TGFbeta type II-type III receptor fusions

Abstract

Certain embodiments are directed to novel heterotrimeric fusions in which the ectodomain of the TGF-β type II receptor (TβP?II) is coupled to the N- and C-terminal ends of the endoglin-domain of the TGF-β type III receptor (TpRIIIE). Certain embodiments are directed to novel heterotrimeric polypeptides in which the ectodomain of the TGF-β type II receptor (TI3RII) is coupled to the N- and C-terminal ends of the endoglin-domain (E domain) of the TGF-β type III receptor (TI3RIII). This trimeric receptor, known as RER, can bind all three TGF-β isoforms with sub-nanomolar affinity and is effective at neutralizing signaling induced by all three TGF-β isoforms, but not other ligands of the TGF-β superfamily, such as activins, growth and differentiation factors (GDFs), and bone morphonogenetic proteins (BMPs).

Claims

1. A TGFβ-binding heterotrimeric fusion protein wherein the fusion protein has an amino acid sequence that is 90% identical to SEQ ID NO: 2.

2. The fusion protein of claim 1, further comprising an amino terminal signal sequence.

3. The fusion protein of claim 1, further comprising an amino terminal or carboxy terminal tag.

4. The fusion protein of claim 3, wherein the tag is a carboxy terminal hexa-histidine.

5. A method of treating a condition related to increased expression TGFβ comprising administering an effective amount of the fusion protein of claim 1 to subject in thereof.

6. The method of claim 5, wherein the condition is a hyperproliferative disorder.

7. The method of claim 6, wherein the hyperproliferative disorder is cancer.

8. The method of claim 5, wherein the condition is fibrosis.

9. A heterotrimeric fusion protein wherein the fusion protein has the amino acid sequence of SEQ ID NO:2.

10. The fusion protein of claim 9, further comprising an amino terminal signal sequence.

11. The fusion protein of claim 9, further comprising an amino terminal or carboxy terminal tag.

12. The fusion protein of claim 11, wherein the tag is a carboxy terminal hexa-Histidine.

.Iadd.13. A fusion protein comprising, in the N-terminal to C-terminal direction: (i) a first portion having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 3; (ii) a second portion having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 4; and (iii) a third portion having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 5..Iaddend.

.Iadd.14. The fusion protein of claim 13, wherein the first portion has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 3..Iaddend.

.Iadd.15. The fusion protein of claim 13, wherein the second portion has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4..Iaddend.

.Iadd.16. The fusion protein of claim 13, wherein the third portion has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 5..Iaddend.

.Iadd.17. The fusion protein of claim 13, wherein the first portion has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 3, the second portion has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4, and the third portion has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 5..Iaddend.

.Iadd.18. The fusion protein of claim 13, wherein the first portion has the amino acid sequence of SEQ ID NO: 3..Iaddend.

.Iadd.19. The fusion protein of claim 13, wherein the second portion has the amino acid sequence of SEQ ID NO: 4..Iaddend.

.Iadd.20. The fusion protein of claim 13, wherein the third portion has the amino acid sequence of SEQ ID NO: 5..Iaddend.

.Iadd.21. The fusion protein of claim 13, wherein the first portion has the amino acid sequence of SEQ ID NO: 3, the second portion has the amino acid sequence of SEQ ID NO: 4, and the third portion has the amino acid sequence of SEQ ID NO: 5..Iaddend.

.Iadd.22. The fusion protein of claim 13, wherein either or both of (i) the first portion and the second portion, and (ii) the second portion and the third portion are joined to one another by way of a polypeptide linker..Iaddend.

.Iadd.23. The fusion protein of claim 13, further comprising an amino terminal or carboxy terminal tag..Iaddend.

.Iadd.24. A nucleic acid encoding the fusion protein of claim 13..Iaddend.

.Iadd.25. The nucleic acid of claim 24, wherein the first portion has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 3..Iaddend.

.Iadd.26. The nucleic acid of claim 24, wherein the second portion has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4..Iaddend.

.Iadd.27. The nucleic acid of claim 24, wherein the third portion has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 5..Iaddend.

.Iadd.28. The nucleic acid of claim 24, wherein the first portion has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 3, the second portion has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4, and the third portion has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 5..Iaddend.

