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  Pharmaceutical Patents  

 

Title:  Treatment of disease by inducing cell apoptosis
United States Patent: 
7,402,567
Issued: 
July 22, 2008

Inventors: 
Chojkier; Mario (San Diego, CA), Buck; Martina (San Diego, CA)
Assignee: 
The Regents of the University of California (Oakland, CA)
The United States of America as represented by the Department of Veterans Affairs, Office of the General Counsel (024) (Washington, DC)

Appl. No.: 
10/415,325
Filed: 
October 26, 2001
PCT Filed: 
October 26, 2001
PCT No.: 
PCT/US01/51123
371(c)(1),(2),(4) Date: 
September 08, 2003
PCT Pub. No.: 
WO02/46218
PCT Pub. Date: 
June 13, 2002


 

Training Courses -- Pharm/Biotech/etc.


Abstract

The present invention relates generally to the treatment and prevention of diseases characterized by excess cell proliferation and/or activation. In particular, the present invention provides compositions and methods to suppress the activation and/or proliferation of various cells. In preferred embodiments, the present invention provides compositions and methods to suppress the activation and/or proliferation of mesenchymally derived cells (including, but not limited to hepatic stellate cells), as well as cells with abnormal growth characteristics. In particularly preferred embodiments, the present invention provides compositions and methods to induce fibrosis.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention relates generally to the treatment and prevention of diseases characterized by excess cell proliferation and/or activation. In particular, the present invention provides compositions and methods to suppress the activation and/or proliferation of various cells, including but not limited to mesenchymal cells. In preferred embodiments, the present invention provides compositions and methods to suppress the activation and/or proliferation of mesenchymally derived cells (including, but not limited to hepatic stellate cells), as well as cells with abnormal growth characteristics. In particularly preferred embodiments, the present invention provides compositions and methods to inhibit or eliminate fibrosis. In alternative preferred embodiments, the present invention provides compositions and methods to induce fibrosis.

The present invention also provides methods and compositions suitable for the suppression of cell activation and/or proliferation. In some preferred embodiments, the present invention also provides methods and compositions suitable for the suppression of hepatic stellate cell activation and/or proliferation. In particularly preferred embodiments, the present invention provides methods for administering C/EBP.beta. with a mutation of Thr.sup.217 to Ala. In other embodiments, the endogenous phosphopeptidases associate with caspases 1 and 8 and result in the inhibition of their activation. In alternative embodiments, the mutant Ala.sup.217 peptides compete with the wild-type peptides, allowing the activation of caspases, resulting in apoptosis.

The modified C/EBP.beta. peptides of the present invention find use with various tissues and cells. It is contemplated that any suitable route of administration will find use with the present invention. Thus, in some embodiments, the peptides are administered using genetic therapy methods, selective peptide delivery systems, or any other suitable method for delivery to the site of interest. In particularly preferred embodiments, the peptides are delivered to hepatic stellate cells. In alternative preferred embodiments, the peptides are administered parenterally while in still further embodiments, the peptides are administered orally or topically.

In some embodiments, the composition(s) of the present invention is/are administered to the subject in a single dose, while in other embodiments, the composition is administered to the subject in multiple doses. In preferred embodiments, the administering is selected from the group consisting of subcutaneous injection, oral administration, intravenous administration, intraarterial administration, intraperitoneal administration, rectal administration, vaginal administration, topical administration, intramuscular administration, intranasal administration, intrapulmonary administration (e.g., inhalation, insufflation, etc.), intratracheal administration, epidermal administration, transdermal administration, subconjunctival administration, intraocular administration, periocular administration, retrobulbar administration, subretinal administration, suprachoroidal administration, intramedullar administration, intracranial administration, intraventricular administration, and intrathecal administration. In alternative embodiments, the administering is administration from a source selected from the group consisting of mechanical reservoirs, devices, implants, and patches. In still further embodiments, the composition is in a form selected from the group consisting of pills, capsules, liquids, gels, powders, suppositories, suspensions, creams, jellies, aerosol sprays, and dietary supplements. Additionally, the peptides may be administered as an ointment, lotion or gel (i.e., for the treatment of skin and mucosal areas). In some embodiments, it is expected that cells in several tissues will contain an expression vector and express the gene of interest (i.e., such that the peptide(s) of interest are expressed in the tissue(s)).

