Modified Fc molecules

[0001] This application claims the benefit of U.S. Provisional Application No. 60/612,680, filed Sep. 24, 2004, which is hereby incorporated by reference.

[0002] The success of the drug Enbrel® (etanercept) brought to fruition the promise of therapeutic agents modified with the constant domain of an antibody. Antibodies comprise two functionally independent parts, a variable domain known as “Fab”, which binds antigen, and a constant domain known as “Fc”, which links to such effector functions as complement activation and attack by phagocytic cells. An Fc has a long serum half-life, whereas an Fab is short-lived. Capon et al. (1989), Nature 337: 525-31. When constructed together with a therapeutic protein, an Fc domain can provide longer half-life or incorporate such functions as Fc receptor binding, protein A binding, complement fixation and perhaps even placental transfer. Id. Table 1 summarizes use of Fc fusion proteins known in the art.

TABLE 1
Fc fusion with therapeutic proteins
	Fusion	Therapeutic
Form of Fc	partner	implications	Reference
IgG1	N-terminus	Hodgkin's disease;	U.S. Pat. No. 5,480,981
	of CD30-L	anaplastic lymphoma;
		T-cell leukemia
Murine Fcγ2a	IL-10	anti-inflammatory;	Zheng et al. (1995), J.
		transplant rejection	Immunol. 154: 5590-600
IgG1	TNF	septic shock	Fisher et al. (1996), N. Engl.
	receptor		J. Med. 334: 1697-1702; Van
			Zee, K. et al. (1996), J.
			Immunol. 156: 2221-30
IgG, IgA, IgM, or	TNF	inflammation,	U.S. Pat. No. 5,808,029,
IgE (excluding	receptor	autoimmune disorders	issued Sep. 15, 1998
the first domain)
IgG1	CD4	AIDS	Capon et al. (1989), Nature
	receptor		337: 525-31
IgG1,	N-terminus	anti-cancer, antiviral	Harvill et al. (1995),
IgG3	of IL-2		Immunotech. 1: 95-105
IgG1	C-terminus	osteoarthritis;	WO 97/23614, published Jul.
	of OPG	bone density	3, 1997
IgG1	N-terminus	anti-obesity	WO 98/28427, filed
	of leptin		Dec. 11, 1997
Human Ig Cγ1	CTLA-4	autoimmune disorders	Linsley (1991), J. Exp. Med.
			174: 561-9

[0003] A much different approach to development of therapeutic agents is peptide library screening. The interaction of a protein ligand with its receptor often takes place at a relatively large interface. However, as demonstrated for human growth hormone and its receptor, only a few key residues at the interface contribute to most of the binding energy. Clackson et al. (1995), Science 267: 383-6. The bulk of the protein ligand merely displays the binding epitopes in the right topology or serves functions unrelated to binding. Thus, molecules of only “peptide” length (2 to 40 amino acids) can bind to the receptor protein of a given large protein ligand. Such peptides may mimic the bioactivity of the large protein ligand (“peptide agonists”) or, through competitive binding, inhibit the bioactivity of the large protein ligand (“peptide antagonists”).

[0004] Phage display peptide libraries have emerged as a powerful method in identifying such peptide agonists and antagonists. See, for example, Scott et al. (1990), Science 249: 386; Devlin et al. (1990), Science 249: 404; U.S. Pat. No. 5,223,409, issued Jun. 29, 1993; U.S. Pat. No. 5,733,731, issued Mar. 31, 1998; U.S. Pat. No. 5,498,530, issued Mar. 12, 1996; U.S. Pat. No. 5,432,018, issued Jul. 11, 1995; U.S. Pat. No. 5,338,665, issued Aug. 16, 1994; U.S. Pat. No. 5,922,545, issued Jul. 13, 1999; WO 96/40987, published Dec. 19, 1996; and WO 98/15833, published Apr. 16, 1998 (each of which is incorporated by reference). In such libraries, random peptide sequences are displayed by fusion with coat proteins of filamentous phage. Typically, the displayed peptides are affinity-eluted against an antibody-immobilized extracellular domain of a receptor. The retained phages may be enriched by successive rounds of affinity purification and repropagation. The best binding peptides may be sequenced to identify key residues within one or more structurally related families of peptides. See, e.g., Cwirla et al. (1997), Science 276: 1696-9, in which two distinct families were identified. The peptide sequences may also suggest which residues may be safely replaced by alanine scanning or by mutagenesis at the DNA level. Mutagenesis libraries may be created and screened to further optimize the sequence of the best binders. Lowman (1997), Ann. Rev. Biophys. Biomol. Struct. 26:401-24.

[0005] Other methods compete with phage display in peptide research. A peptide library can be fused to the carboxyl terminus of the lac repressor and expressed in E. coli. Another E. coli-based method allows display on the cell's outer membrane by fusion with a peptidoglycan-associated lipoprotein (PAL). Hereinafter, these and related methods are collectively referred to as “E. coli display.” Another biological approach to screening soluble peptide mixtures uses yeast for expression and secretion. See Smith et al. (1993), Mol. Pharmacol. 43: 741-8. Hereinafter, the method of Smith et al. and related methods are referred to as “yeast-based screening.” In another method, translation of random RNA is halted prior to ribosome release, resulting in a library of polypeptides with their associated RNA still attached. Hereinafter, this and related methods are collectively referred to as “ribosome display.” Other methods employ chemical linkage of peptides to RNA; see, for example, Roberts & Szostak (1997), Proc. Natl. Acad. Sci. USA 94: 12297-303. Hereinafter, this and related methods are collectively referred to as “RNA-peptide screening.” Chemically derived peptide libraries have been developed in which peptides are immobilized on stable, non-biological materials, such as polyethylene rods or solvent-permeable resins. Another chemically derived peptide library uses photolithography to scan peptides immobilized on glass slides. Hereinafter, these and related methods are collectively referred to as “chemical-peptide screening.” Chemical-peptide screening may be advantageous in that it allows use of D-amino acids and other unnatural analogues, as well as non-peptide elements. Both biological and chemical methods are reviewed in Wells & Lowman (1992), Curr. Opin. Biotechnol. 3: 355-62.

[0006] In the case of known bioactive peptides, rational design of peptide ligands with favorable therapeutic properties can be completed. In such an approach, one makes stepwise changes to a peptide sequence and determines the effect of the substitution upon bioactivity or a predictive biophysical property of the peptide (e.g., solution structure). Hereinafter, these techniques are collectively referred to as “rational design.” In one such technique, one makes a series of peptides in which one replaces a single residue at a time with alanine. This technique is commonly referred to as an “alanine walk” or an “alanine scan.” When two residues (contiguous or spaced apart) are replaced, it is referred to as a “double alanine walk.” The resultant amino acid substitutions can be used alone or in combination to result in a new peptide entity with favorable therapeutic properties.

[0007] Structural analysis of protein-protein interaction may also be used to suggest peptides that mimic the binding activity of large protein ligands. In such an analysis, the crystal structure may suggest the identity and relative orientation of critical residues of the large protein ligand, from which a peptide may be designed. See, e.g., Takasaki et al. (1997), Nature Biotech. 15: 1266-70. Hereinafter, these and related methods are referred to as “protein structural analysis.” These analytical methods may also be used to investigate the interaction between a receptor protein and peptides selected by phage display, which may suggest further modification of the peptides to increase binding affinity.

[0008] Conceptually, one may discover peptide mimetics of any protein using phage display and the other methods mentioned above. These methods have been used for epitope mapping, for identification of critical amino acids in protein-protein interactions, and as leads for the discovery of new therapeutic agents. E.g., Cortese et al. (1996), Curr. Opin. Biotech. 7: 616-21. Peptide libraries are now being used most often in immunological studies, such as epitope mapping. Kreeger (1996), The Scientist 10(13): 19-20.

[0009] Of particular interest here is use of peptide libraries and other techniques in the discovery of pharmacologically active peptides. A number of such peptides identified in the art are summarized in Table 2. The peptides are described in the listed publications, each of which is hereby incorporated by reference. The pharmacologic activity of the peptides is described, and in many instances is followed by a shorthand term therefore in parentheses. Some of these peptides have been modified (e.g., to form C-terminally cross-linked dimers). Typically, peptide libraries were screened for binding to a receptor for a pharmacologically active protein (e.g., EPO receptor). In at least one instance (CTLA4), the peptide library was screened for binding to a monoclonal antibody.

TABLE 2
Pharmacologically active peptides
Form of	Binding partner/
peptide	protein of interesta	Pharmacologic activity	Reference
intrapeptide	EPO receptor	EPO-mimetic	Wrighton et al. (1996),
disulfide-			Science 273: 458-63; U.S.
bonded			Pat. No. 5,773,569, issued
			Jun. 30, 1998 to
			Wrighton et al.
C-terminally	EPO receptor	EPO-mimetic	Livnah et al. (1996),
cross-linked			Science 273: 464-71;
dimer			Wrighton et al. (1997),
			Nature Biotechnology 15:
			1261-5; International
			patent application WO
			96/40772, published Dec.
			19, 1996
linear	EPO receptor	EPO-mimetic	Naranda et al. (1999),
			Proc. Natl. Acad. Sci.
			USA 96: 7569-74; WO
			99/47151, published
			Sep. 23, 1999
linear; C-	c-Mpl	TPO-mimetic	Cwirla et al.(1997)
terminally			Science 276: 1696-9; U.S.
cross-linked			Pat. No. 5,869,451, issued
dimer			Feb. 9, 1999; U.S. Pat. No.
			5,932,946, issued Aug. 3,
			1999; WO 00/24770,
			published May 4, 2000;
			U.S. Pat. App. No.
			2003/0176352, published
			Sept. 18, 2003; WO
			03/031589, published
			Apr. 17, 2003
disulfide-		stimulation of	Paukovits et al. (1984),
linked dimer		hematopoiesis	Hoppe-Seylers Z.
		(“G-CSF-mimetic”)	Physiol. Chem. 365: 303-11;
			Laerum et al. (1988),
			Exp. Hemat. 16: 274-80
alkylene-		G-CSF-mimetic	Bhatnagar et al. (1996), J.
linked dimer			Med. Chem. 39: 3814-9;
			Cuthbertson et al. (1997),
			J. Med. Chem. 40: 2876-82;
			King et al. (1991),
			Exp. Hematol. 19: 481;
			King et al. (1995), Blood
			86 (Suppl. 1): 309a
linear	IL-1 receptor	inflammatory and	U.S. Pat. No. 5,608,035;
		autoimmune diseases	U.S. Pat. No. 5,786,331;
		(“IL-1 antagonist” or	U.S. Pat. No. 5,880,096;
		“IL-1ra-mimetic”)	Yanofsky et al. (1996),
			Proc. Natl. Acad. Sci. 93:
			7381-6; Akeson et al.
			(1996), J. Biol. Chem. 271:
			30517-23; Wiekzorek et
			al. (1997), Pol. J.
			Pharmacol. 49: 107-17;
			Yanofsky (1996), PNAs,
			93: 7381-7386.
linear	Facteur thymique	stimulation of	Inagaki-Ohara et al.
	serique (FTS)	lymphocytes	(1996), Cellular
		(“FTS-mimetic”)	Immunol. 171: 30-40;
			Yoshida (1984), Int. J.
			Immunopharmacol,
			6: 141-6.
intrapeptide	CTLA4 MAb	CTLA4-mimetic	Fukumoto et al. (1998),
disulfide			Nature Biotech. 16: 267-70
bonded
exocyclic	TNF-α receptor	TNF-α antagonist	Takasaki et al. (1997),
			Nature Biotech. 15: 1266-70;
			WO 98/53842,
			published Dec. 3,
			1998
linear	TNF-α receptor	TNF-α antagonist	Chirinos-Rojas ( ), J.
			Imm., 5621-5626.
intrapeptide	C3b	inhibition of	Sahu et al. (1996), J.
disulfide		complement activation;	Immunol. 157: 884-91;
bonded		autoimmune diseases	Morikis et al. (1998),
		(“C3b-antagonist”)	Protein Sci. 7: 619-27
linear	vinculin	cell adhesion	Adey et al. (1997),
		processes-cell growth,	Biochem. J. 324: 523-8
		differentiation, wound
		healing, tumor
		metastasis (“vinculin
		binding”)
linear	C4 binding protein	anti-thrombotic	Linse et al. (1997), J. Biol.
	(C4BP)		Chem. 272: 14658-65
linear	urokinase receptor	processes associated	Goodson et al. (1994),
		with urokinase	Proc. Natl. Acad. Sci. 91:
		interaction with its	7129-33; International
		receptor (e.g.,	application WO
		angiogenesis, tumor	97/35969, published
		cell invasion and	Oct. 2, 1997
		metastasis); (“UKR
		antagonist”)
linear	Mdm2, Hdm2	Inhibition of	Picksley et al. (1994),
		inactivation of p53	Oncogene 9: 2523-9;
		mediated by Mdm2 or	Bottger et al. (1997) J.
		hdm2; anti-tumor	Mol. Biol. 269: 744-56;
		(“Mdm/hdm	Bottger et al. (1996),
		antagonist”)	Oncogene 13: 2141-7
linear	p21WAF1	anti-tumor by	Ball et al. (1997), Curr.
		mimicking the activity	Biol. 7: 71-80
		of p21WAF1
linear	farnesyl transferase	anti-cancer by	Gibbs et al. (1994), Cell
		preventing activation	77: 175-178
		of ras oncogene
linear	Ras effector domain	anti-cancer by	Moodie et al. (1994),
		inhibiting biological	Trends Genet 10: 44-48
		function of the ras	Rodriguez et al. (1994),
		oncogene	Nature 370: 527-532
linear	SH2/SH3 domains	anti-cancer by	Pawson et al (1993),
		inhibiting tumor	Curr. Biol. 3: 434-432
		growth with activated	Yu et al. (1994), Cell
		tyrosine kinases;	76: 933-945; Rickles et al.
		treatment of SH3-	(1994), EMBO J. 13: 5598-5604;
		mediated disease states	Sparks et al. (1994),
		(“SH3 antagonist”)	J. Biol. Chem. 269: 23853-6;
			Sparks et al. (1996),
			Proc. Natl. Acad. Sci. 93:
			1540-4; U.S. Pat. No.
			5,886,150, issued Mar.
			23, 1999; U.S. Pat. No.
			5,888,763, issued Mar.
			30, 1999
linear	p16INK4	anti-cancer by	Fåhraeus et al. (1996),
		mimicking activity of	Curr. Biol. 6: 84-91
		p16; e.g., inhibiting
		cyclin D-Cdk complex
		(“p16-mimetic”)
linear	Src, Lyn	inhibition of Mast cell	Stauffer et al. (1997),
		activation, IgE-related	Biochem. 36: 9388-94
		conditions, type I
		hypersensitivity
		(“Mast cell antagonist”)
linear	Mast cell protease	treatment of	International application
		inflammatory disorders	WO 98/33812, published
		mediated by release of	Aug. 6, 1998
		tryptase-6
		(“Mast cell protease
		inhibitors”)
linear	HBV core antigen	treatment of HBV viral	Dyson & Muray (1995),
	(HBcAg)	infections (“anti-HBV”)	Proc. Natl. Acad. Sci. 92:
			2194-8
linear	selectins	neutrophil adhesion;	Martens et al. (1995), J.
		inflammatory diseases	Biol. Chem. 270: 21129-36;
		(“selectin antagonist”)	European patent
			application EP 0 714 912,
			published Jun. 5, 1996
linear,	calmodulin	calmodulin antagonist	Pierce et al. (1995),
cyclized			Molec. Diversity 1: 259-65;
			Dedman et al. (1993),
			J. Biol. Chem. 268: 23025-30;
			Adey & Kay (1996),
			Gene 169: 133-4
linear,	integrins	tumor-homing;	International
cyclized-		treatment for	applications WO
		conditions related to	95/14714, published
		integrin-mediated	Jun. 1, 1995; WO
		cellular events,	97/08203, published
		including platelet	Mar. 6, 1997; WO
		aggregation,	98/10795, published
		thrombosis, wound	Mar. 19, 1998; WO
		healing, osteoporosis,	99/24462, published
		tissue repair,	May 20, 1999; Kraft et al.
		angiogenesis (e.g., for	(1999), J. Biol. Chem. 274:
		treatment of cancer),	1979-1985
		and tumor invasion
		(“integrin-binding”)
cyclic, linear	fibronectin and	treatment of	WO 98/09985, published
	extracellular matrix	inflammatory and	Mar. 12, 1998
	components of T	autoimmune conditions
	cells and
	macrophages
linear	somatostatin and	treatment or	European patent
	cortistatin	prevention of hormone-	application 0 911 393,
		producing tumors,	published Apr. 28, 1999
		acromegaly, giantism,
		dementia, gastric ulcer,
		tumor growth,
		inhibition of hormone
		secretion, modulation
		of sleep or neural
		activity
linear	bacterial	antibiotic; septic shock;	U.S. Pat. No. 5,877,151,
	lipopolysaccharide	disorders modulatable	issued Mar. 2, 1999
		by CAP37
linear or	pardaxin, mellitin	antipathogenic	WO 97/31019, published
cyclic,			28 Aug. 1997
including D-
amino acids
linear, cyclic	VIP	impotence,	WO 97/40070, published
		neurodegenerative	Oct. 30, 1997
		disorders
linear	CTLs	cancer	EP 0 770 624, published
			May 2, 1997
linear	THF-gamma2		Burnstein (1988),
			Biochem., 27: 4066-71.
linear	Amylin		Cooper (1987), Proc.
			Natl. Acad. Sci., 84: 8628-32.
linear	Adrenomedullin		Kitamura (1993), BBRC,
			192: 553-60.
cyclic, linear	VEGF	anti-angiogenic; cancer,	Fairbrother (1998),
		rheumatoid arthritis,	Biochem., 37: 17754-17764.
		diabetic retinopathy,
		psoriasis (“VEGF
		antagonist”)
cyclic	MMP	inflammation and	Koivunen (1999), Nature
		autoimmune disorders;	Biotech., 17: 768-774.
		tumor growth
		(“MMP inhibitor”)
	HGH fragment	treatment of obesity	U.S. Pat. No. 5,869,452
	Echistatin	inhibition of platelet	Gan (1988), J. Biol.
		aggregation	Chem., 263: 19827-32.
linear	SLE autoantibody	SLE	WO 96/30057, published
			Oct. 3, 1996
	GD1alpha	suppression of tumor	Ishikawa et al. (1998),
		metastasis	FEBS Lett. 441 (1): 20-4
	antiphospholipid	endothelial cell	Blank et al. (1999), Proc.
	beta-2-glycoprotein-	activation,	Natl. Acad. Sci. USA 96:
	I (□2GPI)	antiphospholipid	5164-8
	antibodies	syndrome (APS),
		thromboembolic
		phenomena,
		thrombocytopenia, and
		recurrent fetal loss
linear	T Cell Receptor beta	diabetes	WO 96/11214, published
	chain		Apr. 18, 1996.
		Antiproliferative,	WO 00/01402, published
		antiviral	Jan. 13, 2000.
		anti-ischemic, growth	WO 99/62539, published
		hormone-liberating	Dec. 9, 1999.
		anti-angiogenic	WO 99/61476, published
			Dec. 2, 1999.
linear		Apoptosis agonist;	WO 99/38526, published
		treatment of T cell-	Aug. 5, 1999.
		associated disorders
		(e.g., autoimmune
		diseases, viral infection,
		T cell leukemia, T cell
		lymphoma)
linear	MHC class II	treatment of	U.S. Pat. No. 5,880,103,
		autoimmune diseases	issued Mar. 9, 1999.
linear	androgen R, p75,	proapoptotic, useful in	WO 99/45944, published
	MJD, DCC,	treating cancer	Sep. 16, 1999.
	huntingtin
linear	von Willebrand	inhibition of Factor VIII	WO 97/41220, published
	Factor; Factor VIII	interaction;	Apr. 29, 1997.
		anticoagulants
linear	lentivirus LLP1	antimicrobial	U.S. Pat. No. 5,945,507,
			issued Aug. 31, 1999.
linear	Delta-Sleep	sleep disorders	Graf (1986), Peptides
	Inducing Peptide		7: 1165.
linear	C-Reactive Protein	inflammation and	Barna (1994), Cancer
	(CRP)	cancer	Immunol. Immunother.
			38: 38 (1994).
linear	Sperm-Activating	infertility	Suzuki (1992), Comp.
	Peptides		Biochem. Physiol.
			102B: 679.
linear	angiotensins	hematopoietic factors	Lundergan (1999), J. Periodontal
		for hematocytopenic	Res.
		conditions from cancer,	34(4): 223-228.
		AIDS, etc.
linear	HIV-1 gp41	anti-AIDS	Chan (1998), Cell 93: 681-684.
linear	PKC	inhibition of bone	Moonga (1998), Exp.
		resorption	Physiol. 83: 717-725.
linear	defensins (HNP-1, -2,	antimicrobial	Harvig (1994), Methods
	-3, -4)		Enz. 236: 160-172.
linear	p185HER2/neu, C-	AHNP-mimetic: anti-	Park (2000), Nat.
	erbB-2	tumor	Biotechnol. 18: 194-198.
linear	gp130	IL-6 antagonist	WO 99/60013, published
			Nov. 25, 1999.
linear	collagen, other joint,	autoimmune diseases	WO 99/50282, published
	cartilage, arthritis-		Oct. 7, 1999.
	related proteins
linear	HIV-1 envelope	treatment of	WO 99/51254, published
	protein	neurological	Oct. 14, 1999.
		degenerative diseases
linear	IL-2	autoimmune disorders	WO 00/04048, published
		(e.g., graft rejection,	Jan. 27, 2000; WO
		rheumatoid arthritis)	00/11028, published
			Mar. 2, 2000.
linear, cyclic	various	inflammatory	U.S. Pat. No. 6,660,843
		conditions,
		autoimmune disease,
		others
linear, cyclic	Ang-2	inhibition of	U.S. Pat. App. No.
		angiogenesis (e.g., for	2003/0229023, published
		treatment of tumor)	Dec. 11, 2003; WO
			03/057134, published
			Jul. 17, 2003; U.S.
			2003/0236193, published
			Dec. 25, 2003
	NGF	chronic pain, migraine,	WO 04/026329,
		asthma, hyperactive	published Apr. 1, 2004
		bladder, psoriasis,
		cancer, other conditions
		linked to NGF
	myostatin		U.S. Serial No.
			10/742,379, filed Dec. 19,
			2003; PCT/US03/40781,
			filed Dec. 19, 2003
	BAFF/TALL-1	B-cell mediated	U.S. 2003/0195156,
		autoimmune diseases	published Oct. 16, 2003;
		and cancers (e.g.,	WO 02/092620,
		lupus, B-cell	published Nov. 21, 2002
		lymphoma)
linear	GLP-1	Diabetes, metabolic
		syndrome
aThe protein listed in this column may be bound by the associated peptide (e.g., EPO receptor, IL-1 receptor) or mimicked by the associated peptide. The references listed for each clarify whether the molecule is bound by or mimicked by the peptides.

