WO2003060075A2 - Utilisation du recepteur du tgf-$g(b) de type ii soluble pour la suppression de croissance du cancer du pancreas et de metastase - Google Patents

Utilisation du recepteur du tgf-$g(b) de type ii soluble pour la suppression de croissance du cancer du pancreas et de metastase Download PDF

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WO2003060075A2
WO2003060075A2 PCT/US2002/041003 US0241003W WO03060075A2 WO 2003060075 A2 WO2003060075 A2 WO 2003060075A2 US 0241003 W US0241003 W US 0241003W WO 03060075 A2 WO03060075 A2 WO 03060075A2
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individual
stβrii
pancreatic cancer
cells
tgf
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Murray Korc
Melissa Rowland-Goldsmith
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University of California Berkeley
University of California San Diego UCSD
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University of California San Diego UCSD
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/179—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators

Definitions

  • TGF- ⁇ s Transforming growth factor- ⁇ s
  • TGF- ⁇ s 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- ⁇ s 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- ⁇ s are synthesized as precursors that undergo proteolytic cleavage, leading to the generation of biologically active 25 kD di ers .
  • the mature forms of TGF- ⁇ l and - ⁇ 2 share 70% ar ⁇ ino acid sequence homology, and the mature form of TGF- ⁇ 3 shares 80% homology with the other two TGF- ⁇ s.
  • TGF- ⁇ s enhance the proliferation of cells of mesenchymal origin and inhibit the proliferation of many types of epithelial cells.
  • TGF- ⁇ s act by binding to the type II TGF- ⁇ receptor (T ⁇ RII), 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).
  • T ⁇ RII type II TGF- ⁇ receptor
  • T ⁇ RI Activation of T ⁇ RI 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) .
  • Smad4 Derynck, R., et al., Cell 95: 737- 740, 1998; Attisano, L., and Wrana, J.L., Curr. Opin. Cell Biol. 12: 235-243, 2000.
  • Pancreatic ductal adenocarcinoma 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- ⁇ 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- ⁇ s are most often expressed at high levels by mesenchymal derived rather than epithelial derived cells, and is associated with decreased patient survival.
  • pancreatic cancer cell-derived TGF- ⁇ s 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- ⁇ s may act directly on the cancer cells to enhance the expression of growth promoting genes.
  • TGF- ⁇ s 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 .
  • Smad4 mutations may be enhanced by the presence of Smad4 mutations (Schwarte-Waldhoff I., et al., Proc. Natl. Acad. Sci. USA 97: 9624-9629, 2000).
  • TGF- ⁇ s have been used to suppress the biological actions of TGF- ⁇ s in vivo. These include the use of neutralizing anti-TGF- ⁇ 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 Heat, F. A., Cancer Immunol. Immunother. 41: 302-8, 1995) or antisense strategies to inhibit TGF- ⁇ synthesis (Marzo, A. L., et al., Cancer Res. 57: 3200-7, 1997; Fitzpatrick, D. R., et al .
  • 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- ⁇ molecules and that this overexpression is associated with .a particularly poor prognosis. Accordingly, in order to suppress TGF- ⁇ overexpression, the present inventors prepared a vector encoding sequences corresponding to a soluble TGF- ⁇ receptor that is devoid of signaling activity but that can bind and sequester TGF- ⁇ molecules.
  • pancreatic cancer cell line acts to slow the growth of subcutaneous tumors in nude mice.
  • 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- ⁇ molecules and attenuates the growth and metastatic potential of pancreatic cancer cells.
  • 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.
  • the present invention provides a method for treating pancreatic cancer in an individual, comprising identifying an individual at risk for pancreatic cancer and administering sT ⁇ RII 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 sT ⁇ RII 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 sT ⁇ RII.
  • 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 sT ⁇ RII.