.Iadd.29. The nucleic acid of claim 24, wherein the first portion has the amino acid sequence of SEQ ID NO: 3..Iaddend.

.Iadd.30. The nucleic acid of claim 24, wherein the second portion has the amino acid sequence of SEQ ID NO: 4..Iaddend.

.Iadd.31. The nucleic acid of claim 24, wherein the third portion has the amino acid sequence of SEQ ID NO: 5..Iaddend.

.Iadd.32. The nucleic acid of claim 24, wherein the first portion has the amino acid sequence of SEQ ID NO: 3, the second portion has the amino acid sequence of SEQ ID NO: 4, and the third portion has the amino acid sequence of SEQ ID NO: 5..Iaddend.

.Iadd.33. The nucleic acid of claim 24, wherein either or both of (i) the first portion and the second portion, and (ii) the second portion and the third portion are joined to one another by way of a polypeptide linker..Iaddend.

.Iadd.34. The nucleic acid of claim 24, wherein the fusion protein further comprises an amino terminal or carboxy terminal tag..Iaddend.

.Iadd.35. A vector comprising the nucleic acid of claim 24..Iaddend.

.Iadd.36. A host cell comprising the nucleic acid of claim 24..Iaddend.

.Iadd.37. A host cell comprising the vector of claim 35..Iaddend.

.Iadd.38. A method of producing the fusion protein of claim 13, the method comprising contacting a host cell with a nucleic acid encoding the fusion protein and subsequently isolating the fusion protein from the host cell..Iaddend.

.Iadd.39. A pharmaceutical composition comprising the fusion protein of claim 13 and one or more pharmaceutically acceptable excipients..Iaddend.

.Iadd.40. The pharmaceutical composition of claim 39, wherein the composition is formulated for administration to a human subject..Iaddend.

.Iadd.41. The pharmaceutical composition of claim 40, wherein the composition is formulated for parenteral, subcutaneous, intravenous, intramuscular, or intraperitoneal administration to the human subject..Iaddend.

.Iadd.42. A method of reducing or preventing binding of TGF-β to one or more endogenous TGF-β receptors in a subject, the method comprising administering to the subject the fusion protein of claim 13..Iaddend.

.Iadd.43. The method of claim 42, wherein the subject is a human..Iaddend.

Description

DESCRIPTION OF THE DRAWINGS

(1) The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of the specification embodiments presented herein.

(2) FIG. 1. SPR sensorgrams in which increasing concentrations of the TβRII and TβRIII.sub.E were injected over a SPR sensor surface with immobilized TGF-β2 K25R I92V K94R. The mass normalized sensorgrams are shown in panels a and b; plots of the mass normalized equilibrium response (R.sub.eq) as a function of receptor concentration ([Receptor]), along with fits to R.sub.eg=(R.sub.max×[Receptor])/(K.sub.d+[Receptor]), are shown in panel c.

(3) FIG. 2. SPR sensorgrams in which increasing concentrations of the TGF-β type II receptor ectodomain were injected over immobilized TGF-β2 K25R I92V K94R in the absence (panel a) or presence (panel b) of a saturating concentration (800 nM) of the TGF-β type III receptor endoglin domain. Plots of the mass normalized equilibrium response (R.sub.eq) as a function of receptor concentration ([Receptor]), along with fits to R.sub.eq=(R.sub.max×[Receptor])/(K.sub.d+[Receptor]), are shown in panel c.

(4) FIG. 3. Schematic diagram of the TGFβ:TβRII complex with the TGFβ type III receptor endoglin domain positioned in a manner that it does not sterically overlap with either of the two bound TβRII molecules. The locations on the TβRII N- and C-termini are shown.

(5) FIG. 4. SPR sensorgrams in which increasing concentrations of ER and RER were injected over SPR surfaces with immobilized TGF-β1, -β2, and -β3. The concentrations of injected receptor range from 10 nM downward (in two-fold increments).

(6) FIG. 5. SPR competition binding data in which increasing concentrations of TβRII (R), TβRIII.sub.E-TβRII (ER), and TβRII-TβRIII.sub.E-TβRII (RER) were pre-incubated with 0.8 nM TGF-β3 for 16 h and then injected over a high-density (20000 RU) SPR surface with the TGF-β monoclonal antibody 1D11. Data is presented in terms of the initial slope (which is directly proportional to the concentration of free TGF-β) as a function of the competitor (R, ER, or RER) concentration. Two independent measurements were performed for each of the receptor constructs studied (designated by the a and b suffices in the legend).