[0010] Peptides identified by peptide library screening were for a long time regarded simply as “leads” in development of therapeutic agents rather than as therapeutic agents themselves. Like other proteins and peptides, they would be rapidly removed in vivo either by renal filtration, cellular clearance mechanisms in the reticuloendothelial system, or proteolytic degradation. Francis (1992), Focus on Growth Factors 3: 4-11. As a result, the art used the identified peptides to validate drug targets or as scaffolds for design of organic compounds that might not have been as easily or as quickly identified through chemical library screening. Lowman (1997), Ann. Rev. Biophys. Biomol. Struct. 26:401-24; Kay et al. (1998), Drug Disc. Today 3: 370-8.

[0011] A more recent development is fusion of randomly generated peptides with the Fc domain. See U.S. Pat. No. 6,660,843, issued Dec. 9, 2003 to Feige et al. (incorporated by reference in its entirety). Such molecules have come to be known as “peptibodies.” They include one or more peptides linked to the N-terminus, C-terminus, amino acid sidechains, or to more than one of these sites. Peptibody technology enables design of therapeutic agents that incorporate peptides that target one or more ligands or receptors, tumor-homing peptides, membrane-transporting peptides, and the like. Peptibody technology has proven useful in design of a number of such molecules, including linear and disulfide-constrained peptides, “tandem peptide multimers” (i.e., more than one peptide on a single chain of an Fc domain). See, for example, U.S. Pat. No. 6,660,843; U.S. Pat. App. No. 2003/0195156, published Oct. 16, 2003 (corresponding to WO 02/092620, published Nov. 21, 2002); U.S. Pat. App. No. 2003/0176352, published Sep. 18, 2003 (corresponding to WO 03/031589, published Apr. 17, 2003); U.S. Ser. No. 09/422,838, filed Oct. 22, 1999 (corresponding to WO 00/24770, published May 4, 2000); U.S. Pat. App. No. 2003/0229023, published Dec. 11, 2003; WO 03/057134, published Jul. 17, 2003; U.S. Pat. App. No. 2003/0236193, published Dec. 25, 2003 (corresponding to PCT/US04/010989, filed Apr. 8, 2004); U.S. Ser. No. 10/666,480, filed Sep. 18, 2003 (corresponding to WO 04/026329, published Apr. 1, 2004), each of which is hereby incorporated by reference in its entirety. The art would benefit from further technology enabling such rational design of polypeptide therapeutic agents.

[0012] The present invention concerns a process in which at least one biologically active peptide is incorporated as an internal sequence into an Fc domain. Such an internal sequence may be added by insertion (i.e., between amino acids in the previously existing Fc domain) or by replacement of amino acids in the previously existing Fc domain (i.e., removing amino acids in the previously existing Fc domain and adding peptide amino acids). In the latter case, the number of peptide amino acids added need not correspond to the number of amino acids removed from the previously existing Fc domain; for example, this invention concerns a molecule in which 10 amino acids are removed and 15 amino acids are added. In this invention, pharmacologically active compounds are prepared by a process comprising:

• • [0013] a) selecting at least one peptide that modulates the activity of a protein of interest; and
  • [0014] b) preparing a pharmacologic agent comprising an amino acid sequence of the selected peptide as an internal sequence of an Fc domain.
    This process may be employed to modify an Fc domain that is already linked through an N— or C-terminus or sidechain to a polypeptide (e.g., etanercept) or to a peptide (e.g., as described in U.S. Pat. App. Nos. 2003/0195156, 2003/0176352, 2003/0229023, and 2003/0236193; WO 00/24770; WO 04/026329). The process described throughout may also be employed to modify an Fc domain that is part of an antibody (e.g., adalimumab, epratuzumab, infliximab, Herceptin®, and the like). In this way, different molecules can be produced that have additional functionalities, such as a binding domain to a different epitope or an additional binding domain to the precursor molecule's existing epitope. The peptide can be selected, for example, by phage display (which is preferred), E. coli display, ribosome display, RNA-peptide screening, yeast-based screening, chemical-peptide screening, rational design, or protein structural analysis or may be a naturally occurring peptide (e.g. PTH, GLP-1).

[0015] The invention further relates to molecules comprising an Fc domain modified to comprise a peptide as an internal sequence (preferably in a loop region) of the Fc domain. Molecules comprising an internal peptide sequence are referred to throughout as “Fc internal peptibodies” or “Fc internal peptide molecules.” These molecules are further described herein below.

[0016] The Fc internal peptide molecules may include more than one peptide sequence in tandem in a particular internal region, and they may include further peptides in other internal regions. While the putative loop regions are preferred, insertions in any other non-terminal domains of the Fc are also considered part of this invention. Variants and derivatives of the above compounds (described below) are also encompassed by this invention.

[0017] The compounds of this invention may be prepared by standard synthetic methods, recombinant DNA techniques, or any other methods of preparing peptides and fusion proteins.

[0018] The primary use contemplated for Fc internal peptide molecules is as therapeutic or prophylactic agents. A selected peptide may have activity comparable to—or even greater than—the natural ligand mimicked by the peptide. In addition, certain natural ligand-based therapeutic agents might induce antibodies against the patient's own endogenous ligand. In contrast, the unique sequence of the vehicle-linked peptide avoids this pitfall by having little or typically no sequence identity with the natural ligand. Furthermore, the Fc internal peptibodies may have advantages in refolding and purification over N— or C-terminally linked Fc molecules. Further still, Fc internal peptibodies may be more stable in both thermodynamically, due to the stabilization of chimeric domains, and chemically, due to increased resistance to proteolytic degradation from amino- and carboxy-peptidases. Fc internal peptibodies may also exhibit improved pharmacokinetic properties.

[0019] Although mostly contemplated as therapeutic agents, compounds of this invention may also be useful in screening for such agents. For example, one could use an Fc internal peptibody (e.g., Fc-loop-SH2 domain peptide) in an assay employing anti-Fc coated plates. Fc internal peptibodies may make insoluble peptides soluble and thus useful in a number of assays.

[0020] The compounds of this invention may be used for therapeutic or prophylactic purposes by formulating them with appropriate pharmaceutical carrier materials and administering an effective amount to a patient, such as a human (or other mammal) in need thereof. Other related aspects are also included in the instant invention.

[0021] Numerous additional aspects and advantages of the present invention will become apparent upon consideration of the figures and detailed description of the invention.

[0022] FIGS. 1A, 1B and 1C show loop regions of Fc domains that may be modified in accordance with this invention. In these structural representations of the CH2 and CH3 domains of Fc, the loop regions may be considered any part of the model not shown as β-sheet (flat arrows) or α-helix (cylinder).

[0023] FIG. 1A shows the monomeric rat IgG2a Fc domain (Protein Database file #1I1C, http://www.rcsb.org/pdb/). This figure shows a three dimensional model of rat IgG2a Fc domain monomer from x-ray diffraction crystal structure (pdb #1I1C). Potential Fc loop insertion sites are shown for both CH2 and CH3 domains with the preferred CH3 domain Fc loop insertion site specifically identified.

[0024] FIG. 1B shows the monomeric murine IgG1 Fc domain (Protein Database file #1IGY). This figure shows a three-dimensional model of murine IgG1 Fc domain monomer from x-ray diffraction crystal structure (pdb #1IGY). Potential Fc loop insertion sites are shown for both CH2 and CH3 domains with the preferred CH3 domain Fc loop insertion site specifically identified.

[0025] FIG. 1C shows the monomeric human IgG1 Fc domain (Protein Database file #1H3T). This figure shows a three-dimensional model of human IgG1 Fc domain monomer from x-ray diffraction crystal structure (pdb #1H3T). Potential Fc loop insertion sites are shown for both CH2 and CH3 domains with the preferred CH3 domain Fc loop insertion site specifically identified.

[0026] These structures illustrate the high degree of homology in the secondary and tertiary structural conformations within the Fc domains of different IgG subtypes and between species. The x-ray crystal structure coordinates for these structures can be found in the RCSB Protein Data Bank (http://www.rcsb.org/pdb/).

[0027] FIG. 2A shows a sequence of human IgG1 Fc sequence (SEQ ID NO: 599) used for peptibody fusions with predicted loop sequences in boldface. FIG. 2A shows, in the context of the human IgG1 sequence used for this invention, the Fc loop regions in boldface (SEQ ID NOS: 621, 622, 624, 625, 627, 628, 630, 632, 634, and 636), which are suggested by the structures shown in FIGS. 1A, 1B and 1C. Any, or all of the sites shown in boldface may be suitable for full or partial replacement by or insertion of peptide sequences and are considered part of this invention. Specifically preferred internal sites are underlined (SEQ ID NOS: 623, 626, 629, 631, 633, 635, and 637). One preferred site is SEQ ID NO: 631, between Leu₁₃₉ and Thr₁₄₀ in the DELTK (SEQ ID NO: 630) loop. Potential loop sites in other Ig subtypes are understood in the art based on the alignments provided in FIGS. 2B and 2C.

[0028] FIGS. 2B and 2C show a sequence alignment of human Fc domains from IgA, IgM and IgG subclasses. FIGS. 2B and 2C show exemplary amino acid sequences (SEQ ID NOS: 600 to 607) of human Fc regions from IgA, IgM and IgG subtypes that may be useful in this invention. Also shown in FIGS. 2B and 2C is a consensus sequence (SEQ ID NO: 608).

[0029] FIGS. 2B and 2C also show in boldface the preferred internal sites for peptide addition that correspond to those of the Fc sequence shown in FIG. 2A (SEQ ID NO: 599). In particular, FIGS. 2B and 2C show as such preferred sites the following:

• [0030] SEQ ID NO: 621 as shown in boldface within SEQ ID NOS: 603 to 608;
• [0031] SEQ ID NO: 622 within SEQ ID NOS: 603 to 606 and 608;
• [0032] SEQ ID NO: 638 within SEQ ID NO: 607;
• [0033] SEQ ID NO: 624 within SEQ ID NO: 603 to 608;
• [0034] SEQ ID NO: 625 within SEQ ID NOS: 603 and 604;
• [0035] SEQ ID NO: 639 within SEQ ID NOS: 605 to 608;
• [0036] SEQ ID NO: 627 within SEQ ID NOS: 603 to 605, 607, and 608;
• [0037] SEQ ID NO: 640 within SEQ ID NO: 606;
• [0038] SEQ ID NO: 628 within SEQ ID NOS: 603, 604, and 608;
• [0039] SEQ ID NO: 641 within SEQ ID NO: 605;
• [0040] SEQ ID NO: 642 within SEQ ID NO: 606;
• [0041] SEQ ID NO: 643 within SEQ ID NO: 607;
• [0042] SEQ ID NO: 630 within SEQ ID NO: 603;
• [0043] SEQ ID NO: 644 within SEQ ID NOS: 604 to 608;
• [0044] SEQ ID NO: 632 within SEQ ID NOS: 603, 604, 606, 607, and 608;
• [0045] SEQ ID NO: 645 within SEQ ID NO: 605;
• [0046] SEQ ID NO: 634 within SEQ ID NOS: 603, 604, and 607;
• [0047] SEQ ID NO: 646 within SEQ ID NOS: 605, 606 and 608;
• [0048] SEQ ID NO: 636 within SEQ ID NOS: 603, 604, 606, and 608;
• [0049] SEQ ID NO: 614 within SEQ ID NO: 605; and
• [0050] SEQ ID NO: 620 within SEQ ID NO: 607.

[0051] The sequence alignments of FIGS. 2B and 2C suggest two more potential insertion sites at Q₁₆₇/P₁₆₈ and/or G₁₈₃/S₁₈₄ (using the numbering of SEQ ID NO: 599 in FIG. 2A). These positions correspond to gaps in the IgG sequences where there are 2 and 3 residue insertions found in the aligned IgA and IgM sequences. Other preferred insertion sites correspond to the sequence in FIG. 2A. The preferred insertion sites are underlined FIGS. 2B and 2C and are as follows:

• [0052] H₅₃/E54 in SEQ ID NOS: 603 and 604;
• [0053] H₁₀₀/E₁₀₁ in SEQ ID NO: 605;
• [0054] H₄₉/E₅₀ in SEQ ID NO: 606;
• [0055] Q₅₀/E₅₁ in SEQ ID NO: 607;
• [0056] H₁₁₂/E₁₁₃ in SEQ ID NO: 608;
• [0057] Y₈₁/N₈₂ in SEQ ID NOS: 603 and 604;
• [0058] F₁₂₈/N₁₂₉ in SEQ ID NO: 605;
• [0059] F₇₇/N₇₈ in SEQ ID NO: 606;
• [0060] F₇₈/N₇₉ in SEQ ID NO: 607;
• [0061] F₁₄₀/N₁₄₁ in SEQ ID NO: 608;
• [0062] N₁₁₀/K₁₁₁ in SEQ ID NOS: 603 and 604;
• [0063] N₁₅₇/K₁₅₈ in SEQ ID NO: 605;
• [0064] N₁₀₆/K₁₀₇ in SEQ ID NO: 606;
• [0065] N₁₀₇/K₁₀₈ in SEQ ID NO: 607;
• [0066] N₁₆₉/K₁₇₀ in SEQ ID NO: 608;
• [0067] L₁₄₃/T₁₄₄ in SEQ ID NOS: 603 and 604;
• [0068] M₁₉₀/T₁₉₁ in SEQ ID NO: 605;
• [0069] M₁₃₉/T₁₄₀ in SEQ ID NO: 606;
• [0070] M₁₄₀/T₁₄₁ in SEQ ID NO: 607;
• [0071] M₂₀₄/T₂₀₅ in SEQ ID NO: 608;
• [0072] Q₁₇₁/P₁₇₂ in SEQ ID NOS: 603 and 604;
• [0073] Q₂₁₈/P₂₁₉ in SEQ ID NO: 605;
• [0074] Q₁₆₇/P₁₆₈ in SEQ ID NO: 606;
• [0075] Q₁₆₈/P₁₆₉ in SEQ ID NO: 607;
• [0076] Q₂₃₂/P₂₃₃ in SEQ ID NO: 608;
• [0077] E₁₇₃N₁₇₄ in SEQ ID NOS: 603 and 604;
• [0078] E₂₂₀/N₂₂₁ in SEQ ID NO: 605;
• [0079] E₁₆₉/N₁₇₀ in SEQ ID NO: 606;
• [0080] E₁₇₀/N₁₇₁ in SEQ ID NO: 607;
• [0081] E₂₃₄/N₂₃₅ in SEQ ID NO: 608;
• [0082] S₁₈₆/D₁₈₇ in SEQ ID NOS: 603 and 604;
• [0083] S₂₃₂/D₂₃₃ in SEQ ID NO: 605;
• [0084] S₁₈₁/D₁₈₂ in SEQ ID NO: 606;
• [0085] S₁₈₂/D₁₈₃ in SEQ ID NO: 607;
• [0086] S₂₄₆/D₂₄₇ in SEQ ID NO: 608;
• [0087] G₁₈₈/S₁₈₉ in SEQ ID NOS: 603 and 604;
• [0088] G₂₃₄/S₂₃₅ in SEQ ID NO: 605;
• [0089] G₁₈₃/S₁₈₄ in SEQ ID NO: 606;
• [0090] G₁₈₄/S₁₈₅ in SEQ ID NO: 607;
• [0091] G₂₄₈/S₂₄₉ in SEQ ID NO: 608;
• [0092] G₂₀₅/N₂₀₆ in SEQ ID NOS: 603 and 604;
• [0093] G₂₅₂/N₂₅₃ in SEQ ID NO: 605;
• [0094] G₂₀₁/N₂₀₂ in SEQ ID NO: 606;
• [0095] G₂₀₂/N₂₀₃ in SEQ ID NO: 607; and
• [0096] G₂₆₈/N₂₆₉ in SEQ ID NO: 608.