In alternative embodiments, once the peptides are incorporated into cells, the peptides compete with endogenous C/EBP wild-type protein. In still further embodiments, the presence of mutant C/EBP.beta. peptide(s) results in the induction of stellate cells apoptosis. In other embodiments, the activation and proliferation of hepatic stellate cells is prevented. In particularly preferred embodiments, this prevention of activation and proliferation of hepatic stellate cells results in decreased or complete cessation of fibrous tissue production in the liver. In some embodiments, the methods are used to treat subjects suffering from or suspected of suffering from chronic liver disease (e.g., including but not limited to hepatitis C, hepatitis B, alcoholism, toxic and genetic liver diseases). Furthermore, it is contemplated that the methods of the present invention will find use in the prevention of liver fibrosis associated with liver rejection following liver transplantation.

The present invention provides modified C/EBP.beta. peptides from various species. In some preferred embodiments, the murine C/EBP.beta. peptide comprises a mutation at amino acid 217. In some embodiments, the threonine present in wild-type murine C/EBP.beta. at position 217 (SEQ ID NO:2) is replaced with alanine (SEQ ID NO:3), while in other embodiments, it is replaced with glutamic acid (SEQ ID NO:4). In alternative preferred embodiments, the rat C/EBP.beta. peptide comprises a mutation at amino acid 105. In some embodiments, the serine at position 105 of the wild-type rat C/EBP.beta. (SEQ ID NO:10) is replaced with alanine (SEQ ID NO:11), while in other embodiments, it is replaced with aspartic acid (SEQ ID NO:12). In still further preferred embodiments, the human C/EBP.beta. comprises a mutation at amino acid 266. In some embodiments, the threonine at position 266 of the wild-type human C/EBP.beta. (SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:19, and SEQ ID NO:23) is replaced with alanine (SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:20, and SEQ ID NO:24), while in other embodiments, it is replaced with glutamic acid (SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:21, and SEQ ID NO:25). Other mutations in C/EBP.beta. peptides from various species are contemplated by the present invention. The only requirement of these modified C/EBP.beta. peptides is that they are capable of inducing or prevent apoptosis in a cell or tissue of interest. In particularly preferred embodiments, the induction of apoptosis results in the reduction (i.e., diminution), elimination, and/or prevention of fibrosis in a cell or tissue of interest. In alternative preferred embodiments, the modified C/EBP.beta. peptides induce fibrosis (e.g., to promote wound healing).

In some preferred embodiments, the present invention provides modified CCAAT/Enhancer binding proteins capable of inducing apoptosis. In some preferred embodiments, the modified CCAAT/Enhancer binding protein is selected from the group consisting of modified human CCAAT/Enhancer binding proteins, and modified mouse CCAAT/Enhancer binding proteins. In further preferred embodiments, the protein is encoded by an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:24, and SEQ ID NO:25.

The present invention also provides methods for inducing apoptosis comprising the steps of administering at least one modified CCAAT/Enhancer binding protein to at least one cell. In some preferred embodiments, the cell is a mesenchymal cell. In further embodiments, the cell is selected from the group consisting of hepatic cells, lung cells, kidney cells, skin cells, muscle cells, heart cells, glial cells, ocular cells, and vascular cells. In some particularly preferred embodiments, the administration prevents fibrosis. In alternative preferred embodiments, the administration ameloriates fibrosis.

The present invention also provides methods for inducing apoptosis comprising administering the CCAAT/Enhancer binding protein to a subject under conditions such that the endogenous phosphopeptides of the subject inhibit the activation of at least one caspase of the subject. In some preferred embodiments, the administration results in the apoptosis of selected cells within the subject.

