai. Polyclonal antibody antigen proteins may be used alone or conjugated to conventional carriers to increase immunogenicity and form antisera against peptide-carrier conjugates in animals as described above. To do. The conjugation and immunization of the peptide to the carrier protein can be performed as described (Dymecki et al. (1992) J. Biol. Chem., 267: 4815). Serum is assayed for antibody titers against protein antigens by ELISA or alternatively by dot or spot blotting (Boersma and Van Leeuwen (1994) J. Neurosci. Methods, 51: 317). For example, Green et al. (1982) Cell, 28: 477, shows that the serum reacts strongly with the appropriate peptide.

1 aii. Monoclonal Antibodies Techniques for making monoclonal antibodies are well known and are described in Arnheiter et al. (1981) Nature, 294, 278, preferably any antigen bound to a carrier can be used to make monoclonal antibodies. Monoclonal antibodies are typically obtained from ascites obtained from a hybridoma tissue culture or an animal into which the hybridoma tissue has been introduced. Nevertheless, monoclonal antibodies may be described as “formed against” or “induced by” the protein.

  Monoclonal antibodies are tested for function and specificity after formation by any of a number of means. Similar techniques can be used to test recombinant antibodies produced by phage display or other in vitro selection techniques. Monoclonal antibody-producing hybridomas (or polyclonal sera) can also be screened for antibody binding to the immunogen. Particularly preferred immunological tests include enzyme-linked immunoassay (ELISA), immunoblotting and immunoprecipitation (Voller, (1978) Diagnostic Horizons, 2: 1, Microbiological Associates, Quarterly Publications, Walkersville, MD, MD; J. Clin. Pathol., 31: 507; U.S. Reissue Patent 31,006; British Patent 2,019,408; Butler (1981) Methods Enzymol., 73: 482; Maggio, E. (ed.). (1980) Enzyme Immunoassay, CRC Press, Boca Raton, FL) or LA Immunoassay (RIA) (Weintraub, B., Principles of radioimmunoassays, Seventh Training Counsel on Radioligand, Assay-Assay Techniques, 68-49, The Endocrine. Anything that detects binding of the antibody to the immunogen needs to be labeled to facilitate such detection. Techniques for labeling antibody molecules are well known to those of skill in the art (see Harlow and Lane (1989) Antibodies, Cold Spring Harbor Laboratory, pages 1-726).

(1b) Western (antibody) blotting Western blotting to test the specificity of antibody binding can be carried out using methods well known to those skilled in the art and can be found in Sambrook et al. , Molecular Cloning: A Laboratory Manual, 2nd edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N .; Y. And Ausubel et al. , Short Protocols in Molecular Biology (1999).

Intracellular antibody Intracellular antibody (intracellular binding antibody) LIM2 inhibitors are particularly useful in inhibiting the functional activity of LMO2. The structures and features of these antibodies that enable their function in the intracellular environment are described in PCT / GB2002 / 003512, PCT / GB2003 / 001077, GB 02262728.4 and GB0026727.6. These applications are hereby incorporated by reference. Advantageously, the intracellular antibody (intracellular binding antibody) according to the present invention is an scFv comprising, preferably consisting of, the amino acid sequence shown in FIG.

(Ii) Peptides as LIM2 inhibitors According to the present invention, the term “peptide” refers to 10 or less, 15 or less, 16 or less, 17 or less, 18 or less, 19 or less linked at the N-terminus of each amino acid by amide bonds. Hereinafter, the amino acid is 20 or less, 25 or less, or 50 or less. According to the present invention, advantageously, the term “peptide” according to the present invention refers to 25 or fewer amino acids linked together by amide bonds. Most advantageously, the term “peptide” refers to no more than 20 amino acids linked together at the N-terminus. The peptides according to the invention may be branched or linear. Advantageously, the peptides according to the invention are linear.

  On the other hand, the term “protein” refers to an assembly of one or more polypeptide chains. The “polypeptide chain” defined in the present specification refers to 51 or more amino acids that are joined together by an amide bond at the N-terminus of each amino acid to form a chain. The polypeptide chain of the present invention may be branched or linear. One or more polypeptide chains include protein molecules having secondary and tertiary structural elements. These structural elements determine the structure and function of the resulting protein.

In a preferred embodiment of the invention, the LIM2 inhibitor is a peptide and the consensus sequence:
His / CXXXC
(Wherein C is a zinc bond Cys;
His stands for histidine,
X is any amino acid)
including.

Advantageously, the LIM2 inhibitor is a peptide and the consensus sequence:
HhHis / CXX ¹ Ch
(Wherein h represents a hydrophobic amino acid,
His stands for histidine,
C represents cysteine,
X represents any amino acid, and X ¹ represents a charged amino acid)
including.

  Advantageously, the peptide LIM2 inhibitor according to the present invention is a peptide aptamer (20mer) and comprises the consensus sequence shown above.

LIM domain protein provides an interface interaction ¹⁴ ~ ^16, LIM-only proteins LMO2 It has been previously reported to bind to several partners via the LIM domain.

  We have shown that by modulating LMO2 LIM domain protein-protein interactions, it is possible to regulate important special stages of endothelial remodeling such as tumor angiogenesis.

  The anti-LM02 peptide aptamer according to the present invention is characterized by 207 peptides conserving the cys-xx-cys motif embedded in 20 amino acids containing peptide domains within the Trx protein scaffold. The anti-LM02 peptide aptamers described herein specifically bind to the LMO2 LIM domain and do not bind efficiently to any of the LMO-only proteins. Peptide aptamer analysis shows that LMO2 binding is dependent on the cys / his-xx-cys motif and that the conservation of adjacent hydrophobic residues is dependent on both sides and charged residues in between Yes. As illustrated by the erythropoiesis assay (FIG. 4), the 207 peptide is effective for specific binding (FIG. 3) and inhibition of LMO2 function. Peptide aptamers bind to the LIM2 domain of LMO2, and peptide sequence studies do not reveal any obvious reason for this, as well as any obvious reason for the specificity of LMO2 over other members of the LMO family .

Types of amino acids The charged, polar uncharged and hydrophobic (nonpolar) amino acids described herein are classified in the following table. In the case of amino acid conservative substitutions, the same block of amino acids in the second column, preferably the same line of amino acids in the third column, can be substituted for each other:

(Iii) Protein scaffolds for use in peptide inhibitors according to the present invention Peptide LMO2 inhibitors according to the present invention may be used in unconstrained form or alternatively via one or more linkages to the scaffold. It may be constrained to the N and / or C terminus. Suitable scaffolds may be natural or synthesized. Advantageously, according to the invention, the scaffold is a protein scaffold.

  Suitable scaffolds are well known to those skilled in the art and are selected from the group consisting of immunoglobulin domain based scaffolds, bacterial receptor based scaffolds such as SpA, fibronectin, lipocallin, CTLA4 based Is mentioned. Other suitable scaffolds include those based on fibronectin and affibodies. Other suitable scaffolds include van den Beuken et al. , J .; Mol. Biol. (2001) 310, 591-601 include lipocalins and CTLA4 and scaffolds such as those described in International Publication No. 0069907 (Medical Research Council), which includes, for example, the bacterium GroEL. Based on the ring structure or the ring structure of other chaperone polypeptides.