(7) FIG. 6. Average IC.sub.50 using Mv1Lu PAI1 luciferase reporter cells in 96-well plates. Assays were performed using a four-fold receptor fusion and 1D11 (neutralizing antibody) dilution series and 20 pM TGF-beta 1, 2, or 3 at 37° overnight.

(8) FIGS. 7A-7C. Neutralization curves comparing various traps (RR (RII-RII), RER (RII-BG.sub.E-RII), ER (BG.sub.E-RII), REU (RII-BG.sub.E-BG.sub.U, or alternatively RII-RIII), or EU (BG.sub.E-BG.sub.U, or alternatively RIII) and 1D11 for (A) TGF-β1, (B) TGF-β2, or TGF-β3.

(9) FIGS. 8A-8C. Neutralization curves for various RER preparations relative to (A) TGF-β1, (B) TGF-β2, or (c) TGF-β3

DESCRIPTION

(10) As discussed above, transforming growth factor beta (TGFβ) isoforms (β1, β2, and β3) are homodimeric polypeptides of 25 kDa. TGF-β has nine cysteine residues that are conserved among its family; eight cysteines form four disulfide bonds within the molecule, three of which form a cystine knot structure characteristic of the TGF-β superfamily, while the ninth cysteine forms a disulfide bond with the ninth cysteine of another TGF-β molecule to produce the dimer.

(11) Though a number of TGF-β inhibitors have been reported, none have been approved for clinical use. The novel TGF-β inhibitor described herein—RER—can be produced by artificially fusing together the binding domains of the TGFβ type II receptor and the endoglin domain of the type III receptor. The design of RER—a heterotrimeric fusion in which the ectodomain of the TGF-β type II receptor (R) has been artificially fused onto the N- and C-termini of the endoglin-like domain of the TGF-β type III receptor (E)—was conceived based on the structures of the TGF-βs bound to the their signaling receptors, TβRI and TβRII, and the results of surface plasmon resonance (SPR) binding studies which showed that:

(12) 1. The TGF-β type III receptor endoglin domain binds TGF-β dimers with a stoichiometry of 1:1. This was shown by comparing the maximal mass-normalized SPR response as increasing concentrations of the purified TGF-β type II receptor ectodomain (TβRII or R) and purified TGF-β type III receptor endoglin-like domain (TβRIII.sub.E or E) were injected over immobilized TGF-β2 K25R I92V K94R, a variant of TGF-β2 that binds TβRII with high affinity (FIGS. 1A and 1B) (De Crescenzo et al. J Mol. Biol. 355, 47-62, 2006; Baardsnes et al. Biochemistry 48, 2146-55, 2009). The maximal mass-normalized response for TβRIII.sub.E was found to be approximately one-half of that for TORII, allowing the inventors to infer that TβRIII.sub.E must bind the TGF-β dimer with 1:1 stoichiometry since it is well established through structural studies that TβRII binds TGF-β dimers with 2:1 stoichiometry (FIG. 1C) (Hart et al., Nat Struct Biol. 9, 203-8, 2002; Groppe et al., Mol Cell 29, 157-68, 2008; Radaev et al., Journal of Biological Chemistry 285, 14806-14, 2010).

(13) 2. TβRIII.sub.E binds TGF-β dimers without displacing either of the two bound TβRIIs. This was shown by performing an SPR experiment in which increasing concentrations of TβRII were injected over immobilized TGF-β2 K25R I92V K94R in the absence or presence of a saturating concentration of TβRIII.sub.E (800 nM) (FIGS. 2A and 2B). The data showed that the maximal mass normalized binding response for TβRII was slightly increased in the presence of 800 nM TβRIII.sub.E (FIG. 2C), showing that the two receptors do not compete with one another for binding TGF-β (it is impossible for more than two TβRIIs to bind the TGF-β dimer, and thus the increase in the maximal amplitude is likely caused by an experimental artifact, such as a mismatch in the concentrations of TβRIII.sub.E in the TβRII samples that were injected and the buffer).

