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Title:  150 KDA TGF-B1 accessory receptor acts a negative modulator of TGF-B signaling
United States Patent: 
7,173,002
Issued: 
February 6, 2007

Inventors: 
Philip; Anie (Montreal, CA), Tam; Betty (Ville St. Laurent, CA)
Assignee: 
McGill University (Montreal, CA)
Appl. No.: 
10/475,711
Filed: 
April 24, 2002
PCT Filed: 
April 24, 2002
PCT No.: 
PCT/CA02/00560
371(c)(1),(2),(4) Date: 
May 13, 2004
PCT Pub. No.: 
WO02/085942
PCT Pub. Date: 
October 31, 2002


 

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Abstract

The present invention relates to a TGF-.beta.1 binding protein called r150. This protein has a GPI-anchor contained in r150 itself and not on a tightly associated protein and that it binds TGF-.beta.1 with an affinity comparable to those of the signaling receptors. Furthermore, the released (soluble) form of this protein binds TGF-.beta.1 independent of the types I and II receptors. Also, the soluble form inhibits the binding of TGF-.beta. to its receptor. In addition, evidence that r150 is released from the cell surface by an endogenous phospholipase C is provided. Also, the creation of a mutant human keratinocyte cell line with a defect in GPI synthesis which displays reduced expression of r150 is described. Our results using these mutant keratinocytes suggest that the membrane anchored form of r150 is a negative modulator of TGF-beta responses. These findings, taken together with the observation that r150 forms a heteromeric complex with the signaling receptors, suggest that this accessory receptor in either its membrane anchored or soluble form may antagonize TGF-.beta. responses in human keratinocytes. Experiments with mutants confirmed that TGF.beta.1 activity can be modulated when the expression of the accessory receptor r150 is silenced. The complete nucleic acid and deduced amino acid sequences are now provided. The r150 cloned nucleic acid was used to study overexpression of r150. When r150 gene is overexpressed, TGF.beta. responses are increased. r150 and its derivatives or precursors (fragments, variants and nucleic acids encoding the same) will find a broad clinical utility, knowing that TGF.beta.1 is an important cytokine.

Description of the Invention

BACKGROUND OF THE INVENTION

Transforming growth factor-.beta. (TGF-.beta.) is a 25 kDa multi functional growth factor which plays a central role in the wound healing process (Roberts and Sporn, 1990; O'Kane and Ferguson, 1997). It is an important regulator of the immune response (Letterio and Roberts, 1998), angiogenesis, reepithelialization (Roberts and Sporn, 1990), extracallular matrix protein synthesis and remodeling (Peltonen et al, 1991; Yamamoto et al, 1994). During wound healing, re-epithelialization initiates the repair process which is characterized by recruitment of epidermal stem cells, keratinocyte proliferation and the formation of an epithelial tongue of migrating keratinocytes at the wound edge (Clark, 1996). TGF-.beta. is chemotacfic to keratinocytes and induces the expression of integrins on the migrating epithelium (Helbda, 1988; Zambruno et al, 1995). In spite of its promigratory effect on keratinocytes, TGF-.beta. is a potent inhibitor to epithelial cell proliferation in vitro (Pietenpol et at, 1990) and in vivo (Glick et al, 1993). Targeted deletion of the TGF-.beta.1 gene in keratinocytes causes rapid progression to squamous cell carcinoma (Glick et al, 1994). In addition, the epidermis of transgenic mice expressing a dominant negative TGF-.beta. receptor exhibits a hyperplastic and hyperkeratotic phenotype (Wang et al, 1997). These results support the importance of proper expression of TGF-.beta. and regulation of its function in epidermal development and maintenance of epidermal homeostasis.

TGF-.beta. is a member of the TGF-.beta. superfamily which also include activins, inhibins, bone morphogenic proteins, growth differentiation factor 1 (GDF-1) and glial-derived neurotropic growth factor (GDNF) (Kingsley, 1994).

