U.S. patent application number 10/645761 was filed with the patent office on 2004-04-15 for modified peptides as therapeutic agents.
This patent application is currently assigned to Amgen Inc.. Invention is credited to Boone, Thomas Charles, Cheetham, Janet C., Feige, Ulrich, Liu, Chuan-Fa.
Application Number | 20040071712 10/645761 |
Document ID | / |
Family ID | 26802505 |
Filed Date | 2004-04-15 |
United States Patent
Application |
20040071712 |
Kind Code |
A1 |
Feige, Ulrich ; et
al. |
April 15, 2004 |
Modified peptides as therapeutic agents
Abstract
The present invention concerns fusion of Fc domains with
biologically active peptides and a process for preparing