The present invention relates to chimeric L. monocytogens InIB proteins and their uses in the treatment of injury to the skin, lung, and heart. Federal Support

The present invention arose in part from research funded by NIH/NHLBI ROl HL 073929. The United States government may have certain rights in the invention.

Related Applications

This application claims priority to U.S. Provisional Application 61/122,055 (filed on December 12, 2008) which is incorporated by reference in its entirety.

Background of the Invention

Mechanisms of epithelial and endothelial repair have been the focus of research for skin, lung, liver, kidney, and heart. One of the primary repair pathways involves the up- regulation and release of hepatocyte growth factor (HGF), which activates its receptor c-Met on epithelial and endothelial cells thereby inducing cell survival, cell growth, motility, and morphogenesis. The HGF signaling pathway has been identified as a primary mechanism for normal tissue repair following injury, for tissue maintenance when challenged with toxins, and for regeneration of normal tissue in aging. HGF and its naturally occurring isoforms have been patented for tissue repair processes and for protection of tissues from damage, such as chemotherapeutic agents, in vivo, and for improved regeneration of tissues in situ.

HGF plays a key role in tissue homeostasis and repair in many organs including lung, heart, kidney, liver, and skin. HGF is a pleotropic factor, inducing proliferation, motility and morphogenesis in a cell-type specific manner (Rubin et al. (1993) Biochim. Biophys. Acta 1155: 357-371). HGF, and its receptor c-Met, are required for embryogenesis and organogenesis (Amano et al. (2002) Dev Dyn 223 : 169-179; Birchmeier C and Gherardi E

(1998) Trends Cell Biol. 8: 404-410; Matsumoto et al. (1996) Cancer Chemother. Pharmacol. 38 Suppl: S42-47; Santos et al. (1994) Dev. Biol. 163: 525-529). Knockout mice for HGF or c-Met die in utero from defects in development of the liver and the placenta (Ponzetto et al. (2000) Int. J. Dev. Biol. 44: 645-653). In adults, HGF is involved in tissue homeostasis, especially for cell survival and maintenance of epithelial and endothelial cells in a variety of tissues (Fritsch et al. (1999) Exp. Cell. Res. 248: 391-40; Kagoshima et al. (1992) Eur. J. Biochem. 210: 375-380). The expression and release of HGF from the extracellular matrix are also induced by tissue injury, and HGF plays a key role in tissue repair in lung, liver, kidney, heart, skin, some neurons and hematopoietic progenitor cells (Huh et al. (2004) PNAS 101 : 4477-4482; Kono et al. (1992) 186: 991-998; Maina et al. (1997) Genes & Dev. 11 :

3341-3350; Matsumoto et al. (1992) Crit. Rev. Oncol. 3: 27-54; Matsumoto et al. (1997) Ciba Foundation Symposium 212: 198-211 ; Matsumoto et al. (1993) fe 65: 225-249; Mizuno et al. (1993) Biochem. Biophys. Res. Commun. 194: 178-186; Ohmichi et al. (1992) Am. J. of. Phys 270: L1031-1039; Yanagita et al. (1992) Biochem. Biophys. Res. Commun. 182: 802- 80). HGF has also been shown to be a potent stimulator of growth in culture for a variety of primary cells (Mason et al. (1994) Am J Respir Cell MoI Biol 11 : 561-567; Nakagami e? α/. (2001) Hypertension 37: 581-586; Shimaoka e? α/.(1995) J. Cell. Physiol. 65: 333-338; Takahashi et al. (1995) J. Clin. Invest. 95: 1994-2003; Vila et al. (1995) Lab. Invest. 73: 409- 418). HGF given either exogenously as a protein or via gene therapy mitigates a variety of diseases in liver, kidney, heart, brain, and lung.

HGF activity, signaling through the c-Met receptor, has been shown to mitigate a variety of diseases in liver, kidney, heart, brain, and lung and to increase tissue repair in response to injury in these tissues. HGF also induces normal tissue repair in a mechanism which restores normal tissue structure and prevents the development of extensive scar formation or fibrosis. Scarification, or fibrosis, involves in loss of normal architecture reducing the functional capacity of the organ and has adverse effects on the tissue as a whole. Additionally, aberrant repair processes can become progressive and result in total organ failure. Factors which stimulate normal cellular repair and inhibit apoptosis of normal cells may increase tissue repair from injury and may reduce or inhibit scarification and fibrosis. HGF is a heterodimeric protein containing 20 disulfide bonds in an N-terminal hairpin, four kringle domains, and a serine protease-like domain. HGF is synthesized as a 90 kDa peptide chain containing an N-terminal hairpin, four kringle domains, and a serine protease-like domain. HGF is proteolytically activated by cleavage at R494, resulting in the formation of a 60 kDa heavy chain and a 34 kDa light chain, joined by a disulfide bond (Rubin et al. (1993) Biochim. Biophys. Acta 1155: 357-371). The 20 disulfide bonds in HGF are required for the correct conformation and activity of the protein (Rubing et al. 1993). HGF activation of c-Met, a tyrosine kinase receptor with a single transmembrane domain, results in activation of multiple downstream signaling pathways, most notably p42/p44 MAPK and PI3K/Akt. It has been shown that the combination of these signaling events is required for motility, anti-apoptotic activity, and cell growth (Chan et al. (1993) Exs 65: 67- 79; Cioce et al. (1996) J. Biol. Chem. 271 : 13110-13115; Day et al. (1999) Oncogene 18: 3399-3406).

Despite the obvious potential therapeutic uses of HGF, the complexity of the full- length HGF structure has prevented its development as a pharmaceutical agent. Due to its complex structure, synthesis of full-length HGF has not been successfully produced in sufficient quantities for pharmaceutical use, despite its potential therapeutic uses. Attempts have been made to use smaller fragments of the HGF molecule, such as NKl or NK2, which are a naturally occurring iso forms of HGF containing only the N-terminal hairpin and the first kringle or first and second kringle domains, respectively (Chan et al. (1993) Exs 65: 67-79; Cioce et al. (1996) J. Biol. Chem. 271 : 13110-13115).

NKl and NK2 are partial agonists of the c-Met receptor (Chioce et al. (1996); Day et al. (1999); Rubin (2001) J. Biol. Chem. 276: 32977-32983). NK2 activation of c-Met results in cell motility in transformed cells, but not cell survival or proliferation (Day et al. (1999)). NKl can induce motility, survival and proliferation, but its affinity for c-Met is far reduced from that of the complete factor (Chioce et al. (1996)). However, NKl and NK2 produced in bacteria must still be denatured and refolded to achieve the correct conformation (Stahl et al. (1997) Biochem J. 326 (R 3): 763-772).

The genome of the pathogenic bacterium Listeria monocytogenes encodes a protein that binds the HGF receptor. The process of L. monocytogenes invasion of a host cell involves a "zipper" mechanism, in which the bacterium attaches to the host cell membrane triggering a series of intracellular signaling cascades involving actin cytoskeletal rearrangement, membrane extension of a phagocytic region, and ultimately closure of the membrane around the bacterium (Veiga et al. (2005) Nat. Cell Biol. 894-900). The internalin B protein (InIB) of Listeria monocytogenes was found to bind to and partially activate c-Met, resulting in activation of p42/p44 mitogen activated protein kinase (MAPK) (Veiga and

Cossart (2007) Cell 130: 218-219) and in some cells to also activate phosphatidylinositol 3- kinase (PI3K) (Bierne and Cossart (2002) J. Cell. ScL 115: 3357-3367; Bierne et al. (2000) Cell. Microbiol. 2: 465-476; Bosse et al. (2007) MoI. Cell. Biol. 27: 6615-6628; Ireton et al. (1996) Science 21 A: 780-782). Binding and partial signal transduction was reported with the full-length InIB protein, and expression of InIB correlated with increased invasiveness of the pathogen (Banerjee et al. (2004) MoI. Mircobiol. 52: 257-271; Ireton et al. (1996) Science 274: 780-782; Veiga and Cossart (2007) Cell 130: 218-219). Some DNA synthesis was also observed in transformed (MDCK) cells in response to the full-length InIB protein (Niemann et al. (2008) J. MoI. Biol. 311: 489-500). The structure of a protein segment (amino acids 36 to 321) of InIB in complex with a portion of the c-Met receptor has recently been determined by crystallography (Niemann et al ). This segment of InIB forms stable β-sheet conformation with no disulfide bonds, and binds to the c-Met receptor in an allosteric manner. Unlike full length InIB, this peptide was shown to have no activity for DNA synthesis in MDCK cells (Niemann et al). This segment of InIB is easily produced in standard E. coli protein synthesis systems.

The InIB protein was previously shown to bind to the hepatocyte growth factor (HGF) receptor, c-Met, allowing internalization of the bacterium into the host cell. It was also shown that the binding of Internalin B to c-Met results in a partial activation of the receptor, triggering activation of the MAPK pathway but not of other pathways shown to be required for HGF-induced cell growth. This partial activation, which has been studied only in transformed or immortalized cell types was not shown to improve cell growth or survival.

