# Patent

## Title
Therapeutic peptides

## Abstract

The invention relates to compounds that exhibit improved bioefficacy in multidose administration. More specifically, the invention relates to polypeptides or peptides modified to include an antibody Fc region and one or more water soluble polymers.

## Background
This application was filed as a U.S. National Phase Application pursuant to 35 U.S.C. §371 of International Application No. PCT/US05/24373 which was filed Jul. 8, 2005, which in turn claims benefit under 35 U.S.C. §119 of U.S. Application Ser. No. 60/586,419, which was filed Jul. 8, 2004. The entire disclosure of each of the foregoing applications is incorporated herein by reference in its entirety.


FIELD OF THE INVENTION
Generally, the invention relates to compounds that exhibit improved bioefficacy in multidose administration. More specifically, the invention relates to polypeptides or peptides modified to include an antibody Fc region and one or more water soluble polymers.
BACKGROUND OF THE INVENTION
Recombinant proteins are an emerging class of therapeutic agents. Such recombinant therapeutics have engendered advances in protein formulation and chemical modification. Modifications have been identified that can protect therapeutic proteins, primarily by blocking their exposure to proteolytic enzymes. Protein modifications may also increase the therapeutic protein's stability, circulation time, and biological activity. A review article describing protein modification and fusion proteins is Francis (1992), Focus on Growth Factors 3:4-10 (Mediscript, London), which is hereby incorporated by reference.
One useful modification is combination of a polypeptide with an “Fc” domain of an antibody. Antibodies comprise two functionally independent parts, a variable domain known as “Fab,” which binds antigen, and a constant domain known as “Fc,” which links to such effector functions as complement activation and attack by phagocytic cells. An Fc has a long serum half-life, whereas an Fab is short-lived. Capon et al. (1989), Nature 337: 525-31. When constructed together with a therapeutic protein, an Fc domain can provide longer half-life or incorporate such functions as Fc receptor binding, protein A binding, complement fixation and perhaps even placental transfer. Id. Table 1 summarizes use of Fc fusions known in the art.







TABLE 1







Fc fusion with therapeutic proteins











Fusion
Therapeutic



Form of Fc
partner
implications
Reference





IgG1
N-terminus of
Hodgkin's disease;
U.S. Pat. No. 5,480,981



CD30-L
anaplastic lymphoma;





T-cell leukemia



Murine Fcγ2a
IL-10
anti-inflammatory;
Zheng et al. (1995), J.




transplant rejection
Immunol. 154:





5590-600


IgG1
TNF receptor
septic shock
Fisher et al. (1996), N.





Engl. J. Med. 334: 1697-





1702; Van Zee, K.





et al. (1996). J.





Immunol. 156: 2221-30


IgG, IgA, IgM,
TNF receptor
inflammation, autoimmune
U.S. Pat. No. 5,808,029,


or IgE

disorders
issued Sep. 15, 1998


(excluding the





first domain)





IgG1
CD4 receptor
AIDS
Capon et al. (1989), Nature





337: 525-31


IgG1,
N-terminus
anti-cancer, antiviral
Harvill et al. (1995),


IgG3
of IL-2

Immunotech. 1: 95-105


IgG1
C-terminus of
osteoarthritis;
WO 97/23614, published



OPG
bone density
Jul. 3, 1997


IgG1
N-terminus of
anti-obesity
PCT/US 97/23183, filed



leptin

Dec. 11, 1997


Human Ig
CTLA-4
autoimmune disorders
Linsley (1991), J. Exp.


C□1


Med. 174: 561-9









Polyethylene glycol (“PEG”) fusion proteins and peptides have also been studied for use in pharmaceuticals, on artificial implants, and other applications where biocompatibility is of importance. Various derivatives of PEG have been proposed that have an active moiety for permitting PEG to be attached to pharmaceuticals and implants and to molecules and surfaces generally. For example, PEG derivatives have been proposed for coupling PEG to surfaces to control wetting, static buildup, and attachment of other types of molecules to the surface, including proteins or protein residues.
In other studies, coupling of PEG (“PEGylation”) has been shown to be desirable in overcoming obstacles encountered in clinical use of biologically active molecules. Published PCT Publication No. WO 92/16221 states, for example, that many potentially therapeutic proteins have been found to have a short half life in blood serum.
PEGylation decreases the rate of clearance from the bloodstream by increasing the apparent molecular weight of the molecule. Up to a certain size, the rate of glomerular filtration of proteins is inversely proportional to the size of the protein. The ability of PEGylation to decrease clearance, therefore, is generally not a function of how many PEG groups are attached to the protein, but the overall molecular weight of the altered protein. Decreased clearance can lead to increased efficacy over the non-PEGylated material. See, for example, Conforti et al., Pharm. Research Commun. vol. 19, pg. 287 (1987) and Katre et., Proc. Natl. Acad. Sci. U.S.A. vol. 84, pg. 1487 (1987).
In addition, PEGylation can decrease protein aggregation, (Suzuki et al., Biochem. Biophys. Acta vol. 788, pg. 248 (1984)), alter protein immunogenicity (Abuchowski et al., J. Biol. Chem. vol. 252 pg. 3582 (1977)), and increase protein solubility as described, for example, in PCT Publication No. WO 92/16221.
PEGylation of proteins illustrates some of the problems that have been encountered in attaching PEG to surfaces and molecules. The vast majority of PEGylating reagents react with free primary amino groups of the polypeptide. Most of these free amines are the epsilon amino group of lysine amino acid residues. Typical proteins possess a large number of lysines. Consequently, random attachment of multiple PEG molecules often occurs leading to loss of protein activity.
In addition, if the PEGylated protein is intended for therapeutic use, the multiple species mixture that results from the use of non-specific PEGylation leads to difficulties in the preparation of a product with reproducible and characterizable properties. This non-specific PEGylation makes it difficult to evaluate therapeutics and to establish efficacy and dosing information. Site selective PEGylation of such proteins, however, leads to reproducibly-modified materials that gain the desirable attributes of PEGylation without the loss of activity.
A variety of means have been used to attach the polyethylene glycol molecules to the protein. Amino groups, such as those on lysine residues or at the N-terminus, are convenient for such attachment. For example, Royer (U.S. Pat. No. 4,002,531, incorporated herein by reference in its entirety) states that reductive alkylation was used for attachment of polyethylene glycol molecules to an enzyme. EP 0 539 167 (incorporated herein by reference in its entirety), published Apr. 28, 1993, Wright, “Peg Imidates and Protein Derivates Thereof” states that peptides and organic compounds with free amino group(s) are modified with a derivative of PEG or related water-soluble organic polymers. U.S. Pat. No. 4,904,584 (incorporated herein by reference in its entirety), Shaw, issued Feb. 27, 1990, relates to the modification of lysine residues in proteins for the attachment of polyethylene glycol molecules via reactive amine groups. An increasing number of therapeutic proteins modified in the manner described above to include PEG have been previously described. See also, for example, European patent application publications EP 0 401 384; EP 0 473 268; EP 0 335 423; EP 0 442 724; EP 0 154 316, each of which is incorporated by reference in its entirety.
Other methods for attaching a polymer to a protein involve using a moiety to act as a linking group. Such moieties may, however, be antigenic. A tresyl chloride method involving no linking group is available, but this method may be difficult to use to produce therapeutic products as the use of tresyl chloride may produce toxic by-products. See Francis et al., In: Stability of protein pharmaceuticals: in vivo pathways of degradation and strategies for protein stabilization (Eds. Ahern., T. and Manning, M. C.) Plenum, N.Y., 1991) and Delgado et al., “Coupling of PEG to Protein By Activation With Tresyl Chloride, Applications In Immunoaffinity Cell Preparation”, In: Fisher et al., eds., Separations Using Aqueous Phase Systems, Applications In Cell Biology and Biotechnology, Plenum Press, N.Y. N.Y., 1989 pp. 211-213, each of which is incorporated by reference in its entirety.
In general, the interaction of a protein ligand with its receptor often takes place at a relatively large interface. However, as demonstrated in the case of human growth hormone bound to its receptor, only a few key residues at the interface actually contribute to most of the binding energy. Clackson, T. et al., Science 267:383-386 (1995). This observation and the fact that the bulk of the remaining protein ligand serves only to display the binding epitopes in the right topology makes it possible to find active ligands of much smaller size. 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 et al. (1990), Science 249: 386; Devlin et al. (1990), Science 249: 404; U.S. Pat. No. 5,223,409, issued Jun. 29, 1993; U.S. Pat. No. 5,733,731, issued Mar. 31, 1998; U.S. Pat. No. 5,498,530, issued Mar. 12, 1996; U.S. Pat. No. 5,432,018, issued Jul. 11, 1995; U.S. Pat. No. 5,338,665, issued Aug. 16, 1994; U.S. Pat. No. 5,922,545, issued Jul. 13, 1999; WO 96/40987, published Dec. 19, 1996; and WO 98/15833, published Apr. 16, 1998, each of which is incorporated by reference. In such libraries, random peptide sequences are displayed by fusion with coat proteins of filamentous phage. Typically, the displayed peptides are affinity-eluted against an antibody-immobilized extracellular domain of a receptor. The retained phages may be enriched by successive rounds of affinity purification and repropagation, and the best binding peptides are sequenced to identify key residues within one or more structurally related families of peptides. See, e.g., Cwirla et al. (1997), Science 276: 1696-9, in which two distinct families were identified. The peptide sequences may also suggest which residues may be safely replaced by alanine scanning or by mutagenesis at the DNA level. Mutagenesis libraries may be created and screened to further optimize the sequence of the best binders. Lowman (1997), Ann. Rev. Biophys. Biomol. Struct. 26: 401-24.
Other methods compete with phage display in peptide research. A peptide library can be fused to the carboxyl terminus of the lac repressor and expressed in E. coli. Another E. coli-based method allows display on the cell's outer membrane by fusion with a peptidoglycan-associated lipoprotein (PAL). 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. 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. 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. 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. These and related methods are collectively referred to as “chemical-peptide screening.” Chemical-peptide screening may be advantageous in that it allows use of D-amino acids and other unnatural analogues, as well as non-peptide elements. Both biological and chemical methods are reviewed in Wells & Lowman (1992), Curr. Opin. Biotechnol. 3: 355-62.
In the case of known bioactive peptides, rational design of peptide ligands with favorable therapeutic properties can be carried out. In such an approach, stepwise changes are made to a peptide sequence and the effect of the substitution upon bioactivity or a predictive biophysical property of the peptide (e.g., solution structure) is determined. These techniques are collectively referred to as “rational design.” In one such technique, a series of peptides is made in which a single residue at a time is replaced 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. 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, peptide mimetics of any protein can be identified using phage display and the other methods mentioned above. These methods have also 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 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 therefor 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











Binding





partner/




Form of
protein of
Pharmacologic



peptide
interest1
activity
Reference





intrapeptide
EPO receptor
EPO-mimetic
Wrighton et al. (1996),


disulfide-


Science 273: 458-63; U.S.


bonded


Pat. No. 5,773,569, issued





Jun. 30, 1998 to Wrighton





et al.


C-terminally
EPO receptor
EPO-mimetic
Livnah et al. (1996),


cross-linked


Science 273: 464-71;


dimer


Wrighton et al. (1997),





Nature Biotechnology 15:





1261-5; International patent





application WO 96/40772,





published Dec. 19, 1996


linear
EPO receptor
EPO-mimetic
Naranda et al. (1999), Proc.





Natl. Acad. Sci. USA, 96:





7569-74; WO 99/47151,





published Sep. 23, 1999


linear
c-Mpl
TPO-mimetic
Cwirla et al.(1997) Science





276: 1696-9; U.S. Pat. No.





5,869,451, issued Feb. 9,





1999; U.S. Pat. No.





5,932,946, issued Aug. 3,





1999


C-terminally
c-Mpl
TPO-mimetic
Cwirla et al. (1997),


cross-linked


Science 276: 1696-9


dimer





disulfide-

stimulation of hematopoiesis
Paukovits et al. (1984),


linked dimer

(“G-CSF-mimetic”)
Hoppe-Seylers Z. Physiol.





Chem. 365: 303-11;





Laerum et al. (1988), Exp.





Hemat. 16: 274-80


alkylene-

G-CSF-mimetic
Bhatnagar et al. (1996),


linked dimer


J. Med. Chem. 39: 3814-9;





Cuthbertson et al. (1997), J.





Med. Chem. 40: 2876-82;





King et al. (1991), Exp.





Hematol. 19: 481; King et





al. (1995), Blood 86





(Suppl. 1): 309a


linear
IL-1 receptor
inflammatory and
U.S. Pat. No. 5,608,035;




autoimmune diseases
U.S. Pat. No. 5,786,331;




(“IL-1 antagonist” or
U.S. Pat. No. 5,880,096;




“IL-1ra-mimetic”)
Yanofsky et al. (1996).





Proc. Natl. Acad. Sci. 93:





7381-6; Akeson et al.





(1996), J. Biol. Chem. 271:





30517-23; Wiekzorek et al.





(1997), Pol. J. Pharmacol.





49: 107-17; Yanofsky





(1996), PNAs, 93: 7381-





7386.


linear
Facteur thymique
stimulation of lymphocytes
Inagaki-Ohara et al. (1996),



serique (FTS)
(“FTS-mimetic”)
Cellular Immunol. 171: 30-





40; Yoshida (1984), Int. J.





Immunopharmacol, 6: 141-6.


intrapeptide
CTLA4 MAb
CTLA4-mimetic
Fukumoto et al. (1998),


disulfide


Nature Biotech. 16: 267-70


bonded





exocyclic
TNF-α receptor
TNF-α antagonist
Takasaki et al. (1997),





Nature Biotech. 15: 1266-





70; WO 98/53842,





published Dec. 3, 1998


linear
TNF-α receptor
TNF-α antagonist
Chirinos-Rojas ( ), J.





Imm., 5621-5626.


intrapeptide
C3b
inhibition of complement
Sahu et al. (1996), J.


disulfide

activation; autoimmune
Immunol. 157: 884-91;


bonded

diseases
Morikis et al. (1998),




(“C3b-antagonist”)
Protein Sci. 7: 619-27


linear
vinculin
cell adhesion processes-
Adey et al. (1997).




cell growth, differentiation,
Biochem. J. 324: 523-8




wound healing, tumor





metastasis (“vinculin





binding”)



linear
C4 binding
anti-thrombotic
Linse et al. (1997), J. Biol.



protein (C4BP)

Chem. 272: 14658-65


linear
urokinase receptor
processes associated with
Goodson et al. (1994),




urokinase interaction with its
Proc. Natl. Acad. Sci. 91:




receptor (e.g., angiogenesis,
7129-33; International




tumor cell invasion and
application WO 97/35969,




metastasis); (“UKR
published Oct. 2, 1997




antagonist”)



linear
Mdm2, Hdm2
Inhibition of inactivation of
Picksley et al. (1994),




p53 mediated by Mdm2 or
Oncogene 9: 2523-9;




hdm2; anti-tumor
Bottger et al. (1997) J. Mol.




(“Mdm/hdm antagonist”)
Biol. 269: 744-56; Bottger





et al. (1996), Oncogene 13:





2141-7


linear
p21WAF1
anti-tumor by mimicking the
Ball et al. (1997), Curr.




activity of p21WAF1
Biol. 7: 71-80


linear
farnesyl
anti-cancer by preventing
Gibbs et al. (1994), Cell



transferase
activation of ras oncogene
77: 175-178


linear
Ras effector
anti-cancer by inhibiting
Moodie et al. (1994),



domain
biological function of the ras
Trends Genet 10: 44-48




oncogene
Rodriguez et al. (1994),





Nature 370: 527-532


linear
SH2/SH3
anti-cancer by inhibiting
Pawson et al (1993), Curr.



domains
tumor growth with activated
Biol. 3: 434-432




tyrosine kinases; treatment
Yu et al. (1994), Cell




of SH3-mediated disease
76: 933-945; Rickles et al.




states (“SH3 antagonist”)
(1994), EMBO J. 13: 5598-





5604; Sparks et al. (1994),





J. Biol. Chem. 269: 23853-





6; Sparks et al. (1996),





Proc. Natl. Acad. Sci. 93:





1540-4; U.S. Pat. No.





5,886,150, issued Mar.





23, 1999; U.S. Pat. No.





5,888,763, issued Mar.





30, 1999


linear
p16INK4
anti-cancer by mimicking
Fåhraeus et al. (1996),




activity of p16; e.g.,
Curr. Biol. 6: 84-91




inhibiting cyclin D-Cdk





complex (“p16-mimetic”)



linear
Src, Lyn
inhibition of Mast cell
Stauffer et al. (1997),




activation, IgE-related
Biochem. 36: 9388-94




conditions, type I





hypersensitivity (“Mast cell





antagonist”)



linear
Mast cell protease
treatment of inflammatory
International application




disorders mediated by
WO 98/33812, published




release of tryptase-6
Aug. 6, 1998




(“Mast cell protease





inhibitors”)



linear
HBV core antigen
treatment of HBV viral
Dyson & Muray (1995),



(HBcAg)
infections (“anti-HBV”)
Proc. Natl. Acad. Sci. 92:





2194-8


linear
selectins
neutrophil adhesion;
Martens et al. (1995), J.




inflammatory diseases
Biol. Chem. 270: 21129-




(“selectin antagonist”)
36; European patent





application EP 0 714 912,





published Jun. 5, 1996


linear, cyclized
calmodulin
calmodulin antagonist
Pierce et al. (1995), Molec.





Diversity 1: 259-65;





Dedman et al. (1993), J.





Biol. Chem. 268: 23025-





30; Adey & Kay (1996),





Gene 169: 133-4


linear,
integrins
tumor-homing; treatment for
International applications


cyclized-

conditions related to
WO 95/14714, published




integrin-mediated cellular
Jun. 1, 1995; WO




events, including platelet
97/08203, published Mar.




aggregation, thrombosis,
6, 1997; WO 98/10795,




wound healing,
published Mar. 19, 1998;




osteoporosis, tissue repair,
WO 99/24462, published




angiogenesis (e.g., for
May 20, 1999; Kraft et al.




treatment of cancer), and
(1999), J. Biol. Chem. 274:




tumor invasion
1979-1985




(“integrin-binding”)



cyclic, linear
fibronectin and
treatment of inflammatory
WO 98/09985, published



extracellular
and autoimmune conditions
Mar. 12, 1998



matrix





components of T





cells and





macrophages




linear
somatostatin and
treatment or prevention of
European patent application



cortistatin
hormone-producing tumors,
0 911 393, published Apr.




acromegaly, giantism,
28, 1999




dementia, gastric ulcer,





tumor growth, inhibition of





hormone secretion,





modulation of sleep or





neural activity



linear
bacterial
antibiotic; septic shock;
U.S. Pat. No. 5,877,151,



lipopolysaccharide
disorders modulatable by
issued Mar. 2, 1999




CAP37



linear or
pardaxin, mellitin
antipathogenic
WO 97/31019, published


cyclic,


28 Aug. 1997


including D-





amino acids





linear, cyclic
VIP
impotence,
WO 97/40070, published




neurodegenerative disorders
Oct. 30, 1997


linear
CTLs
cancer
EP 0 770 624, published





May 2, 1997


linear
THF-gamma2

Burnstein (1988),





Biochem., 27: 4066-71.


linear
Amylin

Cooper (1987), Proc. Natl.





Acad. Sci., 84: 8628-32.


linear
Adrenomedullin

Kitamura (1993), BBRC,





192: 553-60.


cyclic, linear
VEGF
anti-angiogenic; cancer,
Fairbrother (1998),




rheumatoid arthritis, diabetic
Biochem., 37: 17754-17764.




retinopathy, psoriasis





(“VEGF antagonist”)



cyclic
MMP
inflammation and
Koivunen (1999), Nature




autoimmune disorders;
Biotech., 17: 768-774.




tumor growth





(“MMP inhibitor”)




HGH fragment
treatment of obesity
U.S. Pat. No. 5,869,452



Echistatin
inhibition of platelet
Gan (1988). J. Biol. Chem.,




aggregation
263: 19827-32.


linear
SLE autoantibody
SLE
WO 96/30057, published





Oct. 3, 1996



GD1alpha
suppression of tumor
Ishikawa et al. (1998),




metastasis
FEBS Lett. 441 (1): 20-4



antiphospholipid
endothelial cell activation,
Blank et al. (1999), Proc.



beta-2-
antiphospholipid syndrome
Natl. Acad. Sci. USA 96:



glycoprotein-I
(APS), thromboembolic
5164-8



(β2GPI)
phenomena,




antibodies
thrombocytopenia, and





recurrent fetal loss



linear
T Cell Receptor
diabetes
WO 96/11214, published



beta chain

Apr. 18, 1996.




Antiproliferative, antiviral
WO 00/01402, published





Jan. 13, 2000.




anti-ischemic, growth
WO 99/62539, published




hormone-liberating
Dec. 9, 1999.




anti-angiogenic
WO 99/61476, published





Dec. 2, 1999.


linear

Apoptosis agonist; treatment
WO 99/38526, published




of T cell-associated
Aug. 5, 1999.




disorders (e.g., autoimmune





diseases, viral infection, T





cell leukemia, T cell





lymphoma)



linear
MHC class II
treatment of autoimmune
U.S. Pat. No. 5,880,103,




diseases
issued Mar. 9, 1999.


linear
androgen R, p75,
proapoptotic, useful in
WO 99/45944, published



MJD, DCC,
treating cancer
Sep. 16, 1999.



huntingtin




linear
von Willebrand
inhibition of Factor VIII
WO 97/41220, published



Factor; Factor VIII
interaction; anticoagulants
Apr. 29, 1997,


linear
lentivirus LLP1
antimicrobial
U.S. Pat. No. 5,945,507,





issued Aug. 31, 1999.


linear
Delta-Sleep
sleep disorders
Graf (1986), Peptides



Inducing Peptide

7: 1165.


linear
C-Reactive
inflammation and cancer
Barna (1994), Cancer



Protein (CRP)

Immunol. Immunother.





38: 38 (1994).


linear
Sperm-Activating
infertility
Suzuki (1992), Comp.



Peptides

Biochem. Physiol.





102B: 679.


linear
angiotensins
hematopoietic factors for
Lundergan (1999), J.




hematocytopenic conditions
Periodontal Res. 34(4):




from cancer, AIDS, etc.
223-228.


linear
HIV-1 gp41
anti-AIDS
Chan (1998), Cell 93:





681-684.


linear
PKC
inhibition of bone resorption
Moonga (1998), Exp.





Physiol. 83: 717-725.


linear
defensins (HNP-
antimicrobial
Harvig (1994), Methods



1, -2, -3, -4)

Enz. 236: 160-172.


linear
p185HER2/neu,
AHNP-mimetic: anti-tumor
Park (2000), Nat.



C-erbB-2

Biotechnol. 18: 194-198.


linear
gp130
IL-6 antagonist
WO 99/60013, published





Nov. 25, 1999.


linear
collagen, other
autoimmune diseases
WO 99/50282, published



joint, cartilage,

Oct. 7, 1999.



arthritis-related





proteins




linear
HIV-1 envelope
treatment of neurological
WO 99/51254, published



protein
degenerative diseases
Oct. 14, 1999.


linear
IL-2
autoimmune disorders (e.g.,
WO 00/04048, published




graft rejection, rheumatoid
Jan. 27, 2000; WO




arthritis)
00/11028, published Mar.





2, 2000.





1The protein listed in this column may be bound by the associated peptide (e.g., EPO receptor, IL-1 receptor) or mimicked by the associated peptide. The references listed for each clarify whether the molecule is bound by or mimicked by the peptides.






Peptides identified by peptide library screening have been regarded as “leads” in development of therapeutic agents rather than being used as therapeutic agents themselves. Like other proteins and peptides, they would be rapidly removed in vivo either by renal filtration, cellular clearance mechanisms in the reticuloendothelial system, or proteolytic degradation. Francis (1992), Focus on Growth Factors 3: 4-11. As a result, the art presently uses the identified peptides to validate drug targets or as scaffolds for design of organic compounds that might not have been as easily or as quickly identified through chemical library screening. Lowman (1997), Ann. Rev. Biophys. Biomol. Struct. 26: 401-24; Kay et al. (1998), Drug Disc. Today 3: 370-8.
In spite of the availability of recombinant therapeutic proteins, previously identified peptide mimetic, and modifications thereof, there thus remains a need in the art to provide additional compounds that have improved bioefficacy, particularly when administered in a multidose regimen.
SUMMARY OF THE INVENTION
The present invention relates to polypeptides or peptides modified to include an antibody Fc region and one or more water soluble polymers.
In one aspect, a substantially homogenous compound is provided comprising a structure set out in Formula I,

[(X1)a—F1—(X2)b]-(L1)c-WSPd  Formula I

wherein:

F1 is a vehicle;
X1 is selected from

    
    
        P1-(L2)e—
        P2-(L3)f-P1-(L2)e—
        P4-(L4)g-P2-(L3)f-P1-(L2)e- and
        P4-(L5)h-P3-(L4)g-P2-(L3)f-P1-(L2)e-
    
    


X2 is selected from:

    
    
        -(L2)e-P1,
        -(L2)e-P1-(L3)f-P2,
        -(L2)e-P1-(L3)f-P2-(L4)g-P3, and
        -(L2)e-P1-(L3)f-P2-(L4)g-P3-(L5)h-P4 
    
    


wherein P1, P2, P3, and P4 are each independently sequences of pharmacologically active peptides;
L1, L2, L3, L4, and L5 are each independently linkers;
a, b, c, e, f, g, and h are each independently 0 or 1,

    
    
        provided that at least one of a and b is 1;
    
    


d is at least 1; and
WSP is a water soluble polymer, the attachment of which is effected at any reactive moiety in F1;
said compound having a property of improved bioefficacy when administered in a multidose regimen. In one aspect, the compound a multimer, and in another aspect, the compound is a dimer.
In one embodiment, the invention provides a compound of Formula I comprising a structure set out in Formula II

[X1—F1]-(L1)c-WSPd  Formula II

wherein F1 is an Fc domain and is attached at the C-terminus of X1, and one or more WSP is attached to the Fc domain, optionally through linker L1. Compounds having this structure are provided as a multimer in one aspect and a dimer in another aspect.

In another embodiment, the invention provides a compound of Formula I comprising a structure set out in Formula III

[F1—X2]-(L1)c-WSPd  Formula III

wherein F1 is an Fc domain and is attached at the N-terminus of X2, and one or more WSP is attached to the Fc domain, optionally through linker L1. Multimers and dimers of a compound having this structure are also provided.

The invention also provides a compound of Formula I comprising a structure set out in Formula IV

[F1-(L2)e-P1]-(L1)c-WSPd  Formula IV

wherein F1 is an Fc domain and is attached at the N-terminus of -(L1)c-P1 and one or more WSP is attached to the Fc domain, optionally through linker L1. Multimers and dimers of a compound having this structure are also provided.

The invention further contemplates a compound of Formula I comprising a structure set out in Formula V

[F1-(L2)e-P1-(L3)f-P2]-(L1)c-WSPd  Formula V

wherein F1 is an Fc domain and is attached at the N-terminus of -L2-P1-L3-P2 and one or more WSP is attached to the Fc domain, optionally through linker L1. Multimers and dimers of a compound having this structure are also provided.

In one aspect, a compound of the invention is provided as described above wherein P1 and/or P2 are independently selected from a peptide set out in any one of Tables 4 through 20. In one aspect, P1 and/or P2 have the same amino acid sequence.
In another aspect, a compound of the invention is provided as described above wherein F1 is an Fc domain. In another aspect, a compound of the invention is provided wherein WSP is PEG. In yet another aspect, a compound as described above is provided wherein F1 is an Fc domain and WSP is PEG.
In another embodiment, a substantially homogenous compound is provided comprising the structure:







(SEQ ID NO: 1)


WSP-Fc-GGGGG-IEGPTLRQWLAARA-GGGGGGGG-IEGPTLRQWLAARA






wherein WSP is a water soluble polymer, said compound having a property of binding to c-Mpl and stimulating platelet production, said compound having a property of improved bioefficacy in multidose administration. Multimers and dimers of a compound having this structure are also contemplated.

In another embodiment, a substantially homogenous compound is provided comprising the structure:







(SEQ ID NO: 2)


PEG-Fc-GGGGG-IEGPTLRQWLAARA-GGGGGGGG-IEGPTLRQWLAARA






said compound having a property of binding to c-Mpl and stimulating platelet production, said compound having a property of improved bioefficacy in multidose administration. Multimers and dimers of a compound having this structure are contemplated.

In one aspect, the PEG component of a compound of the invention has a molecular weight of between about 2 kDa and 100 kDa. In another aspect, the PEG component of a compound of the invention has a molecular weight of between about 6 kDa and 25 kDa.
The invention further provides a composition comprising a compound of the invention wherein the composition comprises at least 50% PEGylated compound. In another aspect, the composition of the invention comprises at least 75% PEGylated compound, at least 85% PEGylated compound, at least 90% PEGylated compound, at least 95% PEGylated compound, and at least 99% PEGylated compound.
In one embodiment, the invention also provides a substantially homogenous compound having the structure:







(SEQ ID NO: 2)


PEG-Fc-GGGGG-IEGPTLRQWLAARA-GGGGGGGG-IEGPTLRQWLAARA






wherein PEG has a molecule weight of about 20 kD, said compound of SEQ ID NO: 2 being a dimer and having a property of binding to c-Mpl and stimulating platelet production, and said compound having a property of improved bioefficacy in multidose administration.

The invention further provides a pharmaceutical composition comprising: (a) a substantially homogenous compound having the structure:







(SEQ ID NO: 2)


PEG-Fc-GGGGG-IEGPTLRQWLAARA-GGGGGGGG-IEGPTLRQWLAARA






wherein PEG has a molecule weight of about 20 kD, (b) at least 95% diPEGylated compound; and (c) a pharmaceutically acceptable diluent, adjuvant or carrier, said composition having a property of improved bioefficacy in multidose administration.

In one embodiment, a pharmaceutical composition is provided comprising: (a) a substantially homogenous compound having the structure:







(SEQ ID NO: 2)


PEG-Fc-GGGGG-IEGPTLRQWLAARA-GGGGGGGG-IEGPTLRQWLAARA






wherein PEG has a molecule weight of about 20 kD and the compound of SEQ ID NO: 2 is a dimer, (b) at least 95% diPEGylated compound; and (c) a pharmaceutically acceptable diluent, adjuvant or carrier, said composition having a property of improved bioefficacy in multidose administration.

