WO2003060075A2 - USE OF SOLUBLE TYPE II TGF-β RECEPTOR TO SUPPRESS PANCREATIC CANCER GROWTH AND METASTASIS - Google Patents

Abstract

A method of treating pancreatic cancer is disclosed, wherein soluble transforming growth factor-β type II receptor is administered to reduce tumor angiogenesis, and to inhibit cancer cell invasiveness and metastasis.

Description

USE OF SOLUBLE TYPE II TGF-β RECEPTOR TO SUPPRESS PANCREATIC CANCER GROWTH AND METASTASIS

BACKGROUND OF THE INVENTION

Transforming growth factor-βs (TGF-βs) are structurally related polypeptide growth factors that regulate many cellular processes, including cell proliferation and differentiation, migration, deposition of the extracellular matrix, immunosuppression, motility, and cell death (Sporn, M.B., and Roberts, A.B., J. Cell Biol., 119: 1017-1021, 1992/ Massague, J. Annu. Rev. Bioche ., 67: 753-791, 1998). TGF-βs enhance the synthesis of matrix proteins, such as proteoglycans, fibronectin, laminin, collagens, tenascin, and vitronectin, increase the synthesis of protease inhibitors while decreasing the synthesis of matrix degrading proteases, and enhance the expression of cell adhesion molecules such as integrins (Kingsley, D.M., Genes Dev. 8: 133-46, 1994; Gold, L., Clinical Reviews in Oncogenesis 10: 303-360, 1999) .

TGF-βs are synthesized as precursors that undergo proteolytic cleavage, leading to the generation of biologically active 25 kD di ers . The mature forms of TGF-βl and -β2 share 70% arαino acid sequence homology, and the mature form of TGF-β3 shares 80% homology with the other two TGF-βs.

TGF-βs enhance the proliferation of cells of mesenchymal origin and inhibit the proliferation of many types of epithelial cells. TGF-βs act by binding to the type II TGF-β receptor (TβRII), which is constitutively active as a ^' serine/threonine kinase (Heldin, C.H., et al . , Nature 390: 465-471, 1997; Massague, J., and Chen, Y.G., Genes Dev. 14: 627-644, 2000). Following ligand binding, TβRII heterodimerizes with and activates type I TGF-β receptor (TβRI) . Activation of TβRI leads to the phosphorylation of Smad2 and Smad3 and induces their heterodimerization with Smad4 (Derynck, R., et al., Cell 95: 737- 740, 1998; Attisano, L., and Wrana, J.L., Curr. Opin. Cell Biol. 12: 235-243, 2000) . These Smad complexes then translocate to the nucleus where they regulate gene transcription (Massague, J., and Wotton D., EMBO J. 19:1745-1754, 2000).

Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease in which non-surgical therapy is ineffective and in which the majority of patients harbor metastatic lesions at presentation precluding the possibility for curative surgical intervention (Warshaw, A.L, and Fernandez, del Castillo C, N. Engl . J. Med. 326: 455-465, 1992) . These cancers frequently overexpress all three mammalian TGF-β isoforms (Friess, H., et al., Gastroenterology 105: 1846-1856, 1993) . This aberrant overexpression occurs in the cancer cells within the tumor mass in spite of the fact that TGF-βs are most often expressed at high levels by mesenchymal derived rather than epithelial derived cells, and is associated with decreased patient survival.

Several alterations that interfere with the ability of TGF- βs to inhibit pancreatic cancer cell growth have been reported, including a high frequency of Smad4 mutations (Hahn, S.A., et al . , Science 271: 350-353, 1996), over-expression of inhibitory Smadδ (Kleeff, J., et al . , Biochem. Biophys. Res. Commun. 255: 268-273, 1999) and Smad7 (Kleeff, J., et al . , Oncogene 18: 5363- 5372, 1999), and under-expression of TβRI (Wagner, M. , et al., Int. J. Cancer 78: 255-260, 1998). Consequently, pancreatic cancer cell-derived TGF-βs cannot act to suppress the growth of the cancer cells. Instead, they may promote pancreatic tumor growth in vivo by acting on the peri-cancerous cellular elements such as endothelial cells and fibroblasts, since these cells do not harbor Smad4 mutations. Furthermore, in pancreatic cells that express high^' levels of Smad7, TGF-βs may act directly on the cancer cells to enhance the expression of growth promoting genes.

Several different types of experiments have suggested that there is a dissociation between the signaling pathways that mediate the growth suppressive effects of TGF-βs and their effects on the expression of genes that modulate the extracellular matrix. Thus, in mink lung epithelial cells, expression of a truncated TβRII renders these cells resistant to TGF-βl-mediated growth inhibitory effects without altering TGF-βl mediated induction of PAI-1 (Chen, R. H., et al . , Science 260: 1335-8, 1993) . Transfection of these cells with a mutant TβRI that lacks the juxtamembrane region preceding the GS domain, leads to TGF-βl mediated PAI-1 production but not to growth inhibition (Saitoh, M., et al . , J. Biol. Che . 271: 2769-75, 1996) .

Over-expression of inhibitory Smad7 makes COLO-357 cells resistant to TGF-βl-mediated growth inhibitory effects without altering TGF-βl mediated induction of PAI-1 (Kleeff, J., et al . , Oncogene 18: 5363-5372, 1999) . Cell lines derived from pulmonary metastases lose TGF-βl-mediated growth inhibitory responses, but still exhibit TGF-βl mediated induction of MMP-9 (Sehgal, I., et al . , Cancer Res. 56: 3359-65, 1996). In this context, the overexpression of TGF-βs in pancreatic cancer cells that frequently harbor Smad4 mutations and overexpress inhibitory Smad molecules may provide a mechanism for the activation of autocrine and paracrine pathways that lead to the expression of genes that promote cancer spread and angiogenesis . Indeed, it has been recently shown that the angiogenic potential of TGF-βs may be enhanced by the presence of Smad4 mutations (Schwarte-Waldhoff I., et al., Proc. Natl. Acad. Sci. USA 97: 9624-9629, 2000).

Several approaches have been used to suppress the biological actions of TGF-βs in vivo. These include the use of neutralizing anti-TGF-β antibodies (Ueki, N., et al . , Biochim. Biophys. Acta 1137: 189-96, 1992; Arteaga, C. L., et al . , J. Clin. Invest. 92: 2569-76, 1993; Hoefer, M. and Anderer, F. A., Cancer Immunol. Immunother. 41: 302-8, 1995) or antisense strategies to inhibit TGF-β synthesis (Marzo, A. L., et al., Cancer Res. 57: 3200-7, 1997; Fitzpatrick, D. R., et al . , Growth Factors 11: 29-44, 1994; Tzai, T. S., et al . , Anticancer Res. 18: 1585-9, 1998), expression of a mutated TGF-βl precursor to inhibit the processing of all three TGF-βs (Lopez, A. R., et al . , Mol. Cell Biol. 12: 1674-9, 1992), -expression of TβRIII, soluble TβRIII, or soluble TβRII to neutralize TGF-β activity (Won, J., et al . , Cancer Res. 59: 1273-7, 1999; Lόpez-Casillas, et al., J. Cell Biol. 124: 557-68, 1994; Bandyopadhyay, A., et al . , Cancer Res. 59: 5041-6, 1999; Ko esli, S., et al . , Eur. J. Bioche . 254: 505-13, 1998), or expression of a dominant-negative version of TβRII to interfere with TGF-β signaling (Oft, M., et al . , Curr. Biol. 8: 1243-52, 1998; Portella, G., et al . , Cell Growth Differ. 9: 393-404, 1998; Yin, J. J., et al . , J. Clin. Invest. 103: 197-206, 1999) .

SUMMARY OF THE INVENTION

Pancreatic cancer is a deadly disease with an extremely poor prognosis. Until the present invention, there have been no effective therapeutic options for this disease. It is known, however, that pancreatic cancer cells overexpress TGF-β molecules and that this overexpression is associated with .a particularly poor prognosis. Accordingly, in order to suppress TGF-β overexpression, the present inventors prepared a vector encoding sequences corresponding to a soluble TGF-β receptor that is devoid of signaling activity but that can bind and sequester TGF- β molecules.

The present inventors have shown that expression of this construct in a pancreatic cancer cell line acts to slow the growth of subcutaneous tumors in nude mice. The present inventors have also shown that expression of this construct has a similar effect in a second pancreatic cancer line and that expression of the soluble receptor attenuates and prevents metastatic spread in a metastatic nude mouse model. The soluble receptor acts as a ""sponge" that soaks up TGF-β molecules and attenuates the growth and metastatic potential of pancreatic cancer cells. Thus, it is herein disclosed that suppression of TGF-β action attenuates the biological aggressiveness of pancreatic cancer cells, thus providing a therapeutic option for the treatment of pancreatic cancer.

This soluble receptor construct may be used to slow tumor growth and prevent cancer spread with minimal side-effects . The construct may be used for gene therapy approaches and for expressing large quantities of the soluble receptor protein for delivery to pancreatic cancer patients. Thus, the present invention provides a method for treating pancreatic cancer in an individual, comprising identifying an individual at risk for pancreatic cancer and administering sTβRII to the individual.

The present invention further provides a method for reducing tumor growth in an individual with pancreatic cancer, comprising identifying an individual at risk for pancreatic cancer and administering to the individual a therapeutically effective amount of sTβRII to the individual.

The present invention further provides a method for inhibiting metastasis in an individual with pancreatic cancer, comprising identifying an individual at risk for pancreatic cancer and administering to the individual a therapeutically effective amount of sTβRII.

The present invention further provides a method for inhibiting tumor angiogenesis in an individual with pancreatic cancer, comprising identifying an individual at risk for pancreatic cancer and administering to the individual a therapeutically effective amount of sTβRII.

The present invention further provides a method for reducing tumor growth in an individual diagnosed with pancreatic cancer, comprising introducing into the individual an expression vector encoding sTβRII, such that an amount of sTβRII effective to reduce tumor growth is expressed in the individual.