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

liiii. Nucleic acid anti-LM02 reagent / LIM2 inhibitor According to the present invention, the LIM2 inhibitor / anti-LOM2 reagent may comprise a nucleic acid molecule. Advantageously, such LIM2 inhibitor is an antisense RNA. Methods for producing such RNA and methods for using them are well known to those skilled in the art.

(C) Inhibition of LMO2 functional activity using a LIM2 inhibitor / anti-LM02 reagent according to the present invention In yet another aspect, the present invention relates to a LIM2 inhibitor according to the present invention or an inhibitor thereof in inhibiting the functional activity of LMO2. The use of a composition comprising:

  The present invention shows that LMO2 has many different roles in mammals. Accordingly, the term “functional activity” of LMO2 as defined herein refers to one or more, several, or all of the actions performed by LMO2 in mammals and described herein. Further, the term “inhibition of LM02 functional activity” refers to substantial inhibition as described herein, or to one or more, several or all of the functions performed by LM02 in a mammal.

  According to the present invention, the term “inhibits” (functional activity of LM02) includes within the scope of the present invention significant inhibition of the functional activity of LM02 as compared to a suitable control. Advantageously, the term “inhibits” (functional activity of LMO2) means 20%, 30%, 40%, 50%, 60%, 70% of the functional activity of LMO2 compared to a suitable control, 80%, 90% inhibition. Most advantageously, the term “inhibits” (functional activity of LM02) refers to a 95, 96, 97, 98, 99 or 100% inhibition of the functional activity of LM02 compared to a suitable control. Suitable controls are well known to those skilled in the art and are described in the detailed description of the invention and also in the examples.

  We have shown that LMO2 has some effect on the conditions of the group consisting of angiogenesis (including pathological and non-pathological angiogenesis), embryogenesis and leukemogenesis.

  More specifically, LMO2 is any one or more of the conditions selected from the group consisting of tumorigenesis, tumor metastasis, LMO2-mediated T cell leukemia, inflammation, ischemia, diabetic retinopathy, wound healing and menstruation. The present inventors consider that there is some influence in the above.

( Ci) Action of LMO2 in mammals:
(Cia) Effect of LMO2 on hematopoietic cell fate By examining T cell tumors arising in the LMO2 gain-of-function transgenic mouse model, we bind in this case to a bi-sequence containing the tandem E box. Similar LMO2-related complexes could be detected ¹⁸ . These observations led to two conclusions. First, since LMO2 appears to be a bridging element of the protein complex detected in red blood cells, elements of the LMO2 complex may alter the overall hematopoietic differentiation and vary by regulating the gene set It seems to be able to regulate the stage-dependent function of. LMO2 results in a distinct action in regulating hematopoietic cell fate. Secondly, the action of LMO2 in T-ALL is probably due to forced formation of protein complexes (a direct result of forced LMO2 expression after chromosomal translocation). Thus, gene regulation via the LMO2 complex can be affected. This can occur by direct DNA binding complex or protein sequestration from normal action ¹³ .

The effect of LMO2 in regulating cell fate in normal hematopoiesis has been demonstrated mainly by gene targeting experiments. Null mutations were made in embryonic stem cells (ES cells) by homologous recombination to produce heterozygous mice. The breeding of these mice indicates that embryos die in stages E9-10 due to the failure of yolk sac erythropoiesis ¹⁹ , indicating that LMO2 is required for primordial erythropoiesis in mouse embryogenesis. Studies on the possible action of LM02 in adult (final) hematopoiesis were performed using chimeric mice ²⁰ and revealed that hematopoiesis does not occur in the absence of the LM02 gene. Thus, LMO2 functions autonomously in cells in the early stages of final hematopoiesis. This important function may be at the level of pluripotent stem cells, at the level of pre-multipotent progeny, or in some cases, the mesoderm gives rise to these precursors, It may be before that.

(Cib) Action of LMO2 in embryogenesis We established a reporter system for LMO2 expression in embryogenesis using the lacZ knock-in of the endogenous LMO2 gene ²¹ , from E8.5, LMO2 It has been found that it has a more specific definitive expression pattern in endothelial cells and blood progenitor cells. In E10.5, LMO2 is expressed in vascular endothelial cells and pluripotent hematopoietic cells when final hematopoiesis is thought to start in the aorta-gonad-medium kidney (AGM) region ^21,22 . Additional expression sites were observed in limb buds and hippocampus. The expression pattern of LMO2 in yolk sac and blood progenitor cells is consistent with its function in primitive and final hematopoiesis. The first embryonic hematopoietic stem cell activity is thought to appear in the AGM region near E10.5, which is derived from the endothelium of large arteries such as the dorsal aorta ^23,24 . In mouse embryogenesis, hemangioblasts (presumed to be a common precursor of endothelium and blood cells) have been proposed to arise from unspecified posterior mesoderm, and thus the early capillary network is these Formed from hemangioblasts. This first process of vasculature in which the capillary network is formed is called angiogenesis. More mature vasculature is formed by remodeling of the early capillary network in a process called angiogenesis ²⁵ . In particular, the early action of LMO2 prior to the identification of hematopoietic stem cells in vasculature construction was investigated by following the fate of LMO2 ES cells in chimeras ²¹ . Non-LM02 ES cells can contribute to the capillary network in pre-E9 chimeric mice. However, in the vicinity of E10, significant vascular tissue disruption is observed in chimeric mice due to the failure of the LMO2ES cell contribution in the endothelium. From E11 onwards, no LMO2ES cells contribute to the endothelium of large arteries. These results indicate that LMO2 is required for angiogenesis but not for angiogenesis. LMO2 is expressed in endothelial cells and blood progenitor cells and has a dual function in hematopoiesis and angiogenesis. Given the close relationship between endothelium and blood cell specification, the action of LMO2 at this critical stage of mouse development is extremely important. This major action of this protein can in turn explain why adult hematopoiesis fails due to LMO2 null mutations.

(Cic) Action of LMO2 in tumor angiogenesis Since ES cells lacking LMO2 are unable to support angiogenesis during tumor growth, we also show that LMO2 protein is essential for tumor angiogenesis ²⁶ . There are many situations in adults where this remodeling process of angiogenesis is required, such as wound healing or menstruation and pathological conditions such as tumor angiogenesis, ischemia, inflammation and diabetic retinopathy ²⁷ . In cancer, angiogenesis is a major part of solid tumor growth and invasion (metastasis). Like all cells, cancer cells require an exchange of oxygen and carbon dioxide, so solid tumors need to form a blood supply to promote ^growth28,29 . This is accomplished by destroying the local vascular endothelium during tumor angiogenesis ^25,30 , allowing the sprouting of the existing endothelium into tumor deposits ³¹ .

This angiogenesis switch occurs when the angiogenesis stimulating factor is relatively increased, and in tumor growth, cancer cells become angiogenic and stimulate the necessary remodeling state. In addition, the concept of a mosaic blood vessel is that most of the blood vessel wall can be formed from tumor cells ³² , and these cells are constantly shed within the lumen of the blood vessel, thus blood circulation contributes to the metastatic population. It is strongly supported by data showing that In addition, so-called hemangioblast-like endothelial precursors are sometimes employed from the bone marrow ³¹ and are another source of vascular wall cells. These features of angiogenesis have been proposed as a major target for treating patients in ^cancer28,29 . Several major molecules that regulate angiogenesis have been identified, including vascular endothelial growth factor ³⁴ , which stimulates the vascular sprouting process but little is known about transcriptional regulators of vascular remodeling. There are also candidates from a study of chromosomal translocations in human leukemia, many of which have been identified as novel genes that are transcription factors (reviewed in ¹ ). The LMO2 gene is an example. This gene is normally expressed in mouse embryonic endothelial cells ²¹ where it is required for angiogenesis after vascularization has occurred. Tumor angiogenesis increases LMO2 expression. Furthermore, in the absence of LMO2, the ES cell tumor model prevents the development of mature vasculature in solid tumors.