  • 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 sT ⁇ RII, such that an amount of sT ⁇ RII 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 sT ⁇ RII, such that an amount of sT ⁇ RII 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 sT ⁇ RII, such that an amount of sT ⁇ RII 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 sT ⁇ RII, such that an amount of sT ⁇ RII effective to reduce the activity of transforming growth factor- ⁇ 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.
  • FIG. 1 Expression of soluble T ⁇ RII transfected COLO-357 cells .
  • RNA (20 ⁇ g/lane) was size fractionated, electrotransferred to a nylon Genescreen membrane, and hybridized with the 32P labeled soluble T ⁇ RII cDNA probe (5 X 105 cpm/ml,
  • the membrane was stripped and reprobed with the 7S cDNA probe (5 X 104 cpm/ml,
  • FIG. 3 Effects of TGF- ⁇ l on cell growth and invasion.
  • TGF- ⁇ l Effects of TGF- ⁇ l on 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- ⁇ l.
  • the respective groups of cells (1 X 105/well) were then added to the upper chambers of a 24-well Transwell unit.
  • Migration across the Matrigel coated polycarbonate membrane (8 ⁇ m pore size) was assessed after 16 hours. The number of cells in 9 separate high power fields
  • FIG. 5 T ⁇ RII immunoreactivity in tumors.
  • FIG. 6 Expression of soluble T ⁇ RII and PAI-1 mRNA transcripts in vivo.
  • Total RNA (20 ⁇ g/lane) was prepared from tumors generated in athymic mice following inoculation with sham transfected COLO-357 cells (S) or clones expressing soluble T ⁇ RII
  • RNA was prepared from tumors (33 ⁇ g/lane) from 3 mice injected with sham transfected cells (sham) and 3 mice injected with pMHsT ⁇ RII 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) .
  • 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 .
  • lysates derived from COLO-357 cells were prepared from tumor tissues derived from sham or pMHsT ⁇ RII 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 ⁇ g) 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 pMHsT ⁇ RII in PANC-1 cells.
  • A) Expression of sT ⁇ RII inRA Northern blot analysis of total RNA (20 ⁇ g/lane) from sham transfected PANC-1 cells (S) or from clones that were stably transfected with pMHsT ⁇ RII (C18 and C 19) was carried out with a human soluble T ⁇ RII cDNA probe (5 X 105 cpm/ml, 1 d exposure) . 7S cDNA was used as a loading control (5 X 10 4 cpm/ml, 2 h exposure) .
  • TGF- ⁇ l Effects of TGF- ⁇ l 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- ⁇ l 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.
  • FIG. 9 T ⁇ RII immunoreacti ity in tumors derived from PANC- 1 cells.
  • An anti-T ⁇ RII antibody recognizing the full-length T ⁇ RII (A, B) and an anti-HA antibody recognizing the HA epitope of the pMHsT ⁇ RII construct (C, D) were used.
  • In the tumor tissue derived from the sham transfected cells there was weak to moderate i munostaining for endogenous T ⁇ RII (A) but undetectable HA immunoreactivity (C) .
  • Strong T ⁇ RII (B) and moderate HA (D) immunoreactivity was present in the tumor tissue derived from cancer cells expressing the pMHsT ⁇ RII construct. Bar scale: 25 ⁇ m.
  • 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 sT ⁇ RII expression in the intra-pancreatic tumors.
  • A) Tumor from sham transfected cells was devoid of HA immunoreactivity.
  • FIG. 12 Relative expression of PAI-1 and uPA.
  • Northern blot analysis was carried out using the PAI-1 cDNA probe (5 x 10 5 cpm/ml, 1 day exposure) .
  • the membrane was reprobed with the uPA cDNA probe (5 x 10 5 cpm/ml, 3 day exposure) .
  • A' 7S cDNA probe was used as a loading control (5 x 10 4 cpm/ml, 1 day exposure) .
  • RNA (20 ⁇ g/lane) was prepared and pooled from intra-pancreatic tumors derived from 4 mice implanted with sham transfected cells (Sham) and 4 mice implanted with pMHsT ⁇ RII transfected cells (Clones) .