The present invention further provides methods for inducing apoptosis comprising administering at least a portion of a modified CCAAT/Enhancer binding protein to at least one cell, wherein the modified CCAAT/Enhancer binding protein is selected from the group consisting of modified murine, modified rat, and modified human CCAAT/Enhancer binding proteins. In some preferred embodiments, the modified murine CCAAT/Enhancer binding protein comprises a mutation at amino acid position 217, wherein the amino acid at position 217 is selected from the group consisting of alanine and glutamic acid. In other preferred embodiments, the modified murine CCAAT/Enhancer binding protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:4. In additional embodiments, the modified human CCAAT/Enhancer binding protein comprises a mutation at amino acid position 266, wherein the amino acid at position 266 is selected from the group consisting of alanine and glutamic acid. In further embodiments, the modified human CCAAT/Enhancer binding protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:11. In yet additional embodiments, the modified rat CCAAT/Enhancer binding protein comprises a mutation at amino acid position 105, wherein the amino acid at position 105 is selected from the group consisting of alanine and aspartic acid. In further embodiments, the modified rat CCAAT/Enhancer binding protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:14 and SEQ ID NO:15. In some preferred embodiments, apoptosis is induced in at least one cell selected from the group consisting of hepatic cells, heart cells, lung cells, kidney cells, ocular cells, neural cells, muscle cells, epithelial cells, endothelial cells, mesenchymal cells, and skin cells. In some preferred embodiments, the cell(s) is/are within a subject. In further embodiments, the subject is a mammal. In some particularly preferred embodiments, the mammal is a human. In additional embodiments, the human is suffering from a fibrosis-related disease, wherein the fibrosis-related disease is selected from the group consisting of hepatic disease, brain damage, myocardial infarction, arteriosclerosis, ocular fibrosis, fibrotic skin conditions, and fibrotic pulmonary disease.

The present invention also provides methods for inducing fibrosis comprising the administration of at least a portion of a modified CCAAT/Enhancer binding protein to at least one tissue, wherein the modified CCAAT/Enhancer binding protein is selected from the group consisting of modified murine, modified rat and modified human CCAAT/Enhancer binding proteins. In some preferred embodiments, prior to the administration of CCAAT/Enhancer binding protein, at least one tissue exhibits impaired wound healing. In some embodiments, the induction of fibrosis provides improved wound healing in at least one tissue. In some particularly preferred embodiments, the modified CCAAT/Enhancer binding protein is the amino acid sequence set forth in SEQ ID NO:4.

DESCRIPTION OF THE INVENTION

The present invention relates generally to the treatment and prevention of diseases characterized by excess cell proliferation and/or activation. In particular, the present invention provides compositions and methods to suppress the activation and/or proliferation of various cells. In preferred embodiments, the present invention provides compositions and methods to suppress the activation and/or proliferation of mesenchymally derived cells (including, but not limited to hepatic stellate cells), as well as cells with abnormal growth characteristics. In particularly preferred embodiments, the present invention provides compositions and methods to inhibit or eliminate fibrosis. In alternative preferred embodiments, the present invention provides compositions and methods to induce fibrosis.

The CCAAT/Enhancer Binding Protein .beta. (C/EBP.beta.) (Descombes et al., Genes Dev., 4:1541-1551 [1990]; and Akira et al., EMBO J., 9:1897-1906 [1990]) mediates the proliferative effects of oxidative stress and of ribosomal protein S-6 kinase (RSK) activated by transforming growth factor (TGF) .alpha. in colonic cancer cells (Chinery et al., Nat. Med., 3:1233-1241 [1997]) and primary mouse hepatocytes (Buck et al., Mol. Cell., 4:1087-1092 [1999]), respectively. However, the mechanisms linking C/EBP.beta. (LAP, NF-IL6) to cell proliferation are unknown. Indeed, an understanding of these mechanisms is not necessary in order to use the present invention.

Activation of the ERK/MAPK signal transduction pathway promotes cell proliferation through several mechanisms, including stimulation of nucleotide synthesis, gene expression, protein synthesis and cell growth (Whitmarsh and Davis, J. Mol. Med., 74:589-607 [2000]). Many of these MAPK's roles in cell growth are mediated by p90RSK (Nebreda and Gavin, Science 286:1309-1310 [1999]). RSK phosphorylates and inactivates the pro-apoptotic protein BAD (Bonni et al., Science 286:1358-1362 [1999]); up-regulates transcription of the anti-apoptotic gene Bcl-2 through phosphorylation and activation of CREB (Bonni et al., supra); and facilitates gene expression by inducing chromatin remodeling via phosphorylation of histone H3 (Sassone-Corsi et al., Science 285:886-891 [1999]).