The present invention further provides a method for inhibiting metastasis in an individual diagnosed with pancreatic cancer, comprising introducing into the individual an expression vector encoding sTβRII, such that an amount of sTβRII effective to inhibit metastasis is expressed in the individual.

The present invention further provides a method for inhibiting tumor angiogenesis in an individual diagnosed with pancreatic cancer, comprising introducing into the individual an expression vector encoding sTβRII, such that an amount of sTβRII effective to inhibit tumor angiogenesis is expressed in the individual . The present invention further provides a method for treating pancreatic cancer in an individual, comprising identifying an individual at risk for pancreatic cancer and introducing into the individual an expression vector, encoding sTβRII, such that an amount of sTβRII effective to reduce the activity of transforming growth factor-β is expressed in the individual.

The present invention further provides a method for preventing disease recurrence following surgery for pancreatic cancer, including prevention of metastases in this clinical setting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Expression of soluble TβRII transfected COLO-357 cells .

A) Northern blotting.

Total RNA was isolated from the sham-transfected cells (S) or from clones transfected with a cDNA encoding soluble TβRII

(C1-C2) . RNA (20 μg/lane) was size fractionated, electrotransferred to a nylon Genescreen membrane, and hybridized with the 32P labeled soluble TβRII cDNA probe (5 X 105 cpm/ml,

1 day exposure) . To confirm equal loading of lanes, the membrane was stripped and reprobed with the 7S cDNA probe (5 X 104 cpm/ml,

2 h exposure) .

B) Immunoblotting. Cell lysates (50 μg/lane) were prepared from the same clones and subjected to 13% SDS PAGE, electrotransferred to a membrane, and blotted with anti-HA antibodies (1:1,000 dilution, 1 in exposure) . To confirm equal loading of lanes, the membrane was stripped and reprobed with an anti-ERK-2 antibody (1:8,000 dilution, 5 s exposure).

C) Detection of secreted soluble TβRII. Concentrated conditioned medium from either the sham (S) or soluble TβRII transfected cells (Cl) was immunoprecipitated with 2 μg anti-HA antibody for 24 hours. The samples were subjected to 13% SDS PAGE, electrotransferred to a membrane, and blotted with biotinylated anti-human TβRII polyclonal antibody (1:5,000 dilution, 1 min exposure) . FIG. 2. Imπrunostaininq of COLO-357 cells and transfected clones . An anti-TβRII antibody recognizing the full-length TβRII (A, B) and an anti-HA antibody recognizing the HA epitope of the pMHsTβRII construct (C, D) were used. In the sham-transfected cells, there was weak im unostaining for endogenous TβRII (A) but undetectable HA immunoreactivity (C) . In contrast, the cancer cells expressing the pMHsTβRII construct (B) and HA (D) exhibited strong immunoreactivity. Scale bar: 25 μm.

FIG. 3. Effects of TGF-βl on cell growth and invasion.

A) Effects of TGF-βl on cell growth. Sham-transfected COLO-357 cells and two clones stably transfected with pMHsTβRII were seeded in 96 well plates (10, 000/well) and incubated for 24 h in DME complete medium. Cells were then placed in serum-free medium in the absence (open boxes) or presence (solid boxes) of 10 pM TGF-βl for 72 h. Growth was determined by the MTT assay. Results are expressed as % of unstimulated control and are the means (±SE) of 8 determinations per experiment from 3 separate experiments. Error bars for the control groups were exceedingly small. *P<0.002 when compared with respective untreated controls.

B) Effects of TGF-βl on invasion. To assess the ability of TGF-βl to induce invasion, COLO-357 cells were pre-incubated for 24 h in serum-free medium containing 0.1% BSA in the absence or presence of 400 pM TGF-βl. The respective groups of cells (1 X 105/well) were then added to the upper chambers of a 24-well Transwell unit. Medium devoid of TGF-βl (open boxes) or containing 400 pM TGF-βl (solid boxes) was then added to the respective lower chambers. Migration across the Matrigel coated polycarbonate membrane (8 μm pore size) was assessed after 16 hours. The number of cells in 9 separate high power fields

(magnification: 200x) were counted in triplicate wells. Data are expressed as the means ± SEM of triplicate determinations from 3 separate experiments. *P<0.01 when compared with the untreated controls .

FIG. 4. Tumor formation in nude mice. Athymic nude mice were injected with COLO-357 cells that were either sham transfected or transfected with pMHsTβRII. Tumors were measured externally on the indicated days and tumor volume was determined by the equation: volume = (1 x h x w) x B/4, where 1 is length, h is height, and w is width of the tumor.

(A) Tumor volume. Results are expressed as % of sham control and are the means ± SEM from three separate experiments, in which tumor volume was determined 28 days after injection of 2 X 106 COLO-357 cells per site (two sites per mouse) . Error bars for the control groups are not shown because they were exceedingly small.

(B) Time course. Data are the means ± SEM from 3 mice injected with sham transfected cells and 6 mice injected with COLO-357 clones expressing pMHsTβRII (6 x 105 cells/site) . However, two mice injected with sham transfected cells were sacrificed after 35 days due to tumor size. Therefore, data from day 56 for these sham derived tumors is the means ± SD. One site injected with clone 1 did not yield a tumor, and this site was excluded from the volume calculations. *P<0.04; ** P<0.001 when compared to the sham control.

FIG. 5. TβRII immunoreactivity in tumors. An anti-TβRII antibody recognizing the full-length TβRII (A, B) and an anti-HA antibody recognizing the HA epitope of the pMHsTβRII construct

(C, D) were used. In the tumor tissue derived from the sham transfected cells, there was weak immunostaining for TβRII (A) but undetectable HA immunoreactivity (C) . Strong TβRII (B) and HA (D) immunoreactivity was present in the tumor tissue derived from cancer cells expressing the pMHsTβRII construct. Scale bars: 25 μm.

FIG. 6. Expression of soluble TβRII and PAI-1 mRNA transcripts in vivo. Total RNA (20 μg/lane) was prepared from tumors generated in athymic mice following inoculation with sham transfected COLO-357 cells (S) or clones expressing soluble TβRII

(C1-C2) .

A) Expression of soluble TβRII. RNA was size fractionated, electrotransferred to a Genescreen nylon membrane, and hybridized with 32P labeled soluble TβRII cDNA (2 x 105 cpm/ml, 1 day exposure) . The membrane was then stripped and probed with 7S cDNA (5 x 104 cpm/ml; 3 h exposure) .

B) Expression of PAI-1. Total RNA prepared was pooled in equal portions with three mice per group. RNA was prepared from tumors (33 μg/lane) from 3 mice injected with sham transfected cells (sham) and 3 mice injected with pMHsTβRII transfected cells (clones) . RNA was size fractionated, electrotransferred to a nylon membrane, and hybridized with the 32P labeled PAI-1 cDNA probe (5 x 105 cpm/ml, 3 day exposure) . To confirm equal loading of lanes, the membranes were stripped and reprobed with the 7S cDNA probe (5 x 104 cpm/ml, 1 day exposure) .

FIG. 7. PECAM-1 expression in tumors derived from COLO-357 cells .

A) Blood vessel frequency in tumor formed by COLO-357 cells. Fifty different high power fields (0.25 mm2 per field) were randomly selected for determination of blood vessel frequency following immunostaining with anti-PECAM-1 antibody. Data are the means ± SEM of 3 separate tumors per group. *P<0.002 and **P<0.0009 when compared with the tumors derived from sham transfected cells.

B) PECAM-1 immunoblotting in lysates derived from COLO-357 cells. Lysates (20 μg/lane) were prepared from tumor tissues derived from sham or pMHsTβRII COLO-357 cells (clones Cl or C2) . Lysates were then subjected to 7.5% SDS PAGE, electrotransferred to a membrane, and blotted with an anti-PECAM-1 antibody (1:3,000 dilution, 5 s exposure) . Human endothelial (E) cell lysates (1.0 μg) served as a positive control. To confirm equal loading of lanes, the membrane was stripped and reprobed with an anti-ERK-2 antibody (1:8000 dilution, 2 s exposure). Exposure time was increased to 10 minutes in order to detect ERK-2 in the case of the endothelial cell lysate.

FIG. 8. Transfection of pMHsTβRII in PANC-1 cells.

A) Expression of sTβRII inRA. Northern blot analysis of total RNA (20 μg/lane) from sham transfected PANC-1 cells (S) or from clones that were stably transfected with pMHsTβRII (C18 and C 19) was carried out with a human soluble TβRII cDNA probe (5 X 105 cpm/ml, 1 d exposure) . 7S cDNA was used as a loading control (5 X 10⁴ cpm/ml, 2 h exposure) .

B) Effects of TGF-βl on cell growth. Sham transfected cells and clones 18 and 19 were seeded in 12 well plates (100,000 cells/well) and incubated for 24 h in DME complete medium. Cells were then placed in serum-free medium in the absence (open boxes) or presence of 10 pM (checkered boxes) or 30 pM (solid boxes) TGF-βl for 48 h. Results are expressed as percent of control and are the means (±SEM) of 3 determinations per experiment from 3 experiments. Error bars for the control groups were exceedingly small. *P<0.01 when compared with respective untreated controls.

C) Tumor growth in subcutaneous model. Athymic nude mice were injected subcutaneously (2 x 10⁶ cells/site; two sites per mouse) with sham transfected cells (5 mice) and clones C18 (4 mice) and C19 (4 mice). Data are the means ± SEM. * P<0.001 when compared to the sham control .

FIG. 9. TβRII immunoreacti ity in tumors derived from PANC- 1 cells. An anti-TβRII antibody recognizing the full-length TβRII (A, B) and an anti-HA antibody recognizing the HA epitope of the pMHsTβRII construct (C, D) were used. In the tumor tissue derived from the sham transfected cells, there was weak to moderate i munostaining for endogenous TβRII (A) but undetectable HA immunoreactivity (C) . Strong TβRII (B) and moderate HA (D) immunoreactivity was present in the tumor tissue derived from cancer cells expressing the pMHsTβRII construct. Bar scale: 25 μm.