(Cii) Assay for testing the functional activity of LMO2 Assays for evaluating the functional activity of LMO2 are well known to those skilled in the art. Suitable assays are null mutations of LMO2 time to inactivate erythroid differentiation pathway, it is known to depend on the function of LMO2 proteins, measuring differentiation into erythroid cells ES cells in vitro ^19,20. LMO2 null mutation does not cause angiogenesis ³⁸ .

  The inventors were able to observe that expression of Trx207 protein in ES cells inhibits the effect on erythropoiesis. Inhibition levels differed between ES clones, with the maximum level reaching ~ 70%. We thought this incomplete inhibition was due to variations in Trx207 protein expression levels in selected ES clones. There is also a problem that arises from expressing a Trx protein with a 20mer peptide (aptamer) attached to the outer loop (data not shown), which means that different scaffolds can be more effective at presenting the peptide. there's a possibility that.

  Thus, anti-LM02 peptide aptamers such as 207 peptide may be more effective in different scaffolds. Indeed, unconstrained peptide aptamers can be very effective. This evidence is provided in the form that the GAL4-207 fusion protein was able to interact with the LMO2 LIM2 domain at a level comparable to the Trx207 protein (FIG. 3).

(Ciii) Delivery of anti-LM02 reagent / LIM2 inhibitor to patient cells In general, LIM2 inhibitors according to the present invention are delivered to cells. Advantageously, it is delivered to the cell nucleus.

( Ciiia) Intracellular expression In order to introduce such molecules into the intracellular environment, the cells are advantageously transfected with a nucleic acid encoding one or more LIM2 inhibitors.

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

  Furthermore, nucleic acids encoding LIM2 inhibitors according to the present invention can be incorporated into cloning vectors for general manipulation and nucleic acid amplification purposes.

  Expression and cloning vectors generally contain nucleic acid sequences 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 an origin of replication or a self-replicating sequence. Such sequences of various bacteria, yeasts and viruses are well known. The origin of replication of plasmid pBR322 is suitable for most gram negative bacteria, the 2m plasmid origin is suitable for yeast, and various viral origins (eg, SV40, polyoma, adenovirus) are suitable for cloning vectors in mammalian cells. Useful. In general, an origin of replication element is not required for mammalian expression vectors, except when used in mammalian cells suitable for high level DNA replication, such as COS cells.

  Most expression vectors are shuttle vectors, that is, they can replicate in at least one class of organisms, but can be transfected into another class of organisms for expression. For example, a vector can be cloned into E. coli and transfected into yeast or mammalian cells, even if it cannot replicate independently of the host cell chromosome. DNA can also be replicated by insertion into the host genome. However, since restriction enzyme digestion is required to cleave the nucleic acid, recovery of genomic DNA is more complex than recovery of exogenously replicated vectors. DNA can be amplified by PCR and can be directly transfected into non-replicating host cells.

  Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the desired nucleic acid. Such a promoter may be an inducible promoter or a constitutive promoter. A promoter is operably linked to a nucleic acid by removing the promoter from the source DNA and inserting the isolated promoter sequence into the vector. Native promoter sequences and multiple heterologous promoters can be used to direct amplification and / or expression of nucleic acids encoding the peptide. The term “functionally coupled” refers to a proximal relationship that allows the elements to perform their functions as intended. A regulatory sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is performed under conditions compatible with the regulatory sequences.

  Gene transcription from vectors in mammalian hosts can be viral genomes such as polyoma virus, adenovirus, infectious epithelioma, bovine papilloma virus, avian sarcoma virus, cytomegalovirus (CMV), retrovirus and simian virus 40 (SV40). It can be regulated by a promoter derived from a source, an actin promoter or a very strong promoter, eg, a heterologous mammalian promoter such as a ribosomal protein promoter, as well as a promoter normally associated with an immunoglobulin sequence.

  Transcription of nucleic acids by higher eukaryotes can be increased by inserting an enhancer sequence into the vector. Enhancers are relatively direction and position independent. Numerous enhancer sequences from mammalian genes (eg, elastase and globin) are well known. Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the late side of the replication origin (the SV40 enhancer from bp 100 to 270 and the CMV early promoter enhancer. The enhancer can be spliced into a vector at the 5 'or 3' position of the desired nucleic acid, but preferably 5 'of the promoter. 'Located at the site.

  Advantageously, the eukaryotic expression vector may comprise a locus regulatory region (LCR). LCR is capable of inducing high level integration site independent expression of transgenes integrated into host cell chromatin, particularly for permanently transfected eukaryotic cell lines in which chromosomal integration of the vector has occurred. This is particularly important if is to be expressed.

  Eukaryotic expression vectors also contain sequences necessary for transcription termination and sequences necessary to stabilize mRNA. Such sequences are usually available from the 5 'and 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments that are transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the LIM2 inhibitor according to the invention.

  Expression vectors that provide for the transient expression of nucleic acids in mammalian cells are particularly useful in the practice of the invention. Transient expression usually uses an expression vector that can be efficiently replicated in the host cell so that the host cell accumulates multiple copies of the expression vector and then synthesizes a high level of the desired gene product. Related to.

(Ciiib) Direct delivery to cells LIM2 inhibitors can be introduced directly into cells by microinjection or delivery using vesicles such as liposomes that can be fused to the cell membrane. Viral membrane fusogenic peptides are advantageously used to facilitate membrane fusion and delivery to the cytoplasm of cells for subsequent translocation to the nucleus.

(Ciiib1) Nuclear localization signal The nuclear localization signal is the signal responsible for targeting the antigen to the nucleus. Such target sequences are described in Baker et al. , 1996, Biol Rev Cam Philos Soc 71, 637-702. Nuclear localization sequences include the binary nuclei exemplified by the SV40 large T antigen consensus sequence PKKKRV (reviewed in Dingwall, et al., 1991, Trends Biochem. Sci. 16, 478-481) or the nucleoplasmin protein. Bipartite nuclear localization (Dingwall, et al., 1987, EMBO J. 6, 69-74; Robbins, et al., 1991, Cell 64, 615-623). The skilled person is aware of other nuclear localization signals suitable for use in targeting the peptides according to the invention to the cell nucleus.

  Antibodies can be introduced directly into cells by microinjection or delivery using vesicles such as liposomes that can be fused to cell membranes. Viral fusion peptides are advantageously used to facilitate membrane fusion and delivery of cells to the cytoplasm.

  Preferably, the anti-LM02 antibody according to the invention is fused or bound to a domain or sequence of a protein responsible for cell membrane transport activity. Preferred transport domains and sequences include the domains and sequences of HIV-1-transactivating protein (Tat), Drosophila antennapedia homodomain protein, and herpes simplex-1 virus VP22 protein. By this means, the anti-LM02 antibody according to the invention can enter the cell or its nucleus when introduced in the vicinity of the cell.