In response to epidermal growth factor, the ERK/MAPK cascade regulates the de novo synthesis of pyrimidine nucleotides by activating carbamoyl phosphate synthetase II (Graves et al., Nature 403:328-332 [2000]). Other mechanisms by which the ERK/MAPK signaling pathway modulates cell survival and the cell cycle (Whitmarsh and Davis, J. Mol. Med., 403:255-256 [1996]) include: a) activation of transcription by phosphorylation of transcriptional factors (Whitmarsh and Davis, J. Mol. Med., 74:589-607 [1996]; Bhatt and Ferrell, Science 286:1362-1365 [1999]) and histone H3 and HMG-14 (Sassone-Corsi et al., supra; and Thomson et al., EMBO J., 17:4779-4793 [1999]); b) stimulation of translation through phosphorylation of initiation factor 4E (eIF-4E) (Pyronnet et al., EMBO J., 18:270-279 [1999]); and c) promotion of DNA replication by increasing expression of cyclin D1 (Lavoie et al., J. Biol. Chem., 271:206086-20616 [1996]). RSK, which is activated by ERK/MAPK phosphorylation, plays an essential role in the ERK/MAPK signaling pathway regulating cell survival and the cell cycle (Bonni et al., supra; Bhatt and Ferrell, supra; Gross et al., Science 286:1365-1367 [1999]; and Sassone-Corsi et al., supra). Thus, the function of C/EBP.beta. in cell survival mediated by RSK was of interest in the development of the present invention.

Primary mouse hepatic stellate cells were used during the development of the present invention, as overproduction of fibrous tissue by these cells (Friedman et al., Proc. Natl. Acad. Sci. USA 82:8681-8685 [1985]; and Ankoma-Sey and Friedman, in Strain and Diehl (eds), Liver Growth and Repair, Chapman & Hall, London, pages 512-537 [1998]) is a critical step in the development of liver cirrhosis following liver injury (Chojkier, in Strain and Diehl (eds.) Liver Growth and Repair, supra, at pages 430-450). Furthermore, these cells remain quiescent, as in the normal liver (Lee et al., J. Clin. Invest., 96:2461-2468 [1995]; and Ankoma-Sey and Friedman, supra), when cultured on an EHS (Matrigel) matrix but are rapidly activated and proliferate with the induction of oxidative stress by either collagen type I or TGF.alpha. in culture (Lee et al., supra; and Friedman et al., J. Biol. Chem., 264:10756-10762), which also modulate C/EBP.beta.'s effects on cell growth (Chinery et al., supra; and Buck et al., [1999], supra). Similarly, stellate cell activation follows the stimulation of oxidative stress both in experimental liver injury with CCl.sub.4 (Houglum et al., J. Clin. Invest., 96:2269-2276 [1995]) and in human liver diseases induced by alcohol, genetic hemochromatosis, porphyria and viral hepatitis (Chojkier, supra). However, until the development of the present invention, it was unknown that the survival dependency of activated hepatic stellate cells requires phosphorylation of C/EBP.beta. (e.g., mouse C/EBP.beta.; (m) C/EBP.beta.) on Thr.sup.217 by RSK. As described in greater detail herein, the hepatotoxin CCl.sub.4 induced activation of RSK, phosphorylation of C/EBP.beta. on Thr.sup.217 and proliferation of stellate cells in normal mice, but caused apoptosis of these cells in C/EBP.beta..sup.-/- and C/EBP.beta.-Ala.sup.217 (a dominant negative non-phosphorylatable mutant) transgenic mice. In other words, following the induction of hepatic oxidative stress with CCl.sub.4, stellate cells from C/EBP.beta..sup.-/- or C/EBP.beta.-Ala.sup.217 (a non-phosphorylatable mutant) transgenic, but not C/EBP.beta..sup.+/+, mice developed apoptosis. Furthermore, both C/EBP.beta.-PThr.sup.217 and the phosphorylation mimic C/EBP.beta.-Glu.sup.217, but not C/EBP.beta.-Ala.sup.217, were found to associate with procaspases 1 and 8 in vitro and in vivo, and inhibit their activation. The data obtained during the development of the present invention indicate that C/EBP.beta. phosphorylation of Thr.sup.217 creates a functional XEXD caspase substrate/inhibitor box (KPhospho-T.sup.217VD) that is mimicked by C/EBP.beta.-Glu.sup.217 (KE.sup.217VD). Consistent with this observation, C/EBP.beta..sup.-/- and C/EBP-Ala.sup.217 stellate cells were rescued from apoptosis by either the cell permeant KE.sup.217VD tetrapeptide or C/EBP.beta.-Glu.sup.217, as described in greater detail herein.