FIG. 10. Tumor metastases in the orthotopic model. The pancreas of a nude mouse was implanted with pancreatic tumor fragments derived from sham-transfected cells, as described below. The arrows point to metastatic lesions in the mesenteric lymph nodes. SI: small intestine; Ce: cecum. Inset: metastatic lesions in the spleen (indicated by arrows) .

FIG. 11. HA immunoreactivity in intra-pancreatic tumors. An anti-HA antibody was used to detect sTβRII expression in the intra-pancreatic tumors. A) Tumor from sham transfected cells was devoid of HA immunoreactivity.

B) Tumor from clone 18C exhibited weak HA immunoreactivity.

C) Tumor from clone 19B exhibited strong HA immunoreactivity. Bar scale: 25 μm.

FIG. 12. Relative expression of PAI-1 and uPA.

A) Subcutaneous model. Total RNA was prepared and pooled from subcutaneous tumors (33 μg/lane) from 3 mice injected with sham transfected cells (sham) and 3 mice injected with pMHsTβRII transfected cells (clones) . Northern blot analysis was carried out using the PAI-1 cDNA probe (5 x 10⁵ cpm/ml, 1 day exposure) . The membrane was reprobed with the uPA cDNA probe (5 x 10⁵ cpm/ml, 3 day exposure) . A' 7S cDNA probe was used as a loading control (5 x 10⁴ cpm/ml, 1 day exposure) .

B) Intra-pancreatic orthotopic model. Total RNA (20 μg/lane) was prepared and pooled from intra-pancreatic tumors derived from 4 mice implanted with sham transfected cells (Sham) and 4 mice implanted with pMHsTβRII transfected cells (Clones) . Total RNA (20 μg/lane) was also prepared from normal pancreatic tissues from 2 mice implanted with pMHsTβRII transfected cells that failed to yield tumors (Normal) . Northern blot analysis was carried- out as in panel A, but with a reduced exposure time following hybridization with the uPA cDNA probe (6 h) .

DETAILED DESCRIPTION OF THE INVENTION

Pancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy that frequently etastasizes and that overexpresses transforming growth factor-betas (TGF-βs) . This overexpression has been correlated with decreased patient survival. TGF-βs bind to a type II TGF-β receptor (TβRII) dimer, which heterotetramerizes with a type I TGF-β receptor (TβRI) dimer, thereby activating downstream signaling.

To determine whether blocking TGF-β actions would suppress pancreatic cancer cell growth in vivo, the present inventors expressed a soluble TβRII construct, encoding a ino acids 1-159 of the extracellular domain of TβRII (SEQ. ID NO. 1), in COLO-357 human pancreatic cancer cells. This cell line expresses all three mammalian TGF-β isoforms and is growth inhibited by TGF-β in vitro.

COLO-357 clones expressing soluble TβRII (sTβRII) were no longer growth inhibited by exogenous TGF-βl and exhibited a marked decrease in their invasive capacity in vitro. When injected subcutaneously into athymic mice, these clones exhibited attenuated growth rates and angiogenesis and decreased levels of PAI-1 mRNA as compared to tumors formed by sham transfected cells. These results indicate that endogenous TGF-βs can confer a growth advantage in vivo to a pancreatic cancer cell line that is growth inhibited in vitro, and indicate that a soluble receptor approach can be used to block these tumorigenic effects of TGF-βs.

To determine whether TGF-βs can act to enhance the metastatic potential of PDAC, PANC-1 human pancreatic cancer cells were transfected with sTβRII. When injected subcutaneously in athymic mice, PANC-1 clones expressing sTβRII exhibited decreased tumor growth in comparison with sham transfected cells, and attenuated expression of plasminogen activator inhibitor 1 (PAI-1), a gene associated with tumor growth.

When tested in an orthotopic mouse model, these clones formed small intra-pancreatic tumors that exhibited a suppressed metastatic capacity and decreased expression of PAI-1 and the metastasis-associated urokinase plasminogen activator (uPA) . These results indicate that TGF-βs act in vivo to enhance the expression of genes that promote the ■ growth and metastasis of pancreatic cancer cells and further support a role for sTβRII in the therapeutic treatment of PDAC.

The following terms are used herein:

"Individual" means any living organism, including humans and other mammals, which produce TGF-β.

"TGF-β" is a family of peptide growth factors.

"TGF-β receptor" is a cell surface protein, of which three types (Type I, Type II, and Type III) are known in mammals. "TGF-β receptor Type II" (or "TβRII") is a membrane-bound protein with an intracellular domain, transmembrane domain, and extracellular domain, which binds TGF-β. Human TGF-β receptor Type II has been determined to have the amino acid sequence shown in U.S. Pat. Nos. 6,001,969; 6,008,011; 6,010,872; 6,046,157; and 6,201,108, all to Lin et al . , and all hereby incorporated by reference in their entireties.

"TGF-β receptor fragment" is a portion or all of a TGF-β receptor molecule that is capable of binding TGF-β.

"Soluble TGF-'β receptor Type II" (or "sTβRII") is a polypeptide comprising a portion or all of the extracellular domain of TβRII, or a variant or derivative thereof, which is soluble and which binds to TGF-β. These polypeptides consist of 159 amino acids or less; preferably, sTβRII is about 159 amino acids. Optionally, ^■ sTβRII may also include stabilizing components as are known in the art as, for example, the Fc immunoglobulin fragment, to stabilize the soluble receptor.

"Variant" refers to polypeptides in which one or more amino acids have been replaced by different amino acids, such that the resulting variant polypeptide is at least 75% homologous, and preferably at least 85% homologous, to the basic sequence as, for example, the sequence of sTβRII as shown in SEQ. ID NO. 1, and wherein the variant polypeptide retains the activity of the basic protein, for example, sTβRII. Homology is defined as the percentage number of amino acids that are identical or constitute conservative substitutions. .Conservative substitutions of amino acids are well known in the art. Representative examples are set forth in Table 1.

TABLE 1

Original Residue Conservative Substitution (s)

Ala Ser

Arg Lys

Asn Gin, His

Asp Glu

Cys Ser

Gin Asn Glu Asp

Gly Pro

His Asn, Gin lie Leu, Val

Leu lie, Val

Lys Arg, Gin, Glu

Met Leu, lie,

Phe Met, Leu, Tyr

Ser Thr

Thr Ser

Trp Tyr

Tyr Trp, Phe

Val lie, Leu

Variants of polypeptides may be generated by conventional techniques, including either random or site-directed mutagenesis of DNA encoding the basic polypeptide. The resultant DNA fragments are then cloned into suitable expression hosts such as E. coli or yeast using conventional technology and clones that retain the desired activity are detected. The term "variant" also includes naturally occurring allelic variants.

"Derivative" refers to a polypeptide that has been derived from the basic sequence by modification, for example by conjugation or co plexing with other chemical moieties or by post-translational modification techniques as would be understood in the art. Such derivatives include amino acid deletions and/or additions to polypeptides or variants thereof wherein said derivatives retain activity of the basic protein, for example, sTβRII. Other derivatives contemplated by the invention include, but are not limited to, modification to side chains, incorporation of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinking agents.

"Therapeutic composition" is defined as comprising sTβRII and a pharmaceutically acceptable carrier. A "pharmaceutically acceptable carrier" is a solid or liquid filler, diluent or encapsulating substance that may be safely used in systemic administration. Depending upon the particular route of administration, a variety of pharmaceutically acceptable carriers, well known in- the art can be used. These carriers include, but are not limited to, sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline, and pyrogen-free water. Preservatives and other additives can also be present. For example, antimicrobial, antioxidant, chelating agents, and inert gases can be added (see, generally, Remington's Pharmaceutical Sciences, 16th Edition, Mack, (1980)).

Polypeptides of the invention may be prepared by any suitable procedure known to those of skill in the art. Recombinant polypeptides of the invention may be produced by culturing a host cell transformed with an expression vector containing nucleic acid encoding a polypeptide, fragment, variant or derivative according to the invention. Recombinant protein may be conveniently prepared by a person skilled in the art using standard protocols as, for example, described in Sambrook, et al., MOLECULAR CLONING. A LABORATORY MANUAL (Cold Spring Harbor Press, 1989) , incorporated herein by reference, in particular Sections 16 and 17; Ausubel et al . , CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (John Wiley & Sons, Inc. 1994-1998), incorporated herein by reference, in particular Chapters 10 and 16; and Coligan et al . , CURRENT PROTOCOLS IN PROTEIN SCIENCE

(John Wiley & Sons, Inc. 1995-1997) which is incorporated by reference herein, in particular Chapters 1, 5 and 6. Examples of vectors suitable for expression of recombinant protein include but are not limited to pGEX, pET-9d, pTrxFus or baculovirus

(available from Invitrogen) . A number of other vectors are available for the production of protein from both full length and partial cDNA and genomic clones, producing both fused or non-fused protein products, depending on the vector used. The resulting proteins are frequently immunologically and functionally similar to the corresponding endogenous proteins.

The obtained polypeptide is purified by methods known in the art. The degree of purification varies depending on the use of the polypeptide. For use in eliciting polyclonal antibodies, for example, the degree of purity may not need to be very high. However, as in some cases impurities may cause adverse reactions, purity of 90-95% is typically preferred and in some instances even required. For the preparation of -a therapeutic composition, however, the degree of purity must be high, as is known in the art.

The present invention provides for the administration of a therapeutic composition comprising sTβRII to an individual diagnosed with pancreatic cancer, or alternatively, diagnosed at risk for pancreatic cancer, to thereby provide for the suppression of intra-pancreatic tumor growth and local as well as distant metastasis, the suppression of angiogenesis, and the inhibition of PAI-1 and uPA overexpression.