There are three widely distributed TGF-.beta. receptors, type I, type II and type III, all of which have been cloned (Roberts and Sporn, 1990; Massague, 1998). The types I and II receptors are both transmembrane serine/threonine kinases that are essential for TGF-.beta. signal transduction. The type III receptor, also known as betaglycan, is a high molecular weight proteoglycan that is not required for signaling, but is believed to play a role in presenting the ligand to the type II receptor (Lopez-Casillas et al, 1993). Endoglin, is another TGF-.beta. receptor predominantly expressed on endothelial cells (Gougos and Letarte, 1990). According to the present model of TGF-.beta. signal transduction, binding of TGF-.beta. to the type II receptor which is a constitutively active kinase, leads to the recruitment and phosphorylation of the type I receptor (Wrana et al, 1994). The activated type I kinase phosphorylates the central intracellular mediators of TGF-.beta. signalling known as the Smad proteins (Heldin et al, 1997). Smad proteins can be classified into three groups: the pathway restricted Smads include the Smad2 and Smad3 which are phosphorylated by the type I receptor of TGF-.beta. or activin, while the Smads 1, 5 and 8 are implicated in BMP signalling. The phosphorylation of the pathway restricted Smads permits their interaction with the common Smad or Smad4 and this heteromeric complex then translocates into the nucleus where it regulates expression of target genes. Finally, there inhibitory Smads which include the Smad 7 and Smad 6 prevent the phosphorlyation of the R-Smads by the type I kinase. (Heldin et al, 1997, Massague, 1998; Wrana and Attisano, 2000)

In blood circulation, TGF-.beta.1 is found bound to the carrier .alpha..sub.2 macroglobulin (.alpha..sub.2M; Webb et al. 1998). .alpha..sub.2M binds many other cytokines and therefore lacks selectivity for TGF-.beta.1. .alpha..sub.2M polymorphism has been associated with Alzheimer's disease, which polymorphism is observed as a deletion in "the bait region" overlapping with TGF-.beta.1 binding domina (Gonias et al. 2000 and Blacker et al 1998).

Although the types I and II receptors are central to TGF-.beta. signaling, it is possible that accessory receptors interacting with the signaling receptors modify TGF-.beta. responses. For example, both endoglin and type III receptor which form heteromeric complexes with the type II receptor have been reported to modulate TGF-.beta. function. When overexpressed in myoblasts, endoglin inhibited while type III receptor enhanced TGF-.beta. responses (Letamendia et al, 1998). In addition, endoglin was shown to antagonize TGF-.beta. mediated growth inhibition of human vascular endothelial cells (Li et al, 2000). Similarly, the newly identified type I-like receptor BAMBI which associates with TGF-.beta. family receptors can inhibit signaling (Onichtchouk et al, 1999).

There are also a number of molecules that can impact TGF-.beta. signal transduction by interacting with one or both of the TGF-.beta. signaling receptors. However, the exact physiological significance of many of these interactions are not clearly defined (for review, Massague, 1998). Three of these interacting proteins: the type II TGF-.beta. receptor interacting protein (TRIP-1) (Chen et al, 1995), B.alpha. (.alpha. subunit of protein phosphatase A) (Griswold-Prenner, 1998), and serine-threonine kinase receptor associated protein (STRAP) (Datta et al, 1998) all contain the highly conserved tryptophan-aspartic acid (WD) repeats. WD domains are important in protein-protein interactions and cellular functions such as cell cycle progression and transmembrane signaling (Neer et al, 1994). TRIP-1 is phosphorylated through its interaction with the type II receptor kinase and exerts an inhibitory effect on TGF-.beta. induced PAI-1 gene transcription, but has no effect on TGF-.beta. mediated growth inhibition (Choy and Derynck, 1998). On the other hand, B.alpha. associates with the type I receptor and positively modulates TGF-.beta. action. Finally, STRAP can interact with both the type I and II receptors and when overexpressed, it exerts an inhibitory effect on TGF-.beta. mediated transcriptional activation. In addition, STRAP can also interact with the inhibitory Smad7, but not Smad6. STRAP's interaction with Smad7 exerts a stabilizing effect on Smad7's association with the activated type I kinase receptor which prevents Smad2/3's association and subsequent phosphorylation (Datta and Moses, 2000).