Results obtained during the development of the present invention and described in further detail herein, indicate that there is a novel C/EBP.beta. mechanism for cell survival downstream of RSK, preventing activation of procaspases 1 and 8 (Thornberry and Lazebnik, Science 281:1312-1316 [1998]). These caspases activate downstream effector procaspases (Earnshaw et al., Ann. Rev. Biochem., 68:383-424 [1999]). Physiologically relevant signaling pathways in stellate cells, such as CCl.sub.4 in mice and collagen type I in culture (Friedman et al., J. Biol. Chem., 264:10756-10762 [1989]; and Rudolph et al., Science 287:1253-1258 [2000]), result in the activation of RSK and phosphorylation of endogenous C/EBP.beta. on Thr.sup.217. C/EBP.beta.-PThr.sup.217, but not unphosphorylated C/EBP.beta., associates with procaspases 1 and 8 (as detected by immunofluorescence, co-immunoprecipitation and direct in vitro association of recombinant proteins) and inhibits their processing, which blocks the apoptotic cascades and allows survival of stellate cells. Thus, the present data provide the first demonstration that phosphorylation of a transcription factor by the ERK/MAPK/RSK pathway stimulates its association with procaspases, preventing their activation. Although it is possible that phosphorylation of C/EBP.beta. on Thr.sup.217 to create a functional XEXD caspase substrate/inhibitor box (Thornberry et al., J. Biol. Chem., 272:17907-17911 [1997]; and Blanchard et al., J. Mol. Biol., 302:9-16 [2000]) explains the anti-apoptotic role of C/EBP.beta., structural analysis is required to elucidate the exact mechanism. Regardless, an understanding of the mechanisms is not necessary in order to use the present invention.

The inhibition of procaspase 1 and 8 activation by C/EBP.beta.-PThr.sup.217 occurs under conditions in which C/EBP.beta. expression is physiologically induced, such as activation of normal stellate cells either in mice treated with the hepatotoxin CCl.sub.4 or in culture on a collagen type I matrix. Moreover, apoptosis is induced under these conditions in stellate cells from C/EBP.beta..sup.-/- mice. These experiments indicate that the findings are not the spurious result of overexpressing C/EBP.beta.. Expression of C/EBP.beta.-Ala.sup.217 in transgenic mice also induced apoptosis of stellate cells upon exposure to growth stimuli (CCl.sub.4 in mice and collagen in culture). In contrast, the phosphorylation mimic C/EBP.beta.-Glu.sup.217 mutant rescued cells from apoptosis induced either by expressing MEKK1, IKB.alpha. or RSK dominant negative mutants or by treating the cells with the proteasome inhibitor lactacystin (Chen et al., Nature 374:386-388 [1995b]; Nakajima et al., Cell 86:465-474 [1996]; and Lee et al., Cell 88:213-222 [1997]). Expression of C/EBP.beta.-Glu.sup.217 in stellate cells also prevented FAS-induced apoptosis, which is mediated by activation of procaspase 8 (Ashkenazi and Dixit, Science 281:1305-1208 [1998]), but not apoptosis induced by serum deprivation, which is mediated by activation of procaspase 9 (Joza et al., Nature 410:549-554 [2001]). These results indicate selective effects of C/EBP.beta.-PThr.sup.217 on apoptosis. C/EBP.beta.'s activation and dimerization domains are not necessary for its association with or inhibition of procaspases 1 and 8. Furthermore, preliminary experiments in stellate cells using reporter genes, which contain C/EBP.beta. binding domains (Descombes et al. supra; and Houglum et al., J. Clin. Invest., 94:808-814 [1994]), did not show differences in transcription activation between C/EBP.beta. wild type and the phosphorylation or Ala.sup.219 mutants.

