TGF-ß RECEPTOR FUSION PROTEINS AND OTHER TGF-ß ANTAGONISTS FOR REDUCING TGF-ß SIGNALING
20190248881 · 2019-08-15
Inventors
Cpc classification
C07K2319/33
CHEMISTRY; METALLURGY
A61P19/08
HUMAN NECESSITIES
C07K2319/30
CHEMISTRY; METALLURGY
C07K2319/70
CHEMISTRY; METALLURGY
C07K2317/24
CHEMISTRY; METALLURGY
A61K47/65
HUMAN NECESSITIES
C07K2317/76
CHEMISTRY; METALLURGY
C07K16/22
CHEMISTRY; METALLURGY
International classification
C07K16/22
CHEMISTRY; METALLURGY
A61P19/08
HUMAN NECESSITIES
A61K47/65
HUMAN NECESSITIES
Abstract
The present invention provides TGF- antagonists and conjugates thereof, as well as methods of using such compositions for attenuating TGF- signaling. These novel compositions and methods may be useful for treating individuals suffering from devastating diseases associated with elevated TGF- signaling, including skeletal disorders, such as osteogenesis imperfecta (OI), and muscular diseases, such as muscular dystrophies.
Claims
1. A method of treating a human patient suffering from a bone disease associated with elevated TGF- signaling, said method comprising administering to said patient a therapeutically effective amount of a composition comprising a fusion protein comprising a TGF- receptor antagonist and a bone-targeting moiety.
2. The method of claim 1, wherein said disease is a disease associated with elevated bone turnover.
3. The method of claim 1, wherein said disease is selected from the group consisting of osteogenesis imperfecta, McCune-Albright syndrome, Gaucher disease, hyperoxaluria, Paget disease of bone, and juvenile Paget disease.
4. The method of claim 3, wherein said disease is osteogenesis imperfecta.
5. (canceled)
6. The method of claim 1, wherein said fusion protein has the amino acid sequence of SEQ ID NO: 28; or a variant of said amino acid sequence.
7. The method of claim 1, wherein said fusion protein has the amino acid sequence of SEQ ID NO: 30; or a variant of said amino acid sequence.
8. A composition comprising a homodimer of a compound of the formula:
(A-L.sup.1-B-L.sup.2-Z), I(a).
(Z-L.sup.2-B-L.sup.1-A), or I(b).
(B-L.sup.1-A-L.sup.2-Z), I(c). wherein each A is independently an RER heterotrimeric fusion polypeptide; wherein each L.sup.1 is independently a linker; wherein each B is independently an Fc domain of an immunoglobulin or is absent; wherein each L.sup.2 is independently a linker or is absent; wherein each Z is independently a bone-targeting moiety or is absent; wherein each A, the RER heterotrimeric fusion polypeptide, independently comprises a polypeptide of the formula: W-L.sup.3-X-L.sup.4-Y, wherein W is a TGF- type II receptor ectodomain or a portion thereof; L.sup.3 is a linker or is absent; X is a TGF- type III receptor endoglin domain or a portion thereof; L.sup.4 is a linker or is absent; Y is a TGF- type II receptor ectodomain or a portion thereof, and wherein at least one of B and Z is present.
9. (canceled)
10. The composition of claim 8, wherein B is present.
11-17. (canceled)
18. The composition of claim 8, wherein Z is present.
19. (canceled)
20. The composition of claim 18, wherein each Z independently comprises a polyanionic peptide, a bisphosphonate, or a peptide having the amino acid sequence of SEQ ID NO: 46 or a variant of said amino acid sequence.
21. The composition of claim 8, wherein the TGF- type II receptor ectodomain, W, is at the N-terminus of the RER heterotrimeric fusion polypeptide and the TGF- type II receptor ectodomain, Y, is at the C-terminus of the RER heterotrimeric fusion polypeptide.
22. The composition of claim 21, wherein: a) the C-terminus of the TGF- type II receptor ectodomain, Y, is covalently joined to the N-terminus of B, Fc domain of an immunoglobulin, via the linker L.sup.1 as in formula I(a); or b) the N-terminus of the TGF- type II receptor ectodomain, W, is covalently joined to the C-terminus of B via the linker L.sup.1 as in formula I(b) or I(c).
23. (canceled)
24. The composition of claim 22, wherein the amino acid sequence of the TGF- type II receptor ectodomain, W, is different than the amino acid sequence of the TGF- type II receptor ectodomain, Y.
25-37. (canceled)
38. The composition of claim 8, comprising the homodimer of a compound of the formula I(a). (A-L.sup.1-B-L.sup.2-Z), wherein A is an RER heterotrimeric fusion polypeptide; wherein L.sup.1 is a linker; wherein B is an Fc domain of an immunoglobulin; wherein L.sup.2 is a linker that is absent; wherein Z is a bone-targeting moiety; wherein A, the RER heterotrimeric fusion polypeptide, comprises a polypeptide of the formula: W-L.sup.3-X-L.sup.4-Y, wherein W is a TGF- type II receptor ectodomain or a portion thereof; L.sup.3 is a linker; X is a TGF- type III receptor endoglin domain or a portion thereof; L.sup.4 is a linker that is absent; and Y is a TGF- type II receptor ectodomain or a portion thereof.
39-68. (canceled)
69. A method of treating a human patient suffering from a disease associated with elevated TGF- signaling, said method comprising administering to said patient a therapeutically effective amount of the composition of claim 8.
70-74. (canceled)
75. The method of claim 69, wherein said disease is osteogenesis imperfecta.
76-110. (canceled)
111. The method of claim 69, wherein said homodimer comprises the amino acid sequence of: a) SEQ ID NO: 29, or a variant of said amino acid sequence; b) SEQ ID NO: 28, or a variant of said amino acid sequence; c) SEQ ID NO: 31, or a variant of said amino acid sequence; or d) SEQ ID NO: 30, or a variant of said amino acid sequence.
112-114. (canceled)
115. A method of improving muscle function in a human patient suffering from a disease associated with elevated TGF- signaling, said method comprising administering to said patient a therapeutically effective amount of the composition of claim 8.
116-119. (canceled)
120. The method of claim 115, wherein said disease is osteogenesis imperfecta.
121-173. (canceled)
174. A method of treating a human patient suffering from a bone disease associated with elevated TGF- signaling, said method comprising administering to said patient a therapeutically effective amount of a TGF- antagonist comprising an antibody or antigen-binding fragment thereof that binds TGF-, wherein said antibody or antigen-binding fragment thereof is conjugated to a targeting moiety that binds a protein or mineral present in human bone tissue.
175-185. (canceled)
186. A method of improving muscle function in a human patient suffering from a disease associated with elevated TGF- signaling, said method comprising administering to said patient a therapeutically effective amount of a TGF- antagonist comprising an antibody or antigen-binding fragment thereof that binds TGF-, wherein said antibody or antigen-binding fragment thereof is conjugated to a targeting moiety that binds a protein or mineral present in human bone tissue.
187-238. (canceled)
239. The method of claim 174, wherein: said antibody or antigen-binding fragment thereof comprises the following complementarity determining regions (CDRs): a CDR-H1 having the amino acid sequence SNVIS (SEQ ID NO: 64); a CDR-H2 having the amino acid sequence GVIPIVDIANYAQRFKG (SEQ ID NO: 65); a CDR-H3 having the amino acid sequence TLGLVLDAMDY (SEQ ID NO: 66); a CDR-L1 having the amino acid sequence RASQSLGSSYLA (SEQ ID NO: 67); a CDR-L2 having the amino acid sequence GASSRAP (SEQ ID NO: 68); and a CDR-L3 having the amino acid sequence QQYADSPIT (SEQ ID NO: 69); or said antibody or antigen binding fragment thereof competitively inhibits the binding of TGF- to an antibody or antigen binding fragment thereof that comprises the following CDRs: a CDR-H1 having the amino acid sequence SNVIS (SEQ ID NO: 64); a CDR-H2 having the amino acid sequence GVIPIVDIANYAQRFKG (SEQ ID NO: 65); a CDR-H3 having the amino acid sequence TLGLVLDAMDY (SEQ ID NO: 66); a CDR-L1 having the amino acid sequence RASQSLGSSYLA (SEQ ID NO: 67); a CDR-L2 having the amino acid sequence GASSRAP (SEQ ID NO: 68); and a CDR-L3 having the amino acid sequence QQYADSPIT (SEQ ID NO: 69).
240-262. (canceled)
263. A kit comprising the pharmaceutical formulation comprising the composition of claim 8, wherein the kit further comprises a package insert instructing a user of said kit to treat a human patient suffering from a disease associated with elevated TGF- signaling and/or elevated turnover, fibrosis, or cancer by administering to said patient a therapeutically effective amount of said pharmaceutical formulation.
264. (canceled)
265. A cell comprising a nucleic acid encoding the composition of claim 8.
266. A method of manufacturing the composition of claim 8, said method comprising: culturing a cell comprising a nucleic acid encoding the composition in a suitable growth medium; and isolating the mature form of the polypeptide encoded by said nucleic acid.
267. The composition of claim 8, wherein: W has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 10; X has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 12; and Y has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 11.
268. The composition of claim 8, wherein the homodimer has the amino acid sequence of SEQ ID NO: 28 or 30.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
[0413] The invention features therapeutic conjugates, such as those that contain transforming growth factor- (TGF-) antagonists, including those bound to a bone-targeting moiety that localizes the antagonist to human bone tissue. Also included are TGF- antagonists that may be used in the absence of a bone-targeting moiety to treat other conditions where lowered TGF- biological activity is desired. TGF- antagonists that may be used in conjunction with the compositions and methods described herein include TGF- receptors, as well as domains and variants thereof. Additionally, TGF- antagonists useful in the context of the compositions and methods described herein include TGF- receptor fusion proteins, such as those that contain one or more TGF- receptor II domains, fragments, or variants thereof bound to one or more TGF- receptor III domains, fragments, or variants thereof. For instance, fusion proteins that may be used in conjunction with the compositions and methods described herein include those that contain one or more ectodomains of TGF- receptor II, such as human or rat TGF- receptor II, bound to one or more endoglin domains of TGF- receptor III, such as human or rat TGF- receptor III. In some embodiments, the TGF- antagonist is a TGF- receptor fusion protein that contains a TGF- receptor II ectodomain bound to a TGF- receptor III endoglin domain, such as a fusion protein in which two TGF- receptor II ectodomain molecules are each independently bound to a single TGF- receptor III endoglin domain molecule. As described herein, the component TGF- receptors or domains, fragments, or variants thereof of a TGF- receptor fusion protein may be bound to one another directly, for instance, by way of an amide bond between each component polypeptide, or indirectly by way of a linker. Similarly, the fusion proteins may be bound to a targeting moiety directly, for instance, by way of an amide bond, or indirectly by way of an Fc domain of an immunoglobulin.
[0414] In addition to a TGF- antagonist, conjugates useful in conjunction with the compositions and methods described herein may contain a targeting moiety bound to the TGF- antagonist, such as a polyanionic peptide capable of binding a mineral present in bone tissue, such as hydroxyapatite. In this way, the TGF- antagonist can be administered to a patient, such as a human patient suffering from a disease associated with elevated osseous TGF- signaling or heightened bone turnover, and may subsequently localize to bone tissue. The invention is based in part on the discovery that this site-selective localization of TGF- antagonists, such as TGF- receptor fusion proteins, to bone tissue promotes the attenuation of TGF- signaling specifically at the site of damaged bone, while preserving TGF- activity in healthy tissues. Administration of the conjugates described herein represents a useful therapeutic strategy for treating, for instance, disorders associated with heightened TGF--mediated osteoclast activity relative to osteoblast activity, such as osteogenesis imperfecta, which is characterized by elevated bone resorption due to the activity of osteoclasts induced by overactive TGF- signal transduction. Additionally, the conjugates described herein can be used to treat muscular dystrophies associated with elevated TGF- signaling. This beneficial activity is due, at least in part, to the ability of the conjugates to suppress TGF- activity selectively at the skeletal-muscular interface, thus restoring muscle function and preserving TGF- activity in healthy tissues.
[0415] The following sections describe, in further detail, various TGF- antagonists, targeting moieties, and linkers that can be used to prepare exemplary conjugates, as well as methods of producing such agents and methods of using the same for the treatment of disorders characterized by elevated TGF- signaling in osseous tissue.
TGF- Antagonists
TGF- Receptors and TGF- Receptor Fusion Proteins
[0416] TGF- antagonists that can be used in conjunction with the compositions and methods described herein include TGF- receptors, as well as domains, fragments, and variants thereof. TGF- receptors, such as TGF- receptors I, II, and III, are capable of binding TGF- isoforms with varying selectivity profiles. By binding TGF-, exogenous receptors administered to a patient, such as a human patient suffering from a skeletal or muscular disease described herein, can sequester TGF- and prevent it from engaging its endogenous TGF- receptor target. In this way, soluble TGF- receptors, and fusion proteins containing these molecules, can inhibit the activation of the TGF- signal transduction pathway. This inhibition of TGF- activity can have important therapeutic phenotypes, particularly at the site of osseous tissue in patients suffering from a disorder characterized by elevated TGF--mediated bone turnover, such as osteogenesis imperfecta, and at the skeletal-muscular interface in patients suffering from muscular dystrophies.
TGF- Isoforms and Endogenous Receptors
[0417] TGF- isoforms (1, 2, and 3) are homodimeric polypeptides of about 25 kDa. These isoforms are secreted in a latent form and only a small percentage of total secreted TGF- isoforms are activated under physiological conditions. TGF- binds to three different cell surface receptors called type I (RI, also referred to herein as TGF- receptor I) type II (RII, also referred to herein as TGF- receptor II), and type III (RIII, also referred to herein as TGF- receptor III).
[0418] RI and RII are serine/threonine kinase receptors. RIII has two TGF- binding sites in its extracellular domain, referred to as the endoglin and uromodulin domains of TGF- receptor III. TGF-1 and TGF-3 bind RII with an affinity that is 200-300 fold higher than TGF-2 (Baardsnes et al., Biochemistry, 48, 2146-55, 2009); accordingly, cells deficient in RIII are 200- to 300-fold less responsive to equivalent concentrations of TGF-2 compared to TGF-1 and TGF--3 (Chiefetz, et al (1990) J. Bio. Chem., 265, 20533-20538). However, in the presence of RIII, cells respond roughly equally to all three TGF- isoforms, consistent with reports that show that RIII can sequester and present the ligand to RII to augment TGF- activity when it is membrane-bound (Chen et al., J. Biol. Chem. 272, 12862-12867, 1997; Lopez-Casillas et al., Cell 73, 1435-1444, 1993; Wang et al., Cell 67, 797-805, 1991; Fukushima et al., J. Biol. Chem. 268, 22710-22715, 1993; Lopez-Casillas et al., J. Cell Biol. 124, 557-568, 1994). Binding of TGF- to RII recruits and activates RI through phosphorylation (Wrana et al., Nature 370, 341-347, 1994). The activated RI phosphorylates intracellular Smad2 and Smad3, which then interact with Smad4 to regulate gene expression in the nucleus (Piek et al., FASEB J. 13, 2105-2124, 1999; Massague and Chen, Genes & Development 14, 627-644, 2000). Through its regulation of gene expression, TGF- has been shown to influence many cellular functions, including bone turnover and osteoclast-mediated bone resorption.
TGF- Receptors as Inhibitors of TGF- Signaling
[0419] Due in part to their ability to bind TGF- and sequester this ligand from its endogenous receptor, exogenous TGF- receptors and domains, fragments, and variants thereof can be used to inhibit TGF- signaling, such as at the site of osseous tissue and at the skeletal-muscular interface. Exemplary TGF- receptor domains that are useful in conjunction with the compositions and methods described herein include TGF- receptor II and III domains, such as the TGF- receptor II ectodomain and TGF- receptor III endoglin domain. The TGF- receptor II ectodomain binds TGF- in a 1:1 stoichiometric ratio, while two molecules of TGF- are bound by a single molecule of the TGF- receptor III ectodomain. The amino acid sequences of various human and rat TGF- receptors are shown in Table 2, below.
TABLE-US-00002 TABLE 2 Amino acid sequences of various human and rat TGF-receptors SEQ ID TGF- NO. Receptor Amino Acid Sequence 1 Full-length MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVN human NDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQK TGF- SCMSNCSITSICEKPQEVCVAVWRKNDENITLE receptor II TVCHDPKLPYHDFILEDAASPKCIMKEKKKPGE TFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIF QVTGISLLPPLGVAISVIIIFYCYRVNRQQKLSST WETGKTRKLMEFSEHCAIILEDDRSDISSTCAN NINHNTELLPIELDTLVGKGRFAEVYKAKLKQNT SEQFETVAVKIFPYEEYASWKTEKDIFSDINLK HENILQFLTAEERKTELGKQYWLITAFHAKGNL QEYLTRHVISWEDLRKLGSSLARGIAHLHSDHT PCGRPKMPIVHRDLKSSNILVKNDLTCCLCDFG LSLRLDPTLSVDDLANSGQVGTARYMAPEVLE SRMNLENVESFKQTDVYSMALVLWEMTSRCN AVGEVKDYEPPFGSKVREHPCVESMKDNVLR DRGRPEIPSFWLNHQGIQMVCETLTECWDHD PEARLTAQCVAERFSELEHLDRLSGRSCSEEKI PEDGSLNTTK 2 Full-length MAVTSHHMIPVMVVLMSACLATAGPEPSTRCE rat LSPINASHPVQALMESFTVLSGCASRGTTGLPR TGF- EVHVLNLRSTDQGPGQRQREVTLHLNPIASVH receptor THHKPIVFLLNSPQPLVWHLKTERLAAGVPRLF III LVSEGSVVQFPSGNFSLTAETEERNFPQENEH LLRWAQKEYGAVTSFTELKIARNIYIKVGEDQV FPPTCNIGKNFLSLNYLAEYLQPKAAEGCVLPS QPHEKEVHIIELITPSSNPYSAFQVDIIVDIRPAQ EDPEVVKNLVLILKCKKSVNWVIKSFDVKGNLK VIAPNSIGFGKESERSMTMTKLVRDDIPSTQEN LMKWALDNGYRPVTSYTMAPVANRFHLRLEN NEEMRDEEVHTIPPELRILLDPDHPPALDNPLF PGEGSPNGGLPFPFPDIPRRGWKEGEDRIPRP KQPIVPSVQLLPDHREPEEVQGGVDIALSVKCD HEKMVVAVDKDSFQTNGYSGMELTLLDPSCKA KMNGTHFVLESPLNGCGTRHRRSTPDGVVYY NSIVVQAPSPGDSSGWPDGYEDLESGDNGFP GDGDEGETAPLSRAGVVVFNCSLRQLRNPSG FQGQLDGNATFNMELYNTDLFLVPSPGVFSVA ENEHVYVEVSVTKADQDLGFAIQTCFLSPYSNP DRMSDYTIIENICPKDDSVKFYSSKRVHFPIPHA EVDKKRFSFLFKSVFNTSLLFLHCELTLCSRKK GSLKLPRCVTPDDACTSLDATMIWTMMQNKKT FTKPLAVVLQVDYKENVPSTKDSSPIPPPPPQI FHGLDTLTVMGIAFAAFVIGALLTGALWYIYSHT GETARRQQVPTSPPASENSSAAHSIGSTQSTP CSSSSTA 3 Full-length MTSHYVIAIFALMSSCLATAGPEPGALCELSPV human SASHPVQALMESFTVLSGCASRGTTGLPQEVH TGF- VLNLRTAGQGPGQLQREVTLHLNPISSVHIHHK receptor SVVFLLNSPHPLVWHLKTERLATGVSRLFLVSE III GSVVQFSSANFSLTAETEERNFPHGNEHLLNW ARKEYGAVTSFTELKIARNIYIKVGEDQVFPPKC NIGKNFLSLNYLAEYLQPKAAEGCVMSSQPQN EEVHIIELITPNSNPYSAFQVDITIDIRPSQEDLE VVKNLILILKCKKSVNWVIKSFDVKGSLKIIAPNSI GFGKESERSMTMTKSIRDDIPSTQGNLVKWAL DNGYSPITSYTMAPVANRFHLRLENNEEMGDE EVHTIPPELRILLDPGALPALQNPPIRGGEGQN GGLPFPFPDISRRVWNEEGEDGLPRPKDPVIP SIQLFPGLREPEEVQGSVDIALSVKCDNEKMIV AVEKDSFQASGYSGMDVTLLDPTCKAKMNGT HFVLESPLNGCGTRPRWSALDGVVYYNSIVIQ VPALGDSSGWPDGYEDLESGDNGFPGDMDE GDASLFTRPEIVVFNCSLQQVRNPSSFQEQPH GNITFNMELYNTDLFLVPSQGVFSVPENGHVY VEVSVTKAEQELGFAIQTCFISPYSNPDRMSHY TIIENICPKDESVKFYSPKRVHFPIPQADMDKKR FSFVFKPVFNTSLLFLQCELTLCTKMEKHPQKL PKCVPPDEACTSLDASIIWAMMQNKKTFTKPLA VIHHEAESKEKGPSMKEPNPISPPIFHGLDTLTV MGIAFAAFVIGALLTGALWYIYSHTGETAGRQQ VPTSPPASENSSAAHSIGSTQSTPCSSSSTA
[0420] The ectodomain of human TGF- receptor II corresponds to residues 24-160 of SEQ ID NO: 1. Human TGF- receptor II ectodomains useful in conjunction with the compositions and methods described herein include those that contain, e.g., from residue 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75 of SEQ ID NO: 1 to residue 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, or 170 of SEQ ID NO: 1. For instance, human TGF- receptor II ectodomains that may be used in conjunction with the compositions and methods described herein include those that contain residues 24-160 of SEQ ID NO: 1, residues 42-159 of SEQ ID NO: 1, as well as those that contain residues 48-159 of SEQ ID NO: 1. Additional examples of TGF- receptor II ectodomains that may be used in conjunction with the compositions and methods described herein include those ectodomains from rat TGF- receptor II, among other mammals.
[0421] The endoglin domain of rat TGF- receptor III corresponds to residues 24-409 of SEQ ID NO: 2. Rat TGF- receptor III endoglin domains useful in conjunction with the compositions and methods described herein include those that contain, e.g., from residue 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 of SEQ ID NO: 2 to residue 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, or 409 of SEQ ID NO: 2. TGF- receptor III endoglin domains useful in conjunction with the compositions and methods described herein may also contain one or more, or all, of the mutations R58H, H116R, C278S, and N337A relative to SEQ ID NO: 2. For instance, rat TGF- receptor III endoglin domains that may be used in conjunction with the compositions and methods described herein include those that contain residues 24-409 of SEQ ID NO: 2, as well as those that contain residues 24-383 of SEQ ID NO: 2. Additional rat TGF- receptor III endoglin domains that may be used in conjunction with the compositions and methods described herein include those that have amino acid sequences that differ from residues 24-409 of SEQ ID NO: 2 by virtue of one or more, or all, of the mutations R58H, H116R, C278S, and N337A, as well as those that have amino acid sequences that differ from residues 24-383 of SEQ ID NO: 2 by virtue of one or more, or all, of the mutations R58H, H116R, C278S, and N337A.
[0422] The endoglin domain of human TGF- receptor III corresponds to residues 21-406 of SEQ ID NO: 3. Human TGF- receptor III endoglin domains useful in conjunction with the compositions and methods described herein include those that contain, e.g., from residue 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 of SEQ ID NO: 3 to residue 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, or 409 of SEQ ID NO: 3. TGF- receptor III endoglin domains useful in conjunction with the compositions and methods described herein may also contain one or more, or all, of the mutations R55H, H113R, C275S, and N334A relative to SEQ ID NO: 3. For instance, human TGF- receptor III endoglin domains that may be used in conjunction with the compositions and methods described herein include those that contain residues 21-406 of SEQ ID NO: 3, as well as those that contain residues 21-380 of SEQ ID NO: 3. Additional human TGF- receptor III endoglin domains that may be used in conjunction with the compositions and methods described herein include those that have amino acid sequences that differ from residues 21-406 of SEQ ID NO: 3 by virtue of one or more, or all, of the mutations R55H, H113R, C275S, and N334A, as well as those that have amino acid sequences that differ from residues 21-380 of SEQ ID NO: 3 by virtue of one or more, or all, of the mutations R55H, H113R, C275S, and N334A.
[0423] The amino acid sequences of various TGF- receptor II and III domains are shown in Table 3, below.
TABLE-US-00003 TABLE 3 Amino acid sequences of various domains of human and rat TGF-receptors SEQ ID NO. TGF-Receptor Domain Amino Acid Sequence 10 Human NGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSI TGF-receptor II CEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDF ectodomain ILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNI (Residues 42-159 of human IFSEEYNTSNPD TGF-receptor II) 11 Human PQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQE TGF-receptor II VCVAVWRKNDENITLETVCHDPKLPYHDFILEDAAS ectodomain PKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYN (Residues 48-159 of human TSNPD TGF-receptor II) 12 Rat GPEPSTRCELSPINASHPVQALMESFTVLSGCASH TGF-receptor III GTTGLPREVHVLNLRSTDQGPGQRQREVTLHLNPI endoglin domain containing ASVHTHHKPIVFLLNSPQPLVWRLKTERLAAGVPR R58H, H116R, C278S, and LFLVSEGSVVQFPSGNFSLTAETEERNFPQENEHL N337A mutations LRWAQKEYGAVTSFTELKIARNIYIKVGEDQVFPPT (Residues 24-383 of rat CNIGKNFLSLNYLAEYLQPKAAEGCVLPSQPHEKE TGF-receptor III containing VHIIELITPSSNPYSAFQVDIIVDIRPAQEDPEVVKNL the foregoing mutations) VLILKSKKSVNWVIKSFDVKGNLKVIAPNSIGFGKES ERSMTMTKLVRDDIPSTQENLMKWALDAGYRPVT SYTMAPVANRFHLRLENNEEMRDEEVHTIPPELRIL LDPD 13 Human GPEPGALCELSPVSASHPVQALMESFTVLSGCASR TGF-receptor III GTTGLPQEVHVLNLRTAGQGPGQLQREVTLHLNPI endoglin domain containing SSVHIHHKSVVFLLNSPHPLVWHLKTERLATGVSRL C275S mutation FLVSEGSVVQFSSANFSLTAETEERNFPHGNEHLL (Residues 21-380 of human NWARKEYGAVTSFTELKIARNIYIKVGEDQVFPPKC TGF-receptor III containing NIGKNFLSLNYLAEYLQPKAAEGCVMSSQPQNEEV the foregoing mutation) HIIELITPNSNPYSAFQVDITIDIRPSQEDLEVVKNLILI LKSKKSVNWVIKSFDVKGSLKIIAPNSIGFGKESERS MTMTKSIRDDIPSTQGNLVKWALDNGYSPITSYTM APVANRFHLRLENNEEMGDEEVHTIPPELRILLDPG
TGF- Receptor Fusion Proteins
[0424] TGF- receptor fusion proteins useful in conjunction with the compositions and methods described herein include those that contain one or more TGF- receptors, or a domain, fragment, or variant thereof, bound to another TGF- receptor, or a domain, fragment, or variant thereof. Exemplary TGF- receptor fusion proteins include those in which two TGF- receptor II ectodomains, such as two human TGF- receptor II ectodomains, are bound to a single TGF- receptor III endoglin domain, such as a rat or human TGF- receptor III endoglin domain. It has been discovered that the endoglin domain of TGF- receptor III binds two TGF- molecules, while the ectodomain of TGF- receptor II binds a single TGF- molecule. Additionally, it has been found that the binding of the TGF- receptor II ectodomain to TGF- occurs at a site that is sterically distal from the site bound by the TGF- receptor III endoglin domain. The binding of TGF- receptor II ectodomain to TGF- thus occurs independently from the binding of TGF- receptor III endoglin domain to TGF-. A multimeric fusion protein containing one or more TGF- receptor II ectodomains bound to one or more TGF- receptor III ectodomains has the capacity to bind TGF- with high affinity by virtue of engaging this ligand at multiple distinct and independent sites. For instance, a trimeric fusion protein containing a TGF- receptor II ectodomain bound to a TGF- receptor III ectodomain, which is in turn bound to another TGF- receptor II ectodomain has the capacity to bind two TGF- molecules per a single fusion protein. Due in part to the binding of the fusion protein to a total of four sites across the ensemble of bound TGF- molecules, the affinity of this interaction is high, as fusion proteins of this structure exhibit low-nanomolar to sub-nanomolar affinity for TGF-. Exemplary TGF- fusion proteins useful in conjunction with the compositions and methods of the invention are described, for instance, in U.S. Pat. No. 9,611,306, the disclosure of which is incorporated herein by reference in its entirety.
[0425] Exemplary TGF- receptor fusion proteins for use in conjunction with the compositions and methods described herein include those having the amino acid sequence of SEQ ID NO: 9, as well as those having at least 70% sequence identity thereto (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or more, sequence identity thereto). The amino acid sequence of SEQ ID NO: 9 is composed of an N-terminal human TGF- receptor II ectodomain (SEQ ID NO: 10) bound to a central rat TGF- receptor III endoglin domain (SEQ ID NO: 12), which is in turn bound to a C-terminal human TGF- receptor II ectodomain (SEQ ID NO: 11).
SEQ ID NO: 9, Exemplary TGF- Receptor Fusion Protein of the Structure:
RII Ectodomain-RIII Endoglin Domain-RII Ectodomain (RER)
[0426]
TABLE-US-00004 NGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKN DENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSD ECNDNIIFSEEYNTSNPDGPEPSTRCELSPINASHPVQALMESFTVLSGC ASHGTTGLPREVHVLNLRSTDQGPGQRQREVTLHLNPIASVHTHHKPIVF LLNSPQPLVWRLKTERLAAGVPRLFLVSEGSVVQFPSGNFSLTAETEERN FPQENEHLLRWAQKEYGAVTSFTELKIARNIYIKVGEDQVFPPTCNIGKN FLSLNYLAEYLQPKAAEGCVLPSQPHEKEVHIIELITPSSNPYSAFQVDI IVDIRPAQEDPEVVKNLVLILKSKKSVNWVIKSFDVKGNLKVIAPNSIGF GKESERSMTMTKLVRDDIPSTQENLMKWALDAGYRPVTSYTMAPVANRFH LRLENNEEMRDEEVHTIPPELRILLDPDPQLCKFCDVRFSTCDNQKSCMS NCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASP KCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
[0427] Additional exemplary TGF- antagonists or conjugates are described below. These TGF- antagonists or conjugates can be used appropriately or interchangeably with the TGF- antagonist constructs and conjugates discussed above or with any of the aspects or embodiments of the invention discussed herein.
[0428] In some instances, the invention features a composition containing a TGF- antagonist, wherein the TGF- antagonist is a fusion protein that comprises a homodimer of a compound of the formula: I(a). (A-L.sup.1-B-L.sup.2-Z), I(b). (Z-L.sup.2-B-L.sup.1-A), or I(c). (B-L.sup.1-A-L.sup.2-Z), where A is an RER heterotrimeric fusion polypeptide; L.sup.1 is a linker; B is an Fc domain of an immunoglobulin or is absent; L.sup.2 is a linker or is absent; Z is a bone-targeting moiety or is absent; and where A, the RER heterotrimeric fusion polypeptide, includes a polypeptide sequence of the formula: W-L.sup.3-X-L.sup.4-Y, where W is a TGF- type II receptor ectodomain or a portion thereof; L.sup.3 is a linker or is absent; X is a TGF- type III receptor endoglin domain or a portion thereof; L.sup.4 is a linker or is absent; Y is a TGF- type II receptor ectodomain or a portion thereof, and where the amino acid sequence of A is not the amino acid sequence of SEQ ID NO: 48.
[0429] Certain aspects of the above composition may vary in ways described below.