[0097] FIG. 2D shows an alignment of human IgG1 Fc domain (Amgen Fc, SEQ ID NO: 609) used for the peptibody platform with rat IgG2A from crystal structure of FcRn/Fc complex (1I1A.pdb, SEQ ID NO: 610 The resulting consensus sequence (SEQ ID NO: 611) is also shown.

[0098] FIG. 3A shows the amino acid sequence (SEQ ID NO: 612) of a human IgG1 Fc domain having insertion of a myostatin binding peptide (SEQ ID NO: 365). Hereinafter, this molecule is referred to as the “myostatin loop peptibody” or “Fc-loop-myo7.” The inserted peptide is shown in boldface and the glycine linkers in italics.

[0099] FIG. 3B shows the amino acid sequence (SEQ ID NO: 613) of a C-terminally linked peptibody referred to as TN8-19-07. This peptibody incorporates the same peptide sequence as Fc-loop-myo7 (SEQ ID NO: 365). The TN8-19-07 peptide is shown in boldface and the glycine and alanine linkers in italics.

[0100] FIG. 3C shows the amino acid sequence (SEQ ID NO: 615) of an Fc internal peptibody referred to hereinafter as Fc-loop-EMP. This peptibody incorporates an EPO-mimetic peptide (SEQ ID NO: 2). The inserted peptide is shown in boldface and the glycine linkers in italics. The cysteines that form a disulfide bond are underlined.

[0101] FIG. 3D shows the amino acid sequence (SEQ ID NO: 616) of an Fc internal peptibody referred to hereinafter as Fc-loop-Amp2. Bioactive peptide (SEQ ID NO: 28) is highlighted in boldface and glycine linkers in italics. There is no disulfide constraint in the AMP-2 peptide insertion.

[0102] FIG. 3E shows the amino acid sequence (SEQ ID NO: 617) of a C-terminally linked peptibody referred to hereinafter as Fc-loop-AMP2-dimer. This tandem-linked therapeutic peptide dimer shows the therapeutic peptide sequence (SEQ ID NO: 28) in boldface and the linkers in italics. This molecule incorporates a tandem peptide dimer of the same peptide sequence as found in Fc-loop-AMP-2.

[0103] FIGS. 4A and 4B shows the expression in E. coli of Fc-loop-myo7 and TN8-19-07 by SDS-PAGE (4-20%). Samples of the crude cell lysate (lys), the insoluble fraction (insol) and the soluble (sol) fraction for both the Fc-loop-Myo7 (#6951) and TN8-19-07 (#6826) are shown in reducing gels. SeeBlue and molecular weight markers (lane 1), whole cell lysate (lane 2), insoluble fraction (lane 3) and insoluble fraction (lane 4).

[0104] FIG. 5 shows a reverse phase, high performance liquid chromatography (RP-HPLC) comparison of the unpurified refold reactions of the Fc-loop-Myo7 (#6951) and TN8-19-07 (#6826). Approximately 10 μg of peptibody was loaded directly from a refold reaction to a Vydak C4 column (5 μM, 300 angstrom, 4.6×250 mm) and eluted with a linear 40-50% ACN gradient at 0.5%/min.

[0105] FIG. 6 shows reversed-phase high performance liquid chromatography (RP-HPLC) comparison of the final, purified pools of Fc-loop TN8-19-07 (#6951) and carboxy-terminal Fc TN8-19-07 (#6826). Loaded 10 μg purified peptibody to Vydak C4 column (5 μM, 300 angstrom, 4.6×250 mm) and eluted with a linear 40-50% ACN gradient at 0.5%/min.

[0106] FIG. 7 shows the analyses of final purified pools of Fc-loop TN8-19-07 (#6951) and carboxy-terminal Fc TN8-19-07 (#6826) by SDS-PAGE (4-20% gel). Five μg of each sample was loaded as follows: #6951 (lane 1), #6826 (lane 2), SeeBlue+markers (lane M), #6951 reduced (lane 3), #6826 reduced (lane 4).

[0107] FIG. 8 shows an in vitro cell-based bioassay for measuring myostatin inhibitory compounds. Fc-loop TN8-19-07 (#6951) retains full inhibitory activity relative to the carboxy-terminal TN8-19-07 peptibody (#6826).

[0108] FIG. 9 shows a western blot analysis of an in vivo stability study for Fc-loop TN8-19-07 (#6951) and the carboxy-terminal TN8-19-07 peptibody (#6826). Sera pools from five mice were evaluated for each time point (0, 4, 24 and 48 hours). Lanes 1-3 are Fc-loop TN8-19-07 standards at 2 ng, 5 ng and 10 ng respectively. Lanes 4 & 5 are the Fc-loop vs. carboxy terminal peptibodies, respectively, at 4 hours. Lanes 6 & 7 are the Fc-loop vs. carboxy terminal peptibodies respectively at 24 hours. Lanes 8 & 9 are the Fc-loop vs. carboxy terminal peptibodies respectively at 48 hours. Lanes 10-12 are the carboxy-terminal peptibody standard at 2 ng, 5 ng and 10 ng, respectively. The gel was a 1 mm 4-12% SDS-PAGE gel run in MES reducing buffer and the western blot was developed using a goat anti-human IgG Fc-HRP conjugate.

[0109] FIG. 10A shows the amino acid sequence (SEQ ID NO: 618) of a human IgG1 Fc domain having insertion of an Ang2 binding peptide (SEQ ID NO: 147). Hereinafter, this molecule is referred to as “Ang2 loop peptibody” or “Fc-loop-Ang2.” Bioactive peptide is highlighted in boldface and Glycine linkers in italics.

[0110] FIG. 10B shows the amino acid sequence (SEQ ID NO: 619) of a C-terminally linked peptibody referred to herein as TN8-Con4. This molecule incorporates the same peptide sequence as Fc-loop-ang2 (SEQ ID NO: 147). The bioactive peptide is highlighted in boldface and the glycine and alanine linkers in italics.

[0111] FIG. 11 shows the expression and distribution in E. coli of the Fc-loop TN8-Con4 (#6888) and carboxy-terminal TN8-Con4 (#5564) peptibodies by SDS-PAGE. Samples of the crude cell lysate (lys), the insoluble fraction (insol) and the soluble (sol) fraction for both the Fc-loop-Tn8-Con4 (#6888) and TN8-Con4 (#5564) are shown in reducing gels.

[0112] FIG. 12 shows a RP-HPLC comparison of Fc-loop Ang2 (#6888) and carboxy-terminal Fc TN8-19-07 (#5564) refold reactions. Loaded 20 μl refold reaction to Vydak C4 column (5 μM, 300 angstrom, 4.6×250 mm) and eluted with a linear 40-50% ACN gradient at 0.5%/min.

[0113] FIG. 13 shows a RP-HPLC comparison of the final purified pools of Fc-loop Ang2 (#6888) and carboxy-terminal Fc TN8-Con4 (#5564). Ten μg purified peptibody was loaded to a Vydak C4 column (5 μM, 300 angstrom, 4.6×250 mm) and eluted with a linear 40-50% ACN gradient at 0.5%/min.

[0114] FIG. 14 shows purified Fc-loop-myo7 and TN8-19-7.

[0115] FIG. 15 shows Biacore binding analysis of Fc-loop-ang2 and Fc-ang2-tandem.

[0116] FIG. 16 shows the results of an in vitro enzyme-linked immunosorbent assay (ELISA) for Fc-loop-ang2, TN8-Con4, and Fc-ang2-tandem.

[0117] FIG. 17 shows the results of a UT7 erythropoietin proliferation assay for Fc-loop-EMP. In the assay, the activity of two different of Fc-loop-EMP molecules is compared to that of epoetin alfa.

[0118] FIG. 18 shows the expression and distribution in E. coli of the Fc-loop TN8-Amp2 (#6875) peptibody by SDS-PAGE. Samples of the crude cell lysate (lys), the insoluble fraction (insol) and the soluble (sol) fraction for the Fc-loop-Amp2 (#6888) are shown in reducing gels.

[0119] FIG. 19 shows an analysis of the final purified pool of Fc-loop AMP 2 (#6875) by SDS-PAGE (4-20% gel). Lane 2 was loaded with 5 μg Fc-loop AMP 2 peptibody; lane 4 with 5 μg reduced Fc-loop AMP 2 peptibody; lanes 1 and 3 with SeeBlue and two molecular weight markers.

[0120] FIG. 20 shows an RP-HPLC analysis of the final purified pool of Fc-loop AMP 2 (#6875). Ten μg purified peptibody was loaded to Vydak C4 column (5 μM, 300 angstrom, 4.6×250 mm) and eluted with a linear 40-50% ACN gradient at 0.5%/min.

[0121] FIG. 21 shows a murine in vivo bioassay of Fc-loop AMP 2 and AMG 531 peptibodies. Mice dosed with a single subcutaneous injection of 50 μg/kg peptibody or carrier alone. See example 9 for assay methodology.

[0122] FIG. 22 shows several strategies for incorporating 2 bioactive peptides into an Fc-loop peptibody.

[0123] FIG. 23 shows SDS-PAGE Gels of purified Fc-loop constructs. Samples (2 ug/lane) were run±reducing buffer on a 4-20% Tris-Glycine SDS-PAGE gel.

[0124] FIG. 24 shows RP-HPLC of Fc-loop constructs.

[0125] Definition of Terms

[0126] The terms used throughout this specification are defined as follows, unless otherwise limited in specific instances.

[0127] When used in connection with an amino acid sequence, the term “comprising” means that a compound may include additional amino acids on either or both of the N— or C-termini of the given sequence.

[0128] “Antibody” or “antibody peptide(s)” refer to an intact antibody, or a binding fragment thereof that competes with the intact antibody for specific binding and includes chimeric, humanized, fully human, and bispecific antibodies. In certain embodiments, binding fragments are produced by recombinant DNA techniques. In additional embodiments, binding fragments are produced by enzymatic or chemical cleavage of intact antibodies. Binding fragments include, but are not limited to, Fab, Fab′, F(ab′)₂, Fv, and single-chain antibodies.

[0129] The term “native Fc” refers to a molecule or sequence comprising the sequence of a non-antigen-binding fragment resulting from digestion of whole antibody, whether in monomeric or multimeric form, into which a peptide sequence may be added by insertion into or replacement of a loop region. The original immunoglobulin source of the native Fc is preferably of human origin and may be any of the immunoglobulins, although IgG1 and IgG2 are preferred Native Fc's are made up of monomeric polypeptides that may be linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent association. The number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4 depending on class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2). One example of a native Fc is a disulfide-bonded dimer resulting from papain digestion of an IgG (see Ellison et al. (1982), Nucleic Acids Res. 10: 4071-9). The term “native Fc” as used herein is generic to the monomeric, dimeric, and multimeric forms.

[0130] The term “Fc variant” refers to a molecule or sequence that is modified from a native Fc but still comprises a binding site for the salvage receptor, FcRn. International applications WO 97/34631 (published 25 Sep. 1997) and WO 96/32478 describe exemplary Fc variants, as well as interaction with the salvage receptor, and are hereby incorporated by reference. Thus, the term “Fc variant” comprises a molecule or sequence that is humanized from a non-human native Fc. Furthermore, a native Fc comprises sites that may be removed because they provide structural features or biological activity that are not required for the fusion molecules of the present invention. Thus, the term “Fc variant” comprises a molecule or sequence that lacks one or more native Fc sites or residues that affect or are involved in (1) disulfide bond formation, (2) incompatibility with a selected host cell (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to an Fc receptor other than a salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC). Fc variants are described in further detail hereinafter.

[0131] The term “Fc domain” encompasses native Fc and Fc variant molecules and sequences as defined above. As with Fc variants and native Fc's, the term “Fc domain” includes molecules in monomeric or multimeric form, whether digested from whole antibody or produced by other means.

[0132] The term “multimer” as applied to Fc domains or molecules comprising Fc domains refers to molecules having two or more polypeptide chains associated covalently, noncovalently, or by both covalent and non-covalent interactions. IgG molecules typically form dimers; IgM, pentamers; IgD, dimers; and IgA, monomers, dimers, trimers, or tetramers. Multimers may be formed by exploiting the sequence and resulting activity of the native Ig source of the Fc or by derivatizing (as defined below) such a native Fc.

[0133] The term “dimer” as applied to Fc domains or molecules comprising Fc domains refers to molecules having two polypeptide chains associated covalently or non-covalently. Exemplary dimers within the scope of this invention are as shown in U.S. Pat. No. 6,660,843, FIG. 2, which is hereby incorporated by reference.

[0134] The terms “derivatizing” and “derivative” or “derivatized” comprise processes and resulting compounds respectively in which (1) the compound has a cyclic portion; for example, cross-linking between cysteinyl residues within the compound; (2) the compound is cross-linked or has a cross-linking site; for example, the compound has a cysteinyl residue and thus forms cross-linked dimers in culture or in vivo; (3) one or more peptidyl linkage is replaced by a non-peptidyl linkage; (4) the N-terminus is replaced by —NRR¹, NRC(O)R¹, —NRC(O)OR¹, —NRS(O)₂R¹, —NHC(O)NHR, a succinimide group, or substituted or unsubstituted benzyloxycarbonyl-NH—, wherein R and R¹ and the ring substituents are as defined hereinafter; (5) the C-terminus is replaced by —C(O)R² or —NR³R⁴ wherein R², R³ and R⁴ are as defined hereinafter; and (6) compounds in which individual amino acid moieties are modified through treatment with agents capable of reacting with selected side chains or terminal residues. Derivatives are further described hereinafter.

[0135] The term “polypeptide” refers to molecules of greater than 40 amino acids, whether existing in nature or not, provided that such molecules are not membrane-bound. Exemplary polypeptides include IL-1ra, leptin, soluble TNF receptors type 1 and type 2 (sTNF-R1, sTNF-R2), KGF, EPO, TPO, G-CSF, darbepoietin, Fab fragments and the like.

[0136] The term “peptide” refers to molecules of 2 to 40 amino acids, with molecules of 3 to 20 amino acids preferred and those of 6 to 15 amino acids most preferred. Exemplary peptides may be randomly generated by any of the methods cited above, carried in a peptide library (e.g., a phage display library), or derived by digestion of proteins.

[0137] The term “randomized” as used to refer to peptide sequences refers to fully random sequences (e.g., selected by phage display methods) and sequences in which one or more residues of a naturally occurring molecule is replaced by an amino acid residue not appearing in that position in the naturally occurring molecule. Exemplary methods for identifying peptide sequences include phage display, E. coli display, ribosome display, yeast-based screening, RNA-peptide screening, chemical screening, rational design, protein structural analysis, and the like.

[0138] The term “pharmacologically active” means that a substance so described is determined to have activity that affects a medical parameter (e.g., blood pressure, blood cell count, cholesterol level) or disease state (e.g., cancer, autoimmune disorders). Thus, pharmacologically active peptides comprise agonistic or mimetic and antagonistic peptides as defined below.

[0139] The terms “-mimetic peptide” and “-agonist peptide” refer to a peptide having biological activity comparable to a protein (e.g., EPO, TPO, G-CSF) that interacts with a protein of interest. These terms further include peptides that indirectly mimic the activity of a protein of interest, such as by potentiating the effects of the natural ligand of the protein of interest; see, for example, the G-CSF-mimetic peptides listed in Table 2 hereof and in Table 7 of U.S. Pat. No. 6,660,843, which is hereby incorporated by reference. Thus, the term “EPO-mimetic peptide” comprises any peptides that can be identified or derived as described in Wrighton et al. (1996), Science 273: 458-63, Naranda et al. (1999), Proc. Natl. Acad. Sci. USA 96: 7569-74, or any other reference in Table 2 identified as having EPO-mimetic subject matter. Those of ordinary skill in the art appreciate that each of these references enables one to select different peptides than actually disclosed therein by following the disclosed procedures with different peptide libraries.

[0140] The term “-antagonist peptide” or “inhibitor peptide” refers to a peptide that blocks or in some way interferes with the biological activity of the associated protein of interest, or has biological activity comparable to a known antagonist or inhibitor of the associated protein of interest. Thus, the term “TNF-antagonist peptide” comprises peptides that can be identified or derived as described in Takasaki et al. (1997), Nature Biotech. 15: 1266-70 or any of the references in Table 2 identified as having TNF-antagonistic subject matter. Those of ordinary skill in the art appreciate that each of these references enables one to select different peptides than actually disclosed therein by following the disclosed procedures with different peptide libraries.

[0141] The term “TPO-mimetic peptide” comprises peptides that can be identified or derived as described in Cwirla et al. (1997), Science 276: 1696-9, U.S. Pat. Nos. 5,869,451 and 5,932,946; U.S. Pat. App. No. 2003/0176352, published Sep. 18, 2003; WO 03/031589, published Apr. 17, 2003 and any other reference in Table 2 identified as having TPO-mimetic subject matter, as well as WO 00/24770, published May 4, 2000, which is hereby incorporated by reference. Those of ordinary skill in the art appreciate that each of these references enables one to select different peptides than actually disclosed therein by following the disclosed procedures with different peptide libraries.

[0142] The term “ang-2-binding peptide” comprises peptides that can be identified or derived as described in U.S. Pat. App. No. 2003/0229023, published Dec. 11, 2003; WO 03/057134, published Jul. 7, 2003; U.S. 2003/0236193, published Dec. 25, 2003; and any other reference in Table 2 identified as having subject matter related to ang-2. Those of ordinary skill in the art appreciate that each of these references enables one to select different peptides than actually disclosed therein by following the disclosed procedures with different peptide libraries.

[0143] The term “NGF-binding peptide” comprises peptides that can be identified or derived as described in WO 04/026329, published Apr. 1, 2004 and any other reference in Table 2 identified as having subject matter related to NGF. Those of ordinary skill in the art appreciate that each of these references enables one to select different peptides than actually disclosed therein by following the disclosed procedures with different peptide libraries.