The use of sTβRII to reduce or prevent metastasis and to suppress tumor growth may be employed as soon as malignant cells are detected, with or without coventional therapeutic methodologies such as chemotherapy agents or radiation. Suitable chemotherapy agents include but are not limited to anti-angiogenic compounds, alkylating compounds, antimetabolites, hormonal agonist/antagonists, monoclonal antibodies for cancer treatment, antiproliferatives, etc., and combinations thereof. Any anti-angiogenic compound can be used. Exemplary anti-angiogenic compounds include O-substituted fumagillol and derivatives thereof, such as TNP-470, described in U.S. Pat. Nos . 5,135,919, 5,698,586, and 5,290,807 to Kishimoto, et al . (hereby incorporated by reference) ; angiostatin and endostatin, described in U.S. Pat. Nos. 5,290,807, 5,639,725 and 5,733,876 to O'Reilly (hereby incorporated by reference) ; thalidomide, as described in U.S. Pat. Nos. 5,629,327 and 5,712,291 to D'Amato (hereby incorporated by reference) ; and other compounds, such as the anti-invasive factor, retinoic acid, and paclitaxel, described in U.S. Pat. No. 5,716,981 to Hunter, et al . , (hereby incorporated by reference) and the metalloproteinase inhibitors described in U.S. Pat. No. 5,713,491 to Murphy, et al (hereby incorporated by reference) . Other well known chemotherapeutic agents may also be used, such as doxorubicin, decarbazine, irinotecan, etoposide phosphate, asparaginase, gemcitabine, carboplatinum, cisplatinum, tomoxifen, methotrexate, ifosfa ide, cyclophosphamide, 5-fluorouracil, vinorelbine tartrate, anastrozole, trastuzumab and combinations thereof. These and other chemotherapeutic agents for the treatment of cancer may be found in the Physicians Desk Reference (hereby incorporated by reference herein) .

The method of prevention or reduction of the establishment, growth and/or metastasis of malignant cells may be used preoperatively and post-operatively as an adjunct to surgery.

It is understood that the dosage of the therapeutic composition of the present invention administered in vivo or in vitro will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the pharmaceutical effect desired. The most preferred dosage will be tailored to the individual subject, as is understood and deter inable by one skilled in the relevant arts. See, e.g., Berkow et al . , eds . , The Merck Manual, 16th edition, Merck and Co., Rahway, N.J. (1992); Goodman et al . , eds., Goodman, and Gilman's The Pharmacological Basis of Therapeutics, 8th edition, Pergamon Press, Inc., Elmsford, N.Y. (1990); Avery's Drug Treatment: Principles and Practice of Clinical Pharmacology and Therapeutics, 3rd edition, ADIS Press, LTD., Williams and Wilkins, Baltimore, Md. (1987) ; Ebadi, Pharmacology, Little, Brown and Co., Boston (1985); Osol et al., eds., Remington's Pharmaceutical Sciences, 17th edition, Mack Publishing Co., Easton, Pa. (1990) ; Katzung Basic and Clinical Pharmacology, Appleton and Lange, Norwalk, Conn., (1992), which references are entirely incorporated herein by reference. Based on the known affinity of the type II TGF-β receptor to its ligands and the present inventors' studies, a serum concentration of about 0.05 to about 0.5 mg/ml may be highly effective in suppressing cancer growth, invasion and metastasis.

The term "therapeutically effective amount" as used herein, means that amount of sTβRII that elicits the biological or medicinal response in a tissue system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation of the symptoms of the disease or disorder being treated.

The total dose required for each treatment can be administered by multiple doses or in a single dose. The diagnostic/pharmaceutical compound or composition can be administered alone or in conjunction with other diagnostics and/or pharmaceuticals directed to the pathology, or directed to other symptoms of the pathology.

The therapeutic composition of the invention may be administered by any of the conventional routes of administration, including oral, rectal, parenteral, sublingual, buccal, intravenous, intra-articular, intramuscular, intra-dermal, subcutaneous, inhalational, intraocular, intraperitoneal, intracerebroventricular, transdermal and the like, or as described in U.S. Pat. No. 5,693,607, the entire contents of which is hereby incorporated by reference. Also, the therapeutic composition of the invention may be in any of several conventional dosage forms, including, but not limited to, tablets, dispersions, suspensions, injections, solutions, capsules, suppositories, aerosols, and transdermal patches. Preferably, the therapeutic composition is administered by subcutaneous or intraperitoneal injection, by regional perfusion to the pancreas or by intra-lesional injection of soluble receptor protein or expression vector by laparoscopy or endoscopic ultrasonography.

The invention also includes recombinant DNA vectors containing a gene encoding sTβRII, or fragments or variants thereof, preferably vectors that target pancreatic cells, as, for example, by targeting overexpressed cell surface receptors such as EGF receptor, FGF receptor, or type Il-β receptor. Preferably, the genes encoding sTβRII are chimeric, encoding a fusion polypeptide comprising sTβRII and an epitope tag. For example, the chimeric gene may encode a sTβRII-heiαagglutinin fusion polypeptide.

The invention also contemplates polyclonal, monoclonal and humanized antibodies against the aforementioned sTβRII polypeptides, fragments, variants and derivatives or, alternatively, against TGF-β. Methods of producing polyclonal antibodies are well known to those skilled in the art. Exemplary protocols which may be used are described for example in Coligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY, (John Wiley & Sons, Inc, 1991) which is incorporated herein by reference, and Ausubel et al . , (1994-1998, supra), in particular Section III of Chapter 11.

Alternatively, monoclonal antibodies may be produced using the standard method as, for example, described in an article by Kohler and Milstein (1975, Nature 256, 495-497) which is herein incorporated by reference.

According to the method of the current invention, large amounts of recombinant sTβRII (or TGF-β), or derivative, variants or fragments thereof, are produced by scale up processes in commercial plants which enables production of a corresponding large quantity of antibodies. The antibodies to recombinant expressed protein can also be produced according to the invention using the standard method available for production of the antibodies to native protein.

The antibodies of the invention may be used for affinity chromatography in isolating natural or recombinant sTβRII (or TGF-β) polypeptides. The antibodies can also be used to screen expression libraries for variant polypeptides of sTβRII (or TGF- β) . Preferably, the antibodies of the invention can be administered to individuals diagnosed with pancreatic cancer, to inhibit binding of TGF-β molecules to endogenously expressed TβRII on the cancer cells and on the adjoining cellular elements, including endothelial cells. Thus, the anti-TβRII antibody could act as mimicker of sTβRII by "soaking-up" TGF-beta molecules." W 03

Preferably, humanized antibodies (XENOMOUSE^®, Abgenix, Inc., Fremont, California; BodeyB., et al., Curr. Pharm. Des. 6:261-76

(2000); Halloran P.F., et al . , Clin. Bioche . 31:353-7 (1998).) are administered as therapeutic agents to treat pancreatic cancer.

Antibodies may administered as described above for therapeutic compositions . Preferably, therapeutic antibodies are administered either subcutaneously or by intravenous injection. Having generally described the invention, the same will be more readily understood through reference to the following examples, which are provided by way of illustration, and are not intended to be limiting of the present invention.

EXAMPLES Materials and Methods Materials. The following materials were purchased from the indicated suppliers: FBS, DMEM medium, trypsin solution, penicillin-streptomycin solution, and Geneticin (G418) from Irvine Scientific (Santa Ana, CA) ; Amplitaq DNA Polymerase from Perkin Elmer (Norwalk, CT) ; restriction enzymes, pMH vector from Boehringer-Manheim (Indianapolis, IN) ; polymerase chain reaction

(PCR) primers from Bio-Synthesis, Inc. (Lewisville, TX) ; TA cloning pCRII vector from Invitrogen (Carlsbad, CA) ; mini plasmid DNA purification kit from Promega (Madison, WI) ; maxi DNA plasmid purification preparation kit and DNA gel extraction kit from Qiagen (Thousand Oaks, CA) ; Sequenase version 1.0 DNA sequencing from USB Specialty Bioc emicals (Cleveland, OH) ; Genescreen membranes from New England Nuclear (Boston, MA) ; random primed labeling kit from Ambion (Austin, TX) ; [³²P] dCTP and [³⁵S] dATP from Amersham (Arlington Heights, IL) ; TE Select D G50 columns from 5Prime-3Prime, Inc. (Boulder, CO) ; DNA and protein molecular weight markers, lipofectamine from Gibco-BRL Life Technologies

(Gaithersburg, MD) ; anti-HA, anti-ERK-2, anti-TβRII antibodies, and protein A agarose from Santa Cruz Biotechnologies, Inc.

(Santa Cruz, CA) ; biotinylated anti-human TβRII polyclonal antibody from R&D Systems, Inc. (Minneapolis, MN) ; horseradish peroxidase-conjugated antibodies from Biorad (Hercules, CA) ; PECAM-1 monoclonal antibody (clone C/70A) from Oncogene Research Products (Cambridge, MA) ; enhanced chemiluminescence (ECL) substrate and Restore Stripping Buffer from Pierce (Rockford, IL) ; Vectastain Universal Elite ABC kit from Vector Labs (Burlingame, CA) ; Immobilon-P nitrocellulose membranes from Millipore Corp. (Bedford, MA); streptavidin-peroxidase from Kirkegaard and Perry Laboratories, Inc. (Gaithersburg, MD) ; centriprep concentrators from Amicon Inc. (Beverly, MA) ; Lab-Tek chamber slides from Nunc, Inc. (Naperville, IL) ; Transwell chambers from Costar (Cambridge, MA) ; Matrigel from Becton Dickinson (Bedford, MD) ; and all other reagents from Sigma Chemicals Co. (St. Louis, MO) . COLO-357 human pancreatic cancer cells were a gift from Dr. R.S. Metzger (Duke University, Durham, NC) . TGF-βl was a gift from Genentech, Inc., (South San Francisco, CA) . Human dermal icrovascular endothelial cells were a gift from Dr. Joyce Bischoff (Children's Hospital, Harvard University Medical School, Boston, MA) and Dr. J. Luo (UC Irvine, Irvine, CA) .