[0430] In some instances, the linker L.sup.1 includes a natural peptidic linker, a synthetic linker, or an amino acid sequence selected from the group comprising SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 402, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 61; or a variant of said amino acid sequences.
[0431] In some instances, B, the Fc domain of an immunoglobulin is present. In some instances, B, the Fc domain of an immunoglobulin is absent. In some instances, B, the Fc domain of an immunoglobulin includes the Fc domain of human IgG, human IgA, human IgM, human IgE, or human IgD; or a variant of said domain. In some instances, the Fc domain of human IgG is IgG1, IgG2, IgG3, or IgG4; or a variant thereof. In some instances, the Fc domain of human includes the amino acid sequence of SEQ ID NO: 47; or a variant of said amino acid sequence.
[0432] In some instances, the linker L.sup.2 is present. In some instances, the linker L.sup.2 is absent. In some instances, the linker L.sup.2 includes a natural peptidic linker, a synthetic linker, or an amino acid sequence selected from the group comprising SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 402, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 61; or a variant of said amino acid sequences.
[0433] In some instances, the bone-targeting moiety, is present. In some instances, the bone-targeting moiety, is absent. In some instances, the bone-targeting moiety includes a polyanionic peptide, a bisphosphonate, or the amino acid sequence of SEQ ID NO: 46; or a variant of said amino acid sequence.
[0434] In some instances, the TGF- type II receptor ectodomain W is at the N-terminus of the RER heterotrimeric fusion polypeptide and the TGF- type II receptor ectodomain Y is at the C-terminus of the RER heterotrimeric fusion polypeptide. In some instances, the C-terminus of the TGF- type II receptor ectodomain Y is covalently joined to the N-terminus of B, Fc domain of an immunoglobulin, via the linker L.sup.1 as in formula I(a). In some instances, the N-terminus of the TGF- type II receptor ectodomain W is covalently joined to the C-terminus of B via the linker L.sup.1 as in formula I(b) or I(c).
[0435] In some instances, the amino acid sequence of the TGF- type II receptor ectodomain W is identical to the amino acid sequence of the TGF- type II receptor ectodomain Y. In some instances, the amino acid sequence of the TGF- type II receptor ectodomain W is different than the amino acid sequence of the TGF- type II receptor ectodomain Y. In some instances, the TGF- type II receptor ectodomains W and/or Y includes an amino acid sequence extending from amino acid residues 22 to 139 of SEQ ID NO: 5, 520 to 631 of SEQ ID NO: 5, 1 to 118 of SEQ ID NO: 9, 479 to 590 of SEQ ID NO: 9, 1 to 118 of SEQ ID NO: 48, 499 to 610 of SEQ ID NO: 48, 1 to 118 of SEQ ID NO: 49, 499 to 610 of SEQ ID NO: 49, 1 to 120 of SEQ ID NO: 50, 501 to 612 of SEQ ID NO: 50, 1 to 120 of SEQ ID NO: 51, 501 to 612 of SEQ ID NO: 51, 1 to 120 of SEQ ID NO: 52, or 510 to 621 of SEQ ID NO: 52; or a variant of said amino acid sequences.
[0436] In some instances, the linker L.sup.3 is present. In some instances, the linker L.sup.3 is absent. In some instances, the linker L.sup.3 includes a natural peptidic linker, a synthetic linker, or an amino acid sequence selected from the group comprising SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 402, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 61; or a variant of said amino acid sequences.
[0437] In some instances, where the TGF- type III receptor endoglin domain X includes an amino acid sequence extending from amino acid residues 157 to 517 of SEQ ID NO: 5, 119 to 478 of SEQ ID NO: 9, 136 to 496 of SEQ ID NO: 48, 136 to 496 of SEQ ID NO: 49, 138 to 500 of SEQ ID NO: 50, 138 to 500 of SEQ ID NO: 51, or 147 to 509 of SEQ ID NO: 52; or a variant of said amino acid sequences. In some instances, the linker L.sup.4 is present. In some instances, where the linker L.sup.4 is absent. In some instances, the linker L.sup.4 includes a natural peptidic linker, a synthetic linker, or an amino acid sequence selected from the group comprising SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 402, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 61; or a variant of said amino acid sequences.
[0438] In some instances, the RER heterotrimeric fusion polypeptide includes an amino acid sequence selected from the group comprising SEQ ID NO: 9, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52; or a variant of said amino acid sequences. In some instances, the RER heterotrimeric fusion polypeptide includes the amino acid sequence of SEQ ID NO: 51; or a variant of said amino acid sequence. In some instances, the RER heterotrimeric fusion polypeptide includes the amino acid sequence of SEQ ID NO: 52; or a variant of said amino acid sequence.
[0439] In some instances, the homodimer includes an amino acid sequence selected from the group comprising SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, and SEQ ID NO: 30; or a variant of said amino acid sequences. In some instances, the homodimer includes an amino acid sequence selected from the group comprising SEQ ID NO: 9, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, and SEQ ID NO: 31; or a variant of said amino acid sequences.
[0440] In a second aspect, In a first aspect, the invention features a composition containing a TGF- antagonist, wherein the TGF- antagonist is a fusion protein that includes a homodimer of a compound of the formula: I(a). (A-L.sup.1-B-L.sup.2-Z); where A is an RER heterotrimeric fusion polypeptide; L.sup.1 is a linker; B is an Fc domain of an immunoglobulin; L.sup.2 is a linker that is absent; Z is a bone-targeting moiety; and A, the RER heterotrimeric fusion polypeptide, includes a polypeptide sequence of the formula: W-L.sup.3-X-L.sup.4-Y, where W is a TGF- type II receptor ectodomain or a portion thereof; L.sup.3 is a linker; X is a TGF- type III receptor endoglin domain or a portion thereof; L.sup.4 is a linker that is absent; and Y is a TGF- type II receptor ectodomain or a portion thereof; and the amino acid sequence of A is not the amino acid sequence of SEQ ID NO: 48.
[0441] In some instances, the homodimer is PCT-0025 having the amino acid sequence of SEQ ID NO: 28; or a variant of said amino acid sequence. In some instances, the homodimer is PCT-0026 having the amino acid sequence of SEQ ID NO: 30; or a variant of said amino acid sequence.
[0442] In another aspect, the invention features a composition containing a TGF- antagonist, wherein the TGF- antagonist is a fusion protein that includes a homodimer of a compound of the formula: II(a). (A-L.sup.1-B-L.sup.2-Z), II(b). (Z-L.sup.2-B-L.sup.1-A), or II(c). (B-L.sup.1-A-L.sup.2-Z), where A is an RER heterotrimeric fusion polypeptide; L.sup.1 is a linker; B is an Fc domain of an immunoglobulin or is absent; L.sup.2 is a linker or is absent; Z is a bone-targeting moiety; A, the RER heterotrimeric fusion polypeptide, includes a polypeptide sequence of the formula: W-L.sup.3-X-L.sup.4-Y, where W is a TGF- type II receptor ectodomain or a portion thereof; L.sup.3 is a linker or is absent; X is a TGF- type III receptor endoglin domain or a portion thereof; L.sup.4 is a linker or is absent; Y is a TGF- type II receptor ectodomain or a portion thereof, and where A includes the amino acid sequence of SEQ ID NO: 48.
[0443] Certain aspects of the above composition may vary in ways described below.
[0444] In some instances, the linker L.sup.1 includes a natural peptidic linker, a synthetic linker, or an amino acid sequence selected from the group comprising SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 402, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 61; or a variant of said amino acid sequences.
[0445] In some instances, the Fc domain of an immunoglobulin is present. In some instances, the Fc domain of an immunoglobulin is absent. In some instances, the Fc domain of an immunoglobulin includes the Fc domain of human IgG, human IgA, human IgM, human IgE, or human IgD; or a variant of said domain. In some instances, the Fc domain of human IgG is IgG1, IgG2, IgG3, or IgG4; or a variant thereof. In some instances, the Fc domain of human includes the amino acid sequence of SEQ ID NO: 47; or a variant of said amino acid sequence.
[0446] In some instances, the linker L.sup.2 is present. In some instances, the linker L.sup.2 is absent. In some instances, the linker L.sup.2 includes a natural peptidic linker, a synthetic linker, or an amino acid sequence selected from the group comprising SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 402, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 61; or a variant of said amino acid sequences.
[0447] In some instances, the bone-targeting moiety includes a polyanionic peptide, a bisphosphonate, or the amino acid sequence of SEQ ID NO: 46; or a variant of said amino acid sequence.
[0448] In some instances, the TGF- type II receptor ectodomain W is at the N-terminus of the RER heterotrimeric fusion polypeptide and the TGF- type II receptor ectodomain Y is at the C-terminus of the RER heterotrimeric fusion polypeptide. In some instances, the C-terminus of the TGF- type II receptor ectodomain Y is covalently joined to the N-terminus of B, Fc domain of an immunoglobulin, via the linker L.sup.1 as in formula I(a). In some instances, the N-terminus of the TGF- type II receptor ectodomain W is covalently joined to the C-terminus of B via the linker L.sup.1 as in formula I(b) or I(c).
[0449] In some instances, the amino acid sequence of the TGF- type II receptor ectodomain W is identical to the amino acid sequence of the TGF- type II receptor ectodomain Y. In some instances, the amino acid sequence of the TGF- type II receptor ectodomain W is different than the amino acid sequence of the TGF- type II receptor ectodomain Y. In some instances, the TGF- type II receptor ectodomains W and/or Y includes an amino acid sequence extending from amino acid residues 22 to 139 of SEQ ID NO: 5, 520 to 631 of SEQ ID NO: 5, 1 to 118 of SEQ ID NO: 9, 479 to 590 of SEQ ID NO: 9, 1 to 118 of SEQ ID NO: 48, 499 to 610 of SEQ ID NO: 48, 1 to 118 of SEQ ID NO: 49, 499 to 610 of SEQ ID NO: 49, 501 to 612 of SEQ ID NO: 50, 501 to 612 of SEQ ID NO: 51, or 510 to 621 of SEQ ID NO: 52; or a variant of said amino acid sequences. In some instances, the TGF- type II receptor ectodomains W and/or Y does not comprise an amino acid sequence extending from amino acid residues 22 to 139 of SEQ ID NO: 5, 520 to 631 of SEQ ID NO: 5, 1 to 118 of SEQ ID NO: 9, 479 to 590 of SEQ ID NO: 9, 1 to 118 of SEQ ID NO: 48, 499 to 610 of SEQ ID NO: 48, 1 to 118 of SEQ ID NO: 49, 499 to 610 of SEQ ID NO: 49, 501 to 612 of SEQ ID NO: 50, 501 to 612 of SEQ ID NO: 51, or 510 to 621 of SEQ ID NO: 52; or a variant of said amino acid sequences.
[0450] In some instances, the linker L.sup.3 is present. In some instances, the linker L.sup.3 is absent. In some instances, the linker L.sup.3 includes a natural peptidic linker, a synthetic linker, or an amino acid sequence selected from the group comprising SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 402, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 61; or a variant of said amino acid sequences.
[0451] In some instances, the TGF- type III receptor endoglin domain X includes an amino acid sequence extending from amino acid residues 157 to 517 of SEQ ID NO: 5, 136 to 496 of SEQ ID NO: 48, or 136 to 496 of SEQ ID NO: 49; or a variant of said amino acid sequences. In some instances, the TGF- type III receptor endoglin domain X does not comprise an amino acid sequence extending from amino acid residues 157 to 517 of SEQ ID NO: 5, 136 to 496 of SEQ ID NO: 48, or 136 to 496 of SEQ ID NO: 49; or a variant of said amino acid sequences.
[0452] In some instances, the linker L.sup.4 is present. In some instances, the linker L.sup.4 is absent. In some instances, the linker L.sup.4 includes a natural peptidic linker, a synthetic linker, or an amino acid sequence selected from the group comprising SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 402, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 61; or a variant of said amino acid sequences.
[0453] In some instances, the RER heterotrimeric fusion polypeptide includes the amino acid sequence of SEQ ID NO: 48; or a variant of said amino acid sequences.
[0454] In some instances, the homodimer includes an amino acid sequence selected from the group comprising SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 32, and SEQ ID NO: 34; or a variant of said amino acid sequences.
[0455] In another aspect, the invention features a composition containing a TGF- antagonist, wherein the TGF- antagonist is a fusion protein that includes a homodimer of a compound of the formula: III(a). (A-L.sup.1-B-L.sup.2-Z), III(b). (Z-L.sup.2-B-L.sup.1-A), or III(c). (B-L.sup.1-A-L.sup.2-Z), where A is an RER heterotrimeric fusion polypeptide; L.sup.1 is a linker; B is an Fc domain of an immunoglobulin or is absent; L.sup.2 is a linker or is absent; Z is a bone-targeting moiety or is absent; and where at least one of the following is present: [0456] a. A, the RER heterotrimeric fusion polypeptide, includes an amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52; or [0457] b. the linker L.sup.1 includes an amino acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38; or [0458] c. the linker L.sup.2 is present and includes an amino acid sequence of SEQ ID NO: 8, or SEQ ID NO: 41; or [0459] d. the linker L.sup.3 is present and includes the amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39: or [0460] e. X, the TGF- type III receptor endoglin domain, includes the amino acid sequence of SEQ ID NO: 44.
[0461] Specific TGF- receptor fusion protein constructs or antagonists of the invention with the D10 bone-targeting moiety are summarized in Table 4, below.
TABLE-US-00005 TABLE 4 TGF- antagonists with the D10 bone-targeting moiety TGF- Antagonist SEQ ID NO. PCT-0015 14 PCT-0019 16 PCT-0020 18 PCT-0021 20 PCT-0022 22 PCT-0023 24 PCT-0024 26 PCT-0025 28 PCT-0026 30 PCT-0017 32 PCT-0018 34
[0462] Specific TGF- receptor fusion protein constructs or antagonists of the invention without the D10 bone-targeting moiety (NT) are summarized in Table 5, below.
TABLE-US-00006 TABLE 5 TGF- antagonists without the D10 bone-targeting moiety TGF- Antagonist SEQ ID NO. PCT-0015NT 15 PCT-0019NT 17 PCT-0020NT 19 PCT-0021NT 21 PCT-0022NT 23 PCT-0023NT 25 PCT-0024NT 27 PCT-0025NT 29 PCT-0026NT 31 PCT-0016NT 33 PCT-0018NT 35
[0463] In some instances, the novel TGF- receptor fusion protein constructs or antagonists of the invention are those with the D10 bone-targeting moiety (SEQ ID NO: 46) and includes the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, or SEQ ID NO: 34, or a variant of said amino acid sequences. The TGF- receptor fusion protein constructs or antagonists with the D10 bone-targeting moiety can be used to treat a variety of disorders associated with elevated TGF- signaling in bone tissue.
[0464] In other instances, the novel TGF- receptor fusion protein constructs or antagonists of the invention are those without the D10 bone-targeting moiety and includes the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, or SEQ ID NO: 35, or a variant of said amino acid sequences. The TGF- receptor fusion protein constructs or antagonists without the D10 bone-targeting moiety can be used to treat a variety of disorders associated with elevated TGF- signaling in both bone tissue and tissues other than bone.
[0465] Other bone-targeting moieties, as described herein, may be used in lieu of the D10 bone-targeting moiety, as appropriate.
[0466] The TGF- antagonist constructs described above may be used appropriately or interchangeably with the compositions and methods of any of the aspects or embodiments of the invention described herein.
Antibodies and Antigen-Binding Fragments Thereof that Bind TGF-
[0467] Examples of TGF- antagonists useful in conjunction with the compositions and methods described herein include antibodies and antigen-binding fragments thereof directed against one or more isoforms of TGF- (such as those described in U.S. Pat. No. 5,571,714, as well as International Patent Application Publication No. WO 1997/013844, the disclosures of each of which are incorporated herein by reference), and antibodies directed against TGF- receptors (such as those described in U.S. Pat. Nos. 5,693,607, 6,008,011, 6,001,969, and 6,010,872, as well as WO 92/00330, WO 93/09228, WO 95/10610, and WO 98/48024, the disclosures of which are incorporated herein by reference).
[0468] Particular TGF- antagonists useful in conjunction with the compositions and methods described herein include anti-TGF- antibody 1D11, as well as antigen-binding fragments thereof and human, humanized, and chimeric variants thereof. Anti-TGF- antibody GC1008, a humanized variant of 1D11, is described in U.S. Pat. No. 9,958,486, the disclosure of which is incorporated herein by reference in its entirety. Anti-TGF- antibody GC1008 contains the following complementarity determining regions (CDRs): [0469] (a) a CDR-H1 having the amino acid sequence SNVIS (SEQ ID NO: 64); [0470] (b) a CDR-H2 having the amino acid sequence GVIPIVDIANYAQRFKG (SEQ ID NO: 65); [0471] (c) a CDR-H3 having the amino acid sequence TLGLVLDAMDY (SEQ ID NO: 66); [0472] (d) a CDR-L1 having the amino acid sequence RASQSLGSSYLA (SEQ ID NO: 67); [0473] (e) a CDR-L2 having the amino acid sequence GASSRAP (SEQ ID NO: 68); and [0474] (f) a CDR-L3 having the amino acid sequence QQYADSPIT (SEQ ID NO: 69).
[0475] Anti-TGF- antibody GC1008 contains a heavy chain variable region having the sequence of SEQ ID NO: 70, and a light chain variable region having the amino acid sequence of SEQ ID NO: 71, shown below:
TABLE-US-00007 GC1008 Heavy chain variable region amino acid sequence (SEQ ID NO: 70) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSSNVISWVRQAPGQGLEWMGG VIPIVDIANYAQRFKGRVTITADESTSTTYMELSSLRSEDTAVYYCASTL GLVLDAMDYWGQGTLVTVSS GC1008 Light chain variable region amino acid sequence (SEQ ID NO: 71) ETVLTQSPGTLSLSPGERATLSCRASQSLGSSYLAWYQQKPGQAPRLLIY GASSRAPGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYADSPITFG QGTRLEIK
[0476] Anti-TGF- antagonists useful in conjunction with the compositions and methods described herein include antibodies and antigen-binding fragments thereof containing one or more, or all, of the CDRs of GC1008, as well as those containing a set of CDRs that each have at least 50% sequence identity (e.g., at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or more, sequence identity) to the CDRs of GC1008, shown above.
[0477] Exemplary anti-TGF- antagonists useful in conjunction with the compositions and methods described herein include monoclonal antibodies and antigen-binding fragments thereof, polyclonal antibodies and antigen-binding fragments thereof, humanized antibodies and antigen-binding fragments thereof, bispecific antibodies and antigen-binding fragments thereof, optimized antibodies and antigen-binding fragments thereof (e.g., affinity-matured antibodies and antigen-binding fragments thereof), dual-variable immunoglobulin domains, single-chain Fv molecules (scFvs), diabodies, triabodies, nanobodies, antibody-like protein scaffolds, Fv fragments, Fab fragments, F(ab).sub.2 molecules, and tandem di-scFVs, among others, such as those that have one or more, or all, of the CDRs of GC1008, as well as those containing a set of CDRs that each have at least 50% sequence identity (e.g., at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or more, sequence identity) to the CDRs of GC1008, shown above.
[0478] Additionally, antibodies and antigen-binding fragments thereof that may be used in conjunction with the compositions and methods described herein include those that bind the same epitope on TGF- as murine antibody 1D11, its humanized counterpart, GC1008, and antibodies or antigen-binding fragments thereof that have the same set of CDRs as 1D11 and GC1008. Exemplary methods that can be used to determine whether an antibody or antigen-binding fragment thereof binds the same epitope on TGF- as a reference antibody, such as 1D11 or GC1008, include competitive binding experiments, such as competitive ELISA experiments or other competitive binding assays known in the art. An antibody or antigen-binding fragment thereof is considered to bind the same epitope on TGF- as a reference antibody, such as 1D11 or GC1008, if the antibody or antigen-binding fragment thereof competitively inhibits the binding of TGF- to the reference antibody. Competitive binding experiments that can be used to determine whether an antibody or antigen-binding fragment thereof binds to the same epitope on TGF- as a reference antibody or antigen-binding fragment thereof are described, for instance, in Nagata et al., Journal of Immunological Methods 292:141-155 (2004), the disclosure of which is incorporated herein by reference in its entirety.
[0479] Thus, antibodies and antigen-binding fragments thereof useful in conjunction with the compositions and methods described herein include those that competitively inhibit the binding of TGF- to an antibody or antigen-binding fragment thereof that contains the following CDRs: [0480] (a) a CDR-H1 having the amino acid sequence SNVIS (SEQ ID NO: 64); [0481] (b) a CDR-H2 having the amino acid sequence GVIPIVDIANYAQRFKG (SEQ ID NO: 65); [0482] (c) a CDR-H3 having the amino acid sequence TLGLVLDAMDY (SEQ ID NO: 66); [0483] (d) a CDR-L1 having the amino acid sequence RASQSLGSSYLA (SEQ ID NO: 67); [0484] (e) a CDR-L2 having the amino acid sequence GASSRAP (SEQ ID NO: 68); and [0485] (f) a CDR-L3 having the amino acid sequence QQYADSPIT (SEQ ID NO: 69).
[0486] Antibodies and antigen-binding fragments thereof that may be used with the compositions and methods described herein include those that competitively inhibit the binding of TGF- to an antibody or antigen-binding fragment thereof having the heavy chain variable region set forth in SEQ ID NO: 70 and/or the light chain variable region set forth in SEQ ID NO: 71.
[0487] Additional TGF- antagonists useful in conjunction with the compositions and methods described herein include anti-TGF- antibody PCT-0011 (with the bone-targeting moiety D10), as well as antigen-binding fragments thereof. Antibodies and antigen-binding fragments thereof that may be used with the compositions and methods described herein include an antibody or antigen-binding fragment thereof having the heavy chain set forth in SEQ ID NO: 62, or a heavy chain having an amino acid sequence that has at least 85% sequence identity (e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or more, sequence identity) to SEQ ID NO: 62, and/or the light chain set forth in SEQ ID NO: 63, or a light chain having an amino acid sequence that has at least 85% sequence identity (e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or more, sequence identity) to SEQ ID NO: 63, that competitively inhibit the binding of TGF- to an anti-TGF- antibody or an antigen-binding fragment thereof, such as 1D11 or GC1008 antibody, or an an antigen-binding fragment thereof.
[0488] Additional TGF- antagonists useful in conjunction with the compositions and methods described herein include anti-TGF- antibody TM1 (LY2382770). The TM1 (LY2382770) antibody sequences are described in detail in, e.g., WO 2005/010049, the disclosure of which is incorporated herein by reference in its entirety.
TGF- Antagonists from TGF- Co-Receptors
[0489] Additional exemplary TGF- antagonists that bind TGF- and inhibit TGF- signaling include peptides from TGF- co-receptors, such as the TGF- co-receptor, CD109. This peptide is described in detail, for instance, in U.S. Pat. No. 7,173,002 and in US 2012/0079614, the disclosures of each of which are incorporated herein by reference in their entirety. This 1428-residue peptide, as well as fragments thereof, have been shown to inhibit TGF- signaling in mammalian cells. Active forms of this peptide may contain a tyrosine (SEQ ID NO: 73) or serine (SEQ ID NO: 75) residue at position 703 within the CD109 sequence. Additionally, fragments of CD109, such as those containing the amino acid sequence of residues 21-1404 or 21-1428, may be used as TGF- antagonist peptides in the context of the conjugates, compositions, and methods described herein. Other fragments of CD109, such as those containing the amino acid sequence WIWLDTNMGYRIYQEFEVT (SEQ ID NO: 72) or WIWLDTNMGSRIYQEFEVT (SEQ ID NO: 74), which correspond to positions 694-712 of SEQ ID NO: 73 and SEQ ID NO: 75, respectively, may be used as TGF- antagonists in the conjugates, compositions, and methods described herein, as these sequences may contain a putative TGF- binding site. Additional fragment of the CD109 peptide that can be used as a TGF- antagonist peptide in the conjugates, compositions, and methods described herein contain the amino acid sequence IDGVYDNAEYAERFMEENEGHIVDIHDFSLGSS (SEQ ID NO: 76), which corresponds to residues 651-683 of SEQ ID NO: 73, which may also contain a putative TGF- binding site.
[0490] Additional fragments of CD109 that can be used in the conjugates, compositions, and methods described herein include a 161-residue portion of this protein that has the amino acid sequence TMENVVHELELYNTGYYLGMFMNSFAVFQECGLWVLTDANLTKDYIDGVYDNAEYAERFMEENEGHIVDIHDFSLGSSPHVRKHFPETWIWLDTNMGSRIYQEFEVTVPDSITSWVATGFVISEDLGLGLTTTPVELQAFQPFFIFLNLPYSVIRGEEFAL (SEQ ID NO: 77). Additional peptidic fragments of CD109 that can be used in the conjugates, compositions, and methods described herein may comprise at least 10, 15, 25, 50, 75, 100, 250, 500, 750, 1000, 1250, 1400 or more contiguous amino acids of SEQ ID NO: 73. Exemplary CD109 fragments that may be used in conjunction with the conjugates, compositions, and methods described herein include those that contain a putative TGF- binding site, such as peptides containing the amino acid sequence RKHFPETWIWLDTNMGYRIYQEFEV (SEQ ID NO: 78), which corresponds to residues 687-711 of SEQ ID NO: 73.
[0491] In addition to the above, peptide antagonists of TGF- useful in conjunction with the conjugates, compositions, and methods described herein include those containing an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, 90%, 95%, 97%, 99%, or greater) to one of the foregoing sequences and/or having one or more conservative amino acid substitutions with respect to one of the foregoing sequences.
[0492] The foregoing antagonistic TGF- peptides are summarized in Table 6, below.
TABLE-US-00008 TABLE 6 Exemplary TGF-antagonist peptide sequences based on CD109 SEQ ID NO. Amino acid sequence 72 WIWLDTNMGYRIYQEFEVT 73 MQGPPLLTAAHLLCVCTAALAVAPGPRFLVTAPGIIRPGGNVTIGVELLEHCPSQVT VKAELLKTASNLTVSVLEAEGVFEKGSFKTLTLPSLPLNSADEIYELRVTGRTQDEIL FSNSTRLSFETKRISVFIQTDKALYKPKQEVKFRIVTLFSDFKPYKTSLNILIKDPKSNL IQQWLSQQSDLGVISKTFQLSSHPILGDWSIQVQVNDQTYYQSFQVSEYVLPKFEV TLQTPLYCSMNSKHLNGTITAKYTYGKPVKGDVTLTFLPLSFWGKKKNITKTFKING SANFSFNDEEMKNVMDSSNGLSEYLDLSSPGPVEILTTVTESVTGISRNVSTNVFFK QHDYIIEFFDYTTVLKPSLNFTATVKVTRADGNQLTLEERRNNVVITVTQRNYTEYW SGSNSGNQKMEAVQKINYTVPQSGTFKIEFPILEDSSELQLKAYFLGSKSSMAVHSL FKSPSKTYIQLKTRDENIKVGSPFELVVSGNKRLKELSYMVVSRGQLVAVGKQNST MFSLTPENSWTPKACVIVYYIEDDGEIISDVLKIPVQLVFKNKIKLYWSKVKAEPSEK VSLRISVTQPDSIVGIVAVDKSVNLMNASNDITMENVVHELELYNTGYYLGMFMNSF AVFQECGLWVLTDANLTKDYIDGVYDNAEYAERFMEENEGHIVDIHDFSLGSSPHV RKHFPETWIWLDTNMGYRIYQEFEVTVPDSITSWVATGFVISEDLGLGLTTTPVELQ AFQPFFIFLNLPYSVIRGEEFALEITIFNYLKDATEVKVIIEKSDKFDILMTSNEINATGH QQTLLVPSEDGATVLFPIRPTHLGEIPITVTALSPTASDAVTQMILVKAEGIEKSYSQS ILLDLTDNRLQSTLKTLSFSFPPNTVTGSERVQITAIGDVLGPSINGLASLIRMPYGCG EQNMINFAPNIYILDYLTKKKQLTDNLKEKALSFMRQGYQRELLYQREDGSFSAFGN YDPSGSTWLSAFVLRCFLEADPYIDIDQNVLHRTYTWLKGHQKSNGEFWDPGRVIH SELQGGNKSPVTLTAYIVTSLLGYRKYQPNIDVQESIHFLESEFSRGISDNYTLALITY ALSSVGSPKAKEALNMLTWRAEQEGGMQFWVSSESKLSDSWQPRSLDIEVAAYA LLSHFLQFQTSEGIPIMRWLSRQRNSLGGFASTQDTTVALKALSEFAALMNTERTNI QVTVTGPSSPSPLAVVQPTAVNISANGFGFAICQLNVVYNVKASGSSRRRRSIQN QEAFDLDVAVKENKDDLNHVDLNVCTSFSGPGRSGMALMEVNLLSGFMVPSEAIS LSETVKKVEYDHGKLNLYLDSVNETQFCVNIPAVRNFKVSNTQDASVSIVDYYEPR RQAVRSYNSEVKLSSCDLCSDVQGCRPCEDGASGSHHHSSVIFIFCFKLLYFMEL WL 74 WIWLDTNMGSRIYQEFEVT 75 MQGPPLLTAAHLLCVCTAALAVAPGPRFLVTAPGIIRPGGNVTIGVELLEHCPSQVT VKAELLKTASNLTVSVLEAEGVFEKGSFKTLTLPSLPLNSADEIYELRVTGRTQDEIL FSNSTRLSFETKRISVFIQTDKALYKPKQEVKFRIVTLFSDFKPYKTSLNILIKDPKSNL IQQWLSQQSDLGVISKTFQLSSHPILGDWSIQVQVNDQTYYQSFQVSEYVLPKFEV TLQTPLYCSMNSKHLNGTITAKYTYGKPVKGDVTLTFLPLSFWGKKKNITKTFKING SANFSFNDEEMKNVMDSSNGLSEYLDLSSPGPVEILTTVTESVTGISRNVSTNVFFK QHDYIIEFFDYTTVLKPSLNFTATVKVTRADGNQLTLEERRNNVVITVTQRNYTEYW SGSNSGNQKMEAVQKINYTVPQSGTFKIEFPILEDSSELQLKAYFLGSKSSMAVHSL FKSPSKTYIQLKTRDENIKVGSPFELVVSGNKRLKELSYMVVSRGQLVAVGKQNST MFSLTPENSWTPKACVIVYYIEDDGEIISDVLKIPVQLVFKNKIKLYWSKVKAEPSEK VSLRISVTQPDSIVGIVAVDKSVNLMNASNDITMENVVHELELYNTGYYLGMFMNSF AVFQECGLWVLTDANLTKDYIDGVYDNAEYAERFMEENEGHIVDIHDFSLGSSPHV RKHFPETWIWLDTNMGSRIYQEFEVTVPDSITSWVATGFVISEDLGLGLTTTPVELQ AFQPFFIFLNLPYSVIRGEEFALEITIFNYLKDATEVKVIIEKSDKFDILMTSNEINATGH QQTLLVPSEDGATVLFPIRPTHLGEIPITVTALSPTASDAVTQMILVKAEGIEKSYSQS ILLDLTDNRLQSTLKTLSFSFPPNTVTGSERVQITAIGDVLGPSINGLASLIRMPYGCG EQNMINFAPNIYILDYLTKKKQLTDNLKEKALSFMRQGYQRELLYQREDGSFSAFGN YDPSGSTWLSAFVLRCFLEADPYIDIDQNVLHRTYTWLKGHQKSNGEFWDPGRVIH SELQGGNKSPVTLTAYIVTSLLGYRKYQPNIDVQESIHFLESEFSRGISDNYTLALITY ALSSVGSPKAKEALNMLTWRAEQEGGMQFWVSSESKLSDSWQPRSLDIEVAAYA LLSHFLQFQTSEGIPIMRWLSRQRNSLGGFASTQDTTVALKALSEFAALMNTERTNI QVTVTGPSSPSPLAVVQPTAVNISANGFGFAICQLNVVYNVKASGSSRRRRSIQNQ EAFDLDVAVKENKDDLNHVDLNVCTSFSGPGRSGMALMEVNLLSGFMVPSEAISLS ETVKKVEYDHGKLNLYLDSVNETQFCVNIPAVRNFKVSNTQDASVSIVDYYEPRRQ AVRSYNSEVKLSSCDLCSDVQGCRPCEDGASGSHHHSSVIFIFCFKLLYFMELWL 76 IDGVYDNAEYAERFMEENEGHIVDIHDFSLGSS 77 TMENVVHELELYNTGYYLGMFMNSFAVFQECGLWVLTDANLTKDYIDGVYDNAEY AERFMEENEGHIVDIHDFSLGSSPHVRKHFPETWIWLDTNMGSRIYQEFEVTVPDSI TSWVATGFVISEDLGLGLTTTPVELQAFQPFFIFLNLPYSVIRGEEFAL 78 RKHFPETWIWLDTNMGYRIYQEFEV
Additional Peptidic and Proteinaceous TGF- Antagonists
[0493] In addition to the above, peptide antagonists capable of binding TGF- for use with the conjugates, compositions, and methods described herein include those described in U.S. Pat. No. 7,723,473, the disclosure of which is incorporated herein by reference in its entirety, as well as peptide antagonists of TGF- containing an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, 90%, 95%, 97%, 99%, or greater) to one of these sequences and/or having one or more conservative amino acid substitutions with respect to one of these sequences. These TGF- antagonists specifically bind to TGF- receptors, which include type I, type II, type III and type V receptors. It has been shown that these peptides, some of which correspond in sequence to amino acid numbers 41-65 of TGF-.sub.1, TGF-.sub.2, and TGF-.sub.3, inhibit the binding of TGF-.sub.1, TGF-.sub.2, and TGF-.sub.3, to TGF- receptors. These peptides have been shown to attenuate TGF--induced growth inhibition and TGF--induced expression of PAI-1. It has also been shown that the W/RXXD motif found within these peptide sequences determines the specificity of activity of the antagonist peptide. These TGF- antagonist peptides are summarized in Table 7, below.
