Title of Invention	"MODIFIED FC MOLECULES"
Abstract	The present invention concerns molecules and a process in which one or more biologically active peptides are incorporated into an Fc domain. In this invention, pharmacologically active compounds may be prepared by a process comprising (a) selecting at least one peptide that modulates the activity of a protein of interest; and (b) preparing a pharmacologic agent comprising an amino acid sequence of the selected peptide in a loop region 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 peptide or to a polypeptide (e.g., etanercept). This process 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, E. coli display, ribosome display, RNA-peptide screening, yeast-based screening, chemical-peptide screening, rational design, or protein structural analysis.

Title of Invention

"MODIFIED FC MOLECULES"

Abstract

The present invention concerns molecules and a process in which one or more biologically active peptides are incorporated into an Fc domain. In this invention, pharmacologically active compounds may be prepared by a process comprising (a) selecting at least one peptide that modulates the activity of a protein of interest; and (b) preparing a pharmacologic agent comprising an amino acid sequence of the selected peptide in a loop region 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 peptide or to a polypeptide (e.g., etanercept). This process 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, E. coli display, ribosome display, RNA-peptide screening, yeast-based screening, chemical-peptide screening, rational design, or protein structural analysis.

Full Text	Modified Fc Molecules This application claims the benefit of U.S. Provisional Application No. 60/612,680, filed September 24, 2004, which is hereby incorporated by reference. Background of the Invention 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 halflife, 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 (Table Removed) Linslev(1991). J. EXD. Med. 174:561-9 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"). Phage display peptide libraries have emerged as a powerful method in identifying such peptide agonists and antagonists. See, for example, Scott etal. (1990), Science 249: 386; Devlin etal. (1990), Science 249: -2.- 404-; U.S. Pat. No. 5,223,409, issued June 29, 1993; U.S. Pat. No. 5,733,731, issued March 31, 1998; U.S. Pat. No. 5,498,530, issued March 12, 1996; U.S. Pat. No. 5,432,018, issued July 11, 1995; U.S. Pat. No. 5,338,665, issued August 16,1994; U.S. Pat. No. 5,922,545, issued July 13,1999; WO 96/40987, published December 19,1996; and WO 98/15833, published April 16,1998 (each of which is incorporated by reference). In such libraries, randompeptide 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- Other methods compete with phage display in peptide research. A peptide library can be fused to the carboxyl terminus of the lac represser 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 Damino 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. 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. 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. 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. 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 (Table Removed) linear GLP-1 Diabetes, metabolic syndrome 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 th-i^eticuloendothelial 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 etal. (1998), Drug Disc. Today 3: 370-8. A more recent development is fusion of randomly generated peptides with the Fc domain. See U.S. Pat. No. 6,660,843, issued December 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 Nterminus, 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, tumorhoming 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 Sept. 18, 2003 (corresponding to WO 03/031589, published April 17, 2003); U.S. Ser. No. 09/422,838, filed October 22, 1999 (corresponding to WO 00/24770, published May 4, 2000); U.S. Pat. App. No. 2003/0229023, published December 11, 2003; WO 03/057134, published July 17, 2003; U.S. Pat. App. No. 2003/0236193, published December 25, 2003 (corresponding to PCT/US04/010989, filed April 8, 2004); U.S. Ser. No. 10/666,480, filed September 18, 2003 (corresponding to WO 04/026329, published April 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. Summary of the Invention 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: a) selecting at least one peptide that modulates the activity of a protein of interest; and 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., adadimumab, 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). 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. The Fc internal peptide molecules may include more than one peptide sequence in tandem in 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. 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. 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. 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. 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. Numerous additional aspects and advantages of the present invention will become apparent upon consideration of the figures and detailed description of the invention. Description of the Figures Figures 1A, IB 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 CHS domains of Fc, the loop regions may be considered any part of the model not shown as p-sheet (flat arrows) or a-helix (cylinder). Figure 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 CHS domain Fc loop insertion site specifically identified. Figure IB shows the monomeric murine IgGl Fc domain (Protein Database file #1IGY). This figure shows a three-dimensional model of murine IgGl Fc domain monomer from x-ray diffraction crystal structure (pdb #1IGY). Potential Fc loop insertion sites are shown for both CH2 and CHS domains with the preferred CH3 domain Fc loop insertion site specifically identified. Figure 1C shows the monomeric human IgGl Fc domain (Protein Database file #1H3T). This figure shows a three-dimensional model of human IgGl Fc domain monomer from x-ray diffraction crystal structure (pdb #1H3T). Potential Fc loop insertion sites are shown for both CH2 and CHS domains with the preferred CH3 domain Fc loop insertion site specifically identified. 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/). Figure 2A shows a sequence of human IgGl Fc sequence (SEQ ID NO: 599) used for peptibody fusions with predicted loop sequences in boldface. Figure 2A shows, in the context of the human IgGl 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 Figures 1A, IB 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 Leui39 and Thr14o 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 Figures 2B and 2C. Figures 2B and 2C show a sequence alignment of human Fc domains from IgA, IgM and IgG subclasses. Figures 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 Figures 2B and 2C is a consensus sequence (SEQ ID NO: 608). Figures 2B and 2C also show in boldface the preferred internal sites for peptide addition that correspond to those of the Fc sequence shown in Figure 2A (SEQ ID NO: 599). In particular, Figures 2B and 2C show as such preferred sites the following: • SEQ ID NO: 621 as shown in boldface within SEQ ID NOS: 603 to 608; • SEQ ID NO: 622 within SEQ ID NOS: 603 to 606 and 608; • SEQ ID NO: 638 within SEQ ID NO: 607; . SEQ ID NO: 624 within SEQ ID NO: 603 to 608; • SEQ ID NO: 625 within SEQ ID NOS: 603 and 604; • SEQ ID NO: 639 within SEQ ID NOS: 605 to 608; • SEQ ID NO: 627 within SEQ ID NOS: 603 to 605,607, and 608; • SEQ ID NO: 640 within SEQ ID NO: 606; • SEQ ID NO: 628 within SEQ ID NOS: 603,604, and 608; • SEQ ID NO: 641 within SEQ ID NO: 605; • SEQ ID NO: 642 within SEQ ID NO: 606; • SEQ ID NO: 643 within SEQ ID NO: 607; t SEQ ID NO: 630 within SEQ ID NO: 603; • SEQ ID NO: 644 within SEQ ID NOS: 604 to 608; • SEQ ID NO: 632 within SEQ ID NOS: 603,604, 606, 607, and 608; • SEQ ID NO: 645 within SEQ ID NO: 605; • SEQ ID NO: 634 within SEQ ID NOS: 603, 604, and 607; • SEQ ID NO: 646 within SEQ ID NOS: 605, 606 and 608; • SEQ ID NO: 636 within SEQ ID NOS: 603,604, 606, and 608; • SEQ ID NO: 614 within SEQ ID NO: 605; and • SEQ ID NO: 620 within SEQ ID NO: 607. The sequence alignments of Figures 2B and 2C suggest two more potential insertion sites at Qi67/Pi68 and/or G183/S184 (using the numbering of SEQ ID NO: 599 in Figure 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 Figure 2A. The preferred insertion sites are underlined Figures 2B and 2C and are as follows: • H53/ E54 in SEQ ID NOS: 603 and 604; • H100/E101in SEQ ID NO: 605; • H49/E5o in SEQ ID NO: 606; • Q50/E51 in SEQ ID NO: 607; • H112/E113 in SEQ ID NO: 608; • Y81/N82 in SEQ ID NOS: 603 and 604; • F128/N129 in SEQ ID NO: 605; • F77/N78 in SEQ ID NO: 606; • F78/N79 in SEQ ID NO: 607; • F140/N141 in SEQ ID NO: 608; • N110/K111 in SEQ ID NOS: 603 and 604; • N157/K158 in SEQ ID NO: 605; • N106 /K107 in SEQ ID NO: 606; • N107K108 in SEQ ID NO: 607; • N169K170 in SEQ ID NO: 608; • L143/T144 in SEQ ID NOS: 603 and 604; • M190/T191 in SEQ ID NO: 605; • M139 /T140 in SEQ ID NO: 606; . M140WT141 in SEQ ID NO: 607; • M204/T2o5 in SEQ ID NO: 608; • Q171P172 in SEQ ID NOS: 603 and 604; • Q218/P219 in SEQ ID NO: 605; • Q167//P168 in SEQ ID NO: 606; • Q168/P169 in SEQ ID NO: 607; • Q232/P233 in SEQ ID NO: 608; • E173N174 in SEQ ID NOS: 603 and 604; • E220/N221 in SEQ ID NO: 605; • E169/N170 in SEQ ID NO: 606; • E170/N171 in SEQ ID NO: 607; • E234/N235 in SEQ ID NO: 608; • S186/D187 in SEQ ID NOS: 603 and 604; • S232/D233 in SEQ ID NO: 605; • S181/D182 in SEQ ID NO: 606; • S182/D183 in SEQ ID NO: 607; • S246/D247 in SEQ ID NO: 608; • G188/S189 in SEQ ID NOS: 603 and 604; • G234/S235 in SEQ ID NO: 605; • G183/S184 in SEQ ID NO: 606; • G184/S185 in SEQ ID NO: 607; • G248/S249 in SEQ ID NO: 608; • G205 / N206 in SEQ ID NOS: 603 and 604; • G2S2/N253 in SEQ ID NO: 605; • G201/N202 in SEQ ID NO: 606; • G202/N203 in SEQ ID NO: 607; and • G268/N269 in SEQ ID NO: 608. Figure 2D shows an alignment of human IgGl Fc domain (Amgen Fc, SEQ ID NO: 609) used for the peptibody platform with rat IgG2A from crystal structure of FcRn/Fc complex (UlA.pdb, SEQ ID NO: 610 The resulting consensus sequence (SEQ ID NO: 611) is also shown. Figure 3A shows the amino acid sequence (SEQ ID NO: 612) of a human IgGl 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 lirmers in italics. Figure 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. Figure 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. Figure 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. Figure 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. Figures 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). Figure 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 ng of peptibody was loaded directly from a refold reaction to a Vydak C4 column (5 uM, 300 angstrom, 4.6 x 250 mm) and eluted with a linear 40-50% ACN gradient at 0.5 %/min. Figure 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 Hg purified peptibody to Vydak C4 column (5 uM, 300 angstrom, 4.6 x 250 mm) and eluted with a linear 40- 50% ACN gradient at 0.5 %/min. Figure 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 ng 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). Figure 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). Figure 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 Fcloop vs. carboxy terminal peptibodies respectively at 24 hours. Lanes 8 & 9 are the Fc-looo vs. carboxv 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. Figure 10A shows the amino acid sequence (SEQ ID NO: 618) of a human IgGl 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. Figure 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. Figure 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. Figure 12 shows a RP-HPLC comparison of Fc-loop Ang2 (#6888) and carboxy-terminal Fc TN8-19-07 (#5564) refold reactions. Loaded 20 jal refold reaction to Vydak C4 column (5 ^M, 300 angstrom, 4.6 x 250 mm) and eluted with a linear 40-50% ACN gradient at 0.5 %/min. Figure 13 shows a RP-HPLC comparison of the final purified pools of Fc-loop Ang2 (#6888) and carboxy-terminal Fc TN8-Con4 (#5564). Ten ng purified peptibody was loaded to a Vydak C4 column (5 M, 300 angstrom, 4.6 x 250 mm) and eluted with a linear 40-50% ACN gradient at 0.5 %/min. Figure 14 shows purified Fc-loop-myo7 and TN8-19-7. Figure 15 shows Biacore binding analysis of Fc-loop-ang2 and Fc-ang2-tandem. Figure 16 shows the results of an in vitro enzyme-linked immunosorbent assay (ELISA) for Fc-loopang2, TN8-Con4, and Fc-ang2-tandem. Figure 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. Figure 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. Figure 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 ng Fc-loop AMP 2 peptibody; lane 4 with 5 ng reduced Fc-loop AMP 2 peptibody; lanes 1 and 3 with SeeBlue and two molecular weight markers. Figure 20 shows an RP-HPLC analysis of the final purified pool of Fc-loop AMP 2 (#6875). Ten ng purified peptibody was loaded to Vydak C4 column (5 \\M., 300 angstrom, 4.6 x 250 mm) and eluted with a linear 40-50% ACN gradient at 0.5 %/min. Figure 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 ng/kg peptibody or carrier alone. See example 9 for assay methodology. Figure 22 shows several strategies for incorporating 2 bioactive peptides into an Fc-loop peptibody. shows SDS-PAGE Gels of purified Fc-loop constructs. Samples (2ug/lane) were run +/- reducing buffer on a 4-20% Tris-Glycine SDS-PAGE gel. Figure 24 shows RP-HPLC of Fc-loop constructs. ' Detailed Description of the Invention Definition of Terms The terms used throughout this specification are defined as follows, unless otherwise limited in specific instances. 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. "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. The term "native Fc" refers to a molecule or sequence comprising the sequence of a non-antigenbinding 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 IgGl 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., IgGl, IgG2, IgG3, IgAl, 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. 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 September 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. 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. 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. 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, Figure 2, which is hereby incorporated by reference. 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 Cterminus 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. 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-lra, leptin, soluble TNF receptors type 1 and type 2 (sTNF-Rl, sTNF-R2), KGF, EPO, TPO, G-CSF, darbepoietin, Fab fragments and the like. 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. 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. 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. 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), Froc. 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. 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. 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 Sept. 18, 2003; WO 03/031589, published April 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. 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 7/17/03; 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. The term "NGF-binding peptide" comprises peptides that can be identified or derived as described in WO 04/026329, published April 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. The term "myostatin-binding peptide" comprises peptides that can be identified or derived as described in U.S. Ser. No. 10/742,379, filed December 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. 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. Structure of compounds In General 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: wherein: F1 is an Fc domain modified so that it comprises at least one X3 in a loop region; Xi and X2 are each independently selected from -(Li)c-P1, -(Li)c-P1-(L2)d -P2, -(L^c-PML^- X3 is independently selected from -(L5)C-P5, -(L5)c-P5-(L6)d -P, -(L5)c-P5-(L6)d-p6-(l7)e-P7, and -(L5)C-P5- (L6)d-P6-(I7)e -P7-(L«)f-P8; P1, P2/ P3, and P4 are each independently sequences of pharmacologically active polypeptides or pharmacologically active peptides; P5, P6, P7, and P8 are each independently sequences of pharmacologically active peptides; L1, L2, L3, L4, L5, L6, U, and L8 are each independently linkers; and a, b, c, d, e, and f are each independently 0 or 1. In preferred embodiments, a and b are both zero—i.e., neither X1 nor X2 groups appear at the Nterminus or C-terminus of the Fc domain. 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. Thus, compound I comprises compounds of the formulae and multimers thereof wherein F1 is attached at the C-terminus of X1; and multimers thereof wherein F1 is attached at the N-terminus of X2; and multimers thereof wherein F1 is attached at the N-terminus of -(UJc-P1; and and multimers thereof wherein F1 is attached at the N-terminus of -L1-P1-L2-P2. Feptides 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. 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. J. 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. 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 (Table Removed) Other 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. 3 TNF receptors use multiple, distinct intracellular molecules for signal transduction including "death domain" of FAS R and 55 kDa TNF-aR 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. 4 The Duffy blood group antigen (DARC) is an erythrocyte receptor that can bind several different chemokines! IL-1 R belongs to the immunoglobulin superfamily but their signal transduction events characteristics remain unclear. 5 The neurotrophic cytokines can associate with NGF/TNF receptors also. Particular proteins of interest as targets for peptide generation in the present invention include the following: (Table Removed) splice variants of molecules preferentially expressed on tumor cells; e.g., CD44, CD30 unglycosylated variants of mucin and Lewis Y surface glycoproteins : CD19, CD20, CD33, CD45 prostate specific membrane antigen and prostate specific cell antigen matrix metalloproteinases (MMPs), both secreted and membrane-bound (e.g., MMP-9) Cathepsins angiopoietin-2 TIE-2 receptor heparanase urokinase plasminogen activator (UPA), UFA receptor parathyroid hormone (PTH), parathyroid hormone-related protein (PTHrP), PTH-RI, PTH- Insulin 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 July 17, 2003; U.S. 2003/0236193, published Dec. 25, 2003; WO 00/24770, published May 4,2000; U.S. 2003/0176352, published Sept. 18,2003; WO 03/031589, published April 17, 2003; U.S. Ser. No. 10/666,480, filed September 18, 2003; WO 04/026329, published April 1, 2004; U.S. Ser. No. 10/742,379, filed December 19, 2003; PCT/US03/40781, filed December 19, 2003, each of which are hereby incorporated by reference. Such peptides may be prepared by methods disclosed in the art. 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 6 STKS may encompass many other TGF-p-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. 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 (Table Removed) This invention requires the presence of at least one Fc domain modified to comprise a peptide sequence. 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 combining 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: 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. 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 (Figure 2A) is one such Fc variant. 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. 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). 5. Sites involved in interaction with complement, such as the Clq binding site, are removed. For example, one may delete or substitute the EKK sequence of human IgGl. Complement recruitment may not be advantageous for the molecules of this invention and so may be avoided with such an Fc variant. 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. 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 IgGl. These sites, as well, are not required for the fusion molecules of the present invention and so may be removed. 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. Preferred Fc variants include the following. In SEQ ID NO: 599 (Figure 2A) the leucine at position 15 may be substituted with glutamate; 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. Additional vehicles 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. Patent 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 US Patent No. 6, 133, 426 to Gonzales et al., issued October 17,2000. 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. 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). 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, Figures 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. 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 al-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 tor 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. 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 April 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 hurnanization). Linkers 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)s), poly(Gly-Ala), and polyalanines. Other specific examples of linkers are: (Gly)3Lys(Gly)4 (SEQ ID NO: 595); (Gly)3AsnGlySer(Gly)2 (SEQ ID NO: 596); (Gly)3Cys(Gly)4 (SEQ ID NO: 597); and 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. 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., Ci-Ce) lower acyl, halogen (e.g., Cl, Br), CN, NH2, phenyl, etc. An exemplary nonpeptide linker is a PEG linker, 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. Derivatives 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. Exemplary derivatives include compounds in which: 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. For citations to references on preparation of cyclized derivatives, see Table 2. 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. 3. One or more peptidyl [-C(O)NR-] linkages (bonds) is replaced by a non-peptidyl linkage. Exemplary nonpeptidyl linkages are -CH2-carbamate [-CH2-OC(O)NR-], phosphonate , -CHb-sulfonamide [-CHa- S(O)2NR-], urea [-NHC(O)NH-], -CH2-secondary amine, and alkylated peptide [-C(O)NR6- wherein R6 is lower alkyl]. 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 -NFfr), -NRQOJR1, -NRC(O)OR1, -NRS(O)2R1, -NHC(O)NHR!, 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. 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-C4alkyl). 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), T. Med. Chem. 39: 3814-9; Alberts et al. (1993) Thirteenth Am. Pep. Symp.. 357-9. 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. 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-aminocontaining residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate. 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. 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. Carboxyl sidechain groups (aspartyl or glutamyl) may be selectively modified by reaction with carbodiimides (R'-N=C=N-R') such as l-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or l-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. 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. 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), T. Med. Chem. 39: 3814-9. 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., l,l-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-l,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. 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 Thx) 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. 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). , .. 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. 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. 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 mole.cules 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). 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. Useful conjugation partners include: • radioisotopes, such as 90Yttrium, 131Iodine, 225Actinium, and 213Bismuth; • ricin A toxin, microbially derived toxins such as Pseudomonas endotoxin (e.g., PE38, PE40), and the like; • partner molecules in capture systems (see below); • biotin, streptavidin (useful as either partner molecules in capture systems or as tracers, especially for diagnostic use); and • cytotoxic agents (e.g., doxorubicin). 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. 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. 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 posttranslational modifications resulting from their rapid proliferation. Thus, one aspect of this invention is a process comprising: a) selecting at least one randomized peptide that specifically binds to a target epitope; and 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. Variants 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. 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. 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 (Met1-peptibody) are contemplated, as are specific binding agents with additional methionine and lysine residues at positions -2 and -1 (Met1-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. 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. 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). 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. 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, MA), the FLAG system (IBI, New Haven, CT), and the 6xHis system (Qiagen, Chatsworth, CA). These systems are capable of producing recrambinant 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. 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, MA). 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. Patent 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. 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. 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. 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 T. Applied Math., 48:1073 (1988). - 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, WI, BLASTP, BLASTN, and FASTA (Altschul et al.. T. 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. 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. For example, using the computer algorithm GAP (Genetics Computer Group, University of Wisconsin, Madison, WI), 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 3X 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., Froc. Natl. Acad. Sci USA, 89:10915-10919 (1992) for the BLOSUM 62 comparison matrix) is also used by the algorithm. In certain embodiments, the parameters for a polypeptide sequence comparison include the following: Algorithm: Needleman et al.. T. Mol. Biol.. 48:443-453 (1970); Comparison matrix: BLOSUM 62 from Henikoff et al., supra (1992); Gap Penalty: 12 Gap Length Penalty: 4 Threshold of Similarity: 0 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. In certain embodiments, the parameters for polynucleotide molecule sequence (as opposed to an amino acid sequence) comparisons include the following: Algorithm: Needleman et al., supra (1970); Comparison matrix: matches = +10, mismatch = 0 Gap Penalty: 50 Gap Length Penalty: 3 The GAP program may also be useful with the above parameters. The aforementioned parameters are the default parameters for polynucleotide molecule comparisons. 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, proteinto- 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). 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. 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 Xi-X2-Xs would be Xa-X2-Xi. 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. Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as a-, 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, Nethylglycine, N-ethylaspargine, hyroxylysine, allo-hydroxylysine, hydroxyproline, isodesmosine, alloisoleucine, N-methylglycine, sarcosine, N-methylisoleucine, N-methylvaline, norvaline, norleucine, orithine, 4- hycftfoxyproline, y-carboxyglutamate, E-N,N,N-trimethyllysine, e-N-acetyllysine, O-phosphoserine, Nacetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, ci-N-methylarginine, and other similar amino acids and amino acids (e.g., 4-hydroxyproline). 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". It will be appreciated that amino acid residues can be divided into classes based on their common side chain properties: 1. Neutral Hydrophobic: Alanine (Ala; A), Valine (Val; V), Leucine (Leu; L), Isoleucine (He; I), Proline (Pro; P), Tryptophan (Trp; W), Phenylalanine (Phe; F), and Methionine (Met, M). 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. 3. Acidic: Aspartic Acid (Asp; D), Glutamic Acid (Glu; E); 4) Basic: Lysine (Lys; K), Arginine (Arg; R), Histidine (His; H). See Lewin, B., Genes V, Oxford University Press (1994), p.11. 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. 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). , The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is understood in the art. Kyte et al., T. 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. 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. 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." Exemplary amino acid substitutions are set forth in Table 10 below. Table 10 Amino Acid Substitutions Original Residues (Table Removed) 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. 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. 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. 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(l):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. 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)). 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. 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. Affinity Maturation 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. Non-Peptide Analogs/Protein Mimetics 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. Fept. 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 Cterminal 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. Covalentlybound 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. 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. Patent 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. Patent 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. Methods of Making 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. 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. 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. 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, biosafety 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. 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. 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. Folypeptides, pp. 335-61 (Katsoyannis and Panayotis eds.); Merrifield (1963), T. 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. Compounds that contain derivatized peptides or which contain non-peptide groups may be synthesized by well-known organic chemistry techniques. Uses of the Compounds 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. 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. 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. Therapeutic uses of TFO-mimetic compounds 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. 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. 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. 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. 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. 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: WO95/26746; WO95/21919; WO95/18858; WO95/21920 and are incorporated herein. 