The immunophilin, FKBP12, interacts with the TGF-.beta. type I receptor and acts as a negative modulator of TGF-.beta. function (Wang et al, 1996). It can interact with unactivated type I receptor and functions to stabilize the quiescent receptor state by protecting phosphorylation sites in the GS domain. Upon ligand stimulation, heteromerization and subsequent phosphorylation of the GS domain by the TGF-.beta. type II kinase results in the release of FKBP12 (Chen et al, 1997; Huse et al, 1999). In contrast, the TGF-.beta. type I receptor associated protein-1 (TRAP-1) interacts only with the activated type I receptor kinase (Charng et al, 1998). TRAP-1 is not phosphorylated by the type I kinase and TRAP-1's interaction is reported to have an inhibitory effect on TGF-.beta. signaling. However, a recent report describes a different function for TRAP-1 (Wurthner et al, 2001). In this study, TRAP-1 was found to associate with inactive TGF-.beta. and activin receptor complexes and upon ligand stimulation, TRAP-1 is released. The conformationally altered TRAP-1 is then believed to associate and subsequently chaperone Smad4 to the activated Smad2. The .alpha. subunit of ras farnesyl protein transferase (FNTA) preferentially interacts with the activated type I receptor and is considered a substrate because it is phosphorylated by the type I kinase and released thereafter (Kawabata et al, 1995). However the functional significance of this phenomenon remains unexplained. The accessory receptors, endoglin and type III receptor which form heteromeric complexes with the type II receptor have also been reported to modulate TGF-.beta. function. When overexpressed in myoblasts, endoglin inhibited while type III receptor enhanced TGF-.beta. responses (Letamendia et al, 1998). Glycosylphosphatidyl inositol (GPI)-anchored proteins which lack transmembrane and cytoplasmic domains have also been shown to bind TGF-.beta.. These proteins have been identified on certain cell lines (Cheifetz and Massague, 1991), but the identity of these GPI-anchored proteins and the role they may play in TGF-.beta. signaling remain unknown. Recently, the present inventors reported the presence of GPI-anchored TGF-.beta. binding proteins on early passage human endometrial stromal cells (Dumont et al, 1995), human skin fibroblasts (Tam and Philip, 1998) and keratinocytes (Tam et al, 1998). On human keratinocytes, they identified a 150 kDa GPI-anchored TGF-.beta.1 binding protein designated as r150 that can form a heteromeric complex with the types I and II TGF-.beta. receptors (Tam et al, 1998). In addition, they demonstrated that upon hydrolysis fom the cell surface by phosphatidylinositol phospholipase C (PIPLC), the soluble form of r150, retains its ability to bind TGF-.beta.1 in the absence of the types I and II receptors. In addition, it was demonstrated that the GPI anchor is contained in a protein with a molecular weight of 150 kDa (Tam et al, 2001). This novel GPI-anchored TGF-.beta.1 binding protein, r150, has the potential to antagonize or potentiate TGF-.beta. action in keratinocytes. In the absence of the cDNA encoding r150, one way to examine the effect of r150's loss in TGF-.beta. signaling is to enzymatically release the binding protein by PIPLC treatment prior to testing for alterations in TGF-.beta. induced responses. However, the efficacy of exogenously added PIPLC is subject to variability, being affected by pH, temperature, and acylation of GPI-anchored proteins (Shukla, 1982; Chen et al, 1998), thus results obtained may be difficult to interpret. In addition, GPI-anchored proteins that are released may get re-synthesized and re-inserted in the plasma membrane soon after PIPLC hydrolysis. Hence, as an alternative, was have created and isolated a keratinocyte cell line that is mutated in GPI anchor biosynthesis. These cells display a significant loss of r150 from their cell surface, thus allowing a comparative examination of TGF-.beta. mediated cellular responses in the GPI anchor deficient cell line versus the parental HaCat cells under stable experimental conditions

GPI-anchored proteins lack transmembrane and cytoplasmic domains, and are attached to the cell membrane via a lipid anchor in which the protein is covalently linked to a glycosyl phosphatidylinositol moiety. GPI-anchored proteins have been reported to have roles in intracellular sorting (Rodriguez-Boulan and Powell, 1992), in transmembrane signaling (Brown, 1993) and to associate with cholesterol and glycosphingolipid-rich membrane microdomains (Brown and London, 1998; Hooper, 1999). Also, the GPI anchor enables a protein to be selectively released from the membrane by phospholipases (Metz et al, 1994; Movahedi and Hooper, 1997). r150 was characterized as GPI-anchored, based on its sensitivity to phosphatidylinositol phospholipase C (PIPLC). However, it is important to rule out other possibilities, namely, (i) r150 is not itself GPI-anchored, but is tightly associated with a protein that is GPI-anchored, and therefore is susceptible to release by PIPLC; (ii) r150 is a complex of two lower molecular weight proteins which became inadvertently cross-linked during the affinity labeling procedure.