The invention also provide a method of treating a hematopoietic disorder comprising administering a compound or composition of the invention in a regimen effective to treat said disorder.



BRIEF DESCRIPTION OF THE FIGURE
Numerous other aspects and advantages of the present invention will therefore be apparent upon consideration of the following detailed description thereof, reference being made to the drawing wherein:
FIG. 1 shows increase in platelet production in vivo in a multidose administration regimen with a PEG-Fc-TMP of the invention, wherein results using a PEG-Fc-TMP are shown with triangles (▴) and results using an Fc-TMP are shown with circles (●). Squares (▪) represent a control.



DETAILED DESCRIPTION OF THE INVENTION
Definition of Terms
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. Of course, these additional amino acids should not significantly interfere with the activity of the compound.
“Substantially homogenous” as used herein with reference to a preparation of the invention means that the preparation includes a single species of a therapeutic compound detectable in the preparation of total therapeutic molecules in the preparation, unless otherwise stated at a specific percentage of total therapeutic molecules. In general, a substantially homogenous preparation is homogenous enough to display the advantages of a homogenous preparation, e.g., ease in clinical application in predictability of lot to lot pharmacokinetics.
“Bioefficacy” refers to the capacity to produce a desired biological effect. Bioefficacy of different compounds, or different dosages of the same compound, or different administrations of the same compound are generally normalized to the amount of compound(s) to permit appropriate comparison.
“Multidose administration” refers to a therapeutic or prophylactic treatment regimen which includes administration of more than one amount of a compound over a period of time.
The term “vehicle” refers to a molecule that prevents degradation and/or increases half-life, reduces toxicity, reduces immunogenicity, or increases biological activity of a therapeutic protein. Exemplary vehicles include an Fc domain as well as a linear polymer; a branched-chain polymer (see, for example, U.S. Pat. Nos. 4,289,872 to Denkenwalter et al., issued Sep. 15, 1981; 5,229,490 to Tam, issued Jul. 20, 1993; WO 93/21259 by Frechet et al., published 28 Oct. 1993); a lipid; a cholesterol group; a carbohydrate or oligosaccharide; or any natural or synthetic protein, polypeptide or peptide that binds to a salvage receptor. Vehicles are further described hereinafter.
The term “native Fc” refers to molecule or sequence comprising the sequence of a non-antigen-binding fragment resulting from digestion of whole antibody, whether in monomeric or multimeric form. The original immunoglobulin source of the native Fc is in one aspect of human origin and may be any of the immunoglobulins. A native Fc is a monomeric polypeptide that may be linked into dimeric or multimeric forms by covalent association (i.e., disulfide bonds), non-covalent association or a combination of both. The number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from one to four depending on class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2). One example of a native Fc is a disulfide-bonded dimer resulting from papain digestion of an IgG. 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 Sep. 1997) and WO 96/32478 describe exemplary Fc variants, as well as interaction with the salvage receptor, and are hereby incorporated by reference. In one aspect, the term “Fc variant” comprises a molecule or sequence that is humanized from a non-human native Fc. In another aspect, 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 Fcs, the term “Fc domain” includes molecules in monomeric or multimeric form, whether digested from whole antibody or produced by other means. In one embodiment, for example, the Fc region can be:







(SEQ ID NO: 1696)


DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED


 


PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK


 


CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK


 


GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG


 


NVFSCSVMHEALHNYTQKSLSLSPGK.






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 terms “derivatizing,” “derivative” or “derivatized” comprise processes and resulting compounds in which, for example and without limitation, (1) the compound has a cyclic portion; for example, cross-linking between cysteinyl residues within the compound; (2) the compound is cross-linked or has a cross-linking site; for example, the compound has a cysteinyl residue and thus forms cross-linked dimers in culture or in vivo; (3) one or more peptidyl linkage is replaced by a non-peptidyl linkage; (4) the N-terminus is replaced by —NRR1, NRC(O)R1, —NRC(O)OR1, —NRS(O)2R1, —NHC(O)NHR, a succinimide group, or substituted or unsubstituted benzyloxycarbonyl-NH—, wherein R and R1 and the ring substituents are as defined hereinafter; (5) the C-terminus is replaced by —C(O)R2 or —NR3R4 wherein R2, R3 and R4 are as defined hereinafter; and (6) compounds in which individual amino acid moieties are modified through treatment with agents capable of reacting with selected side chains or terminal residues. Derivatives are further described hereinafter.
The term “peptide” refers to molecules of 2 to 40 amino acids, molecules of 3 to 20 amino acids, and those of 6 to 15 amino acids. For example, peptides having a size selected from no greater than 35, no greater than 30, no greater than 25, no greater than 20 amino acids and/or no greater than 15 amino acids, are contemplated herein. Exemplary peptides may be randomly generated by any of the methods cited described herein, carried in a peptide library (e.g., a phage display library), derived by digestion of proteins, or chemically synthesized. Peptides include D and L form, either purified or in a mixture of the two forms.
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 Tables 2 and 7. As an example, the term “EPO-mimetic peptide” comprises any peptides that can be identified or derived as described in Wrighton et al. (1996), Science 273: 458-63, Naranda et al. (1999), Proc. Natl. Acad. Sci. USA 96: 7569-74, or any other reference in Table 2 identified as having EPO-mimetic subject matter. Those of ordinary skill in the art appreciate that each of these references enables one to select different peptides than actually disclosed therein by following the disclosed procedures with different peptide libraries.
As another example, the term “TPO-mimetic peptide” or “TMP” 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 and any other reference in Table 2 identified as having TPO-mimetic subject matter, as well as International application WO 00/24770 published May 4, 2000, 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.
As another example, the term “G-CSF-mimetic peptide” comprises any peptides that can be identified or described in Paukovits et al. (1984), Hoppe-Seylers Z. Physiol. Chem. 365: 303-11 or any of the references in Table 2 identified as having G-CSF-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 “CTLA4-mimetic peptide” comprises any peptides that can be identified or derived as described in Fukumoto et al. (1998), Nature Biotech. 16: 267-70. 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 terms “IL-1 antagonist” and “IL-1ra-mimetic peptide” comprises peptides that inhibit or down-regulate activation of the IL-1 receptor by IL-1. IL-1 receptor activation results from formation of a complex among IL-1, IL-1 receptor, and IL-1 receptor accessory protein. IL-1 antagonist or IL-1ra-mimetic peptides bind to IL-1, IL-1 receptor, or IL-1 receptor accessory protein and obstruct complex formation among any two or three components of the complex. Exemplary IL-1 antagonist or IL-1ra-mimetic peptides can be identified or derived as described in U.S. Pat. Nos. 5,608,035, 5,786,331, 5,880,096, or any of the references in Table 2 identified as having IL-1ra-mimetic or IL-1 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 “VEGF-antagonist peptide” comprises peptides that can be identified or derived as described in Fairbrother (1998), Biochem. 37: 17754-64, and in any of the references in Table 2 identified as having VEGF-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 “MMP inhibitor peptide” comprises peptides that can be identified or derived as described in Koivunen (1999), Nature Biotech. 17: 768-74 and in any of the references in Table 2 identified as having MMP inhibitory 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 “myostatin inhibitor peptide” comprises peptides that can be identified by their ability to reduce or block myostatin activity or signaling as demonstrated in in vitro assays such as, for example the pMARE C2C12 cell-based myostatin activity assay or by in vivo animal testing as described in U.S. patent application Publication No US20040181033A1 and PCT application publication No. WO2004/058988. Exemplary myostatin inhibitor pepetides are set out in Tables 21-24.
The term “integrin/adhesion antagonist” comprises peptides that inhibit or down-regulate the activity of integrins, selectins, cell adhesion molecules, integrin receptors, selectin receptors, or cell adhesion molecule receptors. Exemplary integrin/adhesion antagonists comprise laminin, echistatin, the peptides described in Tables 25-28.
The term “bone resorption inhibitor” refers to such molecules as determined by the assays of Examples 4 and 11 of WO 97/23614, which is hereby incorporated by reference in its entirety. Exemplary bone resorption inhibitors include OPG and OPG-L antibody, which are described in WO 97/23614 and WO98/46751, respectively, which are hereby incorporated by reference in their entirety.
The term “nerve growth factor inhibitor” or “nerve growth factor agonist” comprises a peptide that binds to and inhibits nerve growth factor (NGF) activity or signaling. Exemplary peptides of this type are set out in Table 29.
The term “TALL-1 modulating domain” refers to any amino acid sequence that binds to the TALL-1 and comprises naturally occurring sequences or randomized sequences. Exemplary TALL-1 modulating domains can be identified or derived by phage display or other methods mentioned herein. Exemplary peptides of this type are set out in Tables 30 and 31.
The term “TALL-1 antagonist” refers to a molecule that binds to the TALL-1 and increases or decreases one or more assay parameters opposite from the effect on those parameters by full length native TALL-1. Such activity can be determined, for example, by such assays as described in the subsection entitled “Biological activity of AGP-3” in the Materials & Methods section of the patent application entitled, “TNF-RELATED PROTEINS”, WO 00/47740, published Aug. 17, 2000.
The term “Ang 2-antagonist peptide” comprises peptides that can be identified or derived as having Ang-2-antagonistic characteristics. Exemplary peptides of this type are set out in Tables 32-38.
Additionally, physiologically acceptable salts of the compounds of this invention are also contemplated. 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.
The term “WSP” refers to a water soluble polymer which prevents a peptide, protein or other compound to which it is attached from precipitating in an aqueous environment, such as, by way of example, a physiological environment. A more detailed description of various WSP embodiments contemplated by the invention follows.
Structure of Compounds
In General. In preparations in accordance with the invention, a peptide is attached to a vehicle through the N-terminus or C-terminus of the peptide, and the resulting structure further modified with a covalently attached WSP which is attached to the vehicle moiety in the vehicle-peptide product. Thus, the WSP-vehicle-peptide molecules of this invention may be described by the following formula I:

[(X1)a—F1—(X2)b]-(L1)c-WSPd  I

wherein:

F1 is a vehicle;
X1 is selected from

    
    
        P1-(L2)e-
        P2-(L3)f-P1-(L2)e-
        P3-(L4)-P2-(L3)f-P1-(L2)e- and
        P4-(L5)h-P3-(L4)g-P2-(L3)f-P1-(L2)e-
    
    


X2 is selected from:

    
    
        -(L2)e-P1,
        -(L2)e-P1-(L3)f-P2,
        -(L2)e-P1-(L3)f-P2-(L4)g-P3, and
        -(L2)e-P1-(L3)f-P2-(L4)g-P3-(L5)h-P4 
    
    


wherein P1, P2, P3, and P4 are each independently sequences of pharmacologically active peptides;
L1, L2, L3, L4, and L5 are each independently linkers;
a, b, c, e, f, g, and h are each independently 0 or 1,

    
    
        provided that at least one of a and b is 1;
    
    


d is at least 1; and
WSP is a water soluble polymer, the attachment of which is effected at any reactive moiety in F1.
Thus, compound I comprises compounds of the formulae

[X1—F1]-(L1)c-WSPd  II

including multimers thereof, wherein F1 is an Fc domain and is attached at the C-terminus of X1, and one or more WSP is attached to the Fc domain, optionally through linker L1;

[F1—X2]-(L1)c-WSPd  III

including multimers thereof, wherein F1 is an Fc domain and is attached at the N-terminus of X2, and one or more WSP is attached to the Fc domain, optionally through linker L1;

[F1-(L2)e-P1]-(L1)c-WSPd  IV

including multimers thereof, wherein F1 is an Fc domain and is attached at the N-terminus of -(L1)c-P1 and one or more WSP is attached to the Fc domain, optionally through linker L1; and

[F1-(L2)e-P1-(L3)f-P2]-(L1)c-WSPd  V

including multimers thereof, wherein F1 is an Fc domain and is attached at the N-terminus of -L2-P1-L3-P2 and one or more WSP is attached to the Fc domain, optionally through linker L1.

Peptides. Any number of peptides may be used in conjunction with the present invention. Of particular interest are peptides that mimic the activity of EPO, TPO, growth hormone, G-CSF, GM-CSF, IL-1ra, leptin, CTLA4, TRAIL, TGF-α, and TGF-β. Peptide antagonists are also of interest, particularly those antagonistic to the activity of TNF, leptin, any of the interleukins (IL-1, 2, 3, . . . ), and proteins involved in complement activation (e.g., C3b). 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.
By way of example and without limitation, a group of peptides that bind to cytokine receptors are provided. 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 are the CKRs (family I in Table 3). The receptor classification appears in Table 3.







TABLE 3







Cytokine Receptors Classified by Receptor Code








Cytokines (ligands)
Receptor Type










family
subfamily
family
subfamily

















I.
Hematopoietic
1.
IL-2, IL-4, IL-7,
I.
Cytokine R
1.
shared γCr, IL-



cytokines

IL-9, IL-13, IL-15

(CKR)

9R, IL-4R




2.
IL-3, IL-5, GM-CSF


2.
shared GP 140 βR




3.
IL-6, IL-11, IL-


3.
3.shared RP 130,





12, LIF, OSM,



IL-6 R, Leptin R





CNTF, Leptin (OB)








4.
G-CSF, EPO,


4.
“single chain” R,





TPO, PRL, GH



GCSF-R, TPO-R,









GH-R




5.
IL-17, HVS-IL-17


5.
other R2


II.
IL-10 ligands

IL-10, BCRF-1,
II.
IL-10 R







HSV-IL-10






III.
Interferons
1.
IFN-αl, α2, α4, m, t, IFN-β3
III.
Interferon R
1.
IFNAR




2.
IFN-γ


2.
IFNGR


IV.
IL-1 and IL-1
1.
IL-1α, IL-1β, IL-
IV.
IL-1R
1.
IL-1R, IL-1RAcP



like ligands

1Ra








2.
IL-18, IL-18BP


2.
IL-18R, IL-18RAcP


V.
TNF family
1.
TNF-α, TNF-β
3.
NGF/TNF R4

TNF-RI, AGP-3R,





(LT), FASL,CD40 L,



DR4, DR5, OX40,





CD30L, CD27 L, OX40L,



OPG, TACI, CD40,





OPGL, TRAIL, APRIL, AGP-3,



FAS, ODR





BLys, TL5, Ntn-2, KAY,









Neutrokine-α






VI.
Chemokines
1.
α chemokines:
4.
Chemokine R
1.
CXCR





IL-8, GRO α, β,









γ, IF-10, PF-4, SDF-1








2.
β chemokines:


2.
CCR





MIP1α, MIP1β, MCP-1, 2, 3, 4,









RANTES, eotaxin








3.
γ chemokines:


3.
CR





lymphotactin












4.
DARC5


VII.
Growth factors
1.1
SCF, M-CSF, PDGF-AA, AB,
VII.
RKF
1.
TK sub-family





BB, KDR, FLT-1, FLT-3L,


1.1
IgTK III R,VEGF-





VEGF, SSV-PDGF, HGF, SF



RI, VEGF-RII




1.2
FGFα, FGFβ


1.2
IgTK IV R




1.3
EGF, TGF-α, VV-F19 (EGF-


1.3
Cysteine-rich





like)



TK-I




1.4
IGF-I, IGF-II, Insulin


1.4
Cysteine rich









TK-II, IGF-RI




1.5
NGF, BDNF, NT-3, NT-46


1.5
Cysteine knot TK V




2.
TGF-β1, β2, β3


2.
Serine-threonine









kinase subfamily









(STKS)7





1IL-17R - belongs to CKR family but is unassigned to 4 indicated subjamilies.


2Other IFN type I subtypes remain unassigned. Hematopoietic cytokines, IL-10 ligands and interferons do not possess functional intrinsic protein kinases. The signaling molecules for the cytokines are JAK's, STATs and related non-receptor molecules. IL-14. IL-16 and IL-18 have been cloned but according to the receptor code they remain unassigned.


3TNF receptors use multiple, distinct intracellular molecules for signal transduction including “death domain” of FAS R and 55 kDa TNF-□R 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 (7 TM, serpentine) domain receptors. They are G protein-coupled.


4The Duffy blood group antigen (DARC) is an erythrocyte receptor that can bind several different chemokines. IL-1R belongs to the immunoglobulin superfamily but their signal transduction events characteristics remain unclear.


5The neurotrophic cytokines can associate with NGF/TNF receptors also.


6STKS may encompass many other TGF-β-related factors that remain unassigned. The protein kinases are intrinsic part of the intracellular domain of receptor kinase family (RKF). The enzymes participate in the signals transmission via the receptors.






Other proteins of interest as targets for peptide generation in the present invention include the following:

    
    
        (αvβ3
        αVβ1
        Ang-2
        B7
        B7RP1
        CRP1
        Calcitonin
        CD28
        CETP
        cMet
        Complement factor B
        C4b
        CTLA4
        Glucagon
        Glucagon Receptor
        LIPG
        MPL
        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
        TIE-2 receptor
        heparanase
        urokinase plasminogen activator (UPA), UPA receptor
        parathyroid hormone (PTH), parathyroid hormone-related protein (PTHrP),
        PTH-RI, PTH-RII
        Her2
        Her3
        Insulin
        Myostatin
        TALL-1
        Nerve growth factor
        Integrins and receptors
        Selectins and receptors thereof.
        Cell adhesion molecules and receptors thereof.
    
    


Exemplary peptides appear in Tables 4 through 38 below. These peptides may be prepared by any methods disclosed in the art, many of which are discussed herein. In most tables that follow, single letter amino acid abbreviations are used. The X in these sequences (and throughout this specification, unless specified otherwise in a particular instance) means that any of the 20 naturally occurring amino acid residues may be present. Any of these peptides may be linked in tandem (i.e., sequentially), with or without linkers, and a few tandem-linked examples are provided in the table. Linkers are listed as “Λ” and may be any of the linkers described herein. Tandem repeats and linkers are shown separated by dashes for clarity. Any peptide containing a cysteinyl residue may be cross-linked with another Cys-containing peptide, either or both of which may be linked to a vehicle. A few cross-linked examples are provided in the table. Any peptide having more than one Cys residue may form an intrapeptide disulfide bond, as well; see, for example, EPO-mimetic peptides in Table 5. A few examples of intrapeptide disulfide-bonded peptides are specified in the table. Any of these peptides may be derivatized as described herein, and a few derivatized examples are provided in the table. Derivatized peptides in the tables are exemplary rather than limiting, as the associated underivatized peptides may be employed in this invention, as well. For derivatives in which the carboxyl terminus may be capped with an amino group, the capping amino group is shown as —NH2. For derivatives in which amino acid residues are substituted by moieties other than amino acid residues, the substitutions are denoted by σ, which signifies any of the moieties described in Bhatnagar et al. (1996), J. Med. Chem. 39: 3814-9 and Cuthbertson et al. (1997), J. Med. Chem. 40: 2876-82, which are incorporated by reference. The J substituent and the Z substituents (Z5, Z6, . . . Z40) are as defined in U.S. Pat. Nos. 5,608,035, 5,786,331, and 5,880,096, which are incorporated by reference. For the EPO-mimetic sequences (Table 5), the substituents X2 through X11 and the integer “n” are as defined in WO 96/40772, which is incorporated by reference. Also for the EPO-mimetic sequences, the substituents Xna, X1a, X2a, X3a, X4a, X5a and Xca follow the definitions of Xn, X1, X2, X3, X4, X5, and Xc, respectively, of WO 99/47151, which is also incorporated by reference. The substituents “Ψ,” “Θ,” and “+” are as defined in Sparks et al. (1996), Proc. Natl. Acad. Sci. 93: 1540-4, which is hereby incorporated by reference. X4, X5, X6, and X7 are as defined in U.S. Pat. No. 5,773,569, which is hereby incorporated by reference, except that: for integrin-binding peptides, X1, X2, X3, X4, X5, X6, X7, and X8 are as defined in International applications WO 95/14714, published Jun. 1, 1995 and WO 97/08203, published Mar. 6, 1997, which are also incorporated by reference; and for VIP-mimetic peptides, X1, X1′, X1′′, X2, X3, X4, X5, X6 and Z and the integers m and n are as defined in WO 97/40070, published Oct. 30, 1997, which is also incorporated by reference. Xaa and Yaa below are as defined in WO 98/09985, published Mar. 12, 1998, which is incorporated by reference. AA1, AA2, AB1, AB2, and AC are as defined in International application WO 98/53842, published Dec. 3, 1998, which is incorporated by reference. X1, X2, X3, and X4 in Table 17 only are as defined in European application EP 0 911 393, published Apr. 28, 1999. Residues appearing in boldface are D-amino acids. All peptides are linked through peptide bonds unless otherwise noted. Abbreviations are listed at the end of this specification. In the “SEQ ID NO.” column, “NR” means that no sequence listing is required for the given sequence.







TABLE 4







IL-1 antagonist peptide sequences









SEQ



ID


Sequence/structure
NO:





Z11Z7Z8QZ5YZ6Z9Z10
  3


 


XXQZ5YZ6XX
  4


 


Z7XQZ5YZ6XX
  5


 


Z7Z8QZ5YZ6Z9Z10
  6


 


Z11Z7Z8QZ5YZ6Z9Z10
  7


 


Z12Z13Z14Z15Z16Z17Z18Z19Z20Z21Z22Z11Z7Z8QZ5YZ6
  8


Z9Z10L



 


Z23NZ24Z39Z25Z26Z27Z28Z29Z30Z40
  9


 


TANVSSFEWTPYYWQPYALPL
 10


 


SWTDYGYWQPYALPISGL
 11


 


ETPFTWEESNAYYWQPYALPL
 12


 


ENTYSPNWADSMYWQPYALPL
 13


 


SVGEDHNFWTSEYWQPYALPL
 14


 


DGYDRWRQSGERYWQPYALPL
 15


 


FEWTPGYWQPY
 16


 


FEWTPGYWQHY
 17


 


FEWTPGWYQJY
 18


 


AcFEWTPGWYQJY
 19


 


FEWTPGWpYQJY
 20


 


FAWTPGYWQJY
 21


 


FEWAPGYWQJY
 22


 


FEWVPGYWQJY
 23


 


FEWTPGYWQJY
 24


 


AcFEWTPGYWQJY
 25


 


FEWTPaWYQJY
 26


 


FEWTPSarWYQJY
 27


 


FEWTPGYYQPY
 28


 


FEWTPGWWQPY
 29


 


FEWTPNYWQPY
 30


 


FEWTPvYWQJY
 31


 


FEWTPecGYWQJY
 32


 


FEWTPAibYWQJY
 33


 


FEWTSarGYWQJY
 34


 


FEWTPGYWQPY
 35


 


FEWTPGYWQHY
 36


 


FEWTPGWYQJY
 37


 


AcFEWTPGWYQJY
 38


 


FEWTPGW-pY-QJY
 39


 


FAWTPGYWQJY
 40


 


FEWAPGYWQJY
 41


 


FEWVPGYWQJY
 42


 


FEWTPGYWQJY
 43


 


AcFEWTPGYWQJY
 44


 


FEWTPAWYQJY
 45


 


FEWTPSarWYQJY
 46


 


FEWTPGYYQPY
 47


 


FEWTPGWWQPY
 48


 


FEWTPNYWQPY
 49


 


FEWTPVYWQJY
 50


 


FEWTPecGYWQJY
 51


 


FEWTPAibYWQJY
 52


 


FEWTSarGYWQJY
 53


 


FEWTPGYWQPYALPL
 54


 


1NapEWTPGYYQJY
 55


 


YEWTPGYYQJY
 56


 


FEWVPGYYQJY
 57


 


FEWTPSYYQJY
 58


 


FEWTPNYYQJY
 59


 


TKPR
 60


 


RKSSK
 61


 


RKQDK
 62


 


NRKQDK
 63


 


RKQDKR
 64


 


ENRKQDKRF
 65


 


VTKFYF
 66


 


VTKFY
 67


 


VTDFY
 68


 


SHLYWQPYSVQ
 69


 


TLVYWQPYSLQT
 70


 


RGDYWQPYSVQS
 71


 


VHVYWQPYSVQT
 72


 


RLVYWQPYSVQT
 73


 


SRVWFQPYSLQS
 74


 


NMVYWQPYSIQT
 75


 


SVVFWQPYSVQT
 76


 


TFVYWQPYALPL
 77


 


TLVYWQPYSIQR
 78


 


RLVYWQPYSVQR
 79


 


SPVFWQPYSIQI
 80


 


WIEWWQPYSVQS
 81


 


SLIYWQPYSLQM
 82


 


TRLYWQPYSVQR
 83


 


RCDYWQPYSVQT
 84


 


MRVFWQPYSVQN
 85


 


KIVYWQPYSVQT
 86


 


RHLYWQPYSVQR
 87


 


ALVWWQPYSEQI
 88


 


SRVWFQPYSLQS
 89


 


WEQPYALPLE
 90


 


QLVWWQPYSVQR
 91


 


DLRYWQPYSVQV
 92


 


ELVWWQPYSLQL
 93


 


DLVWWQPYSVQW
 94


 


NGNYWQPYSFQV
 95


 


ELVYWQPYSIQR
 96


 


ELMYWQPYSVQE
 97


 


NLLYWQPYSMQD
 98


 


GYEWYQPYSVQR
 99


 


SRVWYQPYSVQR
100


 


LSEQYQPYSVQR
101


 


GGGWWQPYSVQR
102


 


VGRWYQPYSVQR
103


 


VHVYWQPYSVQR
104


 


QARWYQPYSVQR
105


 


VHVYWQPYSVQT
106


 


RSVYWQPYSVQR
107


 


TRVWFQPYSVQR
108


 


GRIWFQPYSVQR
109


 


GRVWFQPYSVQR
110


 


ARTWYQPYSVQR
111


 


ARVWWQPYSVQM
112


 


RLMFYQPYSVQR
113


 


ESMWYQPYSVQR
114


 


HFGWWQPYSVHM
115


 


ARFWWQPYSVQR
116


 


RLVYWQ PYAPIY
117


 


RLVYWQ PYSYQT
118


 


RLVYWQ PYSLPI
119


 


RLVYWQ PYSVQA
120


 


SRVWYQ PYAKGL
121


 


SRVWYQ PYAQGL
122


 


SRVWYQ PYAMPL
123


 


SRVWYQ PYSVQA
124


 


SRVWYQ PYSLGL
125


 


SRVWYQ PYAREL
126


 


SRVWYQ PYSRQP
127


 


SRVWYQ PYFVQP
128


 


EYEWYQ PYALPL
129


 


IPEYWQ PYALPL
130


 


SRIWWQ PYALPL
131


 


DPLFWQ PYALPL
132


 


SRQWVQ PYALPL
133


 


IRSWWQ PYALPL
134


 


RGYWQ PYALPL
135


 


RLLWVQ PYALPL
136


 


EYRWFQ PYALPL
137


 


DAYWVQ PYALPL
138


 


WSGYFQ PYALPL
139


 


NIEFWQ PYALPL
140


 


TRDWVQ PYALPL
141


 


DSSWYQ PYALPL
142


 


IGNWYQ PYALPL
143


 


NLRWDQ PYALPL
144


 


LPEFWQ PYALPL
145


 


DSYWWQ PYALPL
146


 


RSQYYQ PYALPL
147


 


ARFWLQ PYALPL
148


 


NSYFWQ PYALPL
149


 


RFMYWQPYSVQR
150


 


AHLFWQPYSVQR
151


 


WWQPYALPL
152


 


YYQPYALPL
153


 


YFQPYALGL
154


 


YWYQPYALPL
155


 


RWWQPYATPL
156


 


GWYQPYALGF
157


 


YWYQPYALGL
158


 


IWYQPYAMPL
159


 


SNMQPYQRLS
160


 


TFVYWQPY AVGLPAAETACN
161


 


TFVYWQPY SVQMTITGKVTM
162


 


TFVYWQPY SSHXXVPXGFPL
163


 


TFVYWQPY YGNPQWAIHVRH
164


 


TFVYWQPY VLLELPEGAVRA
165


 


TFVYWQPY VDYVWPIPIAQV
166


 


GWYQPYVDGWR
167


 


RWEQPYVKDGWS
168


 


EWYQPYALGWAR
169


 


GWWQPYARGL
170


 


LFEQPYAKALGL
171


 


GWEQPYARGLAG
172


 


AWVQPYATPLDE
173


 


MWYQPYSSQPAE
174


 


GWTQPYSQQGEV
175


 


DWFQPYSIQSDE
176


 


PWIQPYARGFG
177


 


RPLYWQPYSVQV
178


 


TLIYWQPYSVQI
179


 


RFDYWQPYSDQT
180


 


WHQFVQPYALPL
181


 


EWDS VYWQPYSVQ TLLR
182


 


WEQN VYWQPYSVQ SFAD
183


 


SDV VYWQPYSVQ SLEM
184


 


YYDG VYWQPYSVQ VMPA
185


 


SDIWYQ PYALPL
186


 


QRIWWQ PYALPL
187


 


SRIWWQ PYALPL
188


 


RSLYWQ PYALPL
189


 


TIIWEQ PYALPL
190


 


WETWYQ PYALPL
191


 


SYDWEQ PYALPL
192


 


SRIWCQ PYALPL
193


 


EIMFWQ PYALPL
194


 


DYVWQQ PYALPL
195


 


MDLLVQ WYQPYALPL
196


 


GSKVIL WYQPYALPL
197


 


RQGANI WYQPYALPL
198


 


GGGDEP WYQPYALPL
199


 


SQLERT WYQPYALPL
200


 


ETWVRE WYQPYALPL
201


 


KKGSTQ WYQPYALPL
202


 


LQARMN WYQPYALPL
203


 


EPRSQK WYQPYALPL
204


 


VKQKWR WYQPYALPL
205


 


LRRHDV WYQPYALPL
206


 


RSTASI WYQPYALPL
207


 


ESKEDQ WYQPYALPL
208


 


EGLTMK WYQPYALPL
209


 


EGSREG WYQPYALPL
210


 


VIEWWQ PYALPL
211


 


VWYWEQ PYALPL
212


 


ASEWWQ PYALPL
213


 


FYEWWQ PYALPL
214


 


EGWWVQ PYALPL
215


 


WGEWLQ PYALPL
216


 


DYYWEQ PYALPL
217


 


AHTWWQ PYALPL
218


 


FIEWFQ PYALPL
219


 


WLAWEQ PYALPL
220


 


VMEWWQ PYALPL
221


 


ERMWQ PYALPL
222


 


NXXWXX PYALPL
223


 