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. Therapeutic uses of Ang-2 binding molecules Agents that modulate Ang-2 binding activity, or other cellular activity, may be used in combination with other therapeutic agents to enhance their therapeutic effects or decrease potential side effects. In one aspect, the present invention provides reagents and methods useful for treating diseases and conditions characterized by undesirable or aberrant levels of Ang-2 activity in a cell. These diseases include cancers, and other hyperproliferative conditions, such as hyperplasia, psoriasis, contact dermatitis, immunological disorders, and infertility. The present invention also provides methods of treating cancer in an animal, including humans, comprising administering to the animal an effective amount of a specific binding agent, such as a peptibody, that inhibits or decreases Ang-2 activity. The invention is further directed to methods of inhibiting cancer cell growth, including processes of cellular proliferation, invasiveness, and metastasis in biological systems. Methods include use of a compound of the invention as an inhibitor of cancer cell growth. Preferably, the methods are employed to inhibit or reduce cancer cell growth, invasiveness, metastasis, or tumor incidence in living animals, such as mammals. Methods of the invention are also readily adaptable for use in assay systems, e.g., assaying cancer cell growth and properties thereof, as well as identifying compounds that affect cancer cell growth. The cancers treatable by methods of the present invention preferably occur in mammals. Mammals include, for example, humans and other primates, as well as pet or companion animals such as dogs and cats, laboratory animals such as rats, mice and rabbits, and farm animals such as horses, pigs, sheep, and cattle. Tumors or neoplasms include growths of tissue cells in which the multiplication of the cells is uncontrolled and progressive. Some such growths are benign, but others are termed malignant and may lead to death of the organism. Malignant neoplasms or cancers are distinguished from benign growths in that, in addition to exhibiting aggressive cellular proliferation, they may invade surrounding tissues and metastasize. Moreover, malignant neoplasms are characterized in that they show a greater loss of differentiation (greater dedifferentiation), and of their organization relative to one another and their surrounding tissues. This property is also called "anaplasia." Neoplasms treatable by the present invention also include solid tumors, i.e., carcinomas and sarcomas. Carcinomas include those malignant neoplasms derived from epithelial cells that infiltrate (invade) the surrounding tissues and give rise to metastases. Adenocarcinomas are carcinomas derived from glandular tissue, or which form recognizable glandular structures. Another broad category or cancers includes sarcomas, which are tumors whose cells are embedded in a fibrillar or homogeneous substance like embryonic connective tissue. The invention also enables treatment of cancers of the myeloid or lymphoid systems, including leukemias, lymphomas and other cancers that typically do not present as a tumor mass, but are distributed in the vascular or lymphoreticular systems. The ang-2 binding molecules of this invention are thus useful for the treatment of a wide variety of cancers, including solid tumors and leukemias. Types of cancer or tumor cells amenable to treatment according to the invention include, for example, ACTH-producing tumor; acute lymphocytic leukemia; acute nonlymphocytic leukemia; adenoma; cancer of the adrenal cortex; adenocarcinoma of the breast, prostate, and colon; ameloblastoma; apudoma; bladder cancer; brain cancer; branchioma; breast cancer; all forms of bronchogenic carcinoma of the lung; carcinoid heart disease; carcinoma (e.g.. Walker, basal cell, basosquamous, Brown-Pearce, ductal, Ehrlich tumor, Krebs 2, merkel cell, mucinous, non-small cell lung, oat cell, papillary, scirrhous, bronchiolar, bronchogenic, squamous cell, and transitional cell); malignant carcinoid syndrome; immunoproliferative small lung cell carcinoma; cementoma; cervical cancer; chondroblastoma; chondroma; chondrosarcoma; choristoma; chronic lymphocytic leukemia; chronic myelocytic leukemia; colorectal cancer; chordoma; craniopharyngioma; cutaneous T-cell lymphoma; dysgerminoma; endometrial cancer; esophageal cancer; Ewing's sarcoma; fibroma; fibrosarcoma; gallbladder cancer; giant cell tumors; glioma; hairy cell leukemia; hamartoma; head and neck cancer; hepatoma; histiocytic disorders; histiocytosis; Hodgkin's lymphoma; Kaposi's sarcoma; kidney cancer; lipoma; liposarcoma; liver cancer; lung cancer (small and non-small cell); malignant peritoneal effusion; malignant pleural effusion; melanoma; mesenchymoma; mesonephroma; mesothelioma; multiple myeloma; myosarcoma; myxoma; myxosarcoma; neuroblastoma; non-Hodgkin's lymphoma; odontoma; osteoma; osteosarcoma; ovarian cancer; ovarian (germ cell) cancer; pancreatic cancer; papilloma; penile cancer; plasmacytoma; prostate cancer; reticuloendotheliosis; retinoblastoma; skin cancer; soft tissue sarcoma; squamous cell carcinomas; stomach cancer; teratoma; testicular cancer; thymoma; thyroid cancer; trophoblastic neoplasms; uterine cancer; vaginal cancer; cancer of the vulva; Wilms' tumor. Further, the following types of cancers may also be treated: cholangioma; cholesteatoma; cyclindroma; cystadenocarcinoma; cystadenoma; granulosa cell tumor; gynandroblastoma; hidradenoma; islet cell tumor; Leydig cell tumor; papilloma; Sertoli cell tumor; theca cell tumor; leiomyoma; leiomyosarcoma; myoblastoma; myoma; myosarcoma; rhabdomyoma; rhabdomyosarcoma; ependymoma; ganglioneuroma; glioma; medulloblastoma; meningioma; neurilemmoma; neuroblastoma; neuroepithelioma; neurofibroma; neuroma; paraganglioma; paraganglioma nonchromaffin; angiokeratoma; angiolymphoid hyperplasia with eosinophilia; angioma sclerosing; angiomatosis; glomangioma; hemangioendothelioma; hemangioma; hemangiopericytoma; hemangiosarcoma; lymphangioma; lymphangiomyoma; lymphangiosarcoma; pinealoma; carcinosarcoma; chondrosarcoma; cystosarcoma phyllodes; fibrosarcoma; hemangiosarcoma; leiomyosarcoma; leukosarcoma; liposarcoma; lymphangiosarcoma; myosarcoma; myxosarcoma; ovarian carcinoma; rhabdomyosarcoma; sarcoma; neoplasms; nerofibromatosis; and cervical dysplasia. Therapeutic uses of NGF binding molecules The NGF binding molecules may be used in the prevention or treatment of NGF-related diseases and disorders. Such indications include but are not limited to pain (including, but not limited to, inflammatory pain and associated hyperalgesia and allodynia, neuropathic pain and associated hyperalgesia and allodynia, diabetic neuropathy pain, causalgia, sympathetically maintained pain, deafferentation syndromes, acute pain, tension headache, migraine, dental pain, pain from trauma, surgical pain, pain resulting frorn amputation or abscess, causalgia, demyelinating diseases, and trigeminal neuralgia). The peptides and modified peptides of the invention have therapeutic value for the prevention or treatment of other diseases linked to NGF as a causative agent, including, but not limited to, asthma, urge incontinence (i.e., hyperactive bladder), psoriasis, cancer (especially, pancreatic cancer and melanoma), chronic alcoholism, stroke, thalamic pain syndrome, diabetes, acquired immune deficiency syndrome ("AIDS"), toxins and chemotherapy, general headache, migraine, cluster headache, mixedvascular and non-vascular syndromes, general inflammation, arthritis, rheumatic diseases, lupus, osteoarthritis, inflammatory bowel disorders, inflammatory eye disorders, inflammatory or unstable bladder disorders, psoriasis, skin complaints with inflammatory components, sunburn, carditis, dermatitis, myositis, neuritis, collagen vascular diseases, chronic inflammatory conditions, asthma, epithelial tissue damage or dysfunction, herpes simplex, disturbances of visceral motility at respiratory, genitourinary, gastrointestinal or vascular regions, wounds, burns, allergic skin reactions, pruritis, vitiligo, general gastrointestinal disorders, colitis, gastric ulceration, duodenal ulcers, vasomotor or allergic rhinitis, or bronchial disorders. Therapeutic uses of myostatin binding molecules The myostatin binding agents of the present invention bind to myostatin and block or inhibit myostatin signaling within targeted cells. The present invention provides methods and reagents for reducing the amount or activity of myostatin in an animal by administering an effective dosage of one or more myostatin binding agents to the animal. In one aspect, the present invention provides methods and reagents for treating myostatin-related disorders in an animal comprising administering an effective dosage of one or more binding agents to the animal. These myostatin-related disorders include but are not limited to various forms of muscle wasting, as well as metabolic disorders such as diabetes and related disorders, and bone degenerative diseases such as osteoporosis. As shown in the Example 8 of U.S. Ser. No. 10/742,379, exemplary peptibodies of the present invention dramatically increases lean muscle mass in the CD1 nu/nu mouse model. This in vivo activity correlates to the in vitro binding and inhibitory activity described below for the same peptibodies. Muscle wasting disorders include dystrophies such as Duchenne's muscular dystrophy, progressive muscular dystrophy, Becker's type muscular dystrophy, Dejerine-Landouzy muscular dystrophy, Erb's muscular dystrophy, and infantile neuroaxonal muscular dystrophy. For example, blocking myostatin through use of antibodies in vivo improved the dystrophic phenotype of the mdx mouse model of Duchenne muscular dystrophy (Bogdanovich et al. (2002), Nature 420: 28). Use of an exemplary peptibody increases lean muscle mass and increases the ratio of lean muscle to fat in mdx mouse models as described in Example 9 below. Additional muscle wasting disorders arise from chronic disease such as amyotrophic lateral sclerosis, congestive obstructive pulmonary disease, cancer, AIDS, renal failure, and rheumatoid arthritis. For example, cachexia or muscle wasting and loss of body weight was induced in athymic nude mice by a systemically administered myostatin (Zimmers et al., supra). In another example, serum and intramuscular concentrations of myostatin-immunoreactive protein was found to be increased in men exhibiting AIDS-related muscle wasting and was inversely related to fat-free mass (Gonzalez-Cadavid et al. (1998), PNAS USA 95: 14938- 14943). Additional conditions resulting in muscle wasting may arise from inactivity due to disability such as confinement in a wheelchair, prolonged bedrest due to stroke, illness, bone fracture or trauma, and muscular atrophy in a microgravity environment (space flight). For example, plasma myostatin immunoreactive protein was found to increase after prolonged bedrest (Zachwieja et al. J Gravit Physiol. 6(2):11(1999). It was also found that the muscles of rats exposed to a microgravity environment during a space shuttle flight expressed an increased amount of myostatin compared with the muscles of rats which were not exposed (Lalani et al. (2000), T.Endocrin. 167 (3):417-28). In addition, age-related increases in fat to muscle ratios, and age-related muscular atrophy appear to be related to myostatin. For example, the average serum myostatin-immunoreactive protein increased with age in groups of young (19-35 yr old), middle-aged (36-75 yr old), and elderly (76-92 yr old) men and women, while the average muscle mass and fat-free mass declined with age in these groups (Yarasheski et al. J Nutr Aging 6(5):343-8 (2002)). It has also been shown that myostatin gene knockout in mice increased myogenesis and decreased adipogenesis (Lin et al. (2002), Biochem Biophys Res Commun 291(3):701-6, resulting in adults with increased muscle mass and decreased fat accumulation and leptin secretion. Exemplary molecules improve the lean muscle mass to fat ratio in aged mdx mice as shown below. In addition, myostatin has now been found to be expressed at low levels in heart muscle and expression is upregulated after cardiomyocytes after infarct (Sharma et al. (1999), T Cell Fhysiol. 180 (l):l-9). Therefore, reducing myostatin levels in the heart muscle may improve recovery of heart muscle after infarct. Myostatin also appears to influence metabolic disorders including type 2 diabetes, noninsulindependent diabetes mellitus, hyperglycemia, and obesity. For example, lack of myostatin has been shown to improve the obese and diabetic phenotypes of two mouse models (Yen et al. supra). In addition, increasing muscle mass by reducing myostatin levels may improve bone strength and reduce osteoporosis and other degenerative bone diseases. It has been found, for example, that myostatin-deficient mice showed increased mineral content and density of the mouse humerus and increased mineral content of both trabecular and cortical bone at the regions where the muscles attach, as well as increased muscle mass (Hamrick et al. (2002), Calcif Tissue Int 71(1): 63-8). In the present invention, an exemplary peptibody increases the lean muscle mass to fat ratio in mdx mouse models as shown below. The present invention also provides methods and reagents for increasing muscle mass in food animals by administering an effective dosage of the myostatin binding agent to the animal. Since the mature Cterminal myostatin polypeptide is identical in all species tested, myostatin binding agents would be expected to be effective for increasing muscle mass and reducing fat in any agriculturally important species including cattle, chicken, turkeys, and pigs. The myostatin-binding molecules of the present invention may be used alone or in combination with other therapeutic agents to enhance their therapeutic effects or decrease potential side effects. The molecules of the present invention possess one or more desirable but unexpected combination of properties to improve the therapeutic value of the agents. These properties include increased activity, increased solubility, reduced degradation, increased half-life, reduced toxicity, and reduced immunogenicity. Thus the molecules of the present invention are useful for extended treatment regimes. In addition, the properties of hydrophilicity and hydrophobicity of the compounds of the invention are well balanced, thereby enhancing their utility for both in vitro and especially in vivo uses. Specifically, compounds of the invention have an appropriate degree of solubility in aqueous media that permits absorption and bioavailability in the body, while also having a degree of solubility in lipids that permits the compounds to traverse the cell membrane to a putative site of action, such as a particular muscle mass. The myostatin-binding molecules of the present invention are useful for treating a "subject" or any animal, including humans, when administered in an effective dosages in a suitable composition. In addition, the mystatin-binding molecules of the present invention are useful for detecting and quantitating myostatin in a number of assays. These assays are described in detail in U.S. Ser. No. 10/742,379. In general, the myostatin-binding molecules of the present invention are useful as capture agents to bind and immobilize myostatin in a variety of assays, similar to those described, for example, in Asai, ed., Methods in Cell Biology, 37, Antibodies in Cell Biology, Academic Press, Inc., New York (1993). The myostatin-binding molecule may be labeled in some manner or may react with a third molecule such as an anti-binding molecule antibody which is labeled to enable myostatin to be detected and quantitated. For example, a myostatin-binding molecule or a third molecule can be modified with a detectable moiety, such as biotin, which can then be bound by a fourth molecule, such as enzyme-labeled streptavidin, or other proteins. (Akerstrom (1985), T Immunol 135:2589: Chaubert (1997), Mod Fathol 10:585). Throughout any particular assay, incubation and/or washing steps may be required after each combination of reagents. Incubation steps can vary from about 5 seconds to several hours, preferably from about 5 minutes to about 24 hours. However, the incubation time will depend upon the assay format, volume of solution, concentrations, and the like. Usually, the assays will be carried out at ambient temperature, although they can be conducted over a range of temperatures. Therapeutic uses of BAFF-binding molecules. BAFF-binding molecules of this invention may be particularly useful in treatment of B-cell mediated autoimmune diseases. In particular, they may be useful in treating, preventing, ameliorating, diagnosing or prognosing lupus, including systemic lupus erythematosus (SLE), and lupus-associated diseases and conditions. Other preferred indications include B-cell mediated cancers, including B-cell lymphoma. The compounds of this invention can also be used to treat inflammatory conditions of the joints. Inflammatory conditions of a joint are chronic joint diseases that afflict and disable, to varying degrees, millions of people worldwide. Rheumatoid arthritis is a disease of articular joints in which the cartilage and bone are slowly eroded away by a proliferative, invasive connective tissue called pannus, which is derived from the synovial membrane. The disease may involve peri-articular structures such as bursae, tendon sheaths and tendons as well as extra-articular tissues such as the subcutis, cardiovascular system, lungs, spleen, lymph nodes, skeletal muscles, nervous system (central and peripheral) and eyes (Silberberg (1985), Anderson's Pathology, Kissane (ed.), 11:1828). Osteoarthritis is a common joint disease characterized by degenerative changes in articular cartilage and reactive proliferation of bone and cartilage around the joint. Osteoarthritis is a cell-mediated active process that may result from the inappropriate response of chondrocytes to catabolic and anabolic stimuli. Changes in some matrix molecules of articular cartilage reportedly occur in early Osteoarthritis (Thonar et al. (1993), Rheumatic disease clinics of North America, Moskowitz (ed.), 19:635-657 and Shinmei et al. (1992), Arthritis Rheum., 35:1304-1308). TALL-1, TALL-1R and modulators thereof are believed to be useful in the treatment of these and related conditions. BAFF-binding molecules may also be useful in treatment of a number of additional diseases and disorders, including acute pancreatitis; ALS; Alzheimer's disease; asthma; atherosclerosis; autoimmune hemolytic anemia; cancer, particularly cancers related to B cells; cachexia/anorexia; chronic fatigue syndrome; cirrhosis (e.g., primary biliary cirrhosis); diabetes (e.g., insulin diabetes); fever; glomerulonephritis, including IgA glomerulonephritis and primary glomerulonephritis; Goodpasture's syndrome; Guillain-Barre syndrome; graft versus host disease; Hashimoto's thyroiditis; hemorrhagic shock; hyperalgesia; inflammatory bowel disease; inflammatory conditions of a joint, including Osteoarthritis, psoriatic arthritis and rheumatoid arthritis; inflammatory conditions resulting from strain, sprain, cartilage damage, trauma, orthopedic surgery, infection or other disease processes; insulin-dependent diabetes mellitus; ischemic injury, including cerebral ischemia (e.g., brain injury as a result of trauma, epilepsy, hemorrhage or stroke, each of which may lead to neurodegeneration); learning impairment; lung diseases (e.g., ARDS); lupus, particularly systemic lupus erythematosus (SLE); multiple myeloma; multiple sclerosis; Myasthenia gravis; myelogenous (e.g., AML and CML) and other leukemias; myopathies (e.g., muscle protein metabolism, esp. in sepsis); neurotoxicity (e.g., as induced by HIV); osteoporosis; pain; Parkinson's disease; Pemphigus; polymyositis/dermatomyositis; pulmonary inflammation, including autoimmune pulmonary inflammation; pre-term labor; psoriasis; Reiter's disease; reperfusion injury; septic shock; side effects from radiation therapy; Sjogren's syndrome; sleep disturbance; temporal mandibular joint disease; thrombocytopenia, including idiopathic thrombocytopenia and autoimmune neonatal thrombocytopenia; tumor metastasis; uveitis; and vasculitis. Combination Therapy. The therapeutic methods, compositions and compounds of the present invention may also be employed, alone or in combination with other cytokines, soluble Mpl receptor, hematopoietic factors, interleukins, growth factors or antibodies in the treatment of disease states characterized by other symptoms as well as platelet deficiencies. It is anticipated that the inventive compound will prove useful in treating some forms of thrombocytopenia in combination with general stimulators of hematopoiesis, such as IL-3 or GM-CSF. Other megakaryocytic stimulatory factors, i.e., meg-CSF, stem cell factor (SCF), leukemia inhibitory factor (LIF), oncostatin M (OSM), or other molecules with megakaryocyte stimulating activity may also be employed with Mpl ligand. Additional exemplary cytokines or hematopoietic factors for such co-administration include IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-11, colony stimulating factor-1 (CSF-1), SCF, GM-CSF, granulocyte colony stimulating factor (G-CSF), EPO, interferonalpha (IFN-alpha), consensus interferon, IFN-beta, or IFN-gamma. It may further be useful to administer, either simultaneously or sequentially, an effective amount of a soluble mammalian Mpl receptor, which appears to have an effect of causing megakaryocytes to fragment into platelets once the megakaryocytes have reached mature form. Thus, administration of an inventive compound (to enhance the number of mature megakaryoctes) followed by administration of the soluble Mpl receptor (to inactivate the ligand and allow the mature megakaryocytes to produce platelets) is expected to be a particularly effective means of stimulating platelet production. The dosage recited above would be adjusted to compensate for such additional components in the therapeutic composition. Progress of the treated patient can be monitored by conventional methods. In cases where the inventive compounds are added to compositions of platelets and/or megakaryocytes and related cells, the amount to be included will generally be ascertained experimentally by techniques and assays known in the art. An exemplary range of amounts is 0.1 ug—1 mg inventive compound per 106 cells. Pharmaceutical Compositions In General The present invention also provides methods of using pharmaceutical compositions of the inventive compounds. Such pharmaceutical compositions may be for administration for injection, or for oral, pulmonary, nasal, transdermal or other forms of administration. In general, the invention encompasses pharmaceutical compositions comprising effective amounts of a compound of the invention together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. Such compositions include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol); incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes. Hyaluronic acid may also be used, and this may have the effect of promoting sustained duration in the circulation. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the present proteins and derivatives. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA 18042) pages 1435-1712 which are herein incorporated by reference. The compositions may be prepared in liquid form, or may be in dried powder, such as lyophilized form. Implantable sustained release formulations are also contemplated, as are transdermal formulations. Oral dosage forms Contemplated for use herein are oral solid dosage forms, which are described generally in Chapter 89 of Remington's Pharmaceutical Sciences (1990), 18th Ed., Mack Publishing Co. Easton PA 18042, which is herein incorporated by reference. Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets or pellets. Also, liposomal or proteinoid encapsulation may be used to formulate the present compositions (as, for example, proteinoid microspheres reported in U.S. Patent No. 4,925,673). Liposomal encapsulation may be used and the liposomes may be derivatized with various polymers (e.g., U.S. Patent No. 5,013,556). A description of possible solid dosage forms for the therapeutic is given in Chapter 10 of Marshall, K., Modern Pharmaceutics (1979), edited by G. S. Banker and C. T. Rhodes, herein incorporated by reference. In,general, the formulation will include the inventive compound, and inert ingredients which allow for protection against the stomach environment, and release of the biologically active material in the intestine. Also specifically contemplated are oral dosage forms of the above inventive compounds. If necessary, the compounds may be chemically modified so that oral delivery is efficacious. Generally, the chemical modification contemplated is the attachment of at least one moiety to the compound molecule itself, where said moiety permits (a) inhibition of proteolysis; and (b) uptake into the blood stream from the stomach or intestine. Also desired is the increase in overall stability of the compound and increase in circulation time in the body. Moieties useful as covalently attached vehicles in this invention may also be used for this purpose. Examples of such moieties include: PEG, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline. See, for example, Abuchowski and Davis, Soluble Polymer-Enzyme Adducts, Enzymes as Drugs (1981), Hocenberg and Roberts, eds., Wiley- Interscience, New York, NY, , pp 367-83; Newmark, et al. (1982), T. Appl. Biochem. 4:185-9. Other polymers that could be used are poly-l,3-dioxolane and poly-l,3,6-tioxocane. Preferred for pharmaceutical usage, as indicated above, are PEG moieties. For oral delivery dosage forms, it is also possible to use a salt of a modified aliphatic amino acid, such as sodium N-(8-[2-hydroxybenzoyl] amino) caprylate (SNAC), as a carrier to enhance absorption of the therapeutic compounds of this invention. The clinical efficacy of a heparin formulation using SNAC has been demonstrated in a Phase II trial conducted by Emisphere Technologies. See US Patent No. 5,792,451, "Oral drug delivery composition and methods". The compounds of this invention can be included in the formulation as fine multiparticulates in the form of granules or pellets of particle size about 1 mm. The formulation of the material for capsule administration could also be as a powder, lightly compressed plugs or even as tablets. The therapeutic could be prepared by compression. Colorants and flavoring agents may all be included. For example, the protein (or derivative) may be formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible product, such as a refrigerated beverage containing colorants and flavoring agents. One may dilute or increase the volume of the compound of the invention with an inert material. These diluents could include carbohydrates, especially mannitol, a-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch, Certain inorganic salts may also be used as fillers including calcium triphosphate, magnesium carbonate and sodium chloride. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell. Disintegrants may be included in the formulation of the therapeutic into a solid dosage form. Materials used as disintegrants include but are not limited to starch including the commercial disintegrant based on starch, Explotab. Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite may all be used. Another form of the disintegrants are the insoluble cationic exchange resins. Powdered gums may be used as disintegrants and as binders and these can include powdered gums such as agar, Karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants. Binders may be used to hold the therapeutic agent together to form a hard tablet and include materials natural products such as acacia, tragacanth, starch and gelatin. Others include methyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both be used in alcoholic solutions to granulate the therapeutic. An antifrictional agent may be included in the formulation of the therapeutic to prevent sticking during the formulation process. Lubricants may be used as a layer between the therapeutic and the die wall, and these can include but are not limited to; stearic acid including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants may also be used such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000. Glidants that might improve the flow properties of the drug during formulation and to aid rearrangement during compression might be added. The glidants may include starch, talc, pyrogenic silica and hydrated silicoaluminate. To aid dissolution of the compound of this invention into the aqueous environment a surfactant might be added as a wetting agent. Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents might be used and could include benzalkonium chloride or benzethonium chloride. The list of potential nonionic detergents that could be included in the formulation as surfactants are lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants could be present in the formulation of the protein or derivative either alone or as a mixture in different ratios. Additives may also be included in the formulation to enhance uptake of the compound. Additives potentially having this property are for instance the fatty acids oleic acid, linoleic acid and linolenic acid. Controlled release formulation may be desirable. The compound of this invention could be incorporated into an inert matrix which permits release by either diffusion or leaching mechanisms e.g.^ gums. Slowly degenerating matrices may also be incorporated into the formulation, e.g., alginates, polysaccharides. Another form of a controlled release of the compounds of this invention is by a method based on the Oros therapeutic system (Alza Corp.), i.e., the drug is enclosed in a semipermeable membrane which allows water to enter and push drug out through a single small opening due to osmotic effects. Some enteric coatings also have a delayed release effect. Other coatings may be used for the formulation. These include a variety of sugars which could be applied in a coating pan. The therapeutic agent could also be given in a film coated tablet and the materials used in this instance are divided into 2 groups. The first are the nonenteric materials and include methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl-methyl cellulose, sodium carboxy-methyl cellulose, providone and the polyethylene glycols. The second group consists of the enteric materials that are commonly esters of phthalic acid. A mix of materials might be used to provide the optimum film coating. Film coating may be carried out in a pan coater or in a fluidized bed or by compression coating. Pulmonary delivery forms Also contemplated herein is pulmonary delivery of the present protein (or derivatives thereof). The protein (or derivative) is delivered to the lungs of a mammal while inhaling and traverses across the lung epithelial lining to the blood stream. (Other reports of this include Adjei et al., Pharma. Res. (1990) 7: 565-9; Adjei et al. (1990), Internatl. T. Pharmaceutics 63:135-44 (leuprolide acetate); Braquet et al. (1989), T. Cardiovasc. Pharmacol. 13 (suppl.5): s.143-146 (endothelin-1); Hubbard et al. (1989), Annals Int. Med. 3: 206-12 (alantitrypsin); Smith et al. (1989), T. Clin. Invest. 84: 1145-6 (al-proteinase); Oswein et al. (March 1990), "Aerosolization of Proteins", Proc. Symp. Resp. Drug Delivery II, Keystone, Colorado (recombinant human growth hormone); Debs et al. (1988), T. Immunol. 