HK1238658B - Methods and compositions for treatment of disorders with follistatin polypeptides - Google Patents

Description

Methods and compositions for treating conditions using follistatin polypeptides

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. Provisional Application Serial No. 62/007,908, filed on June 4, 2014. The specification of the aforementioned application is incorporated herein in its entirety.

Background of the Invention

The transforming growth factor-β (TGF-β) superfamily contains a variety of growth factors that share common sequence elements and structural motifs. These proteins are known to exert biological effects on a variety of cell types in both vertebrates and invertebrates. Members of the superfamily perform important functions in patterning and tissue specification during embryonic development and can influence a variety of differentiation processes, including adipogenesis, myogenesis, chondrogenesis, cardiogenesis, hematopoiesis, neurogenesis, and epithelial differentiation. The family is divided into two general branches: the BMP/GDF and the TGF-β/activin/BMP10 branches, whose members have different, often complementary, effects. By manipulating the activity of TGF-β family members, important physiological changes can often be induced in organisms. For example, Piedmontese and Belgian Blue cattle breeds carry loss-of-function mutations in the GDF8 (also known as myostatin) gene that cause a significant increase in muscle mass. Grobet et al., Nat. Genet. 1997, 17(1):71-4. Furthermore, in humans, inactive GDF8 alleles are associated with increased muscle mass and reportedly with abnormal strength. Schuelke et al., N Engl J Med 2004, 350:2682-8.

Changes in muscle, bone, cartilage, and other tissues can be achieved by agonizing or antagonizing signaling mediated by suitable TGF-β family members. However, because members of this family can affect more than one tissue, in some patient care situations, therapeutic inhibition of family members needs to be achieved in a local rather than systemic manner. Therefore, there is a need for agents that act as potent modulators of TGF-β signaling and are suitable for local administration.

SUMMARY OF THE INVENTION

In certain aspects, the present disclosure provides follistatin polypeptides designed to inhibit follistatin ligands (e.g., activin A, activin B, GDF8, and GDF11) in the vicinity of the tissue to which the follistatin polypeptide is administered, while having little or no systemic effects on the patient.

The follistatin polypeptides described herein include polypeptides comprising at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the amino acid sequence of any one of SEQ ID NOs: 1-4, 7-16 and 26-43. Optionally, the follistatin polypeptide is designed to dimerize or form higher order multimers. This can be achieved by fusing the follistatin sequence of the follistatin polypeptide to a domain that confers dimerization or multimerization. Examples of such domains are constant domains of immunoglobulins, including, for example, the Fc portion of an immunoglobulin. Optionally, the follistatin portion is directly linked to the heterologous portion, or an intervening sequence, such as a linker, can be used. An example of a linker is the sequence TGGG. Optionally, the follistatin polypeptide can exhibit heparin binding activity in the same manner as human follistatin-288. Alternatively, follistatin may have a masked heparin binding domain in the manner of human follistatin-315. In certain aspects, the present disclosure provides therapeutically optimized follistatin polypeptides comprising a portion of an immunoglobulin constant domain from a human IgG with reduced ADCC or CDC activity compared to native human IgG1. Examples include IgG2, IgG3, IgG4, hybrid IgG2/4, and variants of IgG1, IgG2, IgG3, and IgG4. In certain aspects, the present disclosure provides an optimally active form of follistatin comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 15 or 16, particularly in the case of a dimeric fusion protein (e.g., follistatin-Fc protein), providing superior protein quality and activity over native FST (288) and FST (315) forms.

In certain aspects, the present disclosure provides a polypeptide comprising a first amino acid sequence and a second amino acid sequence, wherein the first amino acid sequence consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 and 16, and wherein the second amino acid sequence comprises a constant domain of an immunoglobulin. Optionally, there is a linker polypeptide located between the first amino acid sequence and the second amino acid sequence. Optionally, the linker polypeptide comprises, consists essentially of, or consists of the sequence TGGG. Optionally, the second amino acid sequence comprises, consists essentially of, or consists of a constant domain of an IgG immunoglobulin. Optionally, the second amino acid sequence comprises, consists essentially of, or consists of a constant domain of an IgG immunoglobulin with reduced ADCC activity compared to human IgG1. Optionally, the second amino acid sequence comprises, consists essentially of, or consists of a constant domain of an IgG immunoglobulin with reduced CDC activity compared to human IgG1. Optionally, the second amino acid sequence comprises, consists essentially of, or consists of a constant domain of an IgG immunoglobulin selected from the group consisting of IgG1, IgG2, and IgG4. Optionally, the second amino acid sequence comprises or consists of an Fc portion of an immunoglobulin (e.g., an IgG immunoglobulin), which can be an immunoglobulin with reduced ADCC, CDC, or both compared to human IgG1, examples of which include IgG2, IgG4, and IgG2/4 hybrids, or various mutations of any of IgG1, IgG2, IgG3, or IgG4. In certain aspects, the present disclosure provides a follistatin polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from SEQ ID NOs: 38-43. In certain aspects, the present disclosure provides follistatin polypeptides comprising, consisting essentially of, or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from SEQ ID NOs: 26-28 and 32-34. In certain aspects, the present disclosure provides follistatin polypeptides comprising, consisting essentially of, or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from SEQ ID NOs: 29-31 and 35-37. Desirable follistatin polypeptides can bind to one or more ligands selected from the group consisting of myostatin, GDF-11, activin A, and activin B with a KD of less than 1 nM, 100 pM, 50 pM, or 10 pM. In certain aspects, any of the above polypeptides may be a dimer, including a heterodimer or a homodimer, or a higher order multimer. Any of the above polypeptides may be incorporated into a pharmaceutical formulation.

In certain aspects, the disclosure provides nucleic acids encoding any of the follistatin polypeptides described herein and cells comprising the nucleic acids, which can be used to produce the follistatin polypeptides.

In certain aspects, the present disclosure provides methods for treating a tissue or organ by directly administering a follistatin polypeptide to the tissue. For example, the present disclosure provides methods for increasing muscle size or strength in a patient, the methods comprising administering an effective amount of a follistatin polypeptide to a target muscle of a patient in need thereof via an intramuscular route, wherein the increase in muscle size or strength occurs in the target muscle, and wherein the follistatin polypeptide does not have a substantial systemic effect on muscle size or strength. The target muscle may be damaged, weakened, or defective, as is the case in a variety of muscle disorders including muscular dystrophy (e.g., Duchenne muscular dystrophy, Becker muscular dystrophy, and fascioscapulohumeral muscular dystrophy), inflammatory muscle disorders (e.g., inclusion body myositis), muscle injury or trauma, muscle disuse (e.g., after prolonged bed rest or limb immobilization), and muscle atrophy or weakening as a result of aging, cancer, or various types of chronic diseases. The methods can also be applied to healthy muscles that require an increase in target muscle size or strength. Additionally, administration of follistatin polypeptides to muscles can result in an overall decrease in body fat and, therefore, can be used to treat obesity or other conditions associated with excess body fat. Optionally, follistatin can be administered directly to adipose tissue. Follistatin polypeptides can be administered to a single target muscle or to more than one target muscle. The methods and follistatin polypeptides can be used to achieve an effect on a target tissue (e.g., muscle) without having a substantial effect on other tissues (e.g., non-target muscles or other organs). Thus, no systemic effects of follistatin are observed. For example, muscle size or strength contralateral to the target muscle may not be substantially increased, or there may be no substantial effect on a metric selected from the group consisting of serum FSH levels, liver size, hematocrit, hemoglobin, and reticulocyte levels in the patient.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the complete, unprocessed amino acid sequence of human follistatin 315 (SEQ ID NO: 3). The leader sequence is in bold italics, the follistatin N-terminal region (FSN) is single-underlined, and the three follistatin domains (FSD) are double-underlined. Specifically, follistatin domain I (FSDI) is in red, follistatin domain II (FSDII) is in blue, and follistatin domain III (FSDIII) is in green.

Figure 2 shows the effect of 4 weeks of subcutaneous treatment with FST(288)-Fc, FST(315)-Fc, or ActRIIB-Fc on the lean tissue mass of mice. The vehicle was Tris-buffered saline. Data are mean ± SEM. *, P < 0.05 relative to TBS by unpaired t-test. #, P < 0.05 relative to the FST group by unpaired t-test. Compared with vehicle control mice, treatment with FST(288)-Fc, FST(315)-Fc, and ActRIIB-Fc resulted in a significant increase in lean tissue mass. The increase in lean tissue mass in ActRIIB-Fc-treated mice was significantly greater than the increase in lean tissue mass observed in FST(288)-Fc or FST(315)-Fc-treated mice.

FIG3 shows the effect of 4 weeks of subcutaneous twice-weekly treatment with FST(288)-Fc, FST(315)-Fc, or ActRIIB-Fc on the grip strength of mice. The vehicle was Tris-buffered saline. Data are mean ± SEM. *, P < 0.05 relative to TBS by unpaired t-test. #, P < 0.05 relative to the FST group by unpaired t-test. ActRIIB-Fc treatment increased the grip strength of mice. No increase in grip strength was observed in mice treated with FST(288)-Fc or FST(315)-Fc.

Figure 4 shows the effect of 4 weeks of subcutaneous injection twice weekly of FST(288)-IgG1, FST(315)-IgG1 or ActRIIB-Fc on the mass of the pectoralis muscle (Pecs), tibialis anterior (TA), gastrocnemius muscle (Gastroc) and femoral muscle of mice. The vehicle was Tris-buffered saline. Data are mean ± SEM. *, P < 0.05 relative to TBS by unpaired t-test. #, P < 0.05 relative to the FST group by unpaired t-test. ActRIIB-Fc treatment significantly increased the mass of the pectoralis muscle, tibialis anterior, gastrocnemius muscle and femoral muscle of mice, but little or no increase in muscle mass was observed in mice treated with FST(288)-IgG1 or FST(315)-IgG1.

Figure 5 shows the effect of 4 weeks of subcutaneous treatment with FST(288)-IgG1 or FST(315)-IgG1 on serum levels of follicle-stimulating hormone (FSH). The vehicle was Tris-buffered saline. Data are mean ± SEM. *, P < 0.05 relative to TBS by unpaired t-test. FST(315)-IgG1 treatment resulted in a significant decrease in serum FSH levels compared to vehicle control mice. In contrast, FST(288)-IgG1 treatment had no effect on serum FSH levels.

Figure 6 shows the effect of 4 weeks of treatment with FST(288)-IgG1, FST(315)-IgG1, or ActRIIB-mFc by subcutaneous injection twice weekly on the lean tissue mass of mice. The vehicle was Tris-buffered saline. Data are mean ± SEM. *, P < 0.05 relative to TBS by unpaired t-test. ActRIIB-mFc treatment resulted in a significant increase in lean tissue mass compared to vehicle control mice. No increase in lean tissue mass was observed in FST(288)-IgG1 or FST(315)-IgG1 treated mice.

Figure 7 shows the effect of 4 weeks of treatment with FST(288)-IgG1, FST(315)-IgG1 or ActRIIB-mFc injected intramuscularly into the right gastrocnemius muscle twice a week on the mass of the gastrocnemius muscle of mice. The vehicle was Tris-buffered saline. The data are mean ± SEM. *, P < 0.05 relative to TBS by unpaired t-test. #, P < 0.05 for injected right gastrocnemius muscle relative to uninjected left gastrocnemius muscle by unpaired t-test. FST(288)-IgG1, FST(315)-IgG1 and ActRIIB-mFc treatment significantly increased the muscle mass of the injected right gastrocnemius muscle. ActRIIB-mFc treatment also significantly increased the muscle mass of the uninjected left gastrocnemius muscle. In contrast, no increase was observed in the uninjected left gastrocnemius muscle in FST(288)-IgG1 or FST(315)-IgG1 treated mice.

FIG8 shows the effect of 3 weeks of treatment with different doses of FST(288)-IgG1 injected intramuscularly into the right gastrocnemius muscle twice weekly on the gastrocnemius muscle mass of mice, expressed as a ratio relative to the uninjected left gastrocnemius muscle. The vehicle was phosphate-buffered saline. Data are mean ± SEM. *, P < 0.05 relative to PBS by unpaired t-test. Increasing doses of FST(288)-IgG1 caused increased hypertrophy of the injected gastrocnemius muscle relative to the uninjected muscle.

FIG9 shows the effect of 4 weeks of treatment with FST(291)-IgG1 injected intramuscularly twice weekly into the left gastrocnemius muscle. The vehicle was phosphate-buffered saline. Data are mean ± SEM. *, P < 0.05 relative to PBS by unpaired t-test. Intramuscular administration of FST(291)-IgG2 resulted in a significant increase in muscle mass in the injected gastrocnemius muscle relative to uninjected muscle and relative to the control.

Detailed Description of the Invention

1. Overview

In certain aspects, the present disclosure relates to follistatin polypeptides. As used herein, the term "follistatin" refers to a family of follistatin (FST) proteins and follistatin-related proteins derived from any species. Follistatin is an autocrine glycoprotein expressed in almost all tissues of higher animals. It was initially isolated from follicular fluid and identified as a protein portion that inhibits the secretion of follicle-stimulating hormone (FSH) from the anterior pituitary, hence its name, FSH inhibitory protein (FSP). Subsequently, its primary function was determined to be binding to and neutralizing members of the TGF-β superfamily, including, for example, activin, a paracrine hormone that increases FSH secretion in the anterior pituitary.