(14) Together, these observations suggest that TGF-β dimers are capable of forming a heterotrimeric complex in which each TGF-β dimer binds two molecules of TβRII and one molecule of TβRIII.sub.E. The structure of the TGF-β bound to TβRII has been reported (Hart et al., Nat Struct Biol. 9, 203-8, 2002; Groppe et al., Mol Cell 29, 157-68, 2008; Radaev et al., Journal of Biological Chemistry 285, 14806-14, 2010), but the structure of TβRIII.sub.E, either alone or bound to TGF-β, has not. This has led to the hybrid structure where the precise structure of TβRIII.sub.E is not known, but its overall positioning between the two bound TβRIIs on the distal ends of the TGF-β dimer is known (FIG. 3).

(15) This hybrid model for binding of TβRII and TβRIII.sub.E led to the construction of the heterotrimeric RER (TβRII-TβRIII.sub.E-TβRII) fusion as a novel inhibitor for binding and sequestering TGF-β. The inclusion of an additional binding domain enhanced the affinity of the fusion for the TGF-βs, especially TGF-β1 and TGF-β3, which bind TβRII with high (K.sub.d ˜120 nM) affinity (Baardsnes et al. Biochemistry 48, 2146-55, 2009; Radaev et al., Journal of Biological Chemistry 285, 14806-14, 2010).

(16) In comparison to the currently described RER, Genzyme's monoclonal antibody GC1008 (the humanized version of the mouse monoclonal antibody 1D11) has been shown to bind the three TGF-β isoforms with a K.sub.d of approximately 5-10 nM (Grütter, et. al., PNAS U.S.A. 105(51): 20251-56, 2008), but it has not proven to be very effective in clinical trials for malignant melanoma and renal cell carcinoma. The reason for the lack of effectiveness might be that GC1008 does not bind the TGF-βs tightly enough to compete against the cell surface TGF-β receptors, which bind the TGF-βs at picomolar to sub-picomolar concentrations.

(17) The polypeptides described herein include high affinity heterotrimeric TGF-β inhibitors, such as RER. As described above RER has been shown to bind all three TGF-β isoforms with low nanomolar affinity to sub-nanomolar affinity. RER is more potent than the monoclonal antibody 1D11. Thus, owing to its enhanced affinity for binding TGF-β, RER more effectively competes against the cell surface receptors for binding TGF-β, and in turn blocking its disease-promoting properties in cancer and fibrosis for example.

(18) An example of an RER amino acid sequence (for example see SEQ ID NO:2) has one or more of the following features:

(19) 1. In certain aspects the TβRII sequence is human (SEQ ID NO:6), while the TβRIII.sub.E sequence can be rat (SEQ ID NO:7). In certain aspects the TβRIII.sub.E sequence can be human (SEQ ID NO:8).

(20) 2. In certain embodiments the N-terminal TβRII sequence of RER extends from residue 42-160 of SEQ ID NO:6, while the C-terminal TβRII sequence of RER extends from residue 48-160 of SEQ ID NO:6.

(21) 3. In certain embodiments the TβRIII.sub.E sequence extends from residue 24-383 of SEQ ID NO:7. In certain aspects, the TβRIII.sub.E sequence includes 1, 2, 3, and/or 4 single amino acid substitutions relative to the wild type rat sequence (SEQ ID NO:7), R58H, H116R, C278S, and N337A.

(22) 4. In certain embodiments there is no linker between TβRIII.sub.E and the C-terminal TβRII domain. In other aspects a Lys-Leu dipeptide encoded by the HindIII restriction site used to join the corresponding DNA fragments together forms a linker. It is contemplated that any dipeptide can be used.

(23) 5. In certain embodiments there is an 18 amino acid linker with the sequence Gly-Leu-Gly-Pro-Val-Glu-Ser-Ser-Pro-Gly-His-Gly-Leu-Asp-Thr-Ala-Ala-Ala (SEQ ID NO:9) that links the C-terminus of the N-terminal TβRII to the N-terminus of TβRIII.sub.E.

(24) 6. In certain embodiments there is a C-terminal hexa-histidine tag (for purification purposes).