TABLE-US-00009 TABLE 7 Exemplary TGF-antagonist peptides SEQ ID NO. Amino acid sequence 79 ANFCLGPCPYIWSLDT 80 ANFCSGPCPYLRSADT 81 PYIWSLDTQY 82 PYLWSSDTQH 83 PYLRSADTTH 84 WSXD x =any AA 85 RSXD x =any AA
[0494] Additional peptidic antagonists of TGF- that can be used in conjunction with the conjugates, compositions, and methods described herein include peptide antagonists described in U.S. Pat. No. 7,057,013, US 2009/0263410, and US 2011/0294734, the disclosures of which are incorporated herein by reference in its entirety, as well as peptide antagonists of TGF- containing an amino acid sequence having at least 50% sequence identity (e.g., at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or greater) to one of these sequences and/or having one or more conservative amino acid substitutions with respect to one of these sequences. These TGF- antagonist peptides are based on the structure of TGF- or a TGF- receptor, and were designed so as to disrupt the binding of endogenous TGF- to a TGF- receptor for the purposes of attenuating TGF- signaling. These synthetic peptides are summarized in Tables 8 and 9, below.
TABLE-US-00010 TABLE 8 Exemplary TGF-antagonist peptides that bind TGF- SEQ ID NO. Amino acid sequence 86 TSLDATMIWTMM 87 SNPYSAFQVDIIVDI 88 TSLMIWTMM 89 TSLDASIIWAMMQN 90 SNPYSAFQVDITID 91 EAVLILQGPPYVSWL 92 LDSLSFQLGLYLSPH
TABLE-US-00011 TABLE 9 Exemplary TGF-antagonist peptides that bind a TGF-receptor SEQ ID NO. Amino acid sequence 93 HANFCLGPCPYIWSL 94 FCLGPCPYIWSLDT 95 HEPKGYHANFCLGPCP
[0495] Additional peptidic antagonists of TGF- that can be used in conjunction with the conjugates, compositions, and methods described herein include peptide antagonists described in US 2009/0263410, the disclosure of which is incorporated herein by reference in its entirety, as well as peptide antagonists of TGF- containing an amino acid sequence having at least 50% sequence identity (e.g., at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or greater) to one of these sequences and/or having one or more conservative amino acid substitutions with respect to one of these sequences. These peptides are summarized in Table 10, below.
TABLE-US-00012 TABLE 10 Exemplary TGF-antagonist peptides that bind TGF- SEQ ID NO. Amino acid sequence 96 TSLDASIIWAMMQN 97 KRIWFIPRSSWYERA 98 KRIWFIPRSSW 99 KRIWFIPRSSW (Amidated at C-terminus) 100 KRIWFIPRSSW (Acetylated at N-terminal K and amidated at C-terminus)
[0496] Additional peptidic antagonists of TGF- that can be used in conjunction with the conjugates, compositions, and methods described herein include peptide antagonists described in US 2011/0294734, the disclosure of which is incorporated herein by reference in its entirety, as well as peptide antagonists of TGF- containing an amino acid sequence having at least 50% sequence identity (e.g., at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or greater) to one of these sequences and/or having one or more conservative amino acid substitutions with respect to one of these sequences. These peptides are summarized in Table 11, below.
TABLE-US-00013 TABLE 11 Exemplary TGF-antagonist peptides SEQ ID NO. Amino acid sequence 101 HANFCLGPCPYIWSL 102 FCLGPCPYIWSLDT 103 TSLDATMIWTMM 104 SNPYSAFQVDIIVDI 105 TSLMIWTMM 106 TSLDASIIWAMMQN 107 SNPYSAFQVDITID 108 EAVLILQGPPYVSWL 109 LDSLSFQLGLYLSPH 110 HEPKGYHANFCLGPCPYIWSLDT 111 WHKYFLRRPLSVRTR 112 RFFTRFPWHYHASRL 113 RKWFLQHRRMPVSVL 114 SGRRHLHRHHIFSLP 115 RLAHSHRHRSHVALT 116 PPYHRFWRGHRHAVQ 117 KRIWFIPRSSWYERA 118 MPLSRYWWLFSHRPR 119 KRIWFIPRSSWYER 120 KRIWFIPRSSWY 121 KRIWFIPRSSW 122 KRIWFIPRSSW (amidated at C-terminus) 123 KRIWFIPRSSW (acetylated at N-terminal K and amidated at C-terminus)
[0497] Additional TGF- antagonists useful in conjunction with the conjugates, compositions, and methods described herein include glycoprotein-A repetitions predominant protein (GARP), as well as well as peptide antagonists of TGF- containing an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, 90%, 95%, 97%, 99%, or greater) to this protein and/or having one or more conservative amino acid substitutions with respect to this protein. The antagonistic activity of this protein is described in detail, for example, in Wang et al., Molecular Biology of the Cell 23:1129-1139 (2012), the disclosure of which is incorporated herein by reference in its entirety. The amino acid sequence of GARP is shown below.
TABLE-US-00014 Glycoprotein-A repetitions predominant protein (GARP): (SEQ ID NO: 124) MRPQILLLLALLTLGLAAQHQDKVPCKMVDKKVSCQVLGLLQVPSVLPPD TETLDLSGNQLRSILASPLGFYTALRHLDLSTNEISFLQPGAFQALTHLE HLSLAHNRLAMATALSAGGLGPLPRVTSLDLSGNSLYSGLLERLLGEAPS LHTLSLAENSLTRLTRHTFRDMPALEQLDLHSNVLMDIEDGAFEGLPRLT HLNLSRNSLTCISDFSLQQLRVLDLSCNSIEAFQTASQPQAEFQLTWLDL RENKLLHFPDLAALPRLIYLNLSNNLIRLPTGPPQDSKGIHAPSEGWSAL PLSAPSGNASGRPLSQLLNLDLSYNEIELIPDSFLEHLTSLCFLNLSRNC LRTFEARRLGSLPCLMLLDLSHNALETLELGARALGSLRTLLLQGNALRD LPPYTFANLASLQRLNLQGNRVSPCGGPDEPGPSGCVAFSGITSLRSLSL VDNEIELLRAGAFLHTPLTELDLSSNPGLEVATGALGGLEASLEVLALQG NGLMVLQVDLPCFICLKRLNLAENRLSHLPAWTQAVSLEVLDLRNNSFSL LPGSAMGGLETSLRRLYLQGNPLSCCGNGWLAAQLHQGRVDVDATQDLIC RFSSQEEVSLSHVRPEDCEKGGLKNINLIIILTFILVSAILLTTLAACCC VRRQKFNQQYKA
[0498] Examples of additional TGF- antagonists useful in conjunction with the conjugates, compositions, and methods described herein include latency associated peptide (see, e.g., WO 91/08291), large latent TGF- (see, e.g., WO 94/09812), fetuin (see, e.g., U.S. Pat. No. 5,821,227), decorin and other proteoglycans such as biglycan, fibromodulin, lumican and endoglin (see, e.g., U.S. Pat. Nos. 5,583,103, 5,654,270, 5,705,609, 5,726,149, 5,824,655 5,830,847, 6,015,693, as well as WO 91/04748, WO 91/10727, WO 93/09800, and WO 94/10187).
[0499] Further examples of TGF- antagonists that may be used in conjunction with the compositions and methods described herein include somatostatin (see, e.g., WO 98/08529), mannose-6-phosphate or mannose-1-phosphate (see, e.g., U.S. Pat. No. 5,520,926), prolactin (see, e.g., WO 97/40848), insulin-like growth factor II (see, e.g., WO 98/17304), IP-10 (see, e.g., WO97/00691), arg-gly-asp containing peptides (see, e.g., U.S. Pat. No. 5,958,411 and WO 93/10808), extracts of plants, fungi and bacteria (see, e.g., EP 813875, JP 8119984, and U.S. Pat. No. 5,693,610), antisense oligonucleotides (see, e.g., U.S. Pat. Nos. 5,683,988, 5,772,995, 5,821,234 and 5,869,462, as well as WO 94/25588), and a host of other proteins involved in TGF- signaling, including SMADs and MADs (see, e.g., EP 874046, WO 97/31020, WO 97/38729, WO 98/03663, WO 98/07735, WO 98/07849, WO 98/45467, WO 98/53068, WO 98/55512, WO 98/56913, WO 98/53830, and WO 99/50296, as well as U.S. Pat. Nos. 5,834,248, 5,807,708, and 5,948,639), in addition to Ski and Sno (see, e.g., G. Vogel, Science, 286:665 (1999) and Stroschein et al., Science, 286:771-74 (1999)) and fragments and derivatives of any of the above molecules that retain the ability to inhibit the activity of TGF-.
Small Molecule TGF- Antagonists
[0500] Additional examples of TGF- antagonists include small molecules that inhibit TGF- signal transduction. These agents can be classified on the basis of the core molecular scaffolds of these molecules. For example, TGF- signaling inhibitors may contain a dihydropyrrlipyrazole, imidazole, pyrazolopyridine, pyrazole, imidazopyridine, triazole, pyridopyrimidine, pyrrolopyrazole, isothiazole, or oxazole functionality as the core structural fragment of the molecule. Some non-limiting examples of small molecule inhibitors of TGF- signaling include ALK5 inhibitor II (also referred to as E-616452), LY364947 (also referred to as ALK5 Inhibitor I, TbR-I Inhibitor, Transforming Growth Factor-b Type I Receptor Kinase Inhibitor), A83-01, and DMH1, known in the art. Other examples of small molecule TGF- antagonists that can be used in conjunction with the compositions and methods described herein include SB431542 (4-(5-Benzol[1,3]dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)-benzamide hydrate, 4-[4-(1,3-Benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide hydrate, 4-[4-(3,4-Methylenedioxyphenyl)-5-(2-pyridyl)-1H-imidazol-2-yl]-benzamide hydrate, an Alk5 inhibitor), Galunisertib (LY2157299, an Alk5 inhibitor), LY2109761 (4-[2-[4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)quinolin-7-yl]oxyethyl]morpholine, an Alk5/TGFRII inhibitor), SB525334 (6-[2-tert-butyl-5-(6-methylpyridin-2-yl)-1H-imidazol-4-yl]quinoxaline, an Alk5 inhibitor), GW788388 (N-(oxan-4-yl)-4-[4-(5-pyridin-2-yl-1H-pyrazol-4-yl)pyridin-2-yl]benzamide, an Alk5 inhibitor), K02288 (3-[6-amino-5-(3,4,5-trimethoxyphenyl)pyridin-3-yl]phenol, an Alk4/Alk5 inhibitor), SD-208 (2-(5-chloro-2-fluorophenyl)-N-pyridin-4-ylpteridin-4-amine, an Alk5 inhibitor), EW-7197 (N-((4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)-2-fluoroaniline, an Alk4/Alk5 inhibitor), and LDN-212854(5-[6-[4-(1-Piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline, an Alk4/Alk5 inhibitor).
[0501] Additional examples of small molecule TGF- antagonists include those that bind TGF- receptors, such as 2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5 napththyridine, [3-(Pyridin-2-yl)-4-(4-quinoyl)]-1H-pyrazole, and 3-(6-Methylpyridin-2-yl)-4-(4-quinolyl)-1-phenylthiocarbamoyl-1H-pyrazole. Other small molecule inhibitors include, but are not limited to, SB-431542, (4-[4-(1,3-Benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide, described in Halder et al., Neoplasia 7(5):509-521 (2005)), SM16, a small molecule inhibitor of TGF receptor ALK5, the structure of which is shown below (Fu, K et al., Arteriosclerosis, Thrombosis and Vascular Biology 28(4):665 (2008)), SB-505124 (an Alk4/Alk5 inhibitor, structure shown below, described in Dacosta Byfield, S., et al., Molecular Pharmacology 65:744-752 (2004)), and 6-bromo-indirubin-3-oxime (described in U.S. Pat. No. 8,298,825), the disclosures of each of which are incorporated herein by reference.
##STR00001##
[0502] Additional examples of small molecule TGF- antagonists include, without limitation, those that are described in, e.g., Callahan, J. F. et al., J. Med. Chem. 45:999-1001 (2002); Sawyer, J. S. et al., J. Med. Chem. 46:3953-3956 (2003); Gellibert, F. et al., J. Med. Chem. 47:4494-4506 (2004); Tojo, M. et al., Cancer Sci. 96:791-800 (2005); Valdimarsdottir, G. et al., APMIS 113:773-389 (2005); Petersen et al., Kidney International 73:705-715 (2008); Yingling, J. M. et al., Nature Rev. Drug Disc. 3:1011-1022 (2004); Byfield, S. D. et al., Mol. Pharmacol., 65:744-752 (2004); Dumont, N, et al., Cancer Cell 3:531-536 (2003); WO 2002/094833; WO 2004/026865; WO 2004/067530; WO 209/032667; WO 2004/013135; WO 2003/097639; WO 2007/048857; WO 2007/018818; WO 2006/018967; WO 2005/039570; WO 2000/031135; WO 1999/058128; U.S. Pat. Nos. 6,509,318; 6,090,383; 6,419,928; 7,223,766; 6,476,031; 6,419,928; 7,030,125; 6,943,191; US 2005/0245520; US 2004/0147574; US 2007/0066632; US 2003/0028905; US 2005/0032835; US 2008/0108656; US 2004/015781; US 2004/0204431; US 2006/0003929; US 2007/0155722; US 2004/0138188; and US 2009/0036382, the disclosures of each which are incorporated by reference as they pertain to TGF- antagonists.
Bone-Targeting Moieties
Collagen-Binding Domains
[0503] A variety of collagen-binding domains can be used in conjunction with the compositions and methods described herein. For instance, a variety of peptides with collagen-binding activity have been described in U.S. Pat. No. 8,450,272, the disclosure of which is incorporated herein by reference in its entirety. Exemplary collagen-binding peptides described therein are summarized below.
TABLE-US-00015 (SEQ ID NO: 125) Pro Val Tyr Pro Ile Gly Thr Glu Lys Glu Pro Asn Asn Ser Lys Glu Thr Ala Ser Gly Pro Ile Val Pro Gly Ile Pro Val Ser Gly Thr Ile Glu Asn Thr Ser Asp Gln Asp Tyr Phe Tyr Phe Asp Val Ile Thr Pro Gly Glu Val Lys Ile Asp Ile Asn Lys Leu Gly Tyr Gly Gly Ala Thr Trp Val Val Tyr Asp Glu Asn Asn Asn Ala Val Ser Tyr Ala Thr Asp Asp Gly Gln Asn Leu Ser Gly Lys Phe Lys Ala Asp Lys Pro Gly Arg Tyr Tyr Ile His Leu Tyr Met Phe Asn Gly Ser Tyr Met Pro Tyr Arg Ile Asn Ile Glu Gly Ser Val Gly Arg (SEQ ID NO: 126) Glu Ile Lys Asp Leu Ser Glu Asn Lys Leu Pro Val Ile Tyr Met His Val Pro Lys Ser Gly Ala Leu Asn Gln Lys Val Val Phe Tyr Gly Lys Gly Thr Tyr Asp Pro Asp Gly Ser Ile Ala Gly Tyr Gln Trp Asp Phe Gly Asp Gly Ser Asp Phe Ser Ser Glu Gln Asn Pro Ser His Val Tyr Thr Lys Lys Gly Glu Tyr Thr Val Thr Leu Arg Val Met Asp Ser Ser Gly Gln Met Ser Glu Lys Thr Met Lys Ile Lys Ile Thr Asp Pro Val Tyr Pro Ile Gly Thr Glu Lys Glu Pro Asn Asn Ser Lys Glu Thr Ala Ser Gly Pro Ile Val Pro Gly Ile Pro Val Ser Gly Thr Ile Glu Asn Thr Ser Asp Gln Asp Tyr Phe Tyr Phe Asp Val Ile Thr Pro Gly Glu Val Lys Ile Asp Ile Asn Lys Leu Gly Tyr Gly Gly Ala Thr Trp Val Val Tyr Asp Glu Asn Asn Asn Ala Val Ser Tyr Ala Thr Asp Asp Gly Gln Asn Leu Ser Gly Lys Phe Lys Ala Asp Lys Pro Gly Arg Tyr Tyr Ile His Leu Tyr Met Phe Asn Gly Ser Tyr Met Pro Tyr Arg Ile Asn Ile Glu Gly Ser Val Gly Arg
[0504] Collagen-binding peptides useful in conjunction with the conjugates and methods described herein also include those having at least 85% sequence identity (e.g., at least 85%, 90%, 95%, 97%, 99%, or greater) to one of the foregoing sequences and/or having one or more conservative amino acid substitutions with respect to one of these sequences.
[0505] Additionally, collagen-binding peptides derived from human glycoprotein VI (GPVI) have been described, for instance, in U.S. Pat. No. 8,084,577, the disclosure of which is incorporated herein by reference in its entirety. Collagen-binding domains of GPVI can be incorporated into conjugates described herein, for instance, using the synthetic chemistry or protein expression methodologies described below. The sequence of the collagen-binding domain of GPVI is described below:
TABLE-US-00016 (SEQ ID NO: 127) Gln Ser Gly Pro Leu Pro Lys Pro Ser Leu Gln Ala Leu Pro Ser Ser Leu Val Pro Leu Glu Lys Pro Val Thr Leu Arg Cys Gln Gly Pro Pro Gly Val Asp Leu Tyr Arg Leu Glu Lys Leu Ser Ser Ser Arg Tyr Gln Asp Gln Ala Val Leu Phe Ile Pro Ala Met Lys Arg Ser Leu Ala Gly Arg Tyr Arg Cys Ser Tyr Gln Asn Gly Ser Leu Trp Ser Leu Pro Ser Asp Gln Leu Glu Leu Val Ala Thr Gly Val Phe Ala Lys Pro Ser Leu Ser Ala Gln Pro Gly Pro Ala Val Ser Ser Gly Gly Asp Val Thr Leu Gln Cys Gln Thr Arg Tyr Gly Phe Asp Gln Phe Ala Leu Tyr Lys Glu Gly Asp Pro Ala Pro Tyr Lys Asn Pro Glu Arg Trp Tyr Arg Ala Ser Phe Pro Ile Ile Thr Val Thr Ala Ala His Ser Gly Thr Tyr Arg Cys Tyr Ser Phe Ser Ser Arg Asp Pro Tyr Leu Trp Ser Ala Pro Ser Asp Pro Leu Glu Leu Val Val Thr
[0506] Collagen-binding peptides useful in conjunction with the conjugates and methods described herein also include those having at least 85% sequence identity (e.g., at least 85%, 90%, 95%, 97%, 99%, or greater) to the foregoing GPVI-derived sequence and/or having one or more conservative amino acid substitutions with respect to this sequence.
[0507] Additionally, collagen-binding peptides derived from human fibronectin can be incorporated into the conjugates described herein (e.g., peptides of about 340 residues corresponding to the amino acid sequence between and including Ala260 and Trp599 of human fibronectin) have been described in detail in WO 2000/049159, the disclosure of which is incorporated herein by reference in its entirety.
[0508] Collagen-binding peptides useful in conjunction with the conjugates and methods described herein also include those having at least 85% sequence identity (e.g., at least 85%, 90%, 95%, 97%, 99%, or greater) to the foregoing fibronectin-derived sequence and/or having one or more conservative amino acid substitutions with respect to this sequence.
[0509] Collagen-binding peptides derived from bone sialoprotein can be incorporated into the conjugates described herein. Such peptide have been described in detail in WO 2005/082941, the disclosure of which is incorporated herein by reference in its entirety. Exemplary sequences derived from the N-terminal domain of bone sialoprotein that bind collagen are summarized below:
TABLE-US-00017 (SEQ ID NO: 128) NGVFKYRPRYFLYKHAYFYPPLKRFPVQ (SEQ ID NO: 129) NGVFKYRPRYFLYK (SEQ ID NO: 130) HAYFYPPLKRFPVQ
[0510] Collagen-binding peptides useful in conjunction with the conjugates and methods described herein also include those having at least 50% sequence identity (e.g., at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or greater) to one of the foregoing sequences and/or having one or more conservative amino acid substitutions with respect to these sequences.
Hydroxyapatite-Binding Domains
[0511] A variety of Hydroxyapatite-binding domains that can be incorporated into conjugates described herein have been identified, for instance, using phage display techniques. Such peptides are described, for example, in U.S. Pat. No. 8,022,040, the disclosure of which is incorporated herein by reference in its entirety. Exemplary hydroxyapatite-binding domains described therein are summarized in Table 12, below.
TABLE-US-00018 TABLE 12 Exemplary hydroxyapatite-binding peptides SEQ ID NO. Amino acid sequence 131 RPHTITN 132 QSSYNPI 133 QTHARHQ 134 ETRTQLL 135 HHQRSPA 136 LQKSPSL 137 PPKDSRG 138 SAKKVFS 139 SQHSTQD 140 TIHSKPA 141 TKDWLPS 142 ANPPLSL 143 AKQTVPV 144 ATFSPPL 145 DQYWGLR 146 EPNHTRF 147 HMLAQTF 148 IGYPVLP 149 KLSAWSF 150 MYPLPAP 151 FTLPTIR 152 SMAAKSS 153 SMYDTHS 154 STLASMR 155 TLMTTPP 156 WLPPRTQ 157 RTPLQPLEDFRP 158 NTTTDIPSPSQF 159 TLDKYTRLLSRY 160 YPIMSHTCCHGV 161 YEPAAAE 162 ANPYHRH 163 ASGPTNV 164 QNYLLPK 165 GTQTPQP 166 HSTGPTR 167 LSKNPLL 168 LSKNPLL 169 KLHASLA 170 PLTQPSH 171 PHNPGKL 172 PTTMTRW 173 VHLTHGQ 174 TLAPTFR 175 VHPRPSL 176 TLLRTQV 177 SSPPRVY 178 SSVPGRP 179 LPFQPPI 180 IQHQAKT 181 LPRDLHATPQQI 182 LTPTMFNMHGVL 183 SIPKMIPTESLL 184 SFQSMSLMTLVV 185 TQTWPQSSSHGL 186 YELQMP 187 AMSQTMTAAIEK 188 GSAGLKYPLYKS 189 INFQFLKPSTTR 190 RHTLPLH 191 NFAMNLR 192 NFAMNLR 193 NPQMQRS 194 NPQMQRS 195 NPQMQRS 196 NYPTLKS 197 NYPTLKS 198 QNPRQIY 199 QNPRQIY 200 QNPRQIY 201 QNPRQIY 202 ETYARPL 203 ETVCASS 204 KPMQFVH 205 KPMQFVH 206 PAKQKAH 207 PTTWGHL 208 PTTWGHL 209 SASGTPS 210 SSYEYHA 211 SSYEYHA 212 STQAHPW 213 TVLGTFP 214 WYPNHLA 215 TTYNSPP 216 MTSQTLR 217 WPANKLSTKSMY 218 WPANKLSTKSMY 219 NPYHPTIPQSVH 220 DKLHRLA 221 QPGLWPS 222 ESLKSIS 223 GSCPPKK 224 GSLFKAL 225 HQWDHKY 226 LSAPMEY 227 MKVHERS 228 FVNLLGQ 229 PIDAFFD 230 PPNMARA 231 PTNKPHT 232 SPNNTRE 233 SPEMKPR 234 SSSMAKM 235 TDHPPKA 236 TLAFQTA 237 APLSLSL 238 HYPTVNF 239 QHNFRGASSSAP 240 HQFPXSNLVWKP 241 LSLRASAATDFQ 242 MQFTPAPSPSDH 243 SVFLPTRHSPDL 244 SVSVGMKPSPRP 245 SVSVGMKPSPRP 246 SVSVGMKPSPRP 247 SVSVGMKPSPRP 248 SVSVGMNAESA 249 RHTLPLH 250 NPQMQRS 251 NYPTLKS 252 NYPTLKS 253 DMRQQRS 254 QNPRQIY 255 QNPRQIY 256 QNPRQIY 257 QNPRQIY 258 QNPRQIY 259 QNPRQIY 260 QNPRQIY 261 QNPRQIY 262 QNPRQIY 263 QTHSSLW 264 ETYQQPL 265 ETYARPL 266 GTSRLFS 267 LTQTLQY 268 KAFDKHG 269 RPMQFVH 270 KPMQFVH 271 KPMQFIH 272 PAKQKAH 273 SASGTPS 274 SSHHHRH 275 SSYEYHA 276 TGPTSLS 277 LRAFPSLPHTVT 278 NPRSQAT 279 HRLGHMS 280 LLPLKFK 281 LPSIHNL 282 KATITGM 283 PDIPLSR 284 PSMKHWR 285 SAKGRAD 286 SRTGAHH 287 SKTSSTS 288 SPNNPRE 289 TLQRMGQ 290 TMTNMAK 291 TTLSPRT 292 TTKNFNK 293 YPKALRN 294 VVKSNGE 295 ITGAY 296 LPLTPLP 297 HSMPHMGTYLLT 298 MQFTPAPSPSDH 299 MPQTLVLPRSLL 300 SSTQVQHTLLQT 301 SWPLYSRDSGLG 302 SVSVGTEAESXA 303 SVSVGMKPSPRP 304 SVSVGMKPSPRP 305 SVSVGMKPSPRP 306 SVSVGMNAESYG 307 THPVVFEDERLF 308 TLPSPLALLTVH 309 WPTYLNPSSLKA 310 ASHNPKL 311 PAKQKAH 312 PAKQKAH 313 SASGTPS 314 TRFYDSL 315 QNPRQIY 316 QNPRQIY 317 QNPRQIY 318 QNPRQIY 319 TGPTSLS 320 TGPTSLS 321 NPQMQRS 322 NPQMQRS 323 NPQMQRS 324 NPQMQRS 325 NPQMQRS 326 KPMQFVH 327 SSYEYHA 328 STQAHPW 329 GTSRLFS 330 NYPTLKS 331 NYPTLKS 332 NYPTLKS 333 NYPTLKS 334 NYPTLKS 335 NYPTLKS 336 NYPTLKS 337 HAPVQPN 338 NPYHPTIPQSVH 339 NPYHPTIPQSVH 340 NPYHPTIPQSVH 341 NPYHPTIPQSVH 342 HQFISPEPFLIS 343 SPNFSWLPLGTTSPNFS 344 WLPLGTT 345 SVSVGMKPSPRP 346 SVSVGMKPSPRP 347 TPLTSPSLVRPQ 348 TPLSYLKGLVTV 349 NPMIMNQ 350 NPMIMNQ 351 NITQLGS 352 HTLLSTT 353 HTLLSTT 354 HTLLSTT 355 LGPGKAF 356 LGPGKAF 357 LGPGKAF 358 LGPGKAF 359 KTSSWAN 360 KMNHMPN 361 SLLTPWL 362 TLGLPML 363 TGLAKT 364 IRLIS 365 LGPGKAF 366 LGPGKAF 367 LGPGKAF 368 DLNYFTLSSKRE 369 DLNYFTLSSKRE 370 TMGFTAPRFPHY 371 TMGFTAPRFPHY 372 TMGFTAPRFPHY 373 TMGFTAPRFPHY 374 HTLLSTT 375 HTLLSTT 376 LASTTHV 377 LGPGKAF 378 LGPGKAF 379 LGPGKAF 380 SLLTPWL 381 NERQMEL 382 NKPLSTL 383 HTLLSTT 384 LKPFSGA 385 LGPGKAF 386 LGPGKAF 387 LGPGKAF 388 LGPGKAF 389 STSAKHW 390 TMGFTAPRFPHY 391 TMGFTAPRFPHY 392 TMGFTAPRFPHY 393 TMGFTAPRFPHY 394 TMGFTAPRFPHY 395 TMGFTAPRFPHY 396 TMGFTAPRFPHY 397 CNYPTLKSC
[0512] Hydroxyapatite-binding peptides useful in conjunction with the conjugates and methods described herein also include those having at least 50% sequence identity (e.g., at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or greater) to one of the foregoing sequences and/or having one or more conservative amino acid substitutions with respect to these sequences.
Polyanionic Peptides
[0513] Exemplary targeting moieties that can be used to localize a TGF- antagonist, such as a TGF- receptor fusion protein described herein, to osseous tissue include polyanionic peptides, such as those that contain one or more amino acids bearing a side-chain substituent selected from the group consisting of carboxylate, sulfonate, phosphonate, and phosphate. For instance, hydroxyapatite-binding targeting moieties include those that feature a plurality of consecutive or discontinuous aspartate or glutamate residues. Polyanionic peptides can bind hydroxyapatite by virtue, for instance, of electrostatic interactions between negatively charged substituents within the peptide, such as one or more carboxylate, sulfonate, phosphonate, or phosphate substituents, among others, to positively charged calcium ions present within hydroxyapatite.
[0514] In some embodiments, the polyanionic peptide contains (e.g., consists of) one or more glutamate residues (e.g., 1-25 glutamate residues, or more, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, or more, glutamate residues). In some embodiments, the polyanionic peptide contains (e.g., consists of) from 3 to 20 glutamate residues (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 glutamate residues). In some embodiments, the polyanionic peptide contains (e.g., consists of) from 5 to 15 glutamate residues (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 glutamate residues). In some embodiments, the polyanionic peptide contains (e.g., consists of) from 8 to 12 glutamate residues (e.g., 8, 9, 10, 11, or 12 glutamate residues). In some embodiments, the polyanionic peptide contains (e.g., consists of) 5 glutamate residues. In some embodiments, the polyanionic peptide contains (e.g., consists of) 6 glutamate residues. In some embodiments, the polyanionic peptide contains (e.g., consists of) 7 glutamate residues. In some embodiments, the polyanionic peptide contains (e.g., consists of) 8 glutamate residues. In some embodiments, the polyanionic peptide contains (e.g., consists of) 9 glutamate residues. In some embodiments, the polyanionic peptide contains (e.g., consists of) 10 glutamate residues. In some embodiments, the polyanionic peptide contains (e.g., consists of) 11 glutamate residues. In some embodiments, the polyanionic peptide contains (e.g., consists of) 12 glutamate residues. In some embodiments, the polyanionic peptide contains (e.g., consists of) 13 glutamate residues. In some embodiments, the polyanionic peptide contains (e.g., consists of) 14 glutamate residues. In some embodiments, the polyanionic peptide contains (e.g., consists of) 15 glutamate residues.