The term "follistatin polypeptide" is used to refer to polypeptides comprising any naturally occurring polypeptide of the follistatin family that retains useful activity, including, for example, ligand binding (e.g., myostatin, GDF-11, activin A, activin B) or heparin binding, as well as any variants thereof (including mutants, fragments, fusions, and peptide mimetics). For example, follistatin polypeptides include polypeptides comprising an amino acid sequence derived from any known follistatin sequence that is at least about 80% identical, preferably at least 85%, 90%, 95%, 97%, 99% or greater identical to the sequence of a follistatin polypeptide. The term "follistatin polypeptide" may refer to a fusion protein comprising any of the above polypeptides and a heterologous (non-follistatin) portion. An amino acid sequence is considered heterologous to follistatin if it is not exclusively present in the long (315 amino acid) form of human follistatin as shown in SEQ ID NO: 3. Provided herein are numerous examples of heterologous moieties that can be immediately adjacent to the follistatin polypeptide portion of the fusion protein by amino acid sequence, or separated by an intervening amino acid sequence (eg, a linker or other sequence).

Based on alternative mRNA splicing and alternative protein glycosylation, follistatin is a single-chain polypeptide with a molecular weight range of 31-49 kDa. The alternatively spliced mRNA encodes two proteins of 315 amino acids (FST315) and 288 amino acids (FST288); follistatin 315 can be further proteolytically degraded to follistatin 303 (FST303). Amino acid sequence analysis shows that the native human follistatin polypeptide contains five domains (from the N-terminal side): a signal sequence peptide (amino acids 1-29 of SEQ ID NO: 1), an N-terminal domain (FSN) (amino acids 30-94 of SEQ ID NO: 1), a follistatin domain I (FSDI) (amino acids 95-164 of SEQ ID NO: 1), a follistatin domain II (FSDII) (amino acids 168-239 of SEQ ID NO: 1), and a follistatin domain III (FSDIII) (amino acids 245-316 of SEQ ID NO: 1). See PNAS, U.S.A., 1988, Vol. 85, No. 12, pp. 4218-4222.

The human follistatin-288 (FST288) precursor has the following amino acid sequence, with the signal peptide in bold, the N-terminal domain (FSN) in single underline, and follistatin domains I-III (FSI, FSII, FSIII) in double underline.

MVRARHQPGGLCLLLLLLCQFMEDRSAQAGNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEE DVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECA LLKARCKEQPELEVQYQGRC KKT CRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHL RKATCLLGRSIGLAYEGKC IKAKS CEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECA MKEAACSSGVLLEVKHSGSC N (SEQ ID NO: 1)

The processed (mature) human follistatin variant FST (288) has the following amino acid sequence, wherein the N-terminal domain is single-underlined and follistatin domains I-III are double-underlined. In addition, it should be understood that either the initial amino acid G or N before the first cysteine can be removed without consequence by processing or intentional removal, and further polypeptides comprising such slightly smaller polypeptides are included.

GNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCEN VDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRC KKT CRDVFC PGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKC IKAK SCEDIQ CTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSC N (SEQ IDNO: 2)

The human follistatin-315 (FST315) precursor has the following amino acid sequence, with the signal peptide in bold, the N-terminal domain (FSN) single-underlined, and follistatin domains I-III (FSI, FSII, FSIII) double-underlined (NCBI accession number AAH04107.1; 344 amino acids).

MVRARHQPGGLCLLLLLLCQFMEDRSAQAGNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEE DVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECA LLKARCKEQPELEVQYQGRC KKT CRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHL RKATCLLGRSIGLAYEGKC IKAKS CEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECA MKEAACSSGVLLEVKHSGSC NSISEDTEEEEEDEDQDYSFPISSILEW (SEQ ID NO: 3)

Processed (mature) human FST (315) has the following amino acid sequence, with the N-terminal domain single-underlined and follistatin domains I-III double-underlined. Furthermore, it should be understood that either the initial amino acid G or N preceding the first cysteine can be removed without consequence by processing or intentional removal, and further encompasses polypeptides comprising such slightly smaller polypeptides.

GNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCEN VDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRC KKT CRDVFC PGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKC IKAK SCEDIQ CTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSC NSISEDTEEEEEDEDQDYSFPISSILEW (SEQ ID NO: 4)

The follistatin polypeptides of the present disclosure may include any naturally occurring domain of the follistatin protein and variants thereof (e.g., mutants, fragments, and peptide mimetics) that retain useful activity. For example, it is well known that FST(315) and FST(288) have high affinity for both activins (activin A and activin B) and myostatin (and the closely related GDF11), and follistatin domains (e.g., FSN and FSD I-III) are thought to be involved in binding to the TGF-β ligands. However, it is believed that each of these three domains may have different affinities for these TGF-β ligands. For example, recent studies have shown that a polypeptide construct comprising only the N-terminal domain (FSN) and two FSDI domains in tandem retains high affinity for myostatin, exhibits little or no affinity for activin when introduced into mice by genetic expression, and promotes systemic muscle growth (Nakatani et al., The FASEB Journal, Vol. 22477-487 (2008)).

In addition, the FSDI domain contains the heparin binding domain of human follistatin, which has the amino acid sequence of KKCRMNKKNKPR (SEQ ID NO: 5). This heparin binding domain can be represented as BBXBXXBBXBXB (SEQ ID NO: 6), where "B" refers to a basic amino acid, specifically lysine (K) or arginine (R). Thus, in certain aspects, the present disclosure includes variant follistatin proteins that exhibit selective binding and/or inhibition of a given TGF-β ligand relative to naturally occurring FST proteins (e.g., maintaining high affinity for myostatin while significantly reducing affinity for activin).

In certain aspects, the disclosure includes polypeptides comprising the FSN domains listed below and, for example, one or more heterologous polypeptides, furthermore, it will be appreciated that either the initial amino acid G or N preceding the first cysteine may be deleted, as in the example described below (SEQ ID NO: 8).

GNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKET(SEQ ID NO: 7)

CWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKET (SEQID NO: 8)

In certain aspects, the disclosure encompasses polypeptides comprising a FSDI domain comprising the minimal core activities of myostatin (and/or GDF11) binding as shown below, along with heparin binding, and, for example, one or more heterologous polypeptides.

CENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRC (SEQ ID NO: 9)

The FSDI sequence can be advantageously maintained in a structural context by being expressed as a polypeptide that also comprises an FSN domain. Thus, the present disclosure includes polypeptides comprising the FSN-FSDI sequence listed below (SEQ ID NO: 10) and, for example, one or more heterologous polypeptides, further recognizing that either the initial amino acid G or N preceding the first cysteine can be removed by processing or intentional removal without consequence, and further including polypeptides comprising such slightly smaller polypeptides.

CWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRC (SEQ ID NO:10)

Nakani et al. showed that the FSN-FSDI-FSDI construct was sufficient to confer systemic muscle growth when genetically expressed in mice, and thus the present disclosure encompasses polypeptides comprising the following amino acid sequence and, for example, one or more heterologous polypeptides.

CWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRC (SEQ ID NO:11)

While the FSDI sequence confers binding to myostatin and GDF11, activins, particularly activin A and activin B, have been shown to be negative regulators of muscle, and thus follistatin polypeptides that inhibit both the myostatin/GDF11 group and the activin A/activin B group may provide a more potent muscle effect. Furthermore, given the findings herein demonstrating low systemic availability of certain follistatin polypeptides, particularly those containing a heparin binding domain, and more particularly those in homodimeric form (e.g., Fc fusions), safety concerns associated with the known effects of activin inhibition on the reproductive axis and other tissues are mitigated. Given that FSDII confers binding to activins A and B, the present disclosure provides polypeptides comprising FSDI and FSDII (SEQ ID NO: 12), as well as the FSN-FSDI-FSDII construct (SEQ ID NO: 13), and, for example, one or more heterologous polypeptides.

CENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKC(SEQ ID NO: 12)

CWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKC (SEQ ID NO:13)

As described in the Examples, a 291 amino acid follistatin polypeptide (representing a truncation of the naturally occurring FST-315) has advantageous properties. Therefore, both the unprocessed (SEQ ID NO: 14) and mature FST (291) (SEQ ID NO: 15) polypeptides are encompassed by the present disclosure and can be conjugated to heterologous proteins. Furthermore, it should be understood that either the initial amino acid G or N preceding the first cysteine can be processed or intentionally removed without consequence, and further encompasses polypeptides comprising such slightly smaller polypeptides, such as the example shown below (SEQ ID NO: 16).

MVRARHQPGGLCLLLLLLCQFMEDRSAQAGNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQY QGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCNSIS (SEQ ID NO: 14)

GNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFCP GSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCNSIS (SEQ ID NO: 15)

CWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFCPG SSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCNSIS (SEQ IDNO: 16)

In certain embodiments, the present invention relates to antagonizing follistatin ligands (also referred to as follistatin ligands) with a subject follistatin polypeptide (e.g., FST-IgG fusion polypeptide). Thus, the compositions and methods of the present disclosure can be used to treat conditions associated with abnormal activity of one or more ligands of follistatin. Exemplary ligands of follistatin include some TGF-β family members, such as activin A, activin B, myostatin (GDF8), and GDF11.

The follistatin protein may be referred to herein as FST. If followed by a number, such as FST(288), this indicates that the protein is the 288 form of follistatin. If present as FST(288)-Fc, this indicates a C-terminal Fc fusion of FST(288), which may or may not include an intervening linker. In this case, Fc can be any immunoglobulin Fc portion, as that term is defined herein. If present as FST(288)-IgG2, this indicates a C-terminal Fc fusion of the Fc portion of human IgG2 with the Fc portion of FST(288).

Activin is a dimeric polypeptide growth factor that belongs to the TGF-β superfamily. There are 3 kinds of activins (A, B and AB), which are homodimers of 2 closely related β subunits (β _A β _A , β B β _B and β _{A β B} ). Other activins C and E have been identified, although the functions of these proteins are not understood. In the TGF-β superfamily, activin is a unique multifunctional factor that can stimulate hormone production in ovarian and placental cells, support neuronal cell survival, affect cell cycle progression positively or negatively according to cell type, and induce mesoderm differentiation at least in amphibian embryos (DePaolo et al., 1991, Proc Soc Ep Biol Med. 198: 500-512; Dyson et al., 1997, Curr Biol. 7: 81-84; Woodruff, 1998, Biochem Pharmacol. 55: 953-963). In addition, it was found that the erythroid differentiation factor (EDF) isolated from stimulated human monocytic leukemia cells was identical to activin A (Murata et al., 1988, PNAS, 85:2434). It has been shown that activin A plays the role of a natural regulator of erythropoiesis in the bone marrow. In several tissues, activin signaling is antagonized by its associated heterodimer (inhibin). For example, during the release of follicle-stimulating hormone (FSH) from the pituitary gland, activin promotes FSH secretion and synthesis, while inhibin prevents FSH secretion and synthesis. Activin is also related to a negative regulator as muscle mass and function, and activin antagonists can promote muscle growth or antagonize muscle loss in vivo. Link and Nishi, Exp Cell Res. 1997 Jun 15;233(2):350-62; He et al, Anat Embryol (Berl). 2005 Jun;209(5):401-7; Souza et al, Mol Endocrinol. 2008 Dec;22(12):2689-702; Am J Physiol Endocrinol Metab. 2009 Jul;297(1):E157-64; Gilson et al, Zhou et al, Cell. 2010 Aug 20;142(4):531-43.

Growth and differentiation factor-8 (GDF8) is also known as myostatin. GDF8 is a negative regulator of skeletal muscle mass. GDF8 is highly expressed in developing and mature skeletal muscle. GDF8 null mutations in transgenic mice are characterized by pronounced skeletal muscle hypertrophy and hyperplasia (McPherron et al., Nature, 1997, 387:83-90). Similar increases in skeletal muscle mass are evident in naturally occurring GDF8 mutations in cattle (Ashmore et al., 1974, Growth, 38:501-507; Swatland and Kieffer, J. Anim. Sci., 1994, 38:752-757; McPherron and Lee, Proc. Natl. Acad. Sci. USA, 1997, 94:12457-12461; and Kambadur et al., Genome Res., 1997, 7:910-915) and, surprisingly, in humans (Schuelke et al., N Engl J Med 2004;350:2682-8). Studies have also shown that muscle wasting associated with human HIV infection is accompanied by increased GDF8 protein expression (Gonzalez-Cadavid et al., PNAS, 1998, 95:14938-43). In addition, GDF8 can regulate the production of muscle-specific enzymes (such as creatine kinase) and regulate myoblast proliferation (WO 00/43781). The GDF8 propeptide can non-covalently bind to the mature GDF8 domain dimer, causing it to lose its biological activity (Miyazono et al., (1988) J. Biol. Chem., 263: 6407-6415; Wakefield et al., (1988) J. Biol. Chem., 263: 7646-7654; and Brown et al., (1990) Growth Factors, 3: 35-43). Other proteins that bind to GDF8 or structurally related proteins and inhibit their biological activity include follistatin and may include follistatin-related proteins (Gamer et al., (1999) Dev. Biol., 208: 222-232).

The terms used in this specification generally have their ordinary meanings in the art, in the context of the present invention, and in the specific context in which each term is used. Certain terms are discussed below or elsewhere in this specification to provide additional guidance to practitioners when describing the compositions and methods of the present invention and how to make and use them. Any application or meaning of a term will be apparent from the specific context in which the term is used.

"About" and "approximately" generally can mean an acceptable degree of error for the amount being measured, given the nature or precision of the measurements. Typically, exemplary degrees of error are typically within twenty percent (20%), preferably 10%, and more preferably 5% of a given value or range of values.