  • Total RNA (20 ⁇ g/lane) was also prepared from normal pancreatic tissues from 2 mice implanted with pMHsT ⁇ RII 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) .
  • Pancreatic ductal adenocarcinoma is a deadly malignancy that frequently etastasizes and that overexpresses transforming growth factor-betas (TGF- ⁇ s) . This overexpression has been correlated with decreased patient survival. TGF- ⁇ s bind to a type II TGF- ⁇ receptor (T ⁇ RII) dimer, which heterotetramerizes with a type I TGF- ⁇ receptor (T ⁇ RI) dimer, thereby activating downstream signaling.
  • T ⁇ RII type II TGF- ⁇ receptor
  • T ⁇ RI type I TGF- ⁇ receptor
  • the present inventors expressed a soluble T ⁇ RII construct, encoding a ino acids 1-159 of the extracellular domain of T ⁇ RII (SEQ. ID NO. 1), in COLO-357 human pancreatic cancer cells.
  • This cell line expresses all three mammalian TGF- ⁇ isoforms and is growth inhibited by TGF- ⁇ in vitro.
  • COLO-357 clones expressing soluble T ⁇ RII were no longer growth inhibited by exogenous TGF- ⁇ l and exhibited a marked decrease in their invasive capacity in vitro.
  • these clones 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.
  • endogenous TGF- ⁇ s 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- ⁇ s.
  • PANC-1 human pancreatic cancer cells were transfected with sT ⁇ RII.
  • PANC-1 clones expressing sT ⁇ RII 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.
  • PAI-1 plasminogen activator inhibitor 1
  • TGF- ⁇ s 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 sT ⁇ RII in the therapeutic treatment of PDAC.
  • “Individual” means any living organism, including humans and other mammals, which produce TGF- ⁇ .
  • TGF- ⁇ is a family of peptide growth factors.
  • TGF- ⁇ receptor is a cell surface protein, of which three types (Type I, Type II, and Type III) are known in mammals.
  • TGF- ⁇ receptor Type II (or “T ⁇ RII") is a membrane-bound protein with an intracellular domain, transmembrane domain, and extracellular domain, which binds TGF- ⁇ .
  • Human TGF- ⁇ 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- ⁇ receptor fragment is a portion or all of a TGF- ⁇ receptor molecule that is capable of binding TGF- ⁇ .
  • Soluble TGF-' ⁇ receptor Type II is a polypeptide comprising a portion or all of the extracellular domain of T ⁇ RII, or a variant or derivative thereof, which is soluble and which binds to TGF- ⁇ .
  • These polypeptides consist of 159 amino acids or less; preferably, sT ⁇ RII is about 159 amino acids.
  • ⁇ sT ⁇ RII 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 sT ⁇ RII as shown in SEQ. ID NO. 1, and wherein the variant polypeptide retains the activity of the basic protein, for example, sT ⁇ RII.
  • 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.
  • 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.
  • suitable expression hosts such as E. coli or yeast using conventional technology and clones that retain the desired activity are detected.
  • 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, sT ⁇ RII. 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 sT ⁇ RII 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.
  • pharmaceutically acceptable 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 .
  • vectors suitable for expression of recombinant protein include but are not limited to pGEX, pET-9d, pTrxFus or baculovirus
  • the obtained polypeptide is purified by methods known in the art.
  • the degree of purification varies depending on the use of the polypeptide.
  • the degree of purity may not need to be very high.
  • purity of 90-95% is typically preferred and in some instances even required.
  • 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 sT ⁇ RII 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.
  • sT ⁇ RII 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 .
  • 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.
  • 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.
  • doxorubicin decarbazine
  • irinotecan etoposide phosphate
  • gemcitabine gemcitabine
  • carboplatinum cisplatinum
  • tomoxifen methotrexate
  • ifosfa ide cyclophosphamide
  • 5-fluorouracil vinorelbine tartrate
  • anastrozole trastuzumab and combinations thereof.