It is now demonstrated that the GPI-anchor is contained in r150 itself and not on a tightly associated protein and that it binds TGF-.beta.1 with an affinity comparable to those of the signaling receptors. Furthermore, the released (soluble) form of this protein binds TGF-.beta. independent of the types I and II receptors. Also, the soluble form inhibits the binding of TGF-.beta. to its receptor. In addititon, we provide evidence that r150 is released from the cell surface by an endogenous phospholipase C. Also, a mutant human keratinocyte cell line with a defect in GPI synthesis was created, which display reduced expression of r150. The results using these mutant keratinocytes suggest that the membrane anchored form of r150 is a negative modulator of TGF-beta responses. These findings, taken together with the observation that r150 forms a heteromeric complex with the signaling receptors, suggest that this accessory receptor in either its membrane anchored or soluble form and its down- or up-regulation may potentiate or antagonize TGF-.beta. responses in human keratinocytes, respectively.

The complete amino acid of a molecule named CD109 was recently disclosed as well as the nucleic acids encoding same (Lin et al. 2002). Sequences comparisons with those of r150 suggest that CD109 is a r150 variant. No definite role has been assigned to CD109 by Lin et al.

SUMMARY OF THE INVENTION

This invention provides a molecule that binds TGF-.beta.1 with a high level of selectivity. This molecule named r150 can be retrieved in a membrane anchored form or as a released free soluble form. Variants and parts of r150 which have the property to bind TGF-.beta.1 are grouped under the name r150-like proteins or peptides. They include those defined in SEQ ID Nos: 2, 4, 8, 10 and 12. Their corresponding coding nucleic acids respectively defined in SEQ ID NOs: 1, 3, 5, 7, 9 and 11.

This invention provides for the use of a protein comprising any one of the following sequences in the making of a medication for inhibiting TGF-.beta.1 activity in a biological tissue SEQ ID Nos: 2, 4, 6, 8, 10 and 12.

Also provided is the use of an antagonist to a protein comprising any one of the following sequences in the making of a medication for increasing TGF-.beta.1 activity in a biological tissue: SEQ ID Nos: 2, 4, 6, 8, 10 and 12.

Also provided is the use of a nucleic acid encoding a protein comprising any one of the following sequences in the making of medication for inhibiting TGF-.beta.1 activity in a biological tissue: SEQ ID Nos: 1, 3, 5, 7, 9 and 11.

Also provided is the use of a molecule which silences the expression of a nucleic encoding a protein comprising any one of the following sequences in the making of medication for increasing TGF-.beta.1 activity in a biological tissue: SEQ ID Nos: 1, 3, 5, 7, 9 and 11. Particularly, the silencing molecule is an antisense nucleic acid.

The present inventors being the first to elucidate the complete nucleic acid sequence of r150 and of its deduced amino acid sequence, this invention provides an isolated nucleic acid encoding a protein comprising any one of the following sequences: SEQ ID Nos: 2, 4, 6, 8, 10 and 12.

In a specific embodiment, the nucleic acid comprises any one of the following nucleotide sequences: SEQ ID No: 1, 3, 5, 7, 9 and 11.

The nucleic acid is particularly one comprising the nucleotide sequence defined in SEQ ID No: 1.

The above nucleic acids may include crude nucleic acids or recombinant vectors; namely expression vectors capable of governing transcription and translation of the crude nucleic acids inserted downstream to a promotor, are preferred tools for producing r150-like proteins.

Recombinant host cells which comprise the nucleic acids or the recombinant vector are other tools. The vectors are normally selected to comprise sequences compatible with the host's machinery. Intervening sequences located 5' and 3' with regard to the crude nucleic acids are adapted or selected by the skilled artisan desirous to produce a particular type of host cells. The signal peptide may be charged also for another one more appropriate for a given cell type.

There host cells may be domesticated and used in a method of producing a r150-like protein. Such a method comprises the steps of: growing a recombinant host cell in a culture medium supporting cell growth and expression of said nucleic acid: recovering the protein from the culture medium or from the cell.