WGNWYQ PYALPL
224


 


TLYWEQ PYALPL
225


 


VWRWEQ PYALPL
226


 


LLWTQ PYALPL
227


 


SRIWXX PYALPL
228


 


SDIWYQ PYALPL
229


 


WGYYXX PYALPL
230


 


TSGWYQ PYALPL
231


 


VHPYXX PYALPL
232


 


EHSYFQ PYALPL
233


 


XXIWYQ PYALPL
234


 


AQLHSQ PYALPL
235


 


WANWFQ PYALPL
236


 


SRLYSQ PYALPL
237


 


GVTFSQ PYALPL
238


 


SIVWSQ PYALPL
239


 


SRDLVQ PYALPL
240


 


HWGH VYWQPYSVQ DDLG
241


 


SWHS VYWQPYSVQ SYPE
242


 


WRDS VYWQPYSVQ PESA
243


 


TWDA VYWQPYSVQ KWLD
244


 


TPPW VYWQPYSVQ SLDP
245


 


YWSS VYWQPYSVQ SVHS
246


 


YWY QPY ALGL
247


 


YWY QPY ALPL
248


 


EWI QPY ATGL
249


 


NWE QPY AKPL
250


 


AFY QPY ALPL
251


 


FLY QPY ALPL
252


 


VCK QPY LEWC
253


 


ETPFTWEESNAYYWQPYALPL
254


 


QGWLTWQDSVDMYWQPYALPL
255


 


FSEAGYTWPENTYWQPYALPL
256


 


TESPGGLDWAKIYWQPYALPL
257


 


DGYDRWRQSGERYWQPYALPL
258


 


TANVSSFEWTPGYWQPYALPL
259


 


SVGEDHNFWTSE YWQPYALPL
260


 


MNDQTSEVSTFP YWQPYALPL
261


 


SWSEAFEQPRNL YWQPYALPL
262


 


QYAEPSALNDWG YWQPYALPL
263


 


NGDWATADWSNY YWQPYALPL
264


 


THDEHI YWQPYALPL
265


 


MLEKTYTTWTPG YWQPYALPL
266


 


WSDPLTRDADL YWQPYALPL
267


 


SDAFTTQDSQAM YWQPYALPL
268


 


GDDAAWRTDSLT YWQPYALPL
269


 


AIIRQLYRWSEM YWQPYALPL
270


 


ENTYSPNWADSM YWQPYALPL
271


 


MNDQTSEVSTFP YWQPYALPL
272


 


SVGEDHNFWTSE YWQPYALPL
273


 


QTPFTWEESNAY YWQPYALPL
274


 


ENPFTWQESNAY YWQPYALPL
275


 


VTPFTWEDSNVF YWQPYALPL
276


 


QIPFTWEQSNAY YWQPYALPL
277


 


QAPLTWQESAAY YWQPYALPL
278


 


EPTFTWEESKAT YWQPYALPL
279


 


TTTLTWEESNAY YWQPYALPL
280


 


ESPLTWEESSAL YWQPYALPL
281


 


ETPLTWEESNAY YWQPYALPL
282


 


EATFTWAESNAY YWQPYALPL
283


 


EALFTWKESTAY YWQPYALPL
284


 


STP-TWEESNAY YWQPYALPL
285


 


ETPFTWEESNAY YWQPYALPL
286


 


KAPFTWEESQAY YWQPYALPL
287


 


STSFTWEESNAY YWQPYALPL
288


 


DSTFTWEESNAY YWQPYALPL
289


 


YIPFTWEESNAY YWQPYALPL
290


 


QTAFTWEESNAY YWQPYALPL
291


 


ETLFTWEESNAT YWQPYALPL
292


 


VSSFTWEESNAY YWQPYALPL
293


 


QPYALPL
294


 


Py-1-NapPYQJYALPL
295


 


TANVSSFEWTPG YWQPYALPL
296


 


FEWTPGYWQPYALPL
297


 


FEWTPGYWQJYALPL
298


 


FEWTPGYYQJYALPL
299


 


ETPFTWEESNAYYWQPYALPL
300


 


FTWEESNAYYWQJYALPL
301


 


ADYL YWQPYA PVTLWV
302


 


GDVAE YWQPYA LPLTSL
303


 


SWTDYG YWQPYA LPISGL
304


 


FEWTPGYWQPYALPL
305


 


FEWTPGYWQJYALPL
306


 


FEWTPGWYQPYALPL
307


 


FEWTPGWYQJYALPL
308


 


FEWTPGYYQPYALPL
309


 


FEWTPGYYQJYALPL
310


 


TANVSSFEWTPGYWQPYALPL
311


 


SWTDYGYWQPYALPISGL
312


 


ETPFTWEESNAYYWQPYALPL
313


 


ENTYSPNWADSMYWQPYALPL
314


 


SVGEDHNFWTSEYWQPYALPL
315


 


DGYDRWRQSGERYWQPYALPL
316


 


FEWTPGYWQPYALPL
317


 


FEWTPGYWQPY
318


 


FEWTPGYWQJY
319


 


EWTPGYWQPY
320


 


FEWTPGWYQJY
321


 


AEWTPGYWQJY
322


 


FAWTPGYWQJY
323


 


FEATPGYWQJY
324


 


FEWAPGYWQJY
325


 


FEWTAGYWQJY
326


 


FEWTPAYWQJY
327


 


FEWTPGAWQJY
328


 


FEWTPGYAQJY
329


 


FEWTPGYWQJA
330


 


FEWTGGYWQJY
331


 


FEWTPGYWQJY
332


 


FEWTJGYWQJY
333


 


FEWTPecGYWQJY
334


 


FEWTPAibYWQJY
335


 


FEWTPSarWYQJY
336


 


FEWTSarGYWQJY
337


 


FEWTPNYWQJY
338


 


FEWTPVYWQJY
339


 


FEWTVPYWQJY
340


 


AcFEWTPGWYQJY
341


 


AcFEWTPGYWQJY
342


 


1Nap-EWTPGYYQJY
343


 


YEWTPGYYQJY
344


 


FEWVPGYYQJY
345


 


FEWTPGYYQJY
346


 


FEWTPSYYQJY
347


 


FEWTPnYYQJY
348


 


SHLY-Nap-QPYSVQM
349


 


TLVY-Nap-QPYSLQT
350


 


RGDY-Nap-QPYSVQS
351


 


NMVY-Nap-QPYSIQT
352


 


VYWQPYSVQ
353


 


VY-Nap-QPYSVQ
354


 


TFVYWQJYALPL
355


 


FEWTPGYYQJ-Bpa
356


 


XaaFEWTPGYYQJ-Bpa
357


 


FEWTPGY-Bpa-QJY
358


 


AcFEWTPGY-Bpa-QJY
359


 


FEWTPG-Bpa-YQJY
360


 


AcFEWTPG-Bpa-YQJY
361


 


AcFE-Bpa-TPGYYQJY
362


 


AcFE-Bpa-TPGYYQJY
363


 


Bpa-EWTPGYYQJY
364


 


AcBpa-EWTPGYYQJY
365


 


VYWQPYSVQ
366


 


RLVYWQPYSVQR
367


 


RLVY-Nap-QPYSVQR
368


 


RLDYWQPYSVQR
369


 


RLVWFQPYSVQR
370


 


RLVYWQPYSIQR
371


 


DNSSWYDSFLL
372


 


DNTAWYESFLA
373


 


DNTAWYENFLL
374


 


PARE DNTAWYDSFLI WC
375


 


TSEY DNTTWYEKFLA SQ
376


 


SQIP DNTAWYQSFLL HG
377


 


SPFI DNTAWYENFLL TY
378


 


EQIY DNTAWYDHFLL SY
379


 


TPFI DNTAWYENFLL TY
380


 


TYTY DNTAWYERFLM SY
381


 


TMTQ DNTAWYENFLL SY
382


 


TI DNTAWYANLVQ TYPQ
383


 


TI DNTAWYERFLA QYPD
384


 


HI DNTAWYEMFLL TYTP
385


 


SQ DNTAWYENFLL SYKA
386


 


QI DNTAWYERFLL QYNA
387


 


NQ DNTAWYESFLL QYNT
388


 


TI DNTAWYENFLL NHNL
389


 


HY DNTAWYERFLQ QGWH
390


 


ETPFTWEESNAYYWQPYALPL
391


 


YIPFTWEESNAYYWQPYALPL
392


 


DGYDRWRQSGERYWQPYALPL
393


 


pY-1Nap-pY-QJYALPL
394


 


TANVSSFEWTPGYWQPYALPL
395


 


FEWTPGYWQJYALPL
396


 


FEWTPGYWQPYALPLSD
397


 


FEWTPGYYQJYALPL
398


 


FEWTPGYWQJY
399


 


AcFEWTPGYWQJY
400


 


AcFEWTPGWYQJY
401


 


AcFEWTPGYYQJY
402


 


AcEEWTPaYWQJY
403


 


AcFEWTPaWYQJY
404


 


AcFEWTPaYYQJY
405


 


FEWTPGYYQJYALPL
406


 


FEWTPGYWQJYALPL
407


 


FEWTPGWYQJYALPL
408


 


TANVSSFEWTPGYWQPYALPL
409


 


AcFEWTPGYWQJY
410


 


AcFEWTPGWYQJY
411


 


AcFEWTPGYYQJY
412


 


AcFEWTPAYWQJY
413


 


AcFEWTPAWYQJY
414


 


AcFEWTPAYYQJY
415
















TABLE 5







EPO-mimetic peptide sequences











SEQ ID



Sequence/structure
NO:






YXCXXGPXTWXCXP
416



YXCXXGPXTWXCXP-YXCXXGPXTWXCXP
417



YXCXXGPXTWXCXP-Λ-YXCXXGPXTWXCXP
418


 







419


 



GGTYSCHFGPLTWVCKPQGG
420



GGDYHCRMGPLTWVCKPLGG
421



GGVYACRMGPITWVCSPLGG
422



VGNYMCHFGPITWVCRPGGG
423



GGLYLCRFGPVTWDCGYKGG
424



GGTYSCHFGPLTWVCKPQGG-
425



GGTYSCHFGPLTWVCKPQGG




GGTYSCHFGPLTWVCKPQGG-Λ-
426



GGTYSCHFGPLTWVCKPQGG




GGTYSCHFGPLTWVCKPQGGSSK
427



GGTYSCHFGPLTWVCKPQGGSSK-
428



GGTYSCHFGPLTWVCKPQGGSSK




GGTYSCHFGPLTWVCKPQGGSSK-Λ-
429



GGTYSCHFGPLTWVCKPQGGSSK



 







430


 



GGTYSCHFGPLTWVCKPQGGSSK(Λ-biotin)
431



CX4X5GPX6TWX7C
432



GGTYSCHGPLTWVCKPQGG
433



VGNYMAHMGPITWVCRPGG
434



GGPHHVYACRMGPLTWIC
435



GGTYSCHFGPLTWVCKPQ
436



GGLYACHMGPMTWVCQPLRG
437



TIAQYICYMGPETWECRPSPKA
438



YSCHFGPLTWVCK
439



YCHFGPLTWVC
440



X3X4X5GPX6TWX7X8
441



YX2X3X4X5GPX6TWX7X8
442



X1YX2X3X4X5GPX6TWX7X8X9X10X11
443



X1YX2CX4X5GPX6TWX7CX9X10X11
444



GGLYLCRFGPVTWDCGYKGG
445



GGTYSCHFGPLTWVCKPQGG
446



GGDYHCRMGPLTWVCKPLGG
447



VGNYMCHFGPITWVCRPGGG
448



GGVYACRMGPITWVCSPLGG
449



VGNYMAHMGPITWVCRPGG
450



GGTYSCHFGPLTWVCKPQ
451



GGLYACHMGPMTWVCQPLRG
452



TIAQYICYMGPETWECRPSPKA
453



YSCHFGPLTWVCK
454



YCHFGPLTWVC
455



SCHFGPLTWVCK
456



(AX2)nX3X4X5GPX6TWX7X8
457



XnCX1X2GWVGX3CX4X5WXC
458
















TABLE 6







TPO-mimetic peptide sequences









SEQ ID


Sequence/structure
NO:





IEGPTLRQWLAARA
459


IEGPTLRQWLAAKA
460


IEGPTLREWLAARA
461


IEGPTLRQWLAARA-Λ-IEGPTLRQWLAARA
462


IEGPTLRQWLAAKA-Λ-IEGPTLRQWLAAKA
463


 






464


 


IEGPTLRQWLAARA-Λ-K(BrAc)-Λ-IEGPTLRQWLAARA
465


IEGPTLRQWLAARA-Λ-K(PEG)-Λ-IEGPTLRQWLAARA
466


 






467


 






468


 


VRDQIXXXL
469


TLREWL
470


GRVRDQVAGW
471


GRVKDQIAQL
472


GVRDQYSWAL
473


ESVREQVMKY
474


SVRSQISASL
475


GVRETVYRHM
476


GVREVIVMHML
477


GRVRDQIWAAL
478


AGVRDQILIWL
479


GRVRDQIMLSL
480


GRVRDQI(X)3L
481


CTLRQWLQGC
482


CTLQEFLEGC
483


CTRTEWLHGC
484


CTLREWLHGGFC
485


CTLREWVFAGLC
486


CTLRQWLILLGMC
487


CTLAEFLASGVEQC
488


CSLQEFLSHGGYVC
489


CTLREFLDPTTAVC
490


CTLKEWLVSHEVWC
491


CTLREWL(X)2-6C
492


REGPTLRQWM
493


EGPTLRQWLA
494


ERGPFWAKAC
495


REGPRCVMWM
496


CGTEGPTLSTWLDC
497


CEQDGPTLLEWLKC
498


CELVGPSLMSWLTC
499


CLTGPFVTQWLYEC
500


CRAGPTLLEWLTLC
501


CADGPTLREWISFC
502


C(X)1-2EGPTLREWL(X)1-2C
503


GGCTLREWLHGGFCGG
504


GGCADGPTLREWISFCGG
505


GNADGPTLRQWLEGRRPKN
506


LAIEGPTLRQWLHGNGRDT
507


HGRVGPTLREWKTQVATKK
508


TIKGPTLRQWLKSREHTS
509


ISDGPTLKEWLSVTRGAS
510


SIEGPTLREWLTSRTPHS
511
















TABLE 7







G-CSF-mimetic peptide sequences











SEQ ID



Sequence/structure
NO:






EEDCK
512


 







513


 



EEDσK
514


 







515


 



PGluEDσK
516


 







517


 



PicSDσK
518


 







519


 



EEDCK-Λ-EEDCK
520



EEDXK-Λ-EEDXK
521
















TABLE 8







TNF-antagonist peptide sequences











SEQ ID



Sequence/structure
NO:






YCFTASENHCY
522



YCFTNSENHCY
523



YCFTRSENHCY
524



FCASENHCY
525



YCASENHCY
526



FCNSENHCY
527



FCNSENRCY
528



FCNSVENRCY
529



YCSQSVSNDCF
530



FCVSNDRCY
531



YCRKELGQVCY
532



YCKEPGQCY
533



YCRKEMGCY
534



FCRKEMGCY
535



YCWSQNLCY
536



YCELSQYLCY
537



YCWSQNYCY
538



YCWSQYLCY
539



DFLPHYKNTSLGHRP
540


 







NR
















TABLE 9







Integrin-binding peptide sequences











SEQ ID



Sequence/structure
NO:






RX1ETX2WX3
541


 



RX1ETX2WX3
542


 



RGDGX
543


 



CRGDGXC
544


 



CX1X2RLDX3X4C
545


 



CARRLDAPC
546


 



CPSRLDSPC
547


 



X1X2X3RGDX4X5X6
548


 



CX2CRGDCX5C
549


 



CDCRGDCFC
550


 



CDCRGDCLC
551


 



CLCRGDCIC
552


 



X1X2DDX4X5X7X8
553


 



X1X2X3DDX4X5X6X7X8
554


 



CWDDGWLC
555


 



CWDDLWWLC
556


 



CWDDGLMC
557


 



CWDDGWMC
558


 



CSWDDGWLC
559


 



CPDDLWWLC
560


 



NGR
NR


 



GSL
NR


 



RGD
NR


 



CGRECPRLCQSSC
561


 



CNGRCVSGCAGRC
562


 



CLSGSLSC
563


 



RGD
NR


 



NGR
NR


 



GSL
NR


 



NGRAHA
564


 



CNGRC
565


 



CDCRGDCFC
566


 



CGSLVRC
567


 



DLXXL
568


 



RTDLDSLRTYTL
569


 



RTDLDSLRTY
570


 



RTDLDSLRT
571


 



RTDLDSLR
572


 



GDLDLLKLRLTL
573


 



GDLHSLRQLLSR
574


 



RDDLHMLRLQLW
575


 



SSDLHALKKRYG
576


 



RGDLKQLSELTW
577


 



RGDLAALSAPPV
578
















TABLE 10







Selectin antagonist peptide sequences











SEQ ID



Sequence/structure
NO:






DITWDQLWDLMK
579


 



DITWDELWKIMN
580


 



DYTWFELWDMMQ
581


 



QITWAQLWNMMK
582


 



DMTWHDLWTLMS
583


 



DYSWHDLWEMMS
584


 



EITWDQLWEVMN
585


 



HVSWEQLWDIMN
586


 



HITWDQLWRIMT
587


 



RNMSWLELWEHMK
588


 



AEWTWDQLWHVMNPAESQ
589


 



HRAEWLALWEQMSP
590


 



KKEDWLALWRIMSV
591


 



ITWDQLWDLMK
592


 



DITWDQLWDLMK
593


 



DITWDQLWDLMK
594


 



DITWDQLWDLMK
595


 



CQNRYTDLVAIQNKNE
596


 



AENWADNEPNNKRNNED
597


 



RKNNKTWTWVGTKKALTNE
598


 



KKALTNEAENWAD
599


 



CQXRYTDLVAIQNKXE
600


 



RKXNXXWTWVGTXKXLTEE
601


 



AENWADGEPNNKXNXED
602


 



CXXXYTXLVAIQNKXE
603


 



RKXXXXWXWVGTXKXLTXE
604


 



AXNWXXXEPNNXXXED
605


 



XKXKTXEAXNWXX
606
















TABLE 11







Antipathogenic peptide sequences











SEQ ID



Sequence/structure
NO:






GFFALIPKIISSPLFKTLLSAVGSALSSSGGQQ
607


 



GFFALIPKIISSPLFKTLLSAVGSALSSSGGQE
608


 



GFFALIPKIISSPLFKTLLSAV
609


 



GFFALIPKIISSPLFKTLLSAV
610


 



KGFFALIPKIISSPLFKTLLSAV
611


 



KKGFFALIPKIISSPLFKTLLSAV
612


 



KKGFFALIPKIISSPLFKTLLSAV
613


 



GFFALIPKIIS
614


 



GIGAVLKVLTTGLPALISWIKRKRQQ
615


 



GIGAVLKVLTTGLPALISWIKRKRQQ
616


 



GIGAVLKVLTTGLPALISWIKRKRQQ
617


 



GIGAVLKVLTTGLPALISWIKR
618


 



AVLKVLTTGLPALISWIKR
619


 



KLLLLLKLLLLK
620


 



KLLLKLLLKLLK
621


 



KLLLKLKLKLLK
622


 



KKLLKLKLKLKK
623


 



KLLLKLLLKLLK
624


 



KLLLKLKLKLLK
625


 



KLLLLK
626


 



KLLLKLLK
627


 



KLLLKLKLKLLK
628


 



KLLLKLKLKLLK
629


 



KLLLKLKLKLLK
630


 



KAAAKAAAKAAK
631


 



KVVVKVVVKVVK
632


 



KVVVKVKVKVVK
633


 



KVVVKVKVKVK
634


 



KVVVKVKVKVVK
635


 



KLILKL
636


 



KVLHLL
637


 



LKLRLL
638


 



KPLHLL
639


 



KLILKLVR
640


 



KVFHLLHL
641


 



HKFRILKL
642


 



KPFHILHL
643


 



KIIIKIKIKIIK
644


 



KIIIKIKIKIIK
645


 



KIIIKIKIKIIK
646


 



KLPIKIKIKIPK
647


 



KIPIKIKIKIVK
648


 



RIIIRIRIRIIR
649


 



RIIIRIRIRIIR
650


 



RIIIRIRIRIIR
651


 



RIVIRIRIRLIR
652


 



RIIVRIRLRIIR
653


 



RIGIRLRVRIIR
654


 



KIVIRIRIRLIR
655


 



RIAVKWRLRFIK
656


 



KIGWKLRVRIIR
657


 



KKIGWLIIRVRR
658


 



RIVIRIRIRLIRIR
659


 



RIIVRIRLRIIRVR
660


 



RIGIRLRVRIIRRV
661


 



KIVIRIRARLIRIRIR
662


 



RIIVKIRLRIIKKIRL
663


 



KIGIKARVRIIRVKII
664


 



RIIVHIRLRIIHHIRL
665


 



HIGIKAHVRIIRVHII
666


 



RIYVKIHLRYIKKIRL
667


 



KIGHKARVHIIRYKII
668


 



RIYVKPHPRYIKKIRL
669


 



KPGHKARPHIIRYKII
670


 



KIVIRIRIRLIRIRIRKIV
671


 



RIIVKIRLRIIKKIRLIKK
672


 



KIGWKLRVRIIRVKIGRLR
673


 



KIVIRIRIRLIRIRIRKIVKVKRIR
674


 



RFAVKIRLRIlKKIRLIKKIRKRVIK
675


 



KAGWKLRVRIIRVKIGRLRKIGWKKRVRIK
676


 



RIYVKPHPRYIKKIRL
677


 



KPGHKARPHIIRYKII
678


 



KIVIRIRIRLIRIRIRKIV
679


 



RIIVKIRLRIIKKIRLIKK
680


 



RIYVSKISIYIKKIRL
681


 



KIVIFTRIRLTSIRIRSIV
682


 



KPIHKARPTIIRYKMI
683


 



cyclicCKGFFALIPKIISSPLFKTLLSAVC
684


 



CKKGFFALIPKIISSPLFKTLLSAVC
685


 



CKKKGFFALIPKIISSPLFKTLLSAVC
686


 



CyclicCRIVIRIRIRLIRIRC
687


 



CyclicCKPGHKARPHIIRYKIIC
688


 



CyclicCRFAVKIRLRIIKKIRLIKKIRKRVIKC
689


 



KLLLKLLL KLLKC
690


 



KLLLKLLLKLLK
691


 



KLLLKLKLKLLKC
692


 



KLLLKLLLKLLK
693
















TABLE 12







VIP-mimetic peptide sequences











SEQ ID



Sequence/Structure
NO:






HSDAVFYDNYTR LRKQMAVKKYLN SILN
694



Nle HSDAVFYDNYTR LRKQMAVKKYLN SILN
695



X1 X1′ X1′′ X2
696



X3 S X4 LN
697


 







698


 



KKYL
699



NSILN
700



KKYL
701



KKYA
702



AVKKYL
703



NSILN
704



KKYV
705



SILauN
706



KKYLNle
707



NSYLN
708



NSIYN
709



KKYLPPNSILN
710



LauKKYL
711



CapKKYL
712



KYL
713



KKYNle
714



VKKYL
715



LNSILN
716



YLNSILN
717



KKYLN
718



KKYLNS
719



KKYLNSI
720



KKYLNSIL
721



KKYL
722



KKYDA
723



AVKKYL
724



NSILN
725



KKYV
726



SILauN
727



NSYLN
728



NSIYN
729



KKYLNle
730



KXYLPPNSILN
731



KKYL
732



KKYDA
733



AVKKYL
734



NSILN
735



KKYV
736



SILauN
737



LauKKYL
738



CapKKYL
739



KYL
740



KYL
741



KKYNle
742



VKKYL
743



LNSILN
744



YLNSILN
745



KKYLNle
746



KKYLN
747



KKYLNS
748



KKYLNSI
749



KKYLNSIL
750



KKKYLD
751



cyclicCKKYLC
752


 







753


 



KKYA
754



WWTDTGLW
755



WWTDDGLW
756



WWDTRGLWVWTI
757



FWGNDGIWLESG
758



DWDQFGLWRGAA
759



RWDDNGLWVVVL
760



SGMWSHYGIWMG
761



GGRWDQAGLWVA
762



KLWSEQGIWMGE
763



CWSMHGLWLC
764



GCWDNTGIWVPC
765



DWDTRGLWVY
766



SLWDENGAWI
767



KWDDRGLWMH
768



QAWNERGLWT
769



QWDTRGLWVA
770



WNVHGIWQE
771



SWDTRGLWVE
772



DWDTRGLWVA
773



SWGRDGLWIE
774



EWTDNGLWAL
775



SWDEKGLWSA
776



SWDSSGLWMD
777
















TABLE 13







Mdm/hdm antagonist peptide sequences











SEQ ID



Sequence/structure
NO:






TFSDLW
778


 



QETFSDLWKLLP
779


 



QPTFSDLWKLLP
780


 



QETFSDYWKLLP
781


 



QPTFSDYWKLLP
782


 



MPRFMDYWEGLN
783


 



VQNFIDYWTQQF
784


 



TGPAFTHYWATF
785


 



IDRAPTFRDHWFALV
786


 



PRPALVFADYWETLY
787


 



PAFSRFWSDLSAGAH
788


 



PAFSRFWSKLSAGAH
789


 



PXFXDYWXXL
790


 



QETFSDLWKLLP
791


 



QPTFSDLWKLLP
792


 



QETFSDYWKLLP
793


 



QPTFSDYWKLLP
794
















TABLE 14







Calmodulin antagonist peptide sequences











SEQ ID



Sequence/structure
NO:






SCVKWGKKEFCGS
795


 



SCWKYWGKECGS
796


 



SCYEWGKLRWCGS
797


 



SCLRWGKWSNCGS
798


 



SCWRWGKYQICGS
799


 



SCVSWGALKLCGS
800


 



SCIRWGQNTFCGS
801


 



SCWQWGNLKICGS
802


 



SCVRWGQLSICGS
803


 



LKKFNARRKLKGAILTTMLAK
804


 



RRWKKNFIAVSAANRFKK
805


 



RKWQKTGHAVRAIGRLSS
806


 



INLKALAALAKKIL
807


 



KIWSILAPLGTTLVKLVA
808


 



LKKLLKLLKKLLKL
809


 



LKWKKLLKLLKKLLKKLL
810


 



AEWPSLTEIKTLSHFSV
811


 



AEWPSPTRVISTTYFGS
812


 



AELAHWPPVKTVLRSFT
813


 



AEGSWLQLLNLMKQMNN
814


 



AEWPSLTEIK
815
















TABLE 15







Mast cell antagonists/Mast cell protease


inhibitor peptide sequences











SEQ ID



Sequence/structure
NO:






SGSGVLKRPLPILPVTR
816


 



RWLSSRPLPPLPLPPRT
817


 



GSGSYDTLALPSLPLHPMSS
818


 



GSGSYDTRALPSLPLHPMSS
819


 



GSGSSGVTMYPKLPPHWSMA
820


 



GSGSSGVRMYPKLPPHWSMA
821


 



GSGSSSMRMVPTIPGSAKHG
822


 



RNR
NR


 



QT
NR


 



RQK
NR


 



NRQ
NR


 



RQK
NR


 



RNRQKT
823


 



RNRQ
824


 



RNRQK
825


 



NRQKT
826


 



RQKT
827
















TABLE 16







SH3 antagonist peptide sequences











SEQ ID



Sequence/structure
NO:






RPLPPLP
828


 



RELPPLP
829


 



SPLPPLP
830


 



GPLPPLP
831


 



RPLPIPP
832


 



RPLPIPP
833


 



RRLPPTP
834


 



RQLPPTP
835


 



RPLPSRP
836


 



RPLPTRP
837


 



SRLPPLP
838


 



RALPSPP
839


 



RRLPRTP
840


 



RPVPPIT
841


 



ILAPPVP
842


 



RPLPMLP
843


 



RPLPILP
844


 



RPLPSLP
845


 



RPLPSLP
846


 



RPLPMIP
847


 



RPLPLIP
848


 



RPLPPTP
849


 



RSLPPLP
850


 



RPQPPPP
851


 



RQLPIPP
852


 



XXXRPLPPLPXP
853


 



XXXRPLPPIPXX
854


 



XXXRPLPPLPXX
855


 



RXXRPLPPLPXP
856


 



RXXRPLPPLPPP
857


 



PPPYPPPPIPXX
858


 



PPPYPPPPVPXX
859


 



LXXRPLPΨP
860


 



ΨXXRPLPXLP
861


 



PPXΘXPPPΨP
862


 



+PPΨPXKPXWL
863


 



RPXΨPΨR+SXP
864


 



PPVPPRPXXTL
865


 



ΨPΨLPΨK
866


 



+ΘDXPLPXLP
867
















TABLE 17







Somatostatin or cortistatin mimetic peptide


sequences









SEQ



ID


Sequence/structure
NO:





X1-X2-Asn-Phe-Phe-Trp-Lys-Thr-Phe-X3-Ser-X4
868


 


Asp Arg Met Pro Cys Arg Asn Phe Phe Trp Lys Thr
869


Phe Ser Ser Cys



 


Met Pro Cys Arg Asn Phe Phe Trp Lys Thr Phe Ser
870


Ser Cys Lys



 


Cys Arg Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys
871


Lys



 


Asp Arg Met Pro Cys Arg Asn Phe Phe Trp Lys Thr
872


Phe Ser Ser Cys



 


Met Pro Cys Lys Asn Phe Phe Trp Lys Thr Phe Ser
873


Ser Cys



 


Cys Arg Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys
874


 


Asp Arg Met Pro Cys Lys Asn Phe Phe Trp Lys Thr
875


Phe Ser Ser Cys



 


Met Pro Cys Lys Asn Phe Phe Trp Lys Thr Phe Ser
876


Ser Cys Lys



 


Cys Lys Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys
877


Lys



 


Asp Arg Met Pro Cys Lys Asn Phe Phe Trp Lys Thr
878


Phe Ser Ser Cys



 


Met Pro Cys Lys Asn Phe Phe Trp Lys Thr Phe Ser
879


Ser Cys



 


Cys Lys Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys
880


 


Asp Arg Met Pro Cys Arg Asn Phe Phe Trp Lys Thr
881


Phe Thr Ser Cys Lys



 


Met Pro Cys Arg Asn Phe Phe Trp Lys Thr Phe Thr
882


Ser Cys Lys



 