Given the importance of HGF in tissue repair and the cost and difficulty associated with producing recombinant HGF, there is great need in the art to be able to effective stimulate the pathways that are activated by HGF. Summary of the Invention

The invention encompasses chimeric proteins comprising at least a first amino acid sequence and a second amino acid sequence comprising one or more domains of InIB protein. The chimeric protein may be tandem dimer and may also have a linker peptide. The first amino acid sequence of the chimeric protein can be the N-terminal hairpin loop of HGF. In one embodiment, the chimeric protein is capable of activating p42/p44 MAPK and Akt. The invention also encompasses nucleic acids and vectors encoding the chimeric protein. In addition, the invention encompasses methods of treating skin injury, lung injury, and ischemic disease by administering a pharmaceutically effective amount of a composition containing the chimeric protein. One embodiment of the invention is a chimeric protein comprising a first amino acid sequence, a linker peptide and a second amino acid sequence comprising one or more domains of InIB protein having at least 95% sequence identity to SEQ ID NO: 1. In another embodiment, the chimeric protein is capable of activating p42/p44 MAPK and Akt. The first amino acid sequence may comprise the N-terminal hairpin loop of hepatocyte growth factor (e.g., the amino acid sequence of SEQ ID NO: 3) or one or more domains of InIB protein having at least 95% sequence identity to SEQ ID NO: 1. In some embodiments, the second amino acid sequence has at least 97%, 98%, 99% or more sequence identity to the amino acid of SEQ ID NO: 1. In some other embodiments, the chimeric protein is a tandem dimer. In some embodiments, the chimeric protein comprises at least (a) the N-terminal hairpin loop of hepatocyte growth factor (e.g., the amino acid sequence of SEQ ID NO: 3) and (b) one or more domains of InIB protein having at least 97% sequence identity to SEQ ID NO: 1, whereby the chimeric protein is capable of activating p42/p44 MAPK and Akt. In yet another embodiment, the chimeric protein comprises at least (a) the N-terminal hairpin loop of hepatocyte growth factor and (b) one or more domains of InIB protein having at least 99% sequence identity to SEQ ID NO: 1, wherein the chimeric protein is capable of activating p42/p44 MAPK and Akt. In yet another embodiment of the invention, the chimeric protein comprises at least (a) the N-terminal hairpin loop of hepatocyte growth factor and (b) one or more domains of InIB protein having at least 99% sequence identity to SEQ ID NO: 7, wherein the chimeric protein is capable of activating p42/p44 MAPK and Akt.

In another embodiment of the invention, the chimeric protein comprises the amino acid sequence of SEQ ID NO: 1 and optionally the amino acid sequence of SEQ ID NO: 3. In an alternate embodiment, the chimeric protein comprises or consists of both the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 3. In yet another embodiment of the invention, the chimeric protein comprises the amino acid sequence of SEQ ID NO: 5. In an alternate embodiment, the chimeric protein comprises the amino acid sequence of SEQ ID NO: 7 and optionally the amino acid sequence of SEQ ID NO: 3. In an alternate embodiment, the chimeric protein comprises or consists of both the amino acid sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO: 3. In yet another embodiment of the invention, the chimeric protein comprises the amino acid sequence of SEQ ID NO: 9.

The invention also encompasses nucleic acids which encode these chimeric proteins. In addition, the invention encompasses nucleic acid molecules which specifically hybridize under stringent conditions to the nucleic acid molecules encoding these chimeric proteins.

The nucleic acid molecules may be inserted into a vector or a host cell. The host cell may be a prokaryote or eukaryote.

One embodiment of the invention is a nucleic acid molecule comprising or consisting of the nucleotide sequence of SEQ ID NO: 2 and optionally the nucleotide sequence of SEQ ID NO: 4. Another embodiment of the invention is a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 6.

Another embodiment of the invention is a nucleic acid molecule comprising or consisting of the nucleotide sequence of SEQ ID NO: 8 and optionally the nucleotide sequence of SEQ ID NO: 4. Another embodiment of the invention is a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 10. The invention also encompasses pharmaceutical compositions containing the chimeric proteins and methods of treating skin injury, lung injury, and ischemic disease. The methods of treating skin injury, lung injury, and ischemic disease encompass administration of a pharmaceutically effective amount of a pharmaceutical composition comprising chimeric proteins of the invention.

One embodiment is a method of treating skin injury comprising administration to a subject in need thereof of a pharmaceutically effective amount of a pharmaceutical composition containing a chimeric protein comprising one or more domains of InIB protein (e.g., a protein comprising the amino acid sequence of SEQ ID NO: 1 or a protein comprising the amino acid sequence of SEQ ID NO: 7). In one embodiment, the protein is capable of activating p42/p44 MAPK and Akt. In another embodiment of the invention, the protein is a tandem dimer. The skin injury may be a dermal burn, cut or combined injury.

Another embodiment is a method of treating lung injury comprising administration to a subject in need thereof of a pharmaceutically effective amount of a pharmaceutical composition containing a chimeric protein comprising one or more domains of InIB protein (e.g., a protein comprising the amino acid sequence of SEQ ID NO: 1 or a protein comprising the amino acid sequence of SEQ ID NO: 7). In another embodiment of the invention, the protein is a tandem dimer. In one embodiment, the protein is capable of activating p42/p44 MAPK and Akt. The lung injury may be a dermal burn, cut or combined injury. Yet another embodiment is a method of treating ischemic disease comprising administration to a subject in need thereof of a pharmaceutically effective amount of a pharmaceutical composition containing a chimeric protein comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 7. In another embodiment of the invention, the protein is a tandem dimer. In one embodiment, the protein is capable of activating p42/p44 MAPK and Akt.

Brief Description of the Drawings

Figure 1 shows the cloning of the InIB segment monomer and tandem dimer. The InIB fragment (MW= 65016.42; MoI Ext. Coef = 36900) amino acids from location 36 to 321 of the original InIB (Accession No. DQ132796) were used to construct a tandem dimer (2x InIB) construct; 6 ^χ His and a VaI were added to the N- terminus. Figure IA shows the nucleotide sequence of L. monocytogenes InIB mRNA, 103-693. The ATF start side and 6xHis were added to the 5' end of the InIB mRNA sequence (codon in bold indicates the start of InIB). Coding sequences for the N-terminal 6xHis, VaI, and the stop codons at the 3' end were printed in uppercases; cloning sites for PmI I and BamH I in bold letters. For the monomer, the sequence was terminated at the second bold and underlined codon. For the tandem dimer, the two codons in bold and underlined were inserted between the repeated sequences. Figure IB shows the protein sequence, amino acids 36 to 321 of InIB with the 6xHis tag (in bold and underlined) and the inserted linker (in bold) was added to the beginning of the second repeat.

Figure 2 shows the expression and purification of the 6><His-InlB fragment, amino acids 36 to 321. The InIB segment known to bind the c-Met receptor was cloned, expressed in E. coli, and purified as described in the Examples. 12.5% SDS PAGE shows fractions during purification: lane 1, whole cell lysate; lane 2, flow through from the Ni-NTA column; lane 3, wash from 6><His binding column; lane 4 to 6, fractions eluted with 20, 60, and 250 mM imidazole. MW, molecular weight markers.

Figure 3 shows the activation of p42/p44 MAPK and cell survival by the InIB monomer, amino acids 36 to 321. Figure 3 A shows a western blot for p42/p44 MAPK. BPAEC were grown to 90% confluence and placed in 0.1% FBS overnight before treatment with the indicated concentrations of the InIB monomer protein for the indicated times. Cell lysates were used in Western blots for phospho-p42/p44 MAPK. The blots were also stripped and blotted for total p42/p44. Figure 3B shows the results of a Neutral Comet assay. BPAEC cells were grown to 80% confluence and placed in 0.01% FBS with the InIB monomer (4.2 mg/ml) or HGF (25 ng/ml) overnight before the addition of Ang II (10 μM). After 16 hours, Neutral Comet assays were performed. Data shows means +/- standard deviation. * indicates p<0.05, n=3. Experiments were performed at least three times. Representative data is shown.

Figure 4 shows the expression and purification of the 6xHis-InlB fragment tandem dimer comprised of a repeat of the InIB fragment sequence. Amino acids 36 to 321 of the InIB protein were cloned with a linker of two amino acids, expressed in E. coli and purified as described in the Examples. 10% SDS PAGE shows fractions during purification: lane 1, whole cell lysate; lane 2, flow through from the Ni-NTA column; lane 3, wash with lysis buffer; lane 4 to 6, fractions eluted with 20, 60, and 250 mM imidazole. MW, molecular weight markers. Figure 5 shows the activation of p42/p44 MAPK and Akt by the InIB fragment tandem dimer. Figure 5 shows a western blot for pMAPK, total MAPK, pAKT and tubulin. BPAEC cells were grown to 80% confluence and placed in 0.01% FBS overnight before treatment with the indicated concentrations of the InIB monomer or tandem dimer at the indicated times. Cell lysates were used in Western blots for phospho-p42/p44 MAK or phospho-Akt. Blots were stripped and blotted for total p42/p44 MAPK or tubulin as loading controls.

Figure 6 shows cell survival by the InIB fragment tandem dimer from Ang II -induced apoptosis. BPAEC cells were grown to 80% confluence and placed in 0.01% FBS overnight. The InIB tandem dimer was added at the indicated concentrations for 1 hour before the addition of Ang II (10 μM). After 16 hours, Neutral Comet assays were performed or DNA was prepared for agarose gel electrophoresis. Figure 6A shows the results of a Neutral Comet assay. Data shows means +/- standard deviation. * indicates p<0.05, n=3. Figure 6B shows the DNA laddering assays. Experiments were performed at least three times. Representative data is shown.

Figure 7 shows cell survival by the InIB fragment tandem dimer and HGF from Ang II -induced apoptosis in lung tissue explants. Figure 7 shows a western blot for activated caspase 3 and β-actin. Ex vivo lung tissue slices were prepared from rat lungs. Tissue was placed in medium with 10% FBS and allowed to incubate for 16 hours. InIB fragment tandem dimer (0.04 μg/ml) or HGF (25 ng/ml) was added for 1 hour before the addition of Ang II (10 μM). After 16 hours, whole lysates were prepared and used for western blotting for activated caspase 3. Blots were stripped and reprobed for β-actin as a loading control.

Figure 8 shows cell survival by the InIB fragment tandem dimer and HGF from bleomycin-induced apoptosis. BPAEC cells were grown to 80% confluence and placed in 0.01% FBS overnight. The InIB tandem dimer (0.04 μg/ml) was then added for 1 hour before the addition of Ang II (10 μM). After 16 hours, DNA was prepared for agarose gel electrophoresis. DNA laddering is shown. The experiment was performed at least three times, and representative data are shown.

Figure 9 shows cell motility induced by the InIB monomer, tandem dimer, and HGF. BPAEC were plated at a density of 5 x 10 ⁴ cells in triplicate in wells of a Transwell dish.

Cells were placed in 10% FBS with either no addition, InIB fragment monomer (4.2 μg/ml), InIB fragment tandem dimer (0.42 μg/ml), or HGF (25 ng/ml). After 16 hours, cells were fixed and stained, and the numbers of cells migrating through the membrane were determined. Data show averages +/- SD; * indicates p<0.05 from basal migration levels, f indicates p<0.05 from InIB monomer-induced migration levels. Experiments were repeated at least 3 times, and representative data are shown.