(25) In one example, an RER expression cassette was inserted downstream of the albumin signal peptide and an engineered NotI cloning site with the sequence Met-Lys-Trp-Val-Thr-Phe-Leu-Leu-Leu-Leu-Phe-Ile-Ser-Gly-Ser-Ala-Phe-Ser-Ala-Ala-Ala (SEQ ID NO:10). The entire albumin signal peptide was placed downstream of the CMV promoter in a modified form of pcDNA3.1 (Invitrogen) as previously described (Zou and Sun, Cell 134, 215-30, 2004).

(26) A plasmid expressing RER construct was transfected into CHO Lec 3.2.8.1 cells (Rosenwald et al., Mol Cell Biol. 9(3):914-24, 1989) and stable transfectants were selected using MSX (Zou and Sun, Cell 134, 215-30, 2004). The stable transfectants were in turn screened for high level expression of the RER fusion by examining the conditioned medium using a polyclonal antibody raised against the rat betaglycan ectodomain. The clone expressing RER at the highest level was expanded and ultimately transferred into serum free medium for production of conditioned medium. The RER was then purified from the conditioned medium by passing it over a NiNTA column, washing it with 25 mM Tris, 100 mM NaCl, and 10 mM imidazole, pH 8 and ultimately by eluting it with the same buffer, but with 300 mM imidazole.

(27) The isolated RER fusion protein was in turn characterized by performing an SPR experiment in which it, together with similarly prepared ER (i.e. the previously described TβRIII.sub.ETβRII fusion (Verona et al., Protein Eng Des Sel. 21, 463-73, 2008), except produced in CHO cells, not bacteria), was injected over a SPR sensor chip with immobilized TGF-β1, -β2, and -β3. This data showed comparable on-rates, but significantly slower off-rates, especially for TGF-β1 and TGF-β3 (FIG. 4). This qualitatively shows that RER binds the TGF-βs with higher affinity than ER; however, the magnitude of this increase proved to be difficult to quantify since the slow association precluded accurate measurement of the equilibrium SPR response, especially at lower concentrations of injected receptor.

(28) To further evaluate affinity, an SPR competition experiment was performed in which the commercially available TGF-β monoclonal antibody 1D11 (R&D Systems) was coupled to an SPR sensor chip at high density (20000 RU) and in turn increasing concentration of R (TβRII), ER (BG.sub.E-RII), or RER(RII-BG.sub.E-RII) were injected in the presence of a fixed low (0.8 nM) concentration of TGF-β3. The initial slope of these sensorgrams (which is a linear function of the free TGF-β3 concentration) was then plotted as a function of the concentration of the receptor fusion (FIG. 5). This showed that RER is indeed a more potent competitor than ER, consistent with the slower dissociation rate for RER compared to ER.

(29) RER polypeptides demonstrate more potent activity relative to similar fusion proteins. For example the average IC.sub.50 [nM] using Mv1Lu PAI1 luciferase reporter cells in 96-well plates is markedly lower for RER polypeptides (FIG. 6). Neutralization curves comparing various receptor fusions (RR (RII-RII, also known as T22d35), RER (RII-BG.sub.E-RII), ER (BG.sub.E-RII), REU (RII-BG.sub.E-BG.sub.U or alternatively RII-RIII), or EU (BG.sub.E-BG.sub.U or alternatively RIII) and 1D11 for (A) TGF-β1, (B) TGF-β2, or TGF-β3 also show an improved activity for RER polypeptides (FIG. 7 and FIG. 8).

(30) I. Linkers

(31) In some embodiments, the invention provides a fusion protein comprising three TGF-β binding domains joined to each other directly or by a linker, such as, e.g., a short peptide linker. In some embodiments, the C terminus of the amino terminal TGF-β binding segment is joined by a peptide linker to the N terminus of the central TGF-β binding segment, and the C terminus of the center TGFβ binding segment may be joined to the N terminus of the carboxy TGFβ binding segment by a second linker. A linker is considered short if it contains 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, to 50 or fewer amino acids.