Cys Arg Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys
883


Lys



 


Asp Arg Met Pro Cys Arg Asn Phe Phe Trp Lys Thr
884


Phe Thr Ser Cys



 


Met Pro Cys Arg Asn Phe Phe Trp Lys Thr Phe Thr
885


Ser Cys



 


Cys Arg Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys
886


 


Asp Arg Met Pro Cys Lys Asn Phe Phe Trp Lys Thr
887


Phe Thr Ser Cys Lys



 


Met Pro Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr
888


Ser Cys Lys



 


Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys
889


Lys



 


Asp Arg Met Pro Cys Lys Asn Phe Phe Trp Lys Thr
890


Phe Thr Ser Cys



 


Met Pro Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr
891


Ser Cys



 


Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys
892
















TABLE 18







UKR antagonist peptide sequences











SEQ ID



Sequence/structure
NO:






AEPMPHSLNFSQYLWYT
893


 



AEHTYSSLWDTYSPLAF
894


 



AELDLWMRHYPLSFSNR
895


 



AESSLWTRYAWPSMPSY
896


 



AEWHPGLSFGSYLWSKT
897


 



AEPALLNWSFFFNPGLH
898


 



AEWSFYNLHLPEPQTIF
899


 



AEPLDLWSLYSLPPLAM
900


 



AEPTLWQLYQFPLRLSG
901


 



AEISFSELMWLRSTPAF
902


 



AELSEADLWTTWFGMGS
903


 



AESSLWRIFSPSALMMS
904


 



AESLPTLTSILWGKESV
905


 



AETLFMDLWHDKHILLT
906


 



AEILNFPLWHEPLWSTE
907


 



AESQTGTLNTLFWNTLR
908


 



AEPVYQYELDSYLRSYY
909


 



AELDLSTFYDIQYLLRT
910


 



AEFFKLGPNGYVYLHSA
911


 



FKLXXXGYVYL
912


 



AESTYHHLSLGYMYTLN
913


 



YHXLXXGYMYT
914
















TABLE 19







Macrophage and/or


T-cell inhibiting peptide sequences











SEQ ID



Sequence/structure
NO:






Xaa-Yaa-Arg
NR


 



Arg-Yaa-Xaa
NR


 



Xaa-Arg-Yaa
NR


 



Yaa-Arg-Xaa
NR


 



Ala-Arg
NR


 



Arg-Arg
NR


 



Asn-Arg
NR


 



Asp-Arg
NR


 



Cys-Arg
NR


 



Gln-Arg
NR


 



Glu-Arg
NR


 



Gly-Arg
NR


 



His-arg
NR


 



Ile-Arg
NR


 



Leu-Arg
NR


 



Lys-Arg
NR


 



Met-Arg
NR


 



Phe-Arg
NR


 



Ser-Arg
NR


 



Thr-Arg
NR


 



Trp-Arg
NR


 



Tyr-Arg
NR


 



Val-Arg
NR


 



Ala-Glu-Arg
NR


 



Arg-Glu-Arg
NR


 



Asn-Glu-Arg
NR


 



Asp-Glu-Arg
NR


 



Cys-Glu-Arg
NR


 



Gln-Glu-Arg
NR


 



Glu-Glu-Arg
NR


 



Gly-Glu-Arg
NR


 



His-Glu-Arg
NR


 



Ile-Glu-Arg
NR


 



Leu-Glu-Arg
NR


 



Lys-Glu-Arg
NR


 



Met-Glu-Arg
NR


 



Phe-Glu-Arg
NR


 



Pro-Glu-Arg
NR


 



Ser-Glu-Arg
NR


 



Thr-Glu-Arg
NR


 



Trp-Glu-Arg
NR


 



Tyr-Glu-Arg
NR


 



Val-Glu-Arg
NR


 



Arg-Ala
NR


 



Arg-Asp
NR


 



Arg-Cys
NR


 



Arg-Gln
NR


 



Arg-Glu
NR


 



Arg-Gly
NR


 



Arg-His
NR


 



Arg-Ile
NR


 



Arg-Leu
NR


 



Arg-Lys
NR


 



Arg-Met
NR


 



Arg-Phe
NR


 



Arg-Pro
NR


 



Arg-Ser
NR


 



Arg-Thr
NR


 



Arg-Trp
NR


 



Arg-Tyr
NR


 



Arg-Val
NR


 



Arg-Glu-Ala
NR


 



Arg-Glu-Asn
NR


 



Arg-Glu-Asp
NR


 



Arg-Glu-Cys
NR


 



Arg-Glu-Gln
NR


 



Arg-Glu-Glu
NR


 



Arg-Glu-Gly
NR


 



Arg-Glu-His
NR


 



Arg-Glu-Ile
NR


 



Arg-Glu-Leu
NR


 



Arg-Glu-Lys
NR


 



Arg-Glu-Met
NR


 



Arg-Glu-Phe
NR


 



Arg-Glu-Pro
NR


 



Arg-Glu-Ser
NR


 



Arg-Glu-Thr
NR


 



Arg-Glu-Trp
NR


 



Arg-Glu-Tyr
NR


 



Arg-Glu-Val
NR


 



Ala-Arg-Glu
NR


 



Arg-Arg-Glu
NR


 



Asn-Arg-Glu
NR


 



Asp-Arg-Glu
NR


 



Cys-Arg-Glu
NR


 



Gln-Arg-Glu
NR


 



Glu-Arg-Glu
NR


 



Gly-Arg-Glu
NR


 



His-Arg-Glu
NR


 



Ile-Arg-Glu
NR


 



Leu-Arg-Glu
NR


 



Lys-Arg-Glu
NR


 



Met-Arg-Glu
NR


 



Phe-Arg-Glu
NR


 



Pro-Arg-Glu
NR


 



Ser-Arg-Glu
NR


 



Thr-Arg-Glu
NR


 



Trp-Arg-Glu
NR


 



Tyr-Arg-Glu
NR


 



Val-Arg-Glu
NR


 



Glu-Arg-Ala,
NR


 



Glu-Arg-Arg
NR


 



Glu-Arg-Asn
NR


 



Glu-Arg-Asp
NR


 



Glu-Arg-Cys
NR


 



Glu-Arg-Gln
NR


 



Glu-Arg-Gly
NR


 



Glu-Arg-His
NR


 



Glu-Arg-Ile
NR


 



Glu-Arg-Leu
NR


 



Glu-Arg-Lys
NR


 



Glu-Arg-Met
NR


 



Glu-Arg-Phe
NR


 



Glu-Arg-Pro
NR


 



Glu-Arg-Ser
NR


 



Glu-Arg-Thr
NR


 



Glu-Arg-Trp
NR


 



Glu-Arg-Tyr
NR


 



Glu-Arg-Val
NR
















TABLE 20







Additional Exemplary Pharmacologically Active


Peptides










SEQ




ID



Sequence/structure
NO:
Activity





VEPNCDIHVMWEWECFERL
915
VEGF-




antagonist


 


GERWCFDGPLTWVCGEES
916
VEGF-




antagonist


 


RGWVEICVADDNGMCVTEAQ
917
VEGF-




antagonist


 


GWDECDVARMWEWECFAGV
918
VEGF-




antagonist


 


GERWCFDGPRAWVCGWEI
919
VEGF-




antagonist


 


EELWCFDGPRAWVCGYVK
920
VEGF-




antagonist


 


RGWVEICAADDYGRCLTEAQ
921
VEGF-




antagonist


 


RGWVEICESDVWGRCL
922
VEGF-




antagonist


 


RGWVEICESDVWGRCL
923
VEGF-




antagonist


 


GGNECDIARMWEWECFERL
924
VEGF-




antagonist


 


RGWVEICAADDYGRCL
925
VEGF-




antagonist


 


CTTHWGFTLC
926
MMP




inhibitor


 


CLRSGXGC
927
MMP




inhibitor


 


CXXHWGFXXC
928
MMP




inhibitor


 


CXPXC
929
MMP




inhibitor


 


CRRHWGFEFC
930
MMP




inhibitor


 


STTHWGFTLS
931
MMP




inhibitor


 


CSLHWGFWWC
932
CTLA4-




mimetic


 


GFVCSGIFAVGVGRC
933
CTLA4-




mimetic


 


APGVRLGCAVLGRYC
934
CTLA4-




mimetic


 


LLGRMK
935
Antiviral




(HBV)


 


ICVVQDWGHHRCTAGHMANLTSHASAI
936
C3b




antagonist


 


ICVVQDWGHHRCT
937
C3b




antagonist


 


CVVQDWGHHAC
938
C3b




antagonist


 


STGGFDDVYDWARGVSSALTTTLVATR
939
Vinculin-




binding


 


STGGFDDVYDWARRVSSALTTTLVATR
940
Vinculin-




binding


 


SRGVNFSEWLYDMSAAMKEASNVFPSRRSR
941
Vinculin-




binding


 


SSQNWDMEAGVEDLTAAMLGLLSTIHSSSR
942
Vinculin-




binding


 


SSPSLYTQFLVNYESAATRIQDLLIASRPSR
943
Vinculin-




binding


 


SSTGWVDLLGALQRAADATRTSIPPSLQNSR
944
Vinculin-




binding


 


DVYTKKELIECARRVSEK
945
Vinculin-




binding


 


EKGSYYPGSGIAQFHIDYNNVS
946
C4BP-




binding


 


SGIAQFHIDYNNVSSAEGWHVN
947
C4BP-




binding


 


LVTVEKGSYYPGSGIAQFHIDYNNVSSAEGWHVN
948
C4BP-




binding


 


SGIAQFHIDYNNVS
949
C4BP-




binding


 


LLGRMK
950
anti-HBV


 


ALLGRMKG
951
anti-HBV


 


LDPAFR
952
anti-HBV


 


CXXRGDC
953
Inhibition




of platelet




aggregation


 


RPLPPLP
954
Src




antagonist


 


PPVPPR
955
Src




antagonist


 


XFXDXWXXLXX
956
Anti-cancer




(particu-




larly for




sarcomas)


 


KACRRLFGPVDSEQLSRDCD
957
p16-mimetic


 


RERWNFDFVTETPLEGDFAW
958
p16-mimetic


 


NRRQTSMTDFYHSNRRLIFS
959
p16-mimetic


 


TSMTDFYHSNRRLIFSNRKP
960
p16-mimetic


 


RRLIF
961
p16-mimetic


 


KRRQTSATDFYHSNRRLIFSRQIKIWFQNRRMKWKK
962
p16-mimetic


 


KRRLIFSNRQIKlWFQNRRMKWKK
963
p16-mimetic


 


Asn Gln Gly Arg His Phe Cys Gly Gly
964
CAP37


Ala Leu Ile His Ala Arg Phe Val Met

mimetic/LPS


Thr Ala Ala Ser Cys Phe Gln

binding


 


Arg His Phe Cys Gly Gly Ala Leu Ile
965
CAP37


His Ala Arg Phe Val Met Thr Ala Ala

mimetic/LPS


Ser Cys

binding


 


Gly Thr Arg Cys Gln Val Ala Gly Trp
966
CAP37


Gly Ser Gln Arg Ser Gly Gly Arg Leu

mimetic/LPS


Ser Arg Phe Pro Arg Phe Val Asn Val

binding


 


WHWRHRIPLQLAAGR
967
carbohy-




drate (GD1




alpha)




mimetic


 


LKTPRV
968
β2GPI Ab




binding


 


NTLKTPRV
969
β2GPI Ab




binding


 


NTLKTPRVGGC
970
β2GPI Ab




binding


 


KDKATF
971
β2GPI Ab




binding


 


KDKATFGCHD
972
β2GPI Ab




binding


 


KDKATFGCHDGC
973
β2GPI Ab




binding


 


TLRVYK
974
β2GPI Ab




binding


 


ATLRVYKGG
975
β2GPI Ab




binding


 


CATLRVYKGG
976
β2GPI Ab




binding


 


INLKALAALAKKIL
977
Membrane-




transport-




ing


 


GWT
NR
Membrane-




transport-




ing


 


GWTLNSAGYLLG
978
Membrane-




transport-




ing


 


GWTLNSAGYLLGKINLKALAALAKKIL
979
Membrane-




transport-




ing


 


CVHAYRS
980
Antiprolif-




erative,




antiviral


 


CVHAYRA
981
Antiprolif-




erative,




antiviral


 


CVHAPRS
982
Antiprolif-




erative,




antiviral


 


CYHAPRA
983
Antiprolif-




erative,




antiviral


 


CYHSYRS
984
Antiprolif-




erative,




antiviral


 


CVHSYRA
985
Antiprolif-




erative,




antiviral


 


CVHSPRS
986
Antiprolif-




erative,




antiviral


 


CVHSPRA
987
Antiprolif-




erative,




antiviral


 


CVHTYRS
988
Antiprolif-




erative,




antiviral


 


CVHTYRA
989
Antiprolif-




erative,




antiviral


 


CVHTPRS
990
Antiprolif-




erative,




antiviral


 


CVHTPRA
991
Antiprolif-




erative,




antiviral


 


HWAWFK
992
anti-ische-




mic, growth




hormone-




liberating
















TABLE 21







MYOSTATIN INHIBITOR PEPTIDES









PEPTI-




BODY
SEQ



NAME
ID
PEPTIDE SEQUENCE





Myostat-
1036
KDKCKMWHWMCKPP


in-TN8-




Con1




 


Myostat-
1037
KDLCAMWHWMCKPP


in-TN8-




Con2




 


Myostat-
1038
KDLCKMWKWMCKPP


in-TN8-




Con3




 


Myostat-
1039
KDLCKMWHWMCKPK


in-TN8-




Con4




 


Myostat-
1040
WYPCYEFHFWCYDL


in-TN8-




Con5




 


Myostat-
1041
WYPCYEGHFWCYDL


in-TN8-




Con6




 


Myostat-
1042
IFGCKWWDVQCYQF


in-TN8-




Con7




 


Myostat-
1043
IFGCKWWDVDCYQF


in-TN8-




Con8




 


Myostat-
1044
ADWCVSPNWFCMVM


in-TN8-




Con9




 


Myostat-
1045
HKFCPWWALFCWDF


in-TN8-




Con10




 


Myostat-
1046
KDLCKMWHWMCKPP


in-TN8-1




 


Myostat-
1047
IDKCAIWGWMCPPL


in-TN8-2




 


Myostat-
1048
WYPCGEFGMWCLNV


in-TN8-3




 


Myostat-
1049
WFTCLWNCDNE


in-TN8-4




 


Myostat-
1050
HTPCPWFAPLCVEW


in-TN8-5




 


Myostat-
1051
KEWCWRWKWMCKPE


in-TN8-6




 


Myostat-
1052
FETCPSWAYFCLDI


in-TN8-7




 


Myostat-
1053
AYKCEANDWGCWWL


in-TN8-8




 


Myostat-
1054
NSWCEDQWHRCWWL


in-TN8-9




 


Myostat-
1055
WSACYAGHFWCYDL


in-TN8-




10




 


Myostat-
1056
ANWCVSPNWFCMVM


in-TN8-




11




 


Myostat-
1057
WTECYQQEFWCWNL


in-TN8-




12




 


Myostat-
1058
ENTCERWKWMCPPK


in-TN8-




13




 


Myostat-
1059
WLPCHQEGFWCMNF


in-TN8-




14




 


Myostat-
1060
STMCSQWHWMCNPF


in-TN8-




15




 


Myostat-
1061
IFGCHWWDVDCYQF


in-TN8-




16




 


Myostat-
1062
IYGCKWWDIQCYDI


in-TN8-




17




 


Myostat-
1063
PDWCIDPDWWCKFW


in-TN8-




18




 


Myostat-
1064
QGHCTRWPWMCPPY


in-TN8-




19




 


Myostat-
1065
WQECYREGFWCLQT


in-TN8-




20




 


Myostat-
1066
WFDCYGPGFKCWSP


in-TN8-




21




 


Myostat-
1067
GVRCPKGHLWCLYP


in-TN8-




22




 


Myostat-
1068
HWACGYWPWSCKWV


in-TN8-




23




 


Myostat-
1069
GPACHSPWWWCVFG


in-TN8-




24




 


Myostat-
1070
TTWCISPMWFCSQQ


in-TN8-




25




 


Myostat-
1071
HKFCPPWAIFCWDF


in-TN8-




26




 


Myostat-
1072
PDWCVSPRWYCNMW


in-TN8-




27




 


Myostat-
1073
VWKCHWFGMDCEPT


in-TN8-




28




 


Myostat-
1074
KKHCQIWTWMCAPK


in-TN8-




29




 


Myostat-
1075
WFQCGSTLFWCYNL


in-TN8-




30




 


Myostat-
1076
WSPCYDHYFYCYTI


in-TN8-




31




 


Myostat-
1077
SWMCGFFKEVCMWV


in-TN8-




32




 


Myostat-
1078
EMLCMIHPVFCNPH


in-TN8-




33




 


Myostat-
1079
LKTCNLWPWMCPPL


in-TN8-




34




 


Myostat-
1080
VVGCKWYEAWCYNK


in-TN8-




35




 


Myostat-
1081
PIHCTQWAWMCPPT


in-TN8-




36




 


Myostat-
1082
DSNCPWYFLSCVIF


in-TN8-




37




 


Myostat-
1083
HIWCNLAMMKCVEM


in-TN8-




38




 


Myostat-
1084
NLQCIYFLGKCIYF


in-TN8-




39




 


Myostat-
1085
AWRCMWFSDVCTPG


in-TN8-




40




 


Myostat-
1086
WFRCFLDADWCTSV


in-TN8-




41




 


Myostat-
1087
EKICQMWSWMCAPP


in-TN8-




42




 


Myostat-
1088
WFYCHLNKSECTEP


in-TN8-




43




 


Myostat-
1089
FWRCAIGIDKCKRV


in-TN8-




44




 


Myostat-
1090
NLGCKWYEVWCFTY


in-TN8-




45




 


Myostat-
1091
IDLCNMWDGMCYPP


in-TN8-




46




 


Myostat-
1092
EMPCNIWGWMCPPV


in-TN8-




47




 


Myostat-
1093
WFRCVLTGIVDWSECFGL


in-TN12-




1




 


Myostat-
1094
GFSCTFGLDEFYVDCSPF


in-TN12-




2




 


Myostat-
1095
LPWCHDQVNADWGFCMLW


in-TN12-




3




 


Myostat-
1096
YPTCSEKFWIYGQTCVLW


in-TN12-




4




 


Myostat-
1097
LGPCPIHHGPWPQYCVYW


in-TN12-




5




 


Myostat-
1098
PFPCETHQISWLGHCLSF


in-TN12-




6




 


Myostat-
1099
HWGCEDLMWSWHPLCRRP


in-TN12-




7




 


Myostat-
1100
LPLCDADMMPTIGFCVAY


in-TN12-




8




 


Myostat-
1101
SHWCETTFWMNYAKCVHA


in-TN12-




9




 


Myostat-
1102
LPKCTHVPFDQGGFCLWY


in-TN12-




10




 


Myostat-
1103
FSSCWSPVSRQDMFCVFY


in-TN12-




11




 


Myostat-
1104
SHKCEYSGWLQPLCYRP


in-TN12-




13




 


Myostat-
1105
PWWCQDNYVQHMLHCDSP


in-TN12-




14




 


Myostat-
1106
WFRCMLMNSFDAFQCVSY


in-TN12-




15




 


Myostat-
1107
PDACRDQPWYMFMGCMLG


in-TN12-




16




 


Myostat-
1108
FLACFVEFELCFDS


in-TN12-




17




 


Myostat-
1109
SAYCIITESDPYVLCVPL


in-TN12-




18




 


Myostat-
1110
PSICESYSTMWLPMCQHN


in-TN12-




19




 


Myostat-
1111
WLDCHDDSWAWTKMCRSH


in-TN12-




20




 


Myostat-
1112
YLNCVMMNTSPFVECYFN


in-TN12-




21




 


Myostat-
1113
YPWCDGFMIQQGITCMFY


in-TN12-




22




 


Myostat-
1114
FDYCTWLNGFKDWKCWSR


in-TN12-




23




 


Myostat-
1115
LPLCNLKEISHVQACVLF


in-TN12-




24




 


Myostat-
1116
SPECAFARWLGIEQCQRD


in-TN12-




25




 


Myostat-
1117
YPQCFNLHLLEWTECDWF


in-TN12-




26




 


Myostat-
1118
RWRCEIYDSEFLPKCWFF


in-TN12-




27




 


Myostat-
1119
LVGCDNVWHRCKLF


in-TN12-




28




 


Myostat-
1120
AGWCHVWGEMFGMGCSAL


in-TN12-




29




 


Myostat-
1121
HHECEWMARWMSLDCVGL


in-TN12-




30




 


Myostat-
1122
FPMCGIAGMKDFDFCVWY


in-TN12-




31




 


Myostat-
1123
RDDCTFWPEWLWKLCERP


in-TN12-




32




 


Myostat-
1124
YNFCSYLFGVSKEACQLP


in-TN12-




33




 


Myostat-
1125
AHWCEQGPWRYGNICMAY


in-TN12-




34




 


Myostat-
1126
NLVCGKISAWGDEACARA


in-TN12-




35




 


Myostat-
1127
HNVCTIMGPSMKWFCWND


in-TN12-




36




 


Myostat-
1128
NDLCAMWGWRNTIWCQNS


in-TN12-




37




 


Myostat-
1129
PPFCQNDNDMLLQSLCKLL


in-TN12-




38




 


Myostat-
1130
WYDCNVPNELLSGLCRLF


in-TN12-




39




 


Myostat-
1131
YGDCDQNHWMWPFTCLSL


in-TN12-




40




 


Myostat-
1132
GWMCHFDLHDWGATCQPD


in-TN12-




41




 


Myostat-
1133
YFHCMFGGHEFEVHCESF


in-TN12-




42




 


Myostat-
1134
AYWCWHGQCVRF


in-TN12-




43




 


Myostat-
1135
SEHWTFTDWDGNEWWVRPF


in-




Linear-1




 


Myostat-
1136
MEMLDSLFELLKDMVPISKA


in-




Linear-2




 


Myostat-
1137
SPPEEALMEWLGWQYGKFT


in-




Linear-3




 


Myostat-
1138
SPENLLNDLYILMTKQEWYG


in-




Linear-4




 


Myostat-
1139
FHWEEGIPFHVVTPYSYDRM


in-




Linear-5




 


Myostat-
1140
KRLLEQFMNDLAELVSGHS


in-




Linear-6




 


Myostat-
1141
DTRDALFQEFYEFVRSRLVI


in-




Linear-7




 


Myostat-
1142
RMSAAPRPLTYRDIMDQYWH


in-




Linear-8




 


Myostat-
1143
NDKAHFFEMFMFDVHNFVES


in-




Linear-9




 


Myostat-
1144
QTQAQKIDGLWELLQSIRNQ


in-Lin-




ear-10




 


Myostat-
1145
MLSEFEEFLGNLVHRQEA


in-Lin-




ear-11




 


Myostat-
1146
YTPKMGSEWTSFWHNRIHYL


in-Lin-




ear-12




 


Myostat-
1147
LNDTLLRELKMVLNSLSDMK


in-Lin-




ear-13




 


Myostat-
1148
FDVERDLMRWLEGFMQSAAT


in-Lin-




ear-14




 


Myostat-
1149
HHGWNYLRKGSAPQWFEAWV


in-Lin-




ear-15




 


Myostat-
1150
VESLHQLQMWLDQKLASGPH


in-Lin-




ear-16




 


Myostat-
1151
RATLLKDFWQLVEGYGDN


in-Lin-




ear-17




 


Myostat-
1152
EELLREFYRFVSAFDY


in-Lin-




ear-18




 


Myostat-
1153
GLLDEFSHFIAEQFYQMPGG


in-Lin-




ear-19




 


Myostat-
1154
YREMSMLEGLLDVLERLQHY


in-Lin-




ear-20




 


Myostat-
1155
HNSSQMLLSELIMLVGSMMQ


in-Lin-




ear-21




 


Myostat-
1156
WREHFLNSDYIRDKLIAIDG


in-Lin-




ear-22




 


Myostat-
1157
QFPFYVFDDLPAQLEYWIA


in-Lin-




ear-23




 


Myostat-
1158
EFFHWLHNHRSEVNHWLDMN


in-Lin-




ear-24




 


Myostat-
1159
EALFQNFFRDVLTLSEREY


in-Lin-




ear-25




 


Myostat-
1160
QYWEQQWMTYFRENGLHVQY


in-Lin-




ear-26




 


Myostat-
1161
NQRMMLEDLWRIMTPMFGRS


in-Lin-




ear-27




 


Myostat-
1162
FLDELKAELSRHYALDDLDE


in-Lin-




ear-29




 


Myostat-
1163
GKLIEGLLNELMQLETFMPD


in-Lin-




ear-30




 


Myostat-
1164
ILLLDEYKKDWKSWF


in-Lin-




ear-31




 


Myostat-
1165
QGHCTRWPWMCPPYGSGSATGGSGSTASSGSGSATG


in-

QGHCTRWPWMCPPY


2xTN8-19




kc




 


Myostat-
1166
WYPCYEGHFWCYDLGSGSTASSGSGSATGWYPCYEG


in-

HFWCYDL


2xTN8-




con6




 


Myostat-
1167
HTPCPWFAPLCVEWGSGSATGGSQSTASSGSGSATGH


in-

TPCPWFAPLCVEW


2xTN8-5




kc




 


Myostat-
1168
PDWCIDPDWWCKFWGSGSATGGSGSTASSGSGSATG


in-

PDWCIDPDWWCKFW


2xTN8-18




kc




 


Myostat-
1169
ANWCVSPNWFCMVMGSGSATGGSGSTASSGSGSAT


in-

GANWCVSPNWFCMVM


2xTN8-11




kc




 


Myostat-
1170
PDWCIDPDWWCKFWGSGSATGGSGSTASSGSGSATG


in-

PDWCIDPDWWCKFW


2xTN8-25




kc




 


Myostat-
1171
HWACGYWPWSCKWVGSGSATGGSGSTASSGSGSAT


in-

GHWACGYWPWSCKWV


2xTN8-23




kc




 


Myostat-
1172
KKHCQIWTWMCAPKGSGSATGGSGSTASSGSGSATG


in-TN8-

QGHCTRWPWMCPPY


29-19 kc




 


Myostat-
1173
QGHCTRWPWMCPPYGSGSATGGSGSTASSGSGSATG


in-TN8-

KKHCQIWTWMCAPK


19-29 kc




 


Myostat-
1174
KKHCQIWTWMCAPKGSGSATGGSGSTASSGSGSATG


in-TN8-

QGHCTRWPWMCPPY


29-19 kn




 


Myostat-
1175
KKHCQIWTWMCAPKGGGGGGGGQGHCTRWPWMCP


in-TN8-

PY


29-19-8g




 


Myostat-
1176
QGHCTRWPWMCPPYGGGGGGKKHCQIWTWMCAPK


in-TN8-




19-29-




6gc
















TABLE 22







MYOSTATIN INHIBITOR PEPTIDES









Affinity- matured
SEQ ID



peptibody
NO:
Peptide sequence





mTN8-19-1
1177
VALHGQCTRWPWMCPPQREG


 


mTN8-19-2
1178
YPEQGLCTRWPWMCPPQTLA


 


mTN8-19-3
1179
GLNQGHCTRWPWMCPPQDSN


 


mTN8-19-4
1180
MITQGQCTRWPWMCPPQPSG


 


mTN8-19-5
1181
AGAQEHCTRWPWMCAPNDWI


 


mTN8-19-6
1182
GVNQGQCTRWRWMCPPNGWE


 


mTN8-19-7
1183
LADHGQCIRWPWMCPPEGWE


 


mTN8-19-8
1184
ILEQAQCTRWPWMCPPQRGG


 


mTN8-19-9
1185
TQTHAQCTRWPWMCPPQWEG


 


mTN8-19-10
1186
VVTQGHCTLWPWMCPPQRWR


 


mTN8-19-11
1187
IYPHDQCTRWPWMCPPQPYP


 


mTN8-19-12
1188
SYWQGQCTRWPWMCPPQWRG


 


mTN8-19-13
1189
MWQQGHCTRWPWMCPPQGWG


 


mTN8-19-14
1190
EFTQWHCTRWPWMCPPQRSQ


 


mTN8-19-15
1191
LDDQWQCTRWPWMCPPQGFS


 


mTN8-19-16
1192
YQTQGLCTRWPWMCPPQSQR


 


mTN8-19-17
1193
ESNQGQCTRWPWMCPPQGGW


 


mTN8-19-18
1194
WTDRGPCTRWPWMCPPQANG


 


mTN8-19-19
1195
VGTQGQCTRWPWMCPPYETG


 


mTN8-19-20
1196
PYEQGKCTRWPWMCPPYEVE


 


mTN8-19-21
1197
SEYQGLCTRWPWMCPPQGWK


 


mTN8-19-22
1198
TFSQGHCTRWPWMCPPQGWG


 


mTN8-19-23
1199
PGAHDHCTRWPWMCPPQSRY


 


mTN8-19-24
1200
VAEEWHCRRWPWMCPPQDWR


 


mTN8-19-25
1201
VGTQGHCTRWPWMCPPQPAG


 


mTN8-19-26
1202
EEDQARCRSWPWMCPPQGWV


 


mTN8-19-27
1203
ADTQGHCTRWPWMCPPQHWF


 


mTN8-19-28
1204
SGPQGHCTRWPWMCAPQGWF


 


mTN8-19-29
1205
TLVQGHCTRWPWMCPPQRWV


 


mTN8-19-30
1206
GMAHGKCTRWAWMCPPQSWK


 


mTN8-19-31
1207
ELYHGQCTRWPWMCPPQSWA


 


mTN8-19-32
1208
VADHGHCTRWPWMCPPQGWG


 


mTN8-19-33
1209
PESQGHCTRWPWMCPPQGWG


 


mTN8-19-34
1210
IPAHGHCTRWPWMCPPQRWR


 


mTN8-19-35
1211
FTVHGHCTRWPWMCPPYGWV


 


mTN8-19-36
1212
PDFPGHCTRWRWMCPPQGWE


 


mTN8-19-37
1213
QLWQGPCTQWPWMCPPKGRY


 


mTN8-19-38
1214
HANDGHCTRWQWMCPPQWGG


 


mTN8-19-39
1215
ETDHGLCTRWPWMCPPYGAR


 


mTN8-19-40
1216
GTWQGLCTRWPWMCPPQGWQ


 


mTN8-19 con1
1217
VATQGQCTRWPWMCPPQGWG


 


mTN8-19 con2
1218
VATQGQCTRWPWMCPPQRWG


 


mTN8 con6-1
1219
QREWYPCYGGHLWCYDLHKA


 


mTN8 con6-2
1220
ISAWYSCYAGHFWCWDLKQK


 


mTN8 con6-3
1221
WTGWYQCYGGHLWCYDLRRK


 


mTN8 con6-4
1222
KTFWYPCYDGHFWCYNLKSS


 


mTN8 con6-5
1223
ESRWYPCYEGHLWCFDLTET
















TABLE 23







MYOSTATIN INHIBITOR PEPTIDES











Affinity





matured
SEQ ID




peptibody
NO:
Peptide Sequence






L2
1224
MEMLDSLFELLKDMVPISKA


 



mL2-Con1
1225
RMEMLESLLELLKEIVPMSKAG


 



mL2-Con2
1226
RMEMLESLLELLKEIVPMSKAR


 



mL2-1
1227
RMEMLESLLELLKDIVPMSKPS


 



mL2-2
1228
GMEMLESLFELLQEIVPMSKAP


 



mL2-3
1229
RMEMLESLLELLKDIVPISNPP


 



mL2-4
1230
RIEMLESLLELLQEIVPISKAE


 



mL2-5
1231
RMEMLQSLLELLKDIVPMSNAR


 



mL2-6
1232
RMEMLESLLELLKEIVPTSNGT


 



mL2-7
1233
RMEMLESLFELLKEIVPMSKAG


 



mL2-8
1234
RMEMLGSLLELLKEIVPMSKAR


 



mL2-9
1235
QMELLDSLFELLKEIVPKSQPA


 



mL2-10
1236
RMEMLDSLLELLKEIVPMSNAR


 



mL2-11
1237
RMEMLESLLELLHEIVPMSQAG


 



mL2-12
1238
QMEMLESLLQLLKEIVPMSKAS


 



mL2-13
1239
RMEMLDSLLELLKDMVPMTTGA


 



mL2-14
1240
RIEMLESLLELLKDMVPMANAS


 



mL2-15
1241
RMEMLESLLQLLNEIVPMSRAR


 



mL2-16
1242
RMEMLESLFDLLKELVPMSKGV


 



mL2-17
1243
RIEMLESLLELLKDLVPIQKAR


 



mL2-18
1244
RMELLESLFELLKDMVPMSDSS


 



mL2-19
1245
RMEMLESLLEVLQEIVPRAKGA


 



mL2-20
1246
RMEMLDSLLQLLNEIVPMSHAR


 



mL2-21
1247
RMEMLESLLELLKDIVPMSNAG


 



mL2-22
1248
RMEMLQSLFELLKGMVPISKAG


 



mL2-23
1249
RMEMLESLLELLKEIVPNSTAA


 



mL2-24
1250
RMEMLQSLLELLKEIVPISKAG


 



mL2-25
1251
RIEMLDSLLELLNELVPMSKAR


 