Figure 10 shows the growth activity of the InIB fragment dimer and monomer, and full length HGF. BPAEC cells were plated at a density of 5 x 10 ⁴ cells/35 mm dish in triplicate and allowed to attach for 6 hours. The cells were placed in 1% FBS with either no addition, InIB monomer (4.2 μg/ml), InIB tandem dimer (0.42 μg/ml), or full length HGF (25 ng/ml). Media with additions was replaced on days 1, 2, and 4. Cells were counted on days 4, 6, 8, and 10. Data shows means +/- standard deviation. * indicates p<0.05 from basal levels, n=3. Representative data is shown. Figure 11 shows the nucleotide sequence of a chimeric protein of the invention comprising the N-terminal hairpin loop of HGF and the InIB tandem dimer.

Figure 12 shows the amino acid sequence and nucleotide of the N-terminal hairpin loop of HGF fragment that may be used in some chimeric proteins of the invention. Figure 12A shows the nucleic acid sequence of human HGF N-terminal hairpin loop corresponding to amino acids 1 to 127. Figure 12B shows the amino acid sequence for human HGF amino acids 1 to 127.

Detailed Description

Applicants discovered that in normal primary endothelial cells a peptide comprising amino acids 36 to 321 of internalin B protein (InIB) induces activation of the MAPK pathway downstream of c-Met binding, thereby inducing cell survival in the presence of apoptotic agents. Applicants further discovered that a modified homodimer of amino acids 36 to 321 of InIB activates both the (p42/p44) MAPK and Akt pathway following c-Met binding. The dimer optimally has the further biological effect of inducing cell growth in addition to survival. Applicants have also shown that this tandem dimer improved signal transduction with activation of Akt, and with the added biological activity of cell growth. This improved signal transduction is also associated with a gain of biological function such as e.g., cell proliferation, cell survival, and improved cell migration. The modified InIB tandem dimer is advantageously suitable as a pharmaceutical agent for tissue repair. In one embodiment of the invention, the modified homodimer of InIB amino acids 36 to 321 is effective for the survival and growth of normal epithelial cells in cell culture and in vivo, and will be effective for all pathways for which HGF has been found to be regenerative and protective. In another embodiment of the invention, this modified peptide may have a therapeutic value equivalent to HGF including tissue repair in skin, liver, kidney, heart, brain, and lung.

Applicants have optimally shown that a modified InIB fragment (e.g., a chimeric InIB protein) containing at least amino acids of 36 to 321 of InIB is able to induce the same pathways as HGF, particular when such a modified peptide is in the form of a tandem dimer. Thus, this invention optimally and advantageously encompasses stimulation of MAPK and optionally Akt without administering HGF.

Chimeric Proteins

The invention encompasses chimeric InIB proteins that contain at least a first amino acid sequence and a second amino acid sequence having one or more domains of InIB protein. The one or more domains of InIB protein (the InIB fragment) may comprise amino acids 36 to 321 of InIB or the amino acid sequence of SEQ ID NO: 1. The chimeric protein may have a linker peptide between the first amino acid sequence and second amino acid sequence. The chimeric protein can also be a tandem dimer. As used herein, the term "chimeric InIB protein" encompasses chimeric proteins comprising constructs of one or more domains of InIB protein, segments, or fragments thereof. The term chimeric InIB protein also encompasses fusion proteins comprising an InIB protein or fragment thereof wherein the protein is not the full-length (i.e., native) InIB. Accordingly, chimeric InIB proteins of the invention encompasses chimeric proteins having one or more fragments InIB that are no more than 286 (i.e., the length SEQ ID NO: 1), 300, 320, 340, 350, 360, 380, 400, 420, 440, 450, 460, 480, 500, 520, 540 or 550 amino acids in length. In one embodiment of the invention, the chimeric protein comprises a construct encompassing only amino acids 36 to 321 of InIB (SEQ ID NO: 1) either as a monomer or a dimer. As used herein, the term "one or more domains of InIB protein" refers to all or part of the N-cap, leucine-rich region repeat and immunoglobulin like domains of the InIB protein. In one embodiment of the invention, these domains encompass amino acids 36 to 321 of InIB (SEQ ID NO: 1).

As used herein "chimeric InIB protein" or "chimeric protein" means a chimeric protein comprising at least a first amino acid sequence and a second amino acid sequence comprising one or more domains of InIB protein having at least 95% or more sequence identity to the amino acid sequence of SEQ ID NO: 1. The term "chimeric InIB protein" also encompasses a chimeric protein comprising at least the amino acid sequence of SEQ ID NO: 3 and one or more domains of InIB protein having at least 95% or more sequence identity to the amino acid sequence of SEQ ID NO: 1. The term "chimeric InIB protein" further encompasses a chimeric protein comprising at least a first amino acid sequence and a second amino acid sequence comprising one or more domains of InIB protein having at least 95% or more sequence identity to the amino acid sequence of SEQ ID NO: 7. The term "chimeric InIB protein" also encompasses a chimeric protein comprising a first amino acid sequence (such as e.g., the amino acid sequence of SEQ ID NO: 1 or 3), a linker peptide and a second amino acid sequence comprising one or more domains of InIB protein having at 95%, 96%, 97%, 98%, 99% or more sequence identity to amino acid sequence of SEQ ID NO: 1.

The chimeric InIB protein is capable of activating p42/p44 MAPK. In a preferred embodiment, the chimeric protein is capable of activating both p42/p44 MAPK and Akt. In one embodiment of the invention, the chimeric InIB protein is a tandem dimer. In some embodiments, additional amino acids are deleted while in other embodiments, the amino acids are substituted. Notwithstanding the deletion(s) and/or substitution(s), the conformation of the InIB chimeric protein remains sufficiently intact so that the protein is capable of inducing activation of the MAPK pathway downstream of c-Met binding (i.e., capable of activating p42/p44 MAPK and Akt). In one embodiment, notwithstanding the deletion(s) and/or substitution(s), the conformation of the InIB chimeric protein remains sufficiently intact so that the protein is a tandem dimer.

One embodiment of the invention is a chimeric InIB protein that has a first amino acid sequence and the amino acid sequence of SEQ ID NO: 1. The chimeric protein may optionally further comprise a linker peptide between the first amino acid sequence and the amino acid sequence of SEQ ID NO: 1. In a preferred embodiment, the first amino acid sequence is at the N-terminus of the one or more domains of InIB protein (e.g., the amino acid sequence of SEQ ID NO: 1). In another preferred embodiment, the first amino acid sequence also is the amino acid sequence of SEQ ID NO: 1.

Another embodiment is a chimeric protein that forms a tandem dimer, is capable of activating both p42/p44 MAPK and Akt and comprises at least a first amino acid sequence and one or more domains of InIB protein having at least 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence of SEQ ID NO: 1. Yet another embodiment is a chimeric protein that is capable of activating both p42/p44 MAPK and Akt and comprises at least a first amino acid sequence and one or more domains of InIB protein having at least 95%, 97%, 99% or more sequence identity to the amino acid sequence of SEQ ID NO: 7. The chimeric protein may be a tandem dimer.

The invention also includes a chimeric InIB protein and comprises at least (1) a first amino acid sequence that aids in the internalization of the one or more domains of InIB protein and (2) one or more domains of InIB protein. Thus, one embodiment of the invention is a chimeric InIB protein, which comprises at least the N-terminal hairpin loop of hepatocyte growth factor and amino acids 36 to 321 of InIB (e.g., the amino acid sequence of SEQ ID NO: 1). In a preferred embodiment, the N-terminal hairpin loop of the hepatocyte growth factor comprises at least the amino acid sequence of SEQ ID NO: 3. In one embodiment, the chimeric InIB protein comprises the N-terminal hairpin loop of hepatocyte growth factor (e.g., the amino acid sequence of SEQ ID NO: 3) and one or more domains of InIB protein (e.g., amino acids 36 to 321 of InIB, the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 7), is capable of activating both p42/p44 MAPK and Akt. In another embodiment, the chimeric InIB protein comprises (1) a first amino acid sequence having at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more sequence identity to the amino acid sequence of SEQ ID NO: 3 and (2) one or more domains of InIB protein having at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more sequence identity to amino acid sequence of SEQ ID NO: 1, wherein the chimeric InIB protein comprises the structure of the N-terminal hairpin loop of the HGF and is capable of activating p42/p44 MAPK and Akt. In an alternate embodiment, the chimeric InIB protein comprises (1) a first amino acid sequence having at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more sequence identity to the amino acid sequence of SEQ ID NO: 3 and (2) one or more domains of InIB protein having at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more sequence identity to amino acid sequence of SEQ ID NO: 7, wherein the chimeric InIB protein comprises the structure of the N-terminal hairpin loop of the HGF and is capable of activating p42/p44 MAPK and Akt.

The invention further includes a chimeric protein comprising a first amino acid sequence, a linker peptide, and a second amino acid sequence comprising one or more domains of InIB protein having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1. In one embodiment of the invention, the first amino acid sequence comprises one or more domains of InIB protein having at least 95, 96, 97, 98, 99% or more sequence identity to the amino acid sequence of SEQ ID NO: 1. In another embodiment, the first amino acid sequence comprises the N-terminal hairpin loop of HGF (e.g., the amino acid sequence of SEQ ID NO: 3). In another embodiment of the invention, the chimeric protein comprises (1) a first amino acid sequence having at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more sequence identity to the amino acid sequence of SEQ ID NO: 3, (2) a linker peptide and (3) a second amino acid sequence comprising one or more domains of InIB protein having at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more sequence identity to amino acid sequence of SEQ ID NO: 1. In another embodiment of the invention, the chimeric protein comprises (1) a first amino acid sequence having at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more sequence identity to the amino acid sequence of SEQ ID NO: 1, (2) a linker peptide and (3) a second amino acid sequence comprising one or more domains of InIB protein having at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more sequence identity to amino acid sequence of SEQ ID NO: 1. In yet another embodiment of the invention, the chimeric protein comprises (1) a first amino acid sequence having at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more sequence identity to the amino acid sequence of SEQ ID NO: 3, (2) a linker peptide and (3) a second amino acid sequence comprising one or more domains of InIB protein having at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more sequence identity to amino acid sequence of SEQ ID NO: 7. The chimeric protein may be capable of activating p42/p44 MAPK and Akt. The chimeric protein can also be a tandem dimer.