  The exogenously added HIV-1-transactivating protein (Tat) can cross the cell membrane to reach the nucleus and transactivate the viral genome. The transport activity was determined by amino acid 37-72 of HIV-Tat (Fawell et al., 1994, Proc. Natl. Acad. Sci. USA 91, 664-668), 37-62 (Anderson et al.,). 1993, Biochem. Biophys. Res. Commun. 194, 876-884) and 49-58 (having the base sequence RKKRRQRRR). Vives et al. (1997), J. et al. Biol Chem 272, 16010-7 identified a sequence consisting of amino acids 48-60 (CGRKKRRQRRRPPPQC) that appears to be important for transport, nuclear localization and transactivation of cellular genes. Intraperitoneal injection of a fusion protein consisting of β-galactosidase and HIV-TAT protein transfection domain delivers biologically active fusion protein to all mouse tissues (Schwarze et al., 1999, Science 285, 1569). -72).

  The third helix of the Drosophila antennapedia homodomain protein has also been shown to have similar properties (reviewed in Prochiantz, A., 1999, Ann NY Acad Sci, 886, 172-9). The domain responsible for the transport of Antennapedia is localized to a peptide of amino acid 16 length rich in basic amino acids having the sequence RQKIKIWFQNRRMKWKK (Derossi, et al., 1994, J Biol Chem, 269, 10444- 50). This peptide has been used to induce biologically active substances in the cytoplasm and nucleus of cells in culture (Theodore, et al., 1995, J. Neurosci 15, 7158-7167). The intracellular translocation of the third helix of the antennapedia homodomain appears to be receptor-independent, and the transport process has been implicated in direct interaction with membrane phospholipids (Derossi et al.,). 1996, J Biol Chem, 271, 18188-93).

Herpes simplex virus VP22 tegument protein is capable of cell-to-cell transport, and VP22 protein expressed in a subpopulation of cells spreads to other cells in the population (Eliot and O'Hare, 1997, Cell 88, 223-33). ). GFP
(Elliot and O'Hare, 1999, Gene Ther 6,1
49-51), a thymidine kinase protein (Dilber et al., 1999, Gene Ther 6,12-21) or p53 (Phelan et al., 1998, Nat Biotechnol 16, 440-3) and VP22 are In this way, it is targeted to cells.

  Specific domains or sequences of proteins that can be transported across the nucleus and / or cell membrane can be identified by mutagenesis or deletion studies. Alternatively, a synthetic or expressed peptide having a candidate sequence can be bound to a reporter and assayed for transport. For example, Vives et al. (1997), J Biol Chem 272, 16010-7, allows synthetic peptides to be bound to fluorescein and transport monitored by fluorescence microscopy. Alternatively, green fluorescent protein can be used as a reporter (Phelan et al., 1998, Nat Biotechnol 16, 440-3).

  Any of the domains or sequences described above or identified as having transport activity can be used to induce immunoglobulins in the cytoplasm or nucleus of a cell. The above antennapedia peptide, also known as penetratin, is preferred as is HIV Tat. The transit peptide can be fused at the N-terminus or C-terminus to a single domain immunoglobulin according to the present invention. N-terminal fusion is preferred.

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

(D) Use of LIM2 inhibitor / anti-LM02 reagent according to the present invention.
In yet another aspect of the invention, there is provided a LIM2 inhibitor according to the invention or a composition comprising it for use in medicine.

  In yet another aspect, the present invention prevents and prevents one or more conditions selected from the group consisting of tumorigenesis, tumor metastasis, LMO2-mediated T cell leukemia, inflammation, ischemia, diabetic retinopathy and wound healing. There is provided the use of one or more LIM2 inhibitors according to the present invention in the manufacture of a medicament for treatment.

  In yet another aspect, the invention provides tumor formation, tumor metastasis, LMO2-mediated T cell leukemia, inflammation, comprising administering one or more LIM2 inhibitors according to the invention to a patient in need of treatment. Methods are provided for preventing and / or treating one or more conditions selected from the group consisting of ischemia, diabetic retinopathy and wound healing.

  The anti-LM02 reagent / LIM2 inhibitor according to the present invention can be used for in vivo therapeutic and prophylactic applications, in vitro and in vivo diagnostic applications, in vitro assays and reagent applications, functional genomics applications and the like.

  LMO2 functions by protein interactions in hematopoiesis, angiogenesis and leukemia induction. Thus, inhibitors of LMO2 are research tools for preventing and / or treating angiogenesis for the former study, in other conditions where tumor growth or vascular remodeling is important, and in treating certain leukemias It becomes. The anti-LM02 peptide aptamers described herein can be used in any of these various situations.

  The therapeutic and prophylactic use of anti-LM02 reagents / LIM2 inhibitors and compositions according to the present invention relates to administering the above to a recipient mammal such as a human. Preferably they involve administration to the mammalian intracellular environment.

  Substantially pure peptides and / or scaffold molecules comprising them of at least 90-95% homogeneity are preferred for administration to mammals, particularly 98-99% or more when the mammal is a human. Uniformity is most preferred for pharmaceutical applications. Once partially or purified to the desired homogeneity, immunoglobulin molecules can be used diagnostically or therapeutically (including in vitro) or using methods well known to those skilled in the art to develop and perform assay procedures. It may be used when

In an exemplary application, the term “prevention” relates to the administration of a protective composition prior to the induction of disease. “Suppression” refers to administration of a composition after a provoking event but before clinical manifestation .

  Selected anti-LM02 / LIM2 inhibitors of the present invention disrupt protein function in vivo and typically prevent inflammatory conditions, tumorigenesis and / or metastasis, LMO2-mediated T cell leukemia, ischemia and diabetic retinopathy Find uses in prevention, suppression or treatment. Anti-LM02 reagents / LIM2 inhibitors have also found use in wound healing and / or regulation of menstruation.

  Anti-LM02 reagent / LIM2 inhibitor according to the present invention, in particular anti-LM02 peptide / LIM2 inhibitor peptide or anti-LM02 intracellular antibody (N and / or C terminus restricted by unbound form or scaffold molecule referred to herein) Advantageously) further comprises a nuclear localization signal. Such molecules are advantageously used as therapeutic agents in treating one or more conditions referred to above.

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

  In general, the selected anti-LM02 reagent / LIM2 inhibitor of the present invention is used in pure form with a pharmacologically suitable carrier. These carriers typically include aqueous or alcohol / aqueous solutions, emulsions or suspensions, both of which contain saline and / or buffered media. Parenteral vehicles include sodium chloride solution, Ringer dextrose, dextrose and sodium chloride, and lactated Ringer. Where necessary to maintain the polypeptide complex in suspension, suitable physiologically acceptable auxiliaries are selected from thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates. be able to.

  Intravenous vehicles include fluid and nutrient replenishers such as Ringer's dextrose-based and electrolyte replenishers. Other additives such as preservatives and antibacterial agents, antioxidants, chelating agents and inert gases may also be included (Mack (1982) Remington's Pharmaceutical Sciences, 16th edition).