(32) Most typically, the linker is a peptide linker that contains 50 or fewer amino acids, e.g., 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 3, 4, 2, or 1 amino acid(s). In certain aspects, the sequence of the peptide linker is a non-TGF-β type II or type III receptor amino acid sequence. In other aspects, the sequence of the peptide linker is additional TGF-β type II or type III receptor amino acid sequence, e.g., the 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, to 50 or fewer amino acids flanking the carboxy an/or amino terminal ends of the binding domains. The term additional in this context refers to amino acids in addition to those that define the segments of the heterotrimeric polypeptide as defined above. In various embodiments, the linker does not contain more than 50, 40, 20, 10, or 5 contiguous amino acids from the native receptor sequences. Typically, the linker will be flexible and allow the proper folding of the joined domains. Amino acids that do not have bulky side groups and charged groups are generally preferred (e.g., glycine, serine, alanine, and threonine). Optionally, the linker may additionally contain one or more adaptor amino acids, such as, for example, those produced as a result of the insertion of restriction sites. Generally, there will be no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 adaptor amino acids in a linker.

(33) In some embodiments, the linker comprises one or more glycines, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, or more glycines. For example, the linker may consist of (GGG)n, where n=1, 2, 3, 4, 5, 6, 7, etc. and optional adaptor amino acids. In certain aspects, the linker is a glycine-serine linker which comprises (GGGS)n, where n=1, 2, 3, 4, 5, etc. In view of the results disclosed herein, the skilled artisan will recognize that any other suitable peptide linker can be used in the fusion proteins of the invention, for example, as described in Alfthan et al., Protein Eng. 8:725-31, 1995; Argos, J. MoI. Biol. 211:943-58, 1990; Crasto et al., Protein Eng., 13:309-12, 2000; Robinson et al., PNAS USA, 95:5929-34, 1998.

(34) II. Nucleic Acids, Vectors, Host Cells

(35) The invention further provides nucleic acids encoding any of the fusion proteins of the invention, vectors comprising such nucleic acids, and host cells comprising such nucleic acids. For example, in an illustrative embodiment, the nucleic acid of the invention comprises the sequence as set forth in SEQ ID NO:1.

(36) Nucleic acids of the invention can be incorporated into a vector, e.g., an expression vector, using standard techniques. The expression vector may then be introduced into host cells using a variety of standard techniques such as liposome-mediated transfection, calcium phosphate precipitation, or electroporation. The host cells according to the present invention can be mammalian cells, for example, Chinese hamster ovary cells, human embryonic kidney cells (e.g., HEK 293), HeLa S3 cells, murine embryonic cells, or NSO cells. However, non-mammalian cells can also be used, including, e.g., bacteria, yeast, insect, and plant cells. Suitable host cells may also reside in vivo or be implanted in vivo, in which case the nucleic acids could be used in the context of in vivo or ex vivo gene therapy.

(37) III. Methods of Making

(38) The invention also provides methods of producing (a) fusion proteins, (b) nucleic acid encoding the same, and (c) host cells and pharmaceutical compositions comprising either the fusion proteins or nucleic acids. For example, a method of producing the fusion protein according to the invention comprises culturing a host cell, containing a nucleic acid that encodes the fusion protein of the invention under conditions resulting in the expression of the fusion protein and subsequent recovery of the fusion protein. In one aspect, the fusion protein is expressed in CHO or HEK 293 cells and purified from the medium using methods known in the art. In some embodiments, the fusion protein is eluted from a column at a neutral pH or above, e.g., pH 7.5 or above, pH 8.0 or above, pH 8.5 or above, or pH 9.0 or above.

(39) The fusion proteins, including variants, as well as nucleic acids encoding the same, can be made using any suitable method, including standard molecular biology techniques and synthetic methods, for example, as described in the following references: Maniatis (1990) Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., and Bodansky et al. (1995) The Practice of Peptide Synthesis, 2nd ed., Spring Verlag, Berlin, Germany). Pharmaceutical compositions can also be made using any suitable method, including for example, as described in Remington: The Science and Practice of Pharmacy, eds. Gennado et al., 21th ed., Lippincott, Williams & Wilkins, 2005).

(40) IV. Pharmaceutical Compositions and Methods of Administration

(41) The invention provides pharmaceutical compositions comprising the fusion proteins of the invention or nucleic acids encoding the fusion proteins.

(42) The fusion protein may be delivered to a cell or organism by means of gene therapy, wherein a nucleic acid sequence encoding the fusion protein is inserted into an expression vector that is administered in vivo or to cells ex vivo, which are then administered in vivo, and the fusion protein is expressed therefrom. Methods for gene therapy to deliver TGF-β antagonists are known (see, e.g., Fakhrai et al., PNAS USA, 93:2909-14, 1996 and U.S. Pat. No. 5,824,655).