140: 3482-8 (interferon-y and tumor necrosis factor a) and Platz et al., U.S. Patent No. 5,284,656 (granulocyte colony stimulating factor). Contemplated for use in the practice of this invention are a wide range of mechanical devices designed for pulmonary delivery of therapeutic products, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art. Some specific examples of commercially available devices suitable for the practice of this invention are the Ultravent nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Missouri; the Acorn II nebulizer, manufactured by Marquest Medical Products, Englewood, Colorado; the Ventolin metered dose inhaler, manufactured by Glaxo Inc., Research Triangle Park, North Carolina; and the Spinhaler powder inhaler, manufactured by Fisons Corp., Bedford, Massachusetts. All such devices require the use of formulations suitable for the dispensing of the inventive compound. Typically, each formulation is specific to the type of device employed and may involve the use of an appropriate propellant material, in addition to diluents, adjuvants and/or carriers useful in therapy. The inventive compound should most advantageously be prepared in particulate form with an average particle size of less than 10 nm (or microns), most preferably 0.5 to 5 ^im, for most effective delivery to the distal lung. Pharmaceutically acceptable carriers include carbohydrates such as trehalose, mannitol, xylitol, sucrose, lactose, and sorbitol. Other ingredients for use in formulations may include DPPC, DOPE, DSPC and DOPC. Natural or synthetic surfactants may be used. PEG may be used (even apart from its use in derivatizing the protein or analog). Dextrans, such as cyclodextran, may be used. Bile salts and other related enhancers may be used. Cellulose and cellulose derivatives may be used. Amino acids may be used, such as use in a buffer formulation. Also, the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated. Formulations suitable for use with a nebulizer, either jet or ultrasonic, -will, typically comprise the inventive compound dissolved in water at a concentration of about 0.1 to 25 mg of biologically active protein per mL of solution. The formulation may also include a buffer and a simple sugar (e.g., for protein stabilization and regulation of osmotic pressure). The nebulizer formulation may also contain a surfactant, to reduce or prevent surface induced aggregation of the protein caused by atomization of the solution in forming the aerosol. Formulations for use with a metered-dose inhaler device will generally comprise a finely divided powder containing the inventive compound suspended in a propellant with the aid of a surfactant. The propellant may be any conventional material employed for this purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, or combinations thereof. Suitable surfactants include sorbitan trioleate and soya lecithin. Oleic acid may also be useful as a surfactant. Formulations for dispensing from a powder inhaler device will comprise a finely divided dry powder containing the inventive compound and may also include a bulking agent, such as lactose, sorbitol, sucrose, mannitol, trehalose, or xylitol in amounts which facilitate dispersal of the powder from the device, e.g., 50 to 90% by weight of the formulation. Other delivery forms Nasal delivery of the inventive compound is also contemplated. Nasal delivery allows the passage of the protein to the blood stream directly after administering the therapeutic product to the nose, without the necessity for deposition of the product in the lung. Formulations for nasal delivery include those with dextran or cyclodextran. Delivery via transport across other mucous membranes is also contemplated. Buccal delivery of the inventive compound is also contemplated. Buccal delivery formulations are known in the art for use with peptides. Dosages The dosage regimen involved in a method for treating the above-described conditions will be determined by the attending physician, considering various factors which modify the action of drugs, e.g. the age, condition, body weight, sex and diet of the patient, the severity of any infection, time of administration and other clinical factors. Generally, the daily regimen should be in the range of 0.1-1000 micrograms of the inventive compound per kilogram of body weight, preferably 0.1-150 micrograms per kilogram. Specific preferred embodiments The inventors have determined preferred peptide sequences for molecules having many different kinds of activity. The inventors have further determined preferred structures of these preferred peptides combined with preferred linkers and vehicles. Preferred structures for these preferred peptides listed in Table 11 below. Linker sequences are shown in bold. Active peptide sequences are shown in bold and areunder lined. Table 11—Preferred embodiments (Table Removed) Working examples The compounds described above may be prepared as described below. These examples comprise preferred embodiments of the invention and are illustrative rather than limiting. Example 1 Preparation of Fc-loop-ang2 In this example of the invention, the disulphide-constrained peptide TN8-Con4 was inserted into the human IgGl Fc-loop domain, defined as the sequence Di37Ei3sLi39Ti4oKi4i (Fig 2A.). TN8-Con4 QEECEWDPWTCEHM (SEQ ID NO: 147) The peptide insertion is between Fc residues Leui39 and Thrwo and includes 2 Gly residues as linkers flanking either side of the inserted peptide (Fig. 10A). The Fc-loop TN8-Con4 construct is labeled with Amgen clone #6888. The carboxy-terminal TN8-Con4 fusion peptibody (Fig. 10B) includes a 5 Gly linker and is labeled with Amgen clone #5564. Both clones #6888 and #5564 were transformed into E. coli by conventional methods known to those familiar in the art. Both clones were found to express at high levels and almost exclusively in the insoluble inclusion body fraction (Fig. 11). The isolated inclusion body fraction (1 g) was solubilized in 6 M guanidine- HC1, 50 mM Tris, 8 mM DTT, pH 9 (10 ml) at room temperature with mixing, for 1 hour. The denatured and reduced peptibodies were refolded from the solubilized inclusion bodies by a 1:25, (v/v) dilution into a refold buffer consisting of 2 M urea, 50 mM Tris, 4 mM cysteine, 1 mM cystamine, pH 8.5. The solubilized peptibodies were added drop wise to the refold buffer at 4 °C with stirring. The refold reactions were allowed to stir for 48 hours, and then aliquots were evaluated by SDS-PAGE and reverse-phase HPLC. The Fc-loop TN8-Con4 (#6888) is considerably more homogeneous in the refold reaction than the carboxy-terminal Fc TN8- Con4 peptibody (#5564) as shown by RP-HPLC (Fig. 12). Purification of the refolded Fc-loop TN8-Con4 and carboxy-terminal Fc TN8-Con4 was achieved using a 2-column process. First a recombinant Protein-A column was equilibrated in 2 M urea, 50 mM Tris, pH 8.5 and loaded with the filtered peptibody refold reaction. The column was then washed with 2 column volumes of equilibration buffer, followed by 2 column volumes of PBS. The peptibody fraction was eluted with 50mM NaOAc, pH3 and quickly neutralized by a 1:4 dilution into 10 mM NaOAc, 50 mM NaCl, pH 5. The diluted Protein-A eluate was again filtered and loaded to an SP Sepharose HP cation exchange column (Pharmacia) equilibrated in 10 mM NaOAc, 50 mM NaCl, pH 5. The peptibody fractions were then eluted with a linear 50- 500 mM NaCl gradient, pooled and concentrated to about 2 mg/ml. The final pools of Fc-loop TN8-Con4 (#6888) and the carboxy-terminal Fc TN8-Con4 (#5564) were evaluated by RP-HPLC (Fig. 13) and SDS-PAGE (Fig. 14). Both RP-HPLC and SDS-PAGE demonstrate that improved product homogeneity is achieved with the Fc-loop TN8-Con4 (#6888) relative to the comparable carboxy-terminal fused peptibody (#5564). Both the Fc-loop TN8-Con4 and carboxy-terminal fused TN8-Con4 peptibodies were further evaluated in an in vitro ELISA for competitive inhibition of the angiopoietin 2 receptor. In this format the peptibody competes with an angiopoietin 2 receptor-Fc fusion for binding to immobilized angiopoietin 2. Binding of the angiopoietin 2 receptor-Fc fusion is monitored by fluorescence using an enzyme-linked immunodetection method and reported as an inhibition constant at 50% inhibition (ICso). This experiment shows that the Fcloop TN8-Con4 is fully active relative to the carboxy-terminal Fc TN8-Con4 (Fig. 15). Stability in vivo of the Fc-loop TN8-Con4 peptibody was compared to the carboxy-terminal TN8-Con4 peptibody in mice. In this study groups of 15 mice were dosed subcutaneously with either peptibody construct at 5 mg/kg. At 4 hours after injection, 5 mice were sacrificed and serum collected. At 24 hours, another 5 mice were harvested and likewise at 48 hours. Each individual serum sample was evaluated by western blot for detectable human IgG-Fc peptibody. Since all the serum within each 5-mouse group was very similar, the groups were pooled to allow representative samples to be run together on a single gel/blot. The result of that analysis (Fig. 16) clearly shows that both the Fc-loop TN8-Con4 peptibody and the carboxyterminal TN8-Con4 peptibody persists in the pooled mouse sera throughout the 48-hour time course with no apparent loss. This result demonstrates that the Fc-loop designed peptibodies are not destabilized in vivo by the peptide insertion. Example 2 Preparation of Fc-loop-myo7 In another embodiment of this invention, a novel, disulphide-constrained peptide TN8-19-7 (U.S. Pat App 2004-0181033-A1, which is incorporated by reference) of the sequence: TN8-19-07 LADHGQCIRWPWMCPPEGWE (SEQ ID NO: 365) was engineered between Leul39 and Thrl40 as an internal fusion in the putative Fc-loop sequence DELTK of an IgGl Fc sequence (Fig 2A). An additional two Gly residues were also added at each end of the TN8-19-07 peptide as flanking linkers. The final Fc-loop TN8-19-07 sequence is given in Figure 3A and is labeled clone # 6951. Alternatively, a carboxy terminal fusion of TN8-19-07 with the same IgGl Fc sequence was prepared to serve as a control (Fig 3B) and labeled clone #6826. The carboxy-terminal fusion included five Gly residues between the Fc and TN8-19-07 to serve as a linker. Both clones #6951 and #6826 were transformed into E. coli by conventional methods used by those familiar in the art, and were found to express at high levels and almost exclusively in the insoluble inclusion body fraction (Fig. 4A and 4B). The isolated inclusion body fraction (1 g) was solubilized in 6 M guanidine- HCl, 50 mM Tris, 8 mM DTT, pH 9 (10 ml) at room temperature with mixing for 1 hour. The denatured and reduced peptibodies were refolded from the solubilized inclusion body fraction by a 1:25 (v/v) dilution into 2 M urea, 50 mM Tris, 4 mM cysteine, 1 mM cystamine, pH 8.5. The solubilized peptibodies were added dropwise to the refold buffer at 4 °C with stirring. The refold reactions were allowed to stir for 48 hours, and then aliquots were evaluated by SDS-PAGE and reverse-phase HPLC. The Fc-loop TN8-19-07 (#6951) was found to be considerably more homogeneous by RP-HPLC (Fig. 5) in the refold reaction than the carboxy-terminal Fc- TN8-19-07 peptibody (#6826). Purification was achieved by a 2-column process. First a recombinant Protein-A column was equilibrated in 2 M urea, 50 mM Tris, pH 8.5 and loaded with the filtered peptibody refold reaction. The column was then washed with 2 column volumes of equilibration buffer, followed by 2 column volumes of PBS. The peptibody fraction was eluted with 50 mM NaOAc, pH 3 and quickly neutralized by a 1:4 dilution into 10 mM NaOAc, 50 mM NaCl, pH 5. The diluted Protein-A eluate was again filtered and loaded to an SP Sepharose HP cation exchange column (Pharmacia) equilibrated in 10 mM NaOAc, 50 mM NaCl, pH 5. The peptibody fractions were then eluted with a linear 50-500 mM NaCl gradient, pooled and concentrated to about 2 mg/ml. The final pools of Fc-loop TN8-19-07 (#6951) and the carboxy-terminal Fc TN8-19-07 (#6826) were evaluated by RP-HPLC (Fig. 6) and SDS-PAGE (Fig. 7). Both RP-HPLC and SDS-PAGE demonstrate that improved homogeneity in the final product is achieved with the Fc-loop TN8-19-07 (#6951) relative to the comparable carboxy-terminal fused peptibody (#6826). An in vitro cell-based assay, which measures the inhibition of myostatin signaling activity, was used to determine the bioactivity of the Fc-loop TN8-19-07 (#6951) compared to the carboxy-terminal fusion (#6826). In this assay, both constructs were titrated against 4 nM myostatin and evaluated for their ability to inhibit the myostatin signaling activity as measured by a luciferase reporter system. The relative peptibody activities are reported as the effective concentration for 50% inhibition (ECso). This experiment shows that the Fc-loop TN8- 19-07 peptibody (#6951) retains full in vitro bioactivity (Fig. 8). Stability in vivo of the Fc-loop TN8-19-07 peptibody was compared to the carboxy-terminal TN8-19-07 peptibody in mice. In this study, groups of 15 mice were dosed subcutaneously with either peptibody construct at 5 mg/kg. At 4 hours post injection 5 mice were sacrificed and serum collected. At 24 hours another 5 mice were harvested and likewise at 48 hours. Each individual serum was evaluated by western blot for detectable human IgG-Fc peptibody. Since all the serum within each 5-mouse group was very similar, the groups were pooled to allow representative samples to be run together on a single gel/blot. The result of that analysis (Fig. 9) clearly shows that the Fc-loop TN8-19-07 peptibody persists in the pooled mouse sera throughout the 48-hour time course with no apparent loss. In contrast, the concentration of the carboxyterminal TN8-19-07 peptibody diminishes steadily through the course of the study until it is nearly undetectable at the 48-hour time point. This result suggests that the Fc-loop design approach may confer additional in vivo stability to the TN8-19-07 peptibody. Example 3 Preparation of TN8-Con4 This molecule was prepared as described above in Example 1 and in U.S. Pat. App. No. 2003/0236192 (also PCT/US04/10989), which is hereby incorporated by reference. Example 4 Preparation of Fc-ang2-tandem This molecule was prepared as described in U.S. 2003/0236193, published Dec. 25, 2003 (also PCT/US04/10989, filed April 8,2004), which is hereby incorporated by reference. Example 5 Preparation of TN8-19-7 This molecule was prepared as described above in example 2 and in U.S. Serial No. 10/742,379, filed Dec. 19,2003 (alsoPCT/USQ3/40781, filed Dec. 19,2003), which is hereby incorporated by reference . Example 6 Preparation of Fc-loop-EMF This molecule was prepared as previously described in example 1. Example 7 Preparation of Fc-loop-Amp2 In another embodiment of this invention a linear, non-constrained peptide, AMP 2 was inserted into the human IgGl Fc -loop domain, defined as the sequence Dis/EissLisgTwoKi/u (Fig. 2A). AMP-2: IEGPTLRQWLAARA (SEQIDNO: 28) The Fc insertion is between Leuisg and Thruo and includes 2 Gly residues as linkers flanking either side of the inserted peptide (Fig. 3D). The Fc-loop AMP 2 construct is labeled as Amgen clone #6875. The Fc-loop AMP 2 clone (#6875) was transformed into E. coli by conventional methods known to those in the art and was found to express at high levels and almost exclusively in the insoluble inclusion body fraction (Fig. 17). The isolated inclusion body fraction (Ig) was solubilized in 6 M guanidine-HCl, 50 mM Tris, 8 mM DTT, pH 9 (10 ml) at room temperature with mixing, for 1 hour. The denatured and reduced peptibody was refolded from the solubilized inclusion body fraction by a 1:25 (v/v) dilution into 2 M urea, 50 mM Tris, 4 mM cysteine, 1 mM cystamine, pH 8.5. The solubilized peptibody was added drop wise to the refold buffer at 4 °C with stirring. The refold reactions were allowed to stir for 48 hours, and then aliquots were evaluated by SDS-PAGE and reversed-phase HPLC Purification was achieved using a 2-column process. First a recombinant Protein-A column was equilibrated in 2 M urea, 50 mM Tris, pH 8.5 and loaded with the filtered peptibody refold reaction. The column was then washed with 2 column volumes of equilibration buffer, followed by 2 column volumes of PBS. The peptibody fraction was eluted with 50 mM NaOAc, pH3 and quickly neutralized by a 1:4 dilution into 10 mM NaOAc, 50 mM NaCl, pH 5. The diluted Protein-A eluate was again filtered and loaded to an SP Sepharose HP cation exchange column (Pharmacia) equilibrated in 10 mM NaOAc, 50 mM NaCl, pH 5. The peptibody fractions were then eluted with a linear 50-500 mM NaCl gradient, pooled and concentrated to about 2 mg/ml. The final pools of Fc-loop AMP 2 (#6875) were evaluated by SDS-PAGE (Fig. 19) and RPHPLC (Fig. 20). The final preparation of Fc-loop AMP 2 was tested in an in vivo mouse bioassay against a carboxyterminal peptibody fusion of two AMP 2 sequences linked in tandem (Fc tandem AMP2). In this comparison, the Fc-tandem-AMP2 has a total valence of four AMP 2 peptides compared to the Fc-loop AMP 2 with only two peptides. The mice received a single subcutaneous injection of 50 ng/kg of either peptibody while their platelet levels were monitored over 15 days (Fig. 21). While the Fc-tandem-AMP2 induced a significant initial platelet increase, the total response was complete by day 9. In contrast, the Fc-loop AMP 2 elicited a much smaller response that peaked at day 8 and persisted for 15 days. These results suggest that the efficacious halflife of the Fc-loop AMP 2 peptibody may be much greater than the conventional carboxy-terminal fused peptibody. The difference in overall amplitude of the response may be a consequence of the greater valence of Fc-tandem-Amp2. Example 8 Preparation of Amp2 This molecule was prepared as described above in example 7, and in U.S. Pat. No. 6,660,843. Example 9 In Vitro cell-based assay and the measurement of myostatin-signaling activity (Figure 8) To quantitate myostatin activity and its blockade, a luciferase reporter system was developed, referred to as pMARE-Luc, which senses Myostatin/Activin signaling strength. The pMARE-luc vector was constructed by subcloning a Smad-responsive CAGA tandem repeat sequence into a basic reporter plasmid pLuc-MCS containing a minimal promoter element (TATA box). The pMARE-luc vector was stably transfected into a skeletal muscle-derived C2C12 cell line (murine). Characterization of myostatin responses of the stable clones led to the identification of C2C12-based clonal reporter cell lines that were capable of detecting both myostatin and Activin signaling activities in 96- well format in a highly sensitive and reproducible manner. Example 10 In Vitro HTRF (homogeneous time-resolved fluorescence) Ang-2 binding assay (Figure 15). Starting from a concentration of 100 nM, Fc-loop peptibody and proper controls were serially diluted in liTRF buffer 3-fold, 9-times across a 96-well plate. Dilutions were then mixed with the following reagents on a 96-well black, round-bottomed assay plate: Streptavidin-Europium (1.6 nM), Biotinylated human angiopoietin-2 (8.0 nM), human Tie2-Fc-APC (10 nM). Assay plate was then incubated at room temperature with shaking for 2 hours. Plate next read on a Rubystar microplate reader (BMG Labtechnologies Inc.). Results were converted to %inhibition, and ICSOs were then calculated by analyzing the %inhibition values in the program GRAFIT 5.0 (IC50, 0-100% parameter). Example 11 In Vivo AMP-2 efficacy assay (Figure 21) Female BDF1 mice are injected subcutaneously with either carrier fluid (1 x PBS with 0.1% BSA), 50 meg/kg of Fc-tandem-AMP2, or 50 meg/kg of Fc-loop-AMP2. The injection volume is 0.2 mL. Blood is collected from each mouse via a puncture of the retro orbital sinus into a heparinized capillary tube, and then transferred to microtainers containing EDTA. Complete blood counts (CBC) including differential white blood cell counts are obtained using an AD VIA 120 blwod analyzer calibrated for mouse blood (Bayer Corp., Tarrytown, NY). Standard bleed days are 0, 3, 5, 7 and 10. Platelet counts are plotted as a function of time post-injection. Example 12 UT-7 EPO Proliferation Assay for EMP Activity (Figure 18) The UT-7Epo proliferation assay uses human megakaryoblastic leukemia cell line that responds murine EPO (mEPO) and human EPO (huEPO) or other EPO like molecules for growth and survival. Growth factors are serially diluted from 1000 ng/ml to 0.488 ng/ml in triplicate, in 100 u,l of 10% FBSIscoves Modified Dulbelcco's medium (IMDM) across the 96 well plate. 15000 cells/well are added to the 96 well plate in 100 jal of 10% FBS IMDM. The total volume per well is 200 ul of media with 15000 cells per well. Cells and media alone are the zero control. Cells are incubated in a humidified chamber at 37 °C. After incubation for 72 hours with the growth factor to be examined, viable cells are determined by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxphenyl)-2-(4-sulfophenyl)2H-tetrazolium, inner salt) incorporation (5+/-1 hr at 37 °C) and growth is measured by O.D. (490 nm absorbance), limits not 4.0 O.D. Reference: Yasusada Miura, Keita Kirito, and Norio Komatsu (1998), "Regulation of Both Erythroid and Megakaryocytic Differentiation of a Human Leukemia Cell Line, UT-7," Acta Haematologica,99:180-184. Example 13 Alternate Fc-Loop Insertion Sites Having proven the feasibility of Fc-Loop insertion-style peptibodies with the L139/T140 insertion site using several different peptides, additional loops were surveyed in the Fc crystal structure. Using Fc-domain homology modeling, twelve different potential insertion sites were selected based on solvent accessible surface exposure, steric constraints within the loop, proximity to the Fc dimer interface and juxtaposition to sites of known effector function, such as the FcRn binding interface. The Fc-Loop sites that were identified as potential insertion sites are detailed and ranked. See Figure 2A and Table 12 below. Table 12 Specific insertion sites for the human IgGl Fc sequence (Table Removed) Of these potential insertion sites, six were expressed using the TN8-19-7 peptide insert (Example 2) and evaluated for refolding efficiency and in vitro activity. An additional construct was added which contained an asymmetric linker system Gly4/Gly6 engineered into the original loop insertion site (L139/T140) previously described. In all, seven new Fc Loop Myostatin constructs were refolded, purified, and tested for activity. The seven new Fc-Loop TN8-19-7 constructs that were tested included insertions in both CH2 and CH3 domains of human IgGl Fc. Specifically, these insertions were: G201/N202 (CH3), E169/N170 (CHS), S181/D182 (CHS), H49/E50 (CH2), L139/T140 (G4-6) (CHS), Y77/N78 (CH2), and K107/A108 (CH2-CH3 linker domain). These constructs were transformed into E. coli by conventional methods known in the art, and were found to express almost exclusively in the insoluble inclusion body fraction. Interestingly, the H49/E50, L139/T140 (G4-6) and Y77/N78 constructs appeared to have the highest levels of expression. The E169/N170, S181/D182, K107/108 showed moderate expression, and the G201/202 construct showed some expression, but very little. Those Fc-Loop TN8-19-7 constructs which expressed well in E. coli were purified by first solubilizing the isolated inclusion body fractions in 6 M Guanidine-HCl, 50 mM Tris, 8 mM DTT pH 9.0 (10 mL per Ig inclusion body) at room temperature with mixing for 1 hour. Then, a variety of refolding conditions were evaluated for each of the denatured and reduced Fc-Loop TN8-19-7 constructs to identify optimal refolding conditions. The three G201/N202, E169/N170, and S181/D182 constructs did not refold well under any of the conditions tested. Of the remaining four Fc-Loop TN8-19-7 constructs, L139/T140 (G4-6) refolded the best, while the remaining three H49/E50, Y77/N78 and K107/A108 refolded with sufficient yield to pursue further purification. Using optimized refold conditions, the denatured and reduced Fc-Loop TN8-19-7 constructs were refolded from the solubilized inclusion body fractions by a 1:25 (v/v) dilution into 4 M Urea, 50 mM Tris-HCl, 0.16 M Arg-HCl, 20% glycerol, 3 M Cystine, 5 mM Cystamine pH 8.5. The solubilized peptibodies were added drop-wise to the refold buffer at 4°C with stirring. The refold reactions were allowed to stir for 72 hours, and subsequently purified chromatographically. Final purification was achieved by a 2-column chromatographic process, as described in Example 2. -The final pools of L139/T140 (G4-6), H49/E50, Y77/N78, and K107/108 were evaluated by RP-HPLC and SDS-PAGE, as illustrated in Figures 23 and 24. The yields from these four constructs are tabulated in Table 13. Table 13 (Table Removed) Among the four analogs purified the L139/T140 (G4-6) insertion site analog was produced with the best purity and in the highest yield. The purified Fc-loop insertion analogs were further analyzed for functional myostatin receptor binding activity using an in vitro cell based inhibition assay, as described in Example 9. The results are shown in Table 14. Table 14 (Table Removed) The purified Fc-loop insertion analogs were further analyzed for FcRn binding using a Biacore assay system. Sample K107/A108 was not tested due to insufficient sample remaining after analysis. The ICso values determined using the in vitro cell based Myostatin inhibition assay, were similar for the L139/T140 (G4-6) insertion and the original Fc-Loop #6951, whereas the Fc-loop insertions at H49/E50 and Y77/N78 showed a reduced ability to inhibit myostatin (Figure 30). The Biacore FcRn binding experiments showed that H49/E50 and Y77/N78 bound the Fc receptor comparably to the control (Fc-Loop-lxTN8-19-7, #6951) with an EC50 of about 680 nM/ but L139/T140 (G4-6) had a lower and more favorable EC5o at around 220 nM In summary, of the six new insertion sites evaluated, three failed to refold efficiently. Among the three new insertion site analogs recovered, K107/A108 folded poorly, H49/E50 (CH2 domain) refolded marginally well, and the two remaining insertions at Y77/N78 (CH2 domain) and the original insertion site of L139/T140 (CHS domain) with an extended, asymmetric linker folded with a higher efficiency. Interestingly, all of these novel insertion site analogs refolded with significantly lower yield than the original Fc-loop construct (#6951) with TN8-19-7 in position L139/T140 using symmetric Gly2 linkers. When tested for retention of FcRn binding capacity, all the Fc-loop molecules appeared similar in affinity with the possible exception of the extended, asymmetric linker construct, which seemed slightly better. This was consistent with the design paradigm to minimize steric interactions between the inserted peptide and the FcRn binding interface. While all the purified Fc-loop constructs were active by the in vitro, cell-based functional assay (Table 14), the original insertion site (L139/T140) and the extended, asymmetric linker insertion at the same site appeared to be the most potent. This work demonstrates that multiple loop domains within the human IgGl Fc, as identified in Figure 23, will tolerate insertion of bioactive peptides while preserving the activity of both the peptide and Fc effector functions such as FcRn binding. Peptide insertion analogs utilizing these Fc-lbop domains can vary significantly in refolding efficiency and peptide activity. Each peptide/insertion combination can be individually optimized to maximize recovery and potency. More preferable would be peptide insertions targeting the underlined sub-domains in Figure 23. Most preferable are the insertion site (L139/T140) and two additional loops in the CH2 domain (H49/E50 and Y77/N78). Abbreviations Abbreviations specific circumstances, Ac AcBpa ACN ADCC Aib bA Bpa BrAc BSA Bzl Cap CTL CTLA4 DARC DCC Dde EDTA used throughout this specification are as defined below, unless otherwise defined in acetyl (used to refer to acetylated residues) acetylatedp-benzoyl-L-phenylalanine acetonitrile antibody-dependent cellular cytotoxicity aminoisobutyric acid beta-alanine p-benzoyl-L-phenylalanine bromoacetyl (BrCH2C(O) Bovine serum albumin Benzyl Caproic acid Cytotoxic T lymphocytes Cytotoxic T lymphocyte antigen 4 Duffy blood group antigen receptor Dicylcohexylcarbodiimide l-(4,4-dimethyl-2,6-dioxo-cyclohexylidene)ethyl ethylene diamine tetraacetic acid EMP Erythropoietin-mimetic peptide ESI-MS Electron spray ionization mass spectrometry EPO Erythropoietin Fmoc fluorenylmethoxycarbonyl G-CSF Granulocyte colony stimulating factor GH Growth hormone HCT hematocrit HGB hemoglobin hGH Human growth hormone HOBt 1-Hydroxybenzotriazole HPLC high performance liquid chromatography IL interleukin IL-R interleukin receptor IL-1R interleukin-1 receptor IL-lra interleukin-1 receptor antagonist Lau Laurie acid LPS lipopolysaccharide LYMPH lymphocytes MALDI-MS Matrix-assisted laser desorption ionization mass spectrometry Me methyl MeO methoxy MES (2-[N-Morpholino]ethanesulfonic acid) MHC major histocompatibility complex MMP matrix metalloproteinase MMPI matrix metalloproteinase inhibitor NaOAc sodium acetate 1-Nap 1-napthylalanine NEUT neutrophils NGF nerve growth factor Me norleucine NMP N-methyl-2-pyrrolidinone PAGE polyacrylamide gel electrophoresis PBS Phosphate-buffered saline Pbf 2,2,4/6/7-pendamethyldihydrobenzofuran-5-sulfonyl PCR polymerase chain reaction Pec pipecolic acid PEG Polyethylene glycol) pGlu pyroglutamic acid Pic picolinic acid PLT platelets pY phosphotyrosine PTFE polytetrafluoroethylene RBC red blood cells RBS ribosome binding site RP-HPLC reversed phase HPLC RT room temperature (25 °C) Sar sarcosine SDS sodium dodecyl sulfate STK serine-threonine kinases t-Boc tert-Butoxycarbonyl tBu tert-Butyl TGF tissue growth factor THF thymic humoral factor TK tyrosine kinase TMP Thrombopoietin-mimetic peptide TNF Tissue necrosis factor TPO Thrombopoietin TRAIL TNF-related apoptosis-inducing ligand Trt trityl UK urokinase UKR urokinase receptor VEGF vascular endothelial cell growth factor VIP vasoactive intestinal peptide WBC white blood cells Amended Claims What is claimed is: 1. A composition of matter of the formula (X')n-F'-(X\ and multimers thereof, wherein: F1 is an Fc domain modified so that it comprises at least one X3 in a loop region, said loop region being in a non-terminal domain of the Fc domain; X1 and X2 are each independently selected from -(L\-P\ -(Lr)e-Pl- X3 is independently selected from -(L5)c-p5/ ~(L\-P^(L\-P6, -(L5)C-PS- P1, P1, P3, and P are each independently sequences of pharmacologically active polypep tides or pharmacologically active peptides; P5, P6, P7, and P8 are each independently sequences of pharmacologically active peptides; " L1, L2, L3, V, L5, L6, L7, and L8 are each independently linkers; and a, b, c, d, e, and f are each independently 0 or 1. 2. The composition of matter of Claim 1 wherein a and b are each 0. 3. The composition of matter of Claim 1 wherein the Fc domain comprises an IgG Fc domain. 4. The composition of matter of Claim 3 wherein the Fc domain comprises a sequence selected from SEQ ID NOS: 599 and 603 to 607. 5. The composition of matter of Claim 1 wherein the Fc domain comprises an IgGl Fc domain. 6. The composition of matter of Claim 5 wherein the IgGl Fc domain comprises SEQ ID NO: 599 and X3 is inserted into or replaces all or part of a sequence selected from SEQ ID NOS: 621,622, 624, 625, 627, 628, 630, 632,634, and 636. 7. The composition of matter of Claim 6 wherein X3 is inserted into or replaces all or part of a sequence selected from SEQ ID NOS: 623, 626, 629, 631, 633,635, and 637. 8. The composition of matter of Claim 7 wherein X3 is inserted at Leu139/Thr140 9. The composition of matter of Claim 5 wherein the IgGl Fc domain comprises SEQ ID NO: 603 and Xs is inserted into or replaces all or part of a sequence selected from SEQ ID NOS: 621, 622, 624, 625, 627, 628, 630, 632, 634, and 636. 10. The composition of matter of Claim 9 wherein X3 is inserted at 11, The composition of matter of Claim 5 wherein the IgGl Fc domain comprises SEQ ID NO: 604 and XJ is inserted into or replaces all or part of a sequence selected from SEQ ID NOS: 621, 622, 624, 625, 627, 628, 632, 634, 636, and 644. 12. The composition of matter of Claim 11 wherein X3 is inserted at NV!0/Km, Lua/Tlw Q171/POT, EJ7/N174/ S186/DW, Gm/Sm, or 13. The composition of matter of Claim 1 wherein the Fc domain comprises an IgG3 Fc domain. 14. The composition of matter of Claim 13 wherein the IgG3 Fc domain comprises SEQ ID NO: 605 and X3 is inserted into or replaces all or part of a sequence selected from SEQ ID NOS: 614, 621, 622, 624, 627, 639, 641, 644, 645, and 646. 15. The composition of matter of Claim 14 wherein X3 is inserted at G/S or G/N. 16. The composition of matter of Claim 1 wherein the Fc domain comprises an IgG2 Fc domain. 17. The composition of matter of Claim 16 wherein the Fc domain comprises SEQ ID NO: 606 and X3 is inserted into or replaces all or part of a sequence selected from SEQ ID NOS: 621, 622, 624, 632, 636, 639, 640, 642, 644, and 646. 18. The composition of matter of Claim 17 wherein X3 is inserted at H The composition of matter of Claim 1 wherein the Fc domain comprises an IgG4 Fc domain. 19. The composition of matter of Claim 19 wherein the Fc domain comprises SEQ ID NO: 607 and X3 is inserted into or replaces all or part of a sequence selected from SEQ ID NOS: 620, 621, 624, 627, 632, 634, 638, 639, 643, and 644. 20. The composition of matter of Claim 20 wherein X"* is inserted at Q/Ej,, 22. The composition of matter of Claim 1 wherein the Fc domain comprises SEQ ID NO: 608 and X3 is inserted into or replaces all or part of a sequence selected from SEQ ID NOS: 621, 622, 628, 624, 627, 632, 636, 639, 644, and 646. 23. The composition of matter of Claim 22 wherein X3 is inserted at 24. The composition of matter of Claim 1 wherein X3 comprises an angiotensin-2 (ang-2) binding peptide sequence. 25. The composition of matter of Claim 24 wherein the ang-2 binding peptide sequence is selected from SEQ ID NOS: 100 to 189. 26. The composition of matter of Claim 25 wherein the ang-2 binding peptide sequence is SEQ ID NO: 147. 27. lite composition of matter of Claim 26 wherein FJ comprises an IgGl Fc domain. 28. The composition of matter of Claim 27 having the sequence of SEQ ID NO: 613. 29. The composition of matter of Claim 1 wherein X3 comprises a myostatin binding peptide sequence. 30. The composition of matter of Claim 29 wherein the myostatin binding peptide sequence is selected from SEQ ID NOS: 218 to 509. 31. The composition of matter of Claim 30 wherein the myostatin binding peptide sequence is SEQ ID NO: 365. 32. The composition of matter of Claim 31 wherein F1 comprises an IgGl Fc domain, 33. The composition of matter of Claim 32 having the sequence SEQ ID NO: 612. 34. The composition of matter of Claim 1 wherein X'1 comprises an erythropoietin-rnimetic (EPO-mimetic) peptide sequence. 35. The composition of matter of Claim 34 wherein the EPO-mimetic peptide sequence is selected from SEQ ID NOS: 1 to 27. 36. The composition of matter of Claim 35 wherein the EPO-mimetic peptide sequence is SEQ ID NO: 2. 37. The composition of matter of Claim 36 wherein F1 comprises an IgGl Fc domain 38. The composition of matter of Claim 37 having the sequence of SEQ ID NO: 615. 39. The composition of matter of Claim 1 wherein X3 comprises a thrombopoietin-mimetic (TPO-mimetic) peptide sequence. 40. The composition of matter of Claim 39 wherein the TPO-mimetic peptide sequence is selected from SEQ ID NOS: 28 to 99. 41. The composition of matter of Claim 40 wherein the TPO-mimetic peptide sequence is SEQ ID NO: 28. 42. The composition of matter of Claim 41 wherein F' comprises an IgGl Fc domain 43. The composition of matter of Claim 42 having the sequence SEQ ID NO: 616. 44. The composition of matter of Claim 1 wherein X3 comprises a nerve growth factor (NGF) binding peptide sequence. 45. The composition of matter of Claim 44 wherein the NGF binding peptide sequence is selected from SEQ ID NOS: 190 to 218, 46. The composition of matter of Claim 1 wherein X3 comprises a 8 cell activating factor (BAFF) binding peptide sequence. 47. The composition of matter of Claim 46 wherein the BAFF binding peptide sequence is selected from SEQ ID NOS: 510 to 594. 48. A DNA encoding a composition of matter of Claim 1. 49. An expression vector comprising the DNA of Claim 48. 50. A host cell comprising the expression vector of Claim 49. 51. The cell of Claim 50, wherein the cell is an E. coll cell, 52. A process for preparing a pharmacologically active compound, which comprises: a. selecting at least one randomized peptide that modulates the activity of a protein of interest; and b. preparing a pharmacologic agent comprising an amino acid sequence of the selected peptide as an internal, non-terminal, sequence of an Fc domain. 53. The process of Claim 52, wherein the internal region of the Fc domain is a loop region. 54. The process of Claim 52, wherein the peptide is selected in a process comprising one or more techniques selected from yeast-based screening, rational design, protein structural analysis, or screening of a phage display library, an E. coli display library, a ribosomal library, or a chemical peptide library. 55. The process of Claim 52, wherein the preparation of the pharmacologic agent is carried out by: a. preparing a gene construct comprising a nucleic acid sequence encoding an Fc domain wherein the amino acid sequence of the selected peptide is inserted into or replaces one or more amino acids within the Fc domain; and b. expressing the gene construct. 56. The process of Claim 55, wherein the gene construct is expressed in an E. coli cell, 57. The process of Claim 52, wherein the protein of interest is a cell surface receptor, 58. The process of Claim 52, wherein the protein of interest has a linear epitope. 59. The process of Claim 52, wherein the protein of interest is a cytokine receptor. 60. The process of Claim 52 wherein the Fc domain is an IgG Fc domain. 61. A modified antibody, comprising an Fc domain modified so that it comprises at least one X3 in a loop region, said loop region being in a non-terminal domain of the Fc domain, wherein: X3 is independently selected from -(L5)C-P5, -(LS)C-P6-(L6)U -P6, -(L5)C-P5- P5, P", P7, and P8 are each independently sequences of pharmacologically active pepticles; L5, L6, L7, and L8 are each independently linkers; and c, d, e, and f are each independently 0 or 1. 62. A process for preparing a modified antibody, which comprises: a) selecting at least one peptide that modulates the activity of a protein of interest; and b) preparing an antibody comprising an amino acid sequence of the selected peptide in a loop region of an Fc domain of the antibody, said loop region being in a non-terminal domain of the Fc domain. 63. A composition substantially as herein described with reference to the foregoing description, tables, examples and the accompanying drawings. 64. A DMA encoding substantially as herein described with reference to the foregoing description, tables, examples and the accompanying drawings. 65. A host cell substantially as herein described with reference to the foregoing description, tables, examples and the accompanying drawings. 66. A process substantially as herein described with reference to the foregoing description, tables, examples and the accompanying drawings.