In another embodiment, the chimeric InIB protein comprises the amino acid sequence of SEQ ID NO: 5. In yet another embodiment, the chimeric InIB protein comprises the amino acid sequence of SEQ ID NO: 5 and is capable of activating p42/p44 MAPK and Akt. In yet another embodiment, the chimeric InIB protein has at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more sequence identity to the amino acid sequence of SEQ ID NO: 5, wherein the protein is capable of activating p42/p44 MAPK and Akt.

In yet another embodiment, the chimeric InIB protein comprises the amino acid sequence of SEQ ID NO: 9. In another embodiment, the chimeric InIB protein comprises the amino acid sequence of SEQ ID NO: 9 and is capable of activating p42/p44 MAPK and Akt. In yet another embodiment, the chimeric InIB protein has at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more sequence identity to the amino acid sequence of SEQ ID NO: 9, wherein the protein is capable of activating p42/p44 MAPK and Akt. The chimeric InIB protein can also include a stabilization domain, which increases the in vitro and in vivo half-life of the fusion polypeptide. As used herein, the term "stabilization domain" refers to an amino acid sequence capable of extending the in vitro and in vivo half-life of a chimeric InIB protein when compared to the InIB alone. The stabilization domain can comprise human proteins (e.g., full length or truncated, soluble proteins from extracellular fragments, etc.) such as human serum albumin, transferrin, or other proteins, which stabilize the in vivo or in vitro half-life of the chimeric toxoid protein. These additional functional domains may themselves serve as linker peptides, for example, for joining at one or more domains of InIB protein to a second protein. Alternatively, they may be located elsewhere in the fusion molecule (e.g., at the amino or carboxy terminus thereof). In alternative embodiments, the stabilization domain is a chemical moiety (e.g., PEG (polyethylene glycol) or a dextran).

The term "chimeric" or "fusion polypeptide" as used herein refers to polypeptides in which: (i) a given functional domain is bound at its carboxy terminus by a non-covalent bond either to the amino terminus of a second protein or to a linker peptide which itself is bound by a non-covalent bond to the amino terminus of the second protein; (ii) a given functional domain (e.g., amino acids 36 to 321 of InIB or the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 7) is bound at its amino terminus by a non-covalent bond either to the carboxy terminus of a second protein (e.g., N-terminal hairpin loop of the hepatocyte growth factor or the amino acid sequence of SEQ ID NO: 3) or to a linker peptide which itself is bound by a non-covalent bond to the carboxy terminus of the second protein. In addition, as used herein, chimeric or fusion peptides are capable of at least activating MAPK and more preferably both p42/p44 MAPK and Akt.

Similarly, "fused" when used in connection with the nucleic acid intermediates of the invention means that the 3'- [or 5'-] terminus of a nucleotide sequence encoding a protein is bound to the respective 3'- [or 5'-] terminus of a nucleotide sequence encoding a second protein, either by a covalent bond or indirectly via a nucleotide linker which itself is covalently bound preferably at its termini to the first functional domain-encoding polynucleotide and optionally, a second functional domain-encoding nucleic acid.

Examples of chimeric or fusion polypeptides of the invention may be represented by, but are not limited by, the following formulas:

R1-L-R2 (i)

R2-L-R1 (ii)

R1-L-R2-L-R1 (iii)

R1-L-R1-L-R2 (iv) R2-L-R1-L-R1 (v) wherein Rl is the amino acid sequence of amino acids 36 to 321 of InIB or the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 7, R2 is second amino acid sequence and/or a stabilizing domain (e.g., human serum albumin), each L is a linker peptide which is bound by a covalent bond to a terminus of Rl and/or R2, whereby the above molecule fragments are read directionally (i.e., with the left side corresponding to the amino terminus and the right side to the carboxy terminus of the molecule). When R2 is the amino acid sequence of the N-terminal hairpin loop of the hepatocyte growth factor or amino acid sequence of SEQ ID NO: 3, R2 is at the N-terminus of chimeric or fusion polypeptides.

The invention includes chimeric and/or fusion polypeptides and salts thereof, comprising at a fusion protein of least one or more domains of InIB protein and at least one second amino acid sequence wherein the fusion protein forms a tandem dimer.

Nucleic Acids

The invention also encompasses nucleic acids that encode the chimeric InIB proteins of the invention. As used herein, the term "nucleic acid" is defined as RNA or DNA that encodes a protein or peptide as defined above, is complementary to a nucleic acid sequence encoding such peptides, hybridizes to nucleic acid molecules that encodes a first amino acid sequence and amino acids 36 to 321 of InIB or the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 7 across the open reading frame under appropriate stringency conditions.

One embodiment of the invention is a nucleic acid that encodes a chimeric InIB protein comprising at least the amino acid sequence SEQ ID NO: 1, wherein the protein is optionally capable of activating p42/p44 MAPK and Akt.

Another embodiment of the invention is a nucleic acid that encodes a chimeric InIB protein comprising at least the amino acid sequence SEQ ID NO: 7, wherein the protein forms a tandem dimer.

Yet another embodiment of the invention is a nucleic acid that encodes a chimeric InIB protein comprising at least (1) the amino acid sequence of SEQ ID NO: 3 and (2) the amino acid sequence of SEQ ID NO: 1. Yet another embodiment of the invention is a nucleic acid that encodes a chimeric

InIB protein comprising at least (1) the amino acid sequence of SEQ ID NO: 3 and (2) the amino acid sequence of SEQ ID NO: 7, wherein the protein forms a tandem dimer.

An alternate embodiment of the invention is a nucleic acid that encodes a chimeric InIB protein comprising the amino sequence of SEQ ID NO: 5. Another embodiment of the invention is a nucleic acid that encodes a chimeric InIB protein comprising the amino sequence of SEQ ID NO: 9.

Another embodiment of the invention is a nucleic acid encoding a chimeric InIB protein having at least a first amino acid sequence with at least 85, 86, 87, 88, 89, 90, 91, 92,

93, 94, 95, 96, 97, 98, 99% or more sequence identity to the amino acid sequence of SEQ ID NO: 3 and one or more domains of InIB protein with at least 85, 86, 87, 88, 89, 90, 91, 92, 93,

94, 95, 96, 97, 98, 99% or more sequence identity to amino acid sequence of SEQ ID NO: 1, wherein the chimeric InIB protein forms a tandem dimer, has the activity of the N-terminal hairpin loop of the hepatocyte growth factor and is capable of activating p42/p44 MAPK and Akt. Another embodiment of the invention is a nucleic acid encoding a chimeric InIB protein having at least a first amino acid sequence with at least 85, 86, 87, 88, 89, 90, 91, 92,

93, 94, 95, 96, 97, 98, 99% or more sequence identity to the amino acid sequence of SEQ ID NO: 3 and one or more domains of InIB protein with at least 85, 86, 87, 88, 89, 90, 91, 92, 93,

94, 95, 96, 97, 98, 99% or more sequence identity to amino acid sequence of SEQ ID NO: 7, wherein the chimeric InIB protein forms a tandem dimer, has the activity of the N-terminal hairpin loop of the hepatocyte growth factor and is capable of activating p42/p44 MAPK and Akt.

Yet another embodiment is a nucleic acid comprising at least the nucleotide sequence of SEQ ID NO: 2, wherein said nucleic acid encodes a chimeric InIB protein that forms a tandem dimer and is capable of at least activating MAPK and more preferably both p42/p44 MAPK and Akt. Another embodiment of the invention, is a nucleic acid comprising a nucleotide sequence having at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more sequence identity with the contiguous nucleotide sequence of the nucleic acid molecule of SEQ ID NO: 2, wherein the nucleic acid encodes a chimeric InIB protein that forms a tandem dimer and is capable of activating p42/p44 MAPK and Akt. The nucleic acid molecule may further comprise the nucleotide sequence of SEQ ID NO: 4. Yet another embodiment is a nucleic acid molecule comprising both nucleotide sequence of SEQ ID NO: 4 and the nucleotide sequence of SEQ ID NO: 2.

Yet another embodiment is a nucleic acid comprising at least the nucleotide sequence of SEQ ID NO: 8, wherein said nucleic acid encodes a chimeric InIB protein that forms a tandem dimer and is capable of at least activating MAPK and more preferably both p42/p44 MAPK and Akt. Another embodiment of the invention, is a nucleic acid comprising a nucleotide sequence having at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more sequence identity with the contiguous nucleotide sequence of the nucleic acid molecule of SEQ ID NO: 8, wherein the nucleic acid encodes a chimeric InIB protein that forms a tandem dimer and is capable of activating p42/p44 MAPK and Akt. The nucleic acid molecule may further comprise the nucleotide sequence of SEQ ID NO: 4. Yet another embodiment is a nucleic acid molecule comprising both nucleotide sequence of SEQ ID NO: 4 and the nucleotide sequence of SEQ ID NO: 8. Yet another embodiment of the invention is a nucleotide encoding a chimeric protein having a first amino acid sequence, a linker peptide and a second amino acid sequence comprising one or more domains of InIB protein having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1. Another embodiment of the invention is a nucleotide encoding a chimeric protein having a first amino acid sequence having at least 95, 96, 97, 98, 99% or more sequence identity to the amino acid sequence of SEQ ID NO: 1 , a linker peptide and a second amino acid sequence comprising one or more domains of InIB protein having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1. An alternate embodiment is a nucleotide encoding a chimeric protein having (1) a first amino acid sequence having at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more sequence identity to the amino acid sequence of SEQ ID NO: 3, (2) a linker peptide and (3) and a second amino acid sequencing comprising one or more domains of InIB protein having at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more sequence identity to amino acid sequence of SEQ ID NO: 1. Preferably the nucleotide encodes chimeric protein may that are capable of activating p42/p44 MAPK and Akt. Another embodiment of the invention is nucleotide having the nucleotide sequence of

SEQ ID NO: 6, wherein said nucleotide encodes a chimeric InIB protein having at least the N- terminal hairpin loop of HGF and one or more domains of InIB protein. Yet another embodiment of the invention is a nucleic acid having at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more sequence identity to nucleotide sequence of SEQ ID NO: 6, wherein said nucleotide encodes a chimeric InIB protein that comprises at least the N- terminal hairpin loop of HGF and is capable of activating p42/p44 MAPK and Akt.