Alternatively, particularly in biological systems, the terms "about" and "approximately" can mean values that are within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a given value. Numerical quantities given herein are approximate and, unless otherwise indicated, are meant to be inferred from the term "about" or "approximately" when not expressly stated.

The methods of the present invention may include steps of comparing sequences to each other, including comparison of a wild-type sequence with one or more mutants (sequence variants). Such comparisons typically include alignments of polymer sequences, for example, using sequence alignment programs and/or algorithms well known in the art (e.g., BLAST, FASTA, and MEGALIGN, etc.). A skilled artisan will readily appreciate that, in alignments where mutations contain insertions or deletions of residues, sequence alignments may introduce "gaps" (typically indicated by dashes or "A") into polymer sequences that do not contain inserted or deleted residues.

"Homologous" in all its grammatical forms and spelling variations refers to the relationship between two proteins that have a "common evolutionary origin", including proteins from the same superfamily of biological species as well as homologous proteins from different biological species. The proteins (and their encoding nucleic acids) have sequence homology, as reflected by their sequence similarity, whether in terms of percent identity or the presence of specific residues or motifs and conserved positions. However, in common usage and in this application, the term "homologous" when modified by an adverb (e.g., "highly") can refer to sequence similarity and may or may not be related to a common evolutionary origin.

The term "sequence similarity" in all its grammatical forms refers to the degree of identity or correspondence between nucleic acid or amino acid sequences that may or may not have a common evolutionary origin.

2. Follistatin peptide

In certain aspects, the present disclosure relates to follistatin polypeptides (e.g., FST-Fc polypeptides), and in particular, truncated forms and variants thereof, exemplified by polypeptides comprising SEQ ID NO: 2, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. Optionally, the fragments, functional variants, and modified forms have similar, identical, or improved biological activity as their corresponding wild-type follistatin polypeptides. For example, the follistatin variants of the present disclosure can bind to and inhibit the function of follistatin ligands (e.g., activin A, activin AB, activin B, and GDF8). Optionally, the follistatin polypeptides regulate the growth of tissues, particularly muscle. Examples of follistatin polypeptides include polypeptides comprising, consisting essentially of, or consisting of the amino acid sequence of any one of SEQ ID NOs: 1-16 and 26-43, and polypeptides comprising, consisting essentially of, or consisting of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 1-16 and 26-43. Variations of these polypeptides can be prepared according to the following guidance. The numbering of the amino acids of the follistatin polypeptide is based on the sequence of SEQ ID NO: 1, regardless of whether the native leader sequence is used.

As described above, follistatin is characterized by three cysteine-rich regions (i.e., FS domains I-III), which are believed to mediate follistatin ligand binding. In addition, researchers have demonstrated that polypeptide constructs containing only one of the three FS-binding domains (e.g., FSDI) retain strong affinity for certain follistatin ligands (e.g., myostatin) and are active in vivo. See Nakatani et al., FASEB Journal, Vol. 22477-487 (2008). Thus, variant follistatin polypeptides of the present disclosure may comprise one or more active portions of a follistatin protein. For example, a construct of the present disclosure may begin at residues corresponding to amino acids 30-95 of SEQ ID NO: 1 and end at positions corresponding to amino acids 316-344 of SEQ ID NO: 1. Other examples include constructs that begin at positions 30-95 of SEQ ID NO: 1 and end at positions corresponding to amino acids 164-167 or 238-244. Other examples may include any one of SEQ ID Nos: 7-16.

The follistatin variations described herein can be combined with each other or with heterologous amino acid sequences in various ways. For example, variant follistatin proteins of the present disclosure include polypeptides comprising one or more FS domains selected from FSDI (amino acids 95-164 of SEQ ID NO: 1 (i.e., SEQ ID NO: 2), FSDII (amino acids 168-239 of SEQ ID NO: 1), or FSDIII (amino acids 245-316 of SEQ ID NO: 1) and polypeptides comprising one or more FS domains selected from FSDI (amino acids 95-164 of SEQ ID NO: 1 (i.e., SEQ ID NO: 2), FSDII (amino acids 168-239 of SEQ ID NO: 1), or FSDIII (amino acids 245-316 of SEQ ID NO: 1). 1) having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identity to one or more FS domains of a sequence (amino acids 245-316 of SEQ ID NO: 1). These FS domains can be combined in any order within the variant follistatin polypeptides of the present disclosure, provided that the recombinant protein retains the desired activity, including, for example, follistatin ligand binding activity (e.g., myostatin) and biological activity (e.g., inducing muscle mass and/or strength). Examples of such follistatin variant polypeptides include, for example, those having, for example, FSDI-FSDII-FSDIII, FSDI-FSDIII, FSDI-FSD Polypeptides having domain structures such as FSD-I-FSDIII, FSDI-FSDII, FSDI-FSDI, FSN-FSDI-FSDII-FSDIII, FSN-FSDI-FSDII, FSN-FSDI-FSDI, FSN-FSDI-FSDIII, and FSN-FSDI-FSDI-FSDIII, and polypeptides obtained by fusing other heterologous polypeptides to the N-terminus or C-terminus of these polypeptides. These domains can be linked directly or via a linker polypeptide. The optional polypeptide linker can be of any sequence and can comprise 1-50, preferably 1-10, and more preferably 1-5 amino acids. In certain aspects, preferred linkers do not contain cysteine amino acids.

In some embodiments, the follistatin variants of the present disclosure have reduced or eliminated binding affinity for one or more follistatin ligands. In certain aspects, the present disclosure provides follistatin variants with reduced or eliminated binding affinity for activin. In certain aspects, the present disclosure provides follistatin variants with reduced or eliminated binding affinity for activin but retain high affinity for myostatin.

In some aspects, the present disclosure provides follistatin variants that do not comprise a sequence corresponding to the FSDII domain or a functionally active FSDII domain. For example, the follistatin polypeptides of the present disclosure may include variants that are partially or completely deleted by partial or complete deletion of the FSDII domain. In some aspects, the follistatin variants include deletion of one or more cysteine residues in the FSDII region or substitution with non-cysteine amino acids.

The follistatin protein of the present disclosure may comprise a signal sequence. The signal sequence may be the native signal sequence of the follistatin protein (e.g., amino acids 1-29 of SEQ ID NO: 1) or a signal sequence from another protein, such as a tissue plasminogen activator (TPA) signal sequence or a honey bee melatin (HBM) signal sequence.

In addition, N-linked glycosylation sites (N-X-S/T) can be added to the follistatin polypeptide and can extend the serum half-life of the FST-Fc fusion protein. The N-X-S/T sequence can generally be introduced at a position outside the ligand binding pocket. The N-X-S/T sequence can be introduced in the linker between the follistatin sequence and the Fc or other fusion components. The site can be introduced with minimal effort by introducing an N in the correct position relative to a pre-existing S or T, or by introducing an S or T at a position equivalent to a pre-existing N. Any S predicted to be glycosylated can be converted to a T without creating an immunogenic site due to the protection provided by glycosylation. Similarly, any T predicted to be glycosylated can be converted to an S. Thus, follistatin variants can include one or more additional non-endogenous N-linked glycosylation consensus sequences.

In certain embodiments, the present disclosure contemplates the creation of functional variants by modifying the structure of follistatin polypeptides for the purpose of improving therapeutic efficacy or stability (e.g., shelf life in vitro and resistance to proteolytic degradation in vivo). Modified follistatin polypeptides can also be generated, for example, by amino acid substitutions, deletions, or additions. For example, it is reasonable to expect that individual substitutions of leucine with isoleucine or valine, aspartic acid with glutamic acid, threonine with serine, or similar substitutions of amino acids with structurally related amino acids (e.g., conservative mutations) will not significantly affect the biological activity of the resulting molecule. Conservative substitutions are substitutions that occur within a family of amino acids that are related in their side chains. Whether changes in the amino acid sequence of a follistatin polypeptide result in a functional homolog can be readily determined by evaluating the ability of the variant follistatin polypeptide to respond in cells in a manner similar to that of the wild-type follistatin polypeptide, or to bind to one or more ligands (e.g., activin or myostatin) in a manner similar to that of wild-type follistatin.

In certain embodiments, the present invention contemplates specific mutations of follistatin polypeptides to alter the glycosylation of the polypeptide. The mutations may be selected to introduce or eliminate one or more glycosylation sites, such as O-linked or N-linked glycosylation sites. Asparagine-linked glycosylation recognition sites generally comprise a tripeptide sequence of asparagine-X-threonine (where "X" is any amino acid), which is specifically recognized by suitable cellular glycosylation enzymes. Changes may also be made by adding one or more serine or threonine residue sequences to the sequence of the wild-type follistatin polypeptide (for O-linked glycosylation sites) or replacing them with one or more serine or threonine residues. Various amino acid substitutions or deletions at one or both of the first or third amino acid positions of the glycosylation recognition site (and/or amino acid deletions at the second position) result in non-glycosylation on the modified tripeptide sequence. Another method for increasing the number of sugar moieties in a follistatin polypeptide is by chemical or enzymatic coupling of glycosides to the follistatin polypeptide. Depending on the coupling method used, the sugar can be attached to: (a) arginine and histidine; (b) free carboxyl groups; (c) free sulfhydryl groups, such as the sulfhydryl group of cysteine; (d) free hydroxyl groups, such as the hydroxyl group of serine, threonine, or hydroxyproline; (e) aromatic residues, such as those of phenylalanine, tyrosine, or tryptophan; or (f) the amide group of glutamine. These methods are described in WO 87/05330 published on September 11, 1987, and in Aplin and Wriston (1981) CRC Crit. Rev. Biochem., pp. 259-306, which are incorporated herein by reference. Removal of one or more sugar moieties present on an ActRIIB polypeptide can be achieved chemically and/or enzymatically. Chemical deglycosylation can include, for example, exposing the follistatin polypeptide to the compound trifluoromethanesulfonic acid or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the amino acid sequence intact. Chemical deglycosylation is further described by Hakimuddin et al. (1987) Arch. Biochem. Biophys. 259:52 and Edge et al. (1981) Anal. Biochem. 118:131. Enzymatic cleavage of sugar moieties on follistatin polypeptides can be accomplished using various endo- and exo-glycosidases as described by Thotakura et al. (1987) Meth. Enzymol. 138:350. The sequence of the follistatin polypeptide can be adjusted, as appropriate, depending on the type of expression system employed, as mammalian, yeast, insect, and plant cells can all introduce different glycosylation patterns that can be influenced by the amino acid sequence of the peptide. Generally, follistatin proteins for use in humans can be expressed in mammalian cell lines that provide appropriate glycosylation, such as HEK293 or CHO cell lines, although other mammalian expression cell lines, yeast cell lines with engineered glycosylation enzymes, and insect cells are also expected to be useful.

The present disclosure also contemplates methods for generating variants, particularly sets of combinatorial variants of follistatin polypeptides (optionally including truncated variants); combinatorial mutant libraries are particularly useful for identifying functional variant sequences. The purpose of screening such combinatorial libraries can be to generate follistatin polypeptide variants with altered properties, such as altered pharmacokinetics or altered ligand binding. Various screening assays are provided below that can be used to evaluate variants. For example, follistatin polypeptide variants can be screened for their ability to bind to a follistatin polypeptide to prevent binding of a follistatin ligand to the follistatin polypeptide.

The activity of follistatin polypeptides or their variants can also be tested in cell-based assays or in vivo assays. For example, the effect of follistatin polypeptide variants on the expression of genes involved in muscle production can be evaluated. This can be performed in the presence of one or more recombinant follistatin ligand proteins (e.g., activin A) as needed, and cells can be transfected to produce follistatin polypeptides and/or their variants, optionally with follistatin ligands. Similarly, follistatin polypeptides can be administered to mice or other animals, and one or more muscle properties, such as muscle mass or strength, can be evaluated. Such assays are well known and conventional in the art. Responsive reporter genes can be used in such cell lines to monitor the effects on downstream signaling.

Combination-derived variants can be prepared that exhibit generally improved selective efficacy relative to naturally occurring follistatin polypeptides. Such variant proteins, when expressed from recombinant DNA constructs, can be used in gene therapy protocols. Similarly, mutagenesis can produce variants with significantly different intracellular half-lives compared to the corresponding wild-type follistatin polypeptide. For example, the altered protein can be rendered more or less stable to proteolytic degradation or other cellular processes that destroy or otherwise inactivate the native follistatin polypeptide. Such variants and genes encoding such variants can be utilized to alter follistatin polypeptide levels by modulating their half-life. For example, a short half-life can produce shorter biological effects and, when part of an inducible expression system, can allow for closer control of recombinant follistatin polypeptide levels within cells.

In certain embodiments, in addition to any modifications naturally present in the follistatin polypeptide, the follistatin polypeptides of the present disclosure may also include post-translational modifications. Such modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Therefore, the modified follistatin polypeptide may contain non-amino acid components, such as polyethylene glycol, lipids, polysaccharides or monosaccharides, and phosphates. The effects of such non-amino acid components on the functionality of the follistatin polypeptide can be tested as described herein for other follistatin polypeptide variants. If the follistatin polypeptide is produced in cells by cleaving a nascent form of the follistatin polypeptide, post-translational processing may also be important for the correct folding and/or function of the protein. Different cells (e.g., CHO, HeLa, MDCK, 293, WI38, NIH-3T3, or HEK293) have specific cellular machinery and unique mechanisms for such post-translational activity, and different cells can be selected to ensure the correct modification and processing of the follistatin polypeptide.