Full Text

Modified Fc Molecules
This application claims the benefit of U.S. Provisional Application No. 60/612,680, filed September 24,
2004, which is hereby incorporated by reference.
Background of the Invention
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 halflife,
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
(Table Removed)
Linslev(1991). J. EXD. Med. 174:561-9
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").
Phage display peptide libraries have emerged as a powerful method in identifying such peptide
agonists and antagonists. See, for example, Scott etal. (1990), Science 249: 386; Devlin etal. (1990), Science 249:
-2.-
404-; U.S. Pat. No. 5,223,409, issued June 29, 1993; U.S. Pat. No. 5,733,731, issued March 31, 1998; U.S. Pat. No.
5,498,530, issued March 12, 1996; U.S. Pat. No. 5,432,018, issued July 11, 1995; U.S. Pat. No. 5,338,665, issued
August 16,1994; U.S. Pat. No. 5,922,545, issued July 13,1999; WO 96/40987, published December 19,1996; and
WO 98/15833, published April 16,1998 (each of which is incorporated by reference). In such libraries, randompeptide 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-
Other methods compete with phage display in peptide research. A peptide library can be fused to the
carboxyl terminus of the lac represser 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 Damino
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.
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.
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.
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.
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
(Table Removed)
linear GLP-1 Diabetes, metabolic syndrome
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
th-i^eticuloendothelial 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 etal. (1998), Drug Disc. Today 3: 370-8.
A more recent development is fusion of randomly generated peptides with the Fc domain. See U.S. Pat.
No. 6,660,843, issued December 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 Nterminus,
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, tumorhoming
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 Sept. 18, 2003 (corresponding to WO
03/031589, published April 17, 2003); U.S. Ser. No. 09/422,838, filed October 22, 1999 (corresponding to WO
00/24770, published May 4, 2000); U.S. Pat. App. No. 2003/0229023, published December 11, 2003; WO
03/057134, published July 17, 2003; U.S. Pat. App. No. 2003/0236193, published December 25, 2003
(corresponding to PCT/US04/010989, filed April 8, 2004); U.S. Ser. No. 10/666,480, filed September 18, 2003
(corresponding to WO 04/026329, published April 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.
Summary of the Invention
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:
a) selecting at least one peptide that modulates the activity of a protein of interest; and
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.,
adadimumab, 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).
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.
The Fc internal peptide molecules may include more than one peptide sequence in tandem in
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.
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.
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.
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.
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.
Numerous additional aspects and advantages of the present invention will become apparent upon
consideration of the figures and detailed description of the invention.
Description of the Figures
Figures 1A, IB 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 CHS domains of Fc, the loop regions may be
considered any part of the model not shown as p-sheet (flat arrows) or a-helix (cylinder).
Figure 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 CHS domain Fc loop insertion site specifically identified.
Figure IB shows the monomeric murine IgGl Fc domain (Protein Database file #1IGY). This figure
shows a three-dimensional model of murine IgGl Fc domain monomer from x-ray diffraction crystal structure
(pdb #1IGY). Potential Fc loop insertion sites are shown for both CH2 and CHS domains with the preferred
CH3 domain Fc loop insertion site specifically identified.
Figure 1C shows the monomeric human IgGl Fc domain (Protein Database file #1H3T). This figure
shows a three-dimensional model of human IgGl Fc domain monomer from x-ray diffraction crystal structure
(pdb #1H3T). Potential Fc loop insertion sites are shown for both CH2 and CHS domains with the preferred
CH3 domain Fc loop insertion site specifically identified.
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/).
Figure 2A shows a sequence of human IgGl Fc sequence (SEQ ID NO: 599) used for peptibody fusions
with predicted loop sequences in boldface. Figure 2A shows, in the context of the human IgGl 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 Figures 1A, IB 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 Leui39 and Thr14o 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 Figures 2B and 2C.
Figures 2B and 2C show a sequence alignment of human Fc domains from IgA, IgM and IgG
subclasses. Figures 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 Figures 2B and
2C is a consensus sequence (SEQ ID NO: 608).
Figures 2B and 2C also show in boldface the preferred internal sites for peptide addition that
correspond to those of the Fc sequence shown in Figure 2A (SEQ ID NO: 599). In particular, Figures 2B and 2C
show as such preferred sites the following:
• SEQ ID NO: 621 as shown in boldface within SEQ ID NOS: 603 to 608;
• SEQ ID NO: 622 within SEQ ID NOS: 603 to 606 and 608;
• SEQ ID NO: 638 within SEQ ID NO: 607;
. SEQ ID NO: 624 within SEQ ID NO: 603 to 608;
• SEQ ID NO: 625 within SEQ ID NOS: 603 and 604;
• SEQ ID NO: 639 within SEQ ID NOS: 605 to 608;
• SEQ ID NO: 627 within SEQ ID NOS: 603 to 605,607, and 608;
• SEQ ID NO: 640 within SEQ ID NO: 606;
• SEQ ID NO: 628 within SEQ ID NOS: 603,604, and 608;
• SEQ ID NO: 641 within SEQ ID NO: 605;
• SEQ ID NO: 642 within SEQ ID NO: 606;
• SEQ ID NO: 643 within SEQ ID NO: 607;
t SEQ ID NO: 630 within SEQ ID NO: 603;
• SEQ ID NO: 644 within SEQ ID NOS: 604 to 608;
• SEQ ID NO: 632 within SEQ ID NOS: 603,604, 606, 607, and 608;
• SEQ ID NO: 645 within SEQ ID NO: 605;
• SEQ ID NO: 634 within SEQ ID NOS: 603, 604, and 607;
• SEQ ID NO: 646 within SEQ ID NOS: 605, 606 and 608;
• SEQ ID NO: 636 within SEQ ID NOS: 603,604, 606, and 608;
• SEQ ID NO: 614 within SEQ ID NO: 605; and
• SEQ ID NO: 620 within SEQ ID NO: 607.
The sequence alignments of Figures 2B and 2C suggest two more potential insertion sites at Qi67/Pi68
and/or G183/S184 (using the numbering of SEQ ID NO: 599 in Figure 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 Figure 2A. The preferred insertion sites are
underlined Figures 2B and 2C and are as follows:
• H53/ E54 in SEQ ID NOS: 603 and 604;
• H100/E101in SEQ ID NO: 605;
• H49/E5o in SEQ ID NO: 606;
• Q50/E51 in SEQ ID NO: 607;
• H112/E113 in SEQ ID NO: 608;
• Y81/N82 in SEQ ID NOS: 603 and 604;
• F128/N129 in SEQ ID NO: 605;
• F77/N78 in SEQ ID NO: 606;
• F78/N79 in SEQ ID NO: 607;
• F140/N141 in SEQ ID NO: 608;
• N110/K111 in SEQ ID NOS: 603 and 604;
• N157/K158 in SEQ ID NO: 605;
• N106 /K107 in SEQ ID NO: 606;
• N107K108 in SEQ ID NO: 607;
• N169K170 in SEQ ID NO: 608;
• L143/T144 in SEQ ID NOS: 603 and 604;
• M190/T191 in SEQ ID NO: 605;
• M139 /T140 in SEQ ID NO: 606;
. M140WT141 in SEQ ID NO: 607;
• M204/T2o5 in SEQ ID NO: 608;
• Q171P172 in SEQ ID NOS: 603 and 604;
• Q218/P219 in SEQ ID NO: 605;
• Q167//P168 in SEQ ID NO: 606;
• Q168/P169 in SEQ ID NO: 607;
• Q232/P233 in SEQ ID NO: 608;
• E173N174 in SEQ ID NOS: 603 and 604;
• E220/N221 in SEQ ID NO: 605;
• E169/N170 in SEQ ID NO: 606;
• E170/N171 in SEQ ID NO: 607;
• E234/N235 in SEQ ID NO: 608;
• S186/D187 in SEQ ID NOS: 603 and 604;
• S232/D233 in SEQ ID NO: 605;
• S181/D182 in SEQ ID NO: 606;
• S182/D183 in SEQ ID NO: 607;
• S246/D247 in SEQ ID NO: 608;
• G188/S189 in SEQ ID NOS: 603 and 604;
• G234/S235 in SEQ ID NO: 605;
• G183/S184 in SEQ ID NO: 606;
• G184/S185 in SEQ ID NO: 607;
• G248/S249 in SEQ ID NO: 608;
• G205 / N206 in SEQ ID NOS: 603 and 604;
• G2S2/N253 in SEQ ID NO: 605;
• G201/N202 in SEQ ID NO: 606;
• G202/N203 in SEQ ID NO: 607; and
• G268/N269 in SEQ ID NO: 608.
Figure 2D shows an alignment of human IgGl Fc domain (Amgen Fc, SEQ ID NO: 609) used for the
peptibody platform with rat IgG2A from crystal structure of FcRn/Fc complex (UlA.pdb, SEQ ID NO: 610 The
resulting consensus sequence (SEQ ID NO: 611) is also shown.
Figure 3A shows the amino acid sequence (SEQ ID NO: 612) of a human IgGl 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
lirmers in italics.
Figure 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.
Figure 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.
Figure 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.
Figure 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.
Figures 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).
Figure 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 ng of
peptibody was loaded directly from a refold reaction to a Vydak C4 column (5 uM, 300 angstrom, 4.6 x 250
mm) and eluted with a linear 40-50% ACN gradient at 0.5 %/min.
Figure 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
Hg purified peptibody to Vydak C4 column (5 uM, 300 angstrom, 4.6 x 250 mm) and eluted with a linear 40-
50% ACN gradient at 0.5 %/min.
Figure 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 ng 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).
Figure 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).
Figure 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 Fcloop
vs. carboxy terminal peptibodies respectively at 24 hours. Lanes 8 & 9 are the Fc-looo vs. carboxv
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.
Figure 10A shows the amino acid sequence (SEQ ID NO: 618) of a human IgGl 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.
Figure 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.
Figure 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.
Figure 12 shows a RP-HPLC comparison of Fc-loop Ang2 (#6888) and carboxy-terminal Fc TN8-19-07
(#5564) refold reactions. Loaded 20 jal refold reaction to Vydak C4 column (5 ^M, 300 angstrom, 4.6 x 250 mm)
and eluted with a linear 40-50% ACN gradient at 0.5 %/min.
Figure 13 shows a RP-HPLC comparison of the final purified pools of Fc-loop Ang2 (#6888) and
carboxy-terminal Fc TN8-Con4 (#5564). Ten ng purified peptibody was loaded to a Vydak C4 column (5 M,
300 angstrom, 4.6 x 250 mm) and eluted with a linear 40-50% ACN gradient at 0.5 %/min.
Figure 14 shows purified Fc-loop-myo7 and TN8-19-7.
Figure 15 shows Biacore binding analysis of Fc-loop-ang2 and Fc-ang2-tandem.
Figure 16 shows the results of an in vitro enzyme-linked immunosorbent assay (ELISA) for Fc-loopang2,
TN8-Con4, and Fc-ang2-tandem.
Figure 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.
Figure 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.
Figure 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 ng Fc-loop AMP 2 peptibody; lane 4 with 5 ng reduced Fc-loop AMP 2
peptibody; lanes 1 and 3 with SeeBlue and two molecular weight markers.
Figure 20 shows an RP-HPLC analysis of the final purified pool of Fc-loop AMP 2 (#6875). Ten ng
purified peptibody was loaded to Vydak C4 column (5 \\M., 300 angstrom, 4.6 x 250 mm) and eluted with a
linear 40-50% ACN gradient at 0.5 %/min.
Figure 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 ng/kg peptibody or carrier alone. See example 9 for assay
methodology.
Figure 22 shows several strategies for incorporating 2 bioactive peptides into an Fc-loop peptibody.
shows SDS-PAGE Gels of purified Fc-loop constructs. Samples (2ug/lane) were run +/- reducing
buffer on a 4-20% Tris-Glycine SDS-PAGE gel.
Figure 24 shows RP-HPLC of Fc-loop constructs. '
Detailed Description of the Invention
Definition of Terms
The terms used throughout this specification are defined as follows, unless otherwise limited in
specific instances.
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.
"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.
The term "native Fc" refers to a molecule or sequence comprising the sequence of a non-antigenbinding
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 IgGl 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., IgGl, IgG2, IgG3, IgAl,
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.
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
September 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.
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.
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.
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, Figure 2, which is hereby incorporated by reference.
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 Cterminus
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.
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-lra, leptin,
soluble TNF receptors type 1 and type 2 (sTNF-Rl, sTNF-R2), KGF, EPO, TPO, G-CSF, darbepoietin, Fab
fragments and the like.
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.
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.
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.
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), Froc. 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.
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.
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 Sept. 18, 2003; WO 03/031589, published April 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.
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 7/17/03; 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.
The term "NGF-binding peptide" comprises peptides that can be identified or derived as described in
WO 04/026329, published April 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.
The term "myostatin-binding peptide" comprises peptides that can be identified or derived as
described in U.S. Ser. No. 10/742,379, filed December 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.
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.
Structure of compounds
In General
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:
wherein:
F1 is an Fc domain modified so that it comprises at least one X3 in a loop region;
Xi and X2 are each independently selected from -(Li)c-P1, -(Li)c-P1-(L2)d -P2, -(L^c-PML^-
X3 is independently selected from -(L5)C-P5, -(L5)c-P5-(L6)d -P*, -(L5)c-P5-(L6)d-p6-(l7)e-P7, and -(L5)C-P5-
(L6)d-P6-(I7)e -P7-(L«)f-P8;
P1, P2/ P3, and P4 are each independently sequences of pharmacologically active polypeptides or
pharmacologically active peptides;
P5, P6, P7, and P8 are each independently sequences of pharmacologically active peptides;
L1, L2, L3, L4, L5, L6, U, and L8 are each independently linkers; and
a, b, c, d, e, and f are each independently 0 or 1.
In preferred embodiments, a and b are both zero—i.e., neither X1 nor X2 groups appear at the Nterminus
or C-terminus of the Fc domain.
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.
Thus, compound I comprises compounds of the formulae
and multimers thereof wherein F1 is attached at the C-terminus of X1;
and multimers thereof wherein F1 is attached at the N-terminus of X2;
and multimers thereof wherein F1 is attached at the N-terminus of -(UJc-P1; and
and multimers thereof wherein F1 is attached at the N-terminus of -L1-P1-L2-P2.
Feptides
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.
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. J. 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.
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
(Table Removed)
Other 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.
3 TNF receptors use multiple, distinct intracellular molecules for signal transduction including "death domain" of FAS R and 55
kDa TNF-aR 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.
4 The Duffy blood group antigen (DARC) is an erythrocyte receptor that can bind several different chemokines! IL-1 R belongs to
the immunoglobulin superfamily but their signal transduction events characteristics remain unclear.
5 The neurotrophic cytokines can associate with NGF/TNF receptors also.
Particular proteins of interest as targets for peptide generation in the present invention include the
following:
(Table Removed)
splice variants of molecules preferentially expressed on tumor cells; e.g., CD44, CD30
unglycosylated variants of mucin and Lewis Y surface glycoproteins :
CD19, CD20, CD33, CD45
prostate specific membrane antigen and prostate specific cell antigen
matrix metalloproteinases (MMPs), both secreted and membrane-bound (e.g., MMP-9)
Cathepsins
angiopoietin-2
TIE-2 receptor
heparanase
urokinase plasminogen activator (UPA), UFA receptor
parathyroid hormone (PTH), parathyroid hormone-related protein (PTHrP), PTH-RI, PTH-
Insulin
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 July 17, 2003; U.S. 2003/0236193, published Dec. 25, 2003; WO
00/24770, published May 4,2000; U.S. 2003/0176352, published Sept. 18,2003; WO 03/031589, published April
17, 2003; U.S. Ser. No. 10/666,480, filed September 18, 2003; WO 04/026329, published April 1, 2004; U.S. Ser.
No. 10/742,379, filed December 19, 2003; PCT/US03/40781, filed December 19, 2003, each of which are hereby
incorporated by reference. Such peptides may be prepared by methods disclosed in the art.
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
6 STKS may encompass many other TGF-p-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.
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
(Table Removed)