[0515] The polyanionic peptide may be a peptide of the formula E.sub.n, wherein E designates a glutamate residue and n is an integer from 1 to 25. For instance, the polyanionic peptide may be of the formula E.sub.1, E.sub.2, E.sub.3, E.sub.4, E.sub.5, E.sub.6, E.sub.7, E.sub.8, E.sub.9, E.sub.10, E.sub.11, E.sub.12, E.sub.13, E.sub.14, E.sub.15, E.sub.16, E.sub.17, E.sub.18, E.sub.19, E.sub.20, E.sub.21, E.sub.22, E.sub.23, E.sub.24, or E.sub.25. In some embodiments, the peptide is a peptide of the formula X.sub.nE.sub.mX.sub.oE.sub.p, wherein E designates a glutamate residue, each X independently designates any naturally-occurring amino acid, m represents an integer from 1 to 25, and n and o each independently represent integers from 0 to 5, and p represents an integer from 1 to 10.
[0516] In some embodiments, the glutamate residues are consecutive. In some embodiments, the glutamate residues are discontinuous.
[0517] In some embodiments, the polyanionic peptide contains (e.g., consists of) one or more aspartate residues (e.g., 1-25 aspartate residues, or more, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, or more, aspartate residues). In some embodiments, the polyanionic peptide contains (e.g., consists of) from 3 to 20 aspartate residues (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 aspartate residues). In some embodiments, the polyanionic peptide contains (e.g., consists of) from 5 to 15 aspartate residues (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 aspartate residues). In some embodiments, the polyanionic peptide contains (e.g., consists of) from 8 to 12 aspartate residues (e.g., 8, 9, 10, 11, or 12 aspartate residues). In some embodiments, the polyanionic peptide contains (e.g., consists of) 5 aspartate residues. In some embodiments, the polyanionic peptide contains (e.g., consists of) 6 aspartate residues. In some embodiments, the polyanionic peptide contains (e.g., consists of) 7 aspartate residues. In some embodiments, the polyanionic peptide contains (e.g., consists of) 8 aspartate residues. In some embodiments, the polyanionic peptide contains (e.g., consists of) 9 aspartate residues. In some embodiments, the polyanionic peptide contains (e.g., consists of) 10 aspartate residues. In some embodiments, the polyanionic peptide contains (e.g., consists of) 11 aspartate residues. In some embodiments, the polyanionic peptide contains (e.g., consists of) 12 aspartate residues. In some embodiments, the polyanionic peptide contains (e.g., consists of) 13 aspartate residues. In some embodiments, the polyanionic peptide contains (e.g., consists of) 14 aspartate residues. In some embodiments, the polyanionic peptide contains (e.g., consists of) 15 aspartate residues.
[0518] The polyanionic peptide may be a peptide of the formula D.sub.n, wherein D designates an aspartate residue and n is an integer from 1 to 25. For instance, the polyanionic peptide may be of the formula D.sub.1, D.sub.2, D.sub.3, D.sub.4, D.sub.5, D.sub.6, D.sub.7, D.sub.8, D.sub.9, D.sub.10, D.sub.11, D.sub.12, D.sub.13, D.sub.14, D.sub.15, D.sub.16, D.sub.17, D.sub.18, D.sub.19, D.sub.20, D.sub.21, D.sub.22, D.sub.23, D.sub.24, or D.sub.25. In some embodiments, the peptide is a peptide of the formula X.sub.nD.sub.mX.sub.oD.sub.p, wherein D designates an aspartate residue, each X independently designates any naturally-occurring amino acid, m represents an integer from 1 to 25, and n and o each independently represent integers from 0 to 5, and p represents an integer from 1 to 10.
[0519] In some embodiments, the aspartate residues are consecutive. In some embodiments, the aspartate residues are discontinuous.
[0520] In some embodiments, the ratio of amino acids bearing a side-chain that is negatively-charged at physiological pH to the total quantity of amino acids in the polyanionic peptide is from about 0.5 to about 2.0.
Bisphosphonates
[0521] Targeting moieties that may be used in conjunction with the compositions and methods described herein include bisphosphonates. Bisphosphonates are pyrophosphate analogues in which the oxygen bridge has been replaced by a carbon with various side chains (PCP). Like pyrophosphate, bisphosphonates bind with high affinity to the bone mineral, hydroxyapatite, due, at least in part, to the strong electrostatic interaction between the anionic phosphonate substituents within these compounds and positively-charged calcium ions within the hydroxyapatite matrix. Bisphosphonates, thus, can be used as targeting moieties to localize a therapeutic agent, such as a TGF- antagonist described herein, to bone tissue. Exemplary bisphosphonates useful in conjunction with the compositions and methods described herein include compounds represented by Formula (I), below,
##STR00002##
[0522] and pharmaceutically acceptable salts thereof, wherein X and Y are each independently hydrogen, halogen, hydroxy, optionally substituted alkoxy, optionally substituted aryloxy, optionally substituted heteroaryloxy, mercapto, optionally substituted alkylthio, optionally substituted arylthio, optionally substituted heteroarylthio, amino, optionally substituted alkylamino, optionally substituted arylamino, optionally substituted heteroarylamino, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, or the like.
[0523] For instance, particular bisphosphonates that may be used as targeting moieties in the conjugates described herein include those set forth in Table 13, below.
TABLE-US-00019 TABLE 13 Exemplary bisphosphonate targeting moieties Bisphosphonate X Y Etidronate
[0524] When used herein in the context of a conjugate, terms for bisphosphonates, such as etidronate, clodronate, tiludronate, pamidronate, neridronate, olpadronate, alendronate, ibandronate, risedronate, and zoledronate, set forth in Table 13, above, refer to a form of the bisphosphonate that is covalently bound to the rest of the conjugate. For instance, a bisphosphonate may be conjugated to a TGF- antagonist described herein, such as a fusion protein containing one or more domains of TGF- receptor II each joined to one or more domains of TGF- receptor III, by modifying one or more substituents of the bisphosphonate to render the molecule compatible with conjugation methods known in the art or described herein. Particularly, to prepare a bisphosphonate for conjugation to a TGF- antagonist described herein, such as by way of a linker, a moiety on the bisphosphonate may be converted to a nucleophile, electrophile, or other reactive species, thereby rendering the bisphosphonate suitable for reaction with a linker or directly with a TGF- antagonist. Exemplary methods for converting bisphosphonate compounds into reactive substrates suitable for conjugation are known in the art and are described, for example, in Uludag et al., Biotechnol. Prog. 16:258-267 (2000), the disclosure of which is incorporated herein by reference in its entirety.
Monoclonal Antibodies
[0525] Exemplary TGF- receptor fusion proteins may be bound to the N-terminal of an Fc domain of an immunoglobulin, either directly or via a hinge linker. Alternatively, exemplary TGF- receptor fusion proteins may be bound to the C-terminal of an Fc domain of an immunoglobulin, either directly or via a hinge linker. A targeting moiety may be bound to the N-terminal of the Fc domain of the immunoglobulin either directly or via a targeting linker. Similarly, a targeting moiety may be bound to the C-terminal of the Fc domain of the immunoglobulin. Finally, the targeting moiety may be bound either directly or via a targeting linker to the C-terminal of the exemplary TGF- receptor fusion proteins.
Fc Domain of an Immunoglobulin
[0526] The Fc domain of the immunoglobulin may comprises the immunoglobulin CH2 and CH3 domain and, optionally, at least a part of the hinge region. The Fc domain may be an IgG, IgM, IgD or IgE immunoglobulin domain or a modified immunoglobulin domain derived, therefrom. The IgG immunoglobulin domain may be selected from IgG1, IgG2, IgG3, or IgG4 domains or from modified domains such as are described in U.S. Pat. No. 5,925,734. The immunoglobulin domain may exhibit effector functions, particularly effector functions selected from ADCC and/or CDC. In some embodiments, however, modified immunoglobulin domains having modified, e.g. at least partially deleted, effector functions may be used.
Signal Peptides
[0527] Conjugates composed of proteinogenic amino acids and that may be used in conjunction with the compositions and methods described herein may contain a signal peptide, such as an N-terminal peptide capable of directing excretion of the conjugate from a mammalian cell. Exemplary signal peptides include the albumin signal peptide, MKWVTFLLLLFISGSAFSAAA (SEQ ID NO: 4) or alpha-lactalbumin peptide, MMSFVSLLLVGILFHATQ (SEQ ID NO: 42). Specific signal peptides, such as those described herein, can improve manufacturing of the TGF- antagonists of the invention, and can be useful for in vivo therapeutic administration of nucleic acids encoding the TGF- antagonists of the invention.
[0528] Exemplary conjugates that contain the albumin signal peptide include those that have the amino acid sequence of SEQ ID NO: 5, as well as those that have at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity thereto). The protein designated by SEQ ID NO: 5 contains a TGF- receptor fusion protein composed of an N-terminal human TGF- receptor II ectodomain, a central rat TGF- receptor III endoglin domain, and a C-terminal TGF- receptor II ectodomain. This TGF- receptor fusion protein is bound at its C-terminus to a decaaspartate (D.sub.10) hydroxyapatite-binding polyanionic peptide by way of a glycine- and serine-containing peptidic linker, and is bound at its N-terminus to the albumin signal peptide of SEQ ID NO: 4.
TABLE-US-00020 Exemplary TGF-antagonist conjugate with signal peptide SEQ ID NO: 5 MKWVTFLLLLFISGSAFSAAANGAVKFPQLCKFCDVRFSTCDNQKSCMSN CSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPK CIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGLGPVESSPGH GLDTAAAGPEPSTRCELSPINASHPVQALMESFTVLSGCASHGTTGLPRE VHVLNLRSTDQGPGQRQREVTLHLNPIASVHTHHKPIVFLLNSPQPLVWR LKTERLAAGVPRLFLVSEGSVVQFPSGNFSLTAETEERNFPQENEHLLRW AQKEYGAVTSFTELKIARNIYIKVGEDQVFPPTCNIGKNFLSLNYLAEYL QPKAAEGCVLPSQPHEKEVHIIELITPSSNPYSAFQVDIIVDIRPAQEDP EVVKNLVLILKSKKSVNWVIKSFDVKGNLKVIAPNSIGFGKESERSMTMT KLVRDDIPSTQENLMKWALDAGYRPVTSYTMAPVANRFHLRLENNEEMRD EEVHTIPPELRILLDPDKLPQLCKFCDVRFSTCDNQKSCMSNCSITSICE KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKK PGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSGDDD DDDDDDD,
Peptide Synthesis Techniques
[0529] Systems and processes for performing solid phase peptide synthesis of conjugates described herein include those that are known in the art and have been described, for instance, in U.S. Pat. Nos. 9,169,287; 9,388,212; 9,206,222; 6,028,172; and 5,233,044, among others, the disclosures of each of which are incorporated herein by reference as they pertain to protocols and techniques for the synthesis of peptides on solid support. Solid phase peptide synthesis is a known process in which amino acid residues are added to peptides that have been immobilized on a solid support, such as a polymeric resin (e.g., a hydrophilic resin, such as a polyethylene-glycol-containing resin, or hydrophobic resin, such as a polystyrene-based resin).
[0530] Peptides, such as those containing protecting groups at amino, hydroxy, thiol, and carboxy substituents, among others, may be bound to a solid support such that the peptide is effectively immobilized on the solid support. For example, the peptides may be bound to the solid support via their C termini, thereby immobilizing the peptides for subsequent reaction in at a resin-liquid interface.
[0531] The process of adding amino acid residues to immobilized peptides can include exposing a deprotection reagent to the immobilized peptides to remove at least a portion of the protection groups from at least a portion of the immobilized peptides. The deprotection reagent exposure step can be configured, e.g., such that side-chain protection groups are preserved, while N-termini protection groups are removed. For instance, an exemplary amino protecting may contain fluorenylmethyloxycarbonyl (Fmoc). A deprotection reagent containing piperidine (e.g., a piperidine solution in an appropriate organic solvent, such as dimethyl formamide (DMF)) may be exposed to the immobilized peptides such that the Fmoc protecting groups are removed from at least a portion of the immobilized peptides. Other protecting groups suitable for the protection of amino substituents include, for instance, the tert-butyloxycarbonyl (Boc) moiety. A deprotection reagent comprising a strong acid, such as trifluoroacetic acid (TFA) may be exposed to immobilized peptides containing a Boc-protected amino substituent so as to remove the Boc protecting group by an ionization process. In this way, peptides can be protected and deprotected at specific sites, such as at one or more side-chains or at the N- or C-terminus of an immobilized peptide so as to append chemical functionality regioselectively at one or more of these positions. This can be used, for instance, to derivatize a side-chain of an immobilized peptide, or to synthesize a peptide, e.g., from the C-terminus to the N-terminus.
[0532] The process of adding amino acid residues to immobilized peptides can include, for instance, exposing protected, activated amino acids to the immobilized peptides such that at least a portion of the activated amino acids are covalently bonded to the immobilized peptides to form newly-bonded amino acid residues. For example, the peptides may be exposed to activated amino acids that react with the deprotected N-termini of the peptides so as to elongate the peptide chain by one amino acid. Amino acids can be activated for reaction with the deprotected peptides by reaction of the amino acid with an agent that enhances the electrophilicity of the carbonyl carbon of the amino acid. For example, phosphonium and uronium salts can, in the presence of a tertiary base (e.g., diisopropylethylamine (DIPEA) and triethylamine (TEA), among others), convert protected amino acids into activated species (for example, BOP, PyBOP, HBTU, and TBTU all generate HOBt esters). Other reagents can be used to help prevent racemization that may be induced in the presence of a base. These reagents include carbodiimides (for example, DCC or WSCDI) with an added auxiliary nucleophile (for example, 1-hydroxy-benzotriazole (HOBt), 1-hydroxy-azabenzotriazole (HOAt), or HOSu) or derivatives thereof. Another reagent that can be utilized to prevent racemization is TBTU. The mixed anhydride method, using isobutyl chloroformate, with or without an added auxiliary nucleophile, can also be used, as well as the azide method, due to the low racemization associated with this reagent. These types of compounds can also increase the rate of carbodiimide-mediated couplings, as well as prevent dehydration of Asn and Gln residues. Typical additional reagents include also bases such as N,N-diisopropylethylamine (DIPEA), triethylamine (TEA) or N-methylmorpholine (NMM). These reagents are described in detail, for instance, in U.S. Pat. No. 8,546,350, the disclosure of which is incorporated herein in its entirety.
[0533] Cyclic peptides can be synthesized using solid-phase peptide synthesis techniques. For instance, a side-chain substituent, such as an amino, carboxy, hydroxy, or thiol moiety can be covalently bound to a resin, leaving the N-terminus and C-terminus of the amino acid exposed in solution. The N- or C-terminus can be chemically protected, for instance, while reactions are carried out that elongate the peptide chain. The termini of the peptide can then be selectively deprotected and coupled to one another while the peptide is immobilized by way of the side-chain linkage to the resin. Techniques and reagents for the synthesis of head-to-tail cyclic peptides are known in the art and are described, for instance, in U.S. Pat. Nos. 9,388,212 and 7,589,170, the disclosures of which are incorporated herein by reference in their entirety.
Linkers for Fusion Protein and Conjugate Preparation
Synthetic Linkers
[0534] A variety of linkers can be used to covalently couple reactive residues within a TGF- antagonist, such as a TGF- receptor or a domain, fragment, or variant thereof, to another TGF- receptor or a domain, fragment, or variant thereof in the production of a TGF- receptor fusion protein, to the Fc domain of an immunoglobulin, or to a bone-targeting moiety, such as a polyanionic peptide that binds hydroxyapatite, in the formation of a therapeutic conjugate as described herein. Exemplary linkers include those that may be cleaved, for instance, by enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage (see, for example, Leriche et al., Bioorg. Med. Chem., 20:571-582, 2012, the disclosure of which is incorporated herein by reference as it pertains to linkers suitable for chemical coupling). Examples of linkers useful for the synthesis of conjugates described herein include those that contain electrophiles, such as Michael acceptors (e.g., maleimides), activated esters, electron-deficient carbonyl compounds, and aldehydes, among others, suitable for reaction with nucleophilic substituents present within antibodies, antigen-binding fragments, and ligands, such as amine and thiol moieties. For instance, linkers suitable for the synthesis of therapeutic conjugates include, without limitation, alkyl, cycloalkyl, and heterocycloalkyl linkers, such as open-chain ethyl, propyl, butyl, hexyl, heptyl, octyl, nonyl, or decyl chains, cyclohexyl groups, cyclopentyl groups, cyclobutyl groups, cyclopropyl groups, piperidinyl groups, morpholino groups, or others containing two reactive moieties (e.g., halogen atoms, aldehyde groups, ester groups, acyl chloride groups, acyl anhydride groups, tosyl groups, mesyl groups, or brosyl groups, among others, that can be displaced by reactive nucleophilic atoms present within a TGF- antagonist peptide and/or bone-targeting moiety), aryl or heteroaryl linkers, such as benzyl, napthyl, or pyridyl groups containing two halomethyl groups that can be displaced by reactive nucleophilic atoms present within a TGF- antagonist peptide and/or bone-targeting moiety. Exemplary linkers include succinimidyl 4-(N-maleimidomethyl)-cyclohexane-L-carboxylate (SMCC), N-succinimidyl iodoacetate (SIA), sulfo-SMCC, m-maleimidobenzoyl-N-hydroxysuccinimidyl ester (MBS), sulfo-MBS, and succinimidyl iodoacetate, among others described, for instance, Liu et al., 18:690-697, 1979, the disclosure of which is incorporated herein by reference as it pertains to linkers for chemical conjugation. Additional linkers include the non-cleavable maleimidocaproyl linkers, which are described by Doronina et al., Bioconjugate Chem. 17:14-24, 2006, the disclosure of which is incorporated herein by reference as it pertains to linkers for chemical conjugation.
[0535] Additional linkers through which one component of a conjugate may be bound to another as described herein include linkers that are covalently bound to one component of the conjugate (e.g., a TGF- receptor or domain, fragment, or variant thereof) on one end of the linker and, on the other end of the linker, contain a chemical moiety formed from a coupling reaction between a reactive substituent present on the linker and a reactive substituent present within the other component of the conjugate (e.g., another TGF- receptor or domain, fragment, or variant thereof, or a hydroxyapatite-binding moiety, such as a polyanionic peptide). Exemplary reactive substituents that may be present within a component of the conjugate include, without limitation, hydroxyl moieties of serine, threonine, and tyrosine residues; amino moieties of lysine residues; carboxyl moieties of aspartic acid and glutamic acid residues; and thiol moieties of cysteine residues, as well as propargyl, azido, haloaryl (e.g., fluoroaryl), haloheteroaryl (e.g., fluoroheteroaryl), haloalkyl, and haloheteroalkyl moieties of non-naturally occurring amino acids. Linkers useful in conjunction with the conjugates described herein include, without limitation, linkers containing chemical moieties formed by coupling reactions as depicted in Table 14 below. Curved lines designate points of attachment to each component of the conjugate.
TABLE-US-00021 TABLE 14 Exemplary chemical moieties formed by coupling reactions in the formation of TGF- antagonist conjugates Exemplary Coupling Reactions Chemical Moiety Formed by Coupling Reactions [3 + 2] Cycloaddition
Peptidic Linkers
[0536] In addition to the synthetic linkers described above, the binding of one component of a TGF- receptor fusion protein to another, or one component of a therapeutic conjugate to another (e.g., a TGF- receptor or TGF- receptor fusion protein to a hydroxyapatite-binding moiety) can be effectuated by way of a peptide linker, also referred to as a peptidic linker. Most typically, the peptide linker contains 50 or fewer amino acids, e.g., 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 3, 4, 2, or 1 amino acid(s). In certain instances, the sequence of the peptide linker is a non-TGF- type II or type III receptor amino acid sequence. In other instances, the sequence of the peptide linker is additional TGF- type II or type III receptor amino acid sequence, e.g., the 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, to 50 or fewer amino acids flanking the carboxy an/or amino terminal ends of the binding domains. TGF- receptor fusion proteins and therapeutic conjugates composed of proteinogenic amino acids in which one or more components are joined by a peptide linker can be prepared, for instance, by expressing a nucleic acid encoding the linker in combination with the components of the fusion protein or conjugate. Exemplary peptide linkers include those that contain one or more glycine residues. Such linkers may be sterically flexible due to the ability of glycine to access a variety of torsional angles.
[0537] For instance, peptide linkers useful in conjunction with the compositions and methods described herein include one or more glycines, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, or more glycines. For example, the linker may comprise (GGG)n, where n=1, 2, 3, 4, 5, 6, 7, etc., such as GGG (SEQ ID NO: 6), and optional adaptor amino acids. Additional examples of peptidic linkers include those that also contain one or more polar amino acids, such as serine or threonine. For instance, linkers useful in conjunction with the compositions and methods described herein include glycine-serine linker, which contain a repeating amino acid sequence of the formula the sequence of (GGGS)n, where n=1, 2, 3, 4, 5, etc. (SEQ ID NO: 60), or the sequence of (GGGGS)n, where n=1, 2, 3, 4, 5, etc. (SEQ ID NO: 61), such as the peptide GGGGS (SEQ ID NO: 7) or GGGGSGGGGSGGGGSG (SEQ ID NO: 8), as well as those that contain one or more cationic or anionic residues, such as a lysine, arginine, aspartate, or glutamate residue.
[0538] Additional peptide linkers useful in conjunction with the compositions and methods described herein include amino acid sequences listed in SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 402, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, and SEQ ID NO: 59.
Methods for the Expression of Conjugates in Host Cells
[0539] In addition to synthetic chemistry techniques such as those described above, TGF- antagonists and conjugates described herein (e.g., protein conjugates wherein the TGF- antagonist is bound to a bone-targeting moiety by one or more peptide bonds) can be expressed in host cells, for instance, by delivering to the host cell a nucleic acid encoding the conjugate protein. The sections that follow describe a variety of established techniques that can be used for the purposes of delivering nucleic acids encoding therapeutic TGF- antagonists and conjugates described herein to a host cell for the purposes of expressing the antagonist and conjugate protein.
Transfection Techniques
[0540] Techniques that can be used to introduce a polynucleotide, such as nucleic acid encoding a TGF- antagonist peptide describe herein, into a cell (e.g., a mammalian cell, such as a human cell) are well known in the art. For instance, electroporation can be used to permeabilize mammalian cells (e.g., human cells) by the application of an electrostatic potential to the cell of interest. Mammalian cells, such as human cells, subjected to an external electric field in this manner are subsequently predisposed to the uptake of exogenous nucleic acids. Electroporation of mammalian cells is described in detail, e.g., in Chu et al., Nucleic Acids Research 15:1311 (1987), the disclosure of which is incorporated herein by reference. A similar technique, Nucleofection, utilizes an applied electric field in order to stimulate the uptake of exogenous polynucleotides into the nucleus of a eukaryotic cell. Nucleofection and protocols useful for performing this technique are described in detail, e.g., in Distler et al., Experimental Dermatology 14:315 (2005), as well as in US 2010/0317114, the disclosures of each of which are incorporated herein by reference.
[0541] Additional techniques useful for the transfection of cells of interest include the squeeze-poration methodology. This technique induces the rapid mechanical deformation of cells in order to stimulate the uptake of exogenous DNA through membranous pores that form in response to the applied stress. This technology is advantageous in that a vector is not required for delivery of nucleic acids into a cell, such as a human cell. Squeeze-poration is described in detail, e.g., in Sharei et al., Journal of Visualized Experiments 81:e50980 (2013), the disclosure of which is incorporated herein by reference.
[0542] Lipofection represents another technique useful for transfection of cells. This method involves the loading of nucleic acids into a liposome, which often presents cationic functional groups, such as quaternary or protonated amines, towards the liposome exterior. This promotes electrostatic interactions between the liposome and a cell due to the anionic nature of the cell membrane, which ultimately leads to uptake of the exogenous nucleic acids, for instance, by direct fusion of the liposome with the cell membrane or by endocytosis of the complex. Lipofection is described in detail, for instance, in U.S. Pat. No. 7,442,386, the disclosure of which is incorporated herein by reference. Similar techniques that exploit ionic interactions with the cell membrane to provoke the uptake of foreign nucleic acids include contacting a cell with a cationic polymer-nucleic acid complex. Exemplary cationic molecules that associate with polynucleotides so as to impart a positive charge favorable for interaction with the cell membrane include activated dendrimers (described, e.g., in Dennig, Topics in Current Chemistry 228:227 (2003), the disclosure of which is incorporated herein by reference) and diethylaminoethyl (DEAE)-dextran, the use of which as a transfection agent is described in detail, for instance, in Gulick et al., Current Protocols in Molecular Biology 40:1:9.2:9.2.1 (1997), the disclosure of which is incorporated herein by reference. Magnetic beads are another tool that can be used to transfect cells in a mild and efficient manner, as this methodology utilizes an applied magnetic field in order to direct the uptake of nucleic acids. This technology is described in detail, for instance, in US 2010/0227406, the disclosure of which is incorporated herein by reference.
[0543] Another useful tool for inducing the uptake of exogenous nucleic acids by cells is laserfection, a technique that involves exposing a cell to electromagnetic radiation of a particular wavelength in order to gently permeabilize the cells and allow polynucleotides to penetrate the cell membrane. This technique is described in detail, e.g., in Rhodes et al., Methods in Cell Biology 82:309 (2007), the disclosure of which is incorporated herein by reference.
[0544] Microvesicles represent another potential vehicle that can be used to modify the genome of a cell according to the methods described herein. For instance, microvesicles that have been induced by the co-overexpression of the glycoprotein VSV-G with, e.g., a genome-modifying protein, such as a nuclease, can be used to efficiently deliver proteins into a cell that subsequently catalyze the site-specific cleavage of an endogenous polynucleotide sequence so as to prepare the genome of the cell for the covalent incorporation of a polynucleotide of interest, such as a gene or regulatory sequence. The use of such vesicles, also referred to as Gesicles, for the genetic modification of eukaryotic cells is described in detail, e.g., in Quinn et al., Genetic Modification of Target Cells by Direct Delivery of Active Protein [abstract]. In: Methylation changes in early embryonic genes in cancer [abstract], in: Proceedings of the 18th Annual Meeting of the American Society of Gene and Cell Therapy; 2015 May 13, Abstract No. 122.
Incorporation of Genes by Gene Editing Techniques
[0545] In addition to the above, a variety of tools have been developed that can be used for the incorporation of exogenous genes, e.g., exogenous genes encoding a TGF- antagonist peptide or conjugate described herein, into cells, such as a human cell. One such method that can be used for incorporating polynucleotides encoding a TGF- antagonist or conjugate described herein into cells involves the use of transposons. Transposons are polynucleotides that encode transposase enzymes and contain a polynucleotide sequence or gene of interest flanked by 5 and 3 excision sites. Once a transposon has been delivered into a cell, expression of the transposase gene commences and results in active enzymes that cleave the gene of interest from the transposon. This activity is mediated by the site-specific recognition of transposon excision sites by the transposase. In some instances, these excision sites may be terminal repeats or inverted terminal repeats. Once excised from the transposon, the gene encoding a TGF- antagonist peptide or conjugate can be integrated into the genome of a mammalian cell by transposase-catalyzed cleavage of similar excision sites that exist within the nuclear genome of the cell. This allows the gene of interest to be inserted into the cleaved nuclear DNA at the complementary excision sites, and subsequent covalent ligation of the phosphodiester bonds that join the gene encoding the TGF- antagonist peptide or conjugate to the DNA of the mammalian cell genome completes the incorporation process. In some cases, the transposon may be a retrotransposon, such that the gene encoding the TGF- antagonist peptide or conjugate is first transcribed to an RNA product and then reverse-transcribed to DNA before incorporation in the mammalian cell genome. Exemplary transposon systems include the piggybac transposon (described in detail in, e.g., WO 2010/085699) and the sleeping beauty transposon (described in detail in, e.g., US 2005/0112764), the disclosures of each of which are incorporated herein by reference as they pertain to transposons for use in gene delivery to a cell of interest, such as a mammalian cell (e.g., a human cell).
[0546] Another tool for the integration of genes encoding TGF- antagonist peptides or conjugates described herein into the genome of a cell, such as a human cell, is the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system, a system that originally evolved as an adaptive defense mechanism in bacteria and archaea against viral infection. The CRISPR/Cas system includes palindromic repeat sequences within plasmid DNA and an associated Cas9 nuclease. This ensemble of DNA and protein directs site specific DNA cleavage of a sequence of interest by first incorporating foreign DNA into CRISPR loci. Polynucleotides containing these foreign sequences and the repeat-spacer elements of the CRISPR locus are in turn transcribed in a host cell to create a guide RNA, which can subsequently anneal to a particular sequence and localize the Cas9 nuclease to this site. In this manner, highly site-specific cas9-mediated DNA cleavage can be engendered in a foreign polynucleotide because the interaction that brings cas9 within close proximity of the DNA molecule of interest is governed by RNA:DNA hybridization. As a result, one can theoretically design a CRISPR/Cas system to cleave any DNA molecule of interest. This technique has been exploited in order to edit eukaryotic genomes (Hwang et al., Nature Biotechnology 31:227 (2013)) and can be used as an efficient means of site-specifically editing cell genomes in order to cleave DNA prior to the incorporation of a gene encoding a gene. The use of CRISPR/Cas to modulate gene expression has been described in, for instance, U.S. Pat. No. 8,697,359, the disclosure of which is incorporated herein by reference as it pertains to the use of the CRISPR/Cas system for genome editing. Alternative methods for site-specifically cleaving genomic DNA prior to the incorporation of a gene of interest in a cell include the use of zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Unlike the CRISPR/Cas system, these enzymes do not contain a guiding polynucleotide to localize to a specific sequence. Sequence specificity is instead controlled by DNA binding domains within these enzymes. The use of ZFNs and TALENs in genome editing applications is described, e.g., in Urnov et al., Nature Reviews Genetics 11:636 (2010); and in Joung et al., Nature Reviews Molecular Cell Biology 14:49 (2013), the disclosure of each of which are incorporated herein by reference as they pertain to compositions and methods for genome editing.
[0547] Additional genome editing techniques that can be used to incorporate polynucleotides encoding a TGF- antagonist or conjugate described herein into the genome of a cell of interest, such as a mammalian cell, include the use of ARCUS meganucleases that can be rationally designed so as to site-specifically cleave genomic DNA. The use of these enzymes for the incorporation of genes encoding a TGF- antagonist peptide or conjugate described herein into the genome of a mammalian cell (e.g., a human cell) is advantageous in view of the defined structure-activity relationships that have been established for such enzymes. Single chain meganucleases can be modified at certain amino acid positions in order to create nucleases that selectively cleave DNA at desired locations, enabling the site-specific incorporation of a gene of interest into the nuclear DNA of a cell, such as a mammalian cell (e.g., a human cell). These single-chain nucleases have been described extensively in, for example, U.S. Pat. Nos. 8,021,867 and 8,445,251, the disclosures of each of which are incorporated herein by reference as they pertain to compositions and methods for genome editing.