In some aspects, functional variants or modified forms of follistatin polypeptides include fusion proteins having at least a portion of a follistatin polypeptide and one or more fusion domains. Well-known examples of the fusion domains include, but are not limited to, polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A, protein G, immunoglobulin heavy chain constant region (Fc), maltose binding protein (MBP), or human serum albumin. Fusion domains can be selected to provide the desired properties. For example, some fusion domains are particularly useful for separating fusion proteins by affinity chromatography. For affinity purification purposes, relevant matrices for affinity chromatography are used, such as glutathione conjugated resins, amylase conjugated resins, and nickel conjugated resins or cobalt conjugated resins. Many of the matrices can be obtained in the form of "kits," such as the Pharmacia GST purification system and the QIAexpress ^™ system (Qiagen) that can be used with (HIS ₆ ) fusion partners. As another example, fusion domains can be selected to facilitate detection of follistatin polypeptides. Examples of the detection domain include various fluorescent proteins (e.g., GFP) and "epitope tags," which are typically short peptide sequences for which specific antibodies can be obtained. Well-known epitope tags for which specific monoclonal antibodies are readily available include FLAG, influenza virus hemagglutinin (HA), and c-myc tags. In some cases, the fusion domain has a protease cleavage site (e.g., a cleavage site for factor Xa or thrombin) that allows the relevant protease to partially digest the fusion protein, thereby releasing the recombinant protein. The released protein can then be separated from the fusion domain by subsequent chromatographic separation. In certain preferred embodiments, the follistatin polypeptide is fused to a domain (a "stabilizer" domain) that stabilizes the follistatin polypeptide in vivo. By "stabilization," it is meant anything that prolongs the serum half-life, whether due to reduced destruction, reduced renal clearance, or other pharmacokinetic effects. Fusions with the Fc portion of immunoglobulins are known to impart desired pharmacokinetic properties to a variety of proteins. Similarly, fusions with human serum albumin can impart desired properties. Other types of fusion domains that can be selected include multimerization (e.g., dimerization, tetramerization) domains and functional domains (which provide additional biological functions, such as further stimulation of muscle growth).

As a specific example, the present disclosure provides a fusion protein containing a follistatin polypeptide fused to a constant domain of an immunoglobulin (e.g., a CH1, CH2, or CH3 domain of an immunoglobulin) or an Fc domain. The Fc domains derived from human IgG1 and IgG2 are provided below (SEQ ID NO: 17 and SEQ ID NO: 18, respectively). As described herein, IgG2, IgG4, or IgG2/4 Fc domains are particularly advantageous for fusing with follistatin polypeptides that retain heparin binding activity because these Fc species have reduced CDC and/or ADCC activity (which is detrimental to cells to which these heparin-binding polypeptides can attach). Other mutations that reduce CDC or ADCC activity are known, and generally, any of these variants are encompassed by the present disclosure and can be used as advantageous components of follistatin fusion proteins. Optionally, the Fc domain of SEQ ID NO: 17 has one or more mutations at residues such as Asp-265, Lys-322, and Asn-434 (numbered according to the corresponding full-length IgG1). In some cases, the mutant Fc domain having one or more of these mutations (e.g., Asp-265 mutation) has reduced ability to bind to Fcγ receptors relative to the wild-type Fc domain. In other cases, the mutant Fc domain having one or more of these mutations (e.g., Asn-434 mutation) has increased ability to bind to MHC class I-associated Fc receptors (FcRN) relative to the wild-type Fc domain.

Examples of human IgG1 and IgG2 amino acid sequences that can be used are shown below:

IgG1

THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 17)

IgG2

VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKT ISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 18)

It will be appreciated that the various components of the fusion protein can be arranged in any manner consistent with the desired functionality. For example, the follistatin polypeptide can be placed at the C-terminus of the heterologous domain, or alternatively, the heterologous domain can be placed at the C-terminus of the follistatin polypeptide. The follistatin polypeptide domain and the heterologous domain need not be contiguous in the fusion protein, and other domains or amino acid sequences may be included at the C-terminus or N-terminus of either domain or between domains.

As used herein, the term "immunoglobulin Fc domain," or simply "Fc," is understood to refer to the carboxyl-terminal portion of an immunoglobulin chain constant region, preferably an immunoglobulin heavy chain constant region, or a portion thereof. For example, an immunoglobulin Fc region may comprise 1) a CH1 domain, a CH2 domain, and a CH3 domain, 2) a CH1 domain and a CH2 domain, 3) a CH1 domain and a CH3 domain, 4) a CH2 domain and a CH3 domain, or 5) a combination of two or more domains and an immunoglobulin hinge region. In a preferred embodiment, an immunoglobulin Fc region comprises at least an immunoglobulin hinge region, a CH2 domain, and a CH3 domain, and preferably lacks a CH1 domain. It is also understood that a follistatin polypeptide may comprise only one immunoglobulin domain, such as a CH1 domain, a CH2 domain, or a CH3 domain. Multiples of these domains provide desirable pharmacokinetic properties, as well as dimerization or higher-order multimerization.

In one embodiment, the heavy chain constant region is derived from an immunoglobulin class IgG (Igγ) (γ subclass 1, 2, 3 or 4). Other classes of immunoglobulins, IgA (Igα), IgD (Igδ), IgE (Igε) and IgM (Igμ), can be used. The selection of suitable immunoglobulin heavy chain constant regions is discussed in detail in U.S. Patent Nos. 5,541,087 and 5,726,044. The selection of specific immunoglobulin heavy chain constant region sequences from certain immunoglobulin classes and subclasses to obtain a particular result is considered to be within the technical level of this area. The portion of the DNA construct encoding the immunoglobulin Fc region preferably includes at least a portion of the hinge domain, preferably at least a portion of the CH ₃ domain of Fc γ or a homologous domain of any one of IgA, IgD, IgE or IgM.

In addition, it is contemplated that amino acid substitutions or deletions within the constant region of the immunoglobulin heavy chain can be used to practice the methods and compositions disclosed herein. One example can be the introduction of amino acid substitutions into the upper CH2 region to generate Fc variants with reduced affinity for Fc receptors (Cole et al. (1997) J. Immunol. 159:3613). Additionally, in many cases, the C-terminal lysine or K can be removed, and thus any polypeptide described herein can omit the C-terminal K present in the Fc domain, such as those shown in SEQ ID NO: 17 or SEQ ID NO: 18.

In certain embodiments, the follistatin polypeptides of the present disclosure contain one or more modifications that stabilize the follistatin polypeptide. For example, the modification extends the in vitro half-life of the follistatin polypeptide, extends the circulatory half-life of the follistatin polypeptide, or reduces proteolytic degradation of the follistatin polypeptide. The stabilizing modifications include, but are not limited to, fusion proteins (including, for example, fusion proteins comprising a follistatin polypeptide and a stabilizer domain), modifications of glycosylation sites (including, for example, addition of glycosylation sites to a follistatin polypeptide), and modifications of sugar moieties (including, for example, removal of sugar moieties from a follistatin polypeptide). In the case of a fusion protein, the follistatin polypeptide is fused to a stabilizer domain (e.g., an IgG molecule (e.g., an Fc domain). As used herein, the term "stabilizer domain" refers not only to a fusion domain (e.g., Fc) in the case of a fusion protein, but also includes non-protein modifications (e.g., sugar moieties) or non-protein polymers (e.g., polyethylene glycol).

In certain embodiments, the present invention provides an isolated and/or purified form of a follistatin polypeptide that is separated from or otherwise substantially free of other proteins.

In certain embodiments, the follistatin polypeptides of the present disclosure (unmodified or modified) can be produced by a variety of techniques known in the art. For example, the follistatin polypeptides can be synthesized using standard protein chemistry techniques, such as those described in Bodansky, M. Principles of Peptide Synthesis, Springer Verlag, Berlin (1993) and Grant GA (ed.), Synthetic Peptides: A User's Guide, WH Freeman and Company, New York (1992). In addition, automated peptide synthesizers are commercially available (e.g., Advanced ChemTech Model 396; Milligen/Biosearch 9600). Alternatively, follistatin polypeptides, fragments or variants thereof can be recombinantly produced using various expression systems well known in the art (e.g., Escherichia coli ( E. coli ), Chinese hamster ovary cells, COS cells, baculovirus) (see also below). In yet another embodiment, modified or unmodified follistatin polypeptides can be produced by digesting a naturally occurring or recombinantly produced full-length follistatin polypeptide using, for example, a protease (e.g., trypsin, thermolysin, chymotrypsin, pepsin, or paired basic amino acid converting enzyme (PACE)). Protease-proteolytic cleavage sites can be identified using computer analysis (using commercially available software, such as MacVector, Omega, PCGene, Molecular Simulation, Inc.). Alternatively, the follistatin polypeptide can be produced from a naturally occurring or recombinantly produced full-length follistatin polypeptide by, for example, standard techniques known in the art, such as chemical cleavage (e.g., cyanogen bromide, hydroxylamine).

3. Nucleic acid encoding follistatin polypeptide

In certain aspects, the present invention provides isolated and/or recombinant nucleic acids encoding any of the follistatin polypeptides disclosed herein. The subject nucleic acids may be single-stranded or double-stranded. The nucleic acids may be DNA or RNA molecules. These nucleic acids may be used, for example, in methods for preparing follistatin polypeptides.

For example, the following sequence encodes the naturally occurring human follistatin precursor polypeptide (SEQ ID NO: 19) (NCBI Accession No. BC004107.2, 1032 bp):

atggtccgcgcgaggcaccagccgggtgggctttgcctcctgctgctgctgctctgccagttcatggaggaccgcagtgcccaggctgggaactgctggctccgtcaagcgaagaacggccgctgccag gtcctgtacaagaccgaactgagcaaggaggagtgctgcagcaccggccggctgagcacctcgtggaccgaggaggacgtgaatgacaacacactcttcaagtggatgattttcaacgggggcgccccc aactgcatcccctgtaaagaaacgtgtgagaacgtggactgtggacctgggaaaaaatgccgaatgaacaagaagaacaaaccccgctgcgtctgcgccccggattgttccaacatcacctggaagggt ccagtctgcgggctggatgggaaaacctaccgcaatgaatgtgcactcctaaaggcaagatgtaaagagcagccagaactggaagtccagtaccaaggcagatgtaaaaagacttgtcgggatgttttc tgtccaggcagctccacatgtgtggtggaccagaccaataatgcctactgtgtgacctgtaatcggatttgcccagagcctgcttcctctgagcaatatctctgtgggaatgatggagtcacctactcc agtgcctgccacctgagaaaggctacctgcctgctgggcagatctattggattagcctatgagggaaagtgtatcaaagcaaagtcctgtgaagatatccagtgcactggtgggaaaaaatgtttatgg gatttcaaggttgggagaggccggtgttccctctgtgatgagctgtgccctgacagtaagtcggatgagcctgtctgtgccagtgacaatgccacttatgccagcgagtgtgccatgaaggaagctgcc tgctcctcaggtgtgctactggaagtaaagcactccggatcttgcaactccatttcggaagacaccgaggaagaggaggaagatgaagaccaggactacagctttcctatatcttctattctagagtgg

The following sequence encodes the mature FST(315) polypeptide (SEQ ID NO: 20).

gggaactgctggctccgtcaagcgaagaacggccgctgccaggtcctgtacaagaccgaactgagcaaggaggagtgctgcagcaccggccggctgagcacctcgtggaccgaggagg acgtgaatgacaacacactcttcaagtggatgattttcaacgggggcgcccccaactgcatcccctgtaaagaaacgtgtgagaacgtggactgtggacctgggaaaaaatgccgaat gaacaagaagaacaaaccccgctgcgtctgcgccccggattgttccaacatcacctggaagggtccagtctgcgggctggatgggaaaacctaccgcaatgaatgtgcactcctaaag gcaagatgtaaagagcagccagaactggaagtccagtaccaaggcagatgtaaaaagacttgtcggggatgttttctgtccaggcagctccacatgtgtggtggaccagaccaataatg cctactgtgtgacctgtaatcggatttgcccagcctgcttcctctgagcaatatctctgtgggaatgatggagtcacctactccagtgcctgccacctgagaaaggctacctgcct gctgggcagatctattggattagcctatgagggaaagtgtatcaaagcaaagtcctgtgaagatatccagtgcactggtgggaaaaaatgtttatgggatttcaaggttgggagaggc cggtgttccctctgtgatgagctgtgccctgacagtaagtcggatgagcctgtctgtgccagtgacaatgccacttatgccagcgagtgtgccatgaaggaagctgcctgctcctcag gtgtgctactggaagtaaagcactccggatcttgcaactccatttcggaagacaccgaggaagaggaggaagatgaagaccaggactacagctttcctatatcttctattctagagtgg

The following sequence encodes the FST(288) polypeptide (SEQ ID NO: 21).

gggaactgctggctccgtcaagcgaagaacggccgctgccaggtcctgtacaagaccgaactgagcaaggaggagtgctgcagcaccggccggctgagcacctcgtgg accgaggaggacgtgaatgacaacacactcttcaagtggatgattttcaacgggggcgcccccaactgcatcccctgtaaagaaacgtgtgagaacgtggactgtgga cctgggaaaaaatgccgaatgaacaagaagaacaaaccccgctgcgtctgcgccccggattgttccaacatcacctggaagggtccagtctgcgggctggatgggaaa acctaccgcaatgaatgtgcactcctaaaggcaagatgtaaagagcagccagaactggaagtccagtaccaaggcagatgtaaaaagacttgtcgggatgttttctgt ccaggcagctccacatgtgtggtggaccagaccaataatgcctactgtgtgacctgtaatcggatttgcccagagcctgcttcctctgagcaatatctctgtgggaat gatggagtcacctactccagtgcctgccacctgagaaaggctacctgcctgctgggcagatctattggattagcctatgagggaaagtgtatcaaagcaaagtcctgt gaagatatccagtgcactggtgggaaaaaatgtttatgggatttcaaggttgggagaggccggtgttccctctgtgatgagctgtgccctgacagtaagtcggatgag cctgtctgtgccagtgacaatgccacttatgccagcgagtgtgccatgaaggaagctgcctgctcctcaggtgtgctactggaagtaaagcactccggatcttgcaac