  • 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.
  • 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 .
  • a serum concentration of about 0.05 to about 0.5 mg/ml may be highly effective in suppressing cancer growth, invasion and metastasis.
  • terapéuticaally effective amount means that amount of sT ⁇ RII 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.
  • 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.
  • 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 sT ⁇ RII, 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- ⁇ receptor.
  • the genes encoding sT ⁇ RII are chimeric, encoding a fusion polypeptide comprising sT ⁇ RII and an epitope tag.
  • the chimeric gene may encode a sT ⁇ RII-hei ⁇ agglutinin fusion polypeptide.
  • the invention also contemplates polyclonal, monoclonal and humanized antibodies against the aforementioned sT ⁇ RII polypeptides, fragments, variants and derivatives or, alternatively, against TGF- ⁇ .
  • 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.
  • 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.
  • recombinant sT ⁇ RII or TGF- ⁇
  • 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 sT ⁇ RII (or TGF- ⁇ ) polypeptides.
  • the antibodies can also be used to screen expression libraries for variant polypeptides of sT ⁇ RII (or TGF- ⁇ ) .
  • the antibodies of the invention can be administered to individuals diagnosed with pancreatic cancer, to inhibit binding of TGF- ⁇ molecules to endogenously expressed T ⁇ RII on the cancer cells and on the adjoining cellular elements, including endothelial cells.
  • the anti-T ⁇ RII antibody could act as mimicker of sT ⁇ RII by "soaking-up" TGF-beta molecules.”
  • humanized antibodies XENOMOUSE ® , Abgenix, Inc., Fremont, California; BodeyB., et al., Curr. Pharm. Des. 6:261-76
  • Antibodies may administered as described above for therapeutic compositions .
  • therapeutic antibodies are administered either subcutaneously or by intravenous injection.
  • therapeutic antibodies are administered either subcutaneously or by intravenous injection.
  • 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) ; [ 32 P] dCTP and [ 35 S] 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
  • TGF- ⁇ l 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) .
  • T ⁇ RII human T ⁇ RII
  • SEQ. ID NO. 2 The complete cDNA of human T ⁇ RII (SEQ. ID NO. 2) was used as the template for PCR amplification of the coding sequence of the extracellular domain of T ⁇ RII (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 T ⁇ RII 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, pMHsT ⁇ RII contained the open reading frame encoding the human soluble T ⁇ RII 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.
  • 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 ⁇ g/ml) and 5% fungazone termed complete medium.
  • DME Dulbecco's modified Eagle's medium
  • FBS penicillin
  • streptomycin 100 ⁇ g/ml
  • 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.
  • 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 ⁇ g/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.
  • DME Dulbecco's modified Eagle's medium
  • FBS penicillin
  • streptomycin 100 ⁇ g/ml
  • fungazone complete medium
  • PANC-1 cells were incubated in serum-free medium (DME containing 0.1% bovine serum albumin, 5 ⁇ g/ml transferrin, 5 ng/ml sodium selenite, antibiotics and fungazone) .
  • DME serum-free medium
  • PANC-1 cells were transfected in a stable manner with the pMHsT ⁇ RII plasmid (10 ⁇ g) , using the lipofectamine method. After expansion of each individual clone, cells were screened for expression of pMHsT ⁇ RII by Northern blotting.
  • cells transfected with the soluble T ⁇ RII 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 ⁇ g/ml) , followed by a 2 hour incubation with protein A agarose (50 ⁇ l) 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 T ⁇ RII polyclonal antibody (1:5,000 dilution) .
  • 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.
  • T ⁇ RII, 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 ⁇ m) from tumor tissue derived from sham transfected or pMHsT ⁇ RII 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 ⁇ g/ml) ; anti-T ⁇ RII antibody (0.2 ⁇ g/ml), recognizing the epitope corresponding to the full-length T ⁇ RII; or anti-PECAM-1 antibody (1:50 dilution), in PBS containing 1% BSA.