This invention requires the presence of at least one Fc domain modified to comprise a peptide
sequence.
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
combining 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:
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.
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 (Figure 2A) is one such Fc variant.
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.
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).
5. Sites involved in interaction with complement, such as the Clq binding site, are removed. For example,
one may delete or substitute the EKK sequence of human IgGl. Complement recruitment may not be
advantageous for the molecules of this invention and so may be avoided with such an Fc variant.
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.
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 IgGl. These sites, as well, are not required for the fusion
molecules of the present invention and so may be removed.
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.
Preferred Fc variants include the following. In SEQ ID NO: 599 (Figure 2A) the leucine at position 15
may be substituted with glutamate; 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.
Additional vehicles 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. Patent 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 US Patent No. 6, 133, 426 to
Gonzales et al., issued October 17,2000.
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.
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).
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, Figures 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.
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 al-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 tor 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.
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 April 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
hurnanization).
Linkers
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)s), poly(Gly-Ala), and polyalanines.
Other specific examples of linkers are:
(Gly)3Lys(Gly)4 (SEQ ID NO: 595);
(Gly)3AsnGlySer(Gly)2 (SEQ ID NO: 596);
(Gly)3Cys(Gly)4 (SEQ ID NO: 597); and
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.
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., Ci-Ce) lower acyl, halogen (e.g., Cl, Br), CN, NH2, phenyl, etc. An exemplary nonpeptide
linker is a PEG linker,
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.
Derivatives
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.
Exemplary derivatives include compounds in which:
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.
For citations to references on preparation of cyclized derivatives, see Table 2.
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.
3. One or more peptidyl [-C(O)NR-] linkages (bonds) is replaced by a non-peptidyl linkage. Exemplary nonpeptidyl
linkages are -CH2-carbamate [-CH2-OC(O)NR-], phosphonate , -CHb-sulfonamide [-CHa-
S(O)2NR-], urea [-NHC(O)NH-], -CH2-secondary amine, and alkylated peptide [-C(O)NR6- wherein R6 is
lower alkyl].
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 -NFfr), -NRQOJR1,
-NRC(O)OR1, -NRS(O)2R1, -NHC(O)NHR!, 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.
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-C4alkyl).
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), T. Med. Chem. 39: 3814-9; Alberts et al. (1993) Thirteenth Am. Pep. Symp..
357-9.
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.
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-aminocontaining
residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal;
chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed
reaction with glyoxylate.
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.
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.
Carboxyl sidechain groups (aspartyl or glutamyl) may be selectively modified by reaction with
carbodiimides (R'-N=C=N-R') such as l-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or l-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.
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.
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), T. Med. Chem. 39: 3814-9.
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., l,l-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-l,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.
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
Thx) 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.
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). , ..
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.
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.
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 mole.cules 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).
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.
Useful conjugation partners include:
• radioisotopes, such as 90Yttrium, 131Iodine, 225Actinium, and 213Bismuth;
• ricin A toxin, microbially derived toxins such as Pseudomonas endotoxin (e.g., PE38, PE40), and
the like;
• partner molecules in capture systems (see below);
• biotin, streptavidin (useful as either partner molecules in capture systems or as tracers, especially
for diagnostic use); and
• cytotoxic agents (e.g., doxorubicin).
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.
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.
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 posttranslational
modifications resulting from their rapid proliferation. Thus, one aspect of this invention is a
process comprising:
a) selecting at least one randomized peptide that specifically binds to a target epitope; and
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.
Variants
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.
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.
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 (Met1-peptibody) are contemplated, as are specific binding agents
with additional methionine and lysine residues at positions -2 and -1 (Met1-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.
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.
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).
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.
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, MA), the FLAG system (IBI,
New Haven, CT), and the 6xHis system (Qiagen, Chatsworth, CA). These systems are capable of producing
recrambinant 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.
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, MA).
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. Patent 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.
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.
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.
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 T. Applied Math., 48:1073 (1988).
- 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, WI, BLASTP, BLASTN, and FASTA (Altschul et al.. T. 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.
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.
For example, using the computer algorithm GAP (Genetics Computer Group, University of Wisconsin,
Madison, WI), 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 3X 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., Froc. Natl. Acad. Sci USA, 89:10915-10919 (1992) for the BLOSUM 62
comparison matrix) is also used by the algorithm.
In certain embodiments, the parameters for a polypeptide sequence comparison include the following:
Algorithm: Needleman et al.. T. Mol. Biol.. 48:443-453 (1970);
Comparison matrix: BLOSUM 62 from Henikoff et al., supra (1992);
Gap Penalty: 12
Gap Length Penalty: 4
Threshold of Similarity: 0
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.
In certain embodiments, the parameters for polynucleotide molecule sequence (as opposed to an
amino acid sequence) comparisons include the following:
Algorithm: Needleman et al., supra (1970);
Comparison matrix: matches = +10, mismatch = 0
Gap Penalty: 50
Gap Length Penalty: 3
The GAP program may also be useful with the above parameters. The aforementioned parameters are
the default parameters for polynucleotide molecule comparisons.
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, proteinto-
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).
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.
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 Xi-X2-Xs would be Xa-X2-Xi. 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.
Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids, unnatural amino acids
such as a-, 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, Nethylglycine,
N-ethylaspargine, hyroxylysine, allo-hydroxylysine, hydroxyproline, isodesmosine, alloisoleucine,
N-methylglycine, sarcosine, N-methylisoleucine, N-methylvaline, norvaline, norleucine, orithine, 4-
hycftfoxyproline, y-carboxyglutamate, E-N,N,N-trimethyllysine, e-N-acetyllysine, O-phosphoserine, Nacetylserine,
N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, ci-N-methylarginine, and other similar
amino acids and amino acids (e.g., 4-hydroxyproline).
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".
It will be appreciated that amino acid residues can be divided into classes based on their common side
chain properties:
1. Neutral Hydrophobic: Alanine (Ala; A), Valine (Val; V), Leucine (Leu; L), Isoleucine (He; I), Proline
(Pro; P), Tryptophan (Trp; W), Phenylalanine (Phe; F), and Methionine (Met, M).
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.
3. Acidic: Aspartic Acid (Asp; D), Glutamic Acid (Glu; E);
4) Basic: Lysine (Lys; K), Arginine (Arg; R), Histidine (His; H).
See Lewin, B., Genes V, Oxford University Press (1994), p.11.
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.
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). ,
The importance of the hydropathic amino acid index in conferring interactive biological function on a
protein is understood in the art. Kyte et al., T. 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.
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.
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."
Exemplary amino acid substitutions are set forth in Table 10 below.
Table 10
Amino Acid Substitutions
Original Residues
(Table Removed)
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.
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.
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.
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(l):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.
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)).
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.
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.
Affinity Maturation
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.
Non-Peptide Analogs/Protein Mimetics
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. Fept. 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 Cterminal
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. Covalentlybound
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.
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.
Patent 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. Patent 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.
Methods of Making
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.
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.
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.
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, biosafety
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.
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.
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. Folypeptides, pp. 335-61 (Katsoyannis and Panayotis eds.); Merrifield (1963), T. 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.
Compounds that contain derivatized peptides or which contain non-peptide groups may be
synthesized by well-known organic chemistry techniques.
Uses of the Compounds
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.
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.
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.
Therapeutic uses of TFO-mimetic compounds
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.
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.
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.
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.
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.
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: WO95/26746; WO95/21919; WO95/18858; WO95/21920 and are incorporated herein.
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.
Therapeutic uses of Ang-2 binding molecules
Agents that modulate Ang-2 binding activity, or other cellular activity, may be used in combination
with other therapeutic agents to enhance their therapeutic effects or decrease potential side effects.
In one aspect, the present invention provides reagents and methods useful for treating diseases and
conditions characterized by undesirable or aberrant levels of Ang-2 activity in a cell. These diseases include
cancers, and other hyperproliferative conditions, such as hyperplasia, psoriasis, contact dermatitis,
immunological disorders, and infertility.
The present invention also provides methods of treating cancer in an animal, including humans,
comprising administering to the animal an effective amount of a specific binding agent, such as a peptibody,
that inhibits or decreases Ang-2 activity. The invention is further directed to methods of inhibiting cancer cell
growth, including processes of cellular proliferation, invasiveness, and metastasis in biological systems.
Methods include use of a compound of the invention as an inhibitor of cancer cell growth. Preferably, the
methods are employed to inhibit or reduce cancer cell growth, invasiveness, metastasis, or tumor incidence in
living animals, such as mammals. Methods of the invention are also readily adaptable for use in assay
systems, e.g., assaying cancer cell growth and properties thereof, as well as identifying compounds that affect
cancer cell growth.
The cancers treatable by methods of the present invention preferably occur in mammals. Mammals
include, for example, humans and other primates, as well as pet or companion animals such as dogs and cats,
laboratory animals such as rats, mice and rabbits, and farm animals such as horses, pigs, sheep, and cattle.
Tumors or neoplasms include growths of tissue cells in which the multiplication of the cells is
uncontrolled and progressive. Some such growths are benign, but others are termed malignant and may lead
to death of the organism. Malignant neoplasms or cancers are distinguished from benign growths in that, in
addition to exhibiting aggressive cellular proliferation, they may invade surrounding tissues and metastasize.
Moreover, malignant neoplasms are characterized in that they show a greater loss of differentiation (greater
dedifferentiation), and of their organization relative to one another and their surrounding tissues. This
property is also called "anaplasia."
Neoplasms treatable by the present invention also include solid tumors, i.e., carcinomas and sarcomas.
Carcinomas include those malignant neoplasms derived from epithelial cells that infiltrate (invade) the
surrounding tissues and give rise to metastases. Adenocarcinomas are carcinomas derived from glandular
tissue, or which form recognizable glandular structures. Another broad category or cancers includes
sarcomas, which are tumors whose cells are embedded in a fibrillar or homogeneous substance like embryonic
connective tissue. The invention also enables treatment of cancers of the myeloid or lymphoid systems,
including leukemias, lymphomas and other cancers that typically do not present as a tumor mass, but are
distributed in the vascular or lymphoreticular systems.
The ang-2 binding molecules of this invention are thus useful for the treatment of a wide variety of
cancers, including solid tumors and leukemias. Types of cancer or tumor cells amenable to treatment
according to the invention include, for example, ACTH-producing tumor; acute lymphocytic leukemia; acute
nonlymphocytic leukemia; adenoma; cancer of the adrenal cortex; adenocarcinoma of the breast, prostate, and
colon; ameloblastoma; apudoma; bladder cancer; brain cancer; branchioma; breast cancer; all forms of
bronchogenic carcinoma of the lung; carcinoid heart disease; carcinoma (e.g.. Walker, basal cell,
basosquamous, Brown-Pearce, ductal, Ehrlich tumor, Krebs 2, merkel cell, mucinous, non-small cell lung, oat
cell, papillary, scirrhous, bronchiolar, bronchogenic, squamous cell, and transitional cell); malignant carcinoid
syndrome; immunoproliferative small lung cell carcinoma; cementoma; cervical cancer; chondroblastoma;
chondroma; chondrosarcoma; choristoma; chronic lymphocytic leukemia; chronic myelocytic leukemia;
colorectal cancer; chordoma; craniopharyngioma; cutaneous T-cell lymphoma; dysgerminoma; endometrial
cancer; esophageal cancer; Ewing's sarcoma; fibroma; fibrosarcoma; gallbladder cancer; giant cell tumors;
glioma; hairy cell leukemia; hamartoma; head and neck cancer; hepatoma; histiocytic disorders; histiocytosis;
Hodgkin's lymphoma; Kaposi's sarcoma; kidney cancer; lipoma; liposarcoma; liver cancer; lung cancer (small
and non-small cell); malignant peritoneal effusion; malignant pleural effusion; melanoma; mesenchymoma;
mesonephroma; mesothelioma; multiple myeloma; myosarcoma; myxoma; myxosarcoma; neuroblastoma;
non-Hodgkin's lymphoma; odontoma; osteoma; osteosarcoma; ovarian cancer; ovarian (germ cell) cancer;
pancreatic cancer; papilloma; penile cancer; plasmacytoma; prostate cancer; reticuloendotheliosis;
retinoblastoma; skin cancer; soft tissue sarcoma; squamous cell carcinomas; stomach cancer; teratoma;
testicular cancer; thymoma; thyroid cancer; trophoblastic neoplasms; uterine cancer; vaginal cancer; cancer of
the vulva; Wilms' tumor.
Further, the following types of cancers may also be treated: cholangioma; cholesteatoma; cyclindroma;
cystadenocarcinoma; cystadenoma; granulosa cell tumor; gynandroblastoma; hidradenoma; islet cell tumor;
Leydig cell tumor; papilloma; Sertoli cell tumor; theca cell tumor; leiomyoma; leiomyosarcoma; myoblastoma;
myoma; myosarcoma; rhabdomyoma; rhabdomyosarcoma; ependymoma; ganglioneuroma; glioma;
medulloblastoma; meningioma; neurilemmoma; neuroblastoma; neuroepithelioma; neurofibroma; neuroma;
paraganglioma; paraganglioma nonchromaffin; angiokeratoma; angiolymphoid hyperplasia with eosinophilia;
angioma sclerosing; angiomatosis; glomangioma; hemangioendothelioma; hemangioma;
hemangiopericytoma; hemangiosarcoma; lymphangioma; lymphangiomyoma; lymphangiosarcoma;
pinealoma; carcinosarcoma; chondrosarcoma; cystosarcoma phyllodes; fibrosarcoma; hemangiosarcoma;
leiomyosarcoma; leukosarcoma; liposarcoma; lymphangiosarcoma; myosarcoma; myxosarcoma; ovarian
carcinoma; rhabdomyosarcoma; sarcoma; neoplasms; nerofibromatosis; and cervical dysplasia.
Therapeutic uses of NGF binding molecules
The NGF binding molecules may be used in the prevention or treatment of NGF-related diseases and
disorders. Such indications include but are not limited to pain (including, but not limited to, inflammatory pain and
associated hyperalgesia and allodynia, neuropathic pain and associated hyperalgesia and allodynia, diabetic
neuropathy pain, causalgia, sympathetically maintained pain, deafferentation syndromes, acute pain, tension
headache, migraine, dental pain, pain from trauma, surgical pain, pain resulting frorn amputation or abscess,
causalgia, demyelinating diseases, and trigeminal neuralgia). The peptides and modified peptides of the invention
have therapeutic value for the prevention or treatment of other diseases linked to NGF as a causative agent,
including, but not limited to, asthma, urge incontinence (i.e., hyperactive bladder), psoriasis, cancer (especially,
pancreatic cancer and melanoma), chronic alcoholism, stroke, thalamic pain syndrome, diabetes, acquired immune
deficiency syndrome ("AIDS"), toxins and chemotherapy, general headache, migraine, cluster headache, mixedvascular
and non-vascular syndromes, general inflammation, arthritis, rheumatic diseases, lupus, osteoarthritis,
inflammatory bowel disorders, inflammatory eye disorders, inflammatory or unstable bladder disorders, psoriasis,
skin complaints with inflammatory components, sunburn, carditis, dermatitis, myositis, neuritis, collagen vascular
diseases, chronic inflammatory conditions, asthma, epithelial tissue damage or dysfunction, herpes simplex,
disturbances of visceral motility at respiratory, genitourinary, gastrointestinal or vascular regions, wounds, burns,
allergic skin reactions, pruritis, vitiligo, general gastrointestinal disorders, colitis, gastric ulceration, duodenal ulcers,
vasomotor or allergic rhinitis, or bronchial disorders.
Therapeutic uses of myostatin binding molecules
The myostatin binding agents of the present invention bind to myostatin and block or inhibit
myostatin signaling within targeted cells. The present invention provides methods and reagents for reducing
the amount or activity of myostatin in an animal by administering an effective dosage of one or more
myostatin binding agents to the animal. In one aspect, the present invention provides methods and reagents
for treating myostatin-related disorders in an animal comprising administering an effective dosage of one or
more binding agents to the animal. These myostatin-related disorders include but are not limited to various
forms of muscle wasting, as well as metabolic disorders such as diabetes and related disorders, and bone
degenerative diseases such as osteoporosis.
As shown in the Example 8 of U.S. Ser. No. 10/742,379, exemplary peptibodies of the present invention
dramatically increases lean muscle mass in the CD1 nu/nu mouse model. This in vivo activity correlates to
the in vitro binding and inhibitory activity described below for the same peptibodies.
Muscle wasting disorders include dystrophies such as Duchenne's muscular dystrophy, progressive
muscular dystrophy, Becker's type muscular dystrophy, Dejerine-Landouzy muscular dystrophy, Erb's
muscular dystrophy, and infantile neuroaxonal muscular dystrophy. For example, blocking myostatin
through use of antibodies in vivo improved the dystrophic phenotype of the mdx mouse model of Duchenne
muscular dystrophy (Bogdanovich et al. (2002), Nature 420: 28). Use of an exemplary peptibody increases
lean muscle mass and increases the ratio of lean muscle to fat in mdx mouse models as described in Example 9
below.
Additional muscle wasting disorders arise from chronic disease such as amyotrophic lateral sclerosis,
congestive obstructive pulmonary disease, cancer, AIDS, renal failure, and rheumatoid arthritis. For example,
cachexia or muscle wasting and loss of body weight was induced in athymic nude mice by a systemically
administered myostatin (Zimmers et al., supra). In another example, serum and intramuscular concentrations
of myostatin-immunoreactive protein was found to be increased in men exhibiting AIDS-related muscle
wasting and was inversely related to fat-free mass (Gonzalez-Cadavid et al. (1998), PNAS USA 95: 14938-
14943). Additional conditions resulting in muscle wasting may arise from inactivity due to disability such as
confinement in a wheelchair, prolonged bedrest due to stroke, illness, bone fracture or trauma, and muscular
atrophy in a microgravity environment (space flight). For example, plasma myostatin immunoreactive protein
was found to increase after prolonged bedrest (Zachwieja et al. J Gravit Physiol. 6(2):11(1999). It was also
found that the muscles of rats exposed to a microgravity environment during a space shuttle flight expressed
an increased amount of myostatin compared with the muscles of rats which were not exposed (Lalani et al.
(2000), T.Endocrin. 167 (3):417-28).
In addition, age-related increases in fat to muscle ratios, and age-related muscular atrophy appear to
be related to myostatin. For example, the average serum myostatin-immunoreactive protein increased with
age in groups of young (19-35 yr old), middle-aged (36-75 yr old), and elderly (76-92 yr old) men and women,
while the average muscle mass and fat-free mass declined with age in these groups (Yarasheski et al. J Nutr
Aging 6(5):343-8 (2002)). It has also been shown that myostatin gene knockout in mice increased myogenesis
and decreased adipogenesis (Lin et al. (2002), Biochem Biophys Res Commun 291(3):701-6, resulting in adults
with increased muscle mass and decreased fat accumulation and leptin secretion. Exemplary molecules
improve the lean muscle mass to fat ratio in aged mdx mice as shown below.
In addition, myostatin has now been found to be expressed at low levels in heart muscle and
expression is upregulated after cardiomyocytes after infarct (Sharma et al. (1999), T Cell Fhysiol. 180 (l):l-9).
Therefore, reducing myostatin levels in the heart muscle may improve recovery of heart muscle after infarct.
Myostatin also appears to influence metabolic disorders including type 2 diabetes, noninsulindependent
diabetes mellitus, hyperglycemia, and obesity. For example, lack of myostatin has been shown to
improve the obese and diabetic phenotypes of two mouse models (Yen et al. supra). In addition, increasing
muscle mass by reducing myostatin levels may improve bone strength and reduce osteoporosis and other
degenerative bone diseases. It has been found, for example, that myostatin-deficient mice showed increased
mineral content and density of the mouse humerus and increased mineral content of both trabecular and
cortical bone at the regions where the muscles attach, as well as increased muscle mass (Hamrick et al. (2002),
Calcif Tissue Int 71(1): 63-8). In the present invention, an exemplary peptibody increases the lean muscle mass
to fat ratio in mdx mouse models as shown below.
The present invention also provides methods and reagents for increasing muscle mass in food animals
by administering an effective dosage of the myostatin binding agent to the animal. Since the mature Cterminal
myostatin polypeptide is identical in all species tested, myostatin binding agents would be expected
to be effective for increasing muscle mass and reducing fat in any agriculturally important species including
cattle, chicken, turkeys, and pigs.
The myostatin-binding molecules of the present invention may be used alone or in combination with
other therapeutic agents to enhance their therapeutic effects or decrease potential side effects. The molecules
of the present invention possess one or more desirable but unexpected combination of properties to improve
the therapeutic value of the agents. These properties include increased activity, increased solubility, reduced
degradation, increased half-life, reduced toxicity, and reduced immunogenicity. Thus the molecules of the
present invention are useful for extended treatment regimes. In addition, the properties of hydrophilicity and
hydrophobicity of the compounds of the invention are well balanced, thereby enhancing their utility for both
in vitro and especially in vivo uses. Specifically, compounds of the invention have an appropriate degree of
solubility in aqueous media that permits absorption and bioavailability in the body, while also having a
degree of solubility in lipids that permits the compounds to traverse the cell membrane to a putative site of
action, such as a particular muscle mass.
The myostatin-binding molecules of the present invention are useful for treating a "subject" or any
animal, including humans, when administered in an effective dosages in a suitable composition.
In addition, the mystatin-binding molecules of the present invention are useful for detecting and
quantitating myostatin in a number of assays. These assays are described in detail in U.S. Ser. No. 10/742,379.
In general, the myostatin-binding molecules of the present invention are useful as capture agents to
bind and immobilize myostatin in a variety of assays, similar to those described, for example, in Asai, ed.,
Methods in Cell Biology, 37, Antibodies in Cell Biology, Academic Press, Inc., New York (1993). The
myostatin-binding molecule may be labeled in some manner or may react with a third molecule such as an
anti-binding molecule antibody which is labeled to enable myostatin to be detected and quantitated. For
example, a myostatin-binding molecule or a third molecule can be modified with a detectable moiety, such as
biotin, which can then be bound by a fourth molecule, such as enzyme-labeled streptavidin, or other proteins.
(Akerstrom (1985), T Immunol 135:2589: Chaubert (1997), Mod Fathol 10:585).
Throughout any particular assay, incubation and/or washing steps may be required after each
combination of reagents. Incubation steps can vary from about 5 seconds to several hours, preferably from
about 5 minutes to about 24 hours. However, the incubation time will depend upon the assay format, volume
of solution, concentrations, and the like. Usually, the assays will be carried out at ambient temperature,
although they can be conducted over a range of temperatures.
Therapeutic uses of BAFF-binding molecules. BAFF-binding molecules of this invention may be
particularly useful in treatment of B-cell mediated autoimmune diseases. In particular, they may be useful in
treating, preventing, ameliorating, diagnosing or prognosing lupus, including systemic lupus erythematosus
(SLE), and lupus-associated diseases and conditions. Other preferred indications include B-cell mediated
cancers, including B-cell lymphoma.
The compounds of this invention can also be used to treat inflammatory conditions of the joints.
Inflammatory conditions of a joint are chronic joint diseases that afflict and disable, to varying degrees,
millions of people worldwide. Rheumatoid arthritis is a disease of articular joints in which the cartilage and
bone are slowly eroded away by a proliferative, invasive connective tissue called pannus, which is derived
from the synovial membrane. The disease may involve peri-articular structures such as bursae, tendon
sheaths and tendons as well as extra-articular tissues such as the subcutis, cardiovascular system, lungs,
spleen, lymph nodes, skeletal muscles, nervous system (central and peripheral) and eyes (Silberberg (1985),
Anderson's Pathology, Kissane (ed.), 11:1828). Osteoarthritis is a common joint disease characterized by
degenerative changes in articular cartilage and reactive proliferation of bone and cartilage around the joint.
Osteoarthritis is a cell-mediated active process that may result from the inappropriate response of
chondrocytes to catabolic and anabolic stimuli. Changes in some matrix molecules of articular cartilage
reportedly occur in early Osteoarthritis (Thonar et al. (1993), Rheumatic disease clinics of North America,
Moskowitz (ed.), 19:635-657 and Shinmei et al. (1992), Arthritis Rheum., 35:1304-1308). TALL-1, TALL-1R and
modulators thereof are believed to be useful in the treatment of these and related conditions.
BAFF-binding molecules may also be useful in treatment of a number of additional diseases and
disorders, including acute pancreatitis; ALS; Alzheimer's disease; asthma; atherosclerosis; autoimmune
hemolytic anemia; cancer, particularly cancers related to B cells; cachexia/anorexia; chronic fatigue syndrome;
cirrhosis (e.g., primary biliary cirrhosis); diabetes (e.g., insulin diabetes); fever; glomerulonephritis, including
IgA glomerulonephritis and primary glomerulonephritis; Goodpasture's syndrome; Guillain-Barre syndrome;
graft versus host disease; Hashimoto's thyroiditis; hemorrhagic shock; hyperalgesia; inflammatory bowel
disease; inflammatory conditions of a joint, including Osteoarthritis, psoriatic arthritis and rheumatoid
arthritis; inflammatory conditions resulting from strain, sprain, cartilage damage, trauma, orthopedic surgery,
infection or other disease processes; insulin-dependent diabetes mellitus; ischemic injury, including cerebral
ischemia (e.g., brain injury as a result of trauma, epilepsy, hemorrhage or stroke, each of which may lead to
neurodegeneration); learning impairment; lung diseases (e.g., ARDS); lupus, particularly systemic lupus
erythematosus (SLE); multiple myeloma; multiple sclerosis; Myasthenia gravis; myelogenous (e.g., AML and
CML) and other leukemias; myopathies (e.g., muscle protein metabolism, esp. in sepsis); neurotoxicity (e.g., as
induced by HIV); osteoporosis; pain; Parkinson's disease; Pemphigus; polymyositis/dermatomyositis;
pulmonary inflammation, including autoimmune pulmonary inflammation; pre-term labor; psoriasis; Reiter's
disease; reperfusion injury; septic shock; side effects from radiation therapy; Sjogren's syndrome; sleep
disturbance; temporal mandibular joint disease; thrombocytopenia, including idiopathic thrombocytopenia
and autoimmune neonatal thrombocytopenia; tumor metastasis; uveitis; and vasculitis.
Combination Therapy. The therapeutic methods, compositions and compounds of the present
invention may also be employed, alone or in combination with other cytokines, soluble Mpl receptor,
hematopoietic factors, interleukins, growth factors or antibodies in the treatment of disease states
characterized by other symptoms as well as platelet deficiencies. It is anticipated that the inventive compound
will prove useful in treating some forms of thrombocytopenia in combination with general stimulators of
hematopoiesis, such as IL-3 or GM-CSF. Other megakaryocytic stimulatory factors, i.e., meg-CSF, stem cell
factor (SCF), leukemia inhibitory factor (LIF), oncostatin M (OSM), or other molecules with megakaryocyte
stimulating activity may also be employed with Mpl ligand. Additional exemplary cytokines or hematopoietic
factors for such co-administration include IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-11, colony
stimulating factor-1 (CSF-1), SCF, GM-CSF, granulocyte colony stimulating factor (G-CSF), EPO, interferonalpha
(IFN-alpha), consensus interferon, IFN-beta, or IFN-gamma. It may further be useful to administer,
either simultaneously or sequentially, an effective amount of a soluble mammalian Mpl receptor, which
appears to have an effect of causing megakaryocytes to fragment into platelets once the megakaryocytes have
reached mature form. Thus, administration of an inventive compound (to enhance the number of mature
megakaryoctes) followed by administration of the soluble Mpl receptor (to inactivate the ligand and allow
the mature megakaryocytes to produce platelets) is expected to be a particularly effective means of stimulating
platelet production. The dosage recited above would be adjusted to compensate for such additional
components in the therapeutic composition. Progress of the treated patient can be monitored by conventional
methods.
In cases where the inventive compounds are added to compositions of platelets and/or
megakaryocytes and related cells, the amount to be included will generally be ascertained experimentally by
techniques and assays known in the art. An exemplary range of amounts is 0.1 ug—1 mg inventive compound
per 106 cells.
Pharmaceutical Compositions
In General
The present invention also provides methods of using pharmaceutical compositions of the inventive
compounds. Such pharmaceutical compositions may be for administration for injection, or for oral, pulmonary,
nasal, transdermal or other forms of administration. In general, the invention encompasses pharmaceutical
compositions comprising effective amounts of a compound of the invention together with pharmaceutically
acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. Such compositions include
diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; additives such as
detergents and solubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium
metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol);
incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid,
polyglycolic acid, etc. or into liposomes. Hyaluronic acid may also be used, and this may have the effect of
promoting sustained duration in the circulation. Such compositions may influence the physical state, stability, rate
of in vivo release, and rate of in vivo clearance of the present proteins and derivatives. See, e.g., Remington's
Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA 18042) pages 1435-1712 which are herein
incorporated by reference. The compositions may be prepared in liquid form, or may be in dried powder, such as
lyophilized form. Implantable sustained release formulations are also contemplated, as are transdermal
formulations.
Oral dosage forms
Contemplated for use herein are oral solid dosage forms, which are described generally in Chapter 89
of Remington's Pharmaceutical Sciences (1990), 18th Ed., Mack Publishing Co. Easton PA 18042, which is
herein incorporated by reference. Solid dosage forms include tablets, capsules, pills, troches or lozenges,
cachets or pellets. Also, liposomal or proteinoid encapsulation may be used to formulate the present compositions (as, for example, proteinoid microspheres reported in U.S. Patent No. 4,925,673). Liposomal
encapsulation may be used and the liposomes may be derivatized with various polymers (e.g., U.S. Patent No.
5,013,556). A description of possible solid dosage forms for the therapeutic is given in Chapter 10 of Marshall,
K., Modern Pharmaceutics (1979), edited by G. S. Banker and C. T. Rhodes, herein incorporated by reference.
In,general, the formulation will include the inventive compound, and inert ingredients which allow for
protection against the stomach environment, and release of the biologically active material in the intestine.
Also specifically contemplated are oral dosage forms of the above inventive compounds. If necessary,