[0144] The term “myostatin-binding peptide” comprises peptides that can be identified or derived as described in U.S. Ser. No. 10/742,379, filed Dec. 19, 2003 and any other reference in Table 2 identified as having subject matter related to myostatin. Those of ordinary skill in the art appreciate that each of these references enables one to select different peptides than actually disclosed therein by following the disclosed procedures with different peptide libraries.

[0145] Additionally, physiologically acceptable salts of the compounds of this invention are also encompassed herein. By “physiologically acceptable salts” is meant any salts that are known or later discovered to be pharmaceutically acceptable. Some specific examples are: acetate; trifluoroacetate; hydrohalides, such as hydrochloride and hydrobromide; sulfate; citrate; tartrate; glycolate; and oxalate.

[0146] Structure of compounds

[0147] In General

[0148] In the compositions of matter prepared in accordance with this invention, the peptide may be attached to the vehicle through the peptide's N-terminus or C-terminus. Thus, the vehicle-peptide molecules of this invention may be described by the following formula I:
(X¹)_a—F¹—(X²)_b
wherein:

[0149] F¹ is an Fc domain modified so that it comprises at least one X³ in a loop region;

[0150] X¹ and X² are each independently selected from -(L¹)_c-P¹, -(L¹)_c-P¹-(L²)_d-P², -(L¹)_c-P¹-(L²)_d-P²-(L³)_e-P³, and -(L¹)_c-P¹-(L²)_d-P²-(L³)_e-P³-(L⁴)_f-P⁴;

[0151] X³ is independently selected from -(L⁵)_c-P⁵, -(L⁵)_c-P⁵-(L⁶)_d-P⁶, -(L⁵)_c-P⁵-(L⁶)_d-P⁶-(L⁷)_e-P⁷, and -(L⁵)_c-P⁵-(L⁶)_d-P⁶-(L⁷)_e-P⁷-(L⁸)_f-P⁸;

[0152] P¹, P², P³, and P⁴ are each independently sequences of pharmacologically active polypeptides or pharmacologically active peptides;

[0153] P⁵, P⁶, P⁷, and P⁸ are each independently sequences of pharmacologically active peptides;

[0154] L¹, L², L³, L⁴, L⁵, L⁶, L⁷, and L⁸ are each independently linkers; and

[0155] a, b, c, d, e, and f are each independently 0 or 1.

[0156] In preferred embodiments, a and b are both zero—i.e., neither X¹ nor X² groups appear at the N-terminus or C-terminus of the Fc domain.

[0157] Those of ordinary skill in the art will appreciate that more than one X³ substituent may be present in the Fc domain, and that the multiple X³ substituents may be different; for example, comprising different P⁵ peptides, different linkers attached to the same peptide sequence, and so on. Likewise, X¹ and X² may be the same or different, and the integers c through f may be different for X¹, X², and X³.

[0158] Thus, compound I comprises compounds of the formulae
X¹—F¹   II
and multimers thereof wherein F¹ is attached at the C-terminus of X¹;
F¹—X²   III
and multimers thereof wherein F¹ is attached at the N-terminus of X²;
F¹-(L¹)_c-P¹   IV
and multimers thereof wherein F¹ is attached at the N-terminus of —(L¹)_c-P¹; and
F¹-(L¹)_c-P¹-(L²)_d-P²   V
and multimers thereof wherein F¹ is attached at the N-terminus of -L¹-P¹-L²-P².

[0159] Peptides

[0160] Any number of peptides may be used in conjunction with the present invention. Preferred peptides bind to angiopoietin-2 (ang-2), myostatin, nerve growth factor (NGF), tumor necrosis factor (TNF), B cell activating factor (BAFF, also referred to as TALL-1) or mimic the activity of EPO, TPO, or G-CSF. Targeting peptides are also of interest, including tumor-homing peptides, membrane-transporting peptides, and the like. All of these classes of peptides may be discovered by methods described in the references cited in this specification and other references.

[0161] Phage display, in particular, is useful in generating peptides for use in the present invention. It has been stated that affinity selection from libraries of random peptides can be used to identify peptide ligands for any site of any gene product. Dedman et al. (1993), J. Biol. Chem. 268: 23025-30. Phage display is particularly well suited for identifying peptides that bind to such proteins of interest as cell surface receptors or any proteins having linear epitopes. Wilson et al. (1998), Can. T. Microbiol. 44: 313-29; Kay et al. (1998), Drug Disc. Today 3: 370-8. Such proteins are extensively reviewed in Herz et al. (1997), J. Receptor & Signal Transduction Res. 17(5): 671-776, which is hereby incorporated by reference. Such proteins of interest are preferred for use in this invention.

[0162] A particularly preferred group of peptides are those that bind to cytokine receptors. Cytokines have recently been classified according to their receptor code. See Inglot (1997), Archivum Immunologiae et Therapiae Experimentalis 45: 353-7, which is hereby incorporated by reference. Among these receptors, most preferred are the CKRs (family I in Table 3). The receptor classification appears in Table 3.

TABLE 3
Cytokine Receptors Classified by Receptor Code
Cytokines (ligands)	Receptor Type
family	subfamily	family	subfamily
I.	Hematopoietic	1.	IL-2, IL-4, IL-7,	I.	Cytokine R	1.	shared γCr, IL-
	cytokines		IL-9, IL-13, IL-		(CKR)		9R, IL-4R
			15
		2.	IL-3, IL-5, GM-			2.	shared GP 140
			CSF				βR
		3.	IL-6, IL-11, IL-			3.	3.shared RP
			12, LIF, OSM,				130, IL-6 R,
			CNTF, Leptin				Leptin R
			(OB)
		4.	G-CSF, EPO,			4.	“single chain”
			TPO, PRL, GH				R, GCSF-R,
							TPO-R, GH-R
		5.	IL-17, HVS-IL-			5.	other Rb
			17
II.	IL-10 ligands		IL-10, BCRF-1,	II.	IL-10 R
			HSV-IL-10
III.	Interferons	1.	IFN-α1, α2, α4,	III.	Interferon R	1.	IFNAR
			m, t, IFN-βc
		2.	IFN-γ			2.	IFNGR
IV.	IL-1 and IL-1	1.	IL-1α, IL-1β,	IV.	IL-1R	1.	IL-1R, IL-
	like ligands		IL-1Ra				1RAcP
		2.	IL-18, IL-18BP			2.	IL-18R, IL-
							18RAcP
V.	TNF family	1.	TNF-α, TNF-β	3.	NGF/TNF Rd		TNF-RI, AGP-3R,
			(LT), FASL,				DR4, DR5, OX40,
			CD40 L,				OPG, TACI, CD40,
			CD30L, CD27				FAS, ODR
			L, OX40L,
			OPGL, TRAIL,
			APRIL, AGP-3,
			BLys, TL5,
			Ntn-2, KAY,
			Neutrokine-α
VI.	Chemokines	1.	α chemokines:	4.	Chemokine R	1.	CXCR
			IL-8, GRO α, β,
			γ, IF-10, PF-4,
			SDF-1
		2.	β chemokines:			2.	CCR
			MIP1α, MIP1β,
			MCP-1,2,3,4,
			RANTES,
			eotaxin
		3.	γ chemokines:			3.	CR
			lymphotactin
						4.	DARCe
VII.	Growth factors	1.1	SCF, M-CSF,	VII.	RKF	1.	TK sub-family
			PDGF-AA, AB,			1.1	IgTK III R,
			BB, KDR, FLT-				VEGF-RI,
			1, FLT-3L,				VEGF-RII,
			VEGF, SSV-
			PDGF, HGF, SF
		1.2	FGFα, FGFβ			1.2	IgTK IV R
		1.3	EGF, TGF-α,			1.3	Cysteine-rich
			VV-F19 (EGF-				TK-I
			like)
		1.4	IGF-I, IGF-II,			1.4	Cysteine rich
			Insulin				TK-II, IGF-RI
		1.5	NGF, BDNF,			1.5	Cysteine knot
			NT-3, NT-4f				TK V
		2.	TGF-β1, β2, β3			2.	Serine
							threonine
							kinase
							subfamily
							(STKS)g
1IL-17R - belongs to CKR family but is unassigned to 4 indicated subfamilies.
2Other IFN type I subtypes remain unassigned. Hematopoietic cytokines, IL-10 ligands and interferons do not possess functional intrinsic protein kinases. The signaling molecules for the cytokines are JAK's, STATs and related non-receptor molecules. IL-14, IL-16 and IL-18 have been cloned but according to the receptor code they remain unassigned.
3TNF receptors use multiple, distinct intracellular molecules for signal transduction including “death domain” of FAS R and 55 kDa TNF-αR that participates in their cytotoxic effects. NGF/TNF R can bind both NGF and related factors as well as TNF ligands. Chemokine receptors are seven transmembrane (7TM, serpentine) domain receptors. They are G protein-coupled.
4The Duffy blood group antigen (DARC) is an erythrocyte receptor that can bind several different chemokines. IL-1R belongs to the immunoglobulin superfamily but their signal transduction events characteristics remain unclear.
5The neurotrophic cytokines can associate with NGF/TNF receptors also.
6STKS may encompass many other TGF-β-related factors that remain unassigned. The protein kinases are intrinsic part of the intracellular domain of receptor kinase family (RKF). The enzymes participate in the signals transmission via the receptors.

[0163] Particular proteins of interest as targets for peptide generation in the present invention include the following:

• • [0164] αvβ3
  • [0165] αVβ1
  • [0166] Ang-2
  • [0167] BAFF/TALL-1
  • [0168] B7
  • [0169] B7RP1
  • [0170] CRP1
  • [0171] Calcitonin
  • [0172] CD28
  • [0173] CETP
  • [0174] cMet
  • [0175] Complement factor B
  • [0176] C4b
  • [0177] CTLA4
  • [0178] Glucagon
  • [0179] Glucagon Receptor
  • [0180] LIPG
  • [0181] MPL
  • [0182] myostatin
  • [0183] splice variants of molecules preferentially expressed on tumor cells; e.g., CD44, CD30
  • [0184] unglycosylated variants of mucin and Lewis Y surface glycoproteins
  • [0185] CD19, CD20, CD33, CD45
  • [0186] prostate specific membrane antigen and prostate specific cell antigen
  • [0187] matrix metalloproteinases (MMPs), both secreted and
  • [0188] membrane-bound (e.g., MMP-9)
  • [0189] Cathepsins
  • [0190] angiopoietin-2
  • [0191] TIE-2 receptor
  • [0192] heparanase
  • [0193] urokinase plasminogen activator (UPA), UPA receptor parathyroid hormone (PTH), parathyroid hormone-related protein (PTHrP), PTH-RI, PTH-RII
  • [0194] Her2
  • [0195] Her3
  • [0196] Insulin

[0197] Exemplary peptides for this invention appear in Tables 4 through 20 of U.S. Pat. No. 6,660,843, which are hereby incorporated by reference. Additional preferred peptides appear in U.S. 2003/0229023, published Dec. 11, 2003; WO 03/057134, published Jul. 17, 2003; U.S. 2003/0236193, published Dec. 25,2003; WO 00/24770, published May 4, 2000; U.S. 2003/0176352, published Sep. 18, 2003; WO 03/031589, published Apr. 17, 2003; U.S. Ser. No. 10/666,480, filed Sep. 18, 2003; WO 04/026329, published Apr. 1, 2004; U.S. Ser. No. 10/742,379, filed Dec. 19, 2003; PCT/US03/40781, filed Dec. 19, 2003, each of which are hereby incorporated by reference. Such peptides may be prepared by methods disclosed in the art.

[0198] Particularly preferred peptides appear in the tables below. Single letter amino acid abbreviations are used. Any of these peptides may be linked in tandem (i.e., sequentially), with or without linkers. Any peptide containing a cysteinyl residue may be cross-linked with another Cys-containing peptide or protein. Any peptide having more than one Cys residue may form an intrapeptide disulfide bond, as well. Any of these peptides may be derivatized as described herein. All peptides are linked through peptide bonds unless otherwise noted.

TABLE 4
EPO-mimetic peptide sequences
	SEQUENCE	SEQ ID NO:
	YXCXXGPXTWXCXP	1
	GGTYSCHFGPLTWVCKPQGG	2
	GGDYHCRMGPLTWVCKPLGG	3
	GGVYACRMGPITWVCSPLGG	4
	VGNYMCHFGPITWVCRPGGG	5
	GGLYLCRFGPVTWDCGYKGG	6
	GGTYSCHFGPLTWVCKPQGGSSK	7
	GGTYSCHGPLTWVCKPQGG	8
	VGNYMAHMGPITWVCRPGG	9
	GGPHHVYACRMGPLTWIC	10
	GGTYSCHFGPLTWVCKPQ	11
	GGLYACHMGPMTWVCQPLRG	12
	TIAQYICYMGPETWECRPSPKA	13
	YSCHFGPLTWVCK	14
	YCHFGPLTWVC	15
	GGLYLCRFGPVTWDCGYKGG	16
	GGTYSCHFGPLTWVCKPQGG	17
	GGDYHCRMGPLTWVCKPLGG	18
	VGNYMCHFGPITWVCRPGGG	19
	GGVYACRMGPITWVCSPLGG	20
	VGNYMAHMGPITWVCRPGG	21
	GGTYSCHFGPLTWVCKPQ	22
	GGLYACHMGPMTWVCQPLRG	23
	TIAQYICYMGPETWECRPSPKA	24
	YSCHFGPLTWVCK	25
	YCHFGPLTWVC	26
	SCHFGPLTWVCK	27

[0199]

TABLE 5
TPO-mimetic peptide sequences
	SEQUENCE	SEQ ID NO:
	IEGPTLRQWLAARA	28
	IEGPTLRQWLAAKA	29
	IEGPTLREWLAARA	30
	TLREWL	31
	GRVRDQVAGW	32
	GRVKDQIAQL	33
	GVRDQVSWAL	34
	ESVREQVMKY	35
	SVRSQISASL	36
	GVRETVYRHM	37
	GVREVIVMHML	38
	GRVRDQIWAAL	39
	AGVRDQILIWL	40
	GRVRDQIMLSL	41
	CTLRQWLQGC	42
	CTLQEFLEGC	43
	CTRTEWLHGC	44
	CTLREWLHGGFC	45
	CTLREWVFAGLC	46
	CTLRQWLILLGMC	47
	CTLAEFLASGVEQC	48
	CSLQEFLSHGGYVC	49
	CTLREFLDPTTAVC	50
	CTLKEWLVSHEVWC	51
	REGPTLRQWM	52
	EGPTLRQWLA	53
	ERGPFWAKAC	54
	REGPRCVMWM	55
	CGTEGPTLSTWLDC	56
	CEQDGPTLLEWLKC	57
	CELVGPSLMSWLTC	58
	CLTGPFVTQWLYEC	59
	CRAGPTLLEWLTLC	60
	CADGPTLREWISFC	61
	GGCTLREWLHGGFCGG	62
	GGCADGPTLREWISFCGG	63
	GNADGPTLRQWLEGRRPKN	64
	LAIEGPTLRQWLHGNGRDT	65
	HGRVGPTLREWKTQVATKK	66
	TIKGPTLRQWLKSREHTS	67
	ISDGPTLKEWLSVTRGAS	68
	SIEGPTLREWLTSRTPHS	69
	GAREGPTLRQWLEWVRVG	70
	RDLDGPTLRQWLPLPSVQ	71
	ALRDGPTLKQWLEYRRQA	72
	ARQEGPTLKEWLFWVRMG	73
	EALLGPTLREWLAWRRAQ	74
	MARDGPTLREWLRTYRMM	75
	WMPEGPTLKQWLFHGRGQ	76
	HIREGPTLRQWLVALRMV	77
	QLGHGPTLRQWLSWYRGM	78
	ELRQGPTLHEWLQHLASK	79
	VGIEGPTLRQWLAQRLNP	80
	WSRDGPTLREWLAWRAVG	81
	AVPQGPTLKQWLLWRRCA	82
	RIREGPTLKEWLAQRRGF	83
	RFAEGPTLREWLEQRKLV	84
	DRFQGPTLREWLAAIRSV	85
	AGREGPTLREWLNMRVWQ	86
	ALQEGPTLRQWLGWGQWG	87
	YCDEGPTLKQWLVCLGLQ	88
	WCKEGPTLREWLRWGFLC	89
	CSSGGPTLREWLQCRRMQ	90
	CSWGGPTLKQWLQCVRAK	91
	CQLGGPTLREWLACRLGA	92
	CWEGGPTLKEWLQCLVER	93
	CRGGGPTLHQWLSCFRWQ	94
	CRDGGPTLRQWLACLQQK	95
	ELRSGPTLKEWLVWRLAQ	96
	GCRSGPTLREWLACREVQ	97
	TCEQGPTLRQWLLCRQGR	98
	QGYCDEGPTLKQWLVCLGLQHS	99

[0200]