  • anti-HA antibody 0.4 ⁇ g/ml
  • anti-T ⁇ RII antibody 0.2 ⁇ g/ml
  • anti-PECAM-1 antibody (1:50 dilution
  • 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 ⁇ g/ml) .
  • T ⁇ RII 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 ⁇ g/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 pMHsT ⁇ RII 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.
  • 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- ⁇ l (10 pM) .
  • the assay was initiated by adding MTT solution at a final concentration of 62.5 ⁇ g MTT/well.
  • 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.
  • the results of the MTT assay correspond with results obtained by cell counting with a hemacytometer or by monitoring [ 3 H] -thymidine incorporation into DNA.
  • PANC-1 sham transfected or pMHsT ⁇ RII 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- ⁇ l. Cell growth was then determined by cell counting using a hemacytometer, and data were expressed as percent of control cell growth.
  • 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- ⁇ l were suspended in 100 ⁇ l serum-free medium containing 0.1% BSA and placed onto this upper compartment. The lower compartment was then filled with 600 ⁇ l of serum-free medium containing 0.5% FBS. TGF- ⁇ l
  • 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).
  • mice 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) .
  • mice Four- to six-week old female nu/nu (nude) mice
  • 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.
  • COLO-357 pancreatic cancer cells were stably transfected with a soluble T ⁇ RII cDNA construct, encoding the entire extracellular domain (amino acids 1-159) of human T ⁇ RII.
  • 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 T ⁇ RII 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 T ⁇ RII (FIG. 1A) .
  • 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 T ⁇ RII antibody.
  • a major band (25 kDa) representing the soluble T ⁇ RII 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) .
  • 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 T ⁇ RII exhibited similar doubling times, ranging from 31 hours for clone 1 to 37 hours for clone 2.
  • TGF- ⁇ l (10 pM) inhibited the growth of sham transfected COLO- 357 cells (30%; P ⁇ 0.002) but was without effect in the soluble T ⁇ RII expressing clones (FIG. 3A) .
  • TGF- ⁇ l 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 T ⁇ RII (FIG. 3B) .
  • expression of soluble T ⁇ RII effectively blocked the biological actions of exogenous TGF- ⁇ l.
  • COLO-357 clones expressing the soluble T ⁇ RII and sham transfected COLO-357 cells exhibited similar doubling times in vitro.
  • the growth inhibitory effect of 10 pM TGF- ⁇ l was completely blocked, indicating that soluble T ⁇ RII efficiently bound and sequestered TGF- ⁇ l.
  • This conclusion is supported by the observation that the stimulatory effect of 400 pM TGF- ⁇ l on cell invasiveness was also completely blocked by soluble T ⁇ RII.
  • TGF- ⁇ l 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- ⁇ l enhances the expression of PAI-1 (Kleeff, J. and Korc, M., J. Biol. Chem. 273: 7495-500, 1998; Chen, R. H., et al .
  • 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- ⁇ l 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- ⁇ s may act directly on pancreatic cancer cells to promote cancer cell invasion.
  • 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 pMHsT ⁇ RII clones or the sham transfected cells were allowed to grow until day 56.
  • tumors derived from the clones never became as large as the sham did on day 35.
  • the tumors derived from sham transfected cells were still progressively growing, whereas the growth of the tumors derived from pMHsT ⁇ RII cells reached a plateau (FIG. 4B) .
  • Tumors from both clones expressed high levels of the soluble T ⁇ RII RNA moiety (FIG. 6A) .
  • tumors from sham transfected cells did not express the soluble T ⁇ RII mRNA transcript (FIG. 6A) .