The following sequence encodes the mature FST(291) polypeptide (SEQ ID NO: 22).

gggactgctggctccgtcaagcgaagaacggccgctgccaggtcctgtacaagaccgaactgagcaaggaggagtgctgcagcaccggccggctgagcacctcgtgga ccgaggaggacgtgaatgacaacacactcttcaagtggatgattttcaacgggggcgcccccaactgcatcccctgtaaagaaacgtgtgagaacgtggactgtggacct gggaaaaaatgccgaatgaacaagaagaacaaaccccgctgcgtctgcgccccggattgttccaacatcacctggaagggtccagtctgcgggctggatgggaaaacct accgcaatgaatgtgcactcctaaaggcaagatgtaaagagcagccagaactggaagtccagtaccaaggcagatgtaaaaagacttgtcggggatgttttctgtccaggc agctccacatgtgtggtggaccagaccaataatgcctactgtgtgacctgtaatcggatttgcccagagcctgcttcctctgagcaatatctctgtgggaatgatggag tcacctactccagtgcctgccacctgagaaaggctacctgcctgctgggcagatctattggattagcctatgagggaaagtgtatcaaagcaaagtcctgtgaagatatc cagtgcactggtgggaaaaaatgtttatgggatttcaaggttgggagaggccggtgttccctctgtgatgagctgtgccctgacagtaagtcggatgagcctgtctgtg ccagtgacaatgccacttatgccagcgagtgtgccatgaaggaagctgcctgctcctcaggtgtgctactggaagtaaagcactccggatcttgcaactccatttcgtgg

In certain aspects, it is further understood that the subject nucleic acids encoding follistatin polypeptides include nucleic acids that are variants of SEQ ID NOs: 19-22. Variant nucleotide sequences include sequences that differ by one or more nucleotide substitutions, additions, or deletions, such as allelic variants; thus, coding sequences may include nucleotide sequences that differ from the coding sequences specified by SEQ ID NOs: 19-22.

In certain embodiments, the present disclosure provides isolated or recombinant nucleic acid sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NOs: 19-22, and in particular those portions thereof derived from follistatin (nucleotides corresponding to amino acids 95-164 of SEQ ID NO: 1). One of ordinary skill in the art will recognize that nucleic acid sequences complementary to SEQ ID NOs: 19-22 and variants of SEQ ID NOs: 19-22 are also within the scope of the present disclosure. In yet another embodiment, the nucleic acid sequences of the present disclosure can be isolated, recombinant and/or fused to heterologous nucleotide sequences, or in a DNA library.

In other embodiments, the nucleic acids of the invention also include nucleotide sequences that hybridize under stringent conditions to the nucleotide sequences specified by SEQ ID NOs: 19-22, complementary sequences of SEQ ID NOs: 19-22, or fragments thereof (eg, nucleotides 19-22).

Those skilled in the art will also readily appreciate that suitable stringency conditions for promoting DNA hybridization can be varied. For example, hybridization can be performed in 6.0 x sodium chloride/sodium citrate (SSC) at about 45°C, followed by a 2.0 x SSC wash at 50°C. For example, the salt concentration of the wash step can be selected from a low stringency of about 2.0 x SSC at 50°C to a high stringency of about 0.2 x SSC at 50°C. In addition, the temperature of the wash step can be increased from a low stringency condition at room temperature (about 22°C) to a high stringency condition at about 65°C. Both temperature and salt can be varied, or the temperature or salt concentration can be kept constant while the other variable is varied. In some embodiments, the present invention provides nucleic acids that are hybridized under low stringency conditions of 6 x SSC at room temperature followed by a 2 x SSC wash at room temperature.

Due to the degeneracy of the genetic code, isolated nucleic acids that are different from the nucleic acids shown in SEQ ID NO: 19-22 are also within the scope of the present disclosure. For example, multiple amino acids are specified with more than one triplet. Codons or synonymous codons specifying the same amino acid (e.g., CAU and CAC are synonymous codons for histidine) can result in "silent" mutations that do not affect the amino acid sequence of the protein. However, it is expected that DNA sequence polymorphisms that do cause changes in the subject protein amino acid sequence may exist in mammalian cells. Those skilled in the art will recognize that due to natural allele variations, these variations may exist in one or more nucleotides (up to about 3-5% nucleotides) of the nucleic acid encoding a particular protein between individuals of a given species. Any and all of the nucleotide variations and resulting amino acid polymorphisms are also within the scope of the present disclosure.

In certain embodiments, in an expression construct, the recombinant nucleic acid of the present disclosure can be effectively linked to one or more regulatory nucleotide sequences. The regulatory nucleotide sequence can generally be suitable for the host cell for expression. Many types of suitable expression vectors and suitable regulatory sequences for various host cells are known in the art. Generally, the one or more regulatory nucleotide sequences can include, but are not limited to, promoter sequences, leader sequences or signal sequences, ribosome binding sites, transcription initiation sequences and termination sequences, translation initiation sequences and termination sequences, and enhancer or activator sequences. The present disclosure contemplates constitutive promoters or inducible promoters known in the art. The promoter can be a naturally occurring promoter or a hybrid promoter that combines elements of more than one promoter. The expression construct can be present on an episome (e.g., plasmid) of the cell, or the expression construct can be inserted into the chromosome. In a preferred embodiment, the expression vector contains a selectable marker gene for the selection of transformed host cells. Selectable marker genes are well known in the art and can change with the host cell employed.

In some aspects, the subject nucleic acid is provided in an expression vector comprising a nucleotide sequence encoding a follistatin polypeptide and operably linked to at least one regulatory sequence. Regulatory sequences are recognized in the art and are selected to direct the expression of the follistatin polypeptide. Thus, the term regulatory sequence includes promoters, enhancers, and other expression control elements. Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology : Methods in Enzymology , Academic Press, San Diego, CA (1990). For example, any of a variety of expression control sequences that control the expression of a DNA sequence when operably linked to a DNA sequence can be used in these vectors to express a DNA sequence encoding a follistatin polypeptide. Such useful expression control sequences include, for example, the early and late promoters of SV40, the tet promoter, the immediate early promoter of adenovirus or cytomegalovirus, the RSV promoter, the lac system, the trp system, the TAC or TRC system, a T7 promoter whose expression is directed by T7 RNA polymerase, the major operator gene and promoter region of bacteriophage λ, the regulatory region of the fd coat protein, the promoter of 3-phosphoglycerate kinase or other glycolytic enzymes, the acid phosphatase promoter (e.g., Pho5), the yeast α-mating factor promoter, the polyhedron promoter of the baculovirus system, and other sequences known to control gene expression in prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. It should be understood that the design of the expression vector may depend on factors such as the choice of the host cell to be transformed and/or the type of protein to be expressed. In addition, the vector copy number, the ability to control the copy number, and the expression of any other proteins encoded by the vector (e.g., antibiotic markers) should also be considered.

The recombinant nucleic acids of the present disclosure can be produced by ligating the cloned gene or portion thereof into a vector suitable for expression in prokaryotes, eukaryotic cells (yeast, avian, insect, or mammalian), or both. Expression vectors for producing recombinant follistatin polypeptides include plasmids and other vectors. For example, suitable vectors include the following types of plasmids for expression in prokaryotes (e.g., E. coli): pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids, and pUC-derived plasmids.

Some mammalian expression vectors contain both prokaryotic sequences that facilitate vector propagation in bacteria and one or more eukaryotic transcription units for expression in eukaryotic cells. pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo, and pHyg derivative vectors are examples of mammalian expression vectors suitable for transfecting eukaryotic cells. Some of these vectors are modified with sequences derived from bacterial plasmids (e.g., pBR322) to facilitate replication in both prokaryotes and eukaryotic cells and selection for drug resistance. Alternatively, viral derivatives such as bovine papillomavirus (BPV-1) or Epstein-Barr virus (pHEBo, pREP derivatives, and p205) can be used to transiently express proteins in eukaryotic cells. Examples of other viral (including retroviral) expression systems can be found in the description of gene therapy delivery systems below. Various methods for plasmid preparation and host biotransformation are well known in the art. For other suitable expression systems and general recombination methods for both prokaryotic and eukaryotic cells, see Molecular Cloning A Laboratory Manual , 2nd edition, edited by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 2001) Chapters 16 and 17. In some cases, it may be necessary to express the recombinant polypeptide by utilizing a baculovirus expression system. Examples of such baculovirus expression systems include pVL derivative vectors (e.g., pVL1392, pVL1393, and pVL941), pAcUW derivative vectors (e.g., pAcUW1), and pBlueBac derivative vectors (e.g., pBlueBac III containing β-gal).

In certain embodiments, vectors for producing the subject follistatin polypeptides in CHO cells can be designed, such as the pcmv-Script vector (Stratagene, La Jolla, Calif.), the pcDNA4 vector (Invitrogen, Carlsbad, Calif.), and the pCI-neo vector (Promega, Madison, Wisc.). Obviously, the subject gene constructs can be used to express the subject follistatin polypeptides in cells propagated in culture, for example, to produce proteins (including fusion proteins or variant proteins) for purification.

The present disclosure further relates to host cells transfected with a recombinant gene comprising one or more coding sequences for the subject follistatin polypeptides (e.g., SEQ ID NOs: 19-22). The host cell can be any prokaryotic or eukaryotic cell. For example, the follistatin polypeptides of the present disclosure can be expressed in bacterial cells (e.g., Escherichia coli), insect cells (e.g., using a baculovirus expression system), yeast, or mammalian cells. Other suitable host cells are known to those skilled in the art.

Therefore, the present disclosure further relates to methods for producing the subject follistatin polypeptide. For example, host cells transfected with an expression vector encoding a follistatin polypeptide can be cultured under suitable conditions that permit expression of the follistatin polypeptide. The follistatin polypeptide can be secreted and isolated from a mixture of cells and culture medium containing the follistatin polypeptide. Alternatively, the follistatin polypeptide can be retained in the cytoplasm or in a membrane fraction, followed by harvesting, lysing the cells, and protein isolation. Cell culture comprises host cells, culture medium, and other by-products. Suitable culture media for cell culture are well known in the art. The subject follistatin polypeptide can be isolated from the cell culture medium, host cells, or both using techniques known in the art for purifying proteins, including ion exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification using antibodies specific for a particular epitope of the follistatin polypeptide. In a preferred embodiment, the follistatin polypeptide is a fusion protein containing a domain that facilitates its purification.

In another embodiment, a fusion gene encoding a purification leader sequence (e.g., a poly-(His)/enterokinase cleavage site sequence on the N-terminus of the desired portion of the recombinant follistatin polypeptide) allows purification of the expressed fusion protein by affinity chromatography using a ^Ni metal resin. The purification leader sequence can then be removed by treatment with enterokinase to yield purified follistatin polypeptide (e.g., see Hochuli et al. (1987) J. Chromatography 411:177; and Janknecht et al., PNAS USA 88:8972).

The technology of preparing fusion genes is well known. Basically, the connection of various DNA fragments encoding different polypeptide sequences is carried out according to conventional techniques, using blunt ends or staggered ends for connection, restriction enzyme digestion to provide suitable ends, filling of sticky ends when appropriate, alkaline phosphatase treatment to avoid unwanted connection, and enzymatic connection. In another embodiment, the fusion gene can be synthesized by conventional techniques, including automatic DNA synthesizers. Alternatively, PCR amplification of the gene fragments can be performed using anchor primers that produce complementary overhangs between two consecutive gene fragments, and the gene fragments can then be annealed to obtain a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology , ed. Ausubel et al., John Wiley & Sons: 1992).

4. Exemplary Therapeutic Applications

In certain embodiments, the compositions of the present disclosure, including, for example, any of FST(288)-IgG1, FST(288)-IgG2, FST(291)-IgG1, FST(291)-IgG2, FST(315)-IgG1, FST(315)-IgG2, and other follistatin polypeptides disclosed herein, can be used to treat or prevent the diseases or conditions described in this section, including diseases or conditions associated with abnormal activity of follistatin polypeptides and/or follistatin ligands (e.g., GDF8). These diseases, conditions, or disorders are generally referred to herein as "follistatin-associated conditions." In certain embodiments, the present disclosure provides methods for treating or preventing an individual in need thereof by administering to the individual a therapeutically effective amount of the follistatin polypeptides described above. These methods are particularly intended for therapeutic and prophylactic treatment of animals, and more particularly humans.

As used herein, a therapeutic agent that "prevents" a disorder or condition refers to a compound that, in a statistical sample, reduces the occurrence of the disorder or condition in treated samples compared to untreated control samples, or delays the onset of one or more symptoms of the disorder or condition or reduces the severity of one or more symptoms of the disorder or condition compared to untreated control samples. As used herein, the term "treating" includes ameliorating or eradicating a condition once it is established.

The follistatin ligand complex plays an important role in tissue growth and early developmental processes, such as the correct formation of various structures, or in one or more post-developmental abilities, including sexual development, pituitary hormone production, and muscle development. Therefore, follistatin-associated conditions include abnormal tissue growth and developmental defects.