TABLE 6
Ang-2 binding peptide sequences
SEQUENCE	SEQ ID NO.
WDPWT	100
WDPWTC	101
Cz2WDPWT (wherein z2 is an acidic or	102
neutral polar amino acid residue)
Cz2WDPWTC (wherein z2 is an acidic or	103
neutral polar amino acid residue)
PIRQEECDWDPWTCEHMWEV	104
TNIQEECEWDPWTCDHMPGK	105
WYEQDACEWDPWTCEHMAEV	106
NRLQEVCEWDPWTCEHMENV	107
AATQEECEWDPWTCEHMPRS	108
LRHQEGCEWDPWTCEHMFDW	109
VPRQKDCEWDPWTCEHMYVG	110
SISHEECEWDPWTCEHMQVG	111
WAAQEECEWDPWTCEHMGRM	112
TWPQDKCEWDPWTCEHMGST	113
GHSQEECGWDPWTCEHMGTS	114
QHWQEECEWDPWTCDHMPSK	115
NVRQEKCEWDPWTCEHMPVR	116
KSGQVECNWDPWTCEHMPRN	117
VKTQEHCDWDPWTCEHMREW	118
AWGQEGCDWDPWTCEHMLPM	119
PVNQEDCEWDPWTCEHMPPM	120
RAPQEDCEWDPWTCAHMDIK	121
HGQNMECEWDPWTCEHMFRY	122
PRLQEECVWDPWTCEHMPLR	123
RTTQEKCEWDPWTCEHMESQ	124
QTSQEDCVWDPWTCDHMVSS	125
QVIGRPCEWDPWTCEHLEGL	126
WAQQEECAWDPWTCDHMVGL	127
LPGQEDCEWDPWTCEHMVRS	128
PMNQVECDWDPWTCEHMPRS	129
FGWSHGCEWDPWTCEHMGST	130
KSTQDDCDWDPWTCEHMVGP	131
GPRISTCQWDPWTCEHMDQL	132
STIGDMCEWDPWTCAHMQVD	133
VLGGQGCEWDPWTCRLLQGW	134
VLGGQGCQWDPWTCSHLEDG	135
TTIGSMCEWDPWTCAHMQGG	136
TKGKSVCQWDPWTCSHMQSG	137
TTIGSMCQWDPWTCAHMQGG	138
WVNEVVCEWDPWTCNHWDTP	139
VVQVGMCQWDPWTCKHMRLQ	140
AVGSQTCEWDPWTCAHLVEV	141
QGMKMFCEWDPWTCAHIVYR	142
TTIGSMCQWDPWTCEHMQGG	143
TSQRVGCEWDPWTCQHLTYT	144
QWSWPPCEWDPWTCQTVWPS	145
GTSPSFCQWDPWTCSHMVQG	146
QEECEWDPWTCEHM	147
QNYKPLDELDATLYEHFIFHYT	148
LNFTPLDELEQTLYEQWTLQQS	149
TKFNPLDELEQTLYEQWTLQHQ	150
VKFKPLDALEQTLYEHWMFQQA	151
VKYKPLDELDEILYEQQTFQER	152
TNFMPMDDLEQRLYEQFILQQG	153
SKFKPLDELEQTLYEQWTLQHA	154
QKFQPLDELEQTLYEQFMLQQA	155
QNFKPMDELEDTLYKQFLFQHS	156
YKFTPLDDLEQTLYEQWTLQHV	157
QEYEPLDELDETLYNQWMFHQR	158
SNFMPLDELEQTLYEQFMLQHQ	159
QKYQPLDELDKTLYDQFMLQQG	160
QKFQPLDELEETLYKQWTLQQR	161
VKYKPLDELDEWLYHQFTLHHQ	162
QKFMPLDELDEILYEQFMFQQS	163
QTFQPLDDLEEYLYEQWIRRYH	164
EDYMPLDALDAQLYEQFILLHG	165
HTFQPLDELEETLYYQWLYDQL	166
YKFNPMDELEQTLYEEFLFQHA	167
TNYKPLDELDATLYEHWILQHS	168
QKFKPLDELEQTLYEQWTLQQR	169
TKFQPLDELDQTLYEQWTLQQR	170
TNFQPLDELDQTLYEQWTLQQR	171
KFNPLDELEETLYEQFTFQQ	172
AGGMRPYDGMLGWPNYDVQA	173
QTWDDPCMHILGPVTWRRCI	174
APGQRPYDGMLGWPTYQRIV	175
SGQLRPCEEIFGCGTQNLAL	176
FGDKRPLECMFGGPIQLCPR	177
GQDLRPCEDMFGCGTKDWYG	178
KRPCEEIFGGCTYQ	179
GFEYCDGMEDPFTFGCDKQT	180
KLEYCDGMEDPFTQGCDNQS	181
LQEWCEGVEDPFTFGCEKQR	182
AQDYCEGMEDPFTFGCEMQK	183
LLDYCEGVQDPFTFGCENLD	184
HQEYCEGMEDPFTFGCEYQG	185
MLDYCEGMDDPFTFGCDKQM	186
LQDYCEGVEDPFTFGCENQR	187
LQDYCEGVEDPFTFGCEKQR	188
FDYCEGVEDPFTFGCDNH	189

[0201]

TABLE 7
NGF-Binding Peptide Sequences
	SEQUENCE	SEQ ID NO.
	TGYTEYTEEWPMGFGYQWSF	190
	TDWLSDFPFYEQYFGLMPPG	191
	FMRFPNPWKLVEPPQGWYYG	192
	VVKAPHFEFLAPPHFHEFPF	193
	FSYIWIDETPSNIDRYMLWL	194
	VNFPKVPEDVEPWPWSLKLY	195
	TWHPKTYEEFALPFFVPEAP	196
	WHFGTPYIQQQPGVYWLQAP	197
	VWNYGPFFMNFPDSTYFLHE	198
	WRIHSKPLDYSHVWFFPADF	199
	FWDGNQPPDILVDWPWNPPV	200
	FYSLEWLKDHSEFFQTVTEW	201
	QFMELLKFFNSPGDSSHHFL	202
	TNVDWISNNWEHMKSFFTED	203
	PNEKPYQMQSWFPPDWPVPY	204
	WSHTEWVPQVWWKPPNHFYV	205
	WGEWINDAQVHMHEGFISES	206
	VPWEHDHDLWEIISQDWHIA	207
	VLHLQDPRGWSNFPPGVLEL	208
	IHGCWFTEEGCVWQ	209
	YMQCQFARDGCPQW	210
	KLQCQYSESGCPTI	211
	FLQCEISGGACPAP	212
	KLQCEFSTSGCPDL	213
	KLQCEFSTQGCPDL	214
	KLQCEFSTSGCPWL	215
	IQGCWFTEEGCPWQ	216
	SFDCDNPWGHVLQSCFGF	217
	SFDCDNPWGHKLQSCFGF	218

[0202]

TABLE 8
Myostatin binding peptide sequences
	SEQUENCE	SEQ ID NO:
	KDKCKMWHWMCKPP	647
	KDLCAMWHWMCKPP	219
	KDLCKMWKWMCKPP	220
	KDLCKMWHWMCKPK	221
	WYPCYEFHFWCYDL	222
	WYPCYEGHFWCYDL	223
	IFGCKWWDVQCYQF	224
	IFGCKWWDVDCYQF	225
	ADWCVSPNWFCMVM	226
	HKFCPWWALFCWDF	227
	KDLCKMWHWMCKPP	228
	IDKCAIWGWMCPPL	229
	WYPCGEFGMWCLNV	230
	WFTCLWNCDNE	231
	HTPCPWFAPLCVEW	232
	KEWCWRWKWMCKPE	233
	FETCPSWAYFCLDI	234
	AYKCEANDWGCWWL	235
	NSWCEDOWHRCWWL	236
	WSACYAGHFWCYDL	237
	ANWCVSPNWFCMVM	238
	WTECYQQEFWCWNL	239
	ENTCERWKWMCPPK	240
	WLPCHQEGFWCMNF	241
	STMCSQWHWMCNPF	242
	IFGCHWWDVDCYQF	243
	IYGCKWWDIQCYDI	244
	PDWCIDPDWWCKFW	245
	QGHCTRWPWMCPPY	246
	WQECYREGFWCLQT	247
	WFDCYGPGFKCWSP	248
	GVRCPKGHLWCLYP	249
	HWACGYWPWSCKWV	250
	GPACHSPWWWCVFG	251
	TTWCISPMWFCSQQ	252
	HKFCPPWAIFCWDF	253
	PDWCVSPRWYCNMW	254
	VWKCHWFGMDCEPT	255
	KKHCQIWTWMCAPK	256
	WFQCGSTLFWCYNL	257
	WSPCYDHYFYCYTI	258
	SWMCGFFKEVCMWV	259
	EMLCMIHPVFCNPH	260
	LKTCNLWPWMCPPL	261
	VVGCKWYEAWCYNK	262
	PIHCTQWAWMCPPT	263
	DSNCPWYFLSCVIF	264
	HIWCNLAMMKCVEM	265
	NLQCIYFLGKCIYF	266
	AWRCMWFSDVCTPG	267
	WFRCFLDADWCTSV	268
	EKICQMWSWMCAPP	269
	WFYCHLNKSECTEP	270
	FWRCAIGIDKCKRV	271
	NLGCKWYEVWCFTY	272
	IDLCNMWDGMCYPP	273
	EMPCNIWGWMCPPV	274
	WFRCVLTGIVDWSECFGL	275
	GFSCTFGLDEFYVDCSPF	276
	LPWCHDQVNADWGFCMLW	277
	YPTCSEKFWIYGQTCVLW	278
	LGPCPIHHGPWPQYCVYW	279
	PFPCETHQISWLGHCLSF	280
	HWGCEDLMWSWHPLCRRP	281
	LPLCDADMMPTIGFCVAY	282
	SHWCETTFWMNYAKCVHA	283
	LPKCTHVPFDQGGFCLWY	284
	FSSCWSPVSRQDMFCVFY	285
	SHKCEYSGWLQPLCYRP	286
	PWWCQDNYVQHMLHCDSP	287
	WFRCMLMNSFDAFQCVSY	288
	PDACRDQPWYMFMGCMLG	289
	FLACFVEFELCFDS	290
	SAYCIITESDPYVLCVPL	291
	PSICESYSTMWLPMCQHN	292
	WLDCHDDSWAWTKMCRSH	293
	YLNCVMMNTSPFVECVFN	294
	YPWCDGFMIQQGITCMFY	295
	FDYCTWLNGFKDWKCWSR	296
	LPLCNLKEISHVQACVLF	297
	SPECAFARWLGIEQCQRD	298
	YPQCFNLHLLEWTECDWF	299
	RWRCEIYDSEFLPKCWFF	300
	LVGCDNVWHRCKLF	301
	AGWCHVWGEMFGMGCSAL	302
	HHECEWMARWMSLDCVGL	303
	FPMCGIAGMKDFDFCVWY	304
	RDDCTFWPEWLWKLCERP	305
	YNFCSYLFGVSKEACQLP	306
	AHWCEQGPWRYGNICMAY	307
	NLVCGKISAWGDEACARA	308
	HNVCTIMGPSMKWFCWND	309
	NDLCAMWGWRNTIWCQNS	310
	PPFCQNDNDMLQSLCKLL	311
	WYDCNVPNELLSGLCRLF	312
	YGDCDQNHWMWPFTCLSL	313
	GWMCHFDLHDWGATCQPD	314
	YFHCMFGGHEFEVHCESF	315
	AYWCWHGQCVRF	316
	SEHWTFTDWDGNEWWVRPF	317
	MEMLDSLFELLKDMVPISKA	318
	SPPEEALMEWLGWQYGKFT	319
	SPENLLNDLYILMTKQEWYG	320
	FHWEEGIPFHVVTPYSYDRM	321
	KRLLEQFMNDLAELVSGHS	322
	DTRDALFQEFYEFVRSRLVI	323
	RMSAAPRPLTYRDIMDQYWH	324
	NDKAHFFEMFMFDVHNFVES	325
	QTQAQKIDGLWELLQSIRNQ	326
	MLSEFEEFLGNLVHRQEA	327
	YTPKMGSEWTSFWHNRIHYL	328
	LNDTLLRELKMVLNSLSDMK	329
	FDVERDLMRWLEGFMQSAAT	330
	HHGWNYLRKGSAPQWFEAWV	331
	VESLHQLQMWLDQKLASGPH	332
	RATLLKDFWQLVEGYGDN	333
	EELLREFYRFVSAFDY	334
	GLLDEFSHFIAEQFYQMPGG	335
	YREMSMLEGLLDVLERLQHY	336
	HNSSQMLLSELIMLVGSMMQ	337
	WREHFLNSDYIRDKLIAIDG	338
	QFPFYVFDDLPAQLEYWIA	339
	EFFHWLHNHRSEVNHWLDMN	340
	EALFQNFFRDVLTLSEREY	341
	QYWEQQWMTYFRENGLHVQY	342
	NQRMMLEDLWRIMTPMFGRS	343
	FLDELKAELSRHYALDDLDE	344
	GKLIEGLLNELMQLETFMPD	345
	ILLLDEYKKDWKSWF	346
	QGHCTRWPWMCPPYGSGSATGGSGST	347
	ASSGSGSATGQGHCTRWPWMCPPY
	WYPCYEGHFWCYDLGSGSTASSGSGSA	348
	TGWYPCYEGHFWCYDL
	HTPCPWFAPLCVEWGSGSATGGSGSTA	349
	SSGSGSATGHTPCPWFAPLCVEW
	PDWCIDPDWWCKFWGSGSATGGSGST	350
	ASSGSGSATGPDWCIDPDWWCKFW
	ANWCVSPNWFCMVMGSGSATGGSGST	351
	ASSGSGSATGANWCVSPNWFCMVM
	PDWCIDPDWWCKFWGSGSATGGSGST	352
	ASSGSGSATGPDWCIDPDWWCKFW
	HWACGYWPWSCKWVGSGSATGGSGST	353
	ASSGSGSATGHWACGYWPWSCKWV
	KKHCQIWTWMCAPKGSGSATGGSGST	354
	ASSGSGSATGQGHCTRWPWMCPPY
	QGHCTRWPWMCPPYGSGSATGGSGST	355
	ASSGSGSATGKKHCQIWTWMCAPK
	KKHCQIWTWMCAPKGSGSATGGSGST	356
	ASSGSGSATGQGHCTRWPWMCPPY
	KKHCQIWTWMCAPKGGGGGGGGQGH	357
	CTRWPWMCPPY
	QGHCTRWPWMCPPYGGGGGGKKHCQ	358
	IWTWMCAPK
	VALHGQCTRWPWMCPPQREG	359
	YPEQGLCTRWPWMCPPQTLA	360
	GLNQGHCTRWPWMCPPQDSN	361
	MITQGQCTRWPWMCPPQPSG	362
	AGAQEHCTRWPWMCAPNDWI	363
	GVNQGQCTRWRWMCPPNGWE	364
	LADHGQCIRWPWMCPPEGWE	365
	ILEQAQCTRWPWMCPPQRGG	366
	TQTHAQCTRWPWMCPPQWEG	367
	VVTQGHCTLWPWMCPPQRWR	368
	IYPHDQCTRWPWMCPPQPYP	369
	SYWQGQCTRWPWMCPPQWRG	370
	MWQQGHCTRWPWMCPPQGWG	371
	EFTQWHCTRWPWMCPPQRSQ	372
	LDDQWQCTRWPWMCPPQGFS	373
	YQTQGLCTRWPWMCPPQSQR	374
	ESNQGQCTRWPWMCPPQGGW	375
	WTDRGPCTRWPWMCPPQANG	376
	VGTQGQCTRWPWMCPPYETG	377
	PYEQGKCTRWPWMCPPYEVE	378
	SEYQGLCTRWPWMCPPQGWK	379
	TFSQGHCTRWPWMCPPQGWG	380
	PGAHDHCTRWPWMCPPQSRY	381
	VAEEWHCRRWPWMCPPQDWR	382
	VGTQGHCTRWPWMCPPQPAG	383
	EEDQAHCRSWPWMCPPQGWV	384
	ADTQGHCTRWPWMCPPQHWF	385
	SGPQGHCTRWPWMCAPQGWF	386
	TLVQGHCTRWPWMCPPQRWV	387
	GMAHGKCTRWAWMCPPQSWK	388
	ELYHGQCTRWPWMCPPQSWA	389
	VADHGHCTRWPWMCPPQGWG	390
	PESQGHCTRWPWMCPPQGWG	391
	IPAHGHCTRWPWMCPPQRWR	392
	FTVHGHCTRWPWMCPPYGWV	393
	PDFPGHCTRWRWMCPPQGWE	394
	QLWQGPCTQWPWMCPPKGRY	395
	HANDGHCTRWQWMCPPQWGG	396
	ETDHGLCTRWPWMCPPYGAR	397
	GTWQGLCTRWPWMCPPQGWQ	398
	VATQGQCTRWPWMCPPQGWG	399
	VATQGQCTRWPWMCPPQRWG	400
	QREWYPCYGGHLWCYDLHKA	401
	ISAWYSCYAGHFWCWDLKQK	402
	WTGWYQCYGGHLWCYDLRRK	403
	KTFWYPCYDGHFWCYNLKSS	404
	ESRWYPCYEGHLWCFDLTET	405
	MEMLDSLFELLKDMVPISKA	406
	RMEMLESLLELLKEIVPMSKAG	407
	RMEMLESLLELLKEIVPMSKAR	408
	RMEMLESLLELLKDIVPMSKPS	409
	GMEMLESLFELLQEIVPMSKAP	410
	RMEMLESLLELLKDIVPISNPP	411
	RIEMLESLLELLQEIVPISKAE	412
	RMEMLQSLLELLKDIVPMSNAR	413
	RMEMLESLLELLKEIVTPTSNGT	414
	RMEMLESLFELLKEIVPMSKAG	415
	RMEMLGSLLELLKEIVPMSKAR	416
	QMELLDSLFELLKEIVPKSQPA	417
	RMEMLDSLLELLKEIVPMSNAR	418
	RMEMLESLLELLHEIVPMSQAG	419
	QMEMLESLLQLLKEIVPMSKAS	420
	RMEMLDSLLELLKDMVPMTTGA	421
	RIEMLESLLELLKDMVPMANAS	422
	RMEMLESLLQLLNEIVPMSRAR	423
	RMEMLESLFDLLKELVPMSKGV	424
	RIEMLESLLELLKDIVPIQKAR	425
	RMELLESLFELLKDMVPMSDSS	426
	RMEMLESLLEVLQEIVPRAKGA	427
	RMEMLDSLLQLLNEIVPMSHAR	428
	RMEMLESLLELLKDIVPMSNAG	429
	RMEMLQSLFELLKGMVPISKAG	430
	RMEMLESLLELLKEIVPNSTAA	431
	RMEMLQSLLELLKEIVPISKAG	432
	RIEMLDSLLELLNELVPMSKAR	433
	HHGWNYLRKGSAPQWFEAWV	434
	QVESLQQLLMWLDQKLASGPQG	435
	RMELLESLFELLKEMVPRSKAV	436
	QAVSLQHLLMWLDQKLASGPQH	437
	DEDSLQQLLMWLDQKLASGPQL	438
	PVASLQQLLIWLDQKLAQGPHA	439
	EVDELQQLLNWLDHKLASGPLQ	440
	DVESLEQLLMWLDHQLASGPHG	441
	QVDSLQQVLLWLEHKLALGPQV	442
	GDESLQHLLMWLEQKLALGPHG	443
	QIEMLESLLDLLRDMVPMSNAF	444
	EVDSLQQLLMWLDQKLASGPQA	445
	EDESLQQLLIYLDKMLSSGPQV	446
	AMDQLHQLLIWLDHKLASGPQA	447
	RIEMLESLLELLDEIALIPKAW	448
	EVVSLQHLLMWLEHKLASGPDG	449
	GGESLQQLLMWLDQQLASGPQR	450
	GVESLQQLLIFLDHMLVSGPHD	451
	NVESLEHLMMWLERLLASGPYA	452
	QVDSLQQLLIWLDHQLASGPKR	453
	EVESLQQLLMWLEHKLAQGPQG	454
	EVDSLQQLLMWLDQKLASGPHA	455
	EVDSLQQLLMWLDQQLASGPQK	456
	GVEQLPQLLMWLEQKLASGPQR	457
	GEDSLQQLLMWLDQQLAAGPQV	458
	ADDSLQQLLMWLDRKLASGPHV	459
	PVDSLQQLLIWLDQKLASGPQG	460
	RATLLKDFWQLVEGYGDN	461
	DWRATLLKEFWQLVEGLGDNLV	462
	QSRATLLKEFWQLVEGLGDKQA	463
	DGRATLLTEFWQLVQGLGQKEA	464
	LARATLLKEFWQLVEGLGEKVV	465
	GSRDTLLKEFWQLVVGLGDMQT	466
	DARATLLKEFWQLVDAYGDRMV	467
	NDRAQLLRDFWQLVDGLGVKSW	468
	GVRETLLYELWYLLKGLGANQG	469
	QARATLLKEFCQLVGCQGDKLS	470
	QERATLLKEFWQLVAGLGQNMR	471
	SGRATLLKEFWQLVQGLGEYRW	472
	TMRATLLKEFWLFVDGQREMQW	473
	GERATLLNDFWQLVDGQGDNTG	474
	DERETLLKEFWQLVHGWGDNVA	475
	GGRATLLKELWQLLEGQGANLV	476
	TARATLLNELVQLVKGYGDKLV	477
	GMRATLLQEFWQLVGGQGDNWM	478
	STRATLLNDLWQLMKGWAEDRG	479
	SERATLLKELWQLVGGWGDNFG	480
	VGRATLLKEFWQLVEGLVGQSR	481
	EIRATLLKEFWQLVDEWREQPN	482
	QLRATLLKEFLQLVHGLGETDS	483
	TQRATLLKEFWQLIEGLGGKHV	484
	HYRATLLKEFWQLVDGLREQGV	485
	QSRVTLLREFWQLVESYRPIVN	486
	LSRATLLNEFWQFVDGQRDKRM	487
	WDRATLLNDFWHLMEELSQKPG	488
	QERATLLKEFWRMVEGLGKNRG	489
	NERATLLREFWQLVGGYGVNQR	490
	YREMSMLEGLLDVLERLQHY	491
	HQRDMSMLWELLDVLDGLRQYS	492
	TQRDMSMLDGLLEVLDQLRQQR	493
	TSRDMSLLWELLEELDRLGHQR	494
	MQHDMSMLYGLVELLESLGHQI	495
	WNRDMRMLESLFEVLDGLRQQV	496
	GYRDMSMLEGLLAVLDRLGPQL	497
	TQRDMSMLEGLLEVLDRLGQQR	498
	WYRDMSMLEGLLEVLDRLGQQR	499
	HNSSQMLLSELIMLVGSMMQ	500
	TQNSRQMLLSDFMMLVGSMIQG	501
	MQTSRHILLSEFMMLVGSIMHG	502
	HDNSRQMLLSDLLHLVGTMIQG	503
	MENSRQNLLRELIMLVGNMSHQ	504
	QDTSRHMLLREFMMLVGEMIQG	505
	DQNSRQMLLSDLMILVGSMIQG	506
	EFFHWLHNHRSEVNHWLDMN	507
	NVFFQWVQKHGRVVYQWLDINV	508
	FDFLQWLQNHRSEVEHWLVMDV	509