Another embodiment of the invention is nucleotide having the nucleotide sequence of SEQ ID NO: 10, wherein said nucleotide encodes a chimeric InIB protein having at least the N-terminal hairpin loop of HGF and one or more domains of InIB protein and wherein the chimeric InIB protein forms a tandem dimer. Yet another embodiment of the invention is a nucleic acid having at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more sequence identity to nucleotide sequence of SEQ ID NO: 10, wherein said nucleotide encodes a chimeric InIB protein that comprises at least the N-terminal hairpin loop of HGF, forms a tandem dimer, and is capable of activating p42/p44 MAPK and Akt. The "nucleic acids" of the invention further include nucleic acid molecules that share at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 2, which encode chimeric InIB proteins that are capable of activating p42/p44 MAPK and Akt. The "nucleic acids" of the invention further include nucleic acid molecules that comprise (1) a segment that shares at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 4 and (2) a segment that shares at least about 85, 86, 87,

88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 2, which encode chimeric InIB proteins that are capable of activating p42/p44 MAPK and Akt. The "nucleic acids" of the invention further include nucleic acid molecules that comprise (1) a segment that shares at least about 85, 86, 87, 88,

89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 4 and (2) a segment that shares at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 8, which encode chimeric InIB proteins that are capable of activating p42/p44 MAPK and Akt. The "nucleic acids" of the invention also include nucleic acid molecules that share at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 6. In addition, the "nucleic acids" of the invention also include nucleic acid molecules that share at least about 85, 86, 87, 88, 89,

90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 10. The "nucleic acid molecules" of the invention also include nucleic acid molecules that comprise (1) a segment that shares at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 4, (2) a linker region and (3) a segment that shares at least about 85, 86, 87, 88, 89, 90,

91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 2, which encode chimeric InIB proteins that are capable of activating p42/p44

MAPK and Akt.

Specifically contemplated are genomic DNA, cDNA, rDNA, mRNA and antisense molecules, as well as nucleic acids based on alternative backbones or including alternative bases whether derived from natural sources or synthesized. Such nucleic acids, however, are defined further as being novel and unobvious over any prior art nucleic acid including that which encodes, hybridizes under appropriate stringency conditions, or is complementary to nucleic acid encoding a protein according to the present invention.

Homology or identity at the nucleotide or amino acid sequence level is determined by BLAST (Basic Local Alignment Search Tool) analysis using the algorithm employed by the programs blastp, blastn, blastx, tblastn and tblastx (Altschul et al. (1997) Nucleic Acids Res. 25, 3389-3402 and Karlin et al. (1990) Proc. Natl. Acad. ScL USA 87, 2264-2268, both fully incorporated by reference) which are tailored for sequence similarity searching. The approach used by the BLAST program is to first consider similar segments, with and without gaps, between a query sequence and a database sequence, then to evaluate the statistical significance of all matches that are identified and finally to summarize only those matches which satisfy a preselected threshold of significance. For a discussion of basic issues in similarity searching of sequence databases, see Altschul et al. (1994) Nature Genetics 6, 119- 129 which is fully incorporated by reference. The search parameters for histogram, descriptions, alignments, expect (i.e., the statistical significance threshold for reporting matches against database sequences), cutoff, matrix and filter (low complexity) are at the default settings. The default scoring matrix used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff et al. (1992) Proc. Natl. Acad. ScL USA 89, 10915-10919, fully incorporated by reference), recommended for query sequences over 85 in length (nucleotide bases or amino acids). For blastn, the scoring matrix is set by the ratios of M (i.e., the reward score for a pair of matching residues) to N (i.e., the penalty score for mismatching residues), wherein the default values for M and N are +5 and -4, respectively. Four blastn parameters were adjusted as follows: Q=IO (gap creation penalty); R=IO (gap extension penalty); wink=l (generates word hits at every wink" ¹ position along the query); and gapw=16 (sets the window width within which gapped alignments are generated). The equivalent Blastp parameter settings were Q=9; R=2; wink=l ; and gapw=32. A Bestfit comparison between sequences, available in the GCG package version 10.0, uses DNA parameters GAP=50 (gap creation penalty) and LEN=3 (gap extension penalty) and the equivalent settings in protein comparisons are GAP=8 and LEN=2.

Another embodiment of the invention is a nucleic acid molecule that hybridizes under stringent conditions to a nucleic acid encoding a chimeric InIB protein having at least a first peptide with at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more sequence identity to amino acid sequence of SEQ ID NO: 3 and second amino acid sequence comprising at least one or more domains of InIB protein with at least 85, 86, 87, 88, 89, 90,

91, 92, 93, 94, 95, 96, 97, 98, 99% or more sequence identity to the amino acid sequence of

SEQ ID NO: 1, wherein the chimeric InIB protein has the activity of the N-terminal hairpin loop of the hepatocyte growth factor and is capable of activating p42/p44 MAPK and Akt.

Yet another embodiment of the invention is a nucleic acid molecule that hybridizes under stringent conditions to a nucleic acid encoding a chimeric InIB protein having at least a first peptide with at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more sequence identity to amino acid sequence of SEQ ID NO: 3, a second amino acid sequence comprising one or more domains of InIB protein with at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more sequence identity to the amino acid sequence of SEQ ID NO: 7, and optionally a linker peptide between the first and second amino acid sequence, wherein the chimeric InIB protein has the activity of the N-terminal hairpin loop of the hepatocyte growth factor and is capable of activating p42/p44 MAPK and Akt.

"Stringent conditions" are those that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS at 50 ⁰ C, or (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer (pH 6.5) with 750 mM NaCl, 75 mM sodium citrate at 42°C. Another example is hybridization in 50% formamide, 5x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42°C, with washes at 42°C in 0.2x SSC and 0.1% SDS. A skilled artisan can readily determine and vary the stringency conditions appropriately to obtain a clear and detectable hybridization signal. Preferred molecules are those that hybridize under the above conditions to the complement of any of the nucleic acids described herein which encode a functional protein. Even more preferred hybridizing molecules are those that hybridize under the above conditions to the complement strand of the open reading frame of any of the chimeric InIB proteins.

As used herein, a nucleic acid molecule is said to be "isolated" when the nucleic acid molecule is substantially separated from contaminant nucleic acid molecules encoding other polypeptides.

The present invention further provides fragments of the encoding nucleic acid molecule. As used herein, a fragment of an encoding nucleic acid molecule refers to a small portion of the entire protein coding sequence. The size of the fragment will be determined by the intended use. Fragments of the encoding nucleic acid molecules of the present invention (i.e., synthetic oligonucleotides) that are used as probes or specific primers for the polymerase chain reaction (PCR), or to synthesize gene sequences encoding proteins of the invention, can easily be synthesized by chemical techniques, for example, the phosphotriester method of Matteucci et al. (1981) J. Am. Chem. Soc. 103, 3185-3191 or using automated synthesis methods. Examples of such probes or primers include, but are not limited to, any of SEQ ID NO: 11 to 14. In addition, larger DNA segments can readily be prepared by well-known methods, such as synthesis of a group of oligonucleotides that define various modular segments of the gene, followed by ligation of oligonucleotides to build the complete modified gene. In a preferred embodiment, the nucleic acid molecule of the present invention contains a contiguous open reading frame of at least about three-thousand and forty-five nucleotides.

The encoding nucleic acid molecules of the present invention may further be modified to contain a detectable label for diagnostic and probe purposes. A variety of such labels is known in the art and can readily be employed with the encoding molecules herein described. Suitable labels include, but are not limited to, biotin, radiolabeled nucleotides, and the like. A skilled artisan can readily employ any such label to obtain labeled variants of the nucleic acid molecules of the invention. Modifications to the primary structure itself by deletion, addition, or alteration of the amino acids incorporated into the protein sequence during translation can be made without destroying the activity of the protein. Such substitutions or other alterations result in proteins having an amino acid sequence encoded by a nucleic acid falling within the contemplated scope of the present invention. The invention encompasses synthetic oligonucleotides having chemical modifications from native nucleic acids, or nucleic acid constructs that express such anti-sense molecules as RNA. The oligonucleotide sequence can be complementary to the nucleic acids encoding chimeric InIB protein. Oligonucleotides may be chemically synthesized by methods known in the art (see

Wagner et al. (1996) Nat. Biotech. 14, 840-844). Oligonucleotides of the invention can be chemically modified from the native phosphodiester structure, in order to increase their intracellular stability and binding affinity. A number of such modifications have been described in the literature, which alter the chemistry of the backbone, sugars, or heterocyclic bases.

The nucleic acid molecules may be operably linked to an expression control sequence or inserted into a vector.

The present invention further provides recombinant DNA molecules (rDNAs) that encode the chimeric InIB proteins of the invention. As used herein, an rDNA molecule is a DNA molecule that has been subjected to molecular manipulation in situ. Methods for generating rDNA molecules are well known in the art, for example, see Sambrook et al. (2005) Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory Press. In the preferred rDNA molecules, a coding DNA sequence is operably linked to expression control sequences and/or vector sequences. The choice of vector and/or expression control sequences to which one of the protein family encoding sequences of the present invention is operably linked depends directly, as is well known in the art, on the functional properties desired, e.g., protein expression, and the host cell to be transformed. A vector contemplated by the present invention is at least capable of directing the replication or insertion into the host chromosome, and preferably also expression, of the structural gene included in the rDNA molecule.

Expression control elements that are used for regulating the expression of an operably linked protein encoding sequence are known in the art and include, but are not limited to, inducible promoters, constitutive promoters, secretion signals, and other regulatory elements. Preferably, the inducible promoter is readily controlled, such as being responsive to a nutrient in the host cell's medium.