Exemplary conditions treated include neuromuscular disorders (e.g., muscular dystrophy and muscle atrophy), congestive obstructive pulmonary disease (and muscle wasting associated with COPD), muscle wasting syndrome, sarcopenia, and cachexia. Other exemplary conditions include muscle degeneration disorders and neuromuscular disorders, tissue repair (e.g., wound healing), and neurodegenerative diseases (e.g., amyotrophic lateral sclerosis).

In certain embodiments, the compositions of the present invention (e.g., FST-Fc polypeptides) are used as a component of a muscular dystrophy treatment. The term "muscular dystrophy" refers to a class of degenerative muscle diseases characterized by the gradual weakening and degeneration of skeletal muscle, and sometimes cardiac and respiratory muscles. Muscular dystrophy is a genetic disorder characterized by progressive muscle wasting and weakness that begins with microscopic muscle changes. As muscle degenerates over time, a person's muscle strength decreases. Exemplary muscular dystrophies that can be treated with regimens including the subject follistatin polypeptides include: Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), Emory-Dejerine muscular dystrophy (EDMD), limb-girdle muscular dystrophy (LGMD), facioscapulohumeral muscular dystrophy (FSH or FSHD) (also known as Landouzy-Dejerine), myotonic dystrophy (MMD) (also known as Steinert's Disease), oculopharyngeal muscular dystrophy (OPMD), distal muscular dystrophy (DD), and congenital muscular dystrophy (CMD).

French neurologist Guillaume Benjamin Amand Duchenne first described Duchenne muscular dystrophy (DMD) in the 1860s. Becker muscular dystrophy (BMD) is named after German physician Peter Emil Becker, who first described this variant of DMD in the 1950s. DMD is one of the most common genetic diseases in males, affecting one in 3,500 boys. DMD occurs when the dystrophin gene, located on the short arm of the X chromosome, is broken. Since males carry only one copy of the X chromosome, they only have one copy of the dystrophin gene. Without dystrophin, muscles are easily damaged during cycles of contraction and relaxation. While muscle regeneration compensates in the early stages of the disease, later in life, muscle progenitor cells are unable to keep up with the ongoing damage, and healthy muscle is replaced by nonfunctional fibrous fatty tissue.

BMD is caused by different mutations in the dystrophin gene. People with BMD have some dystrophin, but either not enough or it's of poor quality. Having some dystrophin protects BMD patients' muscles from degenerating as severely or as quickly as those of people with DMD.

For example, recent studies have shown that blocking or eliminating the function of GDF8 (a follistatin ligand) in vivo can effectively treat at least some symptoms in patients with DMD and BMD. Therefore, the subject follistatin antagonists can be used as GDF8 inhibitors (antagonists) and constitute an alternative method for blocking the function of GDF8 in vivo in patients with DMD and BMD.

Similarly, in other disease conditions where muscle growth is desired, the subject follistatin polypeptides provide an effective method for increasing muscle mass. For example, ALS, also known as Lou Gehrig's disease (motor neuron disease), is a chronic, incurable and unstoppable CNS disease that attacks motor neurons (the CNS component that connects the brain to skeletal muscle). In ALS, motor neurons deteriorate and eventually die, and although the human brain normally remains fully functional and alert, motor commands never reach the muscles. Most people who develop ALS are between the ages of 40 and 70. The earliest motor neurons to weaken are those that go to the arms or legs. ALS patients may have difficulty walking, they may drop things, fall, have slurred speech, and laugh or cry uncontrollably. Eventually, the muscles in the limbs atrophy due to disuse. This muscle weakness can make people debilitated, requiring a wheelchair or becoming unable to get out of bed. Most ALS patients die from respiratory failure or complications of ventilator-assisted ventilation, such as pneumonia, within 3-5 years of onset.

By local administration of the follistatin polypeptides described herein, Charcot-Marie-Tooth Disease (CMT) can be treated. CMT is a genetic disorder that affects peripheral nerves and causes progressive and often localized muscle weakness and degeneration. Aspects of the disease that can be treated include foot deformities (extremely high-arched feet); foot drop (inability to keep the foot parallel to the ground); "slapping" gait (the foot slaps the ground while walking due to foot drop); loss of calf muscles; numbness in the feet; difficulty balancing or weakness in the arms and hands.

The follistatin polypeptides disclosed herein can be used to treat muscles in patients with a variety of systemic muscle disorders, including Lambert-Eaton myasthenic syndrome (LEMS); metabolic dystrophies; spinal muscular atrophy (SMA); dermatomyositis (DM); distal muscular dystrophy (DD); Ehrlich-Dörgens-Göttingen muscular dystrophy (EDMD); endocrine myopathies; Friedreich's Ataxia (FA); hereditary myopathies; mitochondrial myopathies; myasthenia gravis (MG); and polymyositis (PM).

Muscles in patients suffering from post-surgical or disuse atrophy of one or more muscles, including after: hip fracture; total hip arthroplasty (THA); total knee arthroplasty (TKA); or rotator cuff surgery, can be treated with the follistatin polypeptides disclosed herein.

The follistatin polypeptides disclosed herein can be used to treat muscle in patients with a variety of other diseases that cause muscle loss or weakening, including muscle in patients with sarcopenia, cachexia, different types of cancer including lung cancer, colon cancer, and ovarian cancer, patients who rely on long-term ventilatory assistance, diabetes, chronic obstructive pulmonary disease, renal failure, heart failure, trauma and disorders of the peripheral nerves.

The increase in muscle mass induced by follistatin polypeptides may also benefit humans with muscle-wasting disorders. GDF8 expression is negatively correlated with fat-free mass in humans, and increased expression of the GDF8 gene is associated with weight loss in men with AIDS wasting syndrome. By inhibiting GDF8 function in AIDS patients, at least some of the symptoms of AIDS can be alleviated, if not completely eliminated, thereby significantly improving the quality of life of AIDS patients.

5. Pharmaceutical Compositions

In certain embodiments, the compounds of the present invention (e.g., follistatin polypeptides) are formulated with a pharmaceutically acceptable carrier. For example, the follistatin polypeptides can be administered alone or as a component of a pharmaceutical formulation (therapeutic composition). The subject compounds can be formulated for administration in any suitable manner for human or veterinary medicine.

In certain embodiments, the therapeutic methods of the present invention include local, systemic, or local administration of the composition with an implant or device. When administered, the therapeutic composition for the present invention is of course in a physiologically acceptable form without pyrogen. In addition, the composition can be suitably encapsulated or injected in a viscous form to be delivered to the target tissue site (e.g., bone, cartilage, muscle, fat, or neuron), such as a site with tissue damage. Topical administration can be suitable for wound healing and tissue repair. It is also possible to optionally include therapeutically useful substances other than the follistatin polypeptide in the above-mentioned composition, which can be alternatively or additionally administered simultaneously or sequentially with the subject compound (e.g., follistatin polypeptide) in the method of the present invention.

In certain embodiments, the compositions of the present invention may include a matrix that can deliver one or more therapeutic compounds (e.g., follistatin polypeptides) to a target tissue site, provide structure for developing tissue, and, preferably, be absorbed into the body. For example, the matrix can provide a slow release of the follistatin polypeptide. The matrix can be formed from materials currently used for other implantable medical applications.

The choice of matrix material depends on biocompatibility, biodegradability, mechanical properties, cosmetic appearance, and interfacial properties. The specific application of the subject composition will determine the appropriate formulation. Possible matrices for the composition can be biodegradable and chemically defined calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid, and polyanhydrides. Other possible materials are biodegradable and have well-defined biological compositions, such as bone or dermal collagen. Other matrices are composed of pure proteins or extracellular matrix components. Other possible matrices are non-biodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminates, or other ceramics. The matrix can be composed of a combination of any of the above types of materials, such as polylactic acid and hydroxyapatite or collagen and tricalcium phosphate. Bioceramics can vary in composition, such as calcium-aluminate-phosphate, and be processed to change pore size, particle size, particle shape, and biodegradability.

In certain embodiments, the methods of the present invention can be administered orally in the form of, for example, capsules, cachets, pills, tablets, lozenges (using a flavored base, typically sucrose and acacia or tragacanth), powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin or sucrose and acacia), and/or as a mouthwash, etc., each containing a predetermined amount of the active ingredient. The medicament can also be administered as a bolus, electuary, or paste.

In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules, etc.), one or more therapeutic compounds of the present invention may be mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or gum arabic; (3) Wetting agents such as glycerol; (4) disintegrants such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retardants such as paraffin; (6) absorption promoters such as quaternary ammonium compounds; (7) wetting agents such as cetyl alcohol and glyceryl monostearate; (8) absorbents such as kaolin and bentonite; (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets, and pills, the pharmaceutical composition may also contain a buffering agent. Solid compositions of a similar type may also be used as fillers in soft-filled and hard-filled gelatin capsules using excipients such as lactose and high molecular weight polyethylene glycols.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, the liquid dosage form may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers, and emulsifiers, such as ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (specifically cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan and mixtures thereof. In addition to the inert diluent, the oral composition may also include excipients, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, coloring agents, aromatics, and preservatives.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

Certain compositions disclosed herein can be administered topically to the skin or mucous membranes. Topical formulations may additionally include one or more of various agents known to be effective as skin or stratum corneum penetration enhancers. Examples of these are 2-pyrrolidone, N-methyl-2-pyrrolidone, dimethylacetamide, dimethylformamide, propylene glycol, methanol or isopropyl alcohol, dimethyl sulfoxide, and azone. Other agents may also be included to prepare cosmetically acceptable formulations. Examples of these are fats, waxes, oils, dyes, fragrances, preservatives, stabilizers, and surfactants. Keratolytic agents such as those known in the art are also included. Examples are salicylic acid and sulfur.

Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound can be mixed under aseptic conditions with a pharmaceutically acceptable carrier and any preservatives, buffers, or propellants that may be needed. In addition to the subject compound of the present invention (e.g., follistatin polypeptide), ointments, pastes, creams, and gels can contain excipients such as animal and vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicones, bentonite, silicic acid, talc, and zinc oxide, or mixtures thereof.

In addition to the subject compound, powders and sprays may contain excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder, or mixtures of these substances. Sprays may additionally contain customary propellants such as chlorofluorocarbons and volatile unsubstituted hydrocarbons, for example, butane and propane.

In certain embodiments, pharmaceutical compositions suitable for parenteral administration may comprise one or more follistatin polypeptides and one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions or sterile powders (which may be reconstituted into sterile injectable solutions or dispersions prior to use) that may contain antioxidants, buffers, bacteriostatins, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending agents or thickening agents. Examples of suitable aqueous and non-aqueous carriers that can be used in pharmaceutical compositions of the present invention include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate). Appropriate fluidity can be maintained, for example, by using coating materials (e.g., lecithin), by maintaining the desired particle size in the case of dispersants, and by using surfactants.

The compositions of the present invention may also contain adjuvants such as preservatives, wetting agents, emulsifiers, and dispersants. Various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like, may be added to ensure protection against the effects of microorganisms. Isotonic agents (e.g., sugars, sodium chloride, etc.) may also need to be included in the compositions. Additionally, absorption of the injectable form may be prolonged by the addition of substances that delay absorption (e.g., aluminum monostearate and gelatin).

It will be understood that the attending physician will determine the dosage regimen after considering various factors that modify the effects of the subject compounds of the invention (eg, follistatin polypeptides). Various factors will depend on the disease to be treated.

In certain embodiments, the present invention also provides gene therapy for the in vivo production of follistatin polypeptides or other compounds disclosed herein. Such therapies can achieve their therapeutic effects by introducing follistatin polynucleotide sequences into cells or tissues affected by the conditions listed above. Delivery of follistatin polynucleotide sequences can be achieved using recombinant expression vectors (e.g., chimeric viruses or colloidal dispersion systems). Preferred therapeutic delivery of follistatin polynucleotide sequences is using targeted liposomes.

Various viral vectors that can be used for the gene therapy taught herein include adenovirus, herpes virus, vaccinia virus or preferably RNA virus (such as retrovirus). Preferably, the retroviral vector is a derivative of a mouse or avian retrovirus. Examples of retroviral vectors in which a single exogenous gene can be inserted include, but are not limited to, Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV) and Rous sarcoma virus (Roussarcoma virus, RSV). Many other retroviral vectors can incorporate multiple genes. All of these vectors can transfer or integrate genes for selection markers so that transduced cells can be identified and produced. Retroviral vectors can be made target-specific by connecting, for example, sugars, glycolipids or proteins. Preferred targeting is accomplished by using antibodies. Those skilled in the art will recognize that specific polynucleotide sequences can be inserted into the retroviral genome or connected to the viral envelope for target-specific delivery of retroviral vectors containing follistatin polynucleotides. In a preferred embodiment, the vector targets bone, cartilage, muscle or neuronal cells/tissues.

Alternatively, tissue culture cells can be directly transfected with plasmids encoding the retroviral structural genes gag, pol, and env using conventional calcium phosphate transfection. These cells are then transfected with a vector plasmid containing the gene of interest. The resulting cells release the retroviral vector into the culture medium.

Another targeted delivery system for follistatin polynucleotides is a colloidal dispersion system. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. A preferred colloidal system of the present invention is a liposome. Liposomes are artificial membrane vesicles that can be used as delivery vehicles in vitro and in vivo. RNA, DNA, and intact virions can be encapsulated within the aqueous interior and delivered to cells in a biologically active form (see, e.g., Fraley et al., Trends Biochem. Sci., 6:77, 1981). Effective gene transfer methods using liposome vectors are known in the art, see, e.g., Mannino et al., Biotechniques, 6:682, 1988. Liposomes are generally composed of a combination of phospholipids, typically in combination with a steroid (particularly cholesterol). Other phospholipids or other lipids may also be used. The physical properties of liposomes depend on pH, ionic strength, and the presence of divalent cations.