C/EBP.beta.-PThr.sup.217 is mimicked by C/EBP.beta.-Glu.sup.217 (KE.sup.217VD) and therefore, conforms to the current knowledge about the structural requirements for caspase inhibitory/substrate tetrapeptides. These tetrapeptides require an aspartic acid (D) residue at the P1 position (Thornberry and Lazebnik, supra), which is also present in C/EBP.beta. at amino acid D.sup.219 (Cao et al., Genes Develop., 5:1538-1552 [1991]). Using a positional scanning substrate library, it has been shown that the optimal sequence for caspase 8 is I/L/V (P4 position) EXD, although I, V, W, T, P and D are also acceptable at the P4 position, by virtue of being downstream cleavage sites in the apoptotic cascade (Thornberry et al., supra). These findings have been recently corroborated by crystallographic studies of caspase 8. (Blanchard et al., supra). Although group III caspases (including caspase 8) have a preference for small hydrophobic residues at P4, kinetic and crystallographic studies have shown that DEVD, a specific group II inhibitor, containing a hydrophilic residue at this position, interacts favorably with the enzyme in subsite S4, and it is an almost equally potent inhibitor as the specific group III inhibitor IETD (Blanchard et al., supra). The K.sup.216 in C/EBP.beta. has a similar hydrophobicity as D (Boyle et al., Meth. Enzymol., 201:110-149 [1991]). In addition, Blanchard et al. argue that subsite S3 of caspase 8, which interacts with position P3 (but not with P4) of the tetrapeptide, is crucial for determining the specificity, and that the original classification of caspases need to be revised, especially for caspase 8 (Blanchard et al., supra). Like the selective effects of C/EBP.beta.-PThr.sup.217 observed during the development of the present invention, CrmA inhibits procaspases 1 and 8 selectively compared to procaspases 3, 6 and 7 (Zhou et al., J. Biol. Chem., 272:7797-7800 [1997]). However, other reports have suggested that CrmA also inhibits caspases 4, 5, 9 and 10 (Garcia-Calvo et al., J. Biol. Chem., 273:32608-32613 [1998]). In addition, the baculovirus p35 is a potent, albeit less selective, inhibitor of procaspases 1, 3, 6, 8, and 10 (Andrade et al., Immun., 8:451-460 [1998]). In addition, experiments conducted during the development of the present invention have shown that the sequence K-Phospho-T.sup.217VD (or KE.sup.217VD) within C/EBP.beta. is effective in associating with procaspases 1 and 8 and blocking their activation.

The experiments with C/EBP.beta..sup.-/- cells and with dominant negative Ala.sup.217 and dominant positive Glu.sup.217 mutants, including C/EBP.beta.-Ala.sup.217 transgenic mice, strongly support the present invention. The C/EBP.beta.-Ala.sup.217 mutant associates with RSK and acts as a dominant negative, following proliferation of stellate cells in animals and in culture induced by CCl.sub.4 and collagen type I, respectively. The synthetic Ac-KA.sup.217VD-CHO peptide stimulates apoptosis of stellate cells, resembling the dominant negative effects of C/EBP.beta.-Ala.sup.217 and C/EBP.beta.216-253-Ala.sup.217. These effects could result from the inhibition of RSK and/or the facilitation of procaspases 1 and 8 activation, possibly by impeding the binding of C/EBP.beta.-PThr.sup.217 to procaspases 1 and 8, while allowing their self-cleavage. Nonetheless, an understanding of the mechanisms is not necessary in order to use the present invention.

Although rC/EBP.beta. has a naturally occurring Ala for Thr substitution at position 217, rC/EBP.beta. also has a Ser for Ala substitution at position 105 (Buck et al., [1999], supra). It was also observed that endogenous rC/EBP.beta. is phosphorylated on Ser.sup.105 (the RSK rat phosphoacceptor homologue). Expression of the dominant positive rC/EBP.beta.-Asp.sup.105 was sufficient to rescue cells from apoptosis induced by expressing a dominant negative mutant RSK or by treatment with a proteasome inhibitor. Activation of the PKC.alpha. or MAPK signaling pathways results in phosphorylation of rC/EBP.beta. on Ser.sup.105 (Trautwein et al., Nature 364:544-547 [1993]; and Buck et al. [1999], supra). Moreover, stellate cells isolated from rC/EBP.beta.-Asp.sup.105 transgenic mice were refractory to the induction of apoptosis by lactacystin. As described for C/EBP.beta., rC/EBP.beta. peptides containing either P-Ser.sup.105 or its phosphorylation mimic Asp.sup.105, associated with procaspases 1 and 8 in rat stellate cells. The Ac-KKPD.sup.105-CHO tetrapeptide also inhibited activation of procaspase 8. These data indicate that although C/EBP.beta. or rC/EBP.beta. are phosphorylated on different amino acid residues, both phosphorylated proteins are sufficient to rescue cells from apoptosis mediated by caspase 8.