the compounds may be chemically modified so that oral delivery is efficacious. Generally, the chemical
modification contemplated is the attachment of at least one moiety to the compound molecule itself, where
said moiety permits (a) inhibition of proteolysis; and (b) uptake into the blood stream from the stomach or
intestine. Also desired is the increase in overall stability of the compound and increase in circulation time in
the body. Moieties useful as covalently attached vehicles in this invention may also be used for this purpose.
Examples of such moieties include: PEG, copolymers of ethylene glycol and propylene glycol, carboxymethyl
cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline. See, for example, Abuchowski
and Davis, Soluble Polymer-Enzyme Adducts, Enzymes as Drugs (1981), Hocenberg and Roberts, eds., Wiley-
Interscience, New York, NY, , pp 367-83; Newmark, et al. (1982), T. Appl. Biochem. 4:185-9. Other polymers
that could be used are poly-l,3-dioxolane and poly-l,3,6-tioxocane. Preferred for pharmaceutical usage, as
indicated above, are PEG moieties.
For oral delivery dosage forms, it is also possible to use a salt of a modified aliphatic amino acid, such
as sodium N-(8-[2-hydroxybenzoyl] amino) caprylate (SNAC), as a carrier to enhance absorption of the
therapeutic compounds of this invention. The clinical efficacy of a heparin formulation using SNAC has been
demonstrated in a Phase II trial conducted by Emisphere Technologies. See US Patent No. 5,792,451, "Oral
drug delivery composition and methods".
The compounds of this invention can be included in the formulation as fine multiparticulates in the
form of granules or pellets of particle size about 1 mm. The formulation of the material for capsule
administration could also be as a powder, lightly compressed plugs or even as tablets. The therapeutic could
be prepared by compression.
Colorants and flavoring agents may all be included. For example, the protein (or derivative) may be
formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible
product, such as a refrigerated beverage containing colorants and flavoring agents.
One may dilute or increase the volume of the compound of the invention with an inert material. These
diluents could include carbohydrates, especially mannitol, a-lactose, anhydrous lactose, cellulose, sucrose,
modified dextrans and starch, Certain inorganic salts may also be used as fillers including calcium
triphosphate, magnesium carbonate and sodium chloride. Some commercially available diluents are Fast-Flo,
Emdex, STA-Rx 1500, Emcompress and Avicell.
Disintegrants may be included in the formulation of the therapeutic into a solid dosage form. Materials
used as disintegrants include but are not limited to starch including the commercial disintegrant based on
starch, Explotab. Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin,
sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite may all be
used. Another form of the disintegrants are the insoluble cationic exchange resins. Powdered gums may be
used as disintegrants and as binders and these can include powdered gums such as agar, Karaya or
tragacanth. Alginic acid and its sodium salt are also useful as disintegrants.
Binders may be used to hold the therapeutic agent together to form a hard tablet and include materials
natural products such as acacia, tragacanth, starch and gelatin. Others include methyl cellulose (MC),
ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinyl pyrrolidone (PVP) and
hydroxypropylmethyl cellulose (HPMC) could both be used in alcoholic solutions to granulate the therapeutic.
An antifrictional agent may be included in the formulation of the therapeutic to prevent sticking
during the formulation process. Lubricants may be used as a layer between the therapeutic and the die wall,
and these can include but are not limited to; stearic acid including its magnesium and calcium salts,
polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants may also be used
such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights,
Carbowax 4000 and 6000.
Glidants that might improve the flow properties of the drug during formulation and to aid
rearrangement during compression might be added. The glidants may include starch, talc, pyrogenic silica
and hydrated silicoaluminate.
To aid dissolution of the compound of this invention into the aqueous environment a surfactant might
be added as a wetting agent. Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl
sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents might be used and could include
benzalkonium chloride or benzethonium chloride. The list of potential nonionic detergents that could be
included in the formulation as surfactants are lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene
hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid
ester, methyl cellulose and carboxymethyl cellulose. These surfactants could be present in the formulation of
the protein or derivative either alone or as a mixture in different ratios.
Additives may also be included in the formulation to enhance uptake of the compound. Additives
potentially having this property are for instance the fatty acids oleic acid, linoleic acid and linolenic acid.
Controlled release formulation may be desirable. The compound of this invention could be
incorporated into an inert matrix which permits release by either diffusion or leaching mechanisms e.g.^ gums.
Slowly degenerating matrices may also be incorporated into the formulation, e.g., alginates, polysaccharides.
Another form of a controlled release of the compounds of this invention is by a method based on the Oros
therapeutic system (Alza Corp.), i.e., the drug is enclosed in a semipermeable membrane which allows water
to enter and push drug out through a single small opening due to osmotic effects. Some enteric coatings also
have a delayed release effect.
Other coatings may be used for the formulation. These include a variety of sugars which could be
applied in a coating pan. The therapeutic agent could also be given in a film coated tablet and the materials
used in this instance are divided into 2 groups. The first are the nonenteric materials and include methyl
cellulose, ethyl cellulose, hydroxyethyl cellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose,
hydroxypropyl-methyl cellulose, sodium carboxy-methyl cellulose, providone and the polyethylene glycols.
The second group consists of the enteric materials that are commonly esters of phthalic acid.
A mix of materials might be used to provide the optimum film coating. Film coating may be carried
out in a pan coater or in a fluidized bed or by compression coating.
Pulmonary delivery forms
Also contemplated herein is pulmonary delivery of the present protein (or derivatives thereof). The
protein (or derivative) is delivered to the lungs of a mammal while inhaling and traverses across the lung
epithelial lining to the blood stream. (Other reports of this include Adjei et al., Pharma. Res. (1990) 7: 565-9;
Adjei et al. (1990), Internatl. T. Pharmaceutics 63:135-44 (leuprolide acetate); Braquet et al. (1989), T. Cardiovasc.
Pharmacol. 13 (suppl.5): s.143-146 (endothelin-1); Hubbard et al. (1989), Annals Int. Med. 3: 206-12 (alantitrypsin);
Smith et al. (1989), T. Clin. Invest. 84: 1145-6 (al-proteinase); Oswein et al. (March 1990),
"Aerosolization of Proteins", Proc. Symp. Resp. Drug Delivery II, Keystone, Colorado (recombinant human
growth hormone); Debs et al. (1988), T. Immunol. 140: 3482-8 (interferon-y and tumor necrosis factor a) and
Platz et al., U.S. Patent No. 5,284,656 (granulocyte colony stimulating factor).
Contemplated for use in the practice of this invention are a wide range of mechanical devices designed
for pulmonary delivery of therapeutic products, including but not limited to nebulizers, metered dose
inhalers, and powder inhalers, all of which are familiar to those skilled in the art. Some specific examples of
commercially available devices suitable for the practice of this invention are the Ultravent nebulizer,
manufactured by Mallinckrodt, Inc., St. Louis, Missouri; the Acorn II nebulizer, manufactured by Marquest
Medical Products, Englewood, Colorado; the Ventolin metered dose inhaler, manufactured by Glaxo Inc.,
Research Triangle Park, North Carolina; and the Spinhaler powder inhaler, manufactured by Fisons Corp.,
Bedford, Massachusetts.
All such devices require the use of formulations suitable for the dispensing of the inventive
compound. Typically, each formulation is specific to the type of device employed and may involve the use of
an appropriate propellant material, in addition to diluents, adjuvants and/or carriers useful in therapy.
The inventive compound should most advantageously be prepared in particulate form with an
average particle size of less than 10 nm (or microns), most preferably 0.5 to 5 ^im, for most effective delivery
to the distal lung.
Pharmaceutically acceptable carriers include carbohydrates such as trehalose, mannitol, xylitol,
sucrose, lactose, and sorbitol. Other ingredients for use in formulations may include DPPC, DOPE, DSPC
and DOPC. Natural or synthetic surfactants may be used. PEG may be used (even apart from its use in
derivatizing the protein or analog). Dextrans, such as cyclodextran, may be used. Bile salts and other related
enhancers may be used. Cellulose and cellulose derivatives may be used. Amino acids may be used, such as
use in a buffer formulation.
Also, the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of
carriers is contemplated.
Formulations suitable for use with a nebulizer, either jet or ultrasonic, -will, typically comprise the
inventive compound dissolved in water at a concentration of about 0.1 to 25 mg of biologically active protein
per mL of solution. The formulation may also include a buffer and a simple sugar (e.g., for protein
stabilization and regulation of osmotic pressure). The nebulizer formulation may also contain a surfactant, to
reduce or prevent surface induced aggregation of the protein caused by atomization of the solution in forming
the aerosol.
Formulations for use with a metered-dose inhaler device will generally comprise a finely divided
powder containing the inventive compound suspended in a propellant with the aid of a surfactant. The
propellant may be any conventional material employed for this purpose, such as a chlorofluorocarbon, a
hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane,
dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, or combinations thereof.
Suitable surfactants include sorbitan trioleate and soya lecithin. Oleic acid may also be useful as a surfactant.
Formulations for dispensing from a powder inhaler device will comprise a finely divided dry powder
containing the inventive compound and may also include a bulking agent, such as lactose, sorbitol, sucrose,
mannitol, trehalose, or xylitol in amounts which facilitate dispersal of the powder from the device, e.g., 50 to
90% by weight of the formulation.
Other delivery forms
Nasal delivery of the inventive compound is also contemplated. Nasal delivery allows the passage of
the protein to the blood stream directly after administering the therapeutic product to the nose, without the
necessity for deposition of the product in the lung. Formulations for nasal delivery include those with dextran
or cyclodextran. Delivery via transport across other mucous membranes is also contemplated.
Buccal delivery of the inventive compound is also contemplated. Buccal delivery formulations are
known in the art for use with peptides.
Dosages
The dosage regimen involved in a method for treating the above-described conditions will be determined by
the attending physician, considering various factors which modify the action of drugs, e.g. the age, condition, body
weight, sex and diet of the patient, the severity of any infection, time of administration and other clinical factors.
Generally, the daily regimen should be in the range of 0.1-1000 micrograms of the inventive compound per kilogram
of body weight, preferably 0.1-150 micrograms per kilogram.
Specific preferred embodiments
The inventors have determined preferred peptide sequences for molecules having many different
kinds of activity. The inventors have further determined preferred structures of these preferred peptides
combined with preferred linkers and vehicles. Preferred structures for these preferred peptides listed in Table
11 below. Linker sequences are shown in bold. Active peptide sequences are shown in bold and
areunder lined.
Table 11—Preferred embodiments
(Table Removed)
Working examples
The compounds described above may be prepared as described below. These examples comprise
preferred embodiments of the invention and are illustrative rather than limiting.
Example 1
Preparation of Fc-loop-ang2
In this example of the invention, the disulphide-constrained peptide TN8-Con4 was inserted into the
human IgGl Fc-loop domain, defined as the sequence Di37Ei3sLi39Ti4oKi4i (Fig 2A.).
TN8-Con4 QEECEWDPWTCEHM (SEQ ID NO: 147)
The peptide insertion is between Fc residues Leui39 and Thrwo and includes 2 Gly residues as linkers flanking
either side of the inserted peptide (Fig. 10A). The Fc-loop TN8-Con4 construct is labeled with Amgen clone
#6888. The carboxy-terminal TN8-Con4 fusion peptibody (Fig. 10B) includes a 5 Gly linker and is labeled with
Amgen clone #5564.
Both clones #6888 and #5564 were transformed into E. coli by conventional methods known to those
familiar in the art. Both clones were found to express at high levels and almost exclusively in the insoluble
inclusion body fraction (Fig. 11). The isolated inclusion body fraction (1 g) was solubilized in 6 M guanidine-
HC1, 50 mM Tris, 8 mM DTT, pH 9 (10 ml) at room temperature with mixing, for 1 hour. The denatured and
reduced peptibodies were refolded from the solubilized inclusion bodies by a 1:25, (v/v) dilution into a refold
buffer consisting of 2 M urea, 50 mM Tris, 4 mM cysteine, 1 mM cystamine, pH 8.5. The solubilized
peptibodies were added drop wise to the refold buffer at 4 °C with stirring. The refold reactions were allowed
to stir for 48 hours, and then aliquots were evaluated by SDS-PAGE and reverse-phase HPLC. The Fc-loop
TN8-Con4 (#6888) is considerably more homogeneous in the refold reaction than the carboxy-terminal Fc TN8-
Con4 peptibody (#5564) as shown by RP-HPLC (Fig. 12).
Purification of the refolded Fc-loop TN8-Con4 and carboxy-terminal Fc TN8-Con4 was achieved using
a 2-column process. First a recombinant Protein-A column was equilibrated in 2 M urea, 50 mM Tris, pH 8.5
and loaded with the filtered peptibody refold reaction. The column was then washed with 2 column volumes
of equilibration buffer, followed by 2 column volumes of PBS. The peptibody fraction was eluted with 50mM
NaOAc, pH3 and quickly neutralized by a 1:4 dilution into 10 mM NaOAc, 50 mM NaCl, pH 5. The diluted
Protein-A eluate was again filtered and loaded to an SP Sepharose HP cation exchange column (Pharmacia)
equilibrated in 10 mM NaOAc, 50 mM NaCl, pH 5. The peptibody fractions were then eluted with a linear 50-
500 mM NaCl gradient, pooled and concentrated to about 2 mg/ml. The final pools of Fc-loop TN8-Con4
(#6888) and the carboxy-terminal Fc TN8-Con4 (#5564) were evaluated by RP-HPLC (Fig. 13) and SDS-PAGE
(Fig. 14). Both RP-HPLC and SDS-PAGE demonstrate that improved product homogeneity is achieved with
the Fc-loop TN8-Con4 (#6888) relative to the comparable carboxy-terminal fused peptibody (#5564).
Both the Fc-loop TN8-Con4 and carboxy-terminal fused TN8-Con4 peptibodies were further evaluated
in an in vitro ELISA for competitive inhibition of the angiopoietin 2 receptor. In this format the peptibody
competes with an angiopoietin 2 receptor-Fc fusion for binding to immobilized angiopoietin 2. Binding of the
angiopoietin 2 receptor-Fc fusion is monitored by fluorescence using an enzyme-linked immunodetection
method and reported as an inhibition constant at 50% inhibition (ICso). This experiment shows that the Fcloop
TN8-Con4 is fully active relative to the carboxy-terminal Fc TN8-Con4 (Fig. 15).
Stability in vivo of the Fc-loop TN8-Con4 peptibody was compared to the carboxy-terminal TN8-Con4
peptibody in mice. In this study groups of 15 mice were dosed subcutaneously with either peptibody
construct at 5 mg/kg. At 4 hours after injection, 5 mice were sacrificed and serum collected. At 24 hours,
another 5 mice were harvested and likewise at 48 hours. Each individual serum sample was evaluated by
western blot for detectable human IgG-Fc peptibody. Since all the serum within each 5-mouse group was very
similar, the groups were pooled to allow representative samples to be run together on a single gel/blot. The
result of that analysis (Fig. 16) clearly shows that both the Fc-loop TN8-Con4 peptibody and the carboxyterminal
TN8-Con4 peptibody persists in the pooled mouse sera throughout the 48-hour time course with no
apparent loss. This result demonstrates that the Fc-loop designed peptibodies are not destabilized in vivo by
the peptide insertion.
Example 2
Preparation of Fc-loop-myo7
In another embodiment of this invention, a novel, disulphide-constrained peptide TN8-19-7 (U.S. Pat
App 2004-0181033-A1, which is incorporated by reference) of the sequence:
TN8-19-07 LADHGQCIRWPWMCPPEGWE (SEQ ID NO: 365)
was engineered between Leul39 and Thrl40 as an internal fusion in the putative Fc-loop sequence DELTK of
an IgGl Fc sequence (Fig 2A). An additional two Gly residues were also added at each end of the TN8-19-07
peptide as flanking linkers. The final Fc-loop TN8-19-07 sequence is given in Figure 3A and is labeled clone #
6951. Alternatively, a carboxy terminal fusion of TN8-19-07 with the same IgGl Fc sequence was prepared to
serve as a control (Fig 3B) and labeled clone #6826. The carboxy-terminal fusion included five Gly residues
between the Fc and TN8-19-07 to serve as a linker.
Both clones #6951 and #6826 were transformed into E. coli by conventional methods used by those
familiar in the art, and were found to express at high levels and almost exclusively in the insoluble inclusion
body fraction (Fig. 4A and 4B). The isolated inclusion body fraction (1 g) was solubilized in 6 M guanidine-
HCl, 50 mM Tris, 8 mM DTT, pH 9 (10 ml) at room temperature with mixing for 1 hour. The denatured and
reduced peptibodies were refolded from the solubilized inclusion body fraction by a 1:25 (v/v) dilution into 2
M urea, 50 mM Tris, 4 mM cysteine, 1 mM cystamine, pH 8.5. The solubilized peptibodies were added dropwise
to the refold buffer at 4 °C with stirring. The refold reactions were allowed to stir for 48 hours, and then
aliquots were evaluated by SDS-PAGE and reverse-phase HPLC. The Fc-loop TN8-19-07 (#6951) was found to
be considerably more homogeneous by RP-HPLC (Fig. 5) in the refold reaction than the carboxy-terminal Fc-
TN8-19-07 peptibody (#6826).
Purification was achieved by a 2-column process. First a recombinant Protein-A column was
equilibrated in 2 M urea, 50 mM Tris, pH 8.5 and loaded with the filtered peptibody refold reaction. The
column was then washed with 2 column volumes of equilibration buffer, followed by 2 column volumes of
PBS. The peptibody fraction was eluted with 50 mM NaOAc, pH 3 and quickly neutralized by a 1:4 dilution
into 10 mM NaOAc, 50 mM NaCl, pH 5. The diluted Protein-A eluate was again filtered and loaded to an SP
Sepharose HP cation exchange column (Pharmacia) equilibrated in 10 mM NaOAc, 50 mM NaCl, pH 5. The
peptibody fractions were then eluted with a linear 50-500 mM NaCl gradient, pooled and concentrated to
about 2 mg/ml. The final pools of Fc-loop TN8-19-07 (#6951) and the carboxy-terminal Fc TN8-19-07 (#6826)
were evaluated by RP-HPLC (Fig. 6) and SDS-PAGE (Fig. 7). Both RP-HPLC and SDS-PAGE demonstrate that
improved homogeneity in the final product is achieved with the Fc-loop TN8-19-07 (#6951) relative to the
comparable carboxy-terminal fused peptibody (#6826).
An in vitro cell-based assay, which measures the inhibition of myostatin signaling activity, was used to
determine the bioactivity of the Fc-loop TN8-19-07 (#6951) compared to the carboxy-terminal fusion (#6826).
In this assay, both constructs were titrated against 4 nM myostatin and evaluated for their ability to inhibit the
myostatin signaling activity as measured by a luciferase reporter system. The relative peptibody activities are
reported as the effective concentration for 50% inhibition (ECso). This experiment shows that the Fc-loop TN8-
19-07 peptibody (#6951) retains full in vitro bioactivity (Fig. 8).
Stability in vivo of the Fc-loop TN8-19-07 peptibody was compared to the carboxy-terminal TN8-19-07
peptibody in mice. In this study, groups of 15 mice were dosed subcutaneously with either peptibody
construct at 5 mg/kg. At 4 hours post injection 5 mice were sacrificed and serum collected. At 24 hours
another 5 mice were harvested and likewise at 48 hours. Each individual serum was evaluated by western blot
for detectable human IgG-Fc peptibody. Since all the serum within each 5-mouse group was very similar, the
groups were pooled to allow representative samples to be run together on a single gel/blot. The result of that
analysis (Fig. 9) clearly shows that the Fc-loop TN8-19-07 peptibody persists in the pooled mouse sera
throughout the 48-hour time course with no apparent loss. In contrast, the concentration of the carboxyterminal
TN8-19-07 peptibody diminishes steadily through the course of the study until it is nearly
undetectable at the 48-hour time point. This result suggests that the Fc-loop design approach may confer
additional in vivo stability to the TN8-19-07 peptibody.
Example 3
Preparation of TN8-Con4
This molecule was prepared as described above in Example 1 and in U.S. Pat. App. No. 2003/0236192
(also PCT/US04/10989), which is hereby incorporated by reference.
Example 4
Preparation of Fc-ang2-tandem
This molecule was prepared as described in U.S. 2003/0236193, published Dec. 25, 2003 (also
PCT/US04/10989, filed April 8,2004), which is hereby incorporated by reference.
Example 5
Preparation of TN8-19-7
This molecule was prepared as described above in example 2 and in U.S. Serial No. 10/742,379, filed
Dec. 19,2003 (alsoPCT/USQ3/40781, filed Dec. 19,2003), which is hereby incorporated by reference .
Example 6
Preparation of Fc-loop-EMF
This molecule was prepared as previously described in example 1.
Example 7
Preparation of Fc-loop-Amp2
In another embodiment of this invention a linear, non-constrained peptide, AMP 2 was inserted into
the human IgGl Fc -loop domain, defined as the sequence Dis/EissLisgTwoKi/u (Fig. 2A).
AMP-2: IEGPTLRQWLAARA (SEQIDNO: 28)
The Fc insertion is between Leuisg and Thruo and includes 2 Gly residues as linkers flanking either side
of the inserted peptide (Fig. 3D). The Fc-loop AMP 2 construct is labeled as Amgen clone #6875.
The Fc-loop AMP 2 clone (#6875) was transformed into E. coli by conventional methods known to
those in the art and was found to express at high levels and almost exclusively in the insoluble inclusion body
fraction (Fig. 17). The isolated inclusion body fraction (Ig) was solubilized in 6 M guanidine-HCl, 50 mM Tris,
8 mM DTT, pH 9 (10 ml) at room temperature with mixing, for 1 hour. The denatured and reduced peptibody
was refolded from the solubilized inclusion body fraction by a 1:25 (v/v) dilution into 2 M urea, 50 mM Tris, 4
mM cysteine, 1 mM cystamine, pH 8.5. The solubilized peptibody was added drop wise to the refold buffer at
4 °C with stirring. The refold reactions were allowed to stir for 48 hours, and then aliquots were evaluated by
SDS-PAGE and reversed-phase HPLC
Purification was achieved using a 2-column process. First a recombinant Protein-A column was
equilibrated in 2 M urea, 50 mM Tris, pH 8.5 and loaded with the filtered peptibody refold reaction. The
column was then washed with 2 column volumes of equilibration buffer, followed by 2 column volumes of
PBS. The peptibody fraction was eluted with 50 mM NaOAc, pH3 and quickly neutralized by a 1:4 dilution
into 10 mM NaOAc, 50 mM NaCl, pH 5. The diluted Protein-A eluate was again filtered and loaded to an SP
Sepharose HP cation exchange column (Pharmacia) equilibrated in 10 mM NaOAc, 50 mM NaCl, pH 5. The
peptibody fractions were then eluted with a linear 50-500 mM NaCl gradient, pooled and concentrated to
about 2 mg/ml. The final pools of Fc-loop AMP 2 (#6875) were evaluated by SDS-PAGE (Fig. 19) and RPHPLC
(Fig. 20).
The final preparation of Fc-loop AMP 2 was tested in an in vivo mouse bioassay against a carboxyterminal
peptibody fusion of two AMP 2 sequences linked in tandem (Fc tandem AMP2). In this comparison,
the Fc-tandem-AMP2 has a total valence of four AMP 2 peptides compared to the Fc-loop AMP 2 with only
two peptides. The mice received a single subcutaneous injection of 50 ng/kg of either peptibody while their
platelet levels were monitored over 15 days (Fig. 21). While the Fc-tandem-AMP2 induced a significant initial
platelet increase, the total response was complete by day 9. In contrast, the Fc-loop AMP 2 elicited a much
smaller response that peaked at day 8 and persisted for 15 days. These results suggest that the efficacious halflife
of the Fc-loop AMP 2 peptibody may be much greater than the conventional carboxy-terminal fused
peptibody. The difference in overall amplitude of the response may be a consequence of the greater valence of
Fc-tandem-Amp2.
Example 8
Preparation of Amp2
This molecule was prepared as described above in example 7, and in U.S. Pat. No. 6,660,843.
Example 9
In Vitro cell-based assay and the measurement of myostatin-signaling activity (Figure 8)
To quantitate myostatin activity and its blockade, a luciferase reporter system was developed, referred
to as pMARE-Luc, which senses Myostatin/Activin signaling strength. The pMARE-luc vector was
constructed by subcloning a Smad-responsive CAGA tandem repeat sequence into a basic reporter plasmid
pLuc-MCS containing a minimal promoter element (TATA box). The pMARE-luc vector was stably
transfected into a skeletal muscle-derived C2C12 cell line (murine).
Characterization of myostatin responses of the stable clones led to the identification of C2C12-based
clonal reporter cell lines that were capable of detecting both myostatin and Activin signaling activities in 96-
well format in a highly sensitive and reproducible manner.
Example 10
In Vitro HTRF (homogeneous time-resolved fluorescence) Ang-2 binding assay (Figure 15).
Starting from a concentration of 100 nM, Fc-loop peptibody and proper controls were serially diluted
in liTRF buffer 3-fold, 9-times across a 96-well plate. Dilutions were then mixed with the following reagents
on a 96-well black, round-bottomed assay plate: Streptavidin-Europium (1.6 nM), Biotinylated human
angiopoietin-2 (8.0 nM), human Tie2-Fc-APC (10 nM). Assay plate was then incubated at room temperature
with shaking for 2 hours. Plate next read on a Rubystar microplate reader (BMG Labtechnologies Inc.).
Results were converted to %inhibition, and ICSOs were then calculated by analyzing the %inhibition values in
the program GRAFIT 5.0 (IC50, 0-100% parameter).
Example 11
In Vivo AMP-2 efficacy assay (Figure 21)
Female BDF1 mice are injected subcutaneously with either carrier fluid (1 x PBS with 0.1% BSA), 50 meg/kg of
Fc-tandem-AMP2, or 50 meg/kg of Fc-loop-AMP2. The injection volume is 0.2 mL. Blood is collected from each mouse
via a puncture of the retro orbital sinus into a heparinized capillary tube, and then transferred to microtainers containing
EDTA. Complete blood counts (CBC) including differential white blood cell counts are obtained using an AD VIA 120
blwod analyzer calibrated for mouse blood (Bayer Corp., Tarrytown, NY). Standard bleed days are 0, 3, 5, 7 and 10.
Platelet counts are plotted as a function of time post-injection.
Example 12
UT-7 EPO Proliferation Assay for EMP Activity (Figure 18)
The UT-7Epo proliferation assay uses human megakaryoblastic leukemia cell line that responds murine EPO (mEPO) and human EPO (huEPO) or other EPO like molecules for growth and survival.
Growth factors are serially diluted from 1000 ng/ml to 0.488 ng/ml in triplicate, in 100 u,l of 10% FBSIscoves
Modified Dulbelcco's medium (IMDM) across the 96 well plate. 15000 cells/well are added to the 96 well plate in 100 jal of 10% FBS IMDM. The total volume per well is 200 ul of media with 15000 cells per well.
Cells and media alone are the zero control. Cells are incubated in a humidified chamber at 37 °C.
After incubation for 72 hours with the growth factor to be examined, viable cells are determined by
MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxphenyl)-2-(4-sulfophenyl)2H-tetrazolium, inner salt)
incorporation (5+/-1 hr at 37 °C) and growth is measured by O.D. (490 nm absorbance), limits not 4.0 O.D.
Reference: Yasusada Miura, Keita Kirito, and Norio Komatsu (1998), "Regulation of Both Erythroid and
Megakaryocytic Differentiation of a Human Leukemia Cell Line, UT-7," Acta Haematologica,99:180-184.
Example 13
Alternate Fc-Loop Insertion Sites
Having proven the feasibility of Fc-Loop insertion-style peptibodies with the L139/T140 insertion site using
several different peptides, additional loops were surveyed in the Fc crystal structure. Using Fc-domain homology
modeling, twelve different potential insertion sites were selected based on solvent accessible surface exposure, steric
constraints within the loop, proximity to the Fc dimer interface and juxtaposition to sites of known effector function, such
as the FcRn binding interface. The Fc-Loop sites that were identified as potential insertion sites are detailed and ranked.
See Figure 2A and Table 12 below.
Table 12
Specific insertion sites for the human IgGl Fc sequence
(Table Removed)
Of these potential insertion sites, six were expressed using the TN8-19-7 peptide insert (Example 2) and
evaluated for refolding efficiency and in vitro activity. An additional construct was added which contained an
asymmetric linker system Gly4/Gly6 engineered into the original loop insertion site (L139/T140) previously
described. In all, seven new Fc Loop Myostatin constructs were refolded, purified, and tested for activity.
The seven new Fc-Loop TN8-19-7 constructs that were tested included insertions in both CH2 and CH3
domains of human IgGl Fc. Specifically, these insertions were: G201/N202 (CH3), E169/N170 (CHS),
S181/D182 (CHS), H49/E50 (CH2), L139/T140 (G4-6) (CHS), Y77/N78 (CH2), and K107/A108 (CH2-CH3
linker domain). These constructs were transformed into E. coli by conventional methods known in the art, and
were found to express almost exclusively in the insoluble inclusion body fraction. Interestingly, the H49/E50,
L139/T140 (G4-6) and Y77/N78 constructs appeared to have the highest levels of expression. The E169/N170,
S181/D182, K107/108 showed moderate expression, and the G201/202 construct showed some expression, but
very little.
Those Fc-Loop TN8-19-7 constructs which expressed well in E. coli were purified by first solubilizing
the isolated inclusion body fractions in 6 M Guanidine-HCl, 50 mM Tris, 8 mM DTT pH 9.0 (10 mL per Ig
inclusion body) at room temperature with mixing for 1 hour. Then, a variety of refolding conditions were
evaluated for each of the denatured and reduced Fc-Loop TN8-19-7 constructs to identify optimal refolding
conditions. The three G201/N202, E169/N170, and S181/D182 constructs did not refold well under any of the
conditions tested. Of the remaining four Fc-Loop TN8-19-7 constructs, L139/T140 (G4-6) refolded the best,
while the remaining three H49/E50, Y77/N78 and K107/A108 refolded with sufficient yield to pursue further
purification.
Using optimized refold conditions, the denatured and reduced Fc-Loop TN8-19-7 constructs were
refolded from the solubilized inclusion body fractions by a 1:25 (v/v) dilution into 4 M Urea, 50 mM Tris-HCl,
0.16 M Arg-HCl, 20% glycerol, 3 M Cystine, 5 mM Cystamine pH 8.5. The solubilized peptibodies were added
drop-wise to the refold buffer at 4°C with stirring. The refold reactions were allowed to stir for 72 hours, and
subsequently purified chromatographically. Final purification was achieved by a 2-column chromatographic
process, as described in Example 2.
-The final pools of L139/T140 (G4-6), H49/E50, Y77/N78, and K107/108 were evaluated by RP-HPLC
and SDS-PAGE, as illustrated in Figures 23 and 24. The yields from these four constructs are tabulated in
Table 13.
Table 13
(Table Removed)
Among the four analogs purified the L139/T140 (G4-6) insertion site analog was produced with the
best purity and in the highest yield.
The purified Fc-loop insertion analogs were further analyzed for functional myostatin receptor binding
activity using an in vitro cell based inhibition assay, as described in Example 9. The results are shown in Table
14.
Table 14
(Table Removed)
The purified Fc-loop insertion analogs were further analyzed for FcRn binding using a Biacore assay
system. Sample K107/A108 was not tested due to insufficient sample remaining after analysis. The ICso
values determined using the in vitro cell based Myostatin inhibition assay, were similar for the L139/T140
(G4-6) insertion and the original Fc-Loop #6951, whereas the Fc-loop insertions at H49/E50 and Y77/N78
showed a reduced ability to inhibit myostatin (Figure 30). The Biacore FcRn binding experiments showed that
H49/E50 and Y77/N78 bound the Fc receptor comparably to the control (Fc-Loop-lxTN8-19-7, #6951) with an
EC50 of about 680 nM/ but L139/T140 (G4-6) had a lower and more favorable EC5o at around 220 nM
In summary, of the six new insertion sites evaluated, three failed to refold efficiently. Among the three
new insertion site analogs recovered, K107/A108 folded poorly, H49/E50 (CH2 domain) refolded marginally
well, and the two remaining insertions at Y77/N78 (CH2 domain) and the original insertion site of L139/T140
(CHS domain) with an extended, asymmetric linker folded with a higher efficiency. Interestingly, all of these
novel insertion site analogs refolded with significantly lower yield than the original Fc-loop construct (#6951)
with TN8-19-7 in position L139/T140 using symmetric Gly2 linkers.
When tested for retention of FcRn binding capacity, all the Fc-loop molecules appeared similar in
affinity with the possible exception of the extended, asymmetric linker construct, which seemed slightly better.
This was consistent with the design paradigm to minimize steric interactions between the inserted peptide and
the FcRn binding interface.
While all the purified Fc-loop constructs were active by the in vitro, cell-based functional assay (Table
14), the original insertion site (L139/T140) and the extended, asymmetric linker insertion at the same site
appeared to be the most potent.
This work demonstrates that multiple loop domains within the human IgGl Fc, as identified in Figure
23, will tolerate insertion of bioactive peptides while preserving the activity of both the peptide and Fc effector
functions such as FcRn binding. Peptide insertion analogs utilizing these Fc-lbop domains can vary
significantly in refolding efficiency and peptide activity. Each peptide/insertion combination can be
individually optimized to maximize recovery and potency.
More preferable would be peptide insertions targeting the underlined sub-domains in Figure 23. Most
preferable are the insertion site (L139/T140) and two additional loops in the CH2 domain (H49/E50 and
Y77/N78).
Abbreviations
Abbreviations
specific circumstances,
Ac
AcBpa
ACN
ADCC
Aib
bA
Bpa
BrAc
BSA
Bzl
Cap
CTL
CTLA4
DARC
DCC
Dde
EDTA
used throughout this specification are as defined below, unless otherwise defined in
acetyl (used to refer to acetylated residues)
acetylatedp-benzoyl-L-phenylalanine
acetonitrile
antibody-dependent cellular cytotoxicity
aminoisobutyric acid
beta-alanine
p-benzoyl-L-phenylalanine
bromoacetyl (BrCH2C(O)
Bovine serum albumin
Benzyl
Caproic acid
Cytotoxic T lymphocytes
Cytotoxic T lymphocyte antigen 4
Duffy blood group antigen receptor
Dicylcohexylcarbodiimide
l-(4,4-dimethyl-2,6-dioxo-cyclohexylidene)ethyl
ethylene diamine tetraacetic acid
EMP Erythropoietin-mimetic peptide
ESI-MS Electron spray ionization mass spectrometry
EPO Erythropoietin
Fmoc fluorenylmethoxycarbonyl
G-CSF Granulocyte colony stimulating factor
GH Growth hormone
HCT hematocrit
HGB hemoglobin
hGH Human growth hormone
HOBt 1-Hydroxybenzotriazole
HPLC high performance liquid chromatography
IL interleukin
IL-R interleukin receptor
IL-1R interleukin-1 receptor
IL-lra interleukin-1 receptor antagonist
Lau Laurie acid
LPS lipopolysaccharide
LYMPH lymphocytes
MALDI-MS Matrix-assisted laser desorption ionization mass spectrometry
Me methyl
MeO methoxy
MES (2-[N-Morpholino]ethanesulfonic acid)
MHC major histocompatibility complex
MMP matrix metalloproteinase
MMPI matrix metalloproteinase inhibitor
NaOAc sodium acetate
1-Nap 1-napthylalanine
NEUT neutrophils
NGF nerve growth factor
Me norleucine
NMP N-methyl-2-pyrrolidinone
PAGE polyacrylamide gel electrophoresis
PBS Phosphate-buffered saline
Pbf 2,2,4/6/7-pendamethyldihydrobenzofuran-5-sulfonyl
PCR polymerase chain reaction
Pec pipecolic acid
PEG Polyethylene glycol)
pGlu pyroglutamic acid
Pic picolinic acid
PLT platelets
pY phosphotyrosine
PTFE polytetrafluoroethylene
RBC red blood cells
RBS ribosome binding site
RP-HPLC reversed phase HPLC
RT room temperature (25 °C)
Sar sarcosine
SDS sodium dodecyl sulfate
STK serine-threonine kinases
t-Boc tert-Butoxycarbonyl
tBu tert-Butyl
TGF tissue growth factor
THF thymic humoral factor
TK tyrosine kinase
TMP Thrombopoietin-mimetic peptide
TNF Tissue necrosis factor
TPO Thrombopoietin
TRAIL TNF-related apoptosis-inducing ligand
Trt trityl
UK urokinase
UKR urokinase receptor
VEGF vascular endothelial cell growth factor
VIP vasoactive intestinal peptide
WBC white blood cells