L-15
1252
HHGWNYLRKGSAPQWFEAWV


 



mL15-con1
1253
QVESLQQLLMWLDQKLASGPQG


 



mL15-1
1254
RMELLESLFELLKEMVPRSKAV


 



mL15-2
1255
QAVSLQHLLMWLDQKLASGPQH


 



mL15-3
1256
DEDSLQQLLMWLDQKLASGPQL


 



mL15-4
1257
PVASLQQLLIWLDQKLAQGPHA


 



mL15-5
1258
EVDELQQLLNWLDHKLASGPLQ


 



mL15-6
1259
DVESLEQLLMWLDHQLASGPHG


 



mL15-7
1260
QVDSLQQVLLWLEHKLALGPQV


 



mL15-8
1261
GDESLQHLLMWLEQKLALGPHG


 



mL15-9
1262
QIEMLESLLDLLRDMVPMSNAF


 



mL15-10
1263
EVDSLQQLLMWLDQKLASGPQA


 



mL15-11
1264
EDESLQQLLIYLDKMLSSGPQV


 



mL15-12
1265
AMDQLHQLLIWLDHKLASGPQA


 



mL15-13
1266
RIEMLESLLELLDEIALIPKAW


 



mL15-14
1267
EVVSLQHLLMWLEHKLASGPDG


 



mL15-15
1268
GGESLQQLLMWLDQQLASGPQR


 



mL15-16
1269
GVESLQQLLIFLDHMLVSGPHD


 



mL15-17
1270
NVESLEHLMMWLERLLASGPYA


 



mL15-18
1271
QVDSLQQLLIWLDHQLASGPKR


 



mL15-19
1272
EVESLQQLLMWLEHKLAQGPQG


 



mL15-20
1273
EVDSLQQLLMWLDQKLASGPHA


 



mL15-21
1274
EVDSLQQLLMWLDQQLASGPQK


 



mL15-22
1275
GVEQLPQLLMWLEQKLASGPQR


 



mL15-23
1276
GEDSLQQLLMWLDQQLAAGPQV


 



mL15-24
1277
ADDSLQQLLMWLDRKLASGPHV


 



mL15-25
1278
PVDSLQQLLIWLDQKLASGPQG


 



L-17
1279
RATLLKDFWQLVEGYGDN


 



mL17-con1
1280
DWRATLLKEFWQLVEGLGDNLV


 



mL17-con2
1281
QSRATLLKEFWQLVEGLGDKQA


 



mL17-1
1282
DGRATLLTEFWQLVQGLGQKEA


 



mL17-2
1283
LARATLLKEFWQLVEGLGEKVV


 



mL17-3
1284
GSRDTLLKEFWQLVVGLGDMQT


 



mL17-4
1285
DARATLLKEFWQLVDAYGDRMV


 



mL17-5
1286
NDRAQLLRDFWQLVDGLGVKSW


 



mL17-6
1287
GVRETLLYELWYLLKGLGANQG


 



mL17-7
1288
QARATLLKEFCQLVGCQGDKLS


 



mL17-8
1289
QERATLLKEFWQLVAGLGQNMR


 



mL17-9
1290
SGRATLLKEFWQLVQGLGEYRW


 



mL17-10
1291
TMRATLLKEFWLFVDGQREMQW


 



mL17-11
1292
GERATLLNDFWQLVDGQGDNTG


 



mL17-12
1293
DERETLLKEFWQLVHGWGDNVA


 



mL17-13
1294
GGRATLLKELWQLLEGQGANLV


 



mL17-14
1295
TARATLLNELVQLVKGYGDKLV


 



mL17-15
1295
GMRATLLQEFWQLVGGQGDNWM


 



mL17-16
1297
STRATLLNDLWQLMKGWAEDRG


 



mL17-17
1298
SERATLLKELWQLVGGWGDNFG


 



mL17-18
1299
VGRATLLKEFWQLVEGLVGQSR


 



mL17-19
1300
EIRATLLKEFWQLVDEWREQPN


 



mL17-20
1301
QLRATLLKEFLQLVHGLGETDS


 



mL17-21
1302
TQRATLLKEFWQLIEGLGGKHV


 



mL17-22
1303
HYRATLLKEFWQLVDGLREQGV


 



mL17-23
1304
QSRVTLLREFWQLVESYRPIVN


 



mL17-24
1305
LSRATLLNEFWQFVDGQRDKRM


 



mL17-25
1306
WDRATLLNDFWHLMEELSQKPG


 



mL17-26
1307
QERATLLKEFWRMVEGLGKNRG


 



mL17-27
1308
NERATLLREFWQLVGGYGVNQR


 



L-20
1309
YREMSMLEGLLDVLERLQHY


 



mL20-1
1310
HQRDMSMLWELLDVLDGLRQYS


 



mL20-2
1311
TQRDMSMLDGLLEVLDQLRQQR


 



mL20-3
1312
TSRDMSLLWELLEELDRLGHQR


 



mL20-4
1313
MQHDMSMLYGLVELLESLGHQI


 



mL20-5
1314
WNRDMRMLESLFEVLDGLRQQV


 



mL20-6
1315
GYRDMSMLEGLLAVLDRLGPQL


 



mL20 con1
1316
TQRDMSMLEGLLEVLDRLGQQR


 



mL20 con2
1317
WYRDMSMLEGLLEVLDRLGQQR


 



L-21
1318
HNSSQMLLSELIMLVGSMMQ


 



mL21-1
1319
TQNSRQMLLSDFMMLVGSMIQG


 



mL21-2
1320
MQTSRHILLSEFMMLVGSIMHG


 



mL21-3
1321
HDNSRQMLLSDLLBLVGTMIQG


 



mL21-4
1322
MENSRQNLLRELIMLVGNMSHQ


 



mL21-5
1323
QDTSRHMLLREFMMLVGEMIQG


 



mL21 con1
1324
DQNSRQMLLSDLMILVGSMIQG


 



L-24
1325
EFFHWLHNHRSEVNHWLDMN


 



mL24-1
1326
NVFFQWVQKHGRVVYQWLDINV


 



mL24-2
1327
FDFLQWLQNHRSEVEHWLVMDV
















TABLE 24







MYOSTATIN INHIBITOR PEPTIDES








Peptibody



Name
Peptide





2x mTN8-
M-GAQ-WYPCYEGHFWCYDL-


Con6-(N)-
GSGSATGGSGSTASSGSGSATG-WYPCYEGHFWCYDL-


1K
LE-5G-FC (SEQ ID NO: 1328)


 


2x mTN8-
FC-5G-AQ-WYPCYEGHFWCYDL-


Con6-(C)-
GSGSATGGSGSTASSGSGSATG-WYPCYEGHFWCYDL-


1K
LE (SEQ ID NO: 1329)


 


2x mTN8-
M-GAQ-IFGCKWWDVQCYQF-


Con7-(N)-
GSGSATGGSGSTASSGSGSATG-IFGCKWWDVQCYQF-


1K
LE-5G-FC (SEQ ID NO: 1330)


 


2x mTN8-
FC-5G-AQ-IFGCKWWDVQCYQF-


Con7-(C)-
GSGSATGGSGSTASSGSGSATG-IFGCKWWDVQCYQF-


1K
LE (SEQ ID NO: 1331)


 


2x mTN8-
M-GAQ-IFGCKWWDVDCYQF-


Con8-(N)-
GSGSATGGSGSTASSGSGSATG-IFGCKWWDVDCYQF-


1K
LE-5G-FC (SEQ ID NO: 1332)


 


2x mTN8-
FC-5G-AQ-IFGCKWWDVDCYQF-


Con8-(C)-
GSGSATGGSGSTASSGSGSATG-IFGCKWWDVDCYQF-


1K
LE (SEQ ID NO: 1333)


 


2X mTN8-
FC-5G-AQ-


19-7
LADHGQCIRWPWMCPPEGWELEGSGSATGGSGSTASSG



SGSATGLADHGQCIRWPWMCPPEGWE-LE



(SEQ ID NO: 1334)


 


2X mTN8-
FC-5G-AQ-


19-7 ST-GG
LADHGQCIRWPWMCPPEGWEGSGSATGGSGGGASSGSG


de12x LE
SATGLADHGQCIRWPWMCPPEGWE



(SEQ ID NO: 1335)


 


2X mTN8-
FC-5G-AQ-


219-1
SEYQGLCTRWPWMCPPQGWKLEGSGSATGGSGSTASSG



SGSATGSEYQGLCTRWPWMCPPQGWK-LE



(SEQ ID NO: 1336)


 


2X mTN8-
FC-5G-AQ-


19-21 ST-
SEYQGLCTRWPWMCPPQGWKGSGSATGGSGGGASSGS


GG de12x
GSATGSEYQGLCTRWPWMCPPQGWK


LE
(SEQ ID NO: 1337)


 


2X mTN8-
FC-5G-AQ-


19-22
TFSQGHCTRWPWMCPPQGWGLEGSGSATGGSGSTASSG



SGSATGTFSQGHCTRWPWMCPPQGWG-L E



(SEQ ID NO: 1338)


 


2X mTN8-
FC-5G-AQ-


19-32
VADHGHCTRWPWMCPPQGWGLEGSGSATGGSGSTASS



GSGSATGVADHGHCTRWPWMGPPQGWG-LE



(SEQ ID NO: 1339)


 


2X mTN8-
FC-5G-AQ-


19-32 ST-
VADHGHCTRWPWMCPPQGWGGSGSATGGSGGGASSGS


GG de12x
GSATGVADHGHCTRWPWVCPPQGWG


LE
(SEQ ID NO: 1340)


 


2X mTN8-
FC-5G-AQ-


33
PESQGHCTRWPWMCPPQGWGLEGSGSATGGSGSTASSG


19-
SGSATGPESQGHCTRWPWMCPPQGWGLE



(SEQ ID NO: 1341)


 


2X mTN8-
FC-5G-AQ-


19-33 ST-
PESQGHCTRWPWMCPPQGWGGSGSATGGSGGGASSGS


GG de12x
GSATGPESQGHCTRWPWMCP PQGWG


LE
(SEQ ID NO: 1342)
















TABLE 25







Integrin-antagonist peptide sequences











SEQ. ID



Sequence/structure
NO:






CLCRGDCIC
1344


 



CWDDGWLC
1345


 



CWDDLWWLC
1346


 



CWDDGLMC
1347


 



CWDDGWMC
1348


 



CSWDDGWLC
1349


 



CPDDLWWLC
1350


 



NGR
1351


 



GSL
1352


 



RGD
1353


 



CGRECPRLCQSSC
1354


 



CNGRCVSGCAGRC
1355


 



CLSGSLSC
1356


 



GSL
1357


 



NGRAHA
1358


 



CNGRC
1359


 



CDCRGDCFC
1360


 



CGSLVRC
1361


 



DLXXL
1362


 



RTDLDSLRTYTL
1363


 



RTDLDSLRTY
1364


 



RTDLDSLRT
1365


 



RTDLDSLR
1366


 



GDLDLLKLRLTL
1367


 



GDLHSLRQLLSR
1368


 



RDDLHMLRLQLW
1369


 



SSDLHALKKRYG
1370


 



RGDLKQLSELTW
1371


 



CXXRGDC
1372


 



STGGFDDVYDWARGVSSALTTTLVATR
1373


 



STGGFDDVYDWARRVSSALTTTLVATR
1374


 



SRGVNFSEWLYDMSAAMKEASNVFPSRRSR
1375


 



SSQNWDMEAGVEDLTAAMLGLLSTIHSSSR
1376


 



SSPSLYTQFLVNYESAATRIQDLLIASRPSR
1377


 



SSTGWVDLLGALQRAADATRTSIPPSLQNSR
1378


 



DVYTKKELIECARRVSEK
1379


 



RGDGX
1380


 



CRGDGXC
1381


 



CARRLDAPC
1382


 



CPSRLDSPC
1383


 



CDCRGDCFC
1384


 



CDCRGDCLC
1385


 



RGDLAALSAPPV
1386
















TABLE 26







Selectin antagonist peptide sequences











SEQ ID



Sequence/structure
NO:






DITWDQLWDLMK
1387


 



DITWDELWKIMN
1388


 



DYTWFELWDMMQ
1389


 



QITWAQLWNMMK
1390


 



DMTWHDLWTLMS
1391


 



DYSWHDLWEMMS
1392


 



EITWDQLWEVMN
1393


 



HVSWEQLWDIMN
1394


 



HITWDQLWRIMT
1395


 



RNMSWLELWEHMK
1396


 



AEWTWDQLWHVMNPAESQ
1397


 



HRAEWLALWEQMSP
1398


 



KKEDWLALWRIMSV
1399


 



ITWDQLWDLMK
1400


 



DITWDQLWDLMK
1401


 



DITWDQLWDLMK
1402


 



DITWDQLWDLMK
1403


 



CQNRYTDLVAIQNKNE
1404


 



AENWADNEPNNKRNNED
1405


 



RKNNKTWTWVGTKKALTNE
1406


 



KKALTNEAENWAD
1407


 



CQXRYTDLVAIQNKXE
1408


 



AENWADGEPNNKXNXED
1409
















TABLE 27







Vinculin binding peptides











SEQ ID



Sequence/structure
NO:






SSQNWDMEAGVEDLTAAMLGLLSTIHSSSR
1410


 



SSPSLYTQFLVNYESAATRIQDLLIASRPSR
1411


 



SSTGWVDLLGALQRAADATRTSIPPSLQNSR
1412


 



DVYTKKELIECARRVSEK
1413


 



STGGFDDVYDWARGVSSALTTTLVATR
1414


 



STGGFDDVYDWARRVSSALTTTLVATR
1415


 



SRGVNFSEWLYDMSAAMKEASNVFPSRRSR
1416
















TABLE 28







Laminin-related peptide sequences









SEQ ID


Sequence/structure
NO:





YIGSRYIGSR [i.e., (YIGSR)2]
1417


 


YIGSRYIGSRYIGSR [i.e., (YIGSR)3]
1418


 


YIGSRYIGSRYIGSRYIGSR [i.e., (YIGSR)4]
1419


 


YIGSRYIGSRYIGSRYIGSRYIGSR [i.e., (YIGSR)5]
1420


 


IPCNNKGAHSVGLMWWMLAR
1421


 


YIGSRREDVEILDVPDSGR
1422


 


RGDRGDYIGSRRGD
1423


 


YIGSRYIGSRYIGSRYIGSRYIGSR
1424


 


REDVEILDVYIGSRPDSGR
1425


 


YIGSRREDVEILDVPDSGR
1426
















TABLE 29







NGF Modulating Peptides








SEQ ID
Sequence of Peptide Portion of


NO:
Fc-Peptide Fusion Product





1427
TGYTEYTEEWPMGFGYQWSF


 


1428
TDWLSDFPFYEQYFGLMPPG


 


1429
FMRFPNPWKLVEPPQGWYYG


 


1430
VVKAPHFEFLAPPHFHEFPF


 


1431
FSYIWIDETPSNIDRYMLWL


 


1432
VNFPKVPEDVEPWPWSLKLY


 


1433
TWHPKTYEEFALPFFVPEAP


 


1434
WHFGTPYIQQQPGVYWLQAP


 


1435
VWNYGPFFMNFPDSTYFLHE


 


1436
WRIHSKPLDYSHVWFFPADF


 


1437
FWDGNQPPDILVDWPWNPPV


 


1438
FYSLEWLKDHSEFFQTVTEW


 


1439
QFMELLKFFNSPGDSSHHFL


 


1440
TNYDWISNNWEHMKSFFTED


 


1441
PNEKPYQMQSWFPPDWPVPY


 


1442
WSHTEWVPQVWWKPPNHFYV


 


1443
WGEWLNDAQVHMHEGFISES


 


1444
VPWEHDHDLWEIISQDWHIA


 


1445
VLHLQDPRGWSNFPPGVLEL


 


1446
IHGCWFTEEGCVWQ


 


1447
YMQCQFARDGCPQW


 


1448
KLQCQYSESGCPTI


 


1449
FLQCEISGGACPAP


 


1450
KLQCEFSTSGCPDL


 


1451
KLQCEFSTQGCPDL


 


1452
KLQCEFSTSGCPWL


 


1453
IQGCWFTEEGCPWQ


 


1454
SFDCDNPWGHVLQSCFGF


 


1455
SFDCDNPWGHKLQSCFGF
















TABLE 30







TALL MODULATING PEPTIDES











SEQ ID



Sequence/structure
NO:






LPGCKWDLLIKQWVCDPL- Λ -V1
1456


 



V1- Λ - LPGCKWDLLIKQWVCDPL
1457


 



LPGCKWDLLIKQWVCDPL - Λ -
1458



LPGCKWDLLIKQWVCDPL - Λ -V1



 



V1- Λ - LPGCKWDLLIKQWVCDPL - Λ -
1459



LPGCKWDLLIKQWVCDPL



 



SADCYFDILTKSDVCTSS- Λ -V1
1460


 



V1- Λ - SADCYFDILTKSDVCTSS
1461


 



SADCYFDILTKSDVTSS- Λ -
1462



SADCYFDILTKSDVTSS- Λ -V1



 



V1- Λ - SADCYFDILTKSDVTSS - Λ -
1463



SADCYFDILTKSDVTSS



 



FHDCKWDLLTKQWVCHGL- Λ -V1
1464


 



V1- Λ - FHDCKWDLLTKQWVCHGL
1465


 



FHDCKWDLLTKQWVCHGL - Λ -
1466



FHDCKWDLLTKQWVCHGL - Λ -V1



 



V1- Λ - FHDCKWDLLTKQWVCHGL - Λ -
1467



FHDCKWDLLTKQWVCHGL
















TABLE 31







TALL-1 inhibitory peptibodies.










Pepti-




body




SEQ ID



Peptibody
NO
Peptide Sequence





TALL-1-8-1-
1468
MPGTCFPFPW ECTHAGGGGG VDKTHTCPPC


a

PAPELLGGPS VFLFPPKPKD TLMISRTPEV




TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT




KPREEQYNST YRVVSVLTVL HQDWLNGKEY




KCKVSNKALP APIEKTISKA KGQPREPQVY




TLPPSRDELT KNQVSLTCLV KGFYPSDIAV




EWESNGQPEN NYKTTPPVLD SDGSFFLYSK




LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK




SLSLSPGK


 


TALL-1-8-2-
1469
MWGACWPFPW ECFKEGGGGG VDKTHTCPPC


a

PAPELLGGPS VFLFPPKPKD TLMISRTPEV




TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT




KPREEQYNST YRVVSVLTVL HQDWLNGKEY




KCKVSNKALP APIEKTISKA KGQPREPQVY




TLPPSRDELT KNQVSLTCLV KGFYPSDIAV




EWESNGQPEN NYKTTPPVLD SDGSFFLYSK




LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK




SLSLSPGK


 


TALL-1-8-4-
1470
MVPFCDLLTK HCFEAGGGGG VDKTHTCPPC


a

PAPELLGGPS VFLFPPKPKD TLMISRTPEV




TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT




KPREEQYNST YRVVSVLTVL HQDWLNGKEY




KCKVSNKALP APIEKTISKA KGQPREPQVY




TLPPSRDELT KNQVSLTCLV KGFYPSDIAV




EWESNGQPEN NYKTTPPVLD SDGSFFLYSK




LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK




SLSLSPGK


 


TALL-1-12-
1471
MGSRCKYKWD VLTKQCFHHG GGGGVDKTHT


4-a

CPPCPAPELL GGPSVFLFPP KPKDTLMISR




TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH




NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN




GKEYKCKVSN KALPAPIEKT ISKAKGQPRE




PQVYTLPPSR DELTKNQVSL TCLVKGFYPS




DIAVEWESNG QPENNYKTTP PVLDSDGSFF




LYSKLTVDKS RWQQGNVFSC SVMHEALHNH




YTQKSLSLSP GK


 


TALL-1-12-
1472
MLPGCKWDLL IKQWVCDPLG GGGGVDKTHT


3-a

CPPCPAPELL GGPSVFLFPP KPKDTLMISR




TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH




NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN




GKEYKCKVSN KALPAPIEKT ISKAKGQPRE




PQVYTLPPSR DELTKNQVSL TCLVKGEYPS




DIAVEWESNG QPENNYKTTP PVLDSDGSFF




LYSKLTVDKS RWQQGNVFSC SVMHEALHNH




YTQKSLSLSP GK


 


TALL-1-12-
1473
MSADCYFDIL TKSDVCTSSG GGGGVDKTHT


5-a

CPPCPAPELL GGPSVFLFPP KPKDTLMISR




TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH




NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN




GKEYKCKVSN KALPAPIEKT ISKAKGQPRE




PQVYTLPPSR DELTKNQVSL TCLVKGFYPS




DIAVEWESNG QPENNYKTTP PVLDSDGSFF




LYSKLTVDKS RWQQGNVFSC SVMHEALHNH




YTQKSLSLSP GK


 


TALL-1-12-
1474
MSDDCMYDQL TRMFICSNLG GGGGVDKTHT


8-a

CPPCPAPELL GGPSVFLFPP KPKDTLMISR




TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH




NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN




GKEYKCKVSN KALPAPIEKT ISKAKGQPRE




PQVYTLPPSR DELTKNQVSL TCLVKGFYPS




DIAVEWESNG QPENNYKTTP PVLDSDGSFF




LYSKLTVDKS RWQQGNVFSC SVMHEALHNH




YTQKSLSLSP GK


 


TALL-1-12-
1475
MDLNCKYDEL TYKEWCQFNG GGGGVDKTHT


9-a

CPPCPAPELL GGPSVFLFPP KPKDTLMISR




TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH




NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN




GKEYKCKVSN KALPAPIEKT ISKAKGQPRE




PQVYTLPPSR DELTKNQVSL TCLVKGFYPS




DLAVEWESNG QPENNYKTTP PVLDSDGSFF




LYSKLTVDKS RWQQGNVFSC SVMHEALHNH




YTQKSLSLSP GK


 


TALL-1-12-
1476
MFHDCKYDLL TRQMVCHGLG GGGGVDKTHT


10-a

CPPCPAPELL GGPSVFLFPP KPKDTLMISR




TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH




NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN




GKEYKCKVSN KALPAPIEKT ISKAKGQPRE




PQVYTLPPSR DELTKNQVSL TCLVKGFYPS




DIAVEWESNG QPENNYKTTP PVLDSDGSFF




LYSKLTVDKS RWQQGNVFSC SVMHEALHNH




YTQKSLSLSP GK


 


TALL-1-12-
1477
MRNHCFWDHL LKQDICPSPG GGGGVDKTHT


11-a

CPPCPAPELL GGPSVFLFPP KPKDTLMISR




TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH




NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN




GKEYKCKVSN KALPAPIEKT LSKAKGQPRE




PQVYTLPPSR DELTKNQVSL TCLVKGFYPS




DIAVEWESNG QPENNYKTTP PVLDSDGSFF




LYSKLTVDKS RWQQGNVFSC SVMHEALHNH




YTQKSLSLSP GK


 


TALL-1-12-
1478
MANQCWWDSL TKKNVCEFFG GGGGVDKTHT


14-a

CPPCPAPELL GGPSVFLFPP KPKDTLMISR




TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH




NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN




GKEYKCKVSN KALPAPIEKT ISKAKGQPRE




PQVYTLPPSR DELTKNQVSL TCLVKGFYPS




DIAVEWESNG QPENNYKTTP PVLDSDGSFF




LYSKLTVDKS RWQQGNVFSC SVMHEALHNH




YTQKSLSLSP GK


 


TALL-1-
1479
MFHDCKWDLL TKQWVCHGLG GGGGVDKTHT


consensus

CPPCPAPELL GGPSVFLFPP KPKDTLMISR




TPEYTCVVVD VSHEDPEVKF NWYVDGVEVH




NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN




GKEYKCKVSN KALPAPIEKT ISKAKGQPRE




PQVYTLPPSR DELTKNQVSL TCLVKGFYPS




DIAVEWESNG QPENNYKTTP PVLDSDGSFF




LYSKLTVDKS RWQQGNVFSC SVMHEALHNH




YTQKSLSLSP GK


 


TALL-1 12-3
1480
MLPGCKWDLL IKQWVCDPLG SGSATGGSGS


tandem

TASSGSGSAT HMLPGCKWDL LIKQWVCDPL


dimer

GGGGGVDKTH TCPPCPAPEL LGGPSVFLFP




PKPKDTLMIS RTPEVTCVVV DVSHEDPEVK




FNWYVDGVEV HNAKTKPREE QYNSTYRVVS




VLTVLHQDWL NGKEYKCKVS NKALPAPIEK




TISKAKGQPR EPQVYTLPPS RDELTKNQVS




LTCLVKGFYP SDIAVEWESN GQPENNYKTT




PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS




CSVMHEALHN HYTQKSLSLS PGK


 


TALL-1
1481
MFHDCKWDLL TKQWVCHGLG SGSATGGSGS


consensus

TASSGSGSAT HMFHDCKWDL LTKQWVCHGL


tandem

GGGGGVDKTH TCPPCPAPEL LGGPSVFLFP


dimer

PKPKDTLMIS RTPEVTCVVV DVSHEDPEVK




FNWYVDGVEV HNAKTKPREE QYNSTYRVYS




VLTVLHQDWL NGKEYKCKVS NKALPAPIEK




TISKAKGQPR EPQVYTLPPS RDELTKNQVS




LTCLVKGFYP SDIAVEWESN GQPENNYKTT




PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS




CSVMHEALHN HYTQKSLSLS PGK
















TABLE 32







ANG-2 INHIBITOR PEPTIDES











PEPTIDE
SEQ ID NO.
PEPTIDE SEQUENCE






Con4-44
1482
PIRQEECDWDPWTCEHMWEV


 



Con4-40
1483
TNIQEECEWDPWTCDHMPGK


 



Con4-4
1484
WYEQDACEWDPWTCEHMAEV


 



Con4-31
1485
NRLQEVCEWDPWTCEHMENV


 



Con4-C5
1486
AATQEECEWDPWTCEHMPRS


 



Con4-42
1487
LRHQEGCEWDPWTCEHMFDW


 



Con4-35
1488
VPRQKDCEWDPWTCEHMYVG


 



Con4-43
1489
SISHEECEWDPWTCEHMQVG


 



Con4-49
1490
WAAQEECEWDPWTCEHMGRM


 



Con4-27
1491
TWPQDKCEWDPWTCEHMGST


 



Con4-48
1492
GHSQEECGWDPWTCEHMGTS


 



Con4-46
1493
QHWQEECEWDPWTCDHMPSK


 



Con4-41
1494
NVRQEKCEWDPWTCEHMPVR


 



Con4-36
1495
KSGQVECNWDPWTCEHMPRN


 



Con4-34
1496
VKTQEHCDWDPWTCEHMREW


 



Con4-28
1497
AWGQEGCDWDPWTCEHMLPM


 



Con4-39
1498
PVNQEDCEWDPWTCEHMPPM


 



Con4-25
1499
RAPQEDCEWDPWTCAHMDIK


 



Con4-50
1500
HGQNMECEWDPWTCEHMFRY


 



Con4-38
1501
PRLQEECVWDPWTCEHMPLR


 



Con4-29
1502
RTTQEKCEWDPWTCEHMESQ


 



Con4-47
1503
QTSQEDCVWDPWTCDHMVSS


 



Con4-20
1504
QVIGRPCEWDPWTCEHLEGL


 



Con4-45
1505
WAQQEECAWDPWTCDHMVGL


 



Con4-37
1506
LPGQEDCEWDPWTCEHMVRS


 



Con4-33
1507
PMNQVECDWDPWTCEHMPRS


 