In contrast, the non-phosphorylatable rC/EBP.beta.-Ala.sup.105 mutant behaves as a dominant negative (comparable to the C/EBP.beta.-Ala.sup.217 mutant), inducing apoptosis of both mouse and rat stellate cells. The rC/EBP.beta.-PSer.sup.105 sequence is mimicked by KKPD.sup.105, which contains the indispensable D at the P1 position and the highly preferred P at the P2 position as a substrate/inhibitor (XXPD) for granzyme B, as determined by using synthetic substrate libraries (Harris et al., J. Biol. Chem., 273:27364-27373 [1998]). The initiator caspases, caspase 8 and granzyme B, share the tetrapeptide IETD sequence of procaspase 3 as a substrate/inhibitor (Thornberry et al., 1997, supra; Harris et al., supra), indicating that caspase 8 may also recognize the XXPD substrate. This was confirmed by the experimental data obtained during the development of the present invention. The tetrapeptide Ac-KKPD.sup.105-CHO (rC/EBP.beta.) significantly inhibited apoptosis of stellate cells from C/EBP.beta..sup.-/- mice.

The data obtained during the development of the present invention are relevant to diseases that result from the activation of mesenchymal cells, which leads to excessive tissue repair mechanisms (e.g., brain gliosis, liver cirrhosis, and lung and kidney fibrosis). The results described herein indicate that the nonphosphorylatable tetrapeptides of the C/EBP.beta. RSK phosphoacceptor are suitable for therapeutic uses, as they induce apoptosis of various cells following their activation. Thus, for hepatic stellate cells, these tetrapeptides find use in the prevention of the development of liver fibrosis and cirrhosis. Indeed, C/EBP.beta.-Ala.sup.217 transgenic mice are refractory to the induction of liver fibrosis and cirrhosis following the chronic administration of CCl.sub.4. In addition, because IL-1 has been implicated in the pathogenesis of acute neurodegeneration (Rothwell et al., J. Clin. Invest., 100:2648-2652 ([1997]), inhibition of caspase 1 activation and its processing of the IL-1 precursor is contemplated to ameliorate diseases associated with fibrosis and/or apoptosis.

The creation of a functional XEXD caspase inhibitory box by phosphorylation reported herein, may be a prevalent biological mechanism through evolution. Indeed, potential functional XEXD boxes that would be created upon phosphorylation of threonine (Phospho-TVD, analogous to EVD) were identified in more than 22,000 sites and of serine (EV-Phospho-S, analogous to EVD) in more than 27,000 sites in a database containing .about.350,000 proteins. If only 1% of these sequences is phosphorylated in vivo, this would generate functional XEXD caspase inhibitory boxes in .about.500 proteins. However, as indicated herein, an understanding of the mechanisms involved is not necessary in order to use the present invention.

As discussed in greater detail herein, in one embodiment of the present invention, polynucleotide sequences or fragments thereof which encode C/EBP.beta. or fusion proteins or functional equivalents thereof, may be used in recombinant DNA molecules to direct expression of C/EBP.beta. in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be produced and these sequences may be used to clone and express C/EBP.beta..

As will be understood by those of skill in the art, it may be advantageous to produce C/EBP.beta. nucleotide sequences possessing non-naturally occurring codons. For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce a recombinant RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.

The nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter C/EBP.beta.-encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the gene product. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. For example, site-directed mutagenesis may be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, or introduce mutations, and so forth. In another embodiment of the present invention, natural, modified, or recombinant nucleic acid sequences encoding C/EBP.beta. may be ligated to a heterologous sequence to encode a fusion protein. For example, to screen peptide libraries for inhibitors of human C/EBP.beta. activity, it may be useful to encode a chimeric (e.g., human) C/EBP.beta. protein that can be recognized by a commercially available antibody. A fusion protein may also be engineered to contain a cleavage site located between the C/EBP.beta. encoding sequence and the heterologous protein sequence, so that the C/EBP.beta. may be cleaved and purified away from the heterologous moiety.