Construction of a Mammalian Expression Vector. The complete cDNA of human TβRII (SEQ. ID NO. 2) was used as the template for PCR amplification of the coding sequence of the extracellular domain of TβRII (nucleotides 1 - 477, including the signal sequence). PCR was performed using the sense primer, 5'- AAGCTTGCCGCCGCCATGGGTCG, and antisense primer, 5'- CTGGAATTCGTCAGGATTGCTGG . The sense primer introduced a HindiII restriction site and the consensus Kozak translation initiation start site. The antisense primer introduced an EcoRI site.

The PCR fragment was ligated into the pCRII vector. The soluble TβRII coding fragment was isolated after digestion with Hindlll and EcoRV. This gel-purified fragment was subsequently ligated into the HindIII/Eco721 digested pMH expression vector, which is driven by a highly efficient immediate early human CMV promoter sequence and is tagged with the hemagglutinin (HA) epitope at its carboxy terminus. The constructed vector, pMHsTβRII, contained the open reading frame encoding the human soluble TβRII and nucleotides encoding nine amino acids of HA. The sequence and orientation was confirmed by dideoxy chain termination sequencing. The pMH plasmid containing the G418 resistance gene (neomycin) was used for construction of control clones (sham) expressing the vehicle vector alone.

Cell Culture. COLO-357 human pancreatic cancer cells were grown in Dulbecco's modified Eagle's medium (DME) medium, supplemented with 8% FBS, penicillin (100 U/ml) and streptomycin (100 μg/ml) and 5% fungazone termed complete medium. Cells were maintained in monolayer cultures at 370 C in humidified air with 5% C02. The selection medium for the cell lines containing the neomycin resistance gene was supplemented with 0.4 mg/ml G418.

For TGF-βl experiments, cells were incubated overnight in serum-free medium (DME containing 0.1% bovine serum albumin, 5 μg/ml transferrin, 5 ng/ml sodium selenite, antibiotics and fungazone) . To generate cells expressing the human soluble TβRII, COLO-357 cells were transfected in a stable manner with the pMHsTβRII plasmid (10 μg) using lipofectamine as previously reported (Kleeff, J., et al., J. Clin. Invest. 102: 1662-73, 1998) .

After reaching confluence, cells were split 1:10 into selection medium, and single clones were isolated after 3-4 weeks. After expansion of each individual clone, cells from each clone were screened for expression of soluble TβRII by Northern and Western blot analysis. Two clones were selected for further studies .

PANC-1 cells (ATCC, Rockville, MD) were grown in Dulbecco's modified Eagle's medium (DME) supplemented with 8% FBS, penicillin (100 U/ml) , streptomycin (100 μg/ml) , and 5% fungazone (complete medium) and maintained in monolayer culture at 370C in humidified air with 5% C02. To select for cells containing the neomycin resistance gene, the medium was supplemented with 1.25 mg/ml G418. For TGF-βl experiments, cells were incubated in serum-free medium (DME containing 0.1% bovine serum albumin, 5 μg/ml transferrin, 5 ng/ml sodium selenite, antibiotics and fungazone) . PANC-1 cells were transfected in a stable manner with the pMHsTβRII plasmid (10 μg) , using the lipofectamine method. After expansion of each individual clone, cells were screened for expression of pMHsTβRII by Northern blotting.

Immunoblotting and Immunoprecipitation. Exponentially growing human pancreatic and dermal microvascular endothelial cells (50-60% confluent) were washed with ice cold IX PBS and lysed in buffer containing 1% Nonidet P-40, 20 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA, ImM EGTA, 2.5 mM sodium phosphate, 1 mM β-glycerophosphate, ImM sodium vanadate, 1 μg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride. Frozen tumor samples derived from COLO-357 sham transfected or pMHsTβRII clones were homogenized and lysed in the same buffer.

Lysates were subjected to SDS-PAGE and electrotransferred to

Immobilon-P membranes for 40-80 min. After blocking with 5% milk

(lx TTBS with 0.1% Tween-20) , the membranes were incubated with anti-HA monoclonal antibody (1:1200 dilution), anti-CD31 antibody

(1:3000 dilution), washed, and incubated with a secondary horseradish peroxidase conjugated antibody. After washing, bound antibodies were visualized using enhanced chemiluminescence. To confirm equal loading, membranes were stripped for 20 minutes at room temperature in either Restore stripping buffer or 30 minutes at 500 C in buffer containing 2% SDS, 62.5 mM Tris (pH 6.7) and

100 mM 2-mercaptoethanol, and blotted with an anti-ERK-2 antibody

(1:8,000 dilution) .

For immunoprecipitation with the anti-HA antibody, cells transfected with the soluble TβRII expression construct or sham construct were grown to 80% confluency in complete medium and then incubated for 24 hours in serum-free medium. Conditioned medium from the clones or sham transfected cells was concentrated by using a 10,000 molecular weight cutoff filtration membrane.

The concentrated medium was incubated for 12 hours at 40 C with the anti-HA antibody (2 μg/ml) , followed by a 2 hour incubation with protein A agarose (50 μl) at 40 C. Precipitates were washed 3 times with ice-cold PBS, resuspended in 2X loading buffer, and boiled for 5 min at 1000 C. After centrifugation, the supernatants were subjected to western blotting using the biotinylated anti-human TβRII polyclonal antibody (1:5,000 dilution) .

Immunohistochemistry. COLO-357 cells were plated in chamber slides and grown to 70% confluency for 48 hours. The cells were fixed in 1.5% paraformaldehyde for 45 minutes at room temperature (RT) and incubated sequentially for 30 min (RT) with 0.1% Triton X-100, 30 min (RT) with 0.3% hydrogen peroxide/methanol, 30 min (370 C) with 1 mg/ml hyaluronidase, and 40 min (RT) with 10% normal goat serum. Cells were then incubated for 16 hours (40 C) with the highly specific anti-HA antibody (0.4 μg/ml) recognizing the HA epitope encoded by pMHsTβRII or with the highly specific anti-TβRII antibody (0.2 μg/ml) recognizing the epitope corresponding to the full-length . TβRII .

To assess TβRII, HA, and CD31 immunoreactivity, tumors from subcutaneous lesions were removed and immediately divided. Tissues were fixed in 4% formaldehyde and embedded in paraffin wax. Paraffin-embedded sections (4 μm) from tumor tissue derived from sham transfected or pMHsTβRII transfected cells were cut and mounted on poly L-lysine coated glass slides and air-dried overnight at RT. Representative sections of each case were examined by the streptavidin-peroxidase technique using appropriate positive and negative controls. Endogenous peroxidase activity was blocked by incubation for 30 minutes with 0.3% hydrogen peroxidase in methanol. Tissue sections were incubated for 15 minutes (RT) with 10% normal goat serum and then incubated for 16 hours at 40 C with anti-HA antibody (0.4 μg/ml) ; anti-TβRII antibody (0.2 μg/ml), recognizing the epitope corresponding to the full-length TβRII; or anti-PECAM-1 antibody (1:50 dilution), in PBS containing 1% BSA. In addition, tumor samples from the orthotopic model were embedded in OCT compound, frozen in liquid nitrogen, and stored at -800 C. Cryostat sections were then prepared and stained with an ariti-HA antibody (0.25 μg/ml) .

For both cell line and tissue immunohistochemistry, bound TβRII and HA antibodies were detected with biotinylated goat anti-rabbit IgG secondary antibodies and streptavidin-peroxidase complexes, using diaminobenzidine tetrahydrochloride as the substrate. Sections were counterstained with Mayer's hematoxylin. Sections incubated with non-immune rabbit IgG or with secondary antibodies alone did not yield positive immunoreactivity. The frequency of blood vessels in the matrix region of the tumor that was positively stained for PECAM-1 was evaluated morphometrically. Fifty different high power fields were randomly selected for each specimen, with each high power field representing 0.25 mm2 on the microscope grid.

RNA Extraction and Northern Blot Analysis. Total RNA was extracted by the single step acid guanidine thiocyanate-phenol- chloroform method (Chomczynski, P. and Sacchi, N., Anal. Biochem. 162: 156-9, 1987). RNA was size fractionated on 1.2% agarose/1.8 M formaldehyde gels, electrotransferred onto Genescreen nylon membranes, and crosslinked by UV irradiation (Korc, M., et al., J. Clin. Invest. 90: 1352-60, 1992).

The blots were prehybridized and hybridized in 0.75 M NaCl; 5 mM EDTA pH 8.0; 50 mM sodium phosphate; 50% forma ide; 5X Denhardt's solution; 10% dextran sulfate; 1% sodium dodecyl sulfate; and 100 μg/ml salmon sperm DNA with cDNA probes at 420 C. The cDNA probes included a 500 base pair (bp) Hindlll/EcoRI fragment of the human pMHsTβRII cDNA, a 500 bp SacII/Pstl fragment of the human PAI-1, a 1.5 kb PstI fragment of the human uPA (ATCC; Manassas, VA) and a 190 bp BamHI fragment of mouse 7S ribosomal cDNA, which cross-hybridizes with human 7S RNA. The 7S probe was used to confirm equal RNA loading. Membranes were washed under high stringency conditions (washed 2 times in 2 X SSC at room temperature and two times at 550 C in 0.2 X SSPE/1% SDS) . Blots were exposed to Kodak Bio ax MS films in Kodak Bio ax MS cassettes at -800 C.

Cell Growth Assays . To assess the growth inhibitory effects of TGF-β, COLO-357 cell growth was determined by the MTT [3- (4, 5- methylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide] dye reduction assay, which measures the conversion of the MTT tetrazoliu salt into MTT formazan by mitochondrial hexosaminidase (Mosmann, T., J. Immunol. Methods 65: 55-63, 1983; Green, L. M., et al . , J. Immunol. Methods 70: 257-68, 1984).

COLO-357 cells were seeded at a density of 10,000 cells/well in 96-well plates in DME complete medium and incubated for 24 hours prior to incubation for 72 hours in serum-free medium in the absence or presence of TGF-βl (10 pM) . The assay was initiated by adding MTT solution at a final concentration of 62.5 μg MTT/well. (Baldwin, R. L. and Korc, M., Growth Factors 8: 23- 34, 1993; Kleeff, J. and Korc, M., J. Biol. Chem. 273: 7495-500, 1998) .