AC2-Con4
1508
FGWSHGCEWDPWTCEHMGST


 



Con4-32
1509
KSTQDDCDWDPWTCEHMVGP


 



Con4-17
1510
GPRISTCQWDPWTCEHMDQL


 



Con4-8
1511
STIGDMCEWDPWTCAHMQVD


 



AC4-Con4
1512
VLGGQGCEWDPWTCRLLQGW


 



Con4-1
1513
VLGGQGCQWDPWTCSHLEDG


 



Con4-C1
1514
TTIGSMCEWDPWTCAHMQGG


 



Con4-21
1515
TKGKSVCQWDPWTCSHMQSG


 



Con4-C2
1516
TTIGSMCQWDPWTCAHMQGG


 



Con4-18
1517
WVNEVVCEWDPWTCNHWDTP


 



Con4-19
1518
VVQVGMCQWDPWTCKHMRLQ


 



Con4-16
1519
AVGSQTCEWDPWTCAHLVEV


 



Con4-11
1520
QGMKMFCEWDPWTCAHIVYR


 



Con4-C4
1521
TTIGSMCQWDPWTCEHMQGG


 



Con4-23
1522
TSQRVGCEWDPWTCQHLTYT


 



Con4-15
1523
QWSWPPCEWDPWTCQTVWPS


 



Con4-9
1524
GTSPSFCQWDPWTCSHMVQG


 



TN8-Con4*
1525
QEECEWDPWTCEHM
















TABLE 33







ANG-2 INHIBITOR PEPTIDES











Peptide
SEQ ID NO.
Peptide Sequence






L1-1
1526
QNYKPLDELDATLYEHFIFHYT


 



L1-2
1527
LNFTPLDELEQTLYEQWTLQQS


 



L1-3
1528
TKFNPLDELEQTLYEQWTLQHQ


 



L1-4
1529
VKFKPLDALEQTLYEHWMFQQA


 



L1-5
1530
VKYKPLDELDEILYEQQTFQER


 



L1-7
1531
TNFMPMDDLEQRLYEQFILQQG


 



L1-9
1532
SKFKPLDELEQTLYEQWTLQHA


 



L1-10
1533
QKFQPLDELEQTLYEQFMLQQA


 



L1-11
1534
QNFKPMDELEDTLYKQELFQHS


 



L1-12
1535
YKFTPLDDLEQTLYEQWTLQHV


 



L1-13
1536
QEYEPLDELDETLYNQWMFHQR


 



L1-14
1537
SNFMPLDELEQTLYEQFMLQHQ


 



L1-15
1538
QKYQPLDELDKTLYDQFMLQQG


 



L1-16
1539
QKFQPLDELEETLYKQWTLQQR


 



L1-17
1540
VKYKPLDELDEWLYHQFTLHHQ


 



L1-18
1541
QKFMPLDELDEILYEQFMFQQS


 



L1-19
1542
QTFQPLDDLEEYLYEQWIRRYH


 



L1-20
1543
EDYMPLDALDAQLYEQFILLHG


 



L1-21
1544
HTFQPLDELEETLYYQWLYDQL


 



L1-22
1545
YKFNPMDELEQTLYEEFLFQHA


 



AC6-L1
1546
TNYKPLDELDATLYEHWILQHS


 



L1-C1
1547
QKFKPLDELEQTLYEQWTLQQR


 



L1-C2
1548
TKFQPLDELDQTLYEQWTLQQR


 



L1-C3
1549
TNFQPLDELDQTLYEQWTLQQR


 



L1
1550
KFNPLDELEETLYEQFTFQQ
















TABLE 34







ANG-2 INHIBITOR PEPTIDES











Peptide
SEQ ID NO.
Sequence






Con1-1
1551
AGGMRPYDGMLGWPNYDVQA


 



Con1-2
1552
QTWDDPCMHILGPVTWRRCI


 



Con1-3
1553
APGQRPYDGMLGWPTYQRIV


 



Con1-4
1554
SGQLRPCEEIFGCGTQNLAL


 



Con1-5
1555
FGDKRPLECMFGGPIQLCPR


 



Con1-6
1556
GQDLRPCEDMFGCGTKDWYG


 



Con1
1557
KRPCEEIFGGCTYQ
















TABLE 35







ANG-2 INHIBITOR PEPTIDES











Peptide
SEQ ID NO:
Sequence






12-9-1
1558
GFEYCDGMEDPFTFGCDKQT


 



12-9-2
1559
KLEYCDGMEDPFTQGCDNQS


 



12-9-3
1560
LQEWCEGVEDPFTFGCEKQR


 



12-9-4
1561
AQDYCEGMEDPFTFGCEMQK


 



12-9-5
1562
LLDYCEGVQDPFTFGCENLD


 



12-9-6
1563
HQEYCEGMEDPFTFGCEYQG


 



12-9-7
1564
MLDYCEGMDDPFTFGCDKQM


 



12-9-C2
1565
LQDYCEGVEDPFTFGCENQR


 



12-9-C1
1566
LQDYCEGYEDPFTFGCEKQR


 



12-9
1567
FDYCEGVEDPFTFGCDNH
















TABLE 36







Ang-2 Binding Peptides











Peptide
Seq Id No.
Sequence






TN8-8
1568
KRPCEEMWGGCNYD


 



TN8-14
1569
HQICKWDPWTCKHW


 



TN8-Con1
1570
KRPCEEIFGGCTYQ


 



TN8-Con4
1571
QEECEWDPWTCEHM


 



TN12-9
1572
FDYCEGVEDPFTFGCDNH


 



L1
1573
KFNPLDELEETLYEQFTFQQ


 



C17
1574
QYGCDGFLYGCMIN
















TABLE 37







Ang-2 Binding Peptides








Pepti-



body
Peptibody Sequence





L1 (N)
MGAQKFNPLDELEETLYEQFTFQQLEGGGGG-Fc



(SEQ ID NO: 1575)


 


L1 (N)
MKFNPLDELEETLYEQFTFQQLEGGGGG-Fc


WT
(SEQ ID NO: 1576)


 


L1 (N)
MKFNPLDELEETLYEQFTFQQGSGSATGGSGSTASSGSGSAT


1K WT
HLEGGGGG-Fc (SEQ ID NO: 1577)


 


2xL1 (N)
MGAQKFNPLDELEETLYEQFTFQQGGGGGGGGKFNPLDELE



ETLYEQFTFQQLEGGGGG-Fc (SEQ ID NO: 1578)


 


2xL1 (N)
MKFNPLDELEETLYEQFTFQQGGGGGGGKFNPLDELEETLYE


WT
QFTFQQLEGGGGG-Fc (SEQ ID NO: 1579)


 


Con4 (N)
MGAQQEECEWDPWTCEHMLEGGGGG-Fc



(SEQ ID NO: 1580)


 


Con4 (N)
MQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHLEGG


1K-WT
GGG-Fc (SEQ ID NO: 1581)


 


2xCon4
MGAQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATH


(N) 1K
QEECEWDPWTCEHMLEGGGGG-Fc (SEQ ID NO: 1582)


 


L1 (C)
M-Fc-GGGGGAQKFNPLDELEETLYEQFTFQQLE



(SEQ ID NO: 1583)


 


L1 (C)
M-Fc-


1K
GGGGGAQGSGSATGGSGSTASSGSGSATHKFNPLDELEETLY



EQFTFQQLE (SEQ ID NO: 1584)


 


2xL1
M-Fc-


(C)
GGGGGAQKFNPLDELEETLYEQFTFQQGGGGGGGGKFNPLD



ELEETLYEQFTFQQLE (SEQ ID NO: 1585)


 


Con4 (C)
M-Fc-GGGGGAQQEECEWDPWTCEHMLE



(SEQ ID NO: 1586)


 


Con4 (C)
M-Fc-


1K
GGGGGAQGSGSATGGSGSTASSGSGSATHQEECEWDPWTCE



HMLE (SEQ ID NO: 1587)


 


2xCon4
M-Fc-


(C) 1K
GGGGGAQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGS



ATHQEECEWDPWTCEHMLE (SEQ ID NO: 1588)


 


Con4-L1
MGAQEECEWDPWTCEHMGGGGGGGGKFNPLDELEETLYEQ


(N)
FTFQQGSGSATGGSGSTASSGSGSATHLEGGGGG-Fc



(SEQ ID NO: 1589)


 


Con4-L1
M-Fc-


(C)
GGGGGAQGSGSATGGSGSTASSGSGSATHKFNPLDELEETLY



EQFTFQQGGGGGQEECEWDPWTCEHMLE



(SEQ ID NO: 1590)


 


TN-12-9
MGAQ-FDYCEGVEDPFTFGCDNHLE-GGGGG-Fc


(N)
(SEQ ID NO: 1591)


 


C17 (N)
MGAQ-QYGCDGFLYGCMINLE-GGGGG-Fc



(SEQ ID NO: 1592)


 


TN8-8
MGAQ-KRPCEEMWGGCNYDLEGGGGG-Fc


(N)
(SEQ ID NO: 1593)


 


TN8-14
MGAQ-HQICKWDPWTCKHWLEGGGGG-Fc


(N)
(SEQ ID NO: 1594)


 


Con1 (N)
MGAQ-KRPCEEIFGGCTYQLEGGGGG-Fc



(SEQ ID NO: 1595)
















TABLE 38







Ang-2 Binding Peptides









Peptibody Sequence (Seq Id No:)





Con4 



Derived



Affinity-



Matured Pbs



Con4-44 (C)
M-Fc-GGGGGAQ-PIRQEECDWDPWTCEHMWEV-LE



(SEQ ID NO: 1596)


 


Con4-40 (C)
M-Fc-GGGGGAQ-TNIQEECEWDPWTCDHMPGK-LE



(SEQ ID NO: 1597)


 


Con4-4 (C)
M-Fc-GGGGGAQ-WYEQDACEWDPWTCEHMAEV-LE



(SEQ ID NO: 1598)


 


Con4-31 (C)
M-Fc-GGGGGAQ-NRLQEVCEWDPWTCEHMENV-LE



(SEQ ID NO: 1599)


 


Con4-C5 (C)
M-Fc-GGGGGAQ-AATQEECEWDPWTCEHMPRS-LE



(SEQ ID NO: 1600)


 


Con4-42 (C)
M-Fc-GGGGGAQ-LRHQEGCEWDPWTCEHMFDW-LE



(SEQ ID NO: 1602)


 


Con4-35 (C)
M-Fc-GGGGGAQ-VPRQKDCEWDPWTCEHMYVG-LE



(SEQ ID NO: 1602)


 


Con4-43 (C)
M-Fc-GGGGGAQ-SISHEECEWDPWTCEHMQVG-LE



(SEQ ID NO: 1603)


 


Con4-49 (C)
M-Fc-GGGGGAQ-WAAQEECEWDPWTCEHMGRM-LE



(SEQ ID NO: 1604)


 


Con4-27 (C)
M-Fc-GGGGGAQ-TWPQDKCEWDPWTCEHMGST-LE



(SEQ ID NO: 1605)


 


Con4-48 (C)
M-Fc-GGGGGAQ-GHSQEECGWDPWTCEHMGTS-LE



(SEQ ID NO: 1606)


 


Con4-46 (C)
M-Fc-GGGGGAQ-QHWQEECEWDPWTCDHMPSK-LE



(SEQ ID NO: 1607)


 


Con4-41 (C)
M-Fc-GGGGGAQ-NVRQEKCEWDPWTCEHMPVR-LE



(SEQ ID NO: 1608)


 


Con4-36 (C)
M-Fc-GGGGGAQ-KSGQVECNWDPWTCEHMPRN-LE



(SEQ ID NO: 1609)


 


Con4-34 (C)
M-Fc-GGGGGAQ-VKTQEHCDWDPWTCEHMREW-LE



(SEQ ID NO: 1610)


 


Con4-28 (C)
M-Fc-GGGGGAQ-AWGQEGCDWDPWTCEHMLPM-LE



(SEQ ID NO: 1611)


 


Con4-39 (C)
M-Fc-GGGGGAQ-PVNQEDCEWDPWTCEHMPPM-LE



(SEQ ID NO: 1612)


 


Con4-25 (C)
M-Fc-GGGGGAQ-RAPQEDCEWDPWTCAHMDIK-LE



(SEQ ID NO: 1613)


 


Con4-50 (C)
M-Fc-GGGGGAQ-HGQNMECEWDPWTCEHMFRY-LE



(SEQ ID NO: 1614)


 


Con4-38 (C)
M-Fc-GGGGGAQ-PRLQEECVWDPWTCEHMPLR-LE



(SEQ ID NO: 1615)


 


Con4-29 (C)
M-Fc-GGGGGAQ-RTTQEKCEWDPWTCEHMESQ-LE



(SEQ ID NO: 1616)


 


Con4-47 (C)
M-Fc-GGGGGAQ-QTSQEDCVWDPWTCDHMVSS-LE



(SEQ ID NO: 1617)


 


Con4-20 (C)
M-Fc-GGGGGAQ-QVIGRPCEWDPWTCEHLEGL-LE



(SEQ ID NO: 1618)


 


Con4-45 (C)
M-Fc-GGGGGAQ-WAQQEECAWDPWTCDHMVGL-LE



(SEQ ID NO: 1619)


 


Con4-37 (C)
M-Fc-GGGGGAQ-LPGQEDCEWDPWTCEHMVRS-LE



(SEQ ID NO: 1620)


 


Con4-33 (C)
M-Fc-GGGGGAQ-PMNQVECDWDPWTCEHMPRS-LE



(SEQ ID NO: 1621)


 


AC2-Con4
M-Fc-GGGGGAQ-FGWSHGCEWDPWTCEHMGST-LE


(C)
(SEQ ID NO: 1622)


 


Con4-32 (C)
M-Fc-GGGGGAQ-KSTQDDCDWDPWTCEHMVGP-LE



(SEQ ID NO: 1623)


 


Con4-17 (C)
M-Fc-GGGGGAQ-GPRISTCQWDPWTCEHMDQL-LE



(SEQ ID NO: 1624)


 


Con4-8 (C)
M-Fc-GGGGGAQ-STIGDMCEWDPWTCAHMQVD-LE



(SEQ ID NO: 1625)


 


AC4-Con4
M-Fc-GGGGGAQ-VLGGQGCEWDPWTCRLLQGW-LE


(C)
(SEQ ID NO: 1626)


 


Con4-1 (C)
M-Fc-GGGGGAQ-VLGGQGCQWDPWTCSHLEDG-LE



(SEQ ID NO: 1627)


 


Con4-C1 (C)
M-Fc-GGGGGAQ-TTIGSMCEWDPWTCAHMQGG-LE



(SEQ ID NO: 1628)


 


Con4-21 (C)
M-Fc-GGGGGAQ-TKGKSVCQWDPWTCSHMQSG-LE



(SEQ ID NO: 1629)


 


Con4-C2 (C)
M-Fc-GGGGGAQ-TTIGSMCQWDPWTCAHMQGG-LE



(SEQ ID NO: 1630)


 


Con4-18 (C)
M-Fc-GGGGGAQ-WVNEVVCEWDPWTCNHWDTP-LE



(SEQ ID NO: 1631)


 


Con4-19 (C)
M-Fc-GGGGGAQ-VVQVGMCQWDPWTCKHMRLQ-LE



(SEQ ID NO: 1632)


 


Con4-16 (C)
M-Fc-GGGGGAQ-AVGSQTCEWDPWTCAHLVEV-LE



(SEQ ID NO: 1633)


 


Con4-11 (C)
M-Fc-GGGGGAQ-QGMKMFCEWDPWTCAHIVYR-LE



(SEQ ID NO: 1634)


 


Con4-C4 (C)
M-Fc-GGGGGAQ-TTIGSMCQWDPWTCEHMQGG-LE



(SEQ ID NO: 1635)


 


Con4-23 (C)
M-Fc-GGGGGAQ-TSQRVGCEWDPWTCQHLTYT-LE



(SEQ ID NO: 1636)


 


Con4-15 (C)
M-Fc-GGGGGAQ-QWSWPPCEWDPWTCQTVWPS-LE



(SEQ ID NO: 1637)


 


Con4-9 (C)
M-Fc-GGGGGAQ-GTSPSFCQWDPWTCSHMVQG-LE



(SEQ ID NO: 1638)


 


Con4-10 (C)
M-Fc-GGGGGAQ-TQGLHQCEWDPWTCKVLWPS-LE



(SEQ ID NO: 1639)


 


Con4-22 (C)
M-Fc-GGGGGAQ-VWRSQVCQWDPWTCNLGGDW-LE



(SEQ ID NO: 1640)


 


Con4-3 (C)
M-Fc-GGGGGAQ-DKILEECQWDPWTCQFFYGA-LE



(SEQ ID NO: 1641)


 


Con4-5 (C)
M-Fc-GGGGGAQ-ATFARQCQWDPWTCALGGNW-LE



(SEQ ID NO: 1642)


 


Con4-30 (C)
M-Fc-GGGGOAQ-GPAQEECEWDPWTCEPLPLM-LE



(SEQ ID NO: 1643)


 


Con4-26 (C)
M-Fc-GGGGGAQ-RPEDMCSQWDPWTWHLQGYC-LE



(SEQ ID NO: 1644)


 


Con4-7 (C)
M-Fc-GGGGGAQ-LWQLAVCQWDPQTCDHMGAL-LE



(SEQ ID NO: 1645)


 


Con4-12 (C)
M-Fc-GGGGGAQ-TQLVSLCEWDPWTCRLLDGW-LE



(SEQ ID NO: 1646)


 


Con4-13 (C)
M-Fc-GGGGGAQ-MGGAGRCEWDPWTCQLLQGW-LE



(SEQ ID NO: 1647)


 


Con4-14 (C)
M-Fc-GGGGGAQ-MFLPNECQWDPWTCSNLPEA-LE



(SEQ ID NO: 1648)


 


Con4-2 (C)
M-Fc-GGGGGAQ-FGWSHGCEWDPWTCRLLQGW-LE



(SEQ ID NO: 1649)


 


Con4-6 (C)
M-Fc-GGGGGAQ-WPQTEGCQWDPWTCRLLHGW-LE



(SEQ ID NO: 1650)


 


Con4-24 (C)
M-Fc-GGGGGAQ-PDTRQGCQWDPWTCRLYGMW-LE



(SEQ ID NO: 1651)


 


AC1-Con4
M-Fc-GGGGGAQ-TWPQDKCEWDPWTCRLLQGW-LE


(C)
(SEQ ID NO: 1652)


 


AC3-Con4
M-Fc-GGGGGAQ-DKILEECEWDPWTCRLLQGW-LE


(C)
(SEQ ID NO: 1653)


 


AC5-Con4
M-Fc-GGGGGAQ-AATQEECEWDPWTCRLLQGW-LE


(C)
(SEQ ID NO: 1654)


 


L1 Derived



Affinity-



Matured Pbs



L1-7 (N)
MGAQ-TNFMPMDDLEQRLYEQFILQQG-LEGGGGG-Fc



(SEQ ID NO: 1655)


 


AC6-L1 (N)
MGAQ-TNYKPLDELDATLYEHWILQHS LEGGGGG-Fc



(SEQ ID NO: 1656)


 


L1-15 (N)
MGAQ-QKYQPLDELDKTLYDQFMLQQG LEGGGGG-Fc



(SEQ ID NO: 1657)


 


L1-2 (N)
MGAQ-LNFTPLDELEQTLYEQWTLQQS LEGGGGG-Fc



(SEQ ID NO: 1658)


 


L1-10 (N)
MGAQ-QKFQPLDELEQTLYEQFMLQQA LEGGGGG-Fc



(SEQ ID NO: 1659)


 


L1-13 (N)
MGAQ-QEYEPLDELDETLYNQWMFHQR LEGGGGG-Fc



(SEQ ID NO: 1660)


 


L1-5 (N)
MGAQ-VKYKPLDELDEILYEQQTFQER LEGGGGG-Fc



(SEQ ID NO: 1661)


 


L1-C2 (N)
MGAQ-TKFQPLDELDQTLYEQWTLQQR LEGGGGG-Fc



(SEQ ID NO: 1662)


 


L1-C3 (N)
MGAQ-TNFQPLDELDQTLYEQWTLQQR LEGGGGG-Fc



(SEQ ID NO: 1663)


 


L1-11 (N)
MGAQ-QNFKPMDELEDTLYKQFLFQHS LEGGGGG-Fc



(SEQ ID NO: 1664)


 


L1-17 (N)
MGAQ-VKYKPLDELDEWLYHQFTLHHQ LEGGGGG-Fc



(SEQ ID NO: 1665)


 


L1-12 (N)
MGAQ-YKFTPLDDLEQTLYEQWTLQHV LEGGGGG-Fc



(SEQ ID NO: 1666)


 


L1-1 (N)
MGAQ-QNYKPLDELDATLYEHFIFHYT LEGGGGG-Fc



(SEQ ID NO: 1667)


 


L1-4 (N)
MGAQ-VKFKPLDALEQTLYEHWMFQQA LEGGGGG-Fc



(SEQ ID NO: 1668)


 


L1-20 (N)
MGAQ-EDYMPLDALDAQLYEQFILLHG LEGGGGG-Fc



(SEQ ID NO: 1669)


 


L1-22 (N)
MGAQ-YKFNPMDELEQTLYEEFLFQHA LEGGGGG-Fc



(SEQ ID NO: 1670)


 


L1-14 (N)
MGAQ-SNFMPLDELEQTLYEQFMLQHQ LEGGGGG-Fc



(SEQ ID NO: 1671)


 


L1-16 (N)
MGAQ-QKFQPLDELEETLYKQWTLQQR LEGGGGG-Fc



(SEQ ID NO: 1672)


 


L1-18 (N)
MGAQ-QKFMPLDELDEILYEQFMFQQS LEGGGGG-Fc



(SEQ ID NO: 1673)


 


L1-3 (N)
MGAQ-TKFNPLDELEQTLYEQWTLQHQ LEGGGGG-Fc



(SEQ ID NO: 1674)


 


L1-21 (N)
MGAQ-HTFQPLDELEETLYYQWLYDQL LEGGGGG-Fc



(SEQ ID NO: 1675)


 


L1-C1 (N)
MGAQ-QKFKPLDELEQTLYEQWTLQQR LEGGGGG-Fc



(SEQ ID NO: 1676)


 


L1-19 (N)
MGAQ-QTFQPLDDLEEYLYEQWIRRYH LEGGGGG-Fc



(SEQ ID NO: 1677)


 


L1-9 (N)
MGAQ-SKFKPLDELEQTLYEQWTLQHA LEGGGGG-Fc



(SEQ ID NO: 1678)


 


Con1



Derived



Affinity-



Matured Pbs



Con1-4 (C)
M-Fc-GGGGGAQ-SGQLRPCEEIFGCGTQNLAL-LE



(SEQ ID NO: 1679)


 


Con1-1 (C)
M-Fc-GGGGGAQ-AGGMRPYDGMLGWPNYDVQA-LE



(SEQ ID NO: 1680)


 


Con1-6 (C)
M-Fc-GGGGGAQ-GQDLRPCEDMFGCGTKDWYG-LE



(SEQ ID NO: 1681)


 


Con1-3 (C)
M-Fc-GGGGGAQ-APGQRPYDGMLGWPTYQRIV-LE



(SEQ ID NO: 1682)


 


Con1-2 (C)
M-Fc-GGGGGAQ-QTWDDPCMHILGPVTWRRCI-LE



(SEQ ID NO: 1683)


 


Con1-5 (C)
M-Fc-GGGGGAQ-FGDKRPLECMFGGPIQLCPR-LE



(SEQ ID NO: 1684)


 


Parent:
M-Fc-GGGGGAQ-KRPCEEIFGGCTYQ-LE


Con1 (C)
(SEQ ID NO: 1685)


 


12-9



Derived



Affinity-



Matured Pbs



12-9-3 (C)
M-Fc-GGGGGAQ-LQEWCEGVEDPFTFGCEKQR-LE



(SEQ ID NO: 1686)


 


12-9-7 (C)
M-Fc-GGGGGAQ-MLDYCEGMDDPFTFGCDKQM-LE



(SEQ ID NO: 1687)


 


12-9-6 (C)
M-Fc-GGGGGAQ-HQEYCEGMEDPFTFGCEYQG-LE



(SEQ ID NO: 1688)


 


12-9-C2 (C)
M-Fc-GGGGGAQ-LQDYCEGVEDPFTFGCENQR-LE



(SEQ ID NO: 1689)


 


12-9-5 (C)
M-Fc-GGGGGAQ-LLDYCEGVQDPFTFGCENLD-LE



(SEQ ID NO: 1690)


 


12-9-1 (C)
M-Fc-GGGGGAQ-GFEYCDGMEDPFTFGCDKQT-LE



(SEQ ID NO: 1691)


 


12-9-4 (C)
M-Fc-GGGGGAQ-AQDYCEGMEDPFTFGCEMQK-LE



(SEQ ID NO: 1692)


 


12-9-C1 (C)
M-Fc-GGGGGAQ-LQDYCEGVEDPFTFGCEKQR-LE



(SEQ ID NO: 1693)


 


12-9-2 (C)
M-Fc-GGGGGAQ-KLEYCDGMEDPFTQGCDNQS-LE



(SEQ ID NO: 1694)


 


Parent:
M-Fc-GGGGGAQ-FDYCEGVEDPFTFGCDNH-LE


12-9 (C)
(SEQ ID NO: 1695)









In addition to the TMP compounds set out in Table 6, the invention provides numerous other TMP compounds. In one aspect, TMP compounds comprise the following general structure:

TMP1-(L1)n-TMP2 

wherein TMP1 and TMP2 are each independently selected from the group of compounds comprising the core structure:

X2-X3-X4-X5-X6-X7-X8-X9-X10,

wherein,

X2 is selected from the group consisting of Glu, Asp, Lys, and Val;
X3 is selected from the group consisting of Gly and Ala;
X4 is Pro;
X5 is selected from the group consisting of Thr and Ser;
X6 is selected from the group consisting of Leu, Ile, Val, Ala, and Phe;
X7 is selected from the group consisting of Arg and Lys;
X8 is selected from the group consisting of Gln, Asn, and Glu;
X9 is selected from the group consisting of Trp, Tyr, and Phe;
X10 is selected from the group consisting of Leu, Ile, Val, Ala, Phe, Met, and Lys;
L1 is a linker as described herein; and
n is 0 or 1;
and physiologically acceptable salts thereof.
In one embodiment, L1 comprises (Gly)n, wherein n is 1 through 20, and when n is greater than 1, up to half of the Gly residues may be substituted by another amino acid selected from the remaining 19 natural amino acids or a stereoisomer thereof.
In addition to the core structure X2-X10 set forth above for TMP1 and TMP2, other related structures are also possible wherein one or more of the following is added to the TMP1 and/or TMP2 core structure: X1 is attached to the N-terminus and/or X11, X12, X13, and/or X14 are attached to the C-terminus, wherein X1, X12, X13, and X14 are as follows:
X1 is selected from the group consisting of Ile, Ala, Val, Leu, Ser, and Arg;
X11 is selected from the group consisting of Ala, Ile, Val, Leu, Phe, Ser, Thr, Lys, His, and Glu;
X12 is selected from the group consisting of Ala, Ile, Val, Leu, Phe, Gly, Ser, and Gln;
X13 is selected from the group consisting of Arg, Lys, Thr, Val, Asn, Gln, and Gly; and
X14 is selected from the group consisting of Ala, Ile, Val, Leu, Phe, Thr, Arg, Glu, and Gly.
TMP compounds of the invention are made up of, i.e., comprising, at least 9 subunits (X2-X10), wherein X2-X10 comprise the core structure. The X2-X14 subunits are amino acids independently selected from among the 20 naturally-occurring amino acids, however, the invention embraces compounds where X2-X14 are independently selected from the group of atypical, non-naturally occurring amino acids well known in the art. Specific amino acids are identified for each position. For example, X2 may be Glu, Asp, Lys, or Val. Both three-letter and single letter abbreviations for amino acids are used herein; in each case, the abbreviations are the standard ones used for the 20 naturally-occurring amino acids or well-known variations thereof. These amino acids may have either L or D stereochemistry (except for Gly, which is neither L nor D), and the TMPs (as well as all other compounds of the invention) may comprise a combination of stereochemistries. The invention also provides reverse TMP molecules (as well as for all other peptides disclosed herein) wherein the amino terminal to carboxy terminal sequence of the amino acids is reversed. For example, the reverse of a molecule having the normal sequence X1-X2-X3 would be X3-X2-X1. The invention also provides retro-reverse TMP molecules (as well as for all other molecules of the invention described herein) wherein, like a reverse TMP, the amino terminal to carboxy terminal sequence of amino acids is reversed and residues that are normally “L” enantiomers in TMP are altered to the “D” stereoisomer form.
Exemplary TMP compounds of the invention therefore include without limitation the following compounds.