Viral Vectors for Nucleic Acid Delivery
[0548] Viral genomes provide a rich source of vectors that can be used for the efficient delivery of exogenous genes encoding TGF- antagonist peptides and conjugates described herein into the genome of a cell (e.g., a mammalian cell, such as a human cell). Viral genomes are particularly useful vectors for gene delivery because the polynucleotides contained within such genomes are typically incorporated into the genome of a cell by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle, and do not require added proteins or reagents in order to induce gene integration. Examples of viral vectors include AAV, retrovirus, adenovirus (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g. measles and Sendai), positive strand RNA viruses, such as picornavirus and alphavirus, and double stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox). Other viruses useful for delivering polynucleotides encoding TGF- antagonist peptides described herein to a mammalian cell (e.g., a human cell) include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example. Examples of retroviruses include: avian leukosis-sarcoma, mammalian C-type, B-type viruses, D-type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996). Other examples include murine leukemia viruses, murine sarcoma viruses, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus and lentiviruses. Other examples of vectors are described, for example, in U.S. Pat. No. 5,801,030, the disclosure of which is incorporated herein by reference as it pertains to viral vectors for use in gene delivery.
Methods of Therapeutic Treatment
[0549] The present invention is based, in part, on the discovery that muscle weakness in diseases associated with elevated TGF- activity and/or elevated bone turnover can be restored and/or improved through the use of TGF- antagonists. In the case of osteogenesis imperfecta, active TGF- is elevated as a consequence of defective collagen and/or excessive release of TGF- as a result of increased osteoclast activity. The compositions and methods described herein are based, in part, on the finding that bone-derived TGF- binds to TGF- receptors on the surface of adjacent muscle, promoting internal signaling via phosphorylation of SMAD2/3 and inducing transcription of a variety of mRNAs associated with cell function. In muscle, elevated TGF- induces transcription of the Nox4 gene, which encodes NADPH oxidase 4. This enzyme oxidizes the RyR1 calcium channel 1 to yield reactive oxygen species. Oxidation of RyR1 leads to loss of binding by the negative regulator calstabin1, and the ensuing opening of RyR1 causes Ca.sup.2+ to leak from the sarcoplasma reticulum, thereby depleting Ca.sup.2+ stores needed for normal muscle contraction and resulting in decreased muscle strength.
[0550] The compositions and methods described herein can be used to restore and/or improve muscle function in a patient, such as a human patient suffering from a disease associated with elevated TGF- signaling, such as elevated bone turnover (e.g., osteogenesis imperfecta, among others described herein), and a muscle disorder, such as muscular dystrophy. For instance, using the compositions and methods described herein, a TGF- antagonist, such as a TGF- antagonist conjugated to a bone-targeting moiety, may be administered to a patient suffering from a disease associated with elevated TGF- signaling, such as elevated bone turnover (e.g., a human patient suffering from osteogenesis imperfecta), so as to restore and/or improve muscle function in the patient.
[0551] Additionally, the compositions and methods described herein may be used to determine the propensity of a patient (e.g., a human patient suffering from elevated TGF- signaling, osteogenesis imperfecta, or other conditions associated with elevated bone turnover) to respond to TGF- antagonist therapy. Using a method for assessing muscle function (e.g., muscle mass, muscle strength, or muscle quality) described herein or known in the art, a physician may determine that the patient exhibits a level of muscle function that is less than that of a muscle function reference level, such as the level of muscle function of a healthy patient (e.g., a healthy patient of the same gender, age, and/or body mass, among other characteristics, as the patient) or the level of muscle function exhibited by the patient as assessed before the patient was diagnosed as having the disease. A finding that the patient exhibits, for instance, a level of muscle function that is less than that of the muscle function reference level may indicate that the patient is likely to respond to treatment with a TGF- antagonist, such as a TGF- antagonist described herein. Since TGF- antagonism can restore and/or improve muscle function in patients suffering from osteogenesis imperfecta and other disorders associated with elevated bone turnover, patients that exhibit reduced muscle function relative to a muscle function reference level (e.g., the level of muscle function of a healthy patient, such as a healthy patient of the same gender, age, and/or body mass, among other characteristics, as the patient, or the level of muscle function exhibited by the patient as assessed before the patient was diagnosed as having the disease) are particularly likely to benefit from treatment with a TGF- antagonist or conjugate thereof, such as a TGF- antagonist or conjugated described herein.
Routes of Administration
[0552] The TGF- antagonists or conjugates described herein can be administered to a mammalian subject (e.g., a human) suffering from a disease associated with elevated TGF- activity, e.g., heightened bone turnover, and/or muscle wasting, in order, for example, to improve the condition of the patient, e.g. to improve and/or restore muscle function, by attenuating TGF- signaling, including at the site of bone tissue. The compositions described herein (e.g., compositions containing a TGF- antagonist or conjugate thereof of the invention) can be administered to a subject, e.g., via any of the routes of administration described herein, such as subcutaneously, intradermally, intramuscularly, intraperitoneally, intravenously, or orally, or by nasal or by epidural administration. Conjugates described herein can be formulated with excipients, biologically acceptable carriers, and may be optionally conjugated to, admixed with, or co-administered separately (e.g., sequentially) with additional therapeutic agents. The sections that follow describe exemplary conditions that can be treated using the conjugates and pharmaceutical compositions described herein.
Skeletal Disorders
[0553] Diseases and conditions that can be treated using the conjugates described herein include skeletal disorders, such as osteogenesis imperfecta (OI) (for instance, Type I osteogenesis imperfecta, Type II osteogenesis imperfecta, Type III osteogenesis imperfecta, Type IV osteogenesis imperfecta, Type V osteogenesis imperfecta, Type VI osteogenesis imperfecta, Type VII osteogenesis imperfecta, Type VIII osteogenesis imperfecta, Type XI osteogenesis imperfecta, Type X osteogenesis imperfecta, or Type XI osteogenesis imperfecta). These conditions are described, e.g., in Forlino, Nat. Rev. Endo. 7:540 (2011), the disclosure of which is incorporated herein by reference. Osteogenesis imperfecta encompasses a group of congenital bone disorders characterized by deficiencies in one or more proteins involved in bone matrix deposition or homeostasis. Though phenotypes vary among OI types, common symptoms include incomplete ossification of bones and teeth, reduced bone mass, brittle bones, and pathologic fractures. Type-I collagen is one of the most abundant connective tissue proteins in both calcified and non-calcified tissues. Accurate synthesis, post-translational modification, and secretion of type-I collagen are necessary for proper tissue development, maintenance, and repair. Most mutations identified in individuals with osteogenesis imperfecta result in reduced synthesis of type-I collagen, or incorrect synthesis and/or processing of type-I collagen.
[0554] In addition to mutations to the type-I collagen gene, other mutations in genes that participate in the intracellular trafficking and processing of collagens have been identified in individuals suffering from osteogenesis imperfecta. These genes include molecular chaperones, such as FK506 binding protein 10 (FKBPIO) and heat shock protein 47 (HSP47) (Alanay et al., 2010; Christiansen et al., 2010; Kelley et al., 2011). Additional mutations have been identified in intermolecular collagen cross-linking genes, such as procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2), and in members of the collagen prolyl hydroxylase family of genes, including leucine proline-enriched proteoglycan (leprecan) (LEPRE1), peptidylprolyl isomerase B (cyclophilin B) (CYPB), and cartilage associated protein (CRTAP) (Morello et al., 2006; Cabral et al., 2007; Baldridge et al., 2008; van Dijk et al., 2009; Choi et al., 2009; Barnes et al., 2010; Pyott et al., 2011). Mutations aside, proteins such as TGF- and its corresponding receptors are involved in the onset and propagation of osteogenesis imperfecta (Gebken et al., 2000).
[0555] TGF- expression may be regulated by molecules that bind type-I and type-II collagen. In some instances, TGF- expression is regulated by a small leucine rich proteoglycan (SLRP) and/or by decorin. In a certain embodiment, decorin does not bind type-I or type-II collagen in which the 3-hydroxyproline site is absent at position 986 of the type-I and/or type-II collagen molecules.
[0556] The vertebrate skeleton is comprised of bone, which is a living, calcified tissue that provides structure, support, protection, and a source of minerals for regulating ion transport. Bone is a specialized connective tissue that is comprised of both cellular and acellular components. The acellular extracellular matrix (ECM) contains both collagenous and non-collagenous proteins, both of which participate in the calcification process. A correctly secreted and aligned ECM is critical for proper bone formation. Pathology results when any of the ECM proteins are absent, malformed or misaligned, as is evidenced in osteogenesis imperfecta.
[0557] Under normal homeostatic conditions, osteoblasts and osteoclasts work in unison to maintain bone integrity. Pathology results when bone deposition and bone resorption become uncoupled. For example, osteopetrosis is a bone disease characterized by overly dense, hard bone that is a result of unresorptive osteoclasts, while osteoporosis is a bone disorder characterized by brittle, porous bones which can result from increased osteoclast activity. Osteogenesis imperfecta, in particular, can arise as a result of elevated TGF- expression, which causes an increase in osteoclast-mediated bone resorption. The conjugates described herein can be used to suppress bone resorption by attenuating TGF- signaling, for instance specifically at the site of pathological bone tissue. The conjugates described herein provide the advantageous pharmacological property of being able to inhibit TGF- selectively at the site of osseous tissue, thereby restoring bone turnover homeostasis (e.g., in patients suffering from osteogenesis imperfecta) while preserving the effects of TGF- signaling on healthy tissues.
[0558] Several methods can be used to measure and characterize the structure, density, and quality of bone, including histology and histomorphometry, atomic force microscopy, confocal Raman microscopy, nanoindentation, three-point bending test, X-ray imaging, and micro computed tomography (-CT). Using these exemplary techniques, for instance, one of skill in the art can monitor the progression of treatment and the effectiveness of therapy. For instance, an improvement in bone integrity, a slowing of bone resorption, and a restoration of homeostasis of bone turnover among patients suffering from osteogenesis imperfecta (e.g., as determined by one or more of the above methods, or other methods known in the art) can be indicators of effective therapeutic treatment.
[0559] Additional patients in which muscle function may be improved and/or restored using the compositions or methods described herein or diseases and conditions that can be treated with the conjugates described herein include, for instance, renal osteodystrophy, hyperparathyroid induced bone disease, diabetic bone disease, osteoarthritis, steroid induced bone disease, disuse osteoporosis, and Cerebral Palsy, McCune-Albright Syndrome, Gaucher Disease, Hyperoxaluria, Paget Disease of bone, and Juvenile Paget Disease, metastatic bone cancer (e.g., wherein the metastasis is a secondary metastasis to breast cancer or prostate cancer), osteoporosis, fibrous dysplasia, Calmurati-Engleman Disease, Marfan's Syndrome, osteoglophonic dysplasia, autosomal dominant osteopetrosis, osteoporosis, osteoporosis-pseudoglioma syndrome, juvenile, gerodermia osteodysplastica, Duchenne muscular dystrophy, osteosarcoma, osteogenesis imperfecta congenita, microcephaly, cataracts, pseudohypoparathyroidism, Cleidocranial Dysplasia, Dyskeratosis Congenita, Exudative Vitreoretinopathy 1, Schimmelpenning-Feuerstein-Mims Syndrome, Prader-Willi Syndrome, Achondrogenesis, Antley-Bixler Syndrome, Aspartylglucosaminuria, Celiac Disease, Cerebrooculofacioskeletal Syndrome 1, Lysinuric Protein Intolerance, neuropathy, dyskeratosis congenita, Ehlers-Danlos Syndrome, epiphyseal dysplasia, hyaline fibromatosis syndrome, Perrault Syndrome 1, hemochromatosis, homocystinuria (e.g., due to cystathionine beta-synthase deficiency), hypophosphatemic rickets with hypercalciuria, desbuquois dysplasia, multiple pterygium syndrome, lethal congenital contracture syndrome 1, mitochondrial DNA depletion Ssndrome 6 (hepatocerebral Type), Niemann-Pick Disease, osteopetrosis, porphyria, Rothmund-Thomson Syndrome, Wilson Disease, Dent Disease 1, occipital horn syndrome, hyperglycerolemia, hypophosphatemic rickets, Lowe Oculocerebrorenal Syndrome, renal tubulopathy, diabetes mellitus, cerebellar ataxia, vitamin D hydroxylation-deficient rickets, Warburg micro syndrome 1, Stuve-Wiedemann Syndrome, Blue Rubber Bleb Nevus syndrome, Singleton-Merten Syndrome, microcephalic osteodysplastic primordial dwarfism, dysosteosclerosis, Hallermann-Streiff Syndrome, Bruck Syndrome 1, multiple pterygium syndrome (e.g., X-Linked), spondylometaphyseal dysplasia with dentinogenesis imperfecta, Hall-Riggs Mental Retardation Syndrome, infantile multisystem neurologic disease with osseous fragility, acrocephalopolysyndactyly Type III, acroosteolysis, ACTH-independent macronodular adrenal hyperplasia, amino aciduria with mental deficiency, arthropathy, bone fragility (e.g., with craniosynostosis, ocular proptosis, hydrocephalus, and distinctive facial features), brittle cornea syndrome, cerebrotendinous xanthomatosis, Cri-Du-Chat Syndrome, dysplasia epiphysealis hemimelica, autosomal dominant Ehlers-Danlos Syndrome, familial osteodysplasia, Flynn-Aird Syndrome, gerodermia osteodysplastica, glycogen storage disease Ia, Hutchinson-Gilford Progeria Syndrome, Infantile Systemic Hyalinosis, hypertrichotic osteochondrodysplasia, hyperzincemia with functional zinc depletion, hypophosphatasia, autosomal dominant hypophosphatemic rickets, X-linked recessive hypophosphatemic rickets, Lichtenstein Syndrome, macroepiphyseal dysplasia (e.g., with osteoporosis wrinkled skin, and aged appearance), Menkes Disease, Mental Retardation (e.g., X-Linked, Snyder-Robinson type), Jansen type metaphyseal chondrodysplasia, microspherophakia-metaphyseal dysplasia, morquio syndrome a, Morquio Syndrome B, ossified ear cartilages (e.g., with mental deficiency, muscle wasting, and osteocraniostenosis), osteoporosis and oculocutaneous hypopigmentation syndrome, osteoporosis-pseudoglioma syndrome, juvenile osteoporosis, osteosclerosis with ichthyosis and fractures, ovarian dysgenesis 1, ovarian dysgenesis 2, ovarian dysgenesis 3, ovarian dysgenesis 4, pituitary adenoma, polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy, Prader-Willi Habitus, osteopenia, Okamoto type premature aging syndrome, Prieto X-linked mental retardation syndrome, pycnodysostosis, Pyle Disease, Reifenstein Syndrome, autosomal dominant distal renal tubular acidosis, Type 1 Schwartz-Jampel Syndrome, Type 2 Schwartz-Jampel Syndrome, Type 3 Schwartz-Jampel Syndrome, Type 4 Schwartz-Jampel Syndrome, X-linked spondyloepiphyseal dysplasia tarda, and Torg-Winchester Syndrome.
Muscular Disorders
[0560] In addition to treating skeletal disorders, the compositions and methods described herein can be used to treat muscle diseases, such as muscular dystrophies, including Duchenne muscular dystrophy (DMD). DMD represents the most common inherited neuromuscular disease, and is characterized by a lack of dystrophin, muscle wasting, fibrosis, and elevated TGF- signaling (Acua et al., Human Molecular Genetics 23:1237-1249 (2014), the disclosure of which is incorporated herein by reference). Particularly, TGF- signal transduction has been implicated in DMD pathology, and is known to stimulate fibrosis, promote myonecrosis, and inhibit muscle regeneration (Kemaladewi et al., Molecular TherapyNucleic Acids 3:e156 (2014) and Taniguti et al., Muscle & Nerve 43:82-87 (2011), the disclosure of which is incorporated herein by reference). By localizing to bone tissue and inhibiting the activity of TGF- in the proximity of skeletal muscle, the conjugates and pharmaceutical compositions described herein can suppress fibrotic and myonecrotic activity, thereby improving muscle function in patients suffering from muscular dystrophies, such as DMD. As in the case of the treatment of skeletal disorders, the conjugates described herein provide the beneficial property of being able to inhibit TGF- selectively at the site of skeletal-muscular interface, thereby improving muscle function (e.g., in patients suffering from a muscular dystrophy, such as DMD) while preserving the effects of TGF- signaling on healthy tissues.
[0561] In addition to treating DMD, the compositions and methods described herein can be used to treat various other muscular dystrophies, such as inherited muscular dystrophies associated with a laminin-2 deficiency. TGF- inhibition has shown beneficial effects in the treatment of a mouse model of laminin-2-deficient congenital muscular dystrophy. Particularly, it was found that chronic treatment of a mouse model with the TGF- inhibitor, Losartan, significantly increased the lifespan of the mouse, decreased the percentage of fibrotic areas in the muscle, reduced collagen deposits, and significantly improved both the hindlimb and forelimb muscle strength of the mutant mice (see, e.g., Elbaz et al., Ann. Neurol. 71:699-708 (2012), the disclosure of which is incorporated herein by reference).
[0562] Further, the compositions and methods described herein can be used to treat muscular dystrophy caused by mutations in caveolin-3. This form of muscular dystrophy is amenable to treatment with agents that reduce TGF- signaling, as it has been shown that caveolin-3-deficient mice treated with a TGF- receptor type I kinase inhibitor exhibited weight gain and a reduction in hindlimb muscle atrophy (see, e.g., Ohsawa et al., Lab. Invest. 92:1100-1114 (2012), the disclosure of which is incorporated herein by reference).
[0563] The compositions and methods described herein can additionally be used to treat acquired muscle diseases, such as sarcopenia. Sarcopenia is described as the loss of muscle function (e.g., muscle mass) that is characterized by impaired regeneration and increased frailty in older populations. Recent studies have suggested that TGF- signaling plays a significant role in the progression of this condition. It was recently shown that genetically normal, yet aged, sarcopenic muscle had reduced fibrosis and improved muscle function after injury when treated with Losartan (see, e.g., Burks et al., Sci. Transl. Med. 82:82ra37 (2011), the disclosure of which is incorporated herein by reference). Losartan also prevented the loss of muscle fibers in the exaggerated response to immobilization atrophy observed in sarcopenic muscle (Burks et al., 2011). Immobilization atrophy in aged muscle was found to be due to the loss of muscle fibers themselves, rather than to a reduction in fiber diameter. This loss of muscle fibers, the reduction in fibrosis, and the enhanced muscle regeneration with Losartan treatment were attributed to the blockade of both the canonical and non-canonical TGF- signaling pathways. Thus, sarcopenia, and the fibrosis associated with this condition, can be treated with TGF- antagonists. As with the muscular dystrophies described above, the conjugates described herein provide the beneficial property of being able to inhibit TGF- selectively at the site of skeletal-muscular interface, thereby improving muscle function (e.g., in patients suffering from an acquired muscle disease, such as sarcopenia) while preserving the effects of TGF- signaling on healthy tissues.
Methods of Assaying Muscle Function
[0564] The compositions (e.g., compositions containing a TGF- antagonist or conjugate thereof) and methods described herein can be used to treat a mammalian subject (e.g., a human) suffering from a disease associated with elevated TGF- signaling in order to improve muscle function in the subject. For instance, treatment of a patient suffering from a muscular dystrophy, such as DMD, may improve muscle function in the subject. This improvement in muscle function may be assessed, for instance, by any methodology known in the art for measuring muscle strength, muscle quality, muscle mass, and/or the general functional status of the subject. A variety of quantitative or qualitative approaches may be used to assess muscle function (e.g., manual muscle testing, dynamometry, isokinetics, cable tensiometry, muscle mechanography, imaging techniques, functional status assessments, or biochemical assays), examples of which are further described below. Using one or more such approaches to assess muscle function, for instance, one of skill in the art can identify subjects who exhibit reduced muscle function relative to a muscle function reference level (e.g., the level of muscle function of a healthy patient, such as a healthy patient of the same gender, age, and/or body mass, among other characteristics, as the patient) and therefore may benefit from treatment with the compositions described herein. Further, one or more of the methods described herein may be used to monitor changes (e.g., improvements or lack of improvement) in muscle function over time, e.g., to evaluate therapeutic efficacy. Given the range of accepted methodologies available for assessing muscle function, it will be appreciated by one skilled in the art that the particular methodologies used to assess muscle function in a subject may vary based on the skills or judgement of the practitioner carrying out the assessment. In some instances, one or more particular methodologies may be selected based on considerations of a subject's abilities or limitations, as deemed appropriate by a skilled artisan. Methods for assessing muscle function are described, for example, in Waning et al., Nature Medicine 21:1262-1275 (2015), the disclosure of which is incorporated herein by reference as it pertains to methods of assessing muscle function. Exemplary approaches for assessing muscle function are described in further detail, below.
[0565] In some instances, muscle function may be assessed by manual muscle testing (MMT). MMT is a procedure for the evaluation of the function of individual muscles and muscle groups based on the effective performance of a movement in relation to the forces of gravity and manual resistance. Various test positions and procedures for MMT and examples of common grading scales may be used with MMT (e.g., Medical Research Council, Daniels and Worthingham, or Kendall and McCreary grading scale). The particular grading system selected or additional devices (e.g., dynamometer) used during MMT may vary depending on the practitioner and/or the subject. See, for example, Hislop et al. (2013). Daniels and Worthingham's Muscle Testing: Techniques of Manual Examination and Performance Testing. Elsevier Health Sciences., the disclosure of which is incorporated herein by reference.
[0566] In some instances, muscle function may be assessed by dynamometry. Dynamometry includes methods of strength testing that use strength measuring devices (e.g., hand-grip, hand-held, fixed, and isokinetic dynamometers). For example, is some instances, a hand-held dynamometer (HHD) instrument is used to measure muscle function, e.g., during the aforementioned MMT. In some instances, a grip strength test may be used to assess muscle strength (e.g., upper extremity muscle force) using a hand-grip dynamometer. Further, a dynamometer can be used to measure the isometric muscle strength in the shoulder abductors, hip flexors, ankle dorsal flexor, and grip strength bilaterally, for instance. See, for example, Payton, C., & Bartlett, R. (Eds.). (2007). Biomechanical evaluation of movement in sport and exercise: the British Association of Sport and Exercise Sciences guide. Routledge, the disclosure of which is incorporated herein by reference.
[0567] In some instances, muscle function may be assessed by muscle mechanography. Muscle mechanography is a method that can quantitatively assess muscle function based on the performance of movements by the subject such as heel raises, chair rises, single two-legged countermovement jumps, serial one- or two-legged jumps (hopping), or sway on a ground reaction force plate. Muscle mechanography directly measures the applied force vector and calculates measures of muscle force, velocity, power, jump height, and balance or sway (i.e., the change of the center of gravity during a balance test).
[0568] In some instances, muscle function is assessed based on measurements of muscle cross-sectional area, volume, density, or mass using any known or otherwise effective technique that provides muscle area, volume or mass, such as DEXA, or using visual or imaging techniques (e.g., magnetic resonance imaging (MRI) or computed tomography (CT) scans). For example, in some instances, peripheral quantitative computer tomography (pQCT) may be used to measure the cross-sectional area or density of a muscle.
[0569] In some instances, muscle function is assessed based on clinical assays that assess the impact of elevated TGF- on muscles on a biochemical level by testing a muscle biopsy. For example, TGF- elevation can be confirmed via demonstration that the downstream signaling molecules SMAD2 and SMAD3 are activated. This can be measured by immunoblot analysis showing an increased amount of phosphorylated SMAD2 or SMAD3 is present relative to total SMAD2 or SMAD3 in muscle lysates. To assess involvement of NADPH oxidase 4, Nox4 mRNA can be measured using standard RT-PCR in muscle derived from individuals with bone disorders and can be compared to muscle from healthy individuals. Immunoblots of muscle lysates may also be performed to demonstrate oxidation and nitrosylation of RyR1, two downstream consequences of NADPH oxidase 4. Finally, co-immunoprecipation of RyR1 and its associated regulatory protein, Calstabin can be performed. Demonstration that calstabin binding to RyR1 is reduced in muscles from individuals with bone disorders relative to healthy individuals can be used as a surrogate to monitor calcium leak in muscles and associated muscle weakness.
[0570] Other non-limiting examples of methods to assess muscle function include the following: self-selected or usual walking gait speed (e.g., where gait speed is the distance traveled divided by the ambulation time); maximum walking gait speed; step length (e.g., wherein step length is the perpendicular distance between the heel of one foot-strike to the heel of the next foot-strike of the opposite foot); step time (e.g., wherein step time is the time elapsed from floor contact of one foot to floor contact of the next foot); stride length (e.g., wherein stride length is the perpendicular distance between the heel of one foot-strike to the heel of the next foot-strike of the same foot); stride time; base width (e.g., wherein base width is the perpendicular distance from the heel of one foot-strike to the line of progression between two foot-strikes of the opposite foot); step width; stride width; gait cycling time; stance time; swing time; double support phase (e.g., wherein double support phase is the phase of the gait cycle when both feet are in contact with the ground); gait parameters measured on an inclined plane, declined plane, or throughout progressively increased velocity on a treadmill; intraindividual variability for gait measures; chair rise test (e.g., wherein the amount of time to complete 5 chair rises is measured); Katz Index of Independence in Activities of Daily Living; Palliative Performance Scale; Tinetti Gait and Balance Scale; Star Excursion Balance Test; tandem standing and tandem walking to measure balance; single-leg dynamic postural sway on a force plate; single-leg stance time on a hard surface; single-leg stance time on a balance pad; manual muscle testing; isokinetic or isometric measurements of muscle strength; one-repetition maximum strength; maximum rate of force development; electromyography (EMG) of muscle; median activation frequency determined by EMG of soleus and gastrocnemius medialis during plantar-flexion; mean amplitude voltage determined by EMG of muscle; EMG of muscle before or after activity; EMG during stance perturbation, training or jumping; nerve stimulation twitch response (e.g., of the soleus and gastrocnemius muscle); reflex activity during flexion; Hoffman's reflex (H-reflex) or other mechanical stretch reflex; H-reflex measured during 2 tasks such as plantar flexion and stance perturbation; Berg Balance Scale; Short Physical Performance Battery; mechanography; jump height; twenty-meter sprint performance; ten yard sprint performance; forty yard sprint performance; countermovement jump power; bounce-drop jump power; and vertical impact force before jumping.
[0571] Muscle function can be based on one or more muscles or muscle groups in a subject, e.g., muscles associated with fingers, hands, arms, torso, abdominals, shoulders, back, neck, legs, knees, ankle, foot, or toes. For example, in some instances the muscle function may be tested for one or more muscles selected from one or more of the following muscles: pectoralis major, pectoralis minor, serratus anterior, flexor halluces brevis, flexor digitorum brevis, flexor hallucis longus, flexor digitorum longus, extensor digitorum longus and brevis, fibularis tertius, extensor hallucis longus and brevis, tibialis anterior, tibialis posterior, fibularis longus and brevis, triceps brachii and anconeus, latissimus dorsi, teres major, infraspinatus and teres minor, rhomboid and levator scapulae, middle trapezius, lower trapezius, soleus, adductor pollicis, abductor pollicis brevis, opponens pollicis, flexor pollicis longus, flexor pollicis brevis, extensor pollicis longus, extensor pollicis brevis, abductor pollicis longus, abductor digiti minimi, opponens digiti minimi, flexor digiti minimi, lumbricals and interossei, palmaris longus, extensor digitorum, flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, extensor carpi radialis longus, extensor carpi radialis brevis, extensor capri ulnaris, pronator teres, pronator quadratus, supinator and biceps, brachioradialis, coracobrachialis, biceps brachii, brachialis, supraspinatus and middle deltoid, anterior deltoid, posterior deltoid, upper trapezius, supraspinatus, and gastrocnemius.
[0572] In some instances, the muscle function assessment may assess certain bodily movements or other functional manifestations of muscle function, e.g., shoulder shrug, shoulder abduction, elbow flexion or supinated arm, elbow flexion of neutral arm, elbow extension, radial wrist extension, wrist flexion, thumb extension, fifth digit abduction, hip flexion, knee extension, big toe extension, knee flexion, ankle plantar flexion, posture, gripping, jumping, hopping (one feet or two feet), standing up, or sitting down.
[0573] Assessments of muscle function can be performed at any point before, during, or after treatment. In some instances, a muscle function assessment performed prior to treatment may be used for prognostic, diagnostic, or predictive purposes. For example, an individual who displays muscle weakness based on the assessments described herein may be identified as one who may benefit from treatment. Muscle function may also be assessed during or after treatment to monitor changes in muscle function. In some instances, assessments of muscle function at multiple time points before or during treatment. For example, an improvement in muscle function in a subject overtime following administration of the compositions described herein may be an indicator that the treatment is effective or that the subject is responsive to treatment. In contrast, a lack of change or a decrease in muscle function over time following administration of the compositions described herein may be an indicator of lack of therapeutic efficacy
[0574] The results of the muscle function assessment can be used to identify subjects with muscle weakness (e.g., subjects in need of treatment). For example, in instances of quantitative determinations of muscle function, a measurement of muscle function that is lower than a reference value (e.g., muscle function that is lower by about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 80%, about 85%, about 90%, about 95%, about 100%, or more than 100% relative to a reference value) may indicate that the individual is experiencing muscle weakness. In some instances, a measurement of muscle function that is lower than a reference value (e.g., a value that is lower by about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more than 50 relative to a reference value) may indicate that the individual is experiencing muscle weakness. The reference value may be, for instance, a measure of muscle function from one or more control subjects (e.g., a healthy individual or healthy population), a pre-assigned reference value, or a measure of muscle function measured at one or more previous time points in an individual.
[0575] In instances of qualitative assessments that involve (e.g., functional status assessments or MMT), a determination of muscle weakness may be made based on well-known grading scales accepted in the art. In some instances, a lack of an ability to perform a certain movement or physical task may be indicative of muscle weakness.
[0576] The results of the muscle function assessment may also be used to monitor whether treatment is effective in improving muscle function in an individual. For example, in instances of quantitative determinations of muscle function, a measurement of muscle function that is higher than a reference value (e.g., muscle function that is higher by about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 80%, about 85%, about 90%, about 95%, about 100%, or more than 100%) indicates that the individual is responsive to treatment. In some instances, a measurement of muscle function that is higher than a reference value (e.g., a value that is lower by about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more than 50) indicates that the individual is responsive to treatment. In instances of qualitative assessments (e.g., functional status assessments or MMT), a determination of improvements, or lack thereof, in muscle function overtime may be made based on well-known grading scales accepted in the art. In some instances, an ability to perform a certain movement or physical task that could not be performed previously may be indicative of improvements in muscle function.
Assessing Propensity to Benefit from TGF- Antagonist Therapy
[0577] The compositions and methods described herein may be used to determine the propensity of a patient (e.g., a human patient conditions associated with elevated TGF-) signalling to respond to TGF- antagonist therapy. Using a method for assessing muscle function (e.g., muscle mass, muscle strength, or muscle quality) described above or known in the art, a physician may determine that the patient exhibits a level of muscle function that is less than that of a muscle function reference level, such as the level of muscle function of a healthy patient (e.g., a healthy patient of the same gender, age, and/or body mass, among other characteristics, as the patient). A finding that the patient exhibits, for instance, a level of muscle function that is less than that of the muscle function reference level may indicate that the patient is likely to respond to treatment with a TGF- antagonist, such as a TGF- antagonist described herein.
[0578] For example, a physician of skill in the art may assess a patient's likelihood to benefit from TGF- antagonist therapy by determining a level of muscle function exhibited by the patient, such as a level of muscle mass, muscle strength, or muscle quality exhibited by the patient, and comparing the level of muscle function exhibited by the patient to a muscle function reference level. A finding that the patient exhibits a level of muscle function that is less than the muscle function reference level (e.g., by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) indicates that the patient is likely to benefit from TGF- antagonist therapy.