[0203]

TABLE 9
BAFF binding peptide sequences
	SEQUENCE	SEQ ID NO:
	PGTCFPFPWECTHA	510
	WGACWPFPWECFKE	511
	VPFCDLLTKHCFEA	512
	GSRCKYKWDVLTKQCFHH	513
	LPGCKWDLLIKQWVCDPL	514
	SADCYFDILTKSDVCTSS	515
	SDDCMYDQLTRMFICSNL	516
	DLNCKYDELTYKEWCQFN	517
	FHDCKYDLLTRQMVCHGL	518
	RNHCFWDHLLKQDICPSP	519
	ANQCWWDSLTKKNVCEFF	520
	YKGRQQMWDILTRSWVVSL	521
	QQDVGLWWDILTRAWMPNI	522
	QQNAQRVWDLLIRTWVYPQ	523
	GWNEAWWDELTKIWVLEQQ	524
	RITCDTWDSLIKKCVPQQS	525
	GAIMQQFWDSLTKTWLRQS	526
	WLHSGWWDPLTKHWLQQKV	527
	SEWFFWFDPLTRAQQLKFR	528
	GVWFWWFDPLTKQWTQQAG	529
	MQQCKGYYDILTKWCVTNG	530
	LWSKEVWDILTKSWVSQQA	531
	KAAGWWFDWLTKVWVPAP	532
	AYQQTWFWDSLTRLWLSTT	533
	SGQQHFWWDLLTRSWTPST	534
	LGVGQQKWDPLTKQWVSRG	535
	VGKMCQQWDPLIKRTVCVG	536
	CRQGAKFDLLTKQCLLGR	537
	GQAIRHWDVLTKQWVDSQQ	538
	RGPCGSWDLLTKHCLDSQQ	539
	WQWKQQQWDLLTKQMVWVG	540
	PITICRKDLLTKQVVCLD	541
	KTCNGKWDLLTKQCLQQQA	542
	KCLKGKWDLLTKQCVTEV	543
	RCWNGKWDLLTKQCIHPW	544
	NRDMRKWDPLIKQWIVRP	545
	QQAAAATWDLLTKQWLVPP	546
	PEGGPKWDPLTKQQFLPPV	547
	QQTPQQKKWDLLTKQWFTRN	548
	IGSPCKWDLLTKQMICQQT	549
	CTAAGKWDLLTKQCIQQEK	550
	VSQCMKWDLLTKQCLQQGW	551
	VWGTWKWDLLTKQYLPPQQ	552
	GWWEMKWDLLTKQWYRPQQ	553
	TAQQVSKWDLLTKQWLPLA	554
	QLWGTKWDLLTKQYIQQIM	555
	WATSQKWDLLTKQWVQQNM	556
	QQRQCAKWDLLTKQCVLFY	557
	KTTDCKWDLLTKQRICQQV	558
	LLCQQGKWDLLTKQCLKLR	559
	LMWFWKWDLLTKQLVPTF	560
	QQTWAWKWDLLTKQWIGPM	561
	NKELLKWDLLTKQCRGRS	562
	GQQKDLKWDLLTKQYVRQS	563
	PKPCQQKWDLLTKQCLGSV	564
	GQIGWKWDLLTKQWIQQTR	565
	VWLDWKWDLLTKQWIHPQQ	566
	QQEWEYKWDLLTKQWGWLR	567
	HWDSWKWDLLTKQWVVQQA	568
	TRPLQQKWDLLTKQWLRVG	569
	SDQWQQKWDLLTKQWFWDV	570
	QQQTFMKWDLLTKQWIRRH	571
	QQGECRKWDLLTKQCFPGQ	572
	GQQMGWRWDPLIKMCLGPS	573
	QQLDGCKWDLLTKQKVCIP	574
	HGYWQQKWDLLTKQWVSSE	575
	HQQGQCGWDLLTRIYLPCH	576
	LHKACKWDLLTKQCWPMQQ	577
	GPPGSVWDLLTKIWIQQTG	578
	ITQQDWRFDTLTRLWLPLR	579
	QQGGFAAWDVLTKMWITVP	580
	GHGTPWWDALTRIWILGV	581
	VWPWQQKWDLLTKQFVFQD	582
	WQQWSWKWDLLTRQYISSS	583
	NQQTLWKWDLLTKQFITYM	584
	PVYQQGWWDTLTKLYIWDG	585
	WLDGGWRDPLIKRSVQQLG	586
	GHQQQFKWDLLTKQWVQSN	587
	QQRVGQFWDVLTKMFITGS	588
	QQAQGWSYDALIKTWIRWP	589
	GWMHWKWDPLTKQQALPWM	590
	GHPTYKWDLLTKQWILQQM	591
	WNNWSLWDPLTKLWLQQQN	592
	WQWGWKWDLLTKQWVQQQ	593
	GQMGWRWDPLTKMWLGTS	594

[0204] Fc Domains

[0205] This invention requires the presence of at least one Fc domain modified to comprise a peptide sequence.

[0206] As noted above, both native Fc's and Fc variants are suitable Fc domains for use within the scope of this invention. A native Fc may be extensively modified to form an Fc variant in accordance with this invention, provided binding to the salvage receptor is maintained; see, for example WO 97/34631 and WO 96/32478. In such Fc variants, one may remove one or more sites of a native Fc that provide structural features or functional activity not required by the fusion molecules of this invention. One may remove these sites by, for example, substituting or deleting residues, inserting residues into the site, or truncating portions containing the site. The inserted or substituted residues may also be altered amino acids, such as peptidomimetics or D-amino acids. Fc variants may be desirable for a number of reasons, several of which are described below. Exemplary Fc variants include molecules and sequences in which:

• • [0207] 1. Sites involved in disulfide bond formation are removed. Such removal may avoid reaction with other cysteine-containing proteins present in the host cell used to produce the molecules of the invention. For this purpose, the cysteine-containing segment at the N-terminus may be truncated or cysteine residues may be deleted or substituted with other amino acids (e.g., alanyl, seryl). In particular, one may truncate the N-terminal 20-amino acid segment of SEQ ID NO: 599 or delete or substitute the cysteine residues at positions 7 and 10 of SEQ ID NO: 599 Even when cysteine residues are removed, the single chain Fc domains can still form a dimeric Fc domain that is held together non-covalently.
  • [0208] 2. A native Fc is modified to make it more compatible with a selected host cell. For example, one may remove the PA sequence near the N-terminus of a typical native Fc, which may be recognized by a digestive enzyme in E. coli such as proline iminopeptidase. One may also add an N-terminal methionine residue, especially when the molecule is expressed recombinantly in a bacterial cell such as E. coli. The Fc domain of SEQ ID NO: 599 (FIG. 2A) is one such Fc variant.
  • [0209] 3. A portion of the N-terminus of a native Fc is removed to prevent N-terminal heterogeneity when expressed in a selected host cell. For this purpose, one may delete any of the first 20 amino acid residues at the N-terminus, particularly those at positions 1, 2, 3, 4 and 5.
  • [0210] 4. One or more glycosylation sites are removed. Residues that are typically glycosylated (e.g., asparagine) may confer cytolytic response. Such residues may be deleted or substituted with unglycosylated residues (e.g., alanine).
  • [0211] 5. Sites involved in interaction with complement, such as the C1q binding site, are removed. For example, one may delete or substitute the EKK sequence of human IgG1. Complement recruitment may not be advantageous for the molecules of this invention and so may be avoided with such an Fc variant.
  • [0212] 6. Sites are removed that affect binding to Fc receptors other than a salvage receptor. A native Fc may have sites for interaction with certain white blood cells that are not required for the fusion molecules of the present invention and so may be removed.
  • [0213] 7. The ADCC site is removed. ADCC sites are known in the art; see, for example, Molec. Immunol. 29 (5): 633-9 (1992) with regard to ADCC sites in IgG1. These sites, as well, are not required for the fusion molecules of the present invention and so may be removed.
  • [0214] 8. When the native Fc is derived from a non-human antibody, the native Fc may be humanized. Typically, to humanize a native Fc, one will substitute selected residues in the non-human native Fc with residues that are normally found in human native Fc. Techniques for antibody humanization are well known in the art.

[0215] Preferred Fc variants include the following. In SEQ ID NO: 599 (FIG. 2A) the leucine at position 15 may be substituted with glutamate;

[0216] the glutamate at position 99, with alanine; and the lysines at positions 101 and 103, with alanines. In addition, one or more tyrosine residues can be replaced by phenyalanine residues.

[0217] Additional Vehicles

[0218] The invention further embraces molecules covalently modified to include one or more water soluble polymer attachments, such as polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol, as described U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; and 4,179,337. Still other useful polymers known in the art include monomethoxy-polyethylene glycol, dextran, cellulose, or other carbohydrate based polymers, poly-(N-vinyl pyrrolidone)-polyethylene glycol, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol, as well as mixtures of these polymers. Particularly preferred are peptibodies covalently modified with polyethylene glycol (PEG) subunits. Water-soluble polymers may be bonded at specific positions, for example at the amino terminus of the peptibodies, or randomly attached to one or more side chains of the polypeptide. The use of PEG for improving the therapeutic capacity for specific binding agents, e.g. peptibodies, and for humanized antibodies in particular, is described in U.S. Pat. No. 6,133,426 to Gonzales et al., issued Oct. 17, 2000.

[0219] Various means for attaching chemical moieties useful as vehicles are currently available, see, e.g., Patent Cooperation Treaty (“PCT”) International Publication No. WO 96/11953, entitled “N-Terminally Chemically Modified Protein Compositions and Methods,” herein incorporated by reference in its entirety. This PCT publication discloses, among other things, the selective attachment of water soluble polymers to the N-terminus of proteins.

[0220] A preferred polymer vehicle is polyethylene glycol (PEG). The PEG group may be of any convenient molecular weight and may be linear or branched. The average molecular weight of the PEG will preferably range from about 2 kiloDalton (“kD”) to about 100 kD, more preferably from about 5 kDa to about 50 kDa, most preferably from about 5 kD to about 20 kD. The PEG groups will generally be attached to the compounds of the invention via acylation or reductive alkylation through a reactive group on the PEG moiety (e.g., an aldehyde, maleimide, amino, thiol, or ester group) to a reactive group on the inventive compound (e.g., an aldehyde, amino, thiol or ester group).

[0221] A useful strategy for the PEGylation of synthetic peptides consists of combining, through forming a conjugate linkage in solution, a peptide and a PEG moiety, each bearing a special functionality that is mutually reactive toward the other. The peptides can be easily prepared with conventional solid phase synthesis (see, for example, FIGS. 5 and 6 and the accompanying text herein). The peptides are “preactivated” with an appropriate functional group at a specific site. The precursors are purified and fully characterized prior to reacting with the PEG moiety. Ligation of the peptide with PEG usually takes place in aqueous phase and can be easily monitored by reverse phase analytical HPLC. The PEGylated peptides can be easily purified by preparative HPLC and characterized by analytical HPLC, amino acid analysis and laser desorption mass spectrometry.

[0222] Polysaccharide polymers are another type of water soluble polymer which may be used for protein modification. Dextrans are polysaccharide polymers comprised of individual subunits of glucose predominantly linked by α1-6 linkages. The dextran itself is available in many molecular weight ranges, and is readily available in molecular weights from about 1 kD to about 70 kD. Dextran is a suitable water soluble polymer for use in the present invention as a vehicle by itself or in combination with another vehicle (e.g., Fc). See, for example, WO 96/11953 and WO 96/05309. The use of dextran conjugated to therapeutic or diagnostic immunoglobulins has been reported; see, for example, European Patent Publication No. 0 315 456, which is hereby incorporated by reference. Dextran of about 1 kD to about 20 kD is preferred when dextran is used as a vehicle in accordance with the present invention.

[0223] An additional vehicle may also be a protein, polypeptide, peptide, antibody, antibody fragment, or small molecule (e.g., a peptidomimetic compound) capable of binding to a salvage receptor. For example, one could use as a vehicle a polypeptide as described in U.S. Pat. No. 5,739,277, issued Apr. 14, 1998 to Presta et al. Peptides could also be selected by phage display for binding to the FcRn salvage receptor. Such salvage receptor-binding compounds are also included within the meaning of “vehicle” in this invention. Such vehicles should be selected for increased half-life (e.g., by avoiding sequences recognized by proteases) and decreased immunogenicity (e.g., by favoring non-immunogenic sequences, as discovered in antibody humanization).

[0224] Linkers

[0225] Any “linker” group is optional. When present, its chemical structure is not critical, since it serves primarily as a spacer. The linker is preferably made up of amino acids linked together by peptide bonds. Thus, in preferred embodiments, the linker is made up of from 1 to 20 amino acids linked by peptide bonds, wherein the amino acids are selected from the 20 naturally occurring amino acids. Some of these amino acids may be glycosylated, as is well understood by those in the art. In a more preferred embodiment, the 1 to 20 amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. Even more preferably, a linker is made up of a majority of amino acids that are sterically unhindered, such as glycine and alanine. Thus, preferred linkers are polyglycines (particularly (Gly)₄, (Gly)₅), poly(Gly-Ala), and polyalanines.

[0226] Other specific examples of linkers are:

• • [0227] (Gly)₃Lys(Gly)₄ (SEQ ID NO: 595);
  • [0228] (Gly)₃AsnGlySer(Gly)₂ (SEQ ID NO: 596);
  • [0229] (Gly)₃Cys(Gly)₄ (SEQ ID NO: 597); and
  • [0230] GlyProAsnGlyGly (SEQ ID NO: 598).
    To explain the above nomenclature, for example, (Gly)₃Lys(Gly)₄ means Gly-Gly-Gly-Lys-Gly-Gly-Gly-Gly. Combinations of Gly and Ala are also preferred. The linkers shown here are exemplary; linkers within the scope of this invention may be much longer and may include other residues.

[0231] Non-peptide linkers are also possible. For example, alkyl linkers such as —NH—(CH₂)_s—C(O)—, wherein s=2-20 could be used. These alkyl linkers may further be substituted by any non-sterically hindering group such as lower alkyl (e.g., C₁-C₆) lower acyl, halogen (e.g., Cl, Br), CN, NH₂, phenyl, etc. An exemplary non-peptide linker is a PEG linker, VI
wherein n is such that the linker has a molecular weight of 100 to 5000 kD, preferably 100 to 500 kD. The peptide linkers may be altered to form derivatives in the same manner as described above.

[0232] Derivatives

[0233] The invention also provides “derivatives” that include molecules bearing modifications other than, or in addition to, insertions, deletions, or substitutions of amino acid residues. Preferably, the modifications are covalent in nature, and include for example, chemical bonding with polymers, lipids, other organic, and inorganic moieties. Derivatives of the invention may be prepared to increase circulating half-life of a molecule; to improve targeting capacity for the molecule to desired cells, tissues, or organs; to improve the solubility or absorption of a molecule; or to eliminate or attenuate any undesirable side-effect of a molecule.

[0234] Exemplary derivatives include compounds in which:

• • [0235] 1. The compound or some portion thereof is cyclic. For example, the peptide portion may be modified to contain two or more Cys residues (e.g., in the linker), which could cyclize by disulfide bond formation.

[0236] For citations to references on preparation of cyclized derivatives, see Table 2.