Another embodiment of is a vector containing the nucleic acid molecules of the invention (i.e., nucleic acid molecules encoding a chimeric InIB protein having at least a first amino acid sequence and one or more domains of InIB protein). In one embodiment, the vector containing a coding nucleic acid molecule will include a prokaryotic replicon, i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extrachromosomally in a prokaryotic host cell, such as a bacterial host cell, transformed therewith. Such replicons are well known in the art. In addition, vectors that include a prokaryotic replicon may also include a gene whose expression confers a detectable marker such as a drug resistance. Typical bacterial drug resistance genes are those that confer resistance to ampicillin or tetracycline.

Vectors that include a prokaryotic replicon can further include a prokaryotic or bacteriophage promoter capable of directing the expression (transcription and translation) of the coding gene sequences in a bacterial host cell. A promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur. Promoter sequences compatible with bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention.

Any prokaryotic or eukaryotic host can be used to express an rDNA molecule encoding a protein of the invention. In one embodiment, the prokaryotic host is E. coli. One embodiment of the invention is a host cell transformed to contain a nucleic acid of the invention. Transformation of appropriate cell hosts with an rDNA molecule of the present invention is accomplished by well known methods that typically depend on the type of vector used and host system employed. With regard to transformation of prokaryotic host cells, electroporation and salt treatment methods are typically employed, see, for example, Sambrook et al. (2005) Molecular Cloning - A Laboratory Manual, Cold Spring Harbor

Laboratory Press. With regard to transformation of vertebrate cells with vectors containing rDNAs, electroporation, cationic lipid or salt treatment methods are typically employed, see, for example, Graham et al. (1973) Virol. 52, 456; Wigler et al. (1979) Proc. Natl. Acad. ScL USA 76, 1373-1376. Successfully transformed cells, i.e., cells that contain an rDNA molecule of the present invention, can be identified by well known techniques including the selection for a selectable marker. For example, cells resulting from the introduction of an rDNA of the present invention can be cloned to produce single colonies. Cells from those colonies can be harvested, lysed and their DNA content examined for the presence of the rDNA using a method such as that described by Southern (1975) J. MoI. Biol. 98, 503-504 or Berent et al. (1985) Biotech. 3, 208-209 or the proteins produced from the cell assayed via an immunological method.

One skilled in the art would know how to make recombinant nucleic acid molecules that encode chimeric InIB proteins of the invention. Furthermore, one skilled in the art would know how to use these recombinant nucleic acid molecules to obtain the proteins encoded thereby, as described herein for the recombinant nucleic acid molecule, which encodes a chimeric InIB protein of the invention (e.g., a chimeric protein comprising the amino acid of SEQ ID NO: 1 and the amino acid of SEQ ID NO: 3; or a chimeric protein having the amino acid sequence of SEQ ID NO: 9). In accordance with the invention, numerous vector systems for expression of the chimeric InIB protein may be employed. For example, one class of vectors utilizes DNA elements, which are derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV, or MoMLV), Semliki Forest virus, or SV40 virus. Additionally, cells, which have stably integrated the DNA into their chromosomes, may be selected by introducing one or more markers, which allow for the selection of transfected host cells. The marker may provide, for example, prototropy to an auxotrophic host, biocide resistance (e.g., antibiotics) or resistance to heavy metals such as copper or the like. The selectable marker gene can be either directly linked to the DNA sequences to be expressed, or introduced into the same cell by co -trans formation. Additional elements may also be needed for optimal synthesis of mRNA. These elements may include splice signals, as well as transcriptional promoters, enhancers, and termination signals. The cDNA expression vectors incorporating such elements include those described by Okayama (1983) MoI. Cell. Biol. 3, 280-289.

The chimeric InIB protein may be produced by (a) transfecting a cell with an expression vector encoding the chimeric InIB protein; (b) culturing the resulting transfected cell under conditions such that chimeric InIB protein is produced; and (c) recovering the chimeric InIB protein from the cell culture media or the cells themselves.

Once the expression vector or DNA sequence containing the constructs has been prepared for expression, the expression vectors may be transfected or introduced into an appropriate eukaryotic or prokaryotic cell host. Various techniques may be employed to achieve this, such as, for example, protoplast fusion, calcium phosphate precipitation, electroporation, or other conventional techniques. In the case of protoplast fusion, the cells are grown in media and screened for the appropriate activity.

Methods and conditions for culturing the resulting transfected cells and for recovering the chimeric InIB protein so produced are well known to those skilled in the art, and may be varied or optimized depending upon the specific expression vector and host cell employed.

The host cell for expressing the chimeric InIB protein may be prokaryotic or eukaryotic. Exemplary prokaryotic hosts include E. coli, such as E. coli DH5α or BL21. Exemplary eukaryotic hosts include baculovirus vector/insect cell expression systems, yeast shuttle vector/yeast cell expression systems. Methods and conditions for purifying the chimeric InIB protein from the culture media are provided in the invention, but it should be recognized that these procedures can be varied or optimized as is well known to those skilled in the art.

The chimeric InIB protein of the present invention may also be prepared by any known synthetic techniques. Conveniently, the proteins may be prepared using the solid-phase synthetic technique initially described by Merrifield (1965), which is incorporated herein by reference. Other peptide synthesis techniques may be found, for example, in Bodanszky et al. (1976), Peptide Synthesis, Wiley.

The construction of expression vectors that are operable in a variety of hosts is accomplished using appropriate replicons and control sequences, as set forth above. The control sequences, expression vectors, and transformation methods are dependent on the type of host cell used to express the gene and were discussed in detail earlier. Suitable restriction sites can, if not normally available, be added to the ends of the coding sequence so as to provide an excisable gene to insert into these vectors. A skilled artisan can readily adapt any host/expression system known in the art for use with the nucleic acid molecules of the invention to produce recombinant proteins.

Methods of treating tissue injury

The invention also encompasses methods of treating tissue injury (e.g., injury to the skin, lung, and heart) comprising administering an effective amount of a composition comprising a chimeric InIB protein of the invention to a subject in need thereof. In addition, the invention encompasses methods of preventing and/or treating scar formation comprising administering an effective amount of a composition comprising a chimeric InIB protein of the invention to a subject in need thereof.

Like HGF, a chimeric InIB protein induces normal tissue repair in a mechanism which restores normal tissue structure and prevents the development of extensive scar formation or fibrosis. Scarification, or fibrosis, involves in loss of normal architecture reducing the functional capacity of the organ and has adverse effects on the tissue as a whole. Additionally, aberrant repair processes can become progressive and result in total organ failure. Thus, chimeric peptides of the invention induce tissue repair without development of scar formation, loss of functional capacity of the organ or adverse effects on the issue.

One embodiment of the invention is a method of treating skin injury by administration to a subject in need thereof of a pharmaceutically effective amount of a pharmaceutical composition containing a chimeric InIB protein. The method may be used to treat dermal burns, cuts, or combined injury (i.e., both burns and cuts). Another embodiment of the invention is a method of treating lung injury by administration to a subject in need thereof of a pharmaceutically effective amount of a pharmaceutical composition containing a chimeric InIB protein. The method may be used to treat dermal burns, cuts, or combined injury (i.e., both burns and cuts). Yet another embodiment is a method of treating ischemic disease by administration to a subject in need thereof of a pharmaceutically effective amount of a composition containing a chimeric InIB protein.

An alternate embodiment of the invention is a method of treating skin injury by administration to a subject in need thereof of a pharmaceutically effective amount of a pharmaceutical composition comprising a protein comprising the amino acid sequence of SEQ ID NO: 1. Optionally, the protein is further capable of activating p42/p44 MAPK and Akt.

Another embodiment of the invention is a method of treating skin injury by administration to a subject in need thereof of a pharmaceutically effective amount of a pharmaceutical composition comprising a protein comprising the amino acid sequence of SEQ ID NO: 7. Optionally, the protein is further capable of activating p42/p44 MAPK and Akt.

Yet another embodiment of the invention is a method of treating lung injury by administration to a subject in need thereof of a pharmaceutically effective amount of a pharmaceutical composition comprising a protein comprising the amino acid sequence of

SEQ ID NO: 1. Optionally, protein is further capable of activating p42/p44 MAPK and Akt.

Yet another embodiment of the invention is a method of treating lung injury by administration to a subject in need thereof of a pharmaceutically effective amount of a pharmaceutical composition comprising a protein comprising the amino acid sequence of SEQ ID NO: 7. Optionally, protein is further capable of activating p42/p44 MAPK and Akt.

Yet another embodiment of the invention is a method of treating ischemic disease by administration to a subject in need thereof of a pharmaceutically effective amount of a pharmaceutical composition comprising a protein comprising the amino acid sequence of SEQ ID NO: 1. Optionally, the protein is further capable of activating p42/p44 MAPK and Akt.

Yet another embodiment of the invention is a method of treating ischemic disease by administration to a subject in need thereof of a pharmaceutically effective amount of a pharmaceutical composition comprising a protein comprising the amino acid sequence of SEQ ID NO: 7. Optionally, the protein is further capable of activating p42/p44 MAPK and Akt. Additional factors, which stimulate normal cellular repair and inhibit apoptosis of normal cells to further increase tissue repair from injury, reduce or inhibit scarification and fibrosis, may be used in methods of treatments of the invention.

As used herein, the term "therapeutic agent" shall encompass compositions comprising a chimeric InIB protein as formulations containing other pharmaceutically acceptable components such as e.g., pharmaceutically acceptable carriers.

The terms "treating", "treatment," and "therapy" as used herein refer to curative therapy, prophylactic therapy, and preventative therapy.

As used herein, unless stated otherwise, the term composition is meant to encompass, and not limited to, pharmaceutical compositions containing a chimeric InIB protein (e.g., a protein comprising a first amino acid sequence (e.g., the N-terminal hairpin loop of HGF or the amino acid sequence of SEQ ID NO: 3) and the amino acid sequence of amino acids 36 to 321 of InIB or one or more domains of InIB protein having at least 80%, 85%, 90%, 95%, 97%, 99% or more sequence identity to the amino acid sequence of SEQ ID NO: 1. The composition may also contain one or more "excipients" that are "inactive ingredients" or

"compounds" devoid of pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment, or prevention of disease or to affect the structure or any function of the human body.