  • pancreatic cancer cells expressing sT ⁇ RII 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- ⁇ isoforms (Baldwin, R.L., et al . , Int. J. Can. 67: 283-288, 1996)' and exhibit increased in vi tro invasiveness in response to TGF- ⁇
  • FIG. 8A sham transfected PANC-1 cells that were transfected with the pMH empty vector for use as controls, did not express sT ⁇ RII mRNA.
  • the sham-transfected cells exhibited doubling times of approximately 23 hours and were growth inhibited by 10 and 30 pM TGF- ⁇ (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- ⁇ l (FIG. 8B) .
  • the tumorigenicity of pMHsT ⁇ RII expressing PANC-1 cells and sham transfected cells was compared following subcutaneous injection in athymic nude mice. Clones transfected with pMHsT ⁇ RII consistently formed smaller tumors as compared to tumors arising from sham transfected cells.
  • T ⁇ RII immunoreactivity exhibited a heterogeneous pattern of distribution within the tumors (FIG. 9B) , indicating that there was variable but persistent expression of soluble T ⁇ RII in vivo .
  • Example 5 Growth Properties of Soluble T ⁇ RII 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.
  • mice were implanted with sham-derived tissue minces and 8 mice were implanted with pMHsT ⁇ RII 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.
  • mice implanted with pMHsT ⁇ RII expressing clones developed a large primary tumor (1.2 cm), and this mouse developed peritoneal seeding and mesenteric lymph involvement (Table 2) .
  • one mouse implanted with pMHsT ⁇ RII expressing cells developed a medium-sized primary tumor
  • 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
  • mice implanted with pMHsT ⁇ RII expressing clones exhibited metastatic lesions.
  • 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) .
  • the complete cDNA of human T ⁇ RII was used as template for PCR reactions to generate the cDNA inserts encoding residues 4-136 of the extra-cellular domain of T ⁇ RII (sT ⁇ RII) , 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.

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Abstract

L'invention concerne une méthode de traitement du cancer du pancréas, qui consiste à administrer un récepteur du facteur de croissance transformant β de type II soluble afin de réduire une angiogénèse tumorale, et d'inhiber le caractère envahissant des cellules cancéreuses et les métastases.
PCT/US2002/041003 2001-12-21 2002-12-20 Utilisation du recepteur du tgf-$g(b) de type ii soluble pour la suppression de croissance du cancer du pancreas et de metastase Ceased WO2003060075A2 (fr)

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WO2015095628A1 (fr) * 2013-12-19 2015-06-25 Consejo Nacional De Investigaciones Cientificas Y Tecnicas Isoforme du récepteur de type ii au tgf-bêta

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE BIOSIS [Online] ROWLAND-GOLDSMITH ET AL.: 'Soluble type II TGF-beta receptor inhibits TGF-beta signaling in vitro and markedly attenuates tumor formation in vivo', XP002983827 Retrieved from STN Database accession no. PREV200000532461 & PANCREAS vol. 21, no. 4, November 2000, page 473 *
DATABASE MEDLINE [Online] ROWLAND-GOLDSMITH ET AL.: 'Soluble type II transforming growth factor-beta (TGF-beta)receptor inhibits TGF-beta signaling in COLO-357 pancreatic cancer cells in vitro and attenuates tumor formation', XP002983828 Retrieved from STN Database accession no. NLM11555612 & CLINICAL CANCER RESEARCH vol. 7, no. 9, October 2001, pages 2931 - 2940 *
LIN ET AL.: 'The soluble exoplasmic domain of the type II transforming growth factor (TGF)-beta receptor' JOURNAL OF BIOLOGICAL CHEMISTRY vol. 270, no. 6, 10 February 1995, pages 2747 - 2754, XP002983829 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015095628A1 (fr) * 2013-12-19 2015-06-25 Consejo Nacional De Investigaciones Cientificas Y Tecnicas Isoforme du récepteur de type ii au tgf-bêta
US10233227B2 (en) 2013-12-19 2019-03-19 Consejo Nacional De Investigaciones Cientificas Y Tecnicas Isoform of the TGF-beta receptor II

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