The example of lipid that can be used to produce liposomes includes phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides and gangliosides. Illustrative phospholipids include egg yolk phosphatidylcholine (egg phosphatidylcholine), dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine. It is also feasible to target liposomes according to, for example, organ specificity, cell specificity and organelle specificity, and are known in the art.

Examples

Having now generally described the invention, the invention may be more readily understood by reference to the following examples, which are included merely for the purpose of illustrating certain embodiments and implementations of the invention and are not intended to limit the invention.

Example 1: Production of follistatin-Fc protein

Follistatin (FST) is known to have complex pharmacokinetic properties. The shortened form, FST (288), is reported to be more effective at blocking ligands and binds to the cell surface due in part to its unmasked heparin binding domain. FST (315) is considered to be less attracted to the cell surface and less effective due to its acidic C-terminal amino acid sequence (which neutralizes the heparin binding domain). In the literature, follistatin is generally reported to have systemic effects. The applicant sought to determine whether a follistatin construct could be designed that would tend to have an effect in the tissue to which it is administered (e.g., injected muscle), and whether dimerization of follistatin could provide increased tissue retention. The Fc domains of immunoglobulins are known to form dimers. To investigate the effects of follistatin-Fc fusion proteins on muscle and other tissues, and to evaluate the effects of Fc-mediated dimerization on the pharmacokinetic properties of follistatin polypeptides, the applicants produced fusion proteins containing either FST(288) or FST(315) fused to the Fc portion of IgG1. A TGGG linker sequence was selected to link each follistatin polypeptide to the Fc portion.

For each FST-IgG1 construct, the following three leader sequences were considered:

(1) Follistatin leader sequence: mvrarhqpgglcllllllcqfmedrsaqa (SEQ ID NO: 23)

(2) Tissue plasminogen activator (TPA): MDAMKRGLCCVLLLCGAVFVSP (SEQ ID NO: 24)

(3) Bee melittin (HBML): MKFLVNVALVFMVVYISYIYA (SEQ ID NO: 25)

The selected FST-Fc protein incorporates the follistatin leader sequence. The FST(288)-IgG1 fusion has the unprocessed and mature amino acid sequences shown below.

Unprocessed FST(288)-IgG1 (SEQ ID NO: 26)

MVRARHQPGGLCLLLLLLCQFMEDRSAQAGNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDG KTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRRGRCSLCDEL CPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCNTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Mature FST(288)-IgG1 (SEQ ID NO: 27)

GNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEV QYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRRGRCSLCDELCPDSKSDEPVCASD NATYASECAMKEAACSSGVLLEVKHSGSCNTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The initial "GN" sequence can be removed to obtain the following polypeptide. (SEQ ID NO: 28)

CWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQ YQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDN ATYASECAMKEAACSSGVLLEVKHSGSCNTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The FST(315)-IgG1 fusion has the unprocessed and mature amino acid sequences shown below.

Unprocessed FST(315)-IgG1 (SEQ ID NO: 29)

MVRARHQPGGLCLLLLLLCQFMEDRSAQAGNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNEC ALLKARCKEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCAS DNATYASECAMKEAACSSGVLLEVKHSGSCNSISEDTEEEEEDEDQDYSFPISSILEWTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Mature FST(315)-IgG1 (SEQ ID NO: 30)

GNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCK KTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAA CSSGVLLEVKHSGSCNSISEDTEEEEEDEDQDYSFPISSILEWTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The initial "GN" sequence can be removed to obtain the following polypeptide. (SEQ ID NO: 31)

CWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKK TCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAAC SSGVLLEVKHSGSCNSISEDTEEEEEDEDQDYSFPISSILEWTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Proteins were expressed in HEK-293 cells or CHO cells and purified from conditioned media by filtration and protein A chromatography. In some cases, anion exchange and hydrophobic interaction chromatography and/or gel filtration were also employed.

Protein activity was assessed by binding to activin A or GDF11. In each case, the protein bound with a _KD of less than 10 pM.

Example 2: Effects of systemic administration of follistatin-Fc protein on muscle mass and strength in mice

Applicants determined the ability of follistatin-Fc protein to increase muscle mass and strength in wild-type mice following systemic administration. ActRIIB-Fc fusion protein, well known to stimulate a substantial systemic increase in lean muscle mass, was used as a positive control.

C57BL/6 mice were given FST(288)-IgG1 protein, human FST(315)-IgG1 protein, or human ActRIIB-Fc protein (10 mg/kg; subcutaneous (s.c.)) twice/week for 4 weeks. Whole-body nuclear magnetic resonance (NMR) scans were performed on the mice to determine the percentage change in whole-body lean tissue mass. When compared to the vehicle control group, ActRIIB-Fc-treated mice showed a significant (approximately 35%) increase in lean tissue mass. Mice treated with FST(288)-IgG1 or FST(315)-IgG1 protein showed almost no increase in lean tissue mass compared to the control group. See Figure 2. At the end of the study, the pectoralis, tibialis anterior (TA), gastrocnemius, and femoris muscles were cut and weighed. As shown in Figure 4, ActRIIB-Fc treatment was shown to increase muscle mass in each of these muscle groups. In contrast, little or no increase in muscle mass was observed in the FST(288)-IgG1 or FST(315)-IgG1 treated groups. See Figure 2.

During the study, changes in muscle strength were also examined in the mice. Forelimb grip strength was determined by measuring the force exerted by the mice when pulling on a force transducer. Applicants observed that mice treated with ActRIIB-Fc protein showed improved muscle strength. In contrast, no improvement in grip strength was observed in the FST(288)-IgG1 or FST(315)-IgG1 treatment groups. See Figure 3.

In summary, the results demonstrate that systemic administration of ActRIIB-Fc significantly increased both muscle mass and strength in mice compared to vehicle control animals. In contrast, little or no increase in muscle mass or strength was observed in mice treated with either the follistatin-Fc fusion proteins FST(288)-IgG1 or FST(315)-IgG1. Thus, it appears that follistatin-Fc fusion proteins have little or no effect on muscle mass or strength in vivo when administered systemically.

Example 3: Effect of systemic administration of follistatin-Fc protein on FSH levels.

The main characteristic of follistatin is its ability to bind to and inhibit members of the TGF-β superfamily of signaling proteins. Specifically, follistatin is known to be a potent inhibitor of activin activity. Activin is a potent inducer of follicle-stimulating hormone (FSH) production. FSH is synthesized and secreted by gonadotrophs of the anterior pituitary gland and regulates growth and development during puberty and various reproductive processes. To evaluate the systemic effects of follistatin-Fc polypeptides, the effect on FSH levels was evaluated.

Treatment with FST(288)-IgG1 (10 mg/kg; subcutaneous (s.c.) twice/week) resulted in circulating drug levels of 3.836 (± 5.22) μg/mL. Similar treatment with FST(315)-IgG1 resulted in significantly higher serum drug levels of 19.31 (± 1.85) μg/mL. As shown in Figure 5, FST(288)-IgG1 did not have any significant effect on serum levels of FSH, indicating that this FST(288)-IgG1 treatment regimen did not significantly affect systemic activin activity. In contrast, FST(315)-IgG1 treatment resulted in a decrease in circulating FSH levels, indicating that systemic administration of FST(315)-IgG1 has an effect on systemic activin signaling. Collectively, these data suggest that the use of follistatin polypeptides with an unmasked heparin-binding domain fused to an Fc domain that mediates dimerization (e.g., FST(288)-IgG1) produces a protein with little or no systemic activity, whereas FST(315)-IgG1 with a masked heparin-binding domain can be used to achieve systemic effects.

Example 4: Effects of Local Administration of Follistatin-Fc Protein on Muscle Mass and Strength in Mice

Although there were no significant effects following systemic administration, Applicants employed a similar experimental approach to determine whether follistatin could be used to locally increase muscle mass and strength in wild-type mice following intramuscular (i.m.) administration.

C57BL/6 mice were given FST(288)-Fc protein, FST(315)-Fc protein or human ActRIIB-Fc protein (50 micrograms; i.m. into the right gastrocnemius muscle) twice/week for 4 weeks. At different time points after initial treatment, mice were scanned with whole-body nuclear magnetic resonance (NMR) to determine the percentage change in whole-body lean tissue mass. When compared with the vehicle control group, ActRIIB-Fc-treated mice showed a significant increase in lean tissue mass. In contrast, mice treated with FST(288)-Fc or FST(315)-Fc protein did not show a significant increase in lean tissue mass compared with the control group. At the end of the study, the injected right gastrocnemius muscle and the contralateral left gastrocnemius muscle were excised and weighed. As shown in Figure 6, ActRIIB-Fc treatment showed a significant increase in muscle mass in both the right and left gastrocnemius muscles compared with the vehicle-treated mice. Thus, even when administered locally to a single muscle, ActRIIB-Fc exerts a systemic effect on increasing muscle mass. In contrast, both FST(288)-Fc and FST(315)-Fc resulted in an increase in muscle mass in the right gastrocnemius muscle, but had no effect on the mass of the contralateral muscle. Thus, in contrast to the effects observed following systemic administration, it appears that follistatin protein is an effective stimulator of muscle mass when administered directly to muscle. Furthermore, follistatin appears to have a distinct advantage over other agents such as ActRIIB-Fc in that its effect on muscle mass is limited to the site of administration, suggesting that follistatin can be used for targeted therapy of select muscles or muscle groups without affecting the normal growth/activity of surrounding non-target muscles.

Applicants also closely monitored serum levels of follistatin-Fc fusion protein after i.m. administration. Treatment with FST(288)-IgG1 resulted in circulating drug levels of 0.156 (± 0.245) μg/mL. Similar treatment with FST(315)-IgG1 resulted in slightly higher serum drug levels of 3.58 (± 1.73) μg/mL, but these levels were significantly lower than those observed after systemic administration of FST(315)-IgG1. Since both FST(288)-IgG1 and FST(315)-IgG1 circulate in patient serum after i.m. injection at lower levels than observed after systemic administration of FST(288)-IgG1 (i.e., 3.836 (± 5.22) μg/mL), it is expected that neither FST(288)-IgG1 or FST(315)-IgG1 will have a significant effect on serum levels of FSH, just as FST(288)-IgG1 has no such effect after s.c. administration. See Figure 5. Therefore, these data suggest that both FST(288)-IgG1 and FST(315)-IgG1 would be particularly suitable for promoting targeted muscle growth in patients who are reproductively active or who wish to minimize effects on the reproductive system.

Similar experiments were performed to establish a dose-response curve for the effect of FST(288)-IgG1 on muscle mass and quality. Different doses (1-100 μg) were administered i.m. to the right gastrocnemius muscle of C57BL/6 mice twice a week for 4 weeks. As shown in Figure 8, with the larger dose of FST(288)-IgG1, the injected muscle showed a greater selective increase in muscle mass relative to the contralateral muscle. The cross-section of the muscle showed that the increase in muscle mass was the result of myofiber hypertrophy rather than hypoplasia.

Example 5: Fc Optimization of Locally Acting Follistatin-Fc Fusion Protein

As described in the previous examples, follistatin-Fc fusion proteins such as FST(288)-IgG1 and FST(315)-IgG1 have negligible systemic effects in muscle and other tissues, and in particular, the FST(288) form of the protein is active at the site of injection. Applicants and others have demonstrated that FST(288) binds to cells via the heparin binding domain, and that this binding can be abolished by exogenous heparin. Thus, Applicants have determined that immunoglobulin domains known to mediate CDC and ADCC effects on target cells can cause damage to cells treated with heparin-bound follistatin constructs. Such damage can manifest as an immune response in the target tissue or in a reduction in the growth of the target tissue. Applicants therefore generated different forms of follistatin polypeptides using the Fc portion of human IgG2, an example of an IgG constant domain known to abolish the ability to stimulate CDC and ADCC activity. This experiment was conducted to determine whether follistatin-Fc fusion proteins using alternative Fc domains could retain activity.

Applicants prepared fusion proteins containing FST(288) or FST(315) fused to the Fc portion of IgG2. A TGGG linker sequence was selected to link each follistatin polypeptide to the Fc portion.

For each FST-IgG2 construct, the follistatin leader sequence was used.

The FST(288)-IgG2 fusion has the unprocessed and mature amino acid sequences shown below.