After 4 hours, the medium was removed and the dye crystals were dissolved in acidified isopropanol. The optical density was measured at 570 nm and 650 nm with an enzyme linked immunosorbent assay plate reader (Molecular Devices, Menlo Park, CA) . Data were expressed as percent of control cell growth. In pancreatic cancer cells, the results of the MTT assay correspond with results obtained by cell counting with a hemacytometer or by monitoring [³H] -thymidine incorporation into DNA.

PANC-1 sham transfected or pMHsTβRII transfected cells were seeded at a density of 1.0 x 105 cells/well in 12-well plates using DME complete medium. Cells were incubated for 24 hr prior to incubation for 48 hr in serum-free medium in the absence or presence of TGF-βl. Cell growth was then determined by cell counting using a hemacytometer, and data were expressed as percent of control cell growth.

In Vivo Tumorigenicity Assay. To assess the effect of the soluble TβRII on tumorigenicity, 2 x 10⁶ or 6 x 10⁵ COLO-357 cells expressing the empty vector alone (sham) or soluble TβRII were injected subcutaneously into two sites in female athymic (nude) mice. The tumor size was measured weekly until mice were sacrificed 56 days after injection. A portion of the tumor tissue was snap frozen in liquid nitrogen and stored for subsequent RNA and protein analysis. Another portion was prepared for immunohistochemistry studies.

Invasion Assay. The invasive abilities of the COLO-357 sham transfected and soluble TβRII transfected pancreatic cancer cells were measured as previously reported (Albini, A., et al., Cancer Res. 47: 3239-45, 1987) with some modifications (Kleeff, J., et al., Int. J. Cancer 81: 650-7, 1999). Briefly, polycarbonate membranes (8 μm pore size) of the upper compartment of Transwell chambers were coated with 5% Matrigel. COLO-357 cells that were pre-incubated for 24 hours in serum-free medium containing 0.1% BSA in the absence or presence of 400 pM TGF-βl were suspended in 100 μl serum-free medium containing 0.1% BSA and placed onto this upper compartment. The lower compartment was then filled with 600 μl of serum-free medium containing 0.5% FBS. TGF-βl

(400 pM) was added to the lower compartment corresponding to chambers that contained cells previously incubated with TGF-βl. After 16 h, the membranes were fixed in methanol and stained with hematoxylin and eosin. Cells on the upper surface of the filter were carefully removed with a cotton swab, and the cells that had migrated through the membrane to the lower surface of the filter were counted in 9 different fields using a light microscope

(magnification: 200x) . Invasion assays were performed in triplicate.

Growth Characteristics in Subcutaneous and Orthotopic Models. Initially, PANC-1 cells expressing the empty vector alone (sham) or pMHsTβRII were injected subcutaneously into female athymic (nude) mice, as described above. Tumors were measured externally on the indicated days and tumor volume was determined by the equation: volume = (1 x h x w) x B/4, where 1 is length, h is height, and w is width of the tumor. The mice were sacrificed 49 days after injection, when tumor burden in control mice approached the allowable limit.

To generate intra-pancreatic tumors, the subcutaneous tumors were aseptically resected and immediately placed into DME complete media. Three separate tumors from each group (sham, clone 18, or clone 19) were pooled and minced together into pieces of approximately 2 mm³. For each group (sham, clone 18, or clone 19) , nude mice were implanted with three tumor fragments that were introduced into the pancreas via a surgical flap. The mice were anesthetized with a cocktail of xyla-ject and keta-ject (Phoenix Pharm., St. Joseph, MD) , a median incision was made, and the portion of the pancreas near the spleen was exposed (Reyes, G., et al., Cancer Res. 56: 5713-5719, 1996).

Tumor pieces were implanted under a pancreatic flap that was sutured with a 6-0 absorbable suture (ETHICON; Somerville, NJ) . The abdominal wall and skin were then closed with 3-0 silk sutures. After implantation, mice were inspected weekly for tumor formation by palpation. All mice were sacrificed 2 months after implantation. At autopsy, the pancreas and other organs harboring metastatic lesions were resected. All studies with mice were approved by the University of California Irvine Institutional Animal Care and Use Committee (protocol #98-1298) .

Animals. Four- to six-week old female nu/nu (nude) mice

(Harlan, Indianapolis, IN) were used for tumor implantation for both the subcutaneous and orthotopic models. Mice were housed in the University of California, Irvine, animal facility within a sterile environment.

Statistics. Statistical analysis was performed with SigmaStat software (Jandel Scientific, San Raphael, CA) and Prism software (Graphpad Software, Inc., San Diego, CA) . Student's two-sided t test was used when indicated. P<0.05 was taken as the level of significance. Image Quant software (Molecular Dynamics, Sunnyvale, CA) was used to quantitate the intensity of bands from northern blots.

Example 1: Expression of Soluble TβRII in Transfected

COLO-357 Clones.

COLO-357 pancreatic cancer cells were stably transfected with a soluble TβRII cDNA construct, encoding the entire extracellular domain (amino acids 1-159) of human TβRII. Transfected clones were selected after 3-4 weeks of growth in medium supplemented with G418, and subsequent experiments were carried out with 2 independent clones. These clones were selected because they displayed high levels of soluble TβRII mRNA expression by Northern blot analysis (FIG. 1A) . COLO-357 cells transfected with the pMH empty vector carrying the G418 resistance gene served as the control (sham), and did not express the soluble TβRII (FIG. 1A) .

Immunoblotting of cell lysates with the anti-HA antibody, which recognizes the HA epitope at the COOH terminus of the pMH expression vector, confirmed the expression of the soluble TβRII at the protein level (FIG. IB) . To determine whether the soluble TβRII was secreted, conditioned medium from a sham transfected clone and from clone 1 (Cl) was subjected to immunoprecipitation with the anti-HA antibody followed by immunoblotting with the biotinylated anti-human TβRII antibody. A major band (25 kDa) representing the soluble TβRII protein was visible in conditioned medium from the Cl clone, but not from the sham clone. A minor band (35 kDa) was also present in the Cl conditioned medium (FIG. 1C) .

Expression of the soluble TβRII was also confirmed by immunohistochemical analysis. Using the anti-TβRII antibody, there was weak imunostaining for TβRII in the sham transfected cells (FIG. 2A) , representing endogenously expressed TβRII. In contrast, there was strong TβRII immunoreactivity in clone 1 cells (FIG. 2B) and clone 2 (not shown) expressing the transfected soluble TβRII. When the anti-HA antibody was used, sham transfected cells were devoid of immunoreactivity (FIG. 2C) , whereas positive immunostaining for HA was readily evident in the cells transfected with the pMHsTβRII construct (FIG. 2D) .

As the above results indicate, when a cDNA encoding the soluble TβRII was stably transfected into COLO-357 cells in an attempt to suppress the biological actions of cancer cell derived TGF-βs, selected clones expressed high levels of the soluble TβRII. Furthermore, the soluble TβRII was secreted into the conditioned medium where it was present as a major band of 25 kDa and a minor band of 35 kDa.

These findings are consistent with other studies which showed that the extracellular domain of TβRII was detected as multiple bands of 25 to 35 kDa in COS cells (Lin, H. Y., et al.,

J. Biol. Chem. 270: 2747-54, 1995) and EL4 mouse thymoma cells

(Won, J., et al., Cancer Res. 59: 1273-7, 1999). Furthermore, as shown in FIG. 2, HA immunoreactivity was only present in the cancer cells transfected with the pMHsTβRII construct and TβRII immunoreactivity was only present at high levels in the transfected clones. Together, these observations indicate that both selected clones expressed high levels of the soluble TβRII. Example 2: Effects of TGF-βl on Cell Growth and Invasion.

Sham transfected COLO-357 cells exhibited a doubling time of approximately 30 hours, in agreement with previously published growth characteristics of parental COLO-357 cells (Kleeff, J., et al., Biochem. Biophys. Res. Commun. 255: 268-73, 1999). Clones expressing the soluble TβRII exhibited similar doubling times, ranging from 31 hours for clone 1 to 37 hours for clone 2. TGF-βl (10 pM) inhibited the growth of sham transfected COLO- 357 cells (30%; P<0.002) but was without effect in the soluble TβRII expressing clones (FIG. 3A) .

TGF-βl (400 pM) also significantly increased the invasiveness of sham transfected COLO-357 cells in an in vitro cell invasion assay (FIG. 3B) , but was without effect in the clones expressing the soluble TβRII (FIG. 3B) . Thus, expression of soluble TβRII effectively blocked the biological actions of exogenous TGF-βl.

As shown above, COLO-357 clones expressing the soluble TβRII and sham transfected COLO-357 cells exhibited similar doubling times in vitro. However, in the pMHsTβRII expressing clones, the growth inhibitory effect of 10 pM TGF-βl was completely blocked, indicating that soluble TβRII efficiently bound and sequestered TGF-βl. This conclusion is supported by the observation that the stimulatory effect of 400 pM TGF-βl on cell invasiveness was also completely blocked by soluble TβRII.

The mechanisms by which TGF-βl promotes cellular invasion are not known. It has been shown that TGF-βl increases Smad2 expression in COLO-357 cells (Kleeff, J., et al., Dig. Dis. Sci. 44: 1793-802, 1999) and elevated levels of Smad2 are known to enhance cellular motility (Prunier, C, et al . , J. Biol. Chem. 274: 22919-22922, 1999) . It has also been established that TGF- βl enhances the expression of PAI-1 (Kleeff, J. and Korc, M., J. Biol. Chem. 273: 7495-500, 1998; Chen, R. H., et al . , Science 260: 1335-8, 1993; Taipale, J., et al . , Adv. Cancer Res. 75: 87- 134, 1998; Sehgal, I., et al . , Cancer Res. 56: 3359-65, 1996) and increased TGF-β expression correlates with increased PAI-1 levels in pancreatic cancer.