[0579] Upon determining that the patient is likely to benefit from treatment with a TGF- antagonist, such as a TGF- antagonist described herein, the patient may be administered a TGF- antagonist accordingly. The TGF- antagonist may be, for instance, conjugated to a bone-targeting moiety, thereby reducing TGF- signalling in the proximity of the skeletal-muscular interface. In this way, for instance, TGF- signalling in healthy tissues may be preserved. The TGF- antagonist or conjugate thereof may be administered to the patient, for instance, by one or more of the routes of administration described herein, such as subcutaneously, intradermally, intramuscularly, intraperitoneally, intravenously, or orally, or by nasal or by epidural administration. The TGF- antagonist or conjugate thereof may, for instance, be formulated with one or more excipients and/or biologically acceptable carriers, and may be optionally conjugated to, admixed with, or co-administered separately (e.g., sequentially) with one or more additional therapeutic agents
EXAMPLES
[0580] The following examples are put forth so as to provide those of ordinary skill in the art with a description of how the compositions and methods described herein may be used, made, and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.
Example 1
Expression, Surface Plasmon Resonance (SPR), and Neutralization Assay of RER-Fc-D10 TGF- Trap (PCT-0015; SEQ ID NO: 14)
Expression Vector
[0581] The coding region of the TGF- receptor fusion protein RER-Fc-D10 TGF- Trap (PCT-0015) (
Transient Transfection of RER-Fc-D10 TGF- Trap (PCT-0015) Expression in CHO Suspension Cells
[0582] CHO suspension cells were maintained in serum free medium and routinely passed at cell densities between 310.sup.5 to 310.sup.6/ml. For transfection, CHO cells were harvested and suspended at 110.sup.6 cells/ml and one milliliter was plated into each well of a 6 well dish. Transfections were carried out using Lipofectamine 2000 following manufacturer's instructions. Post transfection, cell culture supernatants were analyzed for RER fusion protein expression by immunoblot. Supernatant samples were taken at 24, 48 and 72 hr. The transient transfected pools were subsequently placed under selection using puromycin at 10 g/ml and a cell density of 310.sup.5 cells/ml. After one week of static culture, the selected pools were placed on a shaker platform and cultured until cell densities reached 110.sup.6 cells/ml and viability of >90% for 3 passages. At this stage the cells are deemed to have recovered after phase I and the expression of RER fusion protein was determined.
[0583] Stable pools of RER fusion protein (Pool 2 and Pool 3) were tested for protein expression post selection with puromycin. Cells were seeded at 310.sup.5 cells/ml in serum free medium without puromycin and grown with agitation at 125 rpm at 37 C. and 8% CO.sub.2. Cell viability was determined every other day using Trypan blue exclusion to delineate viable from non-viable cells.
Purification of RER-Fc-D10 TGF- Trap (PCT-0015) Using Protein A Sepharose
[0584] After 10 days of culture the supernatant was clarified by centrifugation and subsequently dialyzed against 1 PBS prior to purification using Protein A Sepharose (
SPR and TGF- Neutralization Testing of PCT-0015 (SEQ ID NO: 14)
[0585] Material purified on Protein A Sepharose shows three major bands migrating with apparent MW around 200 kDa (
[0586] In summary, PCT-0015 HMW (fractions 14 and15) and LMW (fractions 16,17, and 18) fractions show good binding to TGF-1 and TGF-3. HMW shows low binding to TGF-2, and LMW shows little or no binding. By contrast, the monomeric RER domain showed good binding to TGF-2. Taken together, the results indicate that dimeric Fc-fused PCT-0015 has partially lost TGF-2 binding activity. The apparent binding affinities are in the low to sub-nM range.
[0587] TGF- neutralization evaluated using IL-11 release assay indicates that PCT-0015 fractions 15 (HMW) and 17 (LMW) neutralizes TGF-1 and -3 with potencies in sub-nano molar range. These fractions also showed TGF-2 neutralization activity, but EC50s could not be determined as the neutralization window was too small.
TABLE-US-00022 TABLE 15 SPR analysis of SEC fractions: measurements of binding to TGF-s Rmax ka (1/Ms) kd (1/s) KD (M) (RU) TGF-1 1F14 2.88E+05 5.51E04 1.92E09 13.4 1F15 4.20E+05 4.53E04 1.08E09 16.0 1f16 3.58E+05 8.95E04 2.50E09 18.3 1F17 3.58E+05 1.02E04 2.85E09 20.5 1F18 4.84E+05 1.09E04 2.26E09 22.0 TGF-3 1F14 3.38E+05 4.41E04 1.31E09 24.2 1F15 3.66E+05 2.34E04 6.38E09 28.7 1f16 7.27E+05 3.10E04 4.27E09 35.7 1F17 3.42E+05 2.95E04 8.62E09 45.9 1F18 3.15E+05 2.81E04 8.90E09 52.5 TGF-2 1F14 3.81E+05 7.24E04 1.90E09 4.257 1F15 6.36E+05 5.12E04 8.05E09 6.924 1f16 1F17 1F18
Example 2
Purification, SPR, and TGF- Neutralization Assay of PCT-0016NT (SEQ ID NO: 33)
[0588] Purification, SPR, and TGF- neutralization testing of PCT-0016NT (SEQ ID NO: 33) are shown in
[0589] A549 IL-11 neutralization assay indicates high potency for PCT-0016NT, with EC50s in the low pM range. PCT-0016NT is 10-60 fold more potent for TGF-3 and TGF-1, and 100-fold more potent for TGF-2, compared to 1D11. (
TABLE-US-00023 TABLE 16 KD determination of purified peak fraction in SPR assay for PCT-0016NT (SEQ ID NO: 33) Cycle Sample ka (1/Ms) kd (1/s) KD (M) Rmax (RU) TGF-B1 4 PCT0016 5.21E+05 2.58E05 4.96E11 21 6 PCT0016 5.59E+05 1.75E05 3.13E11 21 8 PCT0016 5.80E+05 2.45E05 4.23E11 20 Average: 5.53E+05 2.26E05 4.10E11 21 10 1D11 4.16E+05 4.23E05 1.02E10 102 12 1D11 4.79E+05 4.18E05 8.73E11 92 14 1D11 5.15E+05 3.78E05 7.33E11 87 Average: 4.70E+05 4.06E05 8.73E11 94 TGF-B2 4 PCT0016 4.14E+05 1.59E05 3.85E11 15 6 PCT0016 6.24E+05 1.08E05 1.72E11 13 8 PCT0016 6.10E+05 1.71E05 2.80E11 13 Average: 5.49E+05 1.46E05 2.79E11 14 10 1D11 6.21E+05 5.17E05 8.32E11 49 12 1D11 6.75E+05 5.54E05 8.20E11 47 14 1D11 6.90E+05 4.98E05 7.23E11 46 Average: 6.62E+05 5.23E05 7.92E11 48 TGF-B3 4 PCT0016 4.10E+05 2.93E05 7.14E11 57 6 PCT0016 4.20E+05 2.64E05 6.27E11 58 8 PCT0016 4.37E+05 2.99E05 6.85E11 56 Average: 4.22E+05 2.85E05 6.75E11 57 10 1D11 2.68E+05 2.90E06 1.08E11 296 12 1D11 3.16E+05 5.06E06 1.60E11 263 14 1D11 3.46E+05 2.75E06 7.93E12 243 Average: 3.10E+05 3.57E06 1.16E11 267
Example 3
A549 Cells IL-11 Release Assay for TGF- Neutralization
[0590] A549 lung cancer cells were seeded on 96-well plates (5106 cells/well). The following day, 10 pM TGF- in complete media was incubated with a dilution series of antagonist (range 0.005 to 100 nM) for 30 min at RT. The cells were then treated with 10 pM TGF-antagonist and incubated for 18 h at 37C. Aliquots of conditioned media were added to MSD Streptavidin Gold plates (Meso Scale Diagnostics, Gaithersburg, Md.) coated with 2 g/mL biotinylated mouse anti-human IL-11 antibody (MAB618, R&D Systems, Minneapolis, Minn.) and incubated 18 h at RT. The plates were washed with PBS containing 0.02% Tween 20, then treated with 2 g/mL SULFO-tagged goat anti-human IL-11 antibody (AF-218-NA, R&D Systems) for 1 h at RT. After a final wash, plates were read in a MESO QuickPlex SQ 120 machine (Meso Scale Diagnostics). IL-11 readouts were normalized to cell number/well (CyQUANT, Thermo Fisher Sci) and expressed as percent IL-11 release compared to control cells treated with TGF- alone. Percent of IL-11 released is used as a measurement for TGF- neutralization by the TGF- antagonist.
Example 4
Proposed Signal Peptide Cleavage Site
[0591] Using an alpha-lactalbumin signal peptide promotes cleavage of the signal peptide with a high probability (0.919) at the position indicated below, which is essential for generating the mature form of the TGF- receptor fusion proteins. This leaves two additional amino acids on the mature N-terminus before the asparagine residues of the mature TGF- receptor fusion proteins.
##STR00045##
[0592] Specific signal peptides, such as those described herein, can improve manufacturing of the TGF- receptor fusion proteins of the invention, and can be useful for in vivo therapeutic administration of nucleic acids encoding the TGF- receptor fusion proteins of the invention.
Example 5
PCT-0015 (SEQ ID NO: 14) and PCT-0016NT (SEQ ID NO: 33)
[0593] There are a variety of linkers that may be inserted between the RII ectodomains and RIII endoglin domain of the trimeric TGF- fusion proteins (designated as L.sup.3 in
Example 6
PCT-0020 (SEQ ID NO: 18)
[0594] Another exemplary TGF- fusion protein conjugate (illustrated by Formula a in
[0595] PCT-0020 is more potent than 1D11 antibody, but is less potent than PCT-0016NT by 2-3 fold for TGF-1 and TGF-3 and 17-fold for TGF-2 (Table 17). PCT-0020 is a potent compound from the option 1 series (illustrated by Formula a) but it does not equal to the potency of PCT-0016NT, especially for TGF-2.
TABLE-US-00024 TABLE 17 Comparison of PCT-0020 (SEQ ID NO: 18) potency with PCT-0016NT (SEQ ID NO: 33) and 1D11 Summary EC50 [nM] 1D11 PCT0016 PCT0020 Fold 0020/0016 TGFb1 0.0478 0.0065 0.0171 2.6 TGFb3 0.2249 0.0032 0.0105 3.3 TGFb2 0.5905 0.0041 0.0710 17.4
Example 7
PCT-0021 (SEQ ID NO: 20) and PCT-0022 (SEQ ID NO: 22)
[0596] The linker between the RII ectodomain and RIII endoglin domain (designated as L.sup.3 in
[0597] PCT-0022 includes a human RII sequence as the L.sup.3 linker between the RII ectodomain and RIII endoglin domain. SEC data for PCT-0022 are shown in
[0598] In summary, PCT-0021 is more potent than 1D11 for TGF-1 and TGF-3, whereas PCT-0022 is modestly less potent than 1D11. The SEC fractions contain more than one species, particularly for PCT-0021 FrB14, and this may alter the neutralization efficiency.
TABLE-US-00025 TABLE 18 IC50 for neutralization of the three isoforms of TGF- as compared to 1D11 Compound TGF-1 TGF-3 TGF-2 1D11 0.0722 0.0547 0.492 PCT0021 FrB14 0.0096 0.0198 0.396 PCT0022 FrB13 0.1289 0.2047 2.273
Example 8
PCT-0025 (SEQ ID NO: 28) and PCT-0026 (SEQ ID NO: 30)
[0599] PCT-0025 and PCT-0026 are fully humanized and increase the length of both linker L.sup.1 (hinge linker) and L.sup.3 (shown in Formula a in
[0600]
TABLE-US-00026 TABLE 19 EC50 for neutralization of the three isoforms of TGF- by PCT-0026 as compared to PCT-0020 and 1D11 EC50 Values (picomol/L) 1D11 PCT-0020 PCT-0026 TGF-1 163.9 8.8 1.9 TGF-2 381.9 55.5 105.4 TGF-3 19.0 8.0 4.2
Example 9
Distribution of PCT-0026 (SEQ ID NO: 30) to Bone
[0601] PCT-0026 (SEQ ID NO: 30) was radioactively labeled with zirconium-89 via standard methods. At the start of the study, nude mice (3 per group) were injected intraperitoneally (i.p.) with radiolabeled PCT-0026 (SEQ ID NO: 30) at a concentration of 10 mg/kg. Mice were anesthetized and whole-body images were taken using PET imaging to visualize radioactive distribution at 1 h, 4 h, 24 h, 48 h and 7 days post injection. 7 days post injection, animals were euthanized, femurs were isolated and counted on gamma scintillation counter. Serum was isolated from duplicate mice at 15 min, 1 h, 2 h, 4 h, 24 h, and 48 h post-injection and counted on a gamma scintillation counter. The data is expressed as the percentage of counts relative to total injected counts.
[0602] Positron emission tomography (PET) imaging revealed accumulation of radiolabel within 48 hours post-injection (
Example 10
Administration of a TGF- Antagonist for the Treatment of Diseases Associated with Elevated TGF- Activity
[0603] Using the compositions and methods described herein, a physician of skill in the art can administer to a patient (e.g., a human patient) a conjugate containing a TGF- receptor fusion protein, such as a TGF- receptor fusion protein having the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, and SEQ ID NO: 35, or a TGF- receptor fusion protein having at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or more, sequence identity) thereto. The TGF- receptor fusion protein can be bound to a bone-targeting hydroxyapatite-binding domain, such as a polyanionic peptide of the formula Dn or En, in which D and E designate aspartate and glutamate, respectively, and each n designates an integer from 1 to 25 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25). For example, TGF- receptor fusion protein can be bound to a bone-targeting hydroxyapatite-binding domain of the formula D10. For example, the conjugate may be a protein having the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, and SEQ ID NO: 34, or at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or more, sequence identity) thereto. The patient may be one that is suffering from a disease associated with elevated TGF- activity, muscle weakness, and/or elevated bone turnover relative to a healthy individual not suffering from the disease, such as osteogenesis imperfecta.
[0604] For instance, a physician of skill in the art may assess a patient suffering from osteogenesis imperfecta by first evaluating muscle function in the patient using one or more methodologies, such as manual muscle testing, dynamometry, or muscle mechanography, or imaging techniques to assess muscle-cross sectional area, volume, density, or muscle mass (e.g., MRI or CT scans). A finding that the individual has reduced muscle function relative to a muscle function reference level, such as the level of muscle function in a healthy subject (e.g., a healthy subject of the same gender, age, and/or body mass) can indicate that the patient may be particularly well suited for treatment with a TGF- antagonist capable of improving muscle function. A physician of skill in the art may additionally assess the patient's muscle function over time so as to monitor the progression of the disease during the course of treatment. The physician may administer to the patient a therapeutically effective amount (e.g., an amount sufficient to attenuate TGF- signaling and/or to reduce bone turnover) of a composition containing a TGF- antagonist or conjugate thereof. The TGF- antagonist may be any antagonist described herein, such as, for instance, a TGF- receptor fusion protein, such as a protein having at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, and SEQ ID NO: 35. Optionally, the TGF- antagonist construct may be conjugated to a polyanionic peptide, such as a deca-aspartate peptide, and may have, for instance, at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 5, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, and SEQ ID NO: 34.
[0605] In some other instances, a physician of skill in the art may assess a patient suffering from a bone disease, such as osteogenesis imperfecta, by first evaluating the concentration of one or more biomarkers of bone turnover, such as serum and bone alkaline phosphatase, serum osteocalcin (sOC), serum type I collagen C-telopeptide breakdown products (sCTX), urinary free-deoxypyridinoline (ufDPD), and urinary cross-linked N-telopeptides of type I collagen (uNTX). A finding that one or more of these biomarkers is elevated may signal an elevated bone turnover rate, indicating that the patient may be particularly well suited for treatment with a TGF- antagonist capable of localizing to bone tissue. A physician of skill in the art may additionally assess the patients frequency of, and propensity for, bone fracture so as to monitor the progression of the disease during the course of treatment.
[0606] In some instances, the physician may administer to the patient a therapeutically effective amount (e.g., an amount sufficient to attenuate TGF- signaling) of a composition containing a TGF- antagonist, optionally bound to a bone-targeting moiety. The bone-targeting moiety may be any bone-targeting moiety described herein, such as, for instance, a collagen-binding domain or a hydroxyapatite-binding domain as described herein, e.g., a hydroxyapatite-binding domain containing a deca-poly(Asp) sequence motif.
[0607] The physician may administer to the patient a therapeutically effective amount (e.g., an amount sufficient to attenuate TGF- signaling and/or to reduce bone turnover) of a conjugate containing a TGF- receptor fusion protein, including those bound to a bone-targeting moiety, at a dosing schedule determined by the patients age, weight, gender, and/or severity of the disease. For example, the conjugate may be administered to the subject in one or more doses (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or more) per a specified time interval, such as one or more doses per day, per week, per month, or per year. The patient may be evaluated between doses so as to monitor the effectiveness of the therapy and to increase or reduce the dosage based on the patients response. For example, a reduction in the incidence of bone fractures, an improved ability of the patient to walk, and/or a reduction in the concentration of one or more biomarkers of bone turnover in a sample isolated from the patient may indicate that the therapy is effectively treating the condition.
[0608] The therapy may be administered to the patient by a variety of routes of administration, for instance, as determined by a physician of skill in the art. For example, the therapy may be administered to the patient in one or more repeat doses subcutaneously, intradermally, intramuscularly, intraperitoneally, intravenously, or orally, or by nasal or by epidural administration.
[0609] Prior to the conclusion of therapy, the physician may prescribe progressively lower doses of the conjugate to the patient so as to gradually reduce the concentration of the therapy. The therapy may involve only a single dosing of the therapeutic conjugate. Alternatively, the therapy may continue, for instance, for a period of days, weeks, months, or years prior to completion.
Example 11
Administration of a TGF- Antagonist Conjugated to a Bone-Targeting Hydroxyapatite-Binding Polyanionic Peptide for the Treatment of Osteogenesis Imperfecta
[0610] Using the compositions and methods described herein, a physician of skill in the art can administer to a patient (e.g., a human patient) a conjugate containing a TGF- receptor fusion protein, such as a TGF- receptor fusion protein having the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, and SEQ ID NO: 35, or a TGF- receptor fusion protein having at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or more, sequence identity) thereto. The TGF- receptor fusion protein can be bound to a bone-targeting hydroxyapatite-binding domain, such as a polyanionic peptide of the formula Dn or En, in which D and E designate aspartate and glutamate, respectively, and each n designates an integer from 1 to 25 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25). For example, TGF- receptor fusion protein can be bound to a bone-targeting hydroxyapatite-binding domain of the formula D10. For example, the conjugate may be a protein having the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, and SEQ ID NO: 34, or at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or more, sequence identity) thereto. The patient may be one that is suffering from a disease associated with elevated TGF- activity and/or elevated bone turnover relative to a healthy individual not suffering from the disease, such as osteogenesis imperfecta.
[0611] For instance, a physician of skill in the art may assess a patient suffering from osteogenesis imperfecta by first evaluating the concentration of one or more biomarkers of bone turnover, such as serum and bone alkaline phosphatase, serum osteocalcin (sOC), serum type I collagen C-telopeptide breakdown products (sCTX), urinary free-deoxypyridinoline (ufDPD), and urinary cross-linked N-telopeptides of type I collagen (uNTX). A finding that one or more of these biomarkers is elevated may signal an elevated bone turnover rate, indicating that the patient may be particularly well suited for treatment with a TGF- antagonist capable of localizing to bone tissue. A physician of skill in the art may additionally assess the patients frequency of, and propensity for, bone fracture so as to monitor the progression of the disease during the course of treatment.
[0612] The physician may administer to the patient a therapeutically effective amount (e.g., an amount sufficient to attenuate TGF- signaling and/or to reduce bone turnover) of a conjugate containing a TGF- receptor fusion protein bound to a bone-targeting moiety at a dosing schedule determined by the patient's age, weight, gender, and/or severity of the disease. For example, the conjugate may be administered to the subject in one or more doses (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or more) per a specified time interval, such as one or more doses per day, per week, per month, or per year. The patient may be evaluated between doses so as to monitor the effectiveness of the therapy and to increase or reduce the dosage based on the patients response. For example, a reduction in the incidence of bone fractures, an improved ability of the patient to walk, and/or a reduction in the concentration of one or more biomarkers of bone turnover in a sample isolated from the patient may indicate that the therapy is effectively treating the condition.
[0613] The therapy may be administered to the patient by a variety of routes of administration, for instance, as determined by a physician of skill in the art. For example, the therapy may be administered to the patient in one or more repeat doses subcutaneously, intradermally, intramuscularly, intraperitoneally, intravenously, or orally, or by nasal or by epidural administration.
[0614] Prior to the conclusion of therapy, the physician may prescribe progressively lower doses of the conjugate to the patient so as to gradually reduce the concentration of the therapy. The therapy may involve only a single dosing of the therapeutic conjugate. Alternatively, the therapy may continue, for instance, for a period of days, weeks, months, or years prior to completion.
Example 12
Study of Muscle Function in a Mouse Model of Osteogenesis Imperfecta
[0615] A study is performed to monitor the Nox4 dependent oxidation pathway of RyR1 in muscle from a mouse model of OI with associated muscle weakness. OI models include, but are not limited to, the Brtl+/, oim/, Crtap/ and Jrt+/ mouse models.
[0616] Muscle function may be tested in vivo by testing the forearm grip of OI mice compared to normal mice. Alternatively, muscle weakness may be assessed by measuring the ex vivo specific force of the extensor digitorum longus muscle. TGF- elevation can be confirmed via demonstration that its downstream signaling molecules, SMAD2 and SMAD3 are activated. This may be measured by immunoblot analysis showing an increased amount of phosphorylated SMAD2 or SMAD3 is present relative to total SMAD2 or SMAD3 in muscle lysates. To assess involvement of NADPH oxidase 4, Nox4 mRNA may be measured using standard RT-PCR to confirm increased expression in muscle derived from OI mice relative to muscle from normal mice. Immunoblots of muscle lysates may also be performed to demonstrate oxidation and nitrosylation of RyR1, two downstream consequences of NADPH oxidase 4. Finally, co-immunoprecipation of RyR1 and its associated regulatory protein, calstabin may be performed. Demonstration that calstabin binding to RyR1 is reduced in muscles from OI mice relative to normal mice can be used as a surrogate to monitor calcium leak in muscles and associated muscle weakness. Final demonstration of TGF- involvement in this mechanism can be demonstrating by showing that these parameters are reversed in OI mice treated with a TGF- antagonist.
[0617] Methods
[0618] Grip strength. Forelimb grip strength can be assessed by allowing each mouse to grab a wire mess attached to a force transducer (Bioseb, Bitrolles, France) that records the peak force as the mouse is pulled by the tail horizontally away from the mesh (Bellinger, A. M. et al. Hypernitrosylated ryanodine receptor calcium release channels are leaky in dystrophic muscle. Nat. Med. 15, 325-330, 2009; Bonetto, A., Andersson, D. C. & Waning, D. L. Bonekey Rep. 4, 732; 2015). In this context, a reduction in grip strength relative to a normal mouse is indicative of reduced muscle function. Similarly, in the context of a human, reduced grip strength in a patient relative to a healthy individual is indicative of reduced muscle function.
[0619] Contractility. Ex vivo contractility of the extensor digitorum longus (EDL) muscles can be determined as described (Andersson, D. C. et al. Cell Metab. 14, 196-207, 2011; Bonetto, A., Andersson, D. C. & Waning, D. L. Bonekey Rep. 4, 732; 2015). EDL can be dissected from the hind limbs and stainless-steel hooks, tied to the tendons of the muscles using 4-0 silk sutures and the muscles mounted between a force transducer (Aurora Scientific, Aurora, ON, Canada) and an adjustable hook. The muscles are immersed in a stimulation chamber containing O2/CO2 (95/5%) bubbled Tyrode solution (121 mM NaCl, 5.0 mM KCl, 1.8 mM CaCl2, 0.5 mM MgCl2, 0.4 mM NaH2PO4, 24 mM NaHCO3, 0.1 mM EDTA, 5.5 mM glucose). The muscle is stimulated to contract using a supramaximal stimulus between two platinum electrodes. Data can be collected via Dynamic Muscle Control/Data Acquisition (DMC) and Dynamic Muscle Control Data Analysis (DMA) programs (Aurora Scientific). At the start of each experiment the muscle length can be adjusted to yield the maximum force. The force-frequency relationships can be determined by triggering contraction using incremental stimulation frequencies (0.5-ms pulses at 1-150 Hz for 350 ms at supramaximal voltage). Between stimulations the muscle is allowed to rest for 3 min. At the end of the force measurement, the length (L0) and weight of the muscle are measured and the muscle are snap frozen in liquid N2. To quantify the specific force, the absolute force is normalized to the muscle size, specifically the cross-sectional area, calculated as the muscle weight divided by the length using a muscle density constant of 1.056 kg/m3 (Yamada, T. et al. Arthritis Rheum. 60, 3280-3289; 2009). In this context, a reduction in ex vivo contractility relative to a normal mouse is indicative of reduced muscle function. In the context of a human, measures of muscle function in the terms of muscle force can be determined using, for example, muscle mechanography (e.g., hopping on a force plate) and muscle size, density, volume, or cross-sectional area can be measured using visual or imaging techniques (e.g., magnetic resonance imaging (MRI) or computed tomography (CT) scans). In humans, a decrease in muscle force or muscle size, density, volume, or cross-sectional area in a patient relative to a healthy individual is indicative of reduced muscle function.
[0620] Measurement of protein oxidation and ROS production. To determine channel oxidation the carbonyl groups on the protein side chains can be derivatized to 2,4-dinitrophenylhydrazone (DNP-hydrazone) by reaction with 2,4-dinitrophenylhydrazine (DNPH) (Oxyblot, Millipore, Darmstadt, Germany). The DNP signal on RyR1 can be detected by immunoblotting with an antibody specific to DNP (Millipore, Darmstadt, Germany). Protein carbonyl concentration in tissue lysates can be determined using the OxiSelect Protein Carbonyl ELISA Kit (Cell BioLabs, Inc., San Diego, Calif.). For example, 0.5 mg of EDL lysate can be added to a 96-well protein-binding plate, which is incubated overnight at 4 C. After washing the plate three times with PBS, the protein carbonyl groups are derivatized with DNPH for 45 min at room temperature (in the dark). Plates are developed with a DNP-specific antibody followed by a HRP-conjugated secondary antibody. Protein carbonyl concentration is determined by comparison with a standard curve of oxidized BSA. ROS production is determined in C2C12 myotubes using the OxiSelect in vitro ROS/RNS Assay kit (Cell BioLabs, Inc.). ROS production is measured using 0.25 mg of cell lysate according to the manufacturer's recommendations. For H2O2-treated cells, cells are incubated with 1 mM H2O2 for 30 min before lysis. The investigators are blinded to treatment of subjects. In this context, an increase in protein oxidation and ROS production as determined used these methods is indicative of increased expression of NADPH oxidase 4, which can be associated with reduced muscle function. Increased protein oxidation and ROS production in a mouse model of OI relative to a normal mouse is indicative of reduced muscle function. In the context of humans, similar methods can be used to assess protein oxidation and ROS production in a muscle biopsy, where an increase in protein oxidation and ROS production relative to a healthy individual is indicative of reduced muscle function.
[0621] RyR1 immunoprecipitation and immunoblotting. RyR1 oxidation and nitrosylation and calstabin1 binding can be determined as previously described (Andersson et. al. Cell Metab. 14, 196-207 (2011)). Extensor digitorum longus (EDL) muscles can be isotonically lysed in 0.5 ml of a buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 20 mM NaF, 1.0 mM Na3VO4, and protease inhibitors. C2C12 cells were lysed in NP-40 lysis buffer containing 50 mM Tris-HCl (pH 8.0) 150 mM NaCl, 1.0% NP-40 and protease inhibitors. An anti-RyR antibody (e.g., 4 g anti-RyR1 antibody 5029, a custom antibody against the last nine amino acids (CRKQYEDQLS (SEQ ID NO: 404); a cysteine is added at the N terminus) of the rabbit skeletal muscle RyR1) can be used to immunoprecipitate RyR1 from 250 g of tissue homogenate (Andersson et. al. Cell Metab. 14, 196-207 (2011)). Samples can be incubated with antibody in 0.75 ml of a modified RIPA buffer (50 mM Tris-HCl pH 7.4, 0.9% NaCl, 5.0 mM NaF, 1.0 mM Na3VO4, 1% Triton-X100 and protease inhibitors) for 1 h at 4 C. The immune complexes are incubated with protein A-sepharose beads (Sigma) overnight at 4 C. and the beads are washed twice with modified RIPA buffer. Proteins are separated on 4-12% Bis-Tris gels (Life Technologies) and transferred to nitrocellulose for 1 h at 100 V (Bio-Rad, Hercules, Calif.). After incubation with blocking solution to prevent nonspecific antibody binding, immunoblots are developed with anti-RyR (Affinity Bioreagents, cat. MA3-916, Golden, Colo.; 1:2,000) and anti-Cys-NO antibody (Sigma, cat. N0409, St. Louis, Mo.; 1:2,000) or an anti-calstabin antibody (Santa Cruz Biotechnology, cat. sc-6173, Santa Cruz, Calif.; 1:2,500). Immunoblots are developed and quantified using the Odyssey Infrared Imaging System (LICOR Biosystems, Lincoln, Nebr.) and infrared-labeled secondary antibodies. Detection of pSMAD3, SMAD3, Nox4 (Abcam, Cambridge, UK; 1:1,000 each), GAPDH and tubulin (Sigma; 1:500 each) from mouse muscle, human biopsies, and C2C12 cells can be via lysis in NP-40 buffer and detection and quantification of immobilized proteins can be performed using the Odyssey Infrared Imaging System or GE ImageQuantLAS4000Imaging System (GEHealthcare Bio-sciences, Pittsburgh, Pa.). Increased RyR1 oxidation, nitrosylation, and/or calstabin1 binding in a mouse model of OI relative to a normal mouse is indicative of reduced muscle function. In the context of humans, similar methods can be used to assess RyR1 oxidation, nitrosylation, and/or calstabin1 binding in a muscle biopsy, where an increase in RyR1 oxidation, nitrosylation, and/or calstabin1 binding relative to a healthy individual is indicative of reduced muscle function.
Example 13
Administration of a Conjugate Containing a TGF- Antagonist and a Bone-Targeting Moiety for the Treatment of a Patient Having DMD
[0622] Using the compositions and methods described herein, a physician of skill in the art can administer to a patient (e.g., a human patient) suffering from a muscular dystrophy (e.g., DMD) a conjugate containing a bone-targeting moiety and a TGF- receptor fusion protein, such as a TGF- receptor fusion protein having the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, and SEQ ID NO: 35, or a TGF- receptor fusion protein having at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or more, sequence identity) thereto. The TGF- receptor fusion protein can be bound to a bone-targeting hydroxyapatite-binding domain, such as a polyanionic peptide of the formula Dn or En, in which D and E designate aspartate and glutamate, respectively, and each n designates an integer from 1 to 25 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25). For example, TGF- receptor fusion protein can be bound to a bone-targeting hydroxyapatite-binding domain of the formula D10. For example, the conjugate may be a protein having the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, and SEQ ID NO: 34, or at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or more, sequence identity) thereto. The physician may assess the patient by first evaluating muscle function in the patient using one or more methodologies, such as manual muscle testing, dynamometry, or muscle mechanography, or imaging techniques to assess muscle-cross sectional area, volume, density, or muscle mass (e.g., MRI or CT scans). A finding that the individual has reduced muscle function relative to a control indicates that the patient may be particularly well suited for treatment with a conjugate described herein.
[0623] The physician may administer to the patient a therapeutically effective amount (e.g., an amount sufficient to attenuate TGF- signaling and/or to reduce bone turnover) of a conjugate described herein according to a dosing schedule determined, for instance, by the patient's age, weight, gender, and/or severity of the disease. For example, the conjugate may be administered to the subject in one or more doses (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or more) per a specified time interval, such as one or more doses per day, per week, per month, or per year. The patient may be evaluated between doses so as to monitor the effectiveness of the therapy and to increase or reduce the dosage based on the patient's response. For example, an improvement in one or more, or all, of muscle mass, muscle strength, or muscle quality throughout the course of treatment may indicate that the therapy is effectively treating the muscular dystrophy.