The nucleic acids may be antisense nucleic acids. They may be inserted in a recombinant vector, namely an expression vector and recombinant host cells which comprises such antisense nucleic acids can also be made.

It is further an object of this invention to provide a TGF-.beta.1 binding reagent, which comprises a protein comprising any one of the following sequences: SEQ ID Nos 2, 4, 6, 8, 10 and 12.

Compositions of matter which comprise these reagents and a carrier are other objects of this invention.

The carrier may be a pharmaceutical carrier. Otherwise, it may be a solid support to which r150 is bound to immobilize TGF-.beta.1.

DESCRIPTION OF THE INVENTION

r150 is a TGF-.beta.1 binding molecule. Its complete amino acid sequence as well as the nucleic acid sequence encoding same appear to have been first elucidated by the present inventors. Another group (Lin et al. 2002), using a very different approach (affinity binding to monoclonal antibodies) has found a blood cell surface antigen, which they called CD109. Sequence comparisons show that CD109 (SEQ ID Nos 5 and 6) comprises a 17 amino acid insertion at position 1218 1234 (51 nts). This addition accounts for the difference in amino acids number (1445 for CD109 versus 1428 for r150). Besides that, substitutions of nucleotides are noted: r150 amino acid thr.sup.1224 is changed for a methionine (CD109 amino acid 1241). CD109 shows polymorphism at residue 703 (Schuh et al. 2002). A tyrosine or a serine represent different alleles of CD109. Such polymorphism would presumably exist for r150. It is possible that CD109 or r150 are responsible for the building of an immune response since allo antibodies are retrieved upon administration of CD109 isoforms. It may therefore be implied that an isoform compatible with the recipient subject's tissue may have to be selected as an administrable r150 active ingredient.

r150 binds or sequesters TGF-.beta.1, in its membrane anchored form as well as in its free soluble form (SEQ ID Nos: 4 and 8). As a result, TGF-.beta.1 availability is reduced. The effects induced by TGF-.beta.1 are therefore negatively modulated (or inhibited). Such inhibition may be desirable in conditions where overproduction of TGF-.beta.1 leads to pathological states (cancer is a specific example of such pathology). On the contrary, in other occasions, increasing TGF-.beta.1 activity may be sought. For example, TGF-.beta.1 encourages tissue or organ graft success. Therefore silencing r150 would have for effect to increase TGF-.beta.1 availability and increase graft success.

r150 further appears to be related to .alpha..sub.2 macroglobulin (.alpha..sub.2M) and since the TGF-.beta. binding domain has been determined for .alpha..sub.2M by Webb et al. (1998), the corresponding domain in r150 is presumed to be located a region corresponding to .alpha..sub.2 macroglobulin amino acids 666 706. These corresponds to r150 amino acids 651 683 (SEQ ID No: 10). Therefore, the r150 peptide having the sequence defined in SEQ ID NO: 10 is also contemplated as TGF-.beta.1 binding peptide within the scope of the invention; the nucleic acid encoding this peptide is as well.

Webb et al. (2000) even found the minimal .alpha..sub.2M TGF-.beta.1 binding sequence which appears to be delineated by amino acid 717 and 733. The corresponding strectch in r150 is found between amino acid residues 694 and 712 (SEQ ID No.12).

Gomas et al. (2000) reported that .alpha..sub.2M gene polymorphism has been associated with Alzheimer's disease, which polymorphism is observed as a deletion in "the bait region" overlapping with TGF-.beta.1 binding domain. It is envisageable that r150 could be useful to sequester and neutralize TGF-.beta.1 especially in diseases wherein .alpha..sub.2M is deficient. Any portion of r150 or variants thereof that is capable of binding TGF-.beta.1 activity is intended to be used in the making of a medication or a method or composition for inhibiting TGF-.beta.1 activity. This includes proteins or peptides comprising sequences defined in SEQ ID NOs. 2, 4, 6, 8, 10 and 12. These r150-like proteins or peptides would include any molecule having at least 50% homology with the above sequences. On the opposite, any molecule having an antagonistic activity to the above r150-like proteins or peptides would find a use in the making of a medication or a method or a composition for increasing TGF-.beta.1 activity.