PAI-1 is the main inhibitor of the urokinase-type plasminogen activator system. It promotes cancer cell migration by preventing excessive extracellular matrix degradation by plasmin proteolysis (Andreasen P.A., et al., Cell andMolec. Life Sci. 57: 25-40, 2000), and its reduced expression correlates with attenuated tumorigenicity in Smad4 reconstituted cancer cells (Schwarte-Waldhoff I., et al . , Oncogene 18: 3152-8, 1999). Therefore, it is possible that TGF-βl acts via these mechanisms to promote the motility of COLO-357 cells across a Matrigel membrane. Since pancreatic cancer is a highly invasive malignancy, these observations suggest that TGF-βs may act directly on pancreatic cancer cells to promote cancer cell invasion.

Example 3: Growth Properties of Soluble TβRII Expressing Clones in Vivo.

To compare the tumorigenicity of COLO-357 soluble TβRII expressing cells with sham transfected cells, 2 X 106 cells/site were subcutaneously injected in athymic nude mice (two sites/mouse) . Transfected clones consistently revealed a significant decrease in tumor volume, as compared to tumors arising from sham transfected cells. When tumor volumes from three separate experiments were averaged on day 28 post- injection, there was significant tumor growth inhibition for both Cl (67%) and C2 (62%), when compared to the sham controls (FIG. 4A) .

To determine if tumor growth was also decreased following injection of a lower number of cells, 6 x 105 COLO-357 cells/ site were injected next (two sites per mouse) . Three mice were injected with sham transfected COLO-357 cells, and six mice were injected with pMHsTβRII transfected COLO-357 clones. There was significant inhibition of tumor growth by the transfected clones at 28 (70%; P<0.04) and 35 (75%; P<0.005) days post-injection, when compared to the respective sham controls (FIG. 4B) . One site injected with the pMHsTβRII Cl cells did not form a tumor, even after 56 days. In contrast, all sites injected with sham transfected cells always yielded tumors.

After day 35, two mice each bearing two tumors of the sham transfected cells, had to be sacrificed because the tumor burden reached the maximum allowable limit by our Institutional Animal Care and Use Committee protocol. The remaining mice bearing tumors from either the pMHsTβRII clones or the sham transfected cells were allowed to grow until day 56. Even at time of sacrifice (day 56) , tumors derived from the clones never became as large as the sham did on day 35. At day 56, the tumors derived from sham transfected cells were still progressively growing, whereas the growth of the tumors derived from pMHsTβRII cells reached a plateau (FIG. 4B) .

To confirm the expression of the soluble TβRII in vivo, immunostaining and northern blot analysis were performed next. When using the anti-TβRII antibody, a few cells were weakly positive for endogenous TβRII in the tumors derived from sham transfected cells (FIG. 5A) . As expected, these tumors did not exhibit positive immunostaining when the anti-HA antibody was used (FIG. 5C) . In contrast, strong TβRII (FIG. 5B) and HA (FIG. 5D) immunoreactivity was evident in the cancer cells expressing the pMHsTβRII construct.

Tumors from both clones expressed high levels of the soluble TβRII RNA moiety (FIG. 6A) . In contrast, tumors from sham transfected cells did not express the soluble TβRII mRNA transcript (FIG. 6A) .

Since PAI-1 has been implicated in pancreatic cancer metastasis in humans (Takeuchi Y., et al., Amer. J. of Gastro. 88: 1928-1933, 1993) and is overexpressed in this malignancy (Kleeff, J., et al . , Dig. Dis. Sci. 44: 1793-802, 1999), its expression was analyzed next. In the tumors derived from soluble TβRII clones, there was a 67% decrease in PAI-1 mRNA levels when compared to the sham tumors (FIG. 6B) . Morphometric analysis (FIG. 7A) revealed a statistically significant decrease in the number of cells that stained positive for anti-PECAM-1 antibody in tumors derived from either COLO-357 Cl (6.7 ±0.35) or C2 (5.3 ± 0.37), in comparison with tumors derived from the sham transfected cells (12.2 ± 0.70) . In order to more clearly assess blood vessel mass, immunoblotting with the anti-PECAM-1 antibody was performed next using frozen tumor tissues derived from both COLO-357 cells. Tumors derived from sham transfected COLO-357 cells expressed more PECAM-1 then those derived from pMHsTβRII transfected cells (FIG. 7B) . Human endothelial cells, which are known to express high levels of PECAM-1, served as a positive control and exhibited a strong PECAM-1 signal.

As discussed above, multiple subcutaneous injections of two independent clones expressing soluble TβRII yielded small tumors in nude mice when compared to sham transfected cells. These tumors expressed the soluble TβRII by Northern analysis and exhibited strong HA and TβRII immunoreactivity, thereby confirming in vivo production of soluble TβRII. These tumors also exhibited attenuated PAI-1 expression by comparison with tumors formed by sham transfected cells, indicating that soluble TβRII interfered with TGF-β mediated induction of PAI-1 in vivo.

There was a parallel decrease in tumor angiogenesis as determined by a marked decrease in the number of blood vessels and PECAM-1 levels. Inasmuch as TGF-βl and PAI-1 can promote angiogenesis in vivo (Oft, M., et al., Curr. Biol. 8: 1243-52, 1998; Marzo, A. L., et al . , Cancer Res. 57: 3200-7, 1997; Ueki, N., et al., Biochim. Biophys. Acta 1137: 189-96, 1992; Roberts, A. B., et al., Proc. Natl. Acad. Sci. U.S.A. 83: 4167-71, 1986; Yang, E.Y. and Moses, H. L., J. Cell Biol. Ill: 731-41, 1990; Lambert V., et al . , Faseb Journal 15: 1021-1027, 2001), these findings suggest that the soluble TβRII interferes with proangiogenic pathways in vivo.

As discussed above, COLO-357 pancreatic cancer cells expressing sTβRII exhibit attenuated growth in a subcutaneous, non-metastatic nude mouse model. It was not known, however, whether this attenuated growth could suppress the metastatic potential of pancreatic cancer cells since the subcutaneous mouse model is non-metastatic. Therefore, the growth of PANC-1 human pancreatic cancer cells was tested in a metastatic mouse model. PANC-1 cells were used because they express all three TGF-β isoforms (Baldwin, R.L., et al . , Int. J. Can. 67: 283-288, 1996)' and exhibit increased in vi tro invasiveness in response to TGF-β

(Ellenrieder, V., et al . , Int. J. Cancer, 93: 204-211, 2001). When these cells were stably transfected with a cDNA construct encoding a human sTβRII (pMHsTβRII) there was a marked decrease in their metastatic potential in vivo when compared to sham transfected cells, as discussed below.

Example 4: Effects of pMHsTβRII on TGF-βl Actions and

Subcutaneous Tumor Growth Using PANC-1 Cells Sham transfected PANC-1 cells and clones C18 and C19 stably transfected with pMHsTβRII, encoding the extracellular domain

(amino acids 1-159) of human TβRII, exhibited the endogenous

(-5.2 kb) TβRII mRNA transcript (FIG. 8A) . Clones C18 and C19 expressed, in addition, the (~0.8 kb) sTβRII mRNA transcript

(FIG. 8A) . In contrast, sham transfected PANC-1 cells that were transfected with the pMH empty vector for use as controls, did not express sTβRII mRNA (FIG. 8A) .

The sham-transfected cells exhibited doubling times of approximately 23 hours and were growth inhibited by 10 and 30 pM TGF-β (FIG. 8B) . Although clones C18 and C19 exhibited similar doubling times that ranged from 24 hours to 29 hours, they were not growth inhibited by either concentration of TGF-βl (FIG. 8B) . The tumorigenicity of pMHsTβRII expressing PANC-1 cells and sham transfected cells was compared following subcutaneous injection in athymic nude mice. Clones transfected with pMHsTβRII consistently formed smaller tumors as compared to tumors arising from sham transfected cells. There was significant inhibition (72%) of tumor growth in the pMHsTβRII transfected clones beginning at 7 days post-injection (FIG. 8C) , which was most pronounced at 49 days post-injection (83%) . At this time point, endogenous TβRII immunoreactivity was present in the tumors derived from sham transfected PANC-1 cells (FIG. 9A) . As expected, these tumors did not exhibit any HA immunoreactivity (FIG. 9C) . In contrast, strong TβRII (FIG. 9B) and moderate HA (FIG. 9D) immunoreactivity were evident in the PANC-1 cells expressing the pMHsTβRII construct. In general, TβRII immunoreactivity exhibited a heterogeneous pattern of distribution within the tumors (FIG. 9B) , indicating that there was variable but persistent expression of soluble TβRII in vivo . Example 5: Growth Properties of Soluble TβRII Expressing Clones in an Orthotopic Model Subcutaneous tumors arising from pancreatic cancer cells do not metastasize. Therefore, tissue minces from these tumors were next implanted into the pancreas of nude mice, since this orthotopic model is known to yield metastases (Reyes, G., et al., Cancer Res. 56: 5713-5719, 1996). Tissue fragments from the subcutaneous tumors of three mice previously injected with sham transfected cells were implanted directly into the pancreas of three nude mice as described above. The resulting pancreatic tumors were large (0.8 to 1.1 cm), and formed multiple metastatic lesions, including lesions in the liver, spleen, local lymph nodes and distal lymph nodes.

In contrast, when three mice were implanted with sTβRII derived tumor tissue minces, only one mouse grew a large primary tumor (0.8 cm) and it formed only a few metastatic lesions

(liver, peri-pancreatic lymph nodes) . One mouse formed a small

(0.3 cm) primary tumor and the other mouse did not form a tumor; neither mouse had any metastatic lesions.