  The selected inhibitors of the present invention may be used as separately administered compositions or in combination with other agents. These can include various immunotherapeutic agents such as cyclosporine, methotrexate, adriamycin or cisplatin and immunotoxins. Pharmaceutical compositions may include “cocktails” in which various cytotoxic agents or other agents are combined with an anti-LM02 reagent / LIM2 inhibitor according to the present invention or, optionally, a selected inhibitor combination according to the present invention. it can.

  The route of administration of the pharmaceutical composition according to the present invention may be any of those well known to those skilled in the art. For treatment, selected inhibitor compositions according to the present invention can be administered to any patient by standard techniques. Administration may be by any suitable mode including parenteral, intravenous, intramuscular, intraperitoneal, transdermal, pulmonary route or direct injection using a catheter as appropriate. The dosage and frequency of administration will depend on the patient's age, sex and condition, co-administration of other drugs, contraindications and other parameters that the clinician should consider.

  Selected inhibitors of the present invention can be lyophilized for storage and reconstituted with a suitable carrier at the time of use. Well known lyophilization and reconstitution techniques can be used. It is understood by those skilled in the art that lyophilization and reconstitution can cause varying degrees of loss of functional activity and that the level of use may need to be adjusted upwards for correction.

  Compositions or cocktails thereof containing selected anti-LM02 reagents / LIM2 inhibitors of the present invention can be administered for prophylactic and / or therapeutic treatments. An amount sufficient to effect at least partial inhibition, suppression, modulation, killing or some other measurable parameter of a selected cell population in a therapeutic application is defined as a “therapeutically effective amount” Is done. The amount necessary to achieve this dose depends on the severity of the disease and the general condition of the patient's own immune system, but generally 0.005-5.0 mg of the selected immunoglobulin per kilogram body weight A dose of 0.05 to 2.0 mg / kg / dose is more commonly used. For prophylactic applications, compositions containing selected inhibitor molecules of the present invention or cocktails thereof may be administered in about the same or slightly lower amounts.

  Compositions containing one or more selected anti-LM02 reagent / LIM2 inhibitor molecules according to the present invention are prophylactic and in order to help alter, inactivate, kill or eliminate selected target cell populations of mammals. Can be used for therapeutic situations. Also, the selected repertoire of polypeptides described herein can be used in vitro to selectively kill, delete, or effectively remove target cell populations from a heterogeneous collection of cells. Or it can be used in vitro.

  The invention is described by the following examples, which do not limit the invention.

Example 1: Materials and Methods Screening of LMO2 Peptide Library Using Yeast Two-Hybrid Assay Since the library vector contained the TRP1 gene as a yeast selection marker, selection of yeast TRP1 that is resistant in tryptophan-deficient medium A truncated mouse LMO2 (aa 28-150) was fused to a modified LexA DNA binding domain of the pBTM116 vector in which the marker was replaced with a LEU gene that was resistant in leucine-deficient medium. Using this LexA-LM02 construct as a bait, using the L40 yeast strain, the protocol described in ³⁹ , 3 × 10 ⁶ transformants ³⁵ (kindly Dr. R. Screened by Brent). In this library containing more than 10 ⁹ members, the active site loop of E. coli thioredoxin (Trx) is used as a scaffold displaying the 20mer peptide.

Mammalian two-hybrid assay The yeast Trx-peptide sequence was subcloned into the mammalian VP16 vector pMVN (Clontech) as a fusion with the VP16 activation domain, while the LMO2 bait was fused to the GAL4 DNA binding domain of the pM vector ⁴⁰ . The interaction of LMO2 and peptide was tested in Chinese hamster ovary (CHO) cells. CHO cells were grown in αMEM supplemented with 10% FCS, penicillin and streptomycin. CHO cells were seeded in 6 well plates (3 × 10 ⁵ cells per well) 16-24 hours prior to transfection. Transfection was performed using 10 μl of polyfect transfection reagent (Qiagen), 500 ng each of pM and pVP16 vectors, 500 ng of pG5 luciferase reporter vector, and 50 ng of pRL-CMV (Dual-Luciferase Reporter Assay System, Promega). Cells were harvested 24-36 hours after transfection and a luciferase activity assay was performed by the manufacturer. Three good peptides were subcloned into the pM bait vector as fusions with the GAL4 DNA binding domain and tested for interactions with different portions of LMO2, other LIM-only proteins and other zinc finger proteins.

Generation of ES Cell Lines Expressing Anti-LM02 Peptide Aptamers KZ26 and H16 cell lines have been previously described ²⁰ , LMO2 +/− (one of the alleles of LMO2 has the lacZ gene knocked in) or LMO2−, respectively. /-. AF6 and BB4 cell lines were generated by knocking in Trx207 or Trx, respectively, to one allele of LMO2 of the wild type ES cell line CCB. The 6B23 cell line is a subclone of AF6 in which the CMV-Trx207 expression clone is stably transfected, whereas the G1 and A5 cell lines are stably transfected with EFα-Trx207 and EFα-Trx, respectively. Made from KZ26.

Embryoid Body Differentiation and Erythropoiesis Assay Different in vitro differentiation of ES clones into embryoid bodies (EBs) uses recombinant human vascular endothelial growth factor at 10 ng / ml and mouse erythropoietin at 1 unit / ml. The human basic fibroblast growth factor 2 was used at 25 ng / ml and mouse interleukin 6 was used at 5 ng / ml as previously described ⁴¹ . EBs were formed after 11 days of culture, collected, separated in 1 ml DMEM containing 1.5 mg collagenase for 30 minutes, and cells were analyzed by FACS for expression of the red blood cell marker Ter119.

Example 2: Anti-LM02 peptide aptamers are homologous to elements of the LIM finger Yeast peptide aptamer libraries ^35,36 were screened in vivo using LMO2 baits where LMO2 is fused to the GAL4 DNA binding domain (DBD). . The yeast peptide aptamer library consisted of various sets of clones expressing a random peptide (20mer) incorporated into the outer loop of a bacterial thioredoxin (Trx) protein suitable for interaction between the peptide and the target protein. 250 clones were identified in the yeast screen and 15 of these were designated as specific binders after rescreening. DNA was isolated from these plasmids and sequence data was obtained (Figure 2). A striking feature of these sequences is the presence of the motif cys-xx-cys or his-xx-cys in 13 of 15 clones (Figure 2A). When these peptide aptamers were re-evaluated using a mammalian luciferase 2-hybrid transcriptional activation assay (see below, shown in FIG. 3), proteins from two clones lacking this motif are more stringent than this One assay did not interact with LMO2 bait, but the other was found to show varying activity efficiencies in the two-hybrid assay. A comparison of these derived peptide aptamer sequences, each with the level of luciferase reporter gene transcriptional activity obtained in a mammalian two-hybrid assay, is shown in FIG. 2B (the peptide is a cys / his-xx-cys motif. Aligned). The cys / his-xx-cys motif of the anti-LM02 peptide is highly homologous to a portion of the first LIM finger of the second LIM domain of LM02 (ie, his-leu-glu-cys) Is important (FIG. 2B).