Amended Claims
What is claimed is:
1. A composition of matter of the formula
(X')n-F'-(X\ and multimers thereof, wherein:
F1 is an Fc domain modified so that it comprises at least one X3 in a loop region, said loop region being in a non-terminal domain of the Fc domain;
X1 and X2 are each independently selected from -(L\-P\ -(Lr)e-Pl-
X3 is independently selected from -(L5)c-p5/ ~(L\-P^(L\-P6, -(L5)C-PS-
P1, P1, P3, and P* are each independently sequences of pharmacologically active polypep tides or pharmacologically active peptides;
P5, P6, P7, and P8 are each independently sequences of pharmacologically active peptides; "
L1, L2, L3, V, L5, L6, L7, and L8 are each independently linkers; and
a, b, c, d, e, and f are each independently 0 or 1.
2. The composition of matter of Claim 1 wherein a and b are each 0.
3. The composition of matter of Claim 1 wherein the Fc domain
comprises an IgG Fc domain.
4. The composition of matter of Claim 3 wherein the Fc domain
comprises a sequence selected from SEQ ID NOS: 599 and 603 to 607.
5. The composition of matter of Claim 1 wherein the Fc domain
comprises an IgGl Fc domain.
6. The composition of matter of Claim 5 wherein the IgGl Fc domain
comprises SEQ ID NO: 599 and X3 is inserted into or replaces all or part
of a sequence selected from SEQ ID NOS: 621,622, 624, 625, 627, 628,
630, 632,634, and 636.
7. The composition of matter of Claim 6 wherein X3 is inserted into or
replaces all or part of a sequence selected from SEQ ID NOS: 623, 626,
629, 631, 633,635, and 637.
8. The composition of matter of Claim 7 wherein X3 is inserted at
Leu139/Thr140
9. The composition of matter of Claim 5 wherein the IgGl Fc domain
comprises SEQ ID NO: 603 and Xs is inserted into or replaces all or part
of a sequence selected from SEQ ID NOS: 621, 622, 624, 625, 627, 628,
630, 632, 634, and 636.
10. The composition of matter of Claim 9 wherein X3 is inserted at
11, The composition of matter of Claim 5 wherein the IgGl Fc domain
comprises SEQ ID NO: 604 and XJ is inserted into or replaces all or part
of a sequence selected from SEQ ID NOS: 621, 622, 624, 625, 627, 628,
632, 634, 636, and 644.
12. The composition of matter of Claim 11 wherein X3 is inserted at
NV!0/Km, Lua/Tlw Q171/POT, EJ7/N174/ S186/DW, Gm/Sm, or
13. The composition of matter of Claim 1 wherein the Fc domain
comprises an IgG3 Fc domain.
14. The composition of matter of Claim 13 wherein the IgG3 Fc domain
comprises SEQ ID NO: 605 and X3 is inserted into or replaces all or part
of a sequence selected from SEQ ID NOS: 614, 621, 622, 624, 627, 639,
641, 644, 645, and 646.
15. The composition of matter of Claim 14 wherein X3 is inserted at
G/S or G/N.
16. The composition of matter of Claim 1 wherein the Fc domain
comprises an IgG2 Fc domain.
17. The composition of matter of Claim 16 wherein the Fc domain
comprises SEQ ID NO: 606 and X3 is inserted into or replaces all or part
of a sequence selected from SEQ ID NOS: 621, 622, 624, 632, 636, 639,
640, 642, 644, and 646.
18. The composition of matter of Claim 17 wherein X3 is inserted at H The composition of matter of Claim 1 wherein the Fc domain
comprises an IgG4 Fc domain.
19. The composition of matter of Claim 19 wherein the Fc domain
comprises SEQ ID NO: 607 and X3 is inserted into or replaces all or part
of a sequence selected from SEQ ID NOS: 620, 621, 624, 627, 632, 634,
638, 639, 643, and 644.
20. The composition of matter of Claim 20 wherein X"* is inserted at Q/Ej,,
22. The composition of matter of Claim 1 wherein the Fc domain
comprises SEQ ID NO: 608 and X3 is inserted into or replaces all or part
of a sequence selected from SEQ ID NOS: 621, 622, 628, 624, 627, 632,
636, 639, 644, and 646.
23. The composition of matter of Claim 22 wherein X3 is inserted at
24. The composition of matter of Claim 1 wherein X3 comprises an
angiotensin-2 (ang-2) binding peptide sequence.
25. The composition of matter of Claim 24 wherein the ang-2 binding
peptide sequence is selected from SEQ ID NOS: 100 to 189.
26. The composition of matter of Claim 25 wherein the ang-2 binding
peptide sequence is SEQ ID NO: 147.
27. lite composition of matter of Claim 26 wherein FJ comprises an IgGl
Fc domain.
28. The composition of matter of Claim 27 having the sequence of SEQ ID
NO: 613.
29. The composition of matter of Claim 1 wherein X3 comprises a
myostatin binding peptide sequence.
30. The composition of matter of Claim 29 wherein the myostatin binding
peptide sequence is selected from SEQ ID NOS: 218 to 509.
31. The composition of matter of Claim 30 wherein the myostatin binding
peptide sequence is SEQ ID NO: 365.
32. The composition of matter of Claim 31 wherein F1 comprises an IgGl
Fc domain,
33. The composition of matter of Claim 32 having the sequence SEQ ID
NO: 612.
34. The composition of matter of Claim 1 wherein X'1 comprises an
erythropoietin-rnimetic (EPO-mimetic) peptide sequence.
35. The composition of matter of Claim 34 wherein the EPO-mimetic
peptide sequence is selected from SEQ ID NOS: 1 to 27.
36. The composition of matter of Claim 35 wherein the EPO-mimetic
peptide sequence is SEQ ID NO: 2.
37. The composition of matter of Claim 36 wherein F1 comprises an IgGl
Fc domain
38. The composition of matter of Claim 37 having the sequence of SEQ ID
NO: 615.
39. The composition of matter of Claim 1 wherein X3 comprises a
thrombopoietin-mimetic (TPO-mimetic) peptide sequence.
40. The composition of matter of Claim 39 wherein the TPO-mimetic
peptide sequence is selected from SEQ ID NOS: 28 to 99.
41. The composition of matter of Claim 40 wherein the TPO-mimetic
peptide sequence is SEQ ID NO: 28.
42. The composition of matter of Claim 41 wherein F' comprises an IgGl
Fc domain
43. The composition of matter of Claim 42 having the sequence SEQ ID
NO: 616.
44. The composition of matter of Claim 1 wherein X3 comprises a nerve
growth factor (NGF) binding peptide sequence.
45. The composition of matter of Claim 44 wherein the NGF binding
peptide sequence is selected from SEQ ID NOS: 190 to 218,
46. The composition of matter of Claim 1 wherein X3 comprises a 8 cell
activating factor (BAFF) binding peptide sequence.