A "pharmaceutically acceptable" component is one that is suitable for use with humans, animals, and/or plants without undue adverse side effects (such as e.g., toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio.

A "safe and effective amount" refers to a quantity of a component that is sufficient to yield a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of this invention. By "therapeutically effective amount" is meant an amount of a component effective to yield a desired therapeutic response, e.g., an amount effective to treat the tissue injury (e.g., lung injury, skin injury, or ischemic disease). The specific safe and effective amount or therapeutically effective amount will vary with such factors as the particular condition being treated, the physical condition of the subject, the type of subject being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the specific formulations employed and the structure of the compounds or its derivatives.

Means of application include, but are not limited to direct, indirect, carrier and special means or any combination of means. Direct application of the chimeric InIB protein may be by nasal sprays, nasal drops, nasal ointments, nasal washes, nasal injections, nasal packings, bronchial sprays and inhalers, or indirectly through use of throat lozenges, or through use of mouthwashes or gargles, or through the use of ointments applied to the nasal nares, the bridge of the nose, or the face or any combination of these and similar methods of application. The forms in which the chimeric InIB protein may be administered include but are not limited to lozenges, troches, candies, injectants, chewing gums, tablets, powders, sprays, liquids, ointments, and aerosols.

The therapeutic agent may also be placed in a nasal spray, wherein the nasal spray is the carrier. The nasal spray can be a long acting or timed release spray, and can be manufactured by means well known in the art. An inhalant may also be used, so that the therapeutic agent may reach further down into the bronchial tract, including into the lungs. The therapeutic agent may be added to these substances in a liquid form or in a lyophilized state, whereupon it will be solublized when it meets body fluids such as saliva. The enzyme may also be in a micelle or liposome.

While these methods may be used in any mammalian species such as farm animals including, but not limited to, horses, sheep, pigs, chicken, and cows, the preferred use of compositions is for a human.

The effective dosage rates or amounts of the compositions will depend in part on whether the composition will be used therapeutically or prophylactically, the type of injury, the severity of the injury, the size, and weight of the individual, etc. The duration for use of the composition also depends on whether the use is for prophylactic purposes, wherein the use may be hourly, daily or weekly, for a short time period, or whether the use will be for therapeutic purposes wherein a more intensive regimen of the use of the composition may be needed, such that usage may last for hours, days or weeks, and/or on a daily basis, or at timed intervals during the day. It should be noted that carriers that are classified as "long" or "slow" release carriers (such as, for example, certain nasal sprays or lozenges) could possess or provide a lower concentration of the composition per ml, but over a longer period of time, whereas a "short" or "fast" release carrier (such as, for example, a gargle) could possess or provide a high concentration of composition per ml, but over a shorter period of time. It will furthermore be appreciated that a therapeutically effective amount of a particular composition can be determined by those of ordinary skill in the art with due consideration of the factors pertinent to the subject.

Selection of the preferred effective dose can be determined (e.g., via clinical trials) by a skilled artisan based upon the consideration of several factors which will be known to one of ordinary skill in the art. Such factors include the disease to be treated or prevented, the symptoms involved, the patient's body mass, the patient's immune status and other factors known by the skilled artisan to reflect the accuracy of administered pharmaceutical compositions.

The precise dose to be employed in the formulation will also depend on the route of administration and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

The compositions containing the chimeric InIB protein may also be applied by direct, indirect, carriers and special means or any combination of means. Direct application of the chimeric InIB protein may be by nasal sprays, nasal drops, nasal ointments, nasal washes, nasal injections, nasal packings, bronchial sprays and inhalers, or indirectly through use of throat lozenges, or through use of mouthwashes or gargles, or through the use of ointments applied to the nasal nares, the bridge of the nose, or the face or any combination of these and similar methods of application. The forms in which the chimeric InIB protein may be administered include but are not limited to lozenges, troches, candies, injectants, chewing gums, tablets, powders, sprays, liquids, ointments, and aerosols.

The compositions of the present invention can be administered via parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or buccal routes. For example, an agent may be administered locally to a site of injury via microinfusion. Alternatively, or concurrently, administration may be by the oral route. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. Topical administration may be used. Any common topical formulation such as a solution, suspension, gel, ointment or salve and the like may be employed. Preparation of such topical formulations are described in the art of pharmaceutical formulations as exemplified, for example, by Gennaro et al. (2000) Remington's Pharmaceutical Sciences, Mack Publishing. For topical application, the compositions could also be administered as a powder or spray, particularly in aerosol form.

In one embodiment of the invention, the method comprises administration of the therapeutic agent in a pharmaceutically acceptable carrier. Suitable carriers and their formulations are described in Remington's Pharmaceutical Sciences, 2005, Mack Publishing Co. Typically, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically acceptable carrier include liquids such as saline, Ringer's solution, and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. The formulation may also comprise a lyophilized powder. Further carriers include sustained release preparations such as semi-permeable matrices of solid hydrophobic polymers, which matrices are in the form of shaped articles, e.g., films, liposomes, or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of proinflammatory cytokine inhibitor being administered.

Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore, specifically point out the preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

Examples

The following abbreviations are used throughout: Akt, oncogene activated by retrovirus AKT8, also known as protein kinase B; Ang II, angiotensin II; c-Met, hepatocyte growth factor receptor; FBS, fetal bovine serum; HGF, hepatocyte growth factor; InIB, Internalin B protein of L. monocytogenes; MAPK, mitogen activated protein kinase; NK2, a naturally occurring isoform of HGF; PI3K, phosphatidylinositol 3-kinase.

Cloning and expression of the Listeria monocytogenes InIB: The InIB sequence (GenBank accession no. DQ 132796, nucleotide sequence from 106 to 963) was amplified using the Pfu DNA polymerase (Stratagene, La Jolla, CA), and L. monocytogene genomic DNA, and the primers: forward 5'-ggg aac acg tgG AGA CTA TCA CCG TGT CAA C-3' (SEQ ID NO: 11) and reverse 5'-cgg gat cct att aCT CTT TCA GTG GTT GGG TTA CT-3' (SEQ ID NO: 12), with uppercase bases corresponding to InIB gene sequences and lowercase bases indicating 5 '-extensions with restriction enzyme sites (bold) fox PmI I and Bam HI, respectively. The InIB peptide homodimer was produced by joining the two AcI I cut PCR products which were amplified using the forward or reverse primers described previously and a second reverse primer 5'-ttt gga tec aac gtt CTC TTT CAG TGG TTG GGT TAC TTC-3' (SEQ ID NO: 13) or a second forward primers 5'-ttt gga tec aac gtt CTC TTT CAG TGG TTG GGT TAC TTC-3' (SEQ ID NO: 14), respectively, both containing the AcI I site (bold). PCR condition was as follows: an initial denaturation of 94°C for 3 minutes; 10 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 2:30 minutes; 25 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 2 minutes; with a final extension of 5 minutes at 72 ⁰ C. The PCR products were cloned into pET302/NT-His vector (Invitrogen) using the PmI I and Bam HI sites and insertions were verified by DNA sequencing. The N-terminal 6xHis-tagged recombinant InIB fragment (having one or more domains of InIB protein) was introduced as monomer or tandem dimer into E.coli BL21- CodonPlus cells according the manufacturer's protocol (Stratagene, La Jolla, CA), protein expression was induced at bacterial cell growth of ODgoo = 0.6 to 0.8 with 1 mM IPTG for 3 hours at 37 ⁰ C. Bacterial cells were collected by 10 minutes of centrifugation at 400Og, washed with Lysis buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCl, 10 mM imidazole, pH 8.0), and stored overnight at -20 ⁰ C. On the next day, cells were resuspended in Lysis buffer (0.3 g/ml); 1 mg/ml lysozyme (Novagen) was added and incubated for 30 minutes on ice; Benzonase (lU/ml) was added and incubated for another 15 minutes, then cells were disrupted for 5 ^χ 10s using an ultra sonicator (Heat Systems-Ultrasonics) at 50% output control. Cell debris was removed by centrifugation (30 minutes; 47000 x g); cleared cell lysate was used for protein purification.

Protein purification: Cloned InIB fragment (having one or more domains of InIB protein) as a monomer and as a tandem dimer were purified using Ni-NTA Superflow according to the QIAexpressionist protocol (Qiagen). Briefly, CellThru 10 ml disposable columns were packed by gravity flow with 50% Ni-NTA Superflow (2.5 ml per column; Clontech Laboratories). After equilibration using 20 ml of Lysis buffer, the column was loaded with cell lysate, washed with 10 ml lysis buffer, containing 20 mM and 60 mM imidazole. Proteins were eluted from the column using 10 ml lysis buffer, containing 250 mM imidazole. Column flow-through from each purification step was analyzed by 10 % SDS-PAGE. Imidazole was removed from the eluate by repeated centrifugation (4000 x g; 20 minutes) and washing cycles with Buffer A (50 mM NaH ₂ PO ₄ , 300 mM NaCl, pH 8.0) in an Amicon Ultra- 15 filter unit (10 kDa; Millipore). Endotoxin was removed from this purified and concentrated protein solution using the Detoxi-Gel Endotoxin Removal Gel according to the manufacturer's protocol (Pierce Biotechnology).

Cell Culture: Bovine pulmonary artery endothelial cells (PAEC) purchased from Cell Applications, Inc. Passage 2-8 cells were used for all experiments and were cultured in RPMI 1640 with 10% FBS, 1% penicillin/streptomycin and 0.5% fungizone. Cells were grown in 5% CO ₂ at 37°C in a humidified atmosphere in a culture incubator. HEK293 cells were grown in DMEM with 10% FBS, 1% penicillin/streptomycin and 0.5% fungizone, in 5% CO ₂ at 37°C in a humidified atmosphere in a culture incubator.