• • [0237] 2. The compound is cross-linked or is rendered capable of cross-linking between molecules. For example, the peptide portion may be modified to contain one Cys residue and thereby be able to form an intermolecular disulfide bond with a like molecule. The compound may also be cross-linked through its C-terminus, as in the molecule shown below.
  • [0238] 3. One or more peptidyl [—C(O)NR—] linkages (bonds) is replaced by a non-peptidyl linkage. Exemplary non-peptidyl linkages are —CH₂-carbamate [—CH₂—OC(O)NR—], phosphonate, —CH₂-sulfonamide [—CH₂—S(O)₂NR—], urea [—NHC(O)NH—], —CH₂-secondary amine, and alkylated peptide [—C(O)NR⁶— wherein R⁶ is lower alkyl].
  • [0239] 4. The N-terminus is derivatized. Typically, the N-terminus may be acylated or modified to a substituted amine. Exemplary N-terminal derivative groups include —NRR¹ (other than —NH₂), —NRC(O)R¹, —NRC(O)OR¹, —NRS(O)₂R¹, —NHC(O)NHR¹, succinimide, or benzyloxycarbonyl-NH— (CBZ-NH—), wherein R and R¹ are each independently hydrogen or lower alkyl and wherein the phenyl ring may be substituted with 1 to 3 substituents selected from the group consisting of C₁-C₄ alkyl, C₁-C₄ alkoxy, chloro, and bromo.
  • [0240] 5. The free C-terminus is derivatized. Typically, the C-terminus is esterified or amidated. For example, one may use methods described in the art to add (NH—CH₂—CH₂—NH₂)₂ to compounds of this invention. Likewise, one may use methods described in the art to add —NH₂ to compounds of this invention. Exemplary C-terminal derivative groups include, for example, —C(O)R² wherein R² is lower alkoxy or —NR³R⁴ wherein R³ and R⁴ are independently hydrogen or C₁-C₈ alkyl (preferably C₁-C₄ alkyl).
  • [0241] 6. A disulfide bond is replaced with another, preferably more stable, cross-linking moiety (e.g., an alkylene). See, e.g., Bhatnagar et al. (1996), J. Med. Chem. 39: 3814-9; Alberts et al. (1993) Thirteenth Am. Pep. Symp., 357-9.
  • [0242] 7. One or more individual amino acid residues is modified. Various derivatizing agents are known to react specifically with selected sidechains or terminal residues, as described in detail below.

[0243] Lysinyl residues and amino terminal residues may be reacted with succinic or other carboxylic acid anhydrides, which reverse the charge of the lysinyl residues. Other suitable reagents for derivatizing alpha-amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate.

[0244] Arginyl residues may be modified by reaction with any one or combination of several conventional reagents, including phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginyl residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group.

[0245] Specific modification of tyrosyl residues has been studied extensively, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizole and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.

[0246] Carboxyl sidechain groups (aspartyl or glutamyl) may be selectively modified by reaction with carbodiimides (R′—N═C═N—R′) such as 1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore, aspartyl and glutamyl residues may be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

[0247] Glutaminyl and asparaginyl residues may be deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of this invention.

[0248] Cysteinyl residues can be replaced by amino acid residues or other moieties either to eliminate disulfide bonding or, conversely, to stabilize cross-linking. See, e.g., Bhatnagar et al. (1996), J. Med. Chem. 39: 3814-9.

[0249] Derivatization with bifunctional agents is useful for cross-linking the peptides or their functional derivatives to a water-insoluble support matrix or to other macromolecular vehicles. Commonly used cross-linking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate), and bifunctional maleimides such as bis-N-maleimido-1,8-octane. Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light. Alternatively, reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are employed for protein immobilization.

[0250] Carbohydrate (oligosaccharide) groups may conveniently be attached to sites that are known to be glycosylation sites in proteins. Generally, O-linked oligosaccharides are attached to serine (Ser) or threonine (Thr) residues while N-linked oligosaccharides are attached to asparagine (Asn) residues when they are part of the sequence Asn-X-Ser/Thr, where X can be any amino acid except proline. X is preferably one of the 19 naturally occurring amino acids other than proline. The structures of N-linked and O-linked oligosaccharides and the sugar residues found in each type are different. One type of sugar that is commonly found on both is N-acetylneuraminic acid (referred to as sialic acid). Sialic acid is usually the terminal residue of both N-linked and O-linked oligosaccharides and, by virtue of its negative charge, may confer acidic properties to the glycosylated compound. Such site(s) may be incorporated in the linker of the compounds of this invention and are preferably glycosylated by a cell during recombinant production of the polypeptide compounds (e.g., in mammalian cells such as CHO, BHK, COS). However, such sites may further be glycosylated by synthetic or semi-synthetic procedures known in the art.

[0251] Other possible modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, oxidation of the sulfur atom in Cys, methylation of the alpha-amino groups of lysine, arginine, and histidine side chains. Creighton, Proteins: Structure and Molecule Properties (W. H. Freeman & Co., San Francisco), pp. 79-86 (1983).

[0252] Such derivatized moieties preferably improve one or more characteristics including anti-angiogenic activity, solubility, absorption, biological half life, and the like of the compounds. Alternatively, derivatized moieties may result in compounds that have the same, or essentially the same, characteristics and/or properties of the compound that is not derivatized. The moieties may alternatively eliminate or attenuate any undesirable side effect of the compounds and the like.

[0253] Compounds of the present invention may be changed at the DNA level, as well. The DNA sequence of any portion of the compound may be changed to codons more compatible with the chosen host cell. For E. coli, which is the preferred host cell, optimized codons are known in the art. Codons may be substituted to eliminate restriction sites or to include silent restriction sites, which may aid in processing of the DNA in the selected host cell. The vehicle, linker and peptide DNA sequences may be modified to include any of the foregoing sequence changes.

[0254] Isotope- and toxin-conjugated derivatives. Another set of useful derivatives are the above-described molecules conjugated to toxins, tracers, or radioisotopes. Such conjugation is especially useful for molecules comprising peptide sequences that bind to tumor cells or pathogens. Such molecules may be used as therapeutic agents or as an aid to surgery (e.g., radioimmunoguided surgery or RIGS) or as diagnostic agents (e.g., radioimmunodiagnostics or RID).

[0255] As therapeutic agents, these conjugated derivatives possess a number of advantages. They facilitate use of toxins and radioisotopes that would be toxic if administered without the specific binding provided by the peptide sequence. They also can reduce the side-effects that attend the use of radiation and chemotherapy by facilitating lower effective doses of the conjugation partner.

[0256] Useful conjugation partners include:

• • [0257] radioisotopes, such as ⁹⁰Yttrium, ¹³¹Iodine, ²²⁵Actinium, and ²¹³Bismuth;
  • [0258] ricin A toxin, microbially derived toxins such as Pseudomonas endotoxin (e.g., PE38, PE40), and the like;
  • [0259] partner molecules in capture systems (see below);
  • [0260] biotin, streptavidin (useful as either partner molecules in capture systems or as tracers, especially for diagnostic use); and
  • [0261] cytotoxic agents (e.g., doxorubicin).

[0262] One useful adaptation of these conjugated derivatives is use in a capture system. In such a system, the molecule of the present invention would comprise a benign capture molecule. This capture molecule would be able to specifically bind to a separate effector molecule comprising, for example, a toxin or radioisotope. Both the vehicle-conjugated molecule and the effector molecule would be administered to the patient. In such a system, the effector molecule would have a short half-life except when bound to the vehicle-conjugated capture molecule, thus minimizing any toxic side-effects. The vehicle-conjugated molecule would have a relatively long half-life but would be benign and non-toxic. The specific binding portions of both molecules can be part of a known specific binding pair (e.g., biotin, streptavidin) or can result from peptide generation methods such as those described herein.

[0263] Such conjugated derivatives may be prepared by methods known in the art. In the case of protein effector molecules (e.g., Pseudomonas endotoxin), such molecules can be expressed as fusion proteins from correlative DNA constructs. Radioisotope conjugated derivatives may be prepared, for example, as described for the BEXA antibody (Coulter). Derivatives comprising cytotoxic agents or microbial toxins may be prepared, for example, as described for the BR96 antibody (Bristol-Myers Squibb). Molecules employed in capture systems may be prepared, for example, as described by the patents, patent applications, and publications from NeoRx. Molecules employed for RIGS and RID may be prepared, for example, by the patents, patent applications, and publications from NeoProbe.

[0264] A process for preparing conjugation derivatives is also contemplated. Tumor cells, for example, exhibit epitopes not found on their normal counterparts. Such epitopes include, for example, different post-translational modifications resulting from their rapid proliferation.

[0265] Thus, one aspect of this invention is a process comprising:

• • [0266] a) selecting at least one randomized peptide that specifically binds to a target epitope; and
  • [0267] b) preparing a pharmacologic agent comprising (i) at least one vehicle (Fc domain preferred), (ii) at least one amino acid sequence of the selected peptide or peptides, and (iii) an effector molecule.
    The target epitope is preferably a tumor-specific epitope or an epitope specific to a pathogenic organism. The effector molecule may be any of the above-noted conjugation partners and is preferably a radioisotope.

[0268] Variants

[0269] Variants are also included within the scope of the present invention. Included within variants are insertional, deletional, and substitutional variants. It is understood that a particular molecule of the present invention may contain one, two or all three types of variants. Insertional and substitutional variants may contain natural amino acids, unconventional amino acids (as set forth below), or both.

[0270] In one example, insertional variants are provided wherein one or more amino acid residues, either naturally occurring or unconventional amino acids, supplement a peptide or a peptibody amino acid sequence. Insertions may be located at either or both termini of the protein, or may be positioned within internal regions of the peptibody amino acid sequence. Insertional variants with additional residues at either or both termini can include, for example, fusion proteins and proteins including amino acid tags or labels. Insertional variants include peptides and peptibodies wherein one or more amino acid residues are added to the peptide or peptibody amino acid sequence, or fragment thereof.

[0271] Variants of the invention also include mature peptides and peptibodies wherein leader or signal sequences are removed, and the resulting proteins having additional amino terminal residues, which amino acids may be natural or non-natural. Molecules of this invention (such as peptibodies) with an additional methionyl residue at amino acid position −1 (Met⁻¹-peptibody) are contemplated, as are specific binding agents with additional methionine and lysine residues at positions −2 and −1 (Met⁻²-Lys⁻¹-). Variants having additional Met, Met-Lys, Lys residues (or one or more basic residues, in general) are particularly useful for enhanced recombinant protein production in bacterial host cells.

[0272] The invention also embraces variants having additional amino acid residues that arise from use of specific expression systems. For example, use of commercially available vectors that express a desired polypeptide as part of glutathione-S-transferase (GST) fusion product provides the desired polypeptide having an additional glycine residue at amino acid position −1 after cleavage of the GST component from the desired polypeptide. Variants which result from expression in other vector systems are also contemplated, including those wherein poly-histidine tags are incorporated into the amino acid sequence, generally at the carboxy and/or amino terminus of the sequence.

[0273] Insertional variants also include fusion proteins wherein the amino and/or carboxy termini of the peptide or peptibody is fused to another polypeptide, a fragment thereof or amino acids which are not generally recognized to be part of any specific protein sequence. Examples of such fusion proteins are immunogenic polypeptides, proteins with long circulating half lives, such as immunoglobulin constant regions, marker proteins, proteins or polypeptides that facilitate purification of the desired peptide or peptibody, and polypeptide sequences that promote formation of multimeric proteins (such as leucine zipper motifs that are useful in dimer formation/stability).

[0274] This type of insertional variant generally has all or a substantial portion of the native molecule, linked at the N— or C-terminus, to all or a portion of a second polypeptide. For example, fusion proteins typically employ leader sequences from other species to permit the recombinant expression of a protein in a heterologous host. Another useful fusion protein includes the addition of an immunologically active domain, such as an antibody epitope, to facilitate purification of the fusion protein. Inclusion of a cleavage site at or near the fusion junction will facilitate removal of the extraneous polypeptide after purification. Other useful fusions include linking of functional domains, such as active sites from enzymes, glycosylation domains, cellular targeting signals or transmembrane regions.

[0275] There are various commercially available fusion protein expression systems that may be used in the present invention. Particularly useful systems include but are not limited to the glutathione-S-transferase (GST) system (Pharmacia), the maltose binding protein system (NEB, Beverley, Mass.), the FLAG system (IBI, New Haven, Conn.), and the 6xHis system (Qiagen, Chatsworth, Calif.). These systems are capable of producing recombinant peptides and/or peptibodies bearing only a small number of additional amino acids, which are unlikely to significantly affect the activity of the peptide or peptibody. For example, both the FLAG system and the 6xHis system add only short sequences, both of which are known to be poorly antigenic and which do not adversely affect folding of a polypeptide to its native conformation. Another N-terminal fusion that is contemplated to be useful is the fusion of a Met-Lys dipeptide at the N-terminal region of the protein or peptides. Such a fusion may produce beneficial increases in protein expression or activity.

[0276] Other fusion systems produce polypeptide hybrids where it is desirable to excise the fusion partner from the desired peptide or peptibody. In one embodiment, the fusion partner is linked to the recombinant peptibody by a peptide sequence containing a specific recognition sequence for a protease. Examples of suitable sequences are those recognized by the Tobacco Etch Virus protease (Life Technologies, Gaithersburg, Md.) or Factor Xa (New England Biolabs, Beverley, Mass.).

[0277] The invention also provides fusion polypeptides which comprises all or part of a peptibody or peptide of the present invention, in combination with truncated tissue factor (tTF). tTF is a vascular targeting agent consisting of a truncated form of a human coagulation-inducing protein that acts as a tumor blood vessel clotting agent, as described U.S. Pat. Nos. 5,877,289; 6,004,555; 6,132,729; 6,132,730; 6,156,321; and European Patent No. EP 0988056. The fusion of tTF to the anti-Ang-2 peptibody or peptide, or fragments thereof facilitates the delivery of anti-Ang-2 to target cells.

[0278] In another aspect, the invention provides deletion variants wherein one or more amino acid residues in a peptide or peptibody are removed. Deletions can be effected at one or both termini of the peptibody, or from removal of one or more residues within the peptibody amino acid sequence. Deletion variants necessarily include all fragments of a peptide or peptibody.

[0279] In still another aspect, the invention provides substitution variants of peptides and peptibodies of the invention. Substitution variants include those peptides and peptibodies wherein one or more amino acid residues are removed and replaced with one or more alternative amino acids, which amino acids may be naturally occurring or non-naturally occurring. Substitutional variants generate peptides or peptibodies that are “similar” to the original peptide or peptibody, in that the two molecules have a certain percentage of amino acids that are identical. Substitution variants include substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, amino acids within a peptide or peptibody, wherein the number of substitutions may be up to ten percent or more, of the amino acids of the peptide or peptibody. In one aspect, the substitutions are conservative in nature, however, the invention embraces substitutions that are also non-conservative and also includes unconventional amino acids.

[0280] Identity and similarity of related peptides and peptibodies can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York (1991); and Carillo et al., SIAM J. Applied Math., 48:1073 (1988).

[0281] Preferred methods to determine the relatedness or percent identity of two peptides or polypeptides, or a polypeptide and a peptide, are designed to give the largest match between the sequences tested. Methods to determine identity are described in publicly available computer programs. Preferred computer program methods to determine identity between two sequences include, but are not limited to, the GCG program package, including GAP (Devereux et al., Nucl. Acid. Res., 12:387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis., BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol., 215:403-410 (1990)). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., supra (1990)). The well-known Smith Waterman algorithm may also be used to determine identity.

[0282] Certain alignment schemes for aligning two amino acid sequences may result in the matching of only a short region of the two sequences, and this small aligned region may have very high sequence identity even though there is no significant relationship between the two full-length sequences. Accordingly, in certain embodiments, the selected alignment method (GAP program) will result in an alignment that spans at least ten percent of the full length of the target polypeptide being compared, i.e. at least 40 contiguous amino acids where sequences of at least 400 amino acids are being compared, 30 contiguous amino acids where sequences of at least 300 to about 400 amino acids are being compared, at least 20 contiguous amino acids where sequences of 200 to about 300 amino acids are being compared, and at least 10 contiguous amino acids where sequences of about 100 to 200 amino acids are being compared.

[0283] For example, using the computer algorithm GAP (Genetics Computer Group, University of Wisconsin, Madison, Wis.), two polypeptides for which the percent sequence identity is to be determined are aligned for optimal matching of their respective amino acids (the “matched span”, as determined by the algorithm). In certain embodiments, a gap opening penalty (which is typically calculated as 3× the average diagonal; the “average diagonal” is the average of the diagonal of the comparison matrix being used; the “diagonal” is the score or number assigned to each perfect amino acid match by the particular comparison matrix) and a gap extension penalty (which is usually 1/10 times the gap opening penalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62 are used in conjunction with the algorithm. In certain embodiments, a standard comparison matrix (see Dayhoff et al., Atlas of Protein Sequence and Structure, 5(3)(1978) for the PAM 250 comparison matrix; Henikoff et al., Proc. Natl. Acad. Sci USA, 89:10915-10919 (1992) for the BLOSUM 62 comparison matrix) is also used by the algorithm.

[0284] In certain embodiments, the parameters for a polypeptide sequence comparison include the following:

[0285] Algorithm: Needleman et al., J. Mol. Biol., 48:443-453 (1970);

[0286] Comparison matrix: BLOSUM 62 from Henikoff et al., supra (1992);

[0287] Gap Penalty: 12

[0288] Gap Length Penalty: 4

[0289] Threshold of Similarity: 0

[0290] The GAP program may be useful with the above parameters. In certain embodiments, the aforementioned parameters are the default parameters for polypeptide comparisons (along with no penalty for end gaps) using the GAP algorithm.

[0291] In certain embodiments, the parameters for polynucleotide molecule sequence (as opposed to an amino acid sequence) comparisons include the following:

[0292] Algorithm: Needleman et al., supra (1970);

[0293] Comparison matrix: matches=+10, mismatch=0

[0294] Gap Penalty: 50

[0295] Gap Length Penalty: 3

[0296] The GAP program may also be useful with the above parameters. The aforementioned parameters are the default parameters for polynucleotide molecule comparisons.

[0297] Other exemplary algorithms, gap opening penalties, gap extension penalties, comparison matrices, thresholds of similarity, etc. may be used, including those set forth in the Program Manual, Wisconsin Package, Version 9, September, 1997. The particular choices to be made will be apparent to those of skill in the art and will depend on the specific comparison to be made, such as DNA-to-DNA, protein-to-protein, protein-to-DNA; and additionally, whether the comparison is between given pairs of sequences (in which case GAP or BestFit are generally preferred) or between one sequence and a large database of sequences (in which case FASTA or BLASTA are preferred).

[0298] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology—A Synthesis (2nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference for any purpose.

[0299] The amino acids may have either L or D stereochemistry (except for Gly, which is neither L nor D) and the polypeptides and compositions of the present invention may comprise a combination of stereochemistries. However, the L stereochemistry is preferred. The invention also provides reverse molecules wherein the amino terminal to carboxy terminal sequence of the amino acids is reversed. For example, the reverse of a molecule having the normal sequence X₁—X₂—X₃ would be X₃—X₂—X₁. The invention also provides retro-reverse molecules wherein, as above, the amino terminal to carboxy terminal sequence of amino acids is reversed and residues that are normally “L” enantiomers are altered to the “D” stereoisomer form.