[0624] The conjugate may be administered to the patient by a variety of routes of administration, for instance, as determined by a physician of skill in the art. For example, the conjugate may be administered to the patient in one or more repeat doses subcutaneously, intradermally, intramuscularly, intraperitoneally, intravenously, or orally, or by nasal or by epidural administration. Conjugates described herein can be formulated with excipients, biologically acceptable carriers, and may be optionally conjugated to, admixed with, or co-administered separately (e.g., sequentially) with additional therapeutic agents.
[0625] Prior to the conclusion of therapy, the physician may prescribe progressively lower doses of the conjugate to the patient so as to gradually reduce the concentration of the therapy. In some instances, the therapy may involve only a single dosing of the therapeutic conjugate.
[0626] Alternatively, the therapy may continue, for instance, for a period of days, weeks, months, or years prior to completion.
Example 14
TGF- and Muscle Strength in Mice with Osteogenesis Imperfecta (OI)
[0627] Osteogenesis imperfecta (OI) is a disease attributable to any of a large number of possible mutations of type I collagen. The homozygous murine model (OIM) recapitulates many of the features of human OI including, the skeletal phenotype of severe osteogenesis imperfecta in humans (OI type III). The OIM mice experience spontaneous fractures, reduced bone mineral density, gross changes in skeletal structure, and increased osteoclast activity (Chipman S D. Proc Natl Acad Sci USA. 1993; 90:1701-5). Additionally, OIM mice also have impaired mobility due to reduced muscle strength (Veilleux L N et al. Bone. 2015; 79:52-7; Gentry B A et al. Matrix Biol. 2010; 29(7):638-44).
[0628] RT-PCR of representative TGF- inducible genes confirms that TGF- is elevated in OIM bones relative to WT bones (
Example 15
Immunostaining Detection of Bone-Targeted TGF- Antibody in Skeleton of Mice Treated with Bone-Targeted TGF- Antagonists
[0629] To demonstrate that bone-targeted TGF- antibody is localized in the skeleton, mice treated with a TGF- neutralizing antibody (Fresolimumab, GC1008) (U.S. Pat. No. 9,598,486) containing the bone-targeting moiety D10 (10 aspartate repeat) designated as PCT-0011 at a dose of 5 mg/kg one time weekly, from 12 weeks to 16 weeks of age were euthanized following treatment. Tibia and mandible were isolated and decalcified at 4 oC for 2 weeks in 8% EDTA and 1% formaldehyde. These were embedded in paraffin, sectioned, and processed for IHC using the rabbit anti-human-IgG antibody from Abcam. The DAKO envision-HRP kit was used for the detection. A representative picture of tibia in a 16-week-old mouse taken at 2.5, 10 and 40+ magnification under a light microscope is shown (
Example 16
Mobility Assessments of Mice with Osteogenesis Imperfecta (OIM) Treated with Non-Targeted and Bone-Targeted TGF- Antagonists
[0630] A detailed study was performed to assess the ability of TGF- antagonists with or without a bone-targeting moiety D10 (10 aspartate repeat) to improve muscle functions in OIM mice. OIM mice (2-week-old) were treated with PBS vehicle, or 2, 5 or 10 mg/kg per injection of a neutralizing antibody against TGF- without the bone-targeting moiety D10 (anti-TGF- Antibody 1D11) designated as PCT-001 or the TGF- neutralizing antibody containing the bone-targeting moiety D10 designated as PCT-0011. The study design is shown on
[0631] The open field test used to measure overall mobility/general locomotor activity (
Example 17
Forelimb Grip Strength Assessment of Mice Osteogenesis Imperfecta (OIM) Treated with Non-Targeted and Bone-Targeted TGF- Antagonists
[0632] In the same experiment described in Example 16, a forelimb grip test was performed using a commercial automatic grip strength meter (
Other Embodiments
[0633] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.
[0634] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the invention that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.
[0635] Other embodiments are within the claims.
TABLE-US-00027 Sequences Listing (Full length human TGF-receptor II) SEQ ID NO: 1 MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSC MSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFM CSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNRQQKLSSTWETGKT RKLMEFSEHCAIILEDDRSDISSTCANNINHNTELLPIELDTLVGKGRFAEVYKAKLKQNTSEQFETVAV KIFPYEEYASWKTEKDIFSDINLKHENILQFLTAEERKTELGKQYWLITAFHAKGNLQEYLTRHVISWED LRKLGSSLARGIAHLHSDHTPCGRPKMPIVHRDLKSSNILVKNDLTCCLCDFGLSLRLDPTLSVDDLAN SGQVGTARYMAPEVLESRMNLENVESFKQTDVYSMALVLWEMTSRCNAVGEVKDYEPPFGSKVRE HPCVESMKDNVLRDRGRPEIPSFWLNHQGIQMVCETLTECWDHDPEARLTAQCVAERFSELEHLDR LSGRSCSEEKIPEDGSLNTTK (Full length rat TGF-receptor III) SEQ ID NO: 2 MAVTSHHMIPVMVVLMSACLATAGPEPSTRCELSPINASHPVQALMESFTVLSGCASRGTTGLPREV HVLNLRSTDQGPGQRQREVTLHLNPIASVHTHHKPIVFLLNSPQPLVWHLKTERLAAGVPRLFLVSEG SVVQFPSGNFSLTAETEERNFPQENEHLLRWAQKEYGAVTSFTELKIARNIYIKVGEDQVFPPTCNIG KNFLSLNYLAEYLQPKAAEGCVLPSQPHEKEVHIIELITPSSNPYSAFQVDIIVDIRPAQEDPEVVKNLVL ILKCKKSVNWVIKSFDVKGNLKVIAPNSIGFGKESERSMTMTKLVRDDIPSTQENLMKWALDNGYRPV TSYTMAPVANRFHLRLENNEEMRDEEVHTIPPELRILLDPDHPPALDNPLFPGEGSPNGGLPFPFPDI PRRGWKEGEDRIPRPKQPIVPSVQLLPDHREPEEVQGGVDIALSVKCDHEKMVVAVDKDSFQTNGY SGMELTLLDPSCKAKMNGTHFVLESPLNGCGTRHRRSTPDGVVYYNSIVVQAPSPGDSSGWPDGY EDLESGDNGFPGDGDEGETAPLSRAGVVVFNCSLRQLRNPSGFQGQLDGNATFNMELYNTDLFLVP SPGVFSVAENEHVYVEVSVTKADQDLGFAIQTCFLSPYSNPDRMSDYTIIENICPKDDSVKFYSSKRV HFPIPHAEVDKKRFSFLFKSVFNTSLLFLHCELTLCSRKKGSLKLPRCVTPDDACTSLDATMIWTMMQ NKKTFTKPLAVVLQVDYKENVPSTKDSSPIPPPPPQIFHGLDTLTVMGIAFAAFVIGALLTGALWYIYSH TGETARRQQVPTSPPASENSSAAHSIGSTQSTPCSSSSTA (Full length human TGF-receptor III) SEQ ID NO: 3 MTSHYVIAIFALMSSCLATAGPEPGALCELSPVSASHPVQALMESFTVLSGCASRGTTGLPQEVHVLN LRTAGQGPGQLQREVTLHLNPISSVHIHHKSVVFLLNSPHPLVWHLKTERLATGVSRLFLVSEGSVVQ FSSANFSLTAETEERNFPHGNEHLLNWARKEYGAVTSFTELKIARNIYIKVGEDQVFPPKCNIGKNFLS LNYLAEYLQPKAAEGCVMSSQPQNEEVHIIELITPNSNPYSAFQVDITIDIRPSQEDLEVVKNLILILKCK KSVNWVIKSFDVKGSLKIIAPNSIGFGKESERSMTMTKSIRDDIPSTQGNLVKWALDNGYSPITSYTMA PVANRFHLRLENNEEMGDEEVHTIPPELRILLDPGALPALQNPPIRGGEGQNGGLPFPFPDISRRVWN EEGEDGLPRPKDPVIPSIQLFPGLREPEEVQGSVDIALSVKCDNEKMIVAVEKDSFQASGYSGMDVTL LDPTCKAKMNGTHFVLESPLNGCGTRPRWSALDGVVYYNSIVIQVPALGDSSGWPDGYEDLESGDN GFPGDMDEGDASLFTRPEIVVFNCSLQQVRNPSSFQEQPHGNITFNMELYNTDLFLVPSQGVFSVPE NGHVYVEVSVTKAEQELGFAIQTCFISPYSNPDRMSHYTIIENICPKDESVKFYSPKRVHFPIPQADMD KKRFSFVFKPVFNTSLLFLQCELTLCTKMEKHPQKLPKCVPPDEACTSLDASIIWAMMQNKKTFTKPL AVIHHEAESKEKGPSMKEPNPISPPIFHGLDTLTVMGIAFAAFVIGALLTGALWYIYSHTGETAGRQQV PTSPPASENSSAAHSIGSTQSTPCSSSSTA (Albumin signal peptide) SEQ ID NO: 4 MKWVTFLLLLFISGSAFSAAA (Exemplary TGF-antagonist conjugate) SEQ ID NO: 5 MKWVTFLLLLFISGSAFSAAANGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAV WRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTS NPDGLGPVESSPGHGLDTAAAGPEPSTRCELSPINASHPVQALMESFTVLSGCASHGTTGLPREVHV LNLRSTDQGPGQRQREVTLHLNPIASVHTHHKPIVFLLNSPQPLVWRLKTERLAAGVPRLFLVSEGSV VQFPSGNFSLTAETEERNFPQENEHLLRWAQKEYGAVTSFTELKIARNIYIKVGEDQVFPPTCNIGKN FLSLNYLAEYLQPKAAEGCVLPSQPHEKEVHIIELITPSSNPYSAFQVDIIVDIRPAQEDPEVVKNLVLIL KSKKSVNWVIKSFDVKGNLKVIAPNSIGFGKESERSMTMTKLVRDDIPSTQENLMKWALDAGYRPVT SYTMAPVANRFHLRLENNEEMRDEEVHTIPPELRILLDPDKLPQLCKFCDVRFSTCDNQKSCMSNCSI TSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSD ECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSGDDDDDDDDDD (Polyglycine linker) SEQ ID NO: 6 GGG (G4S linker) SEQ ID NO: 7 GGGGS (linker) SEQ ID NO: 8 GGGGSGGGGSGGGGSG (Exemplary TGF-receptor fusion protein of the structure R.sub.2a-R.sub.3-R.sub.2b or RII ectodomain-RIII endoglin domain-RII ectodomain (RER)) SEQ ID NO: 9 NGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPY HDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGPEPSTRCELSPINASHP VQALMESFTVLSGCASHGTTGLPREVHVLNLRSTDQGPGQRQREVTLHLNPIASVHTHHKPIVFLLNS PQPLVWRLKTERLAAGVPRLFLVSEGSVVQFPSGNFSLTAETEERNFPQENEHLLRWAQKEYGAVT SFTELKIARNIYIKVGEDQVFPPTCNIGKNFLSLNYLAEYLQPKAAEGCVLPSQPHEKEVHIIELITPSSN PYSAFQVDIIVDIRPAQEDPEVVKNLVLILKSKKSVNWVIKSFDVKGNLKVIAPNSIGFGKESERSMTMT KLVRDDIPSTQENLMKWALDAGYRPVTSYTMAPVANRFHLRLENNEEMRDEEVHTIPPELRILLDPDP QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDA ASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (Human R.sub.2 ectodomain - residues 42-159 of human R.sub.2) SEQ ID NO: 10 NGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPY HDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (Human R.sub.2 ectodomain - residues 48-159 of human R.sub.2) SEQ ID NO: 11 PQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILED AASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (Rat R.sub.3 endoglin domain residues 24-383 containing R58H, H116R, C278S, and N337A mutations) SEQ ID NO: 12 GPEPSTRCELSPINASHPVQALMESFTVLSGCASHGTTGLPREVHVLNLRSTDQGPGQRQREVTLHL NPIASVHTHHKPIVFLLNSPQPLVWRLKTERLAAGVPRLFLVSEGSVVQFPSGNFSLTAETEERNFPQ ENEHLLRWAQKEYGAVTSFTELKIARNIYIKVGEDQVFPPTCNIGKNFLSLNYLAEYLQPKAAEGCVLP SQPHEKEVHIIELITPSSNPYSAFQVDIIVDIRPAQEDPEVVKNLVLILKSKKSVNWVIKSFDVKGNLKVIA PNSIGFGKESERSMTMTKLVRDDIPSTQENLMKWALDAGYRPVTSYTMAPVANRFHLRLENNEEMR DEEVHTIPPELRILLDPD (Human R.sub.3 endoglin domain residues 21-380 containing the C275S mutation) SEQ ID NO: 13 GPEPGALCELSPVSASHPVQALMESFTVLSGCASRGTTGLPQEVHVLNLRTAGQGPGQLQREVTLH LNPISSVHIHHKSVVFLLNSPHPLVWHLKTERLATGVSRLFLVSEGSVVQFSSANFSLTAETEERNFPH GNEHLLNWARKEYGAVTSFTELKIARNIYIKVGEDQVFPPKCNIGKNFLSLNYLAEYLQPKAAEGCVM SSQPQNEEVHIIELITPNSNPYSAFQVDITIDIRPSQEDLEVVKNLILILKSKKSVNWVIKSFDVKGSLKIIA PNSIGFGKESERSMTMTKSIRDDIPSTQGNLVKWALDNGYSPITSYTMAPVANRFHLRLENNEEMGD EEVHTIPPELRILLDPG (PCT-0015 without SP, with bone-targeting moiety) SEQ ID NO: 14 NGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPY HDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGLGPVESSPGHGLDTAA AGPEPSTRCELSPINASHPVQALMESFTVLSGCASHGTTGLPREVHVLNLRSTDQGPGQRQREVTL HLNPIASVHTHHKPIVFLLNSPQPLVWRLKTERLAAGVPRLFLVSEGSVVQFPSGNFSLTAETEERNF PQENEHLLRWAQKEYGAVTSFTELKIARNIYIKVGEDQVFPPTCNIGKNFLSLNYLAEYLQPKAAEGCV LPSQPHEKEVHIIELITPSSNPYSAFQVDIIVDIRPAQEDPEVVKNLVLILKSKKSVNWVIKSFDVKGNLK VIAPNSIGFGKESERSMTMTKLVRDDIPSTQENLMKWALDAGYRPVTSYTMAPVANRFHLRLENNEE MRDEEVHTIPPELRILLDPDKLPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDE NITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDTGG GDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGKDDDDDDDDDD (PCT-0015 without SP, without bone-targeting moiety) SEQ ID NO: 15 NGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPY HDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGLGPVESSPGHGLDTAA AGPEPSTRCELSPINASHPVQALMESFTVLSGCASHGTTGLPREVHVLNLRSTDQGPGQRQREVTL HLNPIASVHTHHKPIVFLLNSPQPLVWRLKTERLAAGVPRLFLVSEGSVVQFPSGNFSLTAETEERNF PQENEHLLRWAQKEYGAVTSFTELKIARNIYIKVGEDQVFPPTCNIGKNFLSLNYLAEYLQPKAAEGCV LPSQPHEKEVHIIELITPSSNPYSAFQVDIIVDIRPAQEDPEVVKNLVLILKSKKSVNWVIKSFDVKGNLK VIAPNSIGFGKESERSMTMTKLVRDDIPSTQENLMKWALDAGYRPVTSYTMAPVANRFHLRLENNEE MRDEEVHTIPPELRILLDPDKLPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDE NITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDTGG GDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK (PCT-0019, without SP, with bone-targeting moiety) SEQ ID NO: 16 NGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPY HDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGLGPVESSPGHGLDTAA AGPEPSTRCELSPINASHPVQALMESFTVLSGCASHGTTGLPREVHVLNLRSTDQGPGQRQREVTL HLNPIASVHTHHKPIVFLLNSPQPLVWRLKTERLAAGVPRLFLVSEGSVVQFPSGNFSLTAETEERNF PQENEHLLRWAQKEYGAVTSFTELKIARNIYIKVGEDQVFPPTCNIGKNFLSLNYLAEYLQPKAAEGCV LPSQPHEKEVHIIELITPSSNPYSAFQVDIIVDIRPAQEDPEVVKNLVLILKSKKSVNWVIKSFDVKGNLK VIAPNSIGFGKESERSMTMTKLVRDDIPSTQENLMKWALDAGYRPVTSYTMAPVANRFHLRLENNEE MRDEEVHTIPPELRILLDPDKLPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDE NITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGG GSGGGGSGGGGSGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKDDDDDDDDDD (PCT-0019, without SP, without bone-targeting moiety) SEQ ID NO: 17 NGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPY HDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGLGPVESSPGHGLDTAA AGPEPSTRCELSPINASHPVQALMESFTVLSGCASHGTTGLPREVHVLNLRSTDQGPGQRQREVTL HLNPIASVHTHHKPIVFLLNSPQPLVWRLKTERLAAGVPRLFLVSEGSVVQFPSGNFSLTAETEERNF PQENEHLLRWAQKEYGAVTSFTELKIARNIYIKVGEDQVFPPTCNIGKNFLSLNYLAEYLQPKAAEGCV LPSQPHEKEVHIIELITPSSNPYSAFQVDIIVDIRPAQEDPEVVKNLVLILKSKKSVNWVIKSFDVKGNLK VIAPNSIGFGKESERSMTMTKLVRDDIPSTQENLMKWALDAGYRPVTSYTMAPVANRFHLRLENNEE MRDEEVHTIPPELRILLDPDKLPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDE NITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGG GSGGGGSGGGGSGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (PCT-0020, without SP, with bone-targeting moiety) SEQ ID NO: 18 NGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPY HDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGLGPVESSPGHGLDTAA AGPEPSTRCELSPINASHPVQALMESFTVLSGCASHGTTGLPREVHVLNLRSTDQGPGQRQREVTL HLNPIASVHTHHKPIVFLLNSPQPLVWRLKTERLAAGVPRLFLVSEGSVVQFPSGNFSLTAETEERNF PQENEHLLRWAQKEYGAVTSFTELKIARNIYIKVGEDQVFPPTCNIGKNFLSLNYLAEYLQPKAAEGCV LPSQPHEKEVHIIELITPSSNPYSAFQVDIIVDIRPAQEDPEVVKNLVLILKSKKSVNWVIKSFDVKGNLK VIAPNSIGFGKESERSMTMTKLVRDDIPSTQENLMKWALDAGYRPVTSYTMAPVANRFHLRLENNEE MRDEEVHTIPPELRILLDPDKLPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDE NITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLV IFQVTGISLLPPLGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKDDDDDDDDDD (PCT-0020, without SP, without bone targeting moiety) SEQ ID NO: 19 NGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPY HDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGLGPVESSPGHGLDTAA AGPEPSTRCELSPINASHPVQALMESFTVLSGCASHGTTGLPREVHVLNLRSTDQGPGQRQREVTL HLNPIASVHTHHKPIVFLLNSPQPLVWRLKTERLAAGVPRLFLVSEGSVVQFPSGNFSLTAETEERNF PQENEHLLRWAQKEYGAVTSFTELKIARNIYIKVGEDQVFPPTCNIGKNFLSLNYLAEYLQPKAAEGCV LPSQPHEKEVHIIELITPSSNPYSAFQVDIIVDIRPAQEDPEVVKNLVLILKSKKSVNWVIKSFDVKGNLK VIAPNSIGFGKESERSMTMTKLVRDDIPSTQENLMKWALDAGYRPVTSYTMAPVANRFHLRLENNEE MRDEEVHTIPPELRILLDPDKLPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDE NITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLV IFQVTGISLLPPLGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (PCT-0021, without SP, with bone targeting moiety) SEQ ID NO: 20 NGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPY HDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGLGPVESSPGHGLDTAA AGPEPSTRCELSPINASHPVQALMESFTVLSGCASHGTTGLPREVHVLNLRSTDQGPGQRQREVTL HLNPIASVHTHHKPIVFLLNSPQPLVWRLKTERLAAGVPRLFLVSEGSVVQFPSGNFSLTAETEERNF PQENEHLLRWAQKEYGAVTSFTELKIARNIYIKVGEDQVFPPTCNIGKNFLSLNYLAEYLQPKAAEGCV LPSQPHEKEVHIIELITPSSNPYSAFQVDIIVDIRPAQEDPEVVKNLVLILKSKKSVNWVIKSFDVKGNLK VIAPNSIGFGKESERSMTMTKLVRDDIPSTQENLMKWALDAGYRPVTSYTMAPVANRFHLRLENNEE MRDEEVHTIPPELRILLDPDKLPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDE NITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLV IFQVTGISLLPPLGVAISVIIIDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKDDDDDDDDDD (PCT-0021, without SP, without bone-targeting moiety) SEQ ID NO: 21 NGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPY HDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGLGPVESSPGHGLDTAA AGPEPSTRCELSPINASHPVQALMESFTVLSGCASHGTTGLPREVHVLNLRSTDQGPGQRQREVTL HLNPIASVHTHHKPIVFLLNSPQPLVWRLKTERLAAGVPRLFLVSEGSVVQFPSGNFSLTAETEERNF PQENEHLLRWAQKEYGAVTSFTELKIARNIYIKVGEDQVFPPTCNIGKNFLSLNYLAEYLQPKAAEGCV LPSQPHEKEVHIIELITPSSNPYSAFQVDIIVDIRPAQEDPEVVKNLVLILKSKKSVNWVIKSFDVKGNLK VIAPNSIGFGKESERSMTMTKLVRDDIPSTQENLMKWALDAGYRPVTSYTMAPVANRFHLRLENNEE MRDEEVHTIPPELRILLDPDKLPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDE NITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLV IFQVTGISLLPPLGVAISVIIIDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (PCT-0022, without SP, with bone-targeting moiety) SEQ ID NO: 22 NGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPY HDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLAG PEPSTRCELSPINASHPVQALMESFTVLSGCASHGTTGLPREVHVLNLRSTDQGPGQRQREVTLHLN PIASVHTHHKPIVFLLNSPQPLVWRLKTERLAAGVPRLFLVSEGSVVQFPSGNFSLTAETEERNFPQE NEHLLRWAQKEYGAVTSFTELKIARNIYIKVGEDQVFPPTCNIGKNFLSLNYLAEYLQPKAAEGCVLPS QPHEKEVHIIELITPSSNPYSAFQVDIIVDIRPAQEDPEVVKNLVLILKSKKSVNWVIKSFDVKGNLKVIAP NSIGFGKESERSMTMTKLVRDDIPSTQENLMKWALDAGYRPVTSYTMAPVANRFHLRLENNEEMRD EEVHTIPPELRILLDPDKLPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITL ETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQ VTGISLLPPLGVAISVIIIDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKDDDDDDDDDD (PCT-0022, without SP, without bone-targeting moiety) SEQ ID NO: 23 NGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPY HDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLAG PEPSTRCELSPINASHPVQALMESFTVLSGCASHGTTGLPREVHVLNLRSTDQGPGQRQREVTLHLN PIASVHTHHKPIVFLLNSPQPLVWRLKTERLAAGVPRLFLVSEGSVVQFPSGNFSLTAETEERNFPQE NEHLLRWAQKEYGAVTSFTELKIARNIYIKVGEDQVFPPTCNIGKNFLSLNYLAEYLQPKAAEGCVLPS QPHEKEVHIIELITPSSNPYSAFQVDIIVDIRPAQEDPEVVKNLVLILKSKKSVNWVIKSFDVKGNLKVIAP NSIGFGKESERSMTMTKLVRDDIPSTQENLMKWALDAGYRPVTSYTMAPVANRFHLRLENNEEMRD EEVHTIPPELRILLDPDKLPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITL ETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQ VTGISLLPPLGVAISVIIIDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (PCT-0023, without SP, with bone-targeting moiety) SEQ ID NO: 24 ADNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKL PYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPL AGPEPGALCELSPVSASHPVQALMESFTVLSGCASRGTTGLPQEVHVLNLRTAGQGPGQLQREVTL HLNPISSVHIHHKSVVFLLNSPHPLVWHLKTERLATGVSRLFLVSEGSVVQFSSANFSLTAETEERNFP HGNEHLLNWARKEYGAVTSFTELKIARNIYIKVGEDQVFPPKCNIGKNFLSLNYLAEYLQPKAAEGCV MSSQPQNEEVHIIELITPNSNPYSAFQVDITIDIRPSQEDLEVVKNLILILKSKKSVNWVIKSFDVKGSLKII APNSIGFGKESERSMTMTKSIRDDIPSTQGNLVKWALDNGYSPITSYTMAPVANRFHLRLENNEEMG DEEVHTIPPELRILLDPGALPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENI TLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIF QVTGISLLPPLGVAISVIIIDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKDDDDDDDDDD (PCT-0023, without SP, without bone-targeting moiety) SEQ ID NO: 25 ADNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKL PYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPL AGPEPGALCELSPVSASHPVQALMESFTVLSGCASRGTTGLPQEVHVLNLRTAGQGPGQLQREVTL HLNPISSVHIHHKSVVFLLNSPHPLVWHLKTERLATGVSRLFLVSEGSVVQFSSANFSLTAETEERNFP HGNEHLLNWARKEYGAVTSFTELKIARNIYIKVGEDQVFPPKCNIGKNFLSLNYLAEYLQPKAAEGCV MSSQPQNEEVHIIELITPNSNPYSAFQVDITIDIRPSQEDLEVVKNLILILKSKKSVNWVIKSFDVKGSLKII APNSIGFGKESERSMTMTKSIRDDIPSTQGNLVKWALDNGYSPITSYTMAPVANRFHLRLENNEEMG DEEVHTIPPELRILLDPGALPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENI TLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIF QVTGISLLPPLGVAISVIIIDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (PCT-0024, without SP, with bone-targeting moiety) SEQ ID NO: 26 ADNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKL PYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGLGPVESSPGHGLDT AAAGPEPGALCELSPVSASHPVQALMESFTVLSGCASRGTTGLPQEVHVLNLRTAGQGPGQLQREV TLHLNPISSVHIHHKSVVFLLNSPHPLVWHLKTERLATGVSRLFLVSEGSVVQFSSANFSLTAETEERN FPHGNEHLLNWARKEYGAVTSFTELKIARNIYIKVGEDQVFPPKCNIGKNFLSLNYLAEYLQPKAAEGC VMSSQPQNEEVHIIELITPNSNPYSAFQVDITIDIRPSQEDLEVVKNLILILKSKKSVNWVIKSFDVKGSL KIIAPNSIGFGKESERSMTMTKSIRDDIPSTQGNLVKWALDNGYSPITSYTMAPVANRFHLRLENNEEM GDEEVHTIPPELRILLDPGALPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDEN ITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVI FQVTGISLLPPLGVAISVIIIDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKDDDDDDDDDD (PCT-0024, without SP, without bone-targeting moiety) SEQ ID NO: 27 ADNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKL PYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGLGPVESSPGHGLDT AAAGPEPGALCELSPVSASHPVQALMESFTVLSGCASRGTTGLPQEVHVLNLRTAGQGPGQLQREV TLHLNPISSVHIHHKSVVFLLNSPHPLVWHLKTERLATGVSRLFLVSEGSVVQFSSANFSLTAETEERN FPHGNEHLLNWARKEYGAVTSFTELKIARNIYIKVGEDQVFPPKCNIGKNFLSLNYLAEYLQPKAAEGC VMSSQPQNEEVHIIELITPNSNPYSAFQVDITIDIRPSQEDLEVVKNLILILKSKKSVNWVIKSFDVKGSL KIIAPNSIGFGKESERSMTMTKSIRDDIPSTQGNLVKWALDNGYSPITSYTMAPVANRFHLRLENNEEM GDEEVHTIPPELRILLDPGALPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDEN ITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVI FQVTGISLLPPLGVAISVIIIDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (PCT-0025, without SP, with bone-targeting moiety) SEQ ID NO: 28 ADNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKL PYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPL GVAISVIIIAGPEPGALCELSPVSASHPVQALMESFTVLSGCASRGTTGLPQEVHVLNLRTAGQGPGQ LQREVTLHLNPISSVHIHHKSVVFLLNSPHPLVWHLKTERLATGVSRLFLVSEGSVVQFSSANFSLTAE TEERNFPHGNEHLLNWARKEYGAVTSFTELKIARNIYIKVGEDQVFPPKCNIGKNFLSLNYLAEYLQPK AAEGCVMSSQPQNEEVHIIELITPNSNPYSAFQVDITIDIRPSQEDLEVVKNLILILKSKKSVNWVIKSFD VKGSLKIIAPNSIGFGKESERSMTMTKSIRDDIPSTQGNLVKWALDNGYSPITSYTMAPVANRFHLRLE NNEEMGDEEVHTIPPELRILLDPGALPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVW RKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSN PDLLLVIFQVTGISLLPPLGVAISVIIIDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKDDDDDDDDDD (PCT-0025, without SP, without bone-targeting moiety) SEQ ID NO: 29 ADNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKL PYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPL GVAISVIIIAGPEPGALCELSPVSASHPVQALMESFTVLSGCASRGTTGLPQEVHVLNLRTAGQGPGQ LQREVTLHLNPISSVHIHHKSVVFLLNSPHPLVWHLKTERLATGVSRLFLVSEGSVVQFSSANFSLTAE TEERNFPHGNEHLLNWARKEYGAVTSFTELKIARNIYIKVGEDQVFPPKCNIGKNFLSLNYLAEYLQPK AAEGCVMSSQPQNEEVHIIELITPNSNPYSAFQVDITIDIRPSQEDLEVVKNLILILKSKKSVNWVIKSFD VKGSLKIIAPNSIGFGKESERSMTMTKSIRDDIPSTQGNLVKWALDNGYSPITSYTMAPVANRFHLRLE NNEEMGDEEVHTIPPELRILLDPGALPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVW RKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSN PDLLLVIFQVTGISLLPPLGVAISVIIIDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (PCT-0026, without SP, with bone-targeting moiety) SEQ ID NO: 30 ADNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKL PYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGLGPVESSPGHGLDT AAAGPEPGALCELSPVSASHPVQALMESFTVLSGCASRGTTGLPQEVHVLNLRTAGQGPGQLQREV TLHLNPISSVHIHHKSVVFLLNSPHPLVWHLKTERLATGVSRLFLVSEGSVVQFSSANFSLTAETEERN FPHGNEHLLNWARKEYGAVTSFTELKIARNIYIKVGEDQVFPPKCNIGKNFLSLNYLAEYLQPKAAEGC VMSSQPQNEEVHIIELITPNSNPYSAFQVDITIDIRPSQEDLEVVKNLILILKSKKSVNWVIKSFDVKGSL KIIAPNSIGFGKESERSMTMTKSIRDDIPSTQGNLVKWALDNGYSPITSYTMAPVANRFHLRLENNEEM GDEEVHTIPPELRILLDPGALPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDEN ITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVI FQVTGISLLPPLGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKDDDDDDDDDD (PCT-0026, without SP, without bone-targeting moiety) SEQ ID NO: 31 ADNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKL PYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGLGPVESSPGHGLDT AAAGPEPGALCELSPVSASHPVQALMESFTVLSGCASRGTTGLPQEVHVLNLRTAGQGPGQLQREV TLHLNPISSVHIHHKSVVFLLNSPHPLVWHLKTERLATGVSRLFLVSEGSVVQFSSANFSLTAETEERN FPHGNEHLLNWARKEYGAVTSFTELKIARNIYIKVGEDQVFPPKCNIGKNFLSLNYLAEYLQPKAAEGC VMSSQPQNEEVHIIELITPNSNPYSAFQVDITIDIRPSQEDLEVVKNLILILKSKKSVNWVIKSFDVKGSL KIIAPNSIGFGKESERSMTMTKSIRDDIPSTQGNLVKWALDNGYSPITSYTMAPVANRFHLRLENNEEM GDEEVHTIPPELRILLDPGALPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDEN ITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVI FQVTGISLLPPLGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (PCT-0017, without SP, with bone-targeting moiety) SEQ ID NO: 32 DDDDDDDDDDGGGGSGGGGSGGGGSGGGGSGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGG SGGGGSGGGGSGNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDEN ITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGLGP VESSPGHGLDTAAAGPEPSTRCELSPINASHPVQALMESFTVLSGCASHGTTGLPREVHVLNLRSTD QGPGQRQREVTLHLNPIASVHTHHKPIVFLLNSPQPLVWRLKTERLAAGVPRLFLVSEGSVVQFPSG NFSLTAETEERNFPQENEHLLRWAQKEYGAVTSFTELKIARNIYIKVGEDQVFPPTCNIGKNFLSLNYL AEYLQPKAAEGCVLPSQPHEKEVHIIELITPSSNPYSAFQVDIIVDIRPAQEDPEVVKNLVLILKSKKSVN WVIKSFDVKGNLKVIAPNSIGFGKESERSMTMTKLVRDDIPSTQENLMKWALDAGYRPVTSYTMAPV ANRFHLRLENNEEMRDEEVHTIPPELRILLDPDKLPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKP QEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNII FSEEYNTSNPD (PCT-0016, without SP, no bone-targeting moiety) SEQ ID NO: 33 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGNGAVKFPQLCKFCDVRFSTCD NQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPG ETFFMCSCSSDECNDNIIFSEEYNTSNPDGLGPVESSPGHGLDTAAAGPEPSTRCELSPINASHPVQ ALMESFTVLSGCASHGTTGLPREVHVLNLRSTDQGPGQRQREVTLHLNPIASVHTHHKPIVFLLNSPQ PLVWRLKTERLAAGVPRLFLVSEGSVVQFPSGNFSLTAETEERNFPQENEHLLRWAQKEYGAVTSFT ELKIARNIYIKVGEDQVFPPTCNIGKNFLSLNYLAEYLQPKAAEGCVLPSQPHEKEVHIIELITPSSNPYS AFQVDIIVDIRPAQEDPEVVKNLVLILKSKKSVNWVIKSFDVKGNLKVIAPNSIGFGKESERSMTMTKLV RDDIPSTQENLMKWALDAGYRPVTSYTMAPVANRFHLRLENNEEMRDEEVHTIPPELRILLDPDKLPQ LCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAA SPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (PCT-0018; without SP, with bone-targeting moiety) SEQ ID NO: 34 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGNGAVKFPQLCKFCDVRFSTCD NQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPG ETFFMCSCSSDECNDNIIFSEEYNTSNPDGLGPVESSPGHGLDTAAAGPEPSTRCELSPINASHPVQ ALMESFTVLSGCASHGTTGLPREVHVLNLRSTDQGPGQRQREVTLHLNPIASVHTHHKPIVFLLNSPQ PLVWRLKTERLAAGVPRLFLVSEGSVVQFPSGNFSLTAETEERNFPQENEHLLRWAQKEYGAVTSFT ELKIARNIYIKVGEDQVFPPTCNIGKNFLSLNYLAEYLQPKAAEGCVLPSQPHEKEVHIIELITPSSNPYS AFQVDIIVDIRPAQEDPEVVKNLVLILKSKKSVNWVIKSFDVKGNLKVIAPNSIGFGKESERSMTMTKLV RDDIPSTQENLMKWALDAGYRPVTSYTMAPVANRFHLRLENNEEMRDEEVHTIPPELRILLDPDKLPQ LCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAA SPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSGGGGSGDD DDDDDDDD (PCT-0018, without SP, without bone-targeting moiety) SEQ ID NO: 35 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGNGAVKFPQLCKFCDVRFSTCD NQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPG ETFFMCSCSSDECNDNIIFSEEYNTSNPDGLGPVESSPGHGLDTAAAGPEPSTRCELSPINASHPVQ ALMESFTVLSGCASHGTTGLPREVHVLNLRSTDQGPGQRQREVTLHLNPIASVHTHHKPIVFLLNSPQ PLVWRLKTERLAAGVPRLFLVSEGSVVQFPSGNFSLTAETEERNFPQENEHLLRWAQKEYGAVTSFT ELKIARNIYIKVGEDQVFPPTCNIGKNFLSLNYLAEYLQPKAAEGCVLPSQPHEKEVHIIELITPSSNPYS AFQVDIIVDIRPAQEDPEVVKNLVLILKSKKSVNWVIKSFDVKGNLKVIAPNSIGFGKESERSMTMTKLV RDDIPSTQENLMKWALDAGYRPVTSYTMAPVANRFHLRLENNEEMRDEEVHTIPPELRILLDPDKLPQ LCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAA SPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (Linker) SEQ ID NO: 36 TGGG (Linker) SEQ ID NO: 37 LLLVIFQVTGISLLPPLGGGGS (Linker) SEQ ID NO: 38 LLLVIFQVTGISLLPPLGVAISVIII (Linker) SEQ ID NO: 39 LLLVIFQVTGISLLPPL (Linker) SEQ ID NO: 40 GLGPVESSPGHGLDTAA (Linker) SEQ ID NO: 41 GGGGSGGGGSGGGGSGGGGSG (Alpha-lactalbumin used as signal peptide alternative to albumin) SEQ ID NO: 42 MMSFVSLLLVGILFHATQ (Human R.sub.2a for PCT-0015, PCT-0015NT, PCT-0019, PCT-0019NT, PCT-0021, PCT-0021NT, PCT-0022, PCT-0022NT, PCT-0016NT, PCT-0017, PCT-0018, PCT-0018) SEQ ID NO: 43 NGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPY HDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (Human R.sub.3 for PCT-0023, PCT-0023NT, PCT-0024, PCT-0024NT, PCT-0025, PCT-0025NT, PCT-0026, PCT-0023) SEQ ID NO: 44 AGPEPGALCELSPVSASHPVQALMESFTVLSGCASRGTTGLPQEVHVLNLRTAGQGPGQLQREVTL HLNPISSVHIHHKSVVFLLNSPHPLVWHLKTERLATGVSRLFLVSEGSVVQFSSANFSLTAETEERNFP HGNEHLLNWARKEYGAVTSFTELKIARNIYIKVGEDQVFPPKCNIGKNFLSLNYLAEYLQPKAAEGCV MSSQPQNEEVHIIELITPNSNPYSAFQVDITIDIRPSQEDLEVVKNLILILKSKKSVNWVIKSFDVKGSLKII APNSIGFGKESERSMTMTKSIRDDIPSTQGNLVKWALDNGYSPITSYTMAPVANRFHLRLENNEEMG DEEVHTIPPELRILLDPGAL (Human R.sub.2b for PCT-0015 to PCT-0026) SEQ ID NO: 45 PQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILED AASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (D10 bone-targeting moiety) SEQ ID NO: 46 DDDDDDDDDD (Fc domain of immunoglobulin) SEQ ID NO: 47 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK (RER of PCT-0020) SEQ ID NO: 48 NGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPY HDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGLGPVESSPGHGLDTAA AGPEPSTRCELSPINASHPVQALMESFTVLSGCASHGTTGLPREVHVLNLRSTDQGPGQRQREVTL HLNPIASVHTHHKPIVFLLNSPQPLVWRLKTERLAAGVPRLFLVSEGSVVQFPSGNFSLTAETEERNF PQENEHLLRWAQKEYGAVTSFTELKIARNIYIKVGEDQVFPPTCNIGKNFLSLNYLAEYLQPKAAEGCV LPSQPHEKEVHIIELITPSSNPYSAFQVDIIVDIRPAQEDPEVVKNLVLILKSKKSVNWVIKSFDVKGNLK VIAPNSIGFGKESERSMTMTKLVRDDIPSTQENLMKWALDAGYRPVTSYTMAPVANRFHLRLENNEE MRDEEVHTIPPELRILLDPDKLPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDE NITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (RER of PCT-0022) SEQ ID NO: 49 NGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPY HDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLAG PEPSTRCELSPINASHPVQALMESFTVLSGCASHGTTGLPREVHVLNLRSTDQGPGQRQREVTLHLN PIASVHTHHKPIVFLLNSPQPLVWRLKTERLAAGVPRLFLVSEGSVVQFPSGNFSLTAETEERNFPQE NEHLLRWAQKEYGAVTSFTELKIARNIYIKVGEDQVFPPTCNIGKNFLSLNYLAEYLQPKAAEGCVLPS QPHEKEVHIIELITPSSNPYSAFQVDIIVDIRPAQEDPEVVKNLVLILKSKKSVNWVIKSFDVKGNLKVIAP NSIGFGKESERSMTMTKLVRDDIPSTQENLMKWALDAGYRPVTSYTMAPVANRFHLRLENNEEMRD EEVHTIPPELRILLDPDKLPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITL ETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (RER of PCT-0023) SEQ ID NO: 50 ADNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKL PYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPL AGPEPGALCELSPVSASHPVQALMESFTVLSGCASRGTTGLPQEVHVLNLRTAGQGPGQLQREVTL HLNPISSVHIHHKSVVFLLNSPHPLVWHLKTERLATGVSRLFLVSEGSVVQFSSANFSLTAETEERNFP HGNEHLLNWARKEYGAVTSFTELKIARNIYIKVGEDQVFPPKCNIGKNFLSLNYLAEYLQPKAAEGCV MSSQPQNEEVHIIELITPNSNPYSAFQVDITIDIRPSQEDLEVVKNLILILKSKKSVNWVIKSFDVKGSLKII APNSIGFGKESERSMTMTKSIRDDIPSTQGNLVKWALDNGYSPITSYTMAPVANRFHLRLENNEEMG DEEVHTIPPELRILLDPGALPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENI TLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (RER of PCT-0024 and PCT-0026) SEQ ID NO: 51 ADNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKL PYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGLGPVESSPGHGLDT AAAGPEPGALCELSPVSASHPVQALMESFTVLSGCASRGTTGLPQEVHVLNLRTAGQGPGQLQREV TLHLNPISSVHIHHKSVVFLLNSPHPLVWHLKTERLATGVSRLFLVSEGSVVQFSSANFSLTAETEERN FPHGNEHLLNWARKEYGAVTSFTELKIARNIYIKVGEDQVFPPKCNIGKNFLSLNYLAEYLQPKAAEGC VMSSQPQNEEVHIIELITPNSNPYSAFQVDITIDIRPSQEDLEVVKNLILILKSKKSVNWVIKSFDVKGSL KIIAPNSIGFGKESERSMTMTKSIRDDIPSTQGNLVKWALDNGYSPITSYTMAPVANRFHLRLENNEEM GDEEVHTIPPELRILLDPGALPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDEN ITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (RER of PCT-0025) SEQ ID NO: 52 ADNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKL PYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPL GVAISVIIIAGPEPGALCELSPVSASHPVQALMESFTVLSGCASRGTTGLPQEVHVLNLRTAGQGPGQ LQREVTLHLNPISSVHIHHKSVVFLLNSPHPLVWHLKTERLATGVSRLFLVSEGSVVQFSSANFSLTAE TEERNFPHGNEHLLNWARKEYGAVTSFTELKIARNIYIKVGEDQVFPPKCNIGKNFLSLNYLAEYLQPK AAEGCVMSSQPQNEEVHIIELITPNSNPYSAFQVDITIDIRPSQEDLEVVKNLILILKSKKSVNWVIKSFD VKGSLKIIAPNSIGFGKESERSMTMTKSIRDDIPSTQGNLVKWALDNGYSPITSYTMAPVANRFHLRLE NNEEMGDEEVHTIPPELRILLDPGALPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVW RKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSN PD (Linker) SEQ ID NO: 402 GGGGSGGGGS (Linker) SEQ ID NO: 53 TGGGDGGGGS (Linker) SEQ ID NO: 54 GGGGSLLLVIFQVTGISLLPPLGVAISVIII (Linker) SEQ ID NO: 55 GGGGSLLLVIFQVTGISLLPPL (Linker) SEQ ID NO: 56 GGGGSGGGGSLLLVIFQVTGISLLPPL (Linker) SEQ ID NO: 57 GGGGSGLGPVESSPGHGLDTAA (Linker) SEQ ID NO: 58 GGGGSGGGGSGLGPVESSPGHGLDTAA (Linker) SEQ ID NO: 59 KL (Linker) SEQ ID NO: 60 (GGGS)n, wherein n =1, 2, 3, 4, or 5 (Linker) SEQ ID NO: 61 (GGGGS)n, wherein n =1, 2, 3, 4, or 5 (Heavy chain of PCT-0011 with D10 bone-targeting moiety) SEQ ID NO: 62 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSSNVISWVRQAPGQGLEWMGGVIPIVDIANYAQRFKG RVTITADESTSTTYMELSSLRSEDTAVYYCASTLGLVLDAMDYWGQGTLVTVSSASTKGPSVFPLAP CSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKT YTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS QEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIE KTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKDDDDDDDDDD (Light chain of PCT-0011) SEQ ID NO: 63 ETVLTQSPGTLSLSPGERATLSCRASQSLGSSYLAWYQQKPGQAPRLLIYGASSRAPGIPDRFSGSG SGTDFTLTISRLEPEDFAVYYCQQYADSPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC (Nucleic acid sequence of PCT-0023 construct) SEQ ID NO: 398 TTTAAGCTTGCCGCCACCATGATGTCCTTTGTCTCTCTGCTCCTGGTTGGCATCCTATTCCATGC CACCCAGGCCGACAACGGTGCAGTCAAGTTTCCACAACTGTGTAAATTTTGTGATGTGAGATTTT CCACCTGTGACAACCAGAAATCCTGCATGAGCAACTGCAGCATCACCTCCATCTGTGAGAAGCC ACAGGAAGTCTGTGTGGCTGTATGGAGAAAGAATGACGAGAACATAACACTAGAGACAGTTTGC CATGACCCCAAGCTCCCCTACCATGACTTTATTCTGGAAGATGCTGCTTCTCCAAAGTGCATTAT GAAGGAAAAAAAAAAGCCTGGAGAGACTTTCTTCATGTGTTCCTGTAGCTCTGATGAGTGCAATG ACAACATCATCTTCTCAGAAGAATATAACACCAGCAATCCTGACTTGTTGCTAGTCATATTTCAAG TGACAGGCATCAGCCTCCTGCCACCACTGGCAGGTCCAGAGCCTGGTGCACTGTGTGAACTGTC ACCTGTCAGTGCCTCCCATCCTGTCCAGGCCTTGATGGAGAGCTTCACTGTTTTGTCAGGCTGT GCCAGCAGAGGCACAACTGGGCTGCCACAGGAGGTGCATGTCCTGAATCTCCGCACTGCAGGC CAGGGGCCTGGCCAGCTACAGAGAGAGGTCACACTTCACCTGAATCCCATCTCCTCAGTCCACA TCCACCACAAGTCTGTTGTGTTCCTGCTCAACTCCCCACACCCCCTGGTGTGGCATCTGAAGACA GAGAGACTTGCCACTGGGGTCTCCAGACTGTTTTTGGTGTCTGAGGGTTCTGTGGTCCAGTTTTC ATCAGCAAACTTCTCCTTGACAGCAGAAACAGAAGAAAGGAACTTCCCCCATGGAAATGAACATC TGTTAAATTGGGCCCGAAAAGAGTATGGAGCAGTTACTTCATTCACCGAACTCAAGATAGCAAGA AACATTTATATTAAAGTGGGGGAAGATCAAGTGTTCCCTCCAAAGTGCAACATAGGGAAGAATTT TCTCTCACTCAATTACCTTGCTGAGTACCTTCAACCCAAAGCAGCAGAAGGGTGTGTGATGTCCA GCCAGCCCCAGAATGAGGAAGTACACATCATCGAGCTAATCACCCCCAACTCTAACCCCTACAG TGCTTTCCAGGTGGATATAACAATTGATATAAGACCTTCTCAAGAGGATCTTGAAGTGGTCAAAAA TCTCATCCTGATCTTGAAGTCTAAAAAGTCTGTCAACTGGGTGATCAAATCTTTTGATGTTAAGGG AAGCCTGAAAATTATTGCTCCTAACAGTATTGGCTTTGGAAAAGAGAGTGAAAGATCTATGACAAT GACCAAATCAATAAGAGATGACATTCCTTCAACCCAAGGGAATCTGGTGAAGTGGGCATTTGGACA ATGGCTATAGTCCAATAACTTCATACACAATGGCTCCTGTGGCTAATAGATTTCATCTTCGGCTTG AAAATAATGAGGAGATGGGAGATGAGGAAGTCCACACTATTCCTCCTGAGCTACGGATCCTGCT GGACCCTGGTGCCCTGCCGCAACTTTGCAAGTTCTGCGACGTGCGATTCTCTACGTGCGATAAT CAAAAGTCCTGTATGTCAAACTGCAGTATTACTTCTATTTGTGAGAAGCCTCAGGAGGTTTGTGTC GCGGTCTGGCGGAAAAACGACGAAAATATCACATTGGAAACGGTCTGCCACGACCCCAAACTTC CCTATCATGATTTCATACTTGAGGATGCAGCTTCACCTAAGTGTATTATGAAAGAGAAGAAGAAAC CAGGCGAAACGTTCTTTATGTGCAGTTGCTCCTCCGATGAATGCAATGATAACATCATTTTCTCC GAGGAGTACAATACTTCAAATCCAGACCTCCTTCTCGTCATTTTTCAAGTTACAGGTATTTCACTG CTCCCCCCTCTCGGCGTTGCGATATCAGTTATCATCATCGACAAAACTCACACATGCCCACCGTG CCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACC CTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCT GAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGG GAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGG CTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAA CCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGG ATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACAT CGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCT GGACTCCGACGGCTCCTTCTTCCTCTATTCCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCC TCTCCCTGTCTCCCGGGAAAGACGACGATGATGATGACGATGACGACGATTGATGACTCGAGTTT (Nucleic acid sequence of PCT-0024 construct) SEQ ID NO: 399 TTTAAGCTTGCCGCCACCATGATGTCCTTTGTCTCTCTGCTCCTGGTTGGCATCCTATTCCATGC CACCCAGGCCGACAACGGTGCAGTCAAGTTTCCACAACTGTGTAAATTTTGTGATGTGAGATTTT CCACCTGTGACAACCAGAAATCCTGCATGAGCAACTGCAGCATCACCTCCATCTGTGAGAAGCC ACAGGAAGTCTGTGTGGCTGTATGGAGAAAGAATGACGAGAACATAACACTAGAGACAGTTTGC CATGACCCCAAGCTCCCCTACCATGACTTTATTCTGGAAGATGCTGCTTCTCCAAAGTGCATTAT GAAGGAAAAAAAAAAGCCTGGAGAGACTTTCTTCATGTGTTCCTGTAGCTCTGATGAGTGCAATG ACAACATCATCTTCTCAGAAGAATATAACACCAGCAATCCTGACGGTCTTGGCCCCGTCGAGAGT AGCCCTGGGCATGGCCTTGATACCGCAGCGGCAGGTCCAGAGCCTGGTGCACTGTGTGAACTG TCACCTGTCAGTGCCTCCCATCCTGTCCAGGCCTTGATGGAGAGCTTCACTGTTTTGTCAGGCTG TGCCAGCAGAGGCACAACTGGGCTGCCACAGGAGGTGCATGTCCTGAATCTCCGCACTGCAGG CCAGGGGCCTGGCCAGCTACAGAGAGAGGTCACACTTCACCTGAATCCCATCTCCTCAGTCCAC ATCCACCACAAGTCTGTTGTGTTCCTGCTCAACTCCCCACACCCCCTGGTGTGGCATCTGAAGAC AGAGAGACTTGCCACTGGGGTCTCCAGACTGTTTTTGGTGTCTGAGGGTTCTGTGGTCCAGTTTT CATCAGCAAACTTCTCCTTGACAGCAGAAACAGAAGAAAGGAACTTCCCCCATGGAAATGAACAT CTGTTAAATTGGGCCCGAAAAGAGTATGGAGCAGTTACTTCATTCACCGAACTCAAGATAGCAAG AAACATTTATATTAAAGTGGGGGAAGATCAAGTGTTCCCTCCAAAGTGCAACATAGGGAAGAATT TTCTCTCACTCAATTACCTTGCTGAGTACCTTCAACCCAAAGCAGCAGAAGGGTGTGTGATGTCC AGCCAGCCCCAGAATGAGGAAGTACACATCATCGAGCTAATCACCCCCAACTCTAACCCCTACA GTGCTTTCCAGGTGGATATAACAATTGATATAAGACCTTCTCAAGAGGATCTTGAAGTGGTCAAA AATCTCATCCTGATCTTGAAGTCTAAAAAGTCTGTCAACTGGGTGATCAAATCTTTTGATGTTAAG GGAAGCCTGAAAATTATTGCTCCTAACAGTATTGGCTTTGGAAAAGAGAGTGAAAGATCTATGAC AATGACCAAATCAATAAGAGATGACATTCCTTCAACCCAAGGGAATCTGGTGAAGTGGGCTTTGG ACAATGGCTATAGTCCAATAACTTCATACACAATGGCTCCTGTGGCTAATAGATTTCATCTTCGGC TTGAAAATAATGAGGAGATGGGAGATGAGGAAGTCCACACTATTCCTCCTGAGCTACGGATCCT GCTGGACCCTGGTGCCCTGCCGCAACTTTGCAAGTTCTGCGACGTGCGATTCTCTACGTGCGAT AATCAAAAGTCCTGTATGTCAAACTGCAGTATTACTTCTATTTGTGAGAAGCCTCAGGAGGTTTGT GTCGCGGTCTGGCGGAAAAACGACGAAAATATCACATTGGAAACGGTCTGCCACGACCCCAAAC TTCCCTATCATGATTTCATACTTGAGGATGCAGCTTCACCTAAGTGTATTATGAAAGAGAAGAAGA AACCAGGCGAAACGTTCTTTATGTGCAGTTGCTCCTCCGATGAATGCAATGATAACATCATTTTCT CCGAGGAGTACAATACTTCAAATCCAGACCTCCTTCTCGTCATTTTTCAAGTTACAGGTATTTCAC TGCTCCCCCCTCTCGGCGTTGCGATATCAGTTATCATCATCGACAAAACTCACACATGCCCACCG TGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACA CCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACC CTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGC GGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACT GGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAA AACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCG GGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGAC ATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTG CTGGACTCCGACGGCTCCTTCTTCCTCTATTCCAAGCTCACCGTGGACAAGAGCAGGTGGCAGC AGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAG CCTCTCCCTGTCTCCCGGGAAAGACGACGATGATGATGACGATGACGACGATTGATGACTCGAG TTT (Nucleic acid sequence of PCT-0025 construct) SEQ ID NO: 400 TTTAAGCTTGCCGCCACCATGATGTCCTTTGTCTCTCTGCTCCTGGTTGGCATCCTATTCCATGC CACCCAGGCCGACAACGGTGCAGTCAAGTTTCCACAACTGTGTAAATTTTGTGATGTGAGATTTT CCACCTGTGACAACCAGAAATCCTGCATGAGCAACTGCAGCATCACCTCCATCTGTGAGAAGCC ACAGGAAGTCTGTGTGGCTGTATGGAGAAAGAATGACGAGAACATAACACTAGAGACAGTTTGC CATGACCCCAAGCTCCCCTACCATGACTTTATTCTGGAAGATGCTGCTTCTCCAAAGTGCATTAT GAAGGAAAAAAAAAAGCCTGGAGAGACTTTCTTCATGTGTTCCTGTAGCTCTGATGAGTGCAATG ACAACATCATCTTCTCAGAAGAATATAACACCAGCAATCCTGACTTGTTGCTAGTCATATTTCAAG TGACAGGCATCAGCCTCCTGCCACCACTGGGAGTTGCCATATCTGTCATCATCATCGCAGGTCC AGAGCCTGGTGCACTGTGTGAACTGTCACCTGTCAGTGCCTCCCATCCTGTCCAGGCCTTGATG GAGAGCTTCACTGTTTTGTCAGGCTGTGCCAGCAGAGGCACAACTGGGCTGCCACAGGAGGTG CATGTCCTGAATCTCCGCACTGCAGGCCAGGGGCCTGGCCAGCTACAGAGAGAGGTCACACTT CACCTGAATCCCATCTCCTCAGTCCACATCCACCACAAGTCTGTTGTGTTCCTGCTCAACTCCCC ACACCCCCTGGTGTGGCATCTGAAGACAGAGAGACTTGCCACTGGGGTCTCCAGACTGTTTTTG GTGTCTGAGGGTTCTGTGGTCCAGTTTTCATCAGCAAACTTCTCCTTGACAGCAGAAACAGAAGA AAGGAACTTCCCCCATGGAAATGAACATCTGTTAAATTGGGCCCGAAAAGAGTATGGAGCAGTTA CTTCATTCACCGAACTCAAGATAGCAAGAAACATTTATATTAAAGTGGGGGAAGATCAAGTGTTC CCTCCAAAGTGCAACATAGGGAAGAATTTTCTCTCACTCAATTACCTTGCTGAGTACCTTCAACCC AAAGCAGCAGAAGGGTGTGTGATGTCCAGCCAGCCCCAGAATGAGGAAGTACACATCATCGAGC TAATCACCCCCAACTCTAACCCCTACAGTGCTTTCCAGGTGGATATAACAATTGATATAAGACCTT CTCAAGAGGATCTTGAAGTGGTCAAAAATCTCATCCTGATCTTGAAGTCTAAAAAGTCTGTCAACT GGGTGATCAAATCTTTTGATGTTAAGGGAAGCCTGAAAATTATTGCTCCTAACAGTATTGGCTTTG GAAAAGAGAGTGAAAGATCTATGACAATGACCAAATCAATAAGAGATGACATTCCTTCAACCCAA GGGAATCTGGTGAAGTGGGCTTTGGACAATGGCTATAGTCCAATAACTTCATACACAATGGCTCC TGTGGCTAATAGATTTCATCTTCGGCTTGAAAATAATGAGGAGATGGGAGATGAGGAAGTCCACA CTATTCCTCCTGAGCTACGGATCCTGCTGGACCCTGGTGCCCTGCCGCAACTTTGCAAGTTCTG CGACGTGCGATTCTCTACGTGCGATAATCAAAAGTCCTGTATGTCAAACTGCAGTATTACTTCTAT TTGTGAGAAGCCTCAGGAGGTTTGTGTCGCGGTCTGGCGGAAAAACGACGAAAATATCACATTG GAAACGGTCTGCCACGACCCCAAACTTCCCTATCATGATTTCATACTTGAGGATGCAGCTTCACC TAAGTGTATTATGAAAGAGAAGAAGAAACCAGGCGAAACGTTCTTTATGTGCAGTTGCTCCTCCG ATGAATGCAATGATAACATCATTTTCTCCGAGGAGTACAATACTTCAAATCCAGACCTCCTTCTCG TCATTTTTCAAGTTACAGGTATTTCACTGCTCCCCCCTCTCGGCGTTGCGATATCAGTTATCATCA TCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTT CCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTG GTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAG GTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGC GTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACA AAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCAC AGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCC TGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGA ACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATTCCAAGCTC ACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCT CTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCCGGGAAAGACGACGATGATGATG ACGATGACGACGATTGATGACTCGAGTTT (Nucleic acid sequence of PCT-0026 construct) SEQ ID NO: 401 TTTAAGCTTGCCGCCACCATGATGTCCTTTGTCTCTCTGCTCCTGGTTGGCATCCTATTCCATGC CACCCAGGCCGACAACGGTGCAGTCAAGTTTCCACAACTGTGTAAATTTTGTGATGTGAGATTTT CCACCTGTGACAACCAGAAATCCTGCATGAGCAACTGCAGCATCACCTCCATCTGTGAGAAGCC ACAGGAAGTCTGTGTGGCTGTATGGAGAAAGAATGACGAGAACATAACACTAGAGACAGTTTGC CATGACCCCAAGCTCCCCTACCATGACTTTATTCTGGAAGATGCTGCTTCTCCAAAGTGCATTAT GAAGGAAAAAAAAAAGCCTGGAGAGACTTTCTTCATGTGTTCCTGTAGCTCTGATGAGTGCAATG ACAACATCATCTTCTCAGAAGAATATAACACCAGCAATCCTGACGGTCTTGGCCCCGTCGAGAGT AGCCCTGGGCATGGCCTTGATACCGCAGCGGCAGGTCCAGAGCCTGGTGCACTGTGTGAACTG TCACCTGTCAGTGCCTCCCATCCTGTCCAGGCCTTGATGGAGAGCTTCACTGTTTTGTCAGGCTG TGCCAGCAGAGGCACAACTGGGCTGCCACAGGAGGTGCATGTCCTGAATCTCCGCACTGCAGG CCAGGGGCCTGGCCAGCTACAGAGAGAGGTCACACTTCACCTGAATCCCATCTCCTCAGTCCAC ATCCACCACAAGTCTGTTGTGTTCCTGCTCAACTCCCCACACCCCCTGGTGTGGCATCTGAAGAC AGAGAGACTTGCCACTGGGGTCTCCAGACTGTTTTTGGTGTCTGAGGGTTCTGTGGTCCAGTTTT CATCAGCAAACTTCTCCTTGACAGCAGAAACAGAAGAAAGGAACTTCCCCCATGGAAATGAACAT CTGTTAAATTGGGCCCGAAAAGAGTATGGAGCAGTTACTTCATTCACCGAACTCAAGATAGCAAG AAACATTTATATTAAAGTGGGGGAAGATCAAGTGTTCCCTCCAAAGTGCAACATAGGGAAGAATT TTCTCTCACTCAATTACCTTGCTGAGTACCTTCAACCCAAAGCAGCAGAAGGGTGTGTGATGTCC AGCCAGCCCCAGAATGAGGAAGTACACATCATCGAGCTAATCACCCCCAACTCTAACCCCTACA GTGCTTTCCAGGTGGATATAACAATTGATATAAGACCTTCTCAAGAGGATCTTGAAGTGGTCAAA AATCTCATCCTGATCTTGAAGTCTAAAAAGTCTGTCAACTGGGTGATCAAATCTTTTGATGTTAAG GGAAGCCTGAAAATTATTGCTCCTAACAGTATTGGCTTTGGAAAAGAGAGTGAAAGATCTATGAC AATGACCAAATCAATAAGAGATGACATTCCTTCAACCCAAGGGAATCTGGTGAAGTGGGCTTTGG ACAATGGCTATAGTCCAATAACTTCATACACAATGGCTCCTGTGGCTAATAGATTTCATCTTCGGC TTGAAAATAATGAGGAGATGGGAGATGAGGAAGTCCACACTATTCCTCCTGAGCTACGGATCCT GCTGGACCCTGGTGCCCTGCCGCAACTTTGCAAGTTCTGCGACGTGCGATTCTCTACGTGCGAT AATCAAAAGTCCTGTATGTCAAACTGCAGTATTACTTCTATTTGTGAGAAGCCTCAGGAGGTTTGT GTCGCGGTCTGGCGGAAAAACGACGAAAATATCACATTGGAAACGGTCTGCCACGACCCCAAAC TTCCCTATCATGATTTCATACTTGAGGATGCAGCTTCACCTAAGTGTATTATGAAAGAGAAGAAGA AACCAGGCGAAACGTTCTTTATGTGCAGTTGCTCCTCCGATGAATGCAATGATAACATCATTTTCT CCGAGGAGTACAATACTTCAAATCCAGACCTCCTTCTCGTCATTTTTCAAGTTACAGGTATTTCAC TGCTCCCCCCTCTCGGCGGAGGCGGTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCAC CTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGAT CTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAA GTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCA GTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGG CAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTG ACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGG AGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCG ACGGCTCCTTCTTCCTCTATTCCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGT CTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTG TCTCCCGGGAAAGACGACGATGATGATGACGATGACGACGATTGATGACTCGAGTTT