Nucleic acids encoding the above r150-like proteins or peptides represent an alternative to the direct administration of proteins or peptides. Antisense nucleics would on the opposite silence the expression of r150-like proteins or peptides. All these nucleic acids include recombinants vectors, namely expression vectors, which are available and well known to the skilled artisan.

A very large body of literature describes diseases or disease models involving up and down regulation of TGF-.beta.1 activity. Nowadays, TGF-.beta.1 binding proteins decorin and an anti-TGF antibody are currently under clinical trials. The present r150-like proteins or peptides could represent a valuable and advantageous alternative to these molecules, because of their selectivity for TGF-.beta.1 isoform, combined to their hydrosolubility.

Here is a non-exhaustive list of disease models where alteration of TGF-.beta. action has been shown to be of therapeutic benefit:

Cancer progression:

Note: TGF-.beta. has biphasic effects during tumorigenesis, acting early as a tumor suppressor, but later stimulating cancer progression.

(i) Suppression of tumor progression by TGF-.beta.

Akhurst R. J. and Derynck R. (2001). TGF-.beta. signaling in cancer a double-edged sward. TRENDS in Cell Biology 11: S44 S51.

Welch, Dr. et al (1990). Transforming growth factor-.beta. stimulates mammary adenocarcinoma cell invasion and metastatic potential. Proc. Natl. Acd. Sci. USA 87: 7678 7682

Markowitz, S. et al. (1995) Inactivation of the type II TGF-.beta. receptor in colon cancer cells with microsatellite instability. Science 268,1336 1338.

Massague .J. et al. (2000) TGF-.beta. signaling in growth control, cancer, and heritable disorders. Cell 103, 295 309.

(ii) Stimulation of tumor progression by TGF-.beta.

Hojo, M. et al. (1999) Cyclosporine induces cancer progression by a cell-autonomous mechanism. Nature 397, 530 534.

Yin, J. J. et al. (1999) TGF-.beta. signaling blockade inhibits PTHrP secretion by breast cancer cells and bone metastases development. J. Clin. Invest. 103, 197 206.

Exogenous TGF-.beta.1 promotes wound healing where as inhibiting TGF-.beta.1 activity or enhancing TGF-.beta.3 activity reduces scarring in animal models

Roberts, A. B., and Sporn, M. B. (1996). Transforming growth factor R. A. F. (ed), Plenum Press, New York, p275 308.

Mustoe, T. A., Pierce, G. F., Thomason, A., Gramates, P., Sporn, M. B., and Deuel, T. F. (1987). Accelerated healing of incisional wounds in rats induced by transforming growth factor-.beta.. Science 237: 1333 1336.

Quaglino, D., Nanney, L. B., Ditesheim, J. A., and Davidson, J. M. (1991). Transforming growth factor-.beta. stimulates wound healing and modulates extracellular matrix gene expression in pig skin: incisional wound model. J. Invest. Dermatol. 97: 34 42.

O'Kane, S., and Ferguson, M. W. J. (1997). Transforming growth factor-.beta.s and wound healing. Int. J. Biochem. Cell. Biol. 29:63 78.

Shah, M., Foreman, D. M., and Ferguson, M. W. J. (1995). Neutralization of TGF-.beta.1 and TGF-.beta.2 or exogenous addition of TGF-.beta.3 to cutaneous rat wounds reduces scarring. J. Cell Science 108: 985 1002.

Choi, B-M., Kwak, H-J., Jun, C-D., Park, S-D., Kim, K-Y., Kim, H-R., and Chung, H-T. (1996). Control of scarring in adult wounds using antisense transforming growth factor-.beta.1 oligodeoxynucleotides. Immunol. Cell Biol. 74:144 150.

Blocking TGF-.beta.1 overproduction reduce tissue fibrosis (pulmonary fibrosis, liver cirhosis, glomerulonephritis, scleroderma and atherosclerosis).

Border et al, (1990). Suppression of experimental glomerulonephritis by antiserum against transforming growth factor beta 1. Nature 346 (6282): 371 374

Isaka Y., Brees D. K., lkegaya K., Kaneda Y., Imai E., Noble N. A., Border W. A. (1998). Gene therapy by skeletal muscle expression of decorin prevents fibrotic disease in rat kidney. Nat. Med. 2: 418 423.