IEGPTLRQWLAARA-GPNG-IEGPTLRQWLAARA
(SEQ. ID NO: 993)


 






(SEQ. ID NO: 994)


 


IEGPTLRQCLAARA-GGGGGGGG-IEGPTLRQCLAARA (linear)
(SEQ. ID NO: 995)


IEGPTLRQALAARA-GGGGGGGG-IEGPTLRQALAARA
(SEQ. ID NO: 996)


IEGPTLRQWLAARA-GGGKGGGG-IEGPTLRQWLAARA
(SEQ. ID NO: 997)


IEGPTLRQWLAARA-GGGK(BrAC)GGGG-IEGPTLRQWLAARA
(SEQ. ID NO: 998)


IEGPTLRQWLAARA-GGGCGGGG-IEGPTLRQWLAARA
(SEQ. ID NO: 999)


IEGPTLRQWLAARA-GGGK(PEG)GGGG-IEGPTLRQWLAARA
(SEQ. ID NO: 1000)


IEGPTLRQWLAARA-GGGC(PEG)GGGG-IEGPTLRQWLAARA
(SEQ. ID NO: 1001)


IEGPTLRQWLAARA-GGGNGSGG-IEGPTLRQWLAARA
(SEQ. ID NO: 1002)


 






(SEQ. ID NO: 1003)


 


IEGPTLRQWLAARA-GGGGGGGG-IEGPTLRQWLAARA
(SEQ. ID NO: 1004)


Fc-IEGPTLRQWLAARA-GPNG-IEGPTLRQWLAARA
(SEQ. ID NO: 1005)


Fc-IEGPTLRQWLAARA-GPNG-IEGPTLRQWLAARA-Fc
(SEQ. ID NO: 1006)


IEGPTLRQWLAARA-GGGGGGGG-IEGPTLRQWLAARA-Fc
(SEQ. ID NO: 1007)


Fc-GG-IEGPTLRQWLAARA-GPNG-IEGPTLRQWLAARA
(SEQ. ID NO: 1008)


Fc-IEGPTLRQWLAARA-GGGGGGGG-IEGPTLRQWLAARA
(SEQ. ID NO: 1009)


 






(SEQ. ID NO: 1010)


 


Fc-IEGPTLRQCLAARA-GGGGGGGG-IEGPTLRQCLAARA (linear)
(SEQ. ID NO: 1011)


Fc-IEGPTLRQALAARA-GGGGGGGG-IEGPTLRQALAARA
(SEQ. ID NO: 1012)


FC-IEGPTLRQWLAARA-GGGKGGGG-IEGPTLRQWLAARA
(SEQ. ID NO: 1013)


Fc-IEGPTLRQWLAARA-GGGCGGGG-IEGPTLRQWLAARA
(SEQ. ID NO: 1014)


Fc-IEGPTLRQWLAARA-GGGNGSGG-IEGPTLRQWLAARA
(SEQ. ID NO: 1015)


 






(SEQ. ID NO: 1016)


 


Fc-GGGGG-IEGPTLRQWLAARA-GGGGGGGG-IEGPTLRQWLAARA
(SEQ. ID NO: 1017)









The present invention is also particularly useful with peptides having activity in treatment of:
cancer, wherein the peptide is a VEGF-mimetic or a VEGF receptor antagonist, a HER2 agonist or antagonist, a CD20 antagonist and the like;
asthma, wherein the protein of interest is a CKR3 antagonist, an IL-5 receptor antagonist, and the like;
thrombosis, wherein the protein of interest is a GPIIb antagonist, a GPIIIa antagonist, and the like;
autoimmune diseases and other conditions involving immune modulation, wherein the protein of interest is an IL-2 receptor antagonist, a CD40 agonist or antagonist, a CD40L agonist or antagonist, a thymopoietin mimetic and the like.
Derivatives. The invention also contemplates derivatizing the peptide and/or vehicle portion (as discussed below) of the compounds. Such derivatives may improve the solubility, absorption, biological half life, and the like of the compounds. The moieties may alternatively eliminate or attenuate any undesirable side-effect of the compounds and the like. 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 non-peptidyl linkages are —CH2-carbamate [—CH2-OC(O)NR—], phosphonate, —CH2-sulfonamide [—CH2-S(O)2NR—], urea [—NHC(O)NH—], —CH2-secondary amine, and alkylated peptide [—C(O)NR6- wherein R6 is lower alkyl].
4. The N-terminus is derivatized. Typically, the N-terminus may be acylated or modified to a substituted amine. Exemplary N-terminal derivative groups include —NRR1 (other than —NH2), —NRC(O)R1, —NRC(O)OR1, —NRS(O)2R1, —NHC(O)NHR1, succinimide, or benzyloxycarbonyl-NH— (CBZ-NH—), wherein R and R1 are each independently hydrogen or lower alkyl and wherein the phenyl ring may be substituted with 1 to 3 substituents selected from the group consisting of C1-C4 alkyl, C1-C4 alkoxy, chloro, and bromo.
5. The free C-terminus is derivatized. Typically, the C-terminus is esterified or amidated. For example, one may use methods described in the art to add (NH—CH2-CH2-NH2)2 to compounds of this invention. Likewise, one may use methods described in the art to add —NH2 to compounds of this invention. Exemplary C-terminal derivative groups include, for example, —C(O)R2 wherein R2 is lower alkoxy or —NR3R4 wherein R3 and R4 are independently hydrogen or C1-C8 alkyl (preferably C1-C4 alkyl).
6. A disulfide bond is replaced with another, preferably more stable, cross-linking moiety (e.g., an alkylene). See, e.g., Bhatnagar et al. (1996), J. Med. Chem. 39: 3814-9; Alberts et al. (1993) Thirteenth Am. Pep. Symp., 357-9.
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-amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate.
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 1-cyclohexyl-3-(2-morpholinyl-(4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore, aspartyl and glutamyl residues may be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
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), J. 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., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate), and bifunctional maleimides such as bis-N-maleimido-1,8-octane. Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light. Alternatively, reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are employed for protein immobilization.
Carbohydrate (oligosaccharide) groups may conveniently be attached to sites that are known to be glycosylation sites in proteins. Generally, O-linked oligosaccharides are attached to serine (Ser) or threonine (Thr) residues while N-linked oligosaccharides are attached to asparagine (Asn) residues when they are part of the sequence Asn-X-Ser/Thr, where X can be any amino acid except proline. X is preferably one of the 19 naturally occurring amino acids other than proline. The structures of N-linked and O-linked oligosaccharides and the sugar residues found in each type are different. One type of sugar that is commonly found on both is N-acetylneuraminic acid (referred to as sialic acid). Sialic acid is usually the terminal residue of both N-linked and O-linked oligosaccharides and, by virtue of its negative charge, may confer acidic properties to the glycosylated compound. Such site(s) may be incorporated in the linker of the compounds of this invention and are preferably glycosylated by a cell during recombinant production of the polypeptide compounds (e.g., in mammalian cells such as CHO, BHK, COS). However, such sites may further be glycosylated by synthetic or semi-synthetic procedures known in the art.
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).
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 molecules may be used as therapeutic agents or as an aid to surgery (e.g., radioimmunoguided surgery or RIGS) or as diagnostic agents (e.g., radioimmunodiagnostics or RID).
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.
Vehicles. The invention requires the presence of at least one vehicle attached to a peptide through the N-terminus, C-terminus or a sidechain of one of the amino acid residues. Multiple vehicles may also be used. In one aspect, an Fc domain is the vehicle. The Fc domain may be fused to the N or C termini of the peptides or at both the N and C termini.
In various embodiments of the invention, the Fc component is either a native Fc or an Fc variant. The immunoglobulin source of the native Fc is, in one aspect, of human origin and may, in alternative embodiments, be of any class of immunoglobulin. Native Fc domains are made up of monomeric polypeptides that may be linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and/or non-covalent association. The number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from one to four depending on class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2). One example of a native Fc is a disulfide-bonded dimer resulting from papain digestion of an IgG (see Ellison et al. (1982), Nucleic Acids Res. 10: 4071-9).
It should be noted that Fc monomers will spontaneously dimerize when the appropriate cysteine residues are present, unless particular conditions are present that prevent dimerization through disulfide bond formation. Even if the cysteine residues that normally form disulfide bonds in the Fc dimer are removed or replaced by other residues, the monomeric chains will generally form a dimer through non-covalent interactions. The term “Fc” herein is used to mean any of these forms: the native monomer, the native dimer (disulfide bond linked), modified dimers (disulfide and/or non-covalently linked), and modified monomers (i.e., derivatives).
As noted, Fc variants are suitable vehicles within the scope of this invention. A native Fc may be extensively modified to form an Fc variant, provided binding to the salvage receptor is maintained; see, for example WO 97/34631 and WO 96/32478. In such Fc variants, one may remove one or more sites of a native Fc that provide structural features or functional activity not required by the fusion molecules of this invention. One may remove these sites by, for example, substituting or deleting residues, inserting residues into the site, or truncating portions containing the site. The inserted or substituted residues may also be altered amino acids, such as peptidomimetics or D-amino acids. Fc variants may be desirable for a number of reasons, several of which are described below. Exemplary Fc variants include molecules and sequences in which:
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). 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. 
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 C1q binding site, are removed. For example, one may delete or substitute the EKK sequence of human IgG1. Complement recruitment may not be advantageous for the molecules of this invention and so may be avoided with such an Fc variant.
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 IgG1. 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.
An alternative vehicle would be a protein, polypeptide, peptide, antibody, antibody fragment, or small molecule (e.g., a peptidomimetic compound) capable of binding to a salvage receptor. For example, one could use as a vehicle a polypeptide as described in U.S. Pat. No. 5,739,277, issued Apr. 14, 1998 to Presta et al. Peptides could also be selected by phage display for binding to the FcRn salvage receptor. Such salvage receptor-binding compounds are also included within the meaning of “vehicle” and are within the scope of this invention. Such vehicles should be selected for increased half-life (e.g., by avoiding sequences recognized by proteases) and decreased immunogenicity (e.g., by favoring non-immunogenic sequences, as discovered in antibody humanization).
Variants, analogs or derivatives of the Fc portion may be constructed by, for example, making various substitutions of residues or sequences.
Variant (or analog) polypeptides include insertion variants, wherein one or more amino acid residues supplement an Fc amino acid sequence. Insertions may be located at either or both termini of the protein, or may be positioned within internal regions of the Fc amino acid sequence. Insertion variants, with additional residues at either or both termini, can include for example, fusion proteins and proteins including amino acid tags or labels. For example, the Fc molecule may optionally contain an N-terminal Met, especially when the molecule is expressed recombinantly in a bacterial cell such as E. coli. 
In Fc deletion variants, one or more amino acid residues in an Fc polypeptide are removed. Deletions can be effected at one or both termini of the Fc polypeptide, or with removal of one or more residues within the Fc amino acid sequence. Deletion variants, therefore, include all fragments of an Fc polypeptide sequence.
In Fc substitution variants, one or more amino acid residues of an Fc polypeptide are removed and replaced with alternative residues. In one aspect, the substitutions are conservative in nature and conservative substitutions of this type are well known in the art. Alternatively, the invention embraces substitutions that are also non-conservative.
For example, cysteine residues can be deleted or replaced with other amino acids to prevent formation of some or all disulfide crosslinks of the Fc sequences. Each cysteine residue can be removed and/or substituted with other amino acids, such as Ala or Ser. As another example, modifications may also be made to introduce amino acid substitutions to (1) ablate the Fc receptor binding site; (2) ablate the complement (C1q) binding site; and/or to (3) ablate the antibody dependent cell-mediated cytotoxicity (ADCC) site. Such sites are known in the art, and any known substitutions are within the scope of Fc as used herein. For example, see Molecular Immunology, Vol. 29, No. 5, 633-639 (1992) with regard to ADCC sites in IgG1.
Likewise, one or more tyrosine residues can be replaced by phenylalanine residues. In addition, other variant amino acid insertions, deletions and/or substitutions are also contemplated and are within the scope of the present invention. Conservative amino acid substitutions will generally be preferred. Furthermore, alterations may be in the form of altered amino acids, such as peptidomimetics or D-amino acids.
Fc sequences of the compound may also be derivatized as described herein for peptides, i.e., bearing modifications other than insertion, deletion, or substitution 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, or may be designed to improve targeting capacity for the polypeptide to desired cells, tissues, or organs.
It is also possible to use the salvage receptor binding domain of the intact Fc molecule as the Fc part of a compound of the invention, such as described in WO 96/32478, entitled “Altered Polypeptides with Increased Half-Life.” Additional members of the class of molecules designated as Fc herein are those that are described in WO 97/34631, entitled “Immunoglobulin-Like Domains with Increased Half-Lives.” Both of the published PCT applications cited in this paragraph are hereby incorporated by reference.
WSP components. Compounds of the invention further include at least one WSP. The WPS moiety of the molecule may be branched or unbranched. For therapeutic use of the end-product preparation, the polymer is pharmaceutically acceptable. In general, a desired polymer is selected based on such considerations as whether the polymer conjugate will be used therapeutically, and if so, the desired dosage, circulation time, resistance to proteolysis, and other considerations. In various aspects, the average molecular weight of each water soluble polymer is between about 2 kDa and about 100 kDa, between about 5 kDa and about 50 kDa, between about 12 kDa and about 40 kDa and between about 20 kDa and about 35 kDa. In yet another aspect the molecular weight of each polymer is between about 6 kDa and about 25 kDa. The term “about” as used herein and throughout, indicates that in preparations of a water soluble polymer, some molecules will weigh more, some less, than the stated molecular weight. Generally, the higher the molecular weight or the more branches, the higher the polymer/protein ratio. Other sizes may be used, depending on the desired therapeutic profile including for example, the duration of sustained release; the effects, if any, on biological activity; the ease in handling; the degree or lack of antigenicity and other known effects of a water soluble polymer on a therapeutic protein.
The WSP should be attached to a peptide or protein with consideration given to effects on functional or antigenic domains of the peptide or protein. In general, chemical derivatization may be performed under any suitable condition used to react a protein with an activated polymer molecule. Activating groups which can be used to link the water soluble polymer to one or more proteins include without limitation sulfone, maleimide, sulfhydryl, thiol, triflate, tresylate, azidirine, oxirane and 5-pyridyl. If attached to the peptide by reductive alkylation, the polymer selected should have a single reactive aldehyde so that the degree of polymerization is controlled.
Suitable, clinically acceptable, water soluble polymers include without limitation, PEG, polyethylene glycol propionaldehyde, copolymers of ethylene glycol/propylene glycol, monomethoxy-polyethylene glycol, carboxymethylcellulose, polyacetals, polyvinyl alcohol (PVA), polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, poly (.beta.-amino acids) (either homopolymers or random copolymers), poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers (PPG) and other polyakylene oxides, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (POG) (e.g., glycerol) and other polyoxyethylated polyols, polyoxyethylated sorbitol, or polyoxyethylated glucose, colonic acids or other carbohydrate polymers, Ficoll or dextran and mixtures thereof.
Polysaccharide polymers are another type of water soluble polymer which may be used for protein modification. Dextrans are polysaccharide polymers comprised of individual subunits of glucose predominantly linked by α1-6 linkages. The dextran itself is available in many molecular weight ranges, and is readily available in molecular weights from about 1 kD to about 70 kD. Dextran is a suitable water soluble polymer for use in the present invention as a vehicle by itself or in combination with another vehicle (e.g., Fc). See, for example, WO 96/11953 and WO 96/05309. The use of dextran conjugated to therapeutic or diagnostic immunoglobulins has been reported; see, for example, European Patent Publication No. 0 315 456, which is hereby incorporated by reference. Dextran of about 1 kD to about 20 kD is preferred when dextran is used as a vehicle in accordance with the present invention.
In one embodiment, the WSP is PEG and the invention contemplates preparations wherein a compound is modified to include any of the forms of PEG that have been used to derivatize other proteins, such as and without limitation mono-(C1-C10) alkoxy- or aryloxy-polyethylene glycol. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The PEG group may be of any convenient molecular weight and may be linear or branched. The average molecular weight of PEG contemplated for use in the invention ranges from about 2 kDa to about 100 kDa, from about 5 kDa to about 50 kDa, from about 5 kDa to about 10 kDa. In another aspect, the PEG moiety has a molecular weight from about 6 kDa to about 25 kDa. PEG groups generally are attached to peptides or proteins via acylation or reductive alkylation through a reactive group on the PEG moiety (e.g., an aldehyde, amino, thiol, or ester group) to a reactive group on the target peptide or protein (e.g., an aldehyde, amino, or ester group). Using methods described herein, a mixture of polymer/peptide conjugate molecules can be prepared, and the advantage provided herein is the ability to select the proportion of polymer/peptide conjugate to include in the mixture. Thus, if desired, a mixture of peptides with various numbers of polymer moieties attached (i.e., zero, one or two) can be prepared with a predetermined proportion of polymer/protein conjugate.
A useful strategy for the PEGylation (other methods are discussed in more detail herein) 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. 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.
Linkers. Any “linker” group is optional, whether positioned between peptides, peptide and vehicle or vehicle and WSP. When present, its chemical structure is not critical, since it serves primarily as a spacer. The linker is preferably made up of amino acids linked together by peptide bonds. Thus, in preferred embodiments, the linker is made up of from 1 to 20 amino acids linked by peptide bonds, wherein the amino acids are selected from the 20 naturally occurring amino acids. Some of these amino acids may be glycosylated, as is well understood by those in the art. In a more preferred embodiment, the 1 to 20 amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. Even more preferably, a linker is made up of a majority of amino acids that are sterically unhindered, such as glycine and alanine. Thus, preferred linkers are polyglycines (particularly (Gly)4, (Gly)5), poly(Gly-Ala), and polyalanines. Other specific examples of linkers are:










(Gly)3Lys(GLy)4
(SEQ ID NO: 1018);


 



(Gly)3AsnGlySer(Gly)2
(SEQ ID NO: 1019);


 



(Gly)3Cys(Gly)4
(SEQ ID NO: 1020);


 



and



 



GlyProAsnGlyGly
(SEQ ID NO: 1021).