  The homology of the anti-LM02 peptide aptamer extends outside the cys / his-xx-cys motif. Comparison of various anti-LM02 peptide aptamers (FIG. 2C) shows that the conserved region of the cys / his-xx-cys motif extends several residues on either side of this region and generally It shows that there are two hydrophobic residues on the N-terminal side and one hydrophobic residue on the C-terminal side. More often, it is important that the charged amino acid is present as the second of the two amino acids in the center of cys / his-xx-cys (FIG. 2C), which is characteristic of a corresponding region of the LMO2 LIM2 domain (FIG. 2B). Finally, the val-tyr dipeptide flanking LMO2 LIM2cys / his-xx-cys (the complete element of the LMO2 LIM2 domain is val-tyr-his-leu-glu-cys-phe) is a peptide aptamer 2 Are not conserved in the two that show the greatest interaction with LMO2 (ie, 207 and 209, FIG. 2B). In general, the conserved regions of anti-LM02 peptide aptamers are hydrophobic-hydrophobic-cys-x-charged-cys-hydrophobic residues (FIG. 2C).

Example 3: Anti-LM02 peptide aptamer specifically binds to the second LIM domain of LM02 An anti-LM02 peptide aptamer was isolated from a random peptide library expressed in yeast. The activity and specificity of the interaction with LMO2 was checked in a mammalian assay based on the activity of luciferase reported after the interaction of the anti-LM02 peptide aptamer with the LMO2 protein bait expressed in CHO cells. The activity level results are summarized in FIG. 2B and observed after co-transfection of a clone expressing the DBD-Trx aptamer fusion (designated bait) and a clone expressing LMO2 fused to the VP16 transcriptional activation domain (designated prey). The activity of each peptide aptamer expressed as a fold increase in luciferase activity is shown. The four aptamers that produced the highest levels of activity were made from clones Trx207, 209, 85 and 81.

Various baits, including GAL4 DBD fused to Trx-anti-LM02 peptide aptamer 207 (FIG. 3A), 209 (FIG. 3C) and 85 (FIG. 3D) or anti-LM02 peptide aptamer 207 without Trx scaffold, are VP16 transcription The specificity of binding by anti-LM02 peptide aptamers was evaluated in a mammalian reporter assay that was coexpressed with various target proteins fused to the transactivation domain. These “plays” included LMO2, the individual LIM domains of LMO2 (ie, LIM1 or LIM2), the related LIM-only proteins LMO1 and LMO4, LMO interacting proteins LDB1 and GATA1 (FIG. 3). Anti-LM02 peptide aptamers that bind only to LMO2 or to the second LIM domain of LMO2 (LIM2) can interact slightly with this protein, but Trx207 does not show significant binding to GATA1, Trx209 and 85 The present inventors observed that. In all three cases, binding of LMO2 to the isolated LIM2 domain was much better than binding to intact LMO2. Furthermore, since the anti-LM02 peptide aptamer did not show binding to the zinc finger domain found in estrogen receptor α (zinc finger type C ₄ ) or protein kinase TTK (zinc finger type C ₂ H ₂ ), the LIM domain zinc Specific to fingers.

  Data obtained using the yeast and CHO interaction assays used peptide aptamers in which both the N-terminus and C-terminus were constrained to the thioredoxin protein scaffold. Since the inventors are unaware of the exact sequence requirements for binding to LMO2, the ability of unconstrained peptides was difficult to assess at this stage of analysis. As an intermediate test, a fusion protein bait was designed in which GAL4 DBD was fused only at the N-terminus of 207 peptide. This protein was used in a CHO reporter assay with a prey protein array containing the LIM protein fused to VP16 (FIG. 3B). In this assay, we found that the unconstrained 207 peptide is equally effective in vivo in specifically binding to LMO2, and that this peptide binds to the complete LMO2 protein. It was found to bind to the LIM2 domain of LMO2 more efficiently. These data indicate that the 207 peptide has a sequence that specifically binds to the LIM2 domain region unrelated to structural constraints within Trx.

Example 4: Anti-LM02 peptide aptamer inhibits LM02 protein function.
LMO2 hematopoietic, a protein interaction module that function by protein interactions in each of the functional situations involving angiogenesis and leukemogenesis ^{37 15 and 17.} Possible use of anti-LM02 peptide aptamers that inhibit LMO2 function in an LMO2-dependent in vitro culture assay using differentiation of embryonic stem (ES) cells to erythroid types, well known to be LMO2-dependent ¹⁹ When wild-type ES cells differentiate (FIG. 4A, CCB), about 20-25% of cells differentiate into red blood cells (measured by expression of the red blood cell surface protein Ter119), and approximately the same number indicates that LMO2 +/− ES cells It was obtained using (Fig. 4A, ES clone KZ26 ^20,26). On the other hand, as previously described ²⁰ , no LMO2 mutant ES cells did not undergo erythropoiesis (FIG. 4A, ES clone H16, LMO2 − / −). Under the control of the endogenous LMO2 transcription promoter (LMO2 gene knock-in, FIG. 4A, ES clone AF6) or this LMO2 gene knock-in plus a transgene expressing LMO2 of the CMV promoter (FIG. 4A, ES clone 6B23) or EFα promoter When the Trx207 anti-LM02 peptide aptamer was expressed in differentiating ES cells under the control of a transgene expressing Trx207 (FIG. 4A, ES clone G1), a decrease in the number of red blood cells occurred. There was no loss of erythroid differentiation in control ES clones expressing Trx without peptide insertion (FIG. 4A, ES clones BB4 and A5). Thus, ES clones expressing the Trx207 fusion show inhibition of erythroid colony formation up to about 50-70% of the wild type level (FIG. 4B), which requires the presence of a 207 peptide aptamer. Therefore, the specific absence of erythropoiesis in ES cells expressing anti-LM02 peptide aptamer 207 indicates inhibition of LMO2 function, which is mediated by the peptide sequence represented by 207 peptide. This is shown (FIG. 4).

Example 5: Anti-LM02 antibody (designated anti-LM02 # 14) can block angiogenesis when developing mouse embryos.
Results and Discussion Anti-LMO2 intracellular antibodies were isolated by intracellular capture methods (Tanaka et al., 2003; Tse et al., 2002a; Tse et al., 2002b; Vistin et al., 1999). One intracellular antibody (anti-LM02 # 14) specifically binds to multiple LMO2 LIM domains in a mammalian cell reporter assay (CHN & THR, unpublished). We wanted to determine if anti-LM02 # 14 can interfere with the biological function of LM02. Initially, we used an in vitro ES cell assay that included differentiation of ES into red blood cells or early endothelial cells in culture. As described (Keller et al., 1993), ES cells were transfected with clones expressing anti-LM02 # 14 and these cells and control untransfected ES cells were subjected to growth factor dependent differentiation. Was generated. After the required culture period, the cells were examined for expression of the surface markers Ter119 (erythrocyte-specific surface protein) or CD31 (Pecam, pan-endothelial protein). The presence of Ter119 indicates erythroid differentiation, and the presence of CD31 indicates de novo formation (angiogenesis) of vascular endothelial cells. Angiogenesis does not occur in ES cell cultures.