(43) The fusion protein may be administered to a cell or organism in a pharmaceutical composition that comprises the fusion protein as an active ingredient. Pharmaceutical compositions can be formulated depending upon the treatment being effected and the route of administration. For example, pharmaceutical compositions of the invention can be administered orally, topically, transdermally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, by intracavitary or intravesical instillation, intraocularly, intraarterially, intralesionally, or by application to mucous membranes, such as, that of the nose, throat, and bronchial tubes. The pharmaceutical composition will typically comprise biologically inactive components, such as diluents, excipients, salts, buffers, preservants, etc. Standard pharmaceutical formulation techniques and excipients are well known to persons skilled in the art (see, e.g., Physicians' Desk Reference (PDR) 2005, 59th ed., Medical Economics Company, 2004; and Remington: The Science and Practice of Pharmacy, eds. Gennado et al. 21th ed., Lippincott, Williams & Wilkins, 2005).

(44) Generally, the fusion protein of the invention may be administered as a dose of approximately from 1 μg/kg to 25 mg/kg, depending on the severity of the symptoms and the progression of the disease. The appropriate therapeutically effective dose of an antagonist is selected by a treating clinician and would range approximately from 1 μg/kg to 20 mg/kg, from 1 μg/kg to 10 mg/kg, from 1 μg/kg to 1 mg/kg, from 10 μg/kg to 1 mg/kg, from 10 μg/kg to 100 μg/kg, from 100 μg to 1 mg/kg, and from 500 μg/kg to 5 mg/kg. Effective dosages achieved in one animal may be converted for use in another animal, including human, using conversion factors known in the art (see, e.g., Freireich et al., Cancer Chemother. Reports, 50(4):219-244 (1996)).

(45) V. Therapeutic and Non-Therapeutic Uses

(46) The fusion proteins of the invention may be used to capture or neutralize TGF-β, thus reducing or preventing TGF-β binding to naturally occurring TGF-β receptors.

(47) The invention includes a method of treating a subject (e.g., mammal) by administering to the mammal a fusion protein described herein or a nucleic acid encoding the fusion protein or cells containing a nucleic acid encoding the fusion protein. The mammal can be for example, primate (e.g., human), rodent (e.g., mouse, guinea pig, rat), or others (such as, e.g., dog, pig, rabbit).

(48) The mammal being treated may have or may be at risk for one or more conditions associated with an excess of TGF-β for which a reduction in TGF-β levels may be desirable. Such conditions include, but are not limited to, fibrotic diseases (such as glomerulonephritis, neural scarring, dermal scarring, pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis), lung fibrosis, radiation-induced fibrosis, hepatic fibrosis, myelofibrosis), peritoneal adhesions, hyperproliferative diseases (e.g., cancer), burns, immune-mediated diseases, inflammatory diseases (including rheumatoid arthritis), transplant rejection, Dupuytren's contracture, and gastric ulcers.

(49) In certain embodiments, the fusion proteins, nucleic acids, and cells of the invention are used to treat diseases and conditions associated with the deposition of extracellular matrix (ECM). Such diseases and conditions include, but are not limited to, systemic sclerosis, postoperative adhesions, keloid and hypertrophic scarring, proliferative vitreoretinopathy, glaucoma drainage surgery, corneal injury, cataract, Peyronie's disease, adult respiratory distress syndrome, cirrhosis of the liver, post myocardial infarction scarring, restenosis (e.g., post-angioplasty restenosis), scarring after subarachnoid hemorrahage, multiple sclerosis, fibrosis after laminectomy, fibrosis after tendon and other repairs, scarring due to tatoo removal, biliary cirrhosis (including sclerosing cholangitis), pericarditis, pleurisy, tracheostomy, penetrating CNS injury, eosinophilic myalgic syndrome, vascular restenosis, veno-occlusive disease, pancreatitis and psoriatic arthropathy. In particular, the fusion proteins, and related aspects of the invention are particularly useful for the treatment of peritoneal fibrosis/adhesions. It is well known that antibodies are readily transferred from the peritoneal cavity into circulation. Therefore, intraperitoneal delivery of the fusion protein may provide a highly localized form of treatment for peritoneal disorders like peritoneal fibrosis and adhesions due to the advantageous concentration of the fusion protein within the affected peritoneum.