Western blots: Activation of signal transduction pathways was determined by Western blotting for phosphorylation of ERK1/2 MAPK and Akt. Cells were treated with InIB segment monomer or homodimer at the indicated concentrations and times. Cell lysates (10 μg of protein) were electrophoresed through a 10% SDS-PAGE and electroblotted onto a membrane. Blots were blocked in 5% BSA in TBS/0.1% Tween 20 (TTBS) for one hour at ambient temperature before incubating overnight with 1 : 1000 dilution of primary antibody in TTBS/0.5% BSA at 4°C. Blots were washed 3 x TTBS for 10 minutes, for 30 minutes total. Secondary antibodies were diluted 1 : 1000 in TTBS for onel hour; blots were washed in TBS for at least two hours before exposing to the film. ECL (Amersham) was applied according to the manufacturer's instructions before film exposure. To normalize for total MAPK, total Akt, or tubulin, blots were stripped using BME Stripping Buffer (10% SDS, B- mercaptoethanol, 0.5 M Tris-HCl) and reblotted as above.

Neutral Comet Assay: The neutral comet assay was used to measure double stranded DNA breaks as an indication of apoptosis by Ang II treatment, as previously described (Kitt et al (2001), Free Radic. Biol. Med. 31 : 902-910). Cells were treated with apoptotic stimuli, washed in PBS, pH 7.4, embedded in 1% agarose and placed on a comet slide (Trevigen). Cells were then placed in lysis solution (2.5 M NaCl, 1% Na-lauryl sarcosinate, 100 mM EDTA, 10 mM Tris base, 0.01% Triton X-100) for 30 minutes. The nuclei were subsequently be electrophoresed for 20 minutes at 1 V/cm in 1 xTris/borate/EDTA buffer (TBE, 5><TBE stock is 250 mM Tris, 250 mM Boric acid, 5 mM EDTA), fixed in ethanol, followed by staining with Sybr® Green (Molecular Probes, Eugene, OR) and visualized with an Olympus FV500 series confocal laser scanning using 4Ox magnification at 478 nm excitation and 507 nm emission wavelengths. Between 100 and 150 comets were scored per experiment and assigned into type A, B or C categories, based on their tail moments. Type C comets were defined as apoptotic cells as described by Krown et al. (1996) J. Clin. Invest. 98: 2854-2865. Growth Assay: BPAEC were plated in triplicate at a density of 5 x 10 ⁴ cells/35 mm dish in RPMI containing 10% or 0.5% FBS. To the attached cells, InIB fragment as a monomer or as a tandem dimer was added with new medium on days 1, 2, 4, 6, and 8. Cells were trypsinized and counted using a hemacytometer on day 6.

Migration Assay: BPAEC were trypsinized, washed, and plated at a density of 4 x 10 ⁵ cells/well in 12 well/plate Transwell (Corning, Corning, NY) dishes (12 μm pore size). Cells were allowed to attach for 3 hours, then treated in both upper and lower chambers pretreated with InIB monomer or InIB fragment tandem dimer. After 16 hours, cells attached to the Transwell insert were fixed for 15 minutes in 100% methanol and stained for 1 hour in 0.94% crystal violet (Fisher, Fairlawn, NJ) in 20% methanol. The insert was rinsed in a large volume of H ₂ O, and the upper side of the membrane was cleaned using a cotton swab. Remaining migrated cells were counted by microscopy.

Ex-vivo Cultures: Rat lungs were used for the ex-vivo experiments. Rats were euthanized with sodium pentobarbital or Fatal -plus. The surface of the anterior chest wall and upper abdomen were sterilized with 70% ethanol. After the trachea was exposed, small nick was made to insert a 22 gauge needle with a short piece of polyethelene tube attached. Two lines were tied around the trachea to stabilize and to prevent the tube from slipping out. Through a midline abdominal incision the chest cavity was exposed and the animal was exsanguinated by dissecting the abdominal aorta. The right ventricle was punctured and the lungs were perfused with sterile PBS to remove the blood. Using aseptic technique, the trachea, lungs, and the heart was dissected from the animal.

To obtain lung slices for the ex-vivo culture, the lungs were inflated with 1 % low melting point agarose dissolved in RPMI medium. The agarose was instilled as a liquid into the trachea using a syringe and fully inflated the lungs. The lungs were placed in a sterile cell culture plates and at 4°C for at least 30 minutes to solidify the agarose. The heart was then excised from the lung and each lobe of the lungs was embedded on a cutting board with 1% agarose to prevent any movement. The agarose-filled and embedded lungs were then chopped on a MclLwain tissue chopper (GeneQ Inc. Quebec, Canada) into 500 microns thick slices. The lung explants slices were incubated in a cell culture media containing 10% FBS, 1% penicillin/streptomycin and 0.5% fungizone for an hour in 37°C in a humidified chamber with 5% CO ₂ . Each lung slices were then transferred to a 24 well cell culture plate with a specific treatment group for 16 to 24 hours in a 37°C humidified chamber with 5% CO ₂ .

Statistical analysis: The Student t test was used for analyzing the cell survival and growth data. To perform multiple comparisons, one way ANOVA was used followed by

Holm-Sidak or Tukey postanalysis (SigmaStat Software, 3.1, Point Richmond, CA, USA). A value of p < 0.05 was considered significant.

Example 1

MAPK p42/p44 activation by InIB 36 to 321 correlates with cell survival The amino acid segment from 36 to 321 of InIB was shown to bind the c-Met receptor

(Niemann et al. (2008) J. MoI. Biol. 377: 489-500). This portion of the InIB protein was cloned, together with a 6-His tag for purification (Fig. 1). The resulting InIB fragment monomer (having one or more domains of InIB) was expressed in E. coli and purified on a Ni-NTA column (Fig. 2). The purified monomer was treated for the removal of endotoxin and used to treat primary pulmonary endothelial cells to look for activation of p42/p44 MAPK and Akt. Western blots showed that concentrations of InIB fragment (4.2 μg/ml) resulted in the phosphorylation of MAPK at 15 minutes, although higher concentrations (42 μg/ml) actually had less phosphorylation, suggesting either some self-interaction of the peptide or non-competitive inhibition for the c-Met receptor (Fig. 3A). Activation of Akt by the InIB monomer was not observed.

Recent laboratory findings examining NK2 activity in primary epithelial and endothelial cell culture showed that although NK2 activates only the p42/p44 MAPK pathway and not the PI3K/Akt signaling pathway, this isoform can induce cell survival (data not shown). This is in contrast with findings in transformed cell lines, in which NK2 does not induce cell survival (Day et al. (1999) Oncogene 18: 3399-3406). Therefore, the cell survival activity of InIB fragment tandem dimer in the presence of Ang II, which induces the intrinsic pathway of apoptosis in cells, was examined. Pretreatment of cells for 24 hours with the InIB fragment tandem dimer (4.2 μg/ml) results in significant protection of primary endothelial cells from Ang II-induced apoptosis (from 61% ± 0.06 apoptosis to 28% ± 0.03, p<0.05) (Fig. 3B). Full length HGF caused a further reduction of apoptosis to 11% ± 0.01, p<0.05. Addition of HGF or InIB fragment alone did not statistically alter the apoptosis from basal levels. Example 2

MAPK p42/p44 & PI3K/Akt activation by InIB 36 to 321 modified tandem dimer correlates with cellular proliferation and increased cellular motility

A tandem dimer of the InIB segment (amino acids 36 to 321 of InIB) was cloned with a 6-His tag and purified on the Ni-NTA column (Figs. 1 , 4). Examination of signal transduction in response to the InIB fragment tandem dimer showed that concentrations as low as 0.04 μg/ml induced phosphorylation of both p42/p44 MAPK and Akt (Fig. 5). At the 10-fold higher concentration, the InIB fragment monomer induced only low levels of MAPK phosphorylation, and Akt phosphorylation could not be detected (Fig 5A). The InIB fragment tandem dimer protected cells from apoptosis by Ang II to basal levels (Fig. 6A) and prevented DNA fragmentation (Fig. 6B). Whereas Ang II induced apoptosis to 73% ± 0.06, 24 hours pretreatment with 0.4 or 0.04 μg/ml InIB fragment tandem dimer resulted in 27% ± 0.09 for both concentrations (p<0.05), suggesting that maximal protection is obtained at 0.04 μg/ml. The InIB fragment dimer also prevented Ang II-induced apoptosis in lung tissue explants, preventing the activation of caspase 3 (Fig. 7). The InIB fragment dimer also prevented bleomycin-induced DNA fragmentation (Fig. 8). Activation of both MAPK pathways has been associated with HGF-induced migration (Day et al. (1999) Oncogene 18: 3399-3406), but activation of PI3K has also been shown to participate in migratory pathways. It was found that the InIB tandem dimer induced cell migration to levels similar to that of full length HGF (Fig. 9). This was significantly higher than migration induced by the InIB fragment monomer (133.3 x 10 ⁵ ± 10.5 x 10 ⁵ oftandem dimer to 89.3 x 10 ⁵ ± 14.3 x 10 ⁵ of InIB fragment monomer). Because the peptide dimer activates the PI3K/Akt pathway in addition to p42/p44 MAPK, cell growth in primary endothelial cells was also investigated. It was found that the InIB fragment tandem dimer caused increased proliferation (Fig. 10). The InIB tandem dimer increased cell numbers: at day 6 basal 5 x 10 ⁴ to InIB 8.8 x 10 ⁴ ± 0.29 x 10 ⁴ (p<0.05); at day 8 to InIB 13.75 x 10 ⁴ ± 0.66 x l0 ⁴ (p<0.05); and at day 10 to InIB 18.67 x 10 ⁴ ± 0.63 x lO ⁴ (p<0.05). The observed increase in cell growth was equivalent to that observed for full length HGF at all time points. In agreement with previous findings (Niemann et al. (2008) J. MoI. Biol. YIl: 489-500), increased proliferation by the InIB fragment monomer was not observed (Fig. 10).

While the invention has been described and illustrated herein by references to various specific materials, procedures and examples, it is understood that the invention is not restricted to the particular combinations of materials and procedures selected for that purpose. Numerous variations of such details can be implied as will be appreciated by those skilled in the art. It is intended that the specification and examples be considered as exemplary, only, with the true scope and spirit of the invention being indicated by the following claims. All references, patents, and patent applications referred to in this application are herein incorporated by reference in their entirety.