  FIG. 5 shows the results of in vitro differentiation of ES cells into erythroid lineages. The lacZ gene is knocked in to one allele of Lmo2, leaving it unexpressed for Lmo2 expression but allowing β-galactosidase expression in the control of the Lmo2 promoter (acting as a reporter of Lmo2 expression) The established ES line subclone KZ26 and normal ES cells (CCB) showed almost the same level of differentiation into red blood cells (Yamada et al., 1998). On the other hand, ES cells having a homozygous null mutation of Lmo2 do not undergo erythropoiesis (clone H16) (Yamada et al., 1998), and ES clones that express anti-LM02 peptide aptamers have severe erythropoiesis potential. Inhibited (clone 6B23). The effect of expressing anti-LM02 antibody # 14 in ES cells is to produce the same effect as a null mutation of the Lmo2 gene. ES clone 3A expresses anti-LM02 # 14 under the control of the pEF-BOS promoter. When growth cells were used to induce these cells to differentiate during culture, very low levels (-10% of control) of Ter119-erythroid colonies were seen. We conclude that anti-LM02 # 14 intracellular antibody inhibits Lmo2 function in erythropoiesis.

  As a control for the specificity of anti-LMO2 # 14 inhibition of Lmo2 function in the ES cell assay, we evaluated the development of cells expressing CD31 (Pecam) in the ES differentiation assay. These cells form during the angiogenesis process of ES cells that are less affected by the Lmo2 null mutation (Yamada et al., 2000) (FIG. 6, ES clone H16). Similar levels of CD31 + cells were observed with ES clone KZ26 (heterozygous for Lmo2 gene), 6B23 (expressing anti-Lmo2 aptamer) or anti-LM02 # 14 intracellular antibody 3A. Thus, the data in FIGS. 5 and 6 show that anti-LM02 # 14 intracellular antibodies can affect Lmo2 function in erythropoiesis but not de novo angiogenesis (Gene). The results are equivalent to those defining Lmo2 function in targeting studies (Warren et al., 1994; Yamada et al., 1998; Yamada et al., 2000).

  Lmo2 has some effect on remodeling of vascular endothelium from existing blood vessels (angiogenesis), but has no effect on de novo development of capillaries (angiogenesis) (Yamada et al., 2000). Thus, this function of LM02 is a therapeutic target for clinical signs associated with angiogenesis. To demonstrate that anti-LM02 # 14 intracellular antibodies may be able to block angiogenesis, an angiogenesis-based assay in mouse embryos was used (Yamada et al., 2000). ES cells injected into donor blastocysts and introduced into pseudopregnant recipients generated chimeric embryos, and a substantial proportion of cells were derived from the injected ES cells. When KZ26 ES cells (heterozygous for Lmo2-lacZ knock-in) are injected into blastocysts and the embryos are stained for β-galactosidase activity (specifically blue in Lmo2-expressing cells), we develop Staining was observed in the middle vessel (Yamada et al., 2000). FIG. 7 shows an embryonic day E10.5 chimeric embryo specimen in which endothelial cells of the vascular being remodeled are stained blue due to Lmo2 gene promoter expression (left panel). Similar analysis of chimeric embryos generated using ES cells with a Lmo2 null mutation did not show blue angiogenesis due to failure of capillary network angiogenesis (remodeling) in the absence of Lmo2 ( FIG. 7, right panel). The same findings were obtained when chimeric embryos were generated using ES cells expressing anti-Lmo2 intracellular antibody (FIG. 7, middle panel). Since this intracellular antibody binds to Lmo2 protein in vivo (CHN & THR, unpublished) and that it blocks LMO2 function in ES cell assays (FIGS. 5 and 6), anti-Lmo2 intracellular antibody We concluded that angiogenesis is prevented by binding to Lmo2 and preventing the protein from exerting its function in vascular endothelial cells.

  Our results indicate that anti-LM02 intracellular antibodies (including scFv antibody fragments) can interfere with LMO2 function in the angiogenic segment of angiogenesis, but do not affect de novo vascular endothelial cell formation. Show. The data presented can be inferred for normal situations such as wound healing or other in vivo situations where angiogenesis occurs such as clinical signs such as cancer using a chimeric embryo assay of Lmo2 function in angiogenesis. Using this anti-LM02 intracellular antibody, derivatives thereof or other agents that act similarly (eg, small chemicals derived based on knowledge of anti-LM02 intracellular binding sites) Suggests an important way to manage angiogenesis in various diseases.

  All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method 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 is to be understood that the claimed invention 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. ing.

[Reference]

LIM domain: indicates structure and function. An interesting aspect of LMO2 function is that this small molecule (amino acid 156) may have such a major effect. This is partially explained by ⁵ the structure comprising two LIM domains (B), LIM domain (B) are each themselves comprising two zinc-binding LIM finger, one zinc atom is cysteine (cys) It is coordinated between the histidine (his-) or aspartic acid (asp) residue (a) ⁴² ~ ^44. LIM domain is a protein interaction module ^{14, 15,} LM02 is a normal cell or T-cell leukemia cells of different compositions DNA binding complex ¹⁵ - seems to be the protein partner versatile play a binding action in body ^{18, 45} . The sequence of the peptide that binds to the LMO2 protein is shown. A peptide library containing the 20mer peptide fused to the Trx protein (used as a scaffold) was screened against clones expressing LMO2 bait. A clone encoding the LMO2 interacting Trx-peptide was isolated and the sequence of the peptide was obtained. The isolated peptide sequence is shown. From 3 × 10 ⁶ transformants, 15 plasmids expressing peptide aptamers that interact with LMO2 were isolated. A. Sequence of 15 peptide clones that interact with LMO2 in the yeast two-hybrid assay. Conserved cys (C) and his (H) residues are indicated by bold underlining. B. It is a sequence of 10 peptides that interact with LMO2 in a yeast two-hybrid assay and are also active in a mammalian cell two-hybrid assay using CHO cells. Conserved cysteine (C) and histidine (H) residue is indicated in bold underlined, underlined the hydrophobic residues that are conserved are indicated by the same residues, and motifs LM02 LIM2 . The folding stimulating activity of luciferase formed in a mammalian two-hybrid assay using each Trx peptide is shown in the LHS of the figure (+/− standard deviation). C. 10 is a sequence of 10 peptides that interact with LMO2. Conserved cysteine (C) and histidine (H) residues are indicated by bold underlining. The conserved short aptamer motif is shown at the top of the diagram, and the conserved hydrophobic residues are underlined. C refers to a conserved charged residue of the aptamer motif. Figure 3 shows a mammalian interaction assay of an anti-LM02 peptide aptamer. A clone expressing the GAL4 DBD-Trx-peptide (pM vector) was used as a bait to express various interactions of prey proteins (VP16 fusion vectors) co-transfected into CHO cells with luciferase reporter clones. Tested against clones. Luciferase activity was measured after 24 hours. The values shown are folding stimuli compared to the empty pM bait vector alone. The VP16 pre-clone used was vector only (pVP16 +), LMO2, LMO2 individual LMI domains (LIM1 or LIM2), LMO4, LMO1, protein kinase TTK (zinc finger type C ₂ H ₂ ), estrogen receptor α (zinc finger) type _{C 4) (VP16-ER)} , was Ldb1 or GATA1. (NB: Gal4-207 was not tested against TTK, ERα, Ldb1 and Gata1.) Mammal two-hybrid data for Trx207 Mammal two-hybrid data for 207 peptides not within the scaffold. Mammal two-hybrid data for Trx209 Mammal two-hybrid data for Trx85 2 shows erythroid differentiation of ES cell-derived embryoid bodies. Embryonic stem cells (ES) were cultured in vitro to produce erythroid differentiation, and the number of erythroid cells was determined by expression of the erythrocyte marker Ter119. ES clone is wild type (CCB); null LMO2 cell (H16); heterozygous LMO2 (lacZ gene knock-in LMO2, clone KZ26); ES cell in which Trx207 is knocked in to one allele of LMO2 (AF6) or CMV The same clone stably transfected with promoter-driven Trx207 (6B23); ES cells (BB4) in which Trx is knocked into one allele of LMO2; EFα promoter-driven Trx207 (G1) is stably transfected Heterozygous LMO2 (KZ26) or EFα promoter-driven Trx alone (A5) was stably transfected. A. Percentage of cells expressing Ter119 in isolated 11-day-old embryoid bodies. B. Percentage of inhibition of cells expressing Ter119 in isolated 11 day old embryoid bodies. Figure 5 shows in vitro differentiation of ES into erythroid lineages. 40% methylcellulose, 20% FBS, 100 U / ml penicillin, 100 μg / ml streptomycin, 100 μM MTG, 10 μg / ml human insulin, 2 mM L-glutamine, 10 ng / ml recombinant mouse VEGF, 1 U / Each ES cell was grown for 11 days in IMDM containing ml recombinant mouse Epo, 25 ng / ml recombinant human FGF and 5 ng / ml recombinant mouse IL-6. Embryoid bodies were separated into single ES cells with collagenase A for FACS analysis. FACS analysis was performed using biotinylated anti-mouse red blood cell mAb (Ter-119) and streptavidin-phycoerythrin conjugate. Each assay was performed in duplicate (indicated by black or white squares). De novo generation (angiogenesis) of vascular endothelial cells in the process of ES cell differentiation. Each ES cell was grown as described in FIG. Embryoid bodies were separated with collagenase A and FACS analysis was performed using biotinylated anti-mouse CD31 mAb and streptavidin-phycoerythrin conjugate. Each assay was performed in duplicate (indicated by black squares or white squares). Figure 3 shows β-galactoside expression in ES cell-derived endothelial cells during angiogenesis. ES cells were injected into the inner cell population of C57BL / 6 blastocysts and implanted into female recipients. On day 10.5, embryos were isolated and stained with Xgal as described (Yamada et al., 1998) to obtain full-length photographs. The amino acid sequence of scFv (anti-LM02 antibody) that binds to the LIM2 domain of LMO2 and inhibits LMO2 functional activity is shown. V _H CDRs are underlined and shaded and referred to as (a), (b) and (c). The V _L CDR shades and is referred to as (a), (b), (c). The sequence of the Gly-Ser linker is underlined. The sequence of scFv is referred to as SEQ ID NO: 12.