Unprocessed FST(288)-IgG2 (SEQ ID NO: 32)

MVRARHQPGGLCLLLLLLCQFMEDRSAQAGNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDG KTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDE LCPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCNTGGGVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQ DWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

It is encoded by the following nucleic acid sequence (SEQ ID NO: 44)

atggtccgcgcgaggcaccagccgggtgggctttgcctcctgctgctgctgctctgccagttcatggaggaccgcagtgcccaggctgggaactgctggctc cgtcaagcgaagaacggccgctgccaggtcctgtacaagaccgaactgagcaaggaggagtgctgcagcaccggccggctgagcacctcgtggaccgaggagg acgtgaatgacaacacactcttcaagtggatgattttcaacgggggcgcccccaactgcatcccctgtaaagaaacgtgtgagaacgtggactgtggacctg ggaaaaaatgccgaatgaacaagaagaacaaaccccgctgcgtctgcgccccggattgttccaacatcacctggaagggtccagtctgcgggctggatgggaa aacctaccgcaatgaatgtgcactcctaaaggcaagatgtaaagagcagccagaactggaagtccagtaccaaggcagatgtaaaaagacttgtcgggatgt tttctgtccaggcagctccacatgtgtggtggaccagaccaataatgcctactgtgtgacctgtaatcggatttgcccagagcctgcttcctctgagcaatat ctctgtgggaatgatggagtcacctactccagtgcctgccacctgagaaaggctacctgcctgctgggcagatctattggattagcctatgagggaaagtgt atcaaagcaaagtcctgtgaagatatccagtgcactggtgggaaaaaatgtttatgggatttcaaggttgggagaggccggtgttccctctgtgatgagctgt gccctgacagtaagtcggatgagcctgtctgtgccagtgacaatgccacttatgccagcgagtgtgccatgaaggaagctgcctgctcctcaggtgtgctac tggaagtaaagcactccggatcttgcaacaccggtggtggagtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttcccccc aaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtacgt ggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttccgtgtggtcagcgtcctcaccgtcgtgcaccaggactgg ctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacag gtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtggg agagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcagggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgagaattc

Mature FST(288)-IgG2 (SEQ ID NO: 33)

GNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELE VQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCAS DNATYASECAMKEAACSSGVLLEVKHSGSCNTGGGVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE YKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The initial "GN" sequence can be removed to obtain the following polypeptide. (SEQ ID NO: 34)

CWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQ YQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRRGRCSLCDELCPDSKSDEPVCASD NATYASECAMKEAACSSGVLLEVKHSGSCNTGGGVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE YKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The FST(315)-IgG2 fusion has the unprocessed and mature amino acid sequences shown below.

Unprocessed FST(315)-IgG2 (SEQ ID NO: 35)

MVRARHQPGGLCLLLLLLCQFMEDRSAQAGNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNE CALLKARCKEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRRGRCSLCDELCPDSKSDEPVCA SDNATYASECAMKEAACSSGVLLEVKHSGSCNSISEDTEEEEEDEDQDYSFPISSILEWTGGGVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVS VLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

It is encoded by the following nucleic acid sequence (SEQ ID NO: 45)

atggtccgcgcgaggcaccagccgggtgggctttgcctcctgctgctgctgctctgccagttcatggaggaccgcagtgcccaggctgggaactgctggctccgtca agcgaagaacggccgctgccaggtcctgtacaagaccgaactgagcaaggaggagtgctgcagcaccggccggctgagcacctcgtggaccgaggaggacgtgaatga caacacactcttcaagtggatgattttcaacgggggtgcccccaactgcatcccctgtaaagaaacgtgtgagaacgtggactgtggacctgggaaaaaatgccgaa tgaacaagaagaacaaaccccgctgcgtctgcgccccggattgttccaacatcacctggaagggtccagtctgcgggctggatgggaaaacctaccgcaatgaatgtg cactcctaaaggcaagatgtaaagagcagccagaactggaagtccagtaccaaggcagatgtaaaaagacttgtcgggatgttttctgtccaggcagctccacatgt gtggtggaccagaccaataatgcctactgtgtgacctgtaatcggatttgcccagagcctgcttcctctgagcaatatctctgtgggaatgatggagtcacctactcc agtgcctgccacctgagaaaggctacctgcctgctgggcagatctattggattagcctatgagggaaagtgtatcaaagcaaagtcctgtgaagatatccagtgcact ggtgggaaaaaatgtttatgggatttcaaggttgggagaggccggtgttccctctgtgatgagctgtgccctgacagtaagtcggatgagcctgtctgtgccagtgac aatgccacttatgccagcgagtgtgccatgaaggaagctgcctgctcctcaggtgtgctactggaagtaaagcactccggatcttgcaactccatttcggaagacac cgaggaagaggaggaagatgaagaccaggactacagctttcctatatcttctattctagagtggaccggtggtggagtcgagtgcccaccgtgcccagcaccacctgt ggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagacc ccgaggtccagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttccgtgtggtcagcgtcctca ccgtcgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcag ccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgcc gtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaag agcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgagaattc

Mature FST(315)-IgG2 (SEQ ID NO: 36)

GNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRC KKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEA ACSSGVLLEVKHSGSCNSISEDTEEEEEDEDQDYSFPISSILEWTGGGVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQ DWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The initial "GN" sequence can be removed to obtain the following polypeptide. (SEQ ID NO: 37)

CWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKK TCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAA CSSGVLLEVKHSGSCNSISEDTEEEEEDEDQDYSFPISSILEWTGGGVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD WLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Proteins were expressed in HEK-293 cells or CHO cells and purified from conditioned media by filtration and protein A chromatography. In some cases, anion exchange and hydrophobic interaction chromatography and/or gel filtration were also employed.

Protein activity was assessed by binding to either activin A or GDF11. In each case, the protein bound with a _KD of less than 10 pM. These data demonstrate that follistatin-IgG2 fusion proteins can be prepared and expressed and retain picomolar ligand binding activity.

Example 6: Optimized locally acting follistatin-Fc fusion protein

To evaluate whether an optimal follistatin-Fc fusion protein could be prepared, various truncations between the C-termini of FST(288) and FST(315) were generated. One of these truncations, ending at amino acid 291 and designated FST(291), exhibited superior expression properties compared to the other forms and retained the desired heparin binding activity despite containing a small portion of the masking domain of FST(315). This form was fused to the Fc portion of human IgG1 and IgG2 to yield FST(291)-IgG1 and FST(291)-IgG2.

A TGGG linker sequence was chosen to link each follistatin polypeptide to the Fc portion.

For each FST-IgG1 construct, the follistatin leader sequence was used.

The FST(291)-IgG1 fusion has the unprocessed and mature amino acid sequences shown below.

Unprocessed FST(291)-IgG1 (SEQ ID NO: 38)

MVRARHQPGGLCLLLLLLCQFMEDRSAQAGNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGK TYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELC PDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCNSISTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Mature FST(291)-IgG1 (SEQ ID NO: 39)

GNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQ YQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNA TYASECAMKEAACSSGVLLEVKHSGSCNSISTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The initial "GN" sequence can be removed to obtain the following polypeptide. (SEQ ID NO: 40)

CWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQY QGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNAT YASECAMKEAACSSGVLLEVKHSGSCNSISTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The FST(291)-IgG2 fusion has the unprocessed and mature amino acid sequences shown below.

Unprocessed FST(291)-IgG2 (SEQ ID NO: 41)

MVRARHQPGGLCLLLLLLCQFMEDRSAQAGNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDG KTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRRGRCSLCDEL CPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCNSISTGGGVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVH QDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Mature FST(291)-IgG2 (SEQ ID NO: 42)

GNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEV QYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDN ATYASECAMKEAACSSGVLLEVKHSGSCNSISTGGGVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGK EYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The initial "GN" sequence can be removed to obtain the following polypeptide. (SEQ ID NO: 43)

CWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQY QGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNA TYASECAMKEAACSSGVLLEVKHSGSCNSISTGGGVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE YKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Proteins were expressed in HEK-293 cells or CHO cells and purified from conditioned media by filtration and protein A chromatography. In some cases, anion exchange and hydrophobic interaction chromatography and/or gel filtration were also employed.

Protein activity was assessed by binding to activin A or GDF11. In each case, the protein bound with a _KD of less than 10 pM.

Additional truncation experiments were performed to identify follistatin-IgG2 constructs that, in the presence of a TGGG linker, exhibited optimal ligand and heparin binding activity, resulting in a polypeptide with high potency, a strong tendency to favor retention in the treated tissue, and a low tendency to produce an inflammatory or immune response in the treated tissue. To this end, a series of constructs, designated FST(278)-IgG2, FST(284)-IgG2, FST(291)-IgG2, and FST(303)-IgG2, were prepared and compared to each other and to FST(288)-IgG2 and FST-(315)-IgG2. Heparin binding was evaluated by measuring protein recovered from cells in the presence or absence of heparin, quantified by ELISA, and expressed as the ratio of protein recovered in the presence of heparin to protein recovered in the absence of heparin. As shown in the table below, FST(278)-IgG2, FST(284)-IgG2, FST(288)-IgG2, and FST(291)-IgG2 all showed similar ratios of 3.00-4.00, while FST(303)-IgG2 and FST(315)-IgG2 showed ratios of 1.50 and 0.97, respectively. This suggests that the heparin binding activity is drastically reduced due to the inclusion of more amino acids between positions 291 and 303.

A cell-based reporter gene assay (A-204 reporter gene assay, described in WO/2006/012627) was performed to assess inhibition of activin and GDF1 1. As shown in the table below, constructs extending beyond position 288 provided improved ligand inhibition.

Taking the heparin binding and ligand inhibition data together, it is apparent that with the TGGG linker used herein or a similarly sized linker (e.g., a linker size of 1-10 amino acids, optionally 3-8 amino acids), the FST-IgG2 construct ending at positions 291-302 has enhanced ligand inhibition relative to FST(288)-IgG2 and increased heparin binding relative to FST(315)-IgG2, and that FST(291)-IgG2 represents the optimal protein for local administration and action.

Example 7: Effects of local administration of FST(291)-IgG2 protein on muscle mass and strength in mice

Applicants evaluated the activity of the optimized FST(291)-IgG2 protein to locally increase muscle mass and strength following intramuscular (i.m.) administration in wild-type mice.

C57BL/6 mice were administered vehicle (PBS), FST(291)-IgG2, or control Fc from IgG1 (100 μg in 50 μl; i.m. into the left gastrocnemius muscle) twice/week for 4 weeks. At the end of the study, both the injected left gastrocnemius muscle and the contralateral right gastrocnemius muscle were excised and weighed. As shown in Figure 9, FST(291)-IgG2 treatment showed an increase in muscle mass to a substantial extent in the injected left gastrocnemius muscle compared to vehicle-treated mice, with no effect observed on the contralateral muscle. In addition, the pectoralis and femoris muscles were weighed and showed no change in the results of administration of vehicle or FST(291)-IgG2. Therefore, FST(291)-IgG2 had a limited effect on the injected muscle group and had little or no systemic effect. Similar experiments were performed, but different muscle groups were injected, including the triceps and tibialis anterior muscles. In each case, selective hypertrophy of the injected muscle was observed.

Additional experiments were performed to directly compare the effects of FST(288)-IgG1 and FST(291)-IgG2 on muscle growth. Although both constructs promoted a significant increase in muscle mass in the injected muscle (gastrocnemius), FST(291)-IgG2 caused an approximately 42% increase in the injected muscle relative to the contralateral muscle, while FST(288)-IgG1 caused an approximately 22% increase in the injected muscle relative to the contralateral muscle.

Therefore, these data suggest that FST(291)-IgG2 is the optimal compound to promote targeted muscle growth in patients in need.

Example 8: Effects of local administration of FST(291)-IgG2 protein on muscle in a Duchenne muscular dystrophy mouse model

The effect of FST(291)-IgG2 on muscle mass was evaluated in a mouse model of Duchenne muscular dystrophy. The C57BL/10ScCN-Dmd ^mdx /J ( mdx ) mouse strain is a well-established model of human Duchenne muscular dystrophy (Bulfield, Siller et al., 1984; Partridge 2013).

Two independent studies were conducted using mdx mice and the wild-type background strain C57BL/10SnJ (WT). In the first study, treatment (FST(291)-IgG2 or vehicle control) was initiated when mice reached 6 weeks of age. In the second study, treatment was initiated when mice reached 4 weeks of age. In both studies, mice received 100 μg FST(291)-IgG2 intramuscularly in the left gastrocnemius muscle twice weekly in a fixed volume of 50 μL per injection. Treatment was continued for 4 weeks in 4-week-old mice and for 6 weeks in 6-week-old mice.

At necropsy, the gastrocnemius muscles from the injected (left) and contralateral uninjected (right) legs were excised and weighed. In both studies, the size of the injected gastrocnemius muscles from WT animals treated with FST(291)-IgG2 was significantly larger compared to the contralateral leg and vehicle controls (P<0.001). In both studies, the size of the gastrocnemius muscles normalized to body weight was significantly larger in both WT and mdx mice compared to the contralateral muscles and to vehicle-treated animals. The increase in muscle mass was slightly more significant in younger animals than in older animals. In terms of percentage increase relative to the contralateral muscle, FST(291)-IgG2 increased muscle mass by 34.2% and 16.4% in 6-week-old WT and mdx mice, respectively. An increase in muscle mass of 62.8% and 41.8% was observed in 4-week-old WT and mdx mice, respectively.

These data indicate that blocking activin/myostatin signaling using twice-weekly intramuscular administration of FST(291)-IgG2 increases muscle mass in a mouse model of muscular dystrophy. The increase in muscle mass occurs only locally in the injected muscle.

Incorporated by reference

All publications and patents mentioned herein are incorporated by reference in their entirety to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

While specific embodiments of the subject matter have been discussed, the foregoing description is illustrative and not restrictive. Numerous variations will become apparent to those skilled in the art upon reviewing this specification and the appended claims. The full scope of the invention should be determined by reference to the claims and their full scope of equivalents and the specification, along with such variations.

Claims (7)

1. A polypeptide consisting of the amino acid sequence of SEQ ID NO: 42. 2. A dimer comprising two polypeptides of claim 1. 3. A pharmaceutical formulation comprising the dimer of claim 2. 4. A nucleic acid comprising a nucleic acid sequence encoding the polypeptide of claim 1. 5. A cell comprising the nucleic acid of claim 4. 6. A composition for increasing muscle size or strength in a patient, wherein the composition comprises the polypeptide of claim 1 or the dimer of claim 2. 7. The composition of claim 6, wherein the patient has Duchenne muscular dystrophy.