[0300] Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as cc-, a-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for polypeptides of the present invention. Examples of unconventional amino acids include, without limitation: aminoadipic acid, beta-alanine, beta-aminopropionic acid, aminobutyric acid, piperidinic acid, aminocaprioic acid, aminoheptanoic acid, aminoisobutyric acid, aminopimelic acid, diaminobutyric acid, desmosine, diaminopimelic acid, diaminopropionic acid, N-ethylglycine, N-ethylaspargine, hyroxylysine, allo-hydroxylysine, hydroxyproline, isodesmosine, allo-isoleucine, N-methylglycine, sarcosine, N-methylisoleucine, N-methylvaline, norvaline, norleucine, orithine, 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and amino acids (e.g., 4-hydroxyproline).

[0301] Similarly, unless specified otherwise, the left-hand end of single-stranded polynucleotide sequences is the 5′ end; the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5′ direction. The direction of 5′ to 3′ addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA and which are 5′ to the 5′ end of the RNA transcript are referred to as “upstream sequences”; sequence regions on the DNA strand having the same sequence as the RNA and which are 3′ to the 3′ end of the RNA transcript are referred to as “downstream sequences”.

[0302] It will be appreciated that amino acid residues can be divided into classes based on their common side chain properties:

• • [0303] 1. Neutral Hydrophobic: Alanine (Ala; A), Valine (Val; V), Leucine (Leu; L), Isoleucine (Ile; I), Proline (Pro; P), Tryptophan (Trp; W), Phenylalanine (Phe; F), and Methionine (Met, M).
  • [0304] 2. Neutral Polar: Glycine (Gly; G); Serine (Ser; S), Threonine (Thr; T), Tyrosine (Tyr; Y), Cysteine (Cys; C), Glutamine (Glu; Q), Asparagine (Asn; N), and Norleucine.
  • [0305] 3. Acidic: Aspartic Acid (Asp; D), Glutamic Acid (Glu; E);
  • [0306] 4) Basic: Lysine (Lys; K), Arginine (Arg; R), Histidine (His; H). See Lewin, B., Genes V, Oxford University Press (1994), p. 11.

[0307] Conservative amino acid substitutions may encompass unconventional amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include, without limitation, peptidomimetics and other reversed or inverted forms of amino acid moieties. Non-conservative substitutions may involve the exchange of a member of one of these classes for a member from another class.

[0308] In making such changes, according to certain embodiments, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

[0309] The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is understood in the art. Kyte et al. J. Mol. Biol., 157:105-131 (1982). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, in certain embodiments, the substitution of amino acids whose hydropathic indices are within±2 is included. In certain embodiments, those which are within±1 are included, and in certain embodiments, those within±0.5 are included.

[0310] It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity, particularly where the biologically functional peptibody or peptide thereby created is intended for use in immunological embodiments, as in the present case. In certain embodiments, the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with a biological property of the protein.

[0311] The following hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5) and tryptophan (−3.4). In making changes based upon similar hydrophilicity values, in certain embodiments, the substitution of amino acids whose hydrophilicity values are within±2 is included, in certain embodiments, those which are within±1 are included, and in certain embodiments, those within±0.5 are included. One may also identify epitopes from primary amino acid sequences on the basis of hydrophilicity. These regions are also referred to as “epitopic core regions.”

[0312] Exemplary amino acid substitutions are set forth in Table 10 below.

TABLE 10
Amino Acid Substitutions
Original		Preferred
Residues	Exemplary Substitutions	Substitutions
Ala	Val, Leu, Ile	Val
Arg	Lys, Gln, Asn	Lys
Asn	Gln, Glu, Asp	Gln
Asp	Glu, Gln, Asp	Glu
Cys	Ser, Ala	Ser
Gln	Asn, Glu, Asp	Asn
Glu	Asp, Gln, Asn	Asp
Gly	Pro, Ala	Ala
His	Asn, Gln, Lys, Arg	Arg
Ile	Leu, Val, Met, Ala, Phe,	Leu
	Norleucine
Leu	Norleucine, Ile, Val, Met,	Ile
	Ala, Phe
Lys	Arg, 1,4 Diamino-butyric	Arg
	Acid, Gln, Asn
Met	Leu, Phe, Ile	Leu
Phe	Leu, Val, Ile, Ala, Tyr	Leu
Pro	Ala	Gly
Ser	Thr, Ala, Cys	Thr
Thr	Ser	Ser
Trp	Tyr, Phe	Tyr
Tyr	Trp, Phe, Thr, Ser	Phe
Val	Ile, Met, Leu, Phe, Ala,	Leu
	Norleucine

[0313] A skilled artisan will be able to determine suitable variants of the polypeptide as set forth herein using well-known techniques. In certain embodiments, one skilled in the art may identify suitable areas of the molecule that may be changed without destroying activity by targeting regions not believed to be important for activity. In certain embodiments, one can identify residues and portions of the molecules that are conserved among similar peptides or polypeptides. In certain embodiments, even areas that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure.

[0314] Additionally, one skilled in the art can review structure-function studies identifying residues in similar polypeptides that are important for activity or structure. In view of such a comparison, one can predict the importance of amino acid residues in a protein that correspond to amino acid residues which are important for activity or structure in similar proteins. One skilled in the art may opt for chemically similar amino acid substitutions for such predicted important amino acid residues.

[0315] One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar polypeptides. In view of such information, one skilled in the art may predict the alignment of amino acid residues of an antibody with respect to its three dimensional structure. In certain embodiments, one skilled in the art may choose not to make radical changes to amino acid residues predicted to be on the surface of the protein, since such residues may be involved in important interactions with other molecules. Moreover, one skilled in the art may generate test variants containing a single amino acid substitution at each desired amino acid residue. The variants can then be screened using activity assays known to those skilled in the art. Such variants could be used to gather information about suitable variants. For example, if one discovered that a change to a particular amino acid residue resulted in destroyed, undesirably reduced, or unsuitable activity, variants with such a change may be avoided. In other words, based on information gathered from such routine experiments, one skilled in the art can readily determine the amino acids where further substitutions should be avoided either alone or in combination with other mutations.

[0316] A number of scientific publications have been devoted to the prediction of secondary structure. See Moult J., Curr. Op. in Biotech., 7(4):422-427 (1996), Chou et al., Biochemistry, 13(2):222-245 (1974); Chou et al., Biochemistry, 113(2):211-222 (1974); Chou et al., Adv. Enzymol. Relat. Areas Mol. Biol., 47:45-148 (1978); Chou et al., Ann. Rev. Biochem., 47:251-276 and Chou et al., Biophys. T., 26:367-384 (1979). Moreover, computer programs are currently available to assist with predicting secondary structure. One method of predicting secondary structure is based upon homology modeling. For example, two polypeptides or proteins which have a sequence identity of greater than 30%, or similarity greater than 40% often have similar structural topologies. The recent growth of the protein structural database (PDB) has provided enhanced predictability of secondary structure, including the potential number of folds within a polypeptide's or protein's structure. See Holm et al., Nucl. Acid. Res., 27(1):244-247 (1999). It has been suggested (Brenner et al., Curr. Op. Struct. Biol., 7(3):369-376 (1997)) that there are a limited number of folds in a given polypeptide or protein and that once a critical number of structures have been resolved, structural prediction will become dramatically more accurate.

[0317] Additional methods of predicting secondary structure include “threading” (Jones, D., Curr. Opin. Struct. Biol., 7(3):377-87 (1997); Sippl et al., Structure, 4(1):15-19 (1996)), “profile analysis” (Bowie et al., Science, 253:164-170 (1991); Gribskov et al., Meth. Enzym., 183:146-159 (1990); Gribskov et al., Proc. Nat. Acad. Sci., 84(13):4355-4358 (1987)), and “evolutionary linkage” (See Holm, supra (1999), and Brenner, supra (1997)).

[0318] In certain embodiments, peptibody variants include glycosylation variants wherein one or more glycosylation sites, such as a N-linked glycosylation site, has been added to the peptibody. An N-linked glycosylation site is characterized by the sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated as X may be any amino acid residue except proline. The substitution or addition of amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain. Alternatively, substitutions which eliminate this sequence will remove an existing N-linked carbohydrate chain. Also provided is a rearrangement of N-linked carbohydrate chains wherein one or more N-linked glycosylation sites (typically those that are naturally occurring) are eliminated and one or more new N-linked sites are created.

[0319] Compounds of the present invention may be changed at the DNA level, as well. The DNA sequence of any portion of the compound may be changed to codons more compatible with the chosen host cell. For E. coli, which is the preferred host cell, optimized codons are known in the art. Codons may be substituted to eliminate restriction sites or to include silent restriction sites, which may aid in processing of the DNA in the selected host cell. The vehicle, linker and peptide DNA sequences may be modified to include any of the foregoing sequence changes. Thus, all modifications, substitution, derivitizations, etc. discussed herein apply equally to all aspects of the present invention, including but not limited to peptides, peptide dimers and multimers, linkers, and vehicles.

[0320] Affinity Maturation

[0321] One embodiment of the present invention includes “affinity matured” peptides and peptibodies. This procedure contemplates increasing the affinity or the bio-activity of the peptides and peptibodies of the present invention using phage display or other selection technologies. Based on a consensus sequence (which is generated for a collection of related peptides), directed secondary phage display libraries can be generated in which the “core” amino acids (determined from the consensus sequence) are held constant or are biased in frequency of occurrence. Alternatively, an individual peptide sequence can be used to generate a biased, directed phage display library. Panning of such libraries can yield peptides (which can be converted to peptibodies) with enhanced binding to the target or with enhanced bio-activity.

[0322] Non-Peptide Analogs/Protein Mimetics

[0323] Furthermore, non-peptide analogs of peptides that provide a stabilized structure or lessened biodegradation, are also contemplated. Peptide mimetic analogs can be prepared based on a selected inhibitory peptide by replacement of one or more residues by nonpeptide moieties. Preferably, the nonpeptide moieties permit the peptide to retain its natural confirmation, or stabilize a preferred, e.g., bioactive, confirmation which retains the ability to recognize and bind Ang-2. In one aspect, the resulting analog/mimetic exhibits increased binding affinity for Ang-2. One example of methods for preparation of nonpeptide mimetic analogs from peptides is described in Nachman et al., Regul. Pept. 57:359-370 (1995). If desired, the peptides of the invention can be modified, for instance, by glycosylation, amidation, carboxylation, or phosphorylation, or by the creation of acid addition salts, amides, esters, in particular C-terminal esters, and N-acyl derivatives of the peptides of the invention. The peptibodies also can be modified to create peptide derivatives by forming covalent or noncovalent complexes with other moieties. Covalently-bound complexes can be prepared by linking the chemical moieties to functional groups on the side chains of amino acids comprising the peptibodies, or at the N— or C-terminus.

[0324] In particular, it is anticipated that the peptides can be conjugated to a reporter group, including, but not limited to a radiolabel, a fluorescent label, an enzyme (e.g., that catalyzes a colorimetric or fluorometric reaction), a substrate, a solid matrix, or a carrier (e.g., biotin or avidin). The invention accordingly provides a molecule comprising a peptibody molecule, wherein the molecule preferably further comprises a reporter group selected from the group consisting of a radiolabel, a fluorescent label, an enzyme, a substrate, a solid matrix, and a carrier. Such labels are well known to those of skill in the art, e.g., biotin labels are particularly contemplated. The use of such labels is well known to those of skill in the art and is described in, e.g. U.S. Pat. Nos.3,817,837; 3,850,752; 3,996,345; and 4,277,437. Other labels that will be useful include but are not limited to radioactive labels, fluorescent labels and chemiluminescent labels. U.S. Patents concerning use of such labels include, for example, U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; and 3,996,345. Any of the peptibodies of the present invention may comprise one, two, or more of any of these labels.

[0325] Methods of Making

[0326] The compounds of this invention largely may be made in transformed host cells using recombinant DNA techniques. To do so, a recombinant DNA molecule coding for the peptide is prepared. Methods of preparing such DNA molecules are well known in the art. For instance, sequences coding for the peptides could be excised from DNA using suitable restriction enzymes. Alternatively, the DNA molecule could be synthesized using chemical synthesis techniques, such as the phosphoramidate method. Also, a combination of these techniques could be used.

[0327] The invention also includes a vector capable of expressing the peptides in an appropriate host. The vector comprises the DNA molecule that codes for the peptides operatively linked to appropriate expression control sequences. Methods of effecting this operative linking, either before or after the DNA molecule is inserted into the vector, are well known. Expression control sequences include promoters, activators, enhancers, operators, ribosomal binding sites, start signals, stop signals, cap signals, polyadenylation signals, and other signals involved with the control of transcription or translation.

[0328] The resulting vector having the DNA molecule thereon is used to transform an appropriate host. This transformation may be performed using methods well known in the art.

[0329] Any of a large number of available and well-known host cells may be used in the practice of this invention. The selection of a particular host is dependent upon a number of factors recognized by the art. These include, for example, compatibility with the chosen expression vector, toxicity of the peptides encoded by the DNA molecule, rate of transformation, ease of recovery of the peptides, expression characteristics, bio-safety and costs. A balance of these factors must be struck with the understanding that not all hosts may be equally effective for the expression of a particular DNA sequence. Within these general guidelines, useful microbial hosts include bacteria (such as E. coli sp.), yeast (such as Saccharomyces sp.) and other fungi, insects, plants, mammalian (including human) cells in culture, or other hosts known in the art.

[0330] Next, the transformed host is cultured and purified. Host cells may be cultured under conventional fermentation conditions so that the desired compounds are expressed. Such fermentation conditions are well known in the art. Finally, the peptides are purified from culture by methods well known in the art.

[0331] The compounds may also be made by synthetic methods. For example, solid phase synthesis techniques may be used. Suitable techniques are well known in the art, and include those described in Merrifield (1973), Chem. Polypeptides, pp. 335-61 (Katsoyannis and Panayotis eds.); Merrifield (1963), J. Am. Chem. Soc. 85: 2149; Davis et al. (1985), Biochem. Intl. 10: 394-414; Stewart and Young (1969), Solid Phase Peptide Synthesis; U.S. Pat. No. 3,941,763; Finn et al. (1976), The Proteins (3rd ed.) 2: 105-253; and Erickson et al. (1976), The Proteins (3rd ed.) 2: 257-527. Solid phase synthesis is the preferred technique of making individual peptides since it is the most cost-effective method of making small peptides.

[0332] Compounds that contain derivatized peptides or which contain non-peptide groups may be synthesized by well-known organic chemistry techniques.

[0333] Uses of the Compounds

[0334] In general. The compounds of this invention have pharmacologic activity resulting from their ability to bind to proteins of interest as agonists, mimetics or antagonists of the native ligands of such proteins of interest. The utility of specific compounds is shown in Table 2. The activity of these compounds can be measured by assays known in the art. For the TPO-mimetic and EPO-mimetic compounds, in vivo assays are further described in the Examples section herein.

[0335] In addition to therapeutic uses, the compounds of the present invention are useful in diagnosing diseases characterized by dysfunction of their associated protein of interest. In one embodiment, a method of detecting in a biological sample a protein of interest (e.g., a receptor) that is capable of being activated comprising the steps of: (a) contacting the sample with a compound of this invention; and (b) detecting activation of the protein of interest by the compound. The biological samples include tissue specimens, intact cells, or extracts thereof. The compounds of this invention may be used as part of a diagnostic kit to detect the presence of their associated proteins of interest in a biological sample. Such kits employ the compounds of the invention having an attached label to allow for detection. The compounds are useful for identifying normal or abnormal proteins of interest. For the EPO-mimetic compounds, for example, presence of abnormal protein of interest in a biological sample may be indicative of such disorders as Diamond Blackfan anemia, where it is believed that the EPO receptor is dysfunctional.

[0336] Therapeutic uses of EPO-mimetic molecules The EPO-mimetic compounds of the invention are useful for treating disorders characterized by low red blood cell levels. Included in the invention are methods of modulating the endogenous activity of an EPO receptor in a mammal, preferably methods of increasing the activity of an EPO receptor. In general, any condition treatable by erythropoietin, such as anemia, may also be treated by the EPO-mimetic compounds of the invention. These compounds are administered by an amount and route of delivery that is appropriate for the nature and severity of the condition being treated and may be ascertained by one skilled in the art. Preferably, administration is by injection, either subcutaneous, intramuscular, or intravenous.

[0337] Therapeutic uses of TPO-Mimetic Compounds

[0338] For the TPO-mimetic compounds, one can utilize such standard assays as those described in WO95/26746 entitled “Compositions and Methods for Stimulating Megakaryocyte Growth and Differentiation”. In vivo assays also appear in the Examples hereinafter.

[0339] The conditions to be treated are generally those that involve an existing megakaryocyte/platelet deficiency or an expected megakaryocyte/platelet deficiency (e.g., because of planned surgery or platelet donation). Such conditions will usually be the result of a deficiency (temporary or permanent) of active Mpl ligand in vivo. The generic term for platelet deficiency is thrombocytopenia, and hence the methods and compositions of the present invention are generally available for treating thrombocytopenia in patients in need thereof.

[0340] Thrombocytopenia (platelet deficiencies) may be present for various reasons, including chemotherapy and other therapy with a variety of drugs, radiation therapy, surgery, accidental blood loss, and other specific disease conditions. Exemplary specific disease conditions that involve thrombocytopenia and may be treated in accordance with this invention are: aplastic anemia, idiopathic thrombocytopenia, metastatic tumors which result in thrombocytopenia, systemic lupus erythematosus, splenomegaly, Fanconi's syndrome, vitamin B12 deficiency, folic acid deficiency, May-Hegglin anomaly, Wiskott-Aldrich syndrome, and paroxysmal nocturnal hemoglobinuria. Also, certain treatments for AIDS result in thrombocytopenia (e.g., AZT). Certain wound healing disorders might also benefit from an increase in platelet numbers.

[0341] With regard to anticipated platelet deficiencies, e.g., due to future surgery, a compound of the present invention could be administered several days to several hours prior to the need for platelets. With regard to acute situations, e.g., accidental and massive blood loss, a compound of this invention could be administered along with blood or purified platelets.

[0342] The TPO-mimetic compounds of this invention may also be useful in stimulating certain cell types other than megakaryocytes if such cells are found to express Mpl receptor. Conditions associated with such cells that express the Mpl receptor, which are responsive to stimulation by the Mpl ligand, are also within the scope of this invention.

[0343] The TPO-mimetic compounds of this invention may be used in any situation in which production of platelets or platelet precursor cells is desired, or in which stimulation of the c-Mpl receptor is desired. Thus, for example, the compounds of this invention may be used to treat any condition in a mammal wherein there is a need of platelets, megakaryocytes, and the like. Such conditions are described in detail in the following exemplary sources:

[0344] WO95/26746; WO95/21919; WO95/18858; WO95/21920 and are incorporated herein.

[0345] The TPO-mimetic compounds of this invention may also be useful in maintaining the viability or storage life of platelets and/or megakaryocytes and related cells. Accordingly, it could be useful to include an effective amount of one or more such compounds in a composition containing such cells.

[0346] Therapeutic uses of Ang-2 Binding Molecules