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., C1-C6) lower acyl, halogen (e.g., Cl, Br), CN, NH2, phenyl, etc. An exemplary non-peptide 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.
Peptide production. A peptide having been identified may be made in transformed host cells using recombinant DNA techniques. If the vehicle component is a polypeptide, the peptide-vehicle fusion product may be expressed as one. To do so, a recombinant DNA molecule encoding the peptide is first prepared using methods 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 therefore provides polynucleotides encoding a compound of the invention.
The invention also provides vectors encoding compounds of the invention in an appropriate host. The vector comprises the polynucleotide that encodes the compound operatively linked to appropriate expression control sequences. Methods of effecting this operative linking, either before or after the polynucleotide 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 polynucleotide therein 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, bio-safety and costs. A balance of these factors must be struck with the understanding that not all hosts may be equally effective for the expression of a particular DNA sequence. Within these general guidelines, useful microbial hosts include bacteria (such as E. coli), yeast (such as Saccharomyces) 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.
Depending on the host cell utilized to express a compound of the invention, carbohydrate (oligosaccharide) groups may conveniently be attached to sites that are known to be glycosylation sites in proteins. Generally, O-linked oligosaccharides are attached to serine (Ser) or threonine (Thr) residues while N-linked oligosaccharides are attached to asparagine (Asn) residues when they are part of the sequence Asn-X-Ser/Thr, where X can be any amino acid except proline. X is preferably one of the 19 naturally occurring amino acids not counting 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.
Alternatively, the compounds may be made by synthetic methods. For example, solid phase synthesis techniques may be used. Suitable techniques are well known in the art, and include those described in Merrifield (1973), Chem. Polypeptides, pp. 335-61 (Katsoyannis and Panayotis eds.); Merrifield (1963), J. Am. Chem. Soc. 85: 2149; Davis et al. (1985), Biochem. Intl. 10: 394-414; Stewart and Young (1969), Solid Phase Peptide Synthesis; U.S. Pat. No. 3,941,763; Finn et al. (1976), The Proteins (3rd ed.) 2: 105-253; and Erickson et al. (1976), The Proteins (3rd ed.) 2: 257-527. Solid phase synthesis is the preferred technique of making individual peptides since it is the most cost-effective method of making small peptides.
Compounds that contain derivatized peptides or which contain non-peptide groups are particularly amendable to synthesis by well-known organic chemistry techniques.
WSP modification. For obtaining a compound covalently attached to a WSP, any method described herein or otherwise known in the art is employed. Methods for preparing chemical derivatives of polypeptides will generally comprise the steps of (a) reacting the polypeptide with the activated polymer molecule (such as a reactive ester or aldehyde derivative of the polymer molecule) under conditions whereby the polypeptide becomes attached to one or more polymer molecules, and (b) obtaining the reaction product(s). The optimal reaction conditions will be determined based on known parameters and the desired result. For example, the larger the ratio of polymer molecules:protein, the greater the percentage of attached polymer molecule.
A biologically active molecule can be linked to a polymer through any available functional group using standard methods well known in the art. Examples of functional groups on either the polymer or biologically active molecule which can be used to form such linkages include amine and carboxy groups, thiol groups such as in cysteine resides, aldehydes and ketones, and hydroxy groups as can be found in serine, threonine, tyrosine, hydroxyproline and hydroxylysine residues.
The polymer can be activated by coupling a reactive group such as trichloro-s-triazine [Abuchowski, et al., (1977), J. Biol. Chem. 252:3582-3586, incorporated herein by reference in its entirety], carbonylimidazole [Beauchamp, et al., (1983), Anal. Biochem. 131:25-33, incorporated herein by reference in its entirety], or succinimidyl succinate [Abuchowski, et al., (1984), Cancer Biochem. Biophys. 7:175-186, incorporated herein by reference in its entirety] in order to react with an amine functionality on the biologically active molecule. Another coupling method involves formation of a glyoxylyl group on one molecule and an aminooxy, hydrazide or semicarbazide group on the other molecule to be conjugated [Fields and Dixon, (1968), Biochem. J. 108:883-887; Gaertner, et al., (1992), Bioconjugate Chem. 3:262-268; Geoghegan and Stroh, (1992), Bioconjugate Chem. 3:138-146; Gaertner, et al., (1994), J. Biol. Chem. 269:7224-7230, each of which is incorporated herein by reference in its entirety]. Other methods involve formation of an active ester at a free alcohol group of the first molecule to be conjugated using chloroformate or disuccinimidylcarbonate, which can then be conjugated to an amine group on the other molecule to be coupled [Veronese, et al., (1985), Biochem. and Biotech. 11:141-152; Nitecki, et al., U.S. Pat. No. 5,089,261; Nitecki, U.S. Pat. No. 5,281,698, each of which is incorporated herein by reference in its entirety]. Other reactive groups which may be attached via free alcohol groups are set forth in Wright, EP 0539167A2 (incorporated herein by reference in its entirety), which also describes the use of imidates for coupling via free amine groups.
Another chemistry involves acylation of the primary amines of a target using the NHS-ester of methoxy-PEG (O-[(N-succinimidyloxycarbonyl)-methyl]-O′-methylpolyethylene glycol). Acylation with methoxy-PEG-NHS results in an amide linkage which will eliminate the charge from the original primary amine. Other methods utilize mild oxidation of a target under conditions selected to target the pendant diol of the penultimate glycosyl unit sialic acid for oxidation to an aldehyde. The resultant glycoaldehyde was then reacted with a methoxy-PEG-hydrazide (O-(Hydrazinocarbonylmethyl)-O′-methylpolyethylene glycol) to form a semi-stable hydrazone between PEG and target. The hydrazone is subsequently reduced by sodium cyanoborohydride to produce a stable PEG conjugate. See for example, U.S. Pat. No. 6,586,398 (Kinstler, et al., Jul. 1, 2003), incorporated herein by reference in its entirety.
In specific applications of techniques for chemical modification, for example, U.S. Pat. No. 4,002,531 (incorporated herein by reference in its entirety) states that reductive alkylation was used for attachment of polyethylene glycol molecules to an enzyme. U.S. Pat. No. 4,179,337 (incorporated herein by reference in its entirety) discloses PEG:protein conjugates involving, for example, enzymes and insulin. U.S. Pat. No. 4,904,584 (incorporated herein by reference in its entirety) discloses the modification of the number of lysine residues in proteins for the attachment of polyethylene glycol molecules via reactive amine groups. U.S. Pat. No. 5,834,594 (incorporated herein by reference in its entirety) discloses substantially non-immunogenic water soluble PEG:protein conjugates, involving for example, the proteins IL-2, interferon alpha, and IL-1ra. The methods of Hakimi et al. involve the utilization of unique linkers to connect the various free amino groups in the protein to PEG. U.S. Pat. Nos. 5,824,784 and 5,985,265 (each of which is incorporated herein by reference in its entirety) teach methods allowing for selectively N-terminally chemically modified proteins and analogs thereof, including G-CSF and consensus interferon. Importantly, these modified proteins have advantages as relates to protein stability, as well as providing for processing advantages.
WSP modification is also described in Francis et al., In: Stability of protein pharmaceuticals: in vivo pathways of degradation and strategies for protein stabilization (Eds. Ahern., T. and Manning, M. C.) Plenum, N.Y., 1991 (incorporated herein by reference in its entirety), is used. In still another aspect, the method described in Delgado et al., “Coupling of PEG to Protein By Activation With Tresyl Chloride, Applications In Immunoaffinity Cell Preparation”, In: Fisher et al., eds., Separations Using Aqueous Phase Systems, Applications In Cell Biology and Biotechnology, Plenum Press, N.Y. N.Y., 1989 pp. 211-213 (incorporated herein by reference in its entirety), which involves the use of tresyl chloride, which results in no linkage group between the WSP moiety and the polypeptide moiety. In other aspects, attachment of a WSP is effected through use of N-hydroxy succinimidyl esters of carboxymethyl methoxy polyethylene glycol, as well known in the art.
For other descriptions of modification of a target with a WSP, see, for example, U.S. patent application No. 20030096400; EP 0 442724A2; EP 0154316; EP 0401384; WO 94/13322; U.S. Pat. Nos. 5,362,852; 5,089,261; 5,281,698; 6,423,685; 6,635,646; 6,433,135; International application WO 90/07938; Gaertner and Offord, (1996), Bioconjugate Chem. 7:38-44; Greenwald et al., Crit. Rev Therap Drug Carrier Syst. 2000; 17:101-161; Kopecek et al., J Controlled Release., 74:147-158, 2001; Harris et al., Clin Pharmacokinet. 2001; 40(7):539-51; Zalipsky et al., Bioconjug Chem. 1997; 8:111-118; Nathan et al., Macromolecules. 1992; 25:4476-4484; Nathan et al., Bioconj Chem. 1993; 4:54-62; and Francis et al., Focus on Growth Factors, 3:4-10 (1992), the disclosures of which are incorporated herein by reference in their entirety.
Reductive alkylation. In one aspect, covalent attachment of a WSP is carried out by reductive alkylation chemical modification procedures as provided herein to selectively modify the N-terminal α-amino group, and testing the resultant product for the desired biological characteristic, such as the biological activity assays provided herein.
Reductive alkylation for attachment of a WSP to a protein or peptide exploits differential reactivity of different types of primary amino groups (e.g., lysine versus the N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.
For reductive alkylation, the polymer(s) selected could have a single reactive aldehyde group. A reactive aldehyde is, for example, polyethylene glycol propionaldehyde, which is water stable, or mono C1-C10 alkoxy or aryloxy derivatives thereof (see U.S. Pat. No. 5,252,714, incorporated herein by reference in its entirety). In one approach, reductive alkylation is employed to conjugate a PEG-aldehyde (O-(3-Oxopropyl)-O′-methylpolyethylene glycol) to a primary amine. Under appropriate conditions, this approach has been demonstrated to yield PEG conjugates predominately modified through the α-amine at the protein N-terminus.
An aldehyde functionality useful for conjugating the biologically active molecule can be generated from a functionality having adjacent amino and alcohol groups. In a polypeptide, for example, an N-terminal serine, threonine or hydroxylysine can be used to generate an aldehyde functionality via oxidative cleavage under mild conditions using periodate. These residues, or their equivalents, can be normally present, for example at the N-terminus of a polypeptide, may be exposed via chemical or enzymatic digestion, or may be introduced via recombinant or chemical methods. The reaction conditions for generating the aldehyde typically involve addition of a molar excess of sodium meta periodate and under mild conditions to avoid oxidation at other positions in the protein. The pH is preferably about 7.0. A typical reaction involves the addition of a 1.5 fold molar excess of sodium meta periodate, followed by incubation for 10 minutes at room temperature in the dark.
The aldehyde functional group can be coupled to an activated polymer containing a hydrazide or semicarbazide functionality to form a hydrazone or sernicarbazone linkage. Hydrazide-containing polymers are commercially available, and can be synthesized, if necessary, using standard techniques. PEG hydrazides for use in the invention can be obtained from Shearwater Polymers, Inc., 2307 Spring Branch Road, Huntsville, Ala. 35801 (now part of Nektar Therapeutics, 150 Industrial Road, San Carlos, Calif. 94070-6256). The aldehyde is coupled to the polymer by mixing the solution of the two components together and heating to about 37° C. until the reaction is substantially complete. An excess of the polymer hydrazide is typically used to increase the amount of conjugate obtained. A typical reaction time is 26 hours. Depending on the thermal stability of the reactants, the reaction temperature and time can be altered to provide suitable results. Detailed determination of reaction conditions for both oxidation and coupling is set forth in Geoghegan and Stroh, (1992), Bioconjugate Chem. 3:138-146, and in Geoghegan, U.S. Pat. No. 5,362,852, each of which is incorporated herein by reference in its entirety.
Using reductive alkylation, the reducing agent should be stable in aqueous solution and preferably be able to reduce only the Schiff base formed in the initial process of reductive alkylation. Reducing agents are selected from, and without limitation, sodium borohydride, sodium cyanoborohydride, dimethylamine borate, timethylamine borate and pyridine borate.
The reaction pH affects the ratio of polymer to protein to be used. In general, if the reaction pH is lower than the pKa of a target reactive group, a larger excess of polymer to protein will be desired. If the pH is higher than the target pKa, the polymer:protein ratio need not be as large (i.e., more reactive groups are available, so fewer polymer molecules are needed).
Accordingly, the reaction is performed in one aspect at a pH which allows one to take advantage of the pKa differences between the ε-amino groups of the lysine residues and that of the α-amino group of the N-terminal residue of the protein. By such selective derivatization, attachment of a water soluble polymer to a protein is controlled; the conjugation with the polymer takes place predominantly at the N-terminus of the protein and no significant modification of other reactive groups, such as the lysine side chain amino groups, occurs.
In one aspect, therefore, methods are provided for covalent attachment of a WSP to a target compound and which provide a substantially homogenous preparation of WSP/protein conjugate molecules, in the absence of further extensive purification as is required using other chemical modification chemistries. More specifically, if polyethylene glycol is used, methods described allow for production of an N-terminally PEGylated protein lacking possibly antigenic linkage groups, i.e., the polyethylene glycol moiety is directly coupled to the protein moiety without potentially toxic by-products.
Depending on the method of WSP attachment chosen, the proportion of WSP molecules attached to the target peptide or protein molecule will vary, as will their concentrations in the reaction mixture. In general, the optimum ratio (in terms of efficiency of reaction in that there is no excess unreacted protein or polymer) is determined by the molecular weight of the WSP selected. In addition, when using methods that involve non-specific attachment and later purification of a desired species, the ratio may depend on the number of reactive groups (typically amino groups) available.
Purification. The method of obtaining a substantially homogeneous WSP-modified preparation is, in one aspect, by purification of a predominantly single species of modified compound from a mixture of species. By way of example, a substantially homogeneous species is first separated by ion exchange chromatography to obtain material having a charge characteristic of a single species (even though other species having the same apparent charge may be present), and then the desired species is separated using size exclusion chromatography. Other methods are reported and contemplated by the invention, includes for example, PCT WO 90/04606, published May 3, 1990, which describes a process for fractionating a mixture of PEG-protein adducts comprising partitioning the PEG/protein adducts in a PEG-containing aqueous biphasic system.
Thus, one aspect of the present invention is a method for preparing a WSP-modified compound conjugate comprised of (a) reacting a compound having more than one amino group with a water soluble polymer moiety under reducing alkylation conditions, at a pH suitable to selectively activate the α-amino group at the amino terminus of the protein moiety so that said water soluble polymer selectively attaches to said α-amino group; and (b) obtaining the reaction product. Optionally, and particularly for a therapeutic product, the reaction products are separated from unreacted moieties.
As ascertained by peptide mapping and N-terminal sequencing, a preparation is provided which comprises at least 50% PEGylated peptide in a mixture of PEGylated peptide and unreacted peptide. In other embodiments, preparations are provided which comprises at least 75% PEGylated peptide in a mixture of PEGylated peptide and unreacted peptide; at least 85% PEGylated peptide in a mixture of PEGylated peptide and unreacted peptide; at least 90% PEGylated peptide in a mixture of PEGylated peptide and unreacted peptide; at least 95% PEGylated peptide in a mixture of PEGylated peptide and unreacted peptide; and at least 99% PEGylated peptide in a mixture of PEGylated peptide and unreacted peptide.
Pharmaceutical Compositions. The present invention further provides pharmaceutical compositions comprising a preparation of the invention. Such pharmaceutical compositions may be for administration for injection, or for oral, nasal, transdermal or other forms of administration, including, e.g., by intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intrathecal, intraocular, retrobulbar, intrapulmonary (e.g., aerosolized drugs) or subcutaneous injection (including depot administration for long term release); by sublingual, anal, vaginal, or by surgical implantation, e.g., embedded under the splenic capsule, brain, or in the cornea. The treatment may consist of a single dose or a plurality of doses over a period of time. In general, comprehended by the invention are 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. The pharmaceutical compositions optionally may include still other pharmaceutically acceptable liquid, semisolid, or solid diluents that serve as pharmaceutical vehicles, excipients, or media, including but are not limited to, polyoxyethylene sorbitan monolaurate, magnesium stearate, methyl- and propylhydroxybenzoate, starches, sucrose, dextrose, gum acacia, calcium phosphate, mineral oil, cocoa butter, and oil of theobroma. 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. 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, incorporated herein by reference. Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets or pellets. Alternatively, proteinoid encapsulation may be used (as, for example, proteinoid microspheres reported in U.S. Pat. No. 4,925,673), or liposomal encapsulation may be used, the liposomes optionally derivatized with various polymers (e.g., U.S. Pat. No. 5,013,556). A description of solid dosage forms for therapeutics in general is given in Chapter 10 of Marshall, K., Modern Pharmaceutics (1979), edited by G. S. Banker and C. T. Rhodes, incorporated herein by reference. In general, the formulation will include a preparation of the invention and inert ingredients which allow for protection against the stomach environment, and release of the biologically active material in the intestine.
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. Examples of such moieties include polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline (Abuchowski and Davis, Soluble Polymer-Enzyme Adducts, Enzymes as Drugs, Hocenberg and Roberts, eds., Wiley-Interscience, New York, N.Y., (1981), pp 367-383; Newmark, et al., J. Appl. Biochem. 4:185-189 (1982)). Other polymers that could be used are poly-1,3-dioxolane and poly-1,3,6-tioxocane.
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 compound. The clinical efficacy of a heparin formulation using SNAC has been demonstrated in a Phase II trial conducted by Emisphere Technologies. See U.S. Pat. No. 5,792,451, “Oral drug delivery composition and methods.”
Preparations of the invention can be included in formulation as fine multiparticulates in the form of granules or pellets of particle size about, for example, one mm. The formulation of the material for capsule administration could also be as a powder, lightly compressed plugs or even as tablets. Compositions are optionally prepared by compression.
Colorants and flavoring agents may be included. For example, the preparation 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.
Preparations of the invention are, in one aspect, diluted or increased in the volume with an inert material. Exemplary diluents include carbohydrates, especially mannitol, α-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.
Preparations including disintegrants are further contemplated in solid dosage form compositions. 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. Another form of disintegrant is an insoluble cationic exchange resin. Powdered gums may also 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.
Pharmaceutical compositions including binders are further contemplated to hold the therapeutic agent together to form a hard tablet and exemplary binders include materials from 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 in a pharmaceutical composition is further contemplated to prevent sticking during the formulation process. Lubricants 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 a pharmaceutical composition during formulation and to aid rearrangement during compression are also provided. Exemplary glidants include starch, talc, pyrogenic silica and hydrated silicoaluminate.
To aid dissolution of a composition into the aqueous environment, incorporation of a surfactant as a wetting agent is contemplated. Exemplary surfactants include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents are contemplated, including for example and without limitation, benzalkonium chloride or benzethonium chloride. In another aspect, compositions using as surfactants include 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. Compositions comprising these surfactants, either alone or as a mixture in different ratios, are therefore further provided.
Optionally, additives are included in a pharmaceutical composition to enhance uptake of the compound, such additives including, for example and without limitation, fatty acids oleic acid, linoleic acid and linolenic acid.
Controlled release compositions. In another aspect, controlled release formulation are provided. A preparation of the invention is incorporated into an inert matrix which permits release by either diffusion or leaching mechanisms e.g., gums. Slowly degenerating matrices, e.g., alginates, polysaccharides, may also be incorporated into the formulation. Another form of a controlled release 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 in compositions of the invention, including for example, a variety of sugars which could be applied in a coating pan. The compositions also include a film coated tablet and the materials used in this instance are divided into two groups. The first includes the nonenteric materials, such as and without limitation 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 is also contemplated 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. Also contemplated herein is pulmonary delivery of a preparation of the invention. The compound is delivered to the lungs of a mammal while inhaling and traverses across the lung epithelial lining to the blood stream. Pulmonary delivery is described in Adjei et al., Pharma. Res. (1990) 7: 565-9; Adjei et al. (1990), Internatl. J. Pharmaceutics 63: 135-44; Braquet et al. (1989), J. Cardiovasc. Pharmacol. 13 (supp1.5): s. 143-146; Hubbard et al. (1989), Annals Int. Med. 3: 206-12; Smith et al. (1989), J. Clin. Invest. 84: 1145-6; Oswein et al. (March 1990), “Aerosolization of Proteins”, Proc. Symp. Resp. Drug Delivery II, Keystone, Colo.; Debs et al. (1988), J. Immunol. 140: 3482-8 and Platz et al., U.S. Pat. No. 5,284,656, the disclosures of which are incorporated herein by reference.
Mechanical devices. Also contemplated for practice of the invention is 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. Specific examples of commercially available devices suitable for the practice of this invention are the Ultravent nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the Acorn II nebulizer, manufactured by Marquest Medical Products, Englewood, Colo.; the Ventolin metered dose inhaler, manufactured by Glaxo Inc., Research Triangle Park, N.C.; and the Spinhaler powder inhaler, manufactured by Fisons Corp., Bedford, Mass.
All such devices require the use of formulations suitable for the dispensing a preparation of the invention. 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.
For effective delivery to distal lung, the composition is prepared in particulate form with an average particle size in one aspect of less than 10 μm (or microns), and in an alternative aspect 0.5 to 5 μm.
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.
Nasal delivery. Nasal delivery of preparations of the invention 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 forms. Buccal delivery of the inventive compound is also contemplated. Buccal delivery formulations are known in the art for use with peptides.
Carriers. 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 a compound of the invention). 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.
Other formulations. The use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is also contemplated.
Dosages. The dosage regimen involved in a method for treating a condition described herein 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. In various aspects, the daily regimen is in the range of 0.1-1000 μg of a preparation per kilogram of body weight (calculating the mass of the protein alone, without chemical modification) or 0.1-150 μg/kg.
Preparations of the invention may be administered by an initial bolus followed by a continuous infusion to maintain therapeutic circulating levels of drug product. As another example, the inventive compound may be administered as a one-time dose. Those of ordinary skill in the art will readily optimize effective dosages and administration regimens as determined by good medical practice and the clinical condition of the individual patient. The frequency of dosing will depend on the pharmacokinetic parameters of the agents and the route of administration. The optimal pharmaceutical formulation will be determined by one skilled in the art depending upon the route of administration and desired dosage. See for example, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712, the disclosure of which is hereby incorporated by reference. Such formulations may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the administered agents. Depending on the route of administration, a suitable dose may be calculated according to body weight, body surface area or organ size. Further refinement of the calculations necessary to determine the appropriate dosage for treatment involving each of the above mentioned formulations is routinely made by those of ordinary skill in the art without undue experimentation, especially in light of the dosage information and assays disclosed herein, as well as the pharmacokinetic data observed in the human clinical trials discussed above. Appropriate dosages may be ascertained through use of established assays for determining blood levels dosages in conjunction with appropriate dose-response data. The final dosage regimen will be determined by the attending physician, considering various factors which modify the action of drugs, e.g. the drug's specific activity, the severity of the damage and the responsiveness of the patient, the age, condition, body weight, sex and diet of the patient, the severity of any infection, time of administration and other clinical factors. As studies are conducted, further information will emerge regarding the appropriate dosage levels and duration of treatment for various diseases and conditions.
The therapeutic methods, compositions and compounds of the present invention may also be employed, alone or in combination with other cytokines, soluble c-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 preparations of the invention 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), M-CSF, SCF, GM-CSF, granulocyte colony stimulating factor (G-CSF), EPO, interferon-alpha (IFN-alpha), consensus interferon, IFN-beta, IFN-gamma, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, thrombopoietin (TPO), angiopoietins, for example Ang-1, Ang-2, Ang-4, Ang-Y, the human angiopoietin-like polypeptide, vascular endothelial growth factor (VEGF), angiogenin, bone morphogenic protein-1, bone morphogenic protein-2, bone morphogenic protein-3, bone morphogenic protein-4, bone morphogenic protein-5, bone morphogenic protein-6, bone morphogenic protein-7, bone morphogenic protein-8, bone morphogenic protein-9, bone morphogenic protein-10, bone morphogenic protein-11, bone morphogenic protein-12, bone morphogenic protein-13, bone morphogenic protein-14, bone morphogenic protein-15, bone morphogenic protein receptor IA, bone morphogenic protein receptor IB, brain derived neurotrophic factor, ciliary neutrophic factor, ciliary neutrophic factor receptor, cytokine-induced neutrophil chemotactic factor 1, cytokine-induced neutrophil, chemotactic factor 2α, cytokine-induced neutrophil chemotactic factor 2β, β, endothelial cell growth factor, endothelin 1, epidermal growth factor, epithelial-derived neutrophil attractant, fibroblast growth factor 4, fibroblast growth factor 5, fibroblast growth factor 6, fibroblast growth factor 7, fibroblast growth factor 8, fibroblast growth factor 8b, fibroblast growth factor 8c, fibroblast growth factor 9, fibroblast growth factor 10, fibroblast growth factor acidic, fibroblast growth factor basic, glial cell line-derived neutrophic factor receptor α1, glial cell line-derived neutrophic factor receptor α2, growth related protein, growth related protein α, growth related protein β, growth related protein y, heparin binding epidermal growth factor, hepatocyte growth factor, hepatocyte growth factor receptor, insulin-like growth factor I, insulin-like growth factor receptor, insulin-like growth factor II, insulin-like growth factor binding protein, keratinocyte growth factor, leukemia inhibitory factor, leukemia inhibitory factor receptor α, nerve growth factor nerve growth factor receptor, neurotrophin-3, neurotrophin-4, placenta growth factor, placenta growth factor 2, platelet-derived endothelial cell growth factor, platelet derived growth factor, platelet derived growth factor A chain, platelet derived growth factor AA, platelet derived growth factor AB, platelet derived growth factor B chain, platelet derived growth factor BB, platelet derived growth factor receptor α, platelet derived growth factor receptor β, pre-B cell growth stimulating factor, stem cell factor receptor, TNF, including TNF0, TNF1, TNF2, transforming growth factor α, transforming growth factor β, transforming growth factor β1, transforming growth factor β1.2, transforming growth factor β2, transforming growth factor β3, transforming growth factor β5, latent transforming growth factor β1, transforming growth factor β binding protein I, transforming growth factor β binding protein II, transforming growth factor β binding protein III, tumor necrosis factor receptor type I, tumor necrosis factor receptor type II, urokinase-type plasminogen activator receptor, vascular endothelial growth factor, and chimeric proteins and biologically or immunologically active fragments thereof. It may further be useful to administer, either simultaneously or sequentially, an effective amount of a soluble mammalian c-Mpl, which appears to have an effect of causing megakaryocytes to fragment into platelets once the megakaryocytes have reached mature form. Thus, administration of a preparation of the invention (to enhance the number of mature megakaryocytes) followed by administration of the soluble c-Mpl (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.
Conditions in general. The compounds of this invention have pharmacological 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 compounds, an in vivo assay is 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 compounds. 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 TPO-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 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 or immune thrombocytopenia (ITP), including idiopathic thrombocytopenic purpura associated with breast cancer; HIV associated ITP and HIV-related thrombotic thrombocytopenic purpura; metastatic tumors which result in thrombocytopenia, systemic lupus erythematosus, including neonatal lupus syndrome, splenomegaly, Fanconi's syndrome, vitamin B12 deficiency, folic acid deficiency, May-Hegglin anomaly, Wiskott-Aldrich syndrome, chronic liver disease; myelodysplastic syndrome associated with thrombocytopenia; paroxysmal nocturnal hemoglobinuria, acute profound thrombocytopenia following C7E3 Fab (Abciximab) therapy; alloimmune thrombocytopenia, including maternal alloimmune thrombocytopenia; thrombocytopenia associated with antiphospholipid antibodies and thrombosis; autoimmune thrombocytopenia; drug-induced immune thrombocytopenia, including carboplatin-induced thrombocytopenia, heparin-induced thrombocytopenia; fetal thrombocytopenia; gestational thrombocytopenia; Hughes' syndrome; lupoid thrombocytopenia; accidental and/or massive blood loss; myeloproliferative disorders; thrombocytopenia in patients with malignancies; thrombotic thrombocytopenia purpura, including thrombotic microangiopathy manifesting as thrombotic thrombocytopenic purpura/hemolytic uremic syndrome in cancer patients; autoimmune hemolytic anemia; occult jejunal diverticulum perforation; pure red cell aplasia; autoimmune thrombocytopenia; nephropathia epidemica; rifampicin-associated acute renal failure; Paris-Trousseau thrombocytopenia; neonatal alloimmune thrombocytopenia; paroxysmal nocturnal hemoglobinuria; hematologic changes in stomach cancer; hemolytic uremic syndromes in childhood; hematologic manifestations related to viral infection including hepatitis A virus and CMV-associated thrombocytopenia. Also, certain treatments for AIDS result in thrombocytopenia (e.g., AZT). Certain wound healing disorders might also benefit from an increase in platelet numbers. Also, certain treatments for AIDS result in thrombocytopenia (e.g., AZT). Certain wound healing disorders might also benefit from an increase in platelet numbers.
The TPO-mimetic compounds of this invention may be used in any situation in which production of platelets or platelet precursor cells is desired, or in which stimulation of the c-Mpl receptor is desired. Thus, for example, the compounds of this invention may be used to treat any condition in a mammal wherein there is a need of platelets, megakaryocytes, and the like. Such conditions are described in detail in the following exemplary sources: 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.
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, interferon-alpha (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 megakaryocytes) 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 μg to 1 mg inventive compound per 106 cells.
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 thrombopoietin in vivo. The generic term for platelet deficiency is thrombocytopenia, and the methods and preparations of the present invention are generally available for treating thrombocytopenia in patients in need thereof.
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 preparation or composition of the invention is optionally administered along with blood or purified platelets.
The TPO-mimetic compounds of the invention are useful in stimulating certain cell types other than megakaryocytes if such cells are found to express c-Mpl. Conditions associated with such cells that express the c-Mpl, which are responsive to stimulation by a preparation or composition described herein are also within the scope of this invention.
The following examples are not intended to be limiting but only exemplary of specific embodiments of the invention.
Example 1
Expression Construct Assembly
A polynucleotide encoding a TMP fusion protein comprising a murine Fc region (mFc-TMP) was constructed by combining nucleotide sequences individually encoding murine Fc and a TMP (described in EP01124961A2). In the first round of PCR, the murine Fc-encoding component was amplified with PCR primers 3155-58 (SEQ ID NO: 1022) and 1388-00 (SEQ ID NO: 1023).








3155-58:



CCGGGTAAAGGTGGAGGTGGTGGTATCGA
(SEQ ID NO: 1024)


 


3155-59:



CCACCTCCACCTTTACCCGGAGAGTGGGAG
(SEQ ID NO: 1025)






In a separate reaction, an TMP-encoding polynucleotide was amplified with primers 1209-85 (SEQ ID NO: 1026) and 3155-59 (SEQ ID NO: 1027).










1209-85:




CGTACAGGTTTACGCAAGAAAATGG
(SEQ ID NO: 1028)


 



1388-00:




CTAGTTATTGCTCAGCGG
(SEQ ID NO: 1029)






The resulting PCR fragments were gel purified and combined in a single tube for a second round of PCR with primers 1209-85 (SEQ ID NO: 1030) and 1388-00 (SEQ ID NO: 1031). The PCR product from this second round of amplification was gel purified and digested with restriction enzymes NdeI and XhoI. The digestion fragment was purified and ligated into the vector pAMG21, previously digested with the same enzymes. This ligation mix was transformed via electroporation into the E. coli strain Amgen 393 and plated onto LB+Kanamycin media. Colonies were screened via PCR and DNA sequencing. A positive clone with a nucleotide sequence (SEQ ID NO: 1032) encoding the mFc-TMP fusion protein (SEQ ID NO: 1033) was identified and designated 6397.
Murine Fc-TMP fusion protein-encoding polynucleotide (SEQ ID NO: 1034)








  1
GATTTGATTC TAGATTTGTT TTAACTAATT AAAGGAGGAA



TAACAT


 







Open RF:









ATGGTCGACGGTTG TAAGCCATGC ATTTGTACAG TCCCAGAAGT



ATCATCTGTC


 


101
TTCATCTTCC CCCCAAAGCC CAAGGATGTG CTCACCATTA



CTCTGACTCC


 


151
TAAGGTCACG TGTGTTGTGG TAGACATCAG CAAGGATGAT



CCCGAGGTCC


 


201
AGTTCAGCTG GTTTGTAGAT GATGTGGAGG TGCACACAGC



TCAGACGCAA


 


251
CCCCGGGAGG AGCAGTTCAA CAGCACTTTC CGCTCAGTCA



GTGAACTTCC


 


301
CATCATGCAC CAGGACTGGC TCAATGGCAA GGAGTTCAAA



TGCAGGGTCA


 


351
ACAGTGCAGC TTTCCCTGCC CCCATCGAGA AAACCATCTC



CAAAACCAAA


 


401
GGCAGACCGA AGGCTCCACA GGTGTACACC ATTCCACCTC



CCAAGGAGCA


 


451
GATGGCCAAG GATAAAGTCA GTCTGACCTG CATGATAACA



GACTTCTTCC


 


501
CTGAAGACAT TACTGTGGAG TGGCAGTGGA ATGGGCAGCC



AGCGGAGAAC


 


551
TACAAGAACA CTCAGCCCAT CATGGACACA GATGGCTCTT



ACTTCGTCTA


 


601
CAGCAAGCTC AATGTGCAGA AGAGCAACTG GGAGGCAGGA



AATACTTTCA


 


651
CCTGCTCTGT GTTACATGAG GGCCTGCACA ACCACCATAC



TGAGAAGAGC


 


701
CTCTCCCACT CTCCGGGTAA AGGTGGAGGT GGTGGTATCG



AAGGTCCGAC


 


751
TCTGCGTCAG TGGCTGGCTG CTCGTGCTGG TGGTGGAGGT



GGCGGCGGAG


 


801
GTATTGAGGG CCCAACCCTT CGCCAATGGC TTGCAGCACG



CGCATAA


 







3′ Sequence:









TCTCGAGGATCCG CGGAAAGAAG AAGAAGAAGA AGAAAGCCCG



AAAGG






Murine Fc-TMP protein sequence (SEQ ID NO: 1035)








  1
MVDGCKPCIC TVPEVSSVFI FPPKPKDVLT ITLTPKVTCV



VVDISKDDPE


 


 51
VQFSWFVDDV EVHTAQTQPR EEQFNSTFRS VSELPIMHQD



WLNGKEFKCR


 


101
VNSAAFPAPI EKTISKTKGR PKAPQVYTIP PPKEQMAKDK



VSLTCMITDF


 


151
FPEDITVEWQ WNGQPAENYK NTQPIMDTDG SYFVYSKLNV



QKSNWEAGNT


 


201
FTCSVLHEGL HNHHTEKSLS HSPGKGGGGG IEGPTLRQWL



AARAGGGGGG


 


251
GGIEGPTLRQ WLAARA*






Example 2
Fermentation of Strain 6397
Fermentation of strain 6397 was initiated by inoculation of 500 mL of sterilized Luria broth with a seed culture of the strain in a shake flask. When cell density reached 0.9 at 600 nm, the contents were used to inoculate a 15 L fermentor containing 10 L of complex based growth medium (800 g glycerol, 500 g trypticase, 3 g sodium citrate, 40 g KH2PO4, 20 g (NH4)2SO4, 5 ml Fluka P-2000 antifoam, 10 ml trace metals (ferric chloride 27.0 g/L, zinc chloride 2.00 g/L, cobalt chloride 2.00 g/L, sodium molybdate 2.00 g/L, calcium chloride 1.00 g/L, cupric sulfate 1.90 g/L, boric acid 0.50 g/L, manganese chloride 1.60 g/L, sodium citrate dihydrate 73.5 g/L), 10 ml vitamins (biotin 0.060 g/L, folic acid 0.040 g/L, riboflavin 0.42 g/L, pyridoxine HCl 1.40 g/L, niacin 6.10 g/L, pantothenic acid 5.40 g/L, sodium hydroxide 5.30 ml/L), add water to bring to 10 L). The fermenter was maintained at 37° C. and pH 7 with dissolved oxygen levels kept at a minimum of 30% saturation. When the cell density reached 13.1 OD units at 600 nm, the culture was induced by the addition 10 ml of 0.5 mg/ml N-(3-oxo-hexanoyl) homoserine lactone. At 6 hours post induction, the broth was chilled to 10° C., and the cells were harvested by centrifugation at 4550 g for 60 min at 5° C. The cell paste was then stored at −80° C.
Example 3
Protein Refolding
E. coli paste (300 g) from strain 6397 expressing mFc-TMP was dissolved in 2250 ml lysis buffer (50 mM Tris HCl, 5 mM EDTA, pH 8.0) and passed through a chilled microfluidizer two times at 13,000 PSI. The homogenate was then centrifuged at 11,300 g for 60 minutes at 4° C. The supernatant was discarded, and the pellet was resuspended in 2400 ml of water using a tissue grinder. The homogenate was then centrifuged at 11,300 g for 60 minutes at 4° C. The supernatant was discarded, and the pellet was resuspended in 200 ml volumes of water using a tissue grinder. The homogenate was centrifuged at 27,200 g for 30 minutes at 4° C., and the supernatant was discarded. About 12.5% of the pellet was resuspended in 28 ml 20 mM Tris HCl, pH 8.0, with 35 mg hen egg white lysozyme (Sigma, St Louis, Mo.) using a tissue grinder and incubated at 37° C. for 20 min. Following incubation, the suspension was centrifuged at 27,200 g for 30 minutes at 22° C., and the supernatant was discarded. The pellet was resuspended in 35 ml 8 M guanidine HCl, 50 mM Tris HCl, pH 8.0, after which 350 μl 1 M DTT (Sigma, St Louis, Mo.) was added and material was incubated at 37° C. for 30 minutes. The solution was then centrifuged at 27,200 g for 30 minutes at 22° C. The supernatant was then transferred to 3.5 L of refolding buffer (50 mM Tris base, 160 mM arginine HCl, 3 M urea, 20% glycerol, pH 9.5, 1 mM cysteine, 1 mM cystamine HCl) at 1 ml/min with gentle stirring at 4° C.
Example 4
Construct Purification
After about 40 hours incubation at 4° C. with gentle agitation, the refold solution described in Example 3 was concentrated to 500 μl using a tangential flow ultrafiltration apparatus with a 30 kDa cartridge (Satorius, Goettingen, Germany) followed by diafiltration against 3 L of Q-Buffer A (20 mM Tris HCl, pH 8.0). The concentrated material was filtered through a Whatman GF/A filter and loaded on to an 86 ml Q-Sepharose fast flow column (2.6 cm ID) (Amersham Biosciences, Piscataway, N.J.) at 15 ml/min. After washing the resin with several column volumes of Q-Buffer A, the protein was eluted using a 20 column volume linear gradient to 60% Q-Buffer B (20 mM Tris HCl, 1 M NaCl, pH 8.0) at 10 ml/min. The peak fractions were pooled, and the pool was passed through a Mustang E syringe filter (Pall Corporation, East Hills, N.Y.) at 1 ml/min. The filtered material was filtered a second time through a 0.22 μm cellulose acetate filter and stored at −80° C.
Example 5
Protein PEGylation
To a cooled (4° C.), stirred solution of mFc-TMP (3.5 ml, 0.8 mg/ml) in a 100 mM sodium acetate buffer, pH 5, containing 20 mM NaCNBH3, was added a 3.8-fold molar excess of methoxypolyethylene glycol aldehyde (MPEG) (average molecular weight, 20 kDa) (Nektar). The stirring of the reaction mixture was continued at the same temperature. The extent of the protein modification during the course of the reaction was monitored by SEC HPLC using a Superose 6 HR 10/30 column (Amersham Biosciences) eluted with a 0.05 M phosphate buffer with 0.15 M NaCl, pH 7.0 at 0.4 ml/min. After 16 hours the SEC HPLC analysis indicated that the majority of the protein has been conjugated to MPEG. At this time the reaction mixture was buffer-exchanged into a 20 mM Tris/HCl buffer, pH 8.12. The MPEG-mFc-AMP2 conjugates were isolated by ion exchange chromatography using a 1 ml Hi Trap HP Q column (Amersham Biosciences) equilibrated with a 20 mM Tris/HCl buffer, pH 8.12. The reaction mixture was loaded on the column at a flow rate of 0.5 ml/min and the unreacted MPEG aldehyde was eluted with three column volumes of the starting buffer. A linear 20-column-volume gradient from 0% to 100% 20 mM Tris/HCl buffer, pH 8.12, containing 0.5 M NaCl was used to the elute the protein-polymer conjugates. Fractions (2 ml) collected during ion exchange chromatography separation were analyzed by HPLC SEC as described above. A fraction containing the mono- and di-MPEG-mFc-TMP conjugates in an approximate ratio of 2.3 to 1 (as determined by SEC HPLC) was concentrated, and sterile filtered.
Example 6
In Vivo Testing
BDF1 mice (Charles River Laboratories, Wilmington, Mass.) were divided into groups of 10 and injected on days 0, 21, and 42 subcutaneously with either diluting agent (Dulbecco's PBS with 0.1% bovine serum albumin) or diluting agent with 50 μg test mono- and di-MPEG-mFc-TMP conjugate protein (as described above) per kg animal. Each group was divided in half and bled (140 μl) from the retro-orbital sinus on alternate time points (days 0, 3, 5, 7, 10, 12, 14, 19, 24, 26, 28, 31, 33, 40, 45, 47, 49, 52 and 59). On day 59, mice were anesthetized with isoflurane prior to bleeding. The collected blood was analyzed for a complete and differential count using an ADVIA 120 automated blood analyzer with murine software (Bayer Diagnostics, New York, N.Y.).
Results showed that administration of mono- and di-MPEG-mFc-TMP conjugates on days 0, 21 and 41 resulted in subsequent increases in platelet levels to essentially the same degree with each dose. See FIG. 1. In comparison, administration of mFc-TMP (lacking a PEG moiety) on the same days (i.e., days 0, 21 and 41) also resulted in increased levels of platelets, but the increase following administration on day 21 was significantly lower (i.e., attenuated) than the increase after administration on day 0, and the increase following administration on day 41 was less (i.e., further attenuated) than that observed following administration on day 21. Overall, these results indicate that the Fc-TMP modified to include a WSP (e.g., PEG) moiety was able to induce platelet production that did not decrease when administered in a multiple dosage regimen.
The present invention has been described in terms of particular embodiments found or proposed to comprise preferred modes for the practice of then invention. It will be appreciated by those of ordinary skill in the art that, in light of the present disclosure, numerous modifications and changes can be made in the particular embodiments exemplified without departing from the intended scope of the invention.

## Claims

1. A compound comprising a structure set out in Formula V


[F1-(L2)e-P1-(L3)f-P2]-(L1)c-PEGd  Formula V

wherein:
F1 is an Fc domain;

wherein P1 and P2 are each independently sequences of pharmacologically active peptides;
L1, L2 and L3 are each independently linkers;
c, e and f are each independently 0 or 1;
d is at least 1; and
wherein the Fc domain is attached at the N-terminus of -L2-P1-L3-P2 and one or more PEG is attached to any reactive moiety in the Fc domain, optionally through linker L1;
said compound having a property of improved bioefficacy when administered in a multidose regimen.

2. The compound of claim 1 which is a multimer.

3. The compound of claim 2 which is a dimer.

4. The compound of claim 1 wherein P1 and/or P2 are independently selected from a peptide set out in any one of Tables 4 through 20.

5. The compound of claim 1 wherein P1 and P2 have the same amino acid sequence as SEQ ID NO:459.

6. The compound of claim 1 wherein PEG has a molecular weight of between about 2 kDa and 100 kDa.

7. The compound of claim 6 wherein said PEG has a molecular weight of between about 6 kDa and 25 kDa.

8. A composition comprising the compound of claim 1, wherein said composition comprises the PEGylated compound in an amount selected from the group consisting of: at least 50% PEGylated compound, at least 75% PEGylated compound, at least 85% PEGylated compound, at least 90% PEGylated compound, at least 95% PEGylated compound.

9. A method of treating a disease or disorder comprising administering a composition comprising the compound of claim 1 in an amount effective to treat the disease or disorder.

10. The method of claim 9, wherein said amount is from 0.1-1000 μg/kg—of body weight or from 0.1-150 μg/kg body weight.

11. A pharmaceutical composition comprising the compound of claim 1 in admixture with a pharmaceutically acceptable carrier thereof.