In another embodiment of the present invention, sequences encoding C/EBP.beta. may be synthesized, in whole or in part, using chemical methods well known in the art (See e.g., Caruthers et al. Nucl. Acids Res. Symp. Ser., 215-223 [1980]; and Horn et al., Nucl. Acids Res. Symp. Ser., 225-232 [1980]). Alternatively, the protein itself may be produced using chemical methods to synthesize the amino acid sequence of C/EBP.beta., or a portion thereof. For example, peptide synthesis can be performed using various solid-phase techniques (Roberge et al., Science 269:202-204 [1995]) and automated synthesis may be achieved, for example, using commercially available synthesizers. These synthesized peptide may be substantially purified by preparative high performance liquid chromatography (HPLC), using any suitable method known in the art. The composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure). Additionally, the amino acid sequence of C/EBP.beta. or any part thereof, may be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins, or any part thereof, to produce a variant polypeptide.

As described herein in greater detail, in order to express a biologically active C/EBP.beta. the nucleotide sequences encoding C/EBP.beta. or functional equivalents, may be inserted into appropriate expression vector (i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence). Methods well known to those skilled in the art find use in the construction of expression vectors containing sequences encoding C/EBP.beta. and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. A variety of expression vector/host systems may be utilized to contain and express sequences encoding C/EBP.beta.. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Control elements and regulatory sequences are also contemplated, as appropriate.

In addition, a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a "prepro" form of the protein may also be used to facilitate correct insertion, folding and/or function. Different host cells known in the art which have specific cellular machinery and characteristic mechanisms for such post-translational activities, may be chosen to ensure the correct modification and processing of the foreign protein.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express C/EBP.beta. may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type. Any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase, adenine phosphoribosyltransferase, and various other suitable systems known in the art. Also, antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection. Although the presence/absence of marker gene expression suggests that the gene of interest is also present, its presence and expression may need to be confirmed. For example, if the sequence encoding C/EBP.beta. is inserted within a marker gene sequence, recombinant cells containing sequences encoding human C/EBP.beta. can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding C/EBP.beta. under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.

Alternatively, host cells which contain the nucleic acid sequence encoding and express C/EBP.beta. may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include membrane, solution, or chip-based technologies for the detection and/or quantification of nucleic acid or protein.

A variety of protocols for detecting and measuring the expression of human C/EBP.beta., using either polyclonal or monoclonal antibodies specific for the protein known in the art find use in the present invention. Examples include enzyme-linked immunosorbant assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).

A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding C/EBP.beta. include oligolabeling, nick translation, end-labeling or PCR amplification using a labeled nucleotide. Alternatively, the sequences encoding C/EBP.beta. or any portions thereof may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commercially available kit. Suitable reporter molecules or labels, which may be used, include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like.

Host cells transformed with nucleotide sequences encoding C/EBP.beta. may be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The protein produced by a recombinant cell may be secreted or contained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides which encode C/EBP.beta. may be designed to contain signal sequences which direct secretion of portions of C/EBP.beta. through a prokaryotic or eukaryotic cell membrane. Other recombinant constructions may be used to join sequences encoding C/EBP.beta. to nucleotide sequences encoding a polypeptide domain which will facilitate purification of soluble proteins. Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, Wash.). The inclusion of cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen, San Diego, Calif.) between the purification domain and human C/EBP.beta. may be used to facilitate purification.

In addition to recombinant production, fragments of C/EBP.beta. may be produced by direct peptide synthesis using solid-phase techniques that are well known in the art (See e.g., Merrifield, J. Am. Chem. Soc., 85:2149-2154 [1963]). Protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved by any commercially available synthesizer suitable for the project. In addition, various fragments of C/EBP.beta. may be chemically synthesized separately and combined using chemical methods to produce the full length molecule.
 

Claim 1 of 9 Claims

1. A method for inducing apoptosis comprising administering a composition to hepatic stellate cells, wherein said composition comprises N-acetyl-KAVD-C-aldehyde peptide (Ac-KAVD-CHO) consisting of residues 216-219 of SEQ ID NO:3 or N-acetyl-KKPA-C-aldehyde peptide (Ac-KKPA-CHO) consisting of residues 102-105 of SEQ ID NO:14.
 

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