(50) The fusion proteins, nucleic acids, and cells of the invention are also useful to treat conditions where promotion of re-epithelialization is beneficial. Such conditions include, but are not limited to: diseases of the skin, such as venous ulcers, ischaemic ulcers (pressure sores), diabetic ulcers, graft sites, graft donor sites, abrasions and burns; diseases of the bronchial epithelium, such as asthma and ARDS; diseases of the intestinal epithelium, such as mucositis associated with cytotoxic treatment, esophagial ulcers (reflex disease), stomach ulcers, and small intestinal and large intestinal lesions (inflammatory bowel disease).

(51) Still further uses of the fusion proteins, nucleic acids, and cells of the invention are in conditions in which endothelial cell proliferation is desirable, for example, in stabilizing atherosclerotic plaques, promoting healing of vascular anastomoses, or in conditions in which inhibition of smooth muscle cell proliferation is desirable, such as in arterial disease, restenosis and asthma.

(52) The fusion proteins, nucleic acids, and cells of the invention are also useful in the treatment of hyperproliferative diseases, such as cancers including, but not limited to, breast, prostate, ovarian, stomach, renal (e.g., renal cell carcinoma), pancreatic, colorectal, skin, lung, thyroid, cervical and bladder cancers, glioma, glioblastoma, mesothelioma, melanoma, as well as various leukemias and sarcomas, such as Kaposi's Sarcoma, and in particular are useful to treat or prevent recurrences or metastases of such tumors. In particular embodiments, the fusion proteins, nucleic acids, and cells of the invention are useful in methods of inhibiting cyclosporin-mediated metastases. It will of course be appreciated that in the context of cancer therapy, “treatment” includes any medical intervention resulting in the slowing of tumor growth or reduction in tumor metastases, as well as partial remission of the cancer in order to prolong life expectancy of a patient. In one embodiment, the invention is a method of treating cancer comprising administering a fusion protein, nucleic acid or cells of the invention. In particular embodiments, the condition is renal cancer, prostate cancer or melanoma.

(53) The fusion proteins, nucleic acids, and cells of the invention are also useful for treating, preventing and reducing the risk of occurrence of renal insufficiencies including, but not limited to, diabetic (type I and type II) nephropathy, radiational nephropathy, obstructive nephropathy, diffuse systemic sclerosis, pulmonary fibrosis, allograft rejection, hereditary renal disease (e.g., polycystic kidney disease, medullary sponge kidney, horseshoe kidney), nephritis, glomerulonephritis, nephrosclerosis, nephrocalcinosis, systemic lupus erythematosus, Sjogren's syndrome, Berger's disease, systemic or glomerular hypertension, tubulointerstitial nephropathy, renal tubular acidosis, renal tuberculosis, and renal infarction. In particular embodiments, the fusion proteins, nucleic acids and cells of the invention are combined with antagonists of the renin-angiotensin-aldosterone system including, but not limited to, renin inhibitors, angiotensin-converting enzyme (ACE) inhibitors, Ang Ii receptor antagonists (also known as “Ang Il receptor blockers”), and aldosterone antagonists (see, for example, WO 2004/098637).

(54) The fusion proteins, nucleic acids, and cells of the invention are also useful to enhance the immune response to macrophage-mediated infections, such as those caused by Leishmania spp., Trypanosoma cruzi, Mycobacterium tuberculosis and Mycobacterium leprae, as well as the protozoan Toxoplasma gondii, the fungi Histoplasma capsulatum, Candida albicans, Candida parapsilosis, and Cryptococcus neoformans, and Rickettsia, for example, R. prowazekii, R. coronii, and R. tsutsugamushi. They are also useful to reduce immunosuppression caused, for example, by tumors, AIDS or granulomatous diseases.

(55) In addition, without being bound to any particular theory, it is also believed that the fusion proteins of the invention, because they lack an immunoglobulin domain (unlike TGF-β antibodies and TGF-β receptor-Fc fusion proteins) may not be as susceptible to clearance from sites of action by the immune system (e.g., in conditions or diseases of the lung).