Next, a larger experiment was carried out in which four mice were implanted with sham-derived tissue minces and 8 mice were implanted with pMHsTβRII expressing clones (Table 2) . All four mice implanted with sham-derived tissue minces grew large pancreatic tumors (0.8 to 1.2 cm), and three of the mice exhibited tumor spread to multiple sites, including liver, spleen, adrenals, perirectum, and kidneys (Table 2) . The lymph nodes adjacent to the aorta, omentum, mesentery, mesenteric and stomach also contained metastatic foci. An example of a pancreatic tumor exhibiting metastases to the mesenteric lymph nodes and spleen is shown in FIG. 10.

In contrast, only one of the 8 mice implanted with pMHsTβRII expressing clones developed a large primary tumor (1.2 cm), and this mouse developed peritoneal seeding and mesenteric lymph involvement (Table 2) . In addition, one mouse implanted with pMHsTβRII expressing cells developed a medium-sized primary tumor

(approximately 0.8 cm in diameter), three mice developed very small (approximately 0.3 cm in diameter) primary tumors, and three mice did not form any tumors (Table 2) . None of these 7 mice developed any metastases. Thus, altogether, only 2 of 11

(18%) mice implanted with pMHsTβRII expressing clones exhibited metastatic lesions.

Table 2 : Primary and Metastatic Lesions

H-= large tumor; ++=medium tumor; +=small tumor; M-metastatic site; M/M=bilateral metastasis As expected, tumors forming after orthotopic implantation of sham transfected cells did not exhibit HA immunoreactivity (FIG. 11A) . Furthermore, the tumor from the mouse implanted with pMHsTβRII expressing clones that developed metastases exhibited weak HA immunoreactivity (FIG. 11B) . In contrast, in the other mice implanted with pMHsTβRII expressing clones (which were not metastatic) , all the tumors exhibited strong HA immunoreactivity (FIG. 11C) .

PAI-1 and uPA are growth and metastasis associated genes that are overexpressed in PDAC (Cantero, D., et al . , Br. J. Cancer 75: 388-395, 1997; Wang, W., et al . , Oncogene 18: 4554-4563, 1999; Kleeff, J., et al . , Dig. Dis. Sci. 44: 1793-1802, 1999) . Therefore, their expression in both the subcutaneous and orthotopic models was analyzed next (FIG. 12A, B) .

Both mRNA moieties were expressed at high levels in tumors from sham transfected cells, irrespective of the model (FIG. 12A, B) . In contrast, their expression was below the level of detection in the normal pancreas (FIG. 12B) . Levels of uPA mRNA (~2.4 kb) were especially elevated in the orthotopic model, since a brief exposure time (6h) and less RNA (20 μg/lane) were required in this model by comparison with the subcutaneous model (FIG. 12) .

In both models, there was a 55-60% decrease in the PAI-1 mRNA transcript levels ( 2.2 kb and 3 kb) in the tumors derived from pMHsTβRII expressing clones when compared with the corresponding sham tumors (FIG. 12A, B) . In contrast, only the intra-pancreatic model exhibited decreased uPA mRNA levels (53%) with the pMHsTβRII expressing tumors (FIG. 12A, B) .

As shown above, expression of the soluble TβRII in transfected clones was confirmed by Northern blotting and by demonstrating that transfected cells were not growth inhibited by TGF-βl in vitro. These observations indicate that expression of the soluble TβRII interfered with the ability of exogenous TGF-βl to activate the endogenous TβRII. Compared to sham transfected cells, PANC-1 clones expressing pMHsTβRII, as confirmed by immunostaining, yielded small tumors in the subcutaneous model. PANC-1 clones expressing pMHsTβRII also formed smaller primary tumors and exhibited decreased metastatic potential in the orthotopic model when compared with the sham transfected cells. The only pMHsTβRII expressing clone that yielded metastases exhibited relatively weak HA immunoreactivity, indicating that strong and persistent expression of pMHsTβRII, rather . than weak and heterogeneous expression, may be required to attenuate the metastatic potential in the orthotopic model. These observations indicate that sTβRII has the potential to be a potent suppressor of PANC-1-derived tumor growth and metastasis.

In addition, there was a marked up-regulation of uPA and PAI-1 in the subcutaneous and intra-pancreatic tumors that formed with sham-transfected PANC-1 cells, by comparison to the levels observed in the normal pancreas. Although the exact site of expression within the tumor mass was not determined, in the case of uPA this up-regulation was especially marked in the orthotopic model .

Furthermore, expression of pMHsTβRII was associated with decreased PAI-1 expression in both models, as well as with- decreased levels of uPA mRNA in the orthotopic model. Inasmuch as targeted deletion of either PAI-1 or uPA results in attenuated tumor formation and growth in mice (Gutierrez, L.S., et al . , Cancer Res. 60: 5839-5847, 2000), the findings of the present inventors suggest that sTβRII may suppress tumor growth and invasion by attenuating the expression of PAI-1 and uPA, and that this phenomenon may be especially important in the orthotopic model .

The present results suggest that there are several potential beneficial consequences to blocking TGF-β actions in vivo by the sTβRII approach. These advantages include suppression of intra-pancreatic tumor growth and local as well as distant metastasis, suppression of angiogenesis, suppression of cancer cell invasiveness, inhibition of PAI-1 and uPA overexpression and, potentially, suppression of uPA-mediated TGF-β activation. Together these findings indicate that sTβRII targets many of the deleterious aspects that occur as a consequences of TGF-β overexpression in PDAC and that sTβRII can ultimately have a distinct therapeutic benefit in the treatment of this malignancy. Example 6: Expressing sTβRII in E. coll

The complete cDNA of human TβRII was used as template for PCR reactions to generate the cDNA inserts encoding residues 4-136 of the extra-cellular domain of TβRII (sTβRII) , using the primers: sense 5 ' -CACGTTCAGAAGTCGGTTAAT and anti-sense 5'- GTCAGGATTGCTGGTGGTATATTC. The DNA inserts were digested with BamHI/Hindlll and sub-cloned into pTrcHisC to construct pTrcHisRII. The plasmid DNA was transformed into E. coll BL-21. Authenticity was confirmed by DNA sequence analysis. It was then determined that the approximately 20 kDa sTβRII was expressed principally in the soluble protein fractions of E. coli lysate, not in the insoluble inclusion bodies, which will facilitate large scale preparation of the protein. Furthermore, initial preparations revealed an ability to block TGF-βl mediated growth inhibition in COLO-357 pancreatic cancer cells.

All patents, patent applications, journal articles and other publications mentioned in this specification are incorporated herein in their entireties by reference.

While this invention has been described in detail with reference to a certain preferred embodiments, it should be appreciated that the present invention is not limited to those precise embodiments. Rather, in view of the present disclosure which describes the current best mode for practicing the invention, many modifications and variations would present themselves to those of skill in the art without departing from the scope and spirit of this invention. The scope of the invention is, therefore, indicated by the following claims rather than by the foregoing description. All changes, modifications, and variations coming within the meaning and range of equivalency of the claims are to be considered within their scope.

Claims

WHAT IS CLAIMED IS:

1. A method for inhibiting metastasis in an individual with pancreatic cancer, comprising: identifying an individual at risk for pancreatic cancer; and administering to the individual a therapeutically effective amount of soluble TGF-β receptor Type II ("sTβRII") .

2. The method of claim 1, wherein the sTβRII is administered by regional perfusion to the pancreas.

3. The method of claim 1, wherein the sTβRII is administered by intraperitoneal injection.

4. The method of claim 1, wherein the individual is a human.

5. A method for inhibiting cancer cell invasiveness in an individual with pancreatic cancer, comprising: identifying an individual at risk for pancreatic cancer; and administering to the individual a therapeutically effective amount of sTβRII.

6. A method for inhibiting tumor angiogenesis in an individual with pancreatic cancer, comprising: identifying an individual at risk for pancreatic cancer; and administering to the individual a therapeutically effective amount of sTβRII.

7. A method for treating pancreatic cancer in an individual, comprising: identifying an individual at risk for pancreatic cancer; and administering sTβRII to the individual.

8. The method of claim 7, wherein the sTβRII is administered by regional perfusion to the pancreas.

9. The method of claim 7, wherein the sTβRII is administered by intraperitoneal injection.

10. The method of claim 7, wherein the individual is a human.

11. A method for preventing tumor recurrence in an individual following surgery for pancreatic cancer, comprising administering to the individual a therapeutically effective amount of sTβRII.

12. A method for inhibiting metastasis in an individual diagnosed with pancreatic cancer, comprising introducing into the individual an expression vector encoding sTβRII, such that an amount of sTβRII effective to inhibit metastasis is expressed in the individual .

13. The method of claim 12, wherein the expression vector is introduced into the vicinity of the pancreatic cancer in the individual .

14. A method for inhibiting tumor angiogenesis in an individual diagnosed with pancreatic cancer, comprising introducing into the individual an expression vector encoding sTβRII, such that an amount of sTβRII effective to inhibit tumor angiogenesis is expressed in the individual.

15. The method of claim 14, wherein the expression vector is introduced into the vicinity of the pancreatic cancer in the individual .

16. A method for treating pancreatic cancer in an individual, comprising: identifying an individual at risk for pancreatic cancer; and introducing into the individual an expression vector encoding sTβRII, such that an amount of sTβRII effective to reduce the activity of transforming growth factor-β is expressed in the individual.

17. A method for inhibiting cancer cell invasiveness in an individual diagnosed with pancreatic cancer, comprising introducing into the individual an expression vector encoding sTβRII, such that an amount of sTβRII effective to inhibit cancer cell invasiveness is expressed in the individual.

18. A method for preventing tumor recurrence in an individual following surgery for pancreatic cancer, comprising introducing into the individual an expression vector encoding sTβRII, such that an amount of sTβRII effective to tumor recurrence is expressed in the individual.

19. A therapeutic composition useful for inhibiting metastasis in an individual with pancreatic cancer, comprising sTβRII and a pharmaceutically acceptable carrier.

20. A therapeutic composition useful for inhibiting angiogenesis in an individual with pancreatic cancer, comprising sTβRII and a pharmaceutically acceptable carrier.

21. A therapeutic composition useful for treating pancreatic cancer in an individual, comprising sTβRII and a pharmaceutically acceptable carrier.