47. The composition of matter of Claim 46 wherein the BAFF binding
peptide sequence is selected from SEQ ID NOS: 510 to 594.
48. A DNA encoding a composition of matter of Claim 1.
49. An expression vector comprising the DNA of Claim 48.
50. A host cell comprising the expression vector of Claim 49.
51. The cell of Claim 50, wherein the cell is an E. coll cell,
52. A process for preparing a pharmacologically active compound, which
comprises:
a. selecting at least one randomized peptide that modulates the
activity of a protein of interest; and
b. preparing a pharmacologic agent comprising an amino acid
sequence of the selected peptide as an internal, non-terminal, sequence
of an Fc domain.
53. The process of Claim 52, wherein the internal region of the Fc domain
is a loop region.
54. The process of Claim 52, wherein the peptide is selected in a process
comprising one or more techniques selected from yeast-based
screening, rational design, protein structural analysis, or screening of a
phage display library, an E. coli display library, a ribosomal library, or
a chemical peptide library.
55. The process of Claim 52, wherein the preparation of the pharmacologic
agent is carried out by:
a. preparing a gene construct comprising a nucleic acid sequence
encoding an Fc domain wherein the amino acid sequence of the
selected peptide is inserted into or replaces one or more amino acids
within the Fc domain; and
b. expressing the gene construct.
56. The process of Claim 55, wherein the gene construct is expressed in an
E. coli cell,
57. The process of Claim 52, wherein the protein of interest is a cell surface
receptor,
58. The process of Claim 52, wherein the protein of interest has a linear
epitope.
59. The process of Claim 52, wherein the protein of interest is a cytokine
receptor.
60. The process of Claim 52 wherein the Fc domain is an IgG Fc domain.
61. A modified antibody, comprising an Fc domain modified so that it
comprises at least one X3 in a loop region, said loop region being in a
non-terminal domain of the Fc domain, wherein:
X3 is independently selected from -(L5)C-P5, -(LS)C-P6-(L6)U -P6, -(L5)C-P5-
P5, P", P7, and P8 are each independently sequences of pharmacologically active pepticles;
L5, L6, L7, and L8 are each independently linkers; and c, d, e, and f are each independently 0 or 1.
62. A process for preparing a modified antibody, which comprises:
a) selecting at least one peptide that modulates the activity of a
protein of interest; and
b) preparing an antibody comprising an amino acid sequence of the
selected peptide in a loop region of an Fc domain of the antibody, said
loop region being in a non-terminal domain of the Fc domain.
63. A composition substantially as herein described with reference to the foregoing
description, tables, examples and the accompanying drawings.
64. A DMA encoding substantially as herein described with reference to the foregoing
description, tables, examples and the accompanying drawings.
65. A host cell substantially as herein described with reference to the foregoing description,
tables, examples and the accompanying drawings.
66. A process substantially as herein described with reference to the foregoing description,
tables, examples and the accompanying drawings.

Documents:

2963-delnp-2007-Abstract-(02-05-2013).pdf

2963-delnp-2007-abstract.pdf

2963-delnp-2007-Assignment-(02-05-2013).pdf

2963-delnp-2007-Claims-(02-05-2013).pdf

2963-delnp-2007-Claims-(22-07-2014).pdf

2963-delnp-2007-claims.pdf

2963-delnp-2007-Correspondance Others-(02-05-2013).pdf

2963-delnp-2007-Correspondance Others-(07-05-2013).pdf

2963-delnp-2007-Correspondence Others-(12-03-2013).pdf

2963-delnp-2007-Correspondence Others-(13-03-2013).pdf

2963-delnp-2007-Correspondence Others-(22-07-2014).pdf

2963-delnp-2007-Correspondence-others (23-09-2008).pdf

2963-delnp-2007-Correspondence-Others-(23-08-2013).pdf

2963-delnp-2007-Correspondence-Others-(28-05-2013).pdf

2963-delnp-2007-correspondence-others.pdf

2963-delnp-2007-description (complete).pdf

2963-delnp-2007-Drawings-(07-05-2013).pdf

2963-delnp-2007-drawings.pdf

2963-delnp-2007-form-1.pdf

2963-delnp-2007-Form-18 (23-09-2008).pdf

2963-delnp-2007-form-2.pdf

2963-delnp-2007-Form-3-(12-03-2013).pdf

2963-delnp-2007-Form-3-(28-05-2013).pdf

2963-delnp-2007-form-3.pdf

2963-delnp-2007-form-5.pdf

2963-delnp-2007-GPA (23-09-2008).pdf

2963-delnp-2007-GPA-(22-07-2014).pdf

2963-delnp-2007-pct-210.pdf

2963-delnp-2007-pct-220.pdf

2963-delnp-2007-pct-304.pdf

2963-delnp-2007-pct-401.pdf

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Patent Number

263272

Indian Patent Application Number

2963/DELNP/2007

PG Journal Number

43/2014

Publication Date

24-Oct-2014

Grant Date

16-Oct-2014

Date of Filing

20-Apr-2007

Name of Patentee

AMGEN INC.

Applicant Address

M/S 28-2-C, ONE AMGEN CENTER DRIVE, THOUSAND OAKS, CALIFORNIA 91320-1799, USA

Inventors:

#	Inventor's Name	Inventor's Address
1	GEGG, COLIN	3357 CORNING STREET, NEWBURY PARK, CALIFORNIA 91320, USA
2	SITNEY, KAREN C	4205 GOODLAND AVENUE, STUDIO CITY, CALIFORNIA 91604, USA
3	XIONG, FEI	2757 AUTUMN RIDGE DRIVE, THOUSAND OAKS, CALIFORNIA 91362, USA

PCT International Classification Number

C07K 19/00

PCT International Application Number

PCT/US2005/034273

PCT International Filing date

2005-09-23

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	60/612,680	2004-09-24	U.S.A.