Claims (30)

  A LIM2 inhibitor capable of binding to the LIM2 domain of LMO2 and inhibiting the functional activity of LMO2. Peptide, consensus sequence:
His / CXXXC
(Wherein C is a zinc bond Cys;
His stands for histidine,
X is any amino acid)
The LIM2 inhibitor according to claim 1, comprising: Peptide, consensus sequence:
HhHis / CXX ¹ Ch
(Wherein h represents a hydrophobic amino acid,
His stands for histidine,
C represents cysteine,
X represents any amino acid, and X ¹ represents a charged amino acid)
The LIM2 inhibitor according to claim 2, comprising:   The LIM2 inhibitor according to claim 2 or 3, which is a peptide aptamer.   The LIM2 inhibitor according to claim 3 or 4, wherein the peptide is restricted within the scaffold.   The LIM2 inhibitor according to any one of claims 2 to 5, wherein the peptide is constrained in the scaffold at least at the N-terminus of the peptide.   The LIM2 inhibitor according to any one of claims 2 to 5, wherein the peptide is constrained in the scaffold at least at the C-terminus of the peptide.   The LIM2 inhibitor according to any one of claims 5 to 7, wherein the peptide is constrained within a scaffold of thioredoxin protein (Trx).   Peptide aptamer consisting of clone 207, clone 209, clone 85, clone 81, clone 63, clone 72, clone 247, clone 90, clone 69 and clone 202 shown in FIG. The LIM2 inhibitor according to any one of claims 1 to 8, which is selected from the group.   The LIM2 inhibitor according to claim 9, which is selected from the group consisting of peptide aptamers consisting of clone 207, clone 209, and clone 85 shown in Fig. 2 and designated in SEQ ID NOs: 2, 3, 4 respectively.   The LIM2 inhibitor according to claim 1, which is an antibody.   12. The LIM2 antibody inhibitor of claim 11, comprising at least one CDR having the amino acid sequence depicted in FIG. 8 and specified in SEQ ID NOs: 12 (a), 12 (b) and 12 (c).   9. All three CDRs having the amino acid sequences shown in FIG. 8 and designated respectively in SEQ ID NOs: 12 (a), 12 (b) and 12 (c), together form an antigen binding site. 12. The anti-LM02 antibody according to 12.   The anti-LM02 antibody (LIM2 inhibitor) according to any one of claims 11 to 13, which is scFv or dAb.   The anti-LM02 antibody (LIM2 inhibitor) according to claim 14, which is scFv.   The anti-LM02 antibody (LIM2 inhibitor) according to any one of claims 11 to 15, which is an intracellular antibody (intracellular binding antibody).   A nucleic acid encoding the anti-LM02 antibody (LIM2 inhibitor) according to any one of claims 11 to 16.   A vector comprising the nucleic acid according to claim 17.   A host cell comprising the vector of claim 18.   The LIM2 inhibitor according to any one of claims 1 to 19, further comprising a nuclear localization signal.   21. The LIM2 inhibitor according to any one of claims 1 to 20, which is fused or conjugated to a domain or sequence of a protein responsible for cell membrane transport activity.   One wherein the domain or sequence of the protein responsible for cell membrane transport activity is selected from the group consisting of HIV-1-transactivating protein (Tat), Drosophila antennapedia homeodomain protein and herpes simplex virus type 1 VP22 protein The LIM2 inhibitor according to claim 21, which is as described above.   23. A composition comprising one or more LIM2 inhibitors according to any one of claims 1-22 and a pharmaceutically acceptable carrier, diluent or excipient.   Use of the LIM2 inhibitor according to any one of claims 1 to 19 or the composition according to claim 20 for inhibiting the functional activity of LMO2.   Use of a LIM2 inhibitor according to any one of claims 1 to 19 or a composition according to claim 20 for the manufacture of a medicament for inhibiting the functional activity of LMO2.   24. A LIM2 inhibitor according to any one of claims 1 to 16 or a composition according to claim 23 for use in medicine.   Claims in the manufacture of a medicament for preventing and / or treating one or more conditions selected from the group consisting of tumorigenesis, tumor metastasis, inflammation, LMO2-mediated T cell leukemia and diabetic retinopathy 24. Use of one or more LIM2 inhibitors according to any one of 1 to 16 or a composition according to claim 23.   17. Tumor formation, tumor metastasis, LMO2-mediated T cell leukemia, inflammation, imagination, comprising administering one or more LIM2 inhibitors according to any one of claims 1-16 to a patient in need of treatment. A method for preventing and / or treating any one or more conditions selected from the group consisting of blood, diabetic retinopathy.   28. Use according to claim 26 or method according to claim 27, wherein the condition is tumorigenesis and / or tumor metastasis. 28. The use according to claim 26 or the method according to claim 27, wherein the condition is LM02 T cell mediated leukemia.

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