Isaka Y., Akagi Y., Ando Y., Tsujie M., Sudo T., Ohno N., Border W. A., Noble N. A., Kaneda Y., Hori M., and Imai E. (1999). Gene therapy by transforming growth factor-beta receptor-IgG Fc chimera suppressed extracellular matrix accumulation in experimental glomerulonephritis [see comments]. Kidney Int. 55:465 475.

Khalil, N. and Greenberg A H (1991). The role of TGF-.beta. in pulmonary fibrosis. Ciba Found Symp. 157: 194 207.

Yamamoto, T., Takagawa S., Katayama I., and Nishioka K. (1999). Anti-Sclerotic effect of transforming growth factor-beta antibody in a mouse model of bleomycin-induced scleroderma. Clin Immunol, 92(1):6 13.

Gressner, A. M., Weiskirchen, R., Breitkopf K., and Dooley S. (2002). Roles of TGF-beta in hepatic fibrosis. Front Biosci (7): d793 807.

Sheppard D. (2001). Integrin-mediated activation of transforming growth factor-beta(1) in pulmonary fibrosis. Chest 120(1 Suppl):49S-53S.

McCaffrey, T. A. (2000). TGF-.beta.s and TGF-.beta. receptors in atherosclerosis. Cytokine. Growth Factor Rev. 11: 103 114.

TGF-beta has tissue protective effects (against ischemia reperfusion injury) in the heart, brain and kidney.

Lefer A M., Ma X-L, Weyrich A S, Scalia R. (1993). Mechanism of the cardioprotective effect of TGF-.beta.1 in feline myocardial ischemia and reperfusion. Proc. Natl. Acad. Sci. USA 90: 1018 1022.

Lefer A M, Tsao P, Aoki N, Palladino M A. (1990). Mediation of cardioprotection by transforming growth factor-.beta.. Science 249: 61 64.

McNeill H. Williams C, Guan J, Dragunow M, Lawlor P, Sirimanne E, Nikolics K,

Gluckman P. (1994). Neuronal rescue with transforming growth factor-beta 1 after hypoxic-ischaemio brain injury. Neuroreport 5: 901 904.

Mehta J L, Yang B C, Strates B S, Mehta P. (1999). Role of TGF-beta1 in platelet-mediated cardioprotection during ischemia-reperfusion in isolated rat hearts. Growth Factors 16: 179 190.

Recombinant hosts comprising the above nucleic acids or recombinant expression vectors can be used as a biological machinery in the production of the r150-like proteins or peptides. The elucidation of the nucleic acid sequence of r150 therefore leads to a method of producing these proteins or peptides by recombinant technology.

A variety of TGF-.beta.1 binding reagents and compositions may be derived from the present invention.

First, peptides such as those defined in SEQ ID. Nos: 10 and 12 may be used as such as a TGF-.beta.1 binding reagent. Larger molecules like those defined in SEQ ID Nos 2, 4, 6 and 8 could be conjugated (through their anchoring region) to a carrier. The carrier may take the form, for example, of a solid or semi-solid medium (beads, chromatography columns, plates, etc.), to immobilize TGF-.beta.1. Pharmaceutical compositions would take any suitable form, depending on the selected route of administration. A r150-like protein or peptide (SEQ ID Nos: 2, 4, 6, 8, 10 and 12) would be formulated with a pharmaceutically acceptable carrier. Doses equivalents those used by intravenous route for decorin and/or the TGF-antibody can be produced.


Claim 1 of 2 Claims

1. A method for inhibiting TGF-.beta.1 activity in a biological tissue of an animal comprising an administration thereto of an effective amount of a protein comprising a sequence selected from the group consisting of: a) SEQ ID NO:2 b) amino acids 694 712 of SEQ ID NO:2; c) amino acids 651 683 of SEQ ID NO:2 d) a protein sequence having a tyrosine at position 703 of SEQ ID NO:2; e) a protein sequence having amino acids 21 to 1428 of SEQ ID NO:2 f) a protein sequence having amino acids 21 to 1404 of SEQ ID NO:2; and g) a protein sequence having a methionine instead of threonine at position 1224 of SEQ ID NO:2 thereby inhibiting TGF-.beta.l activity in a biological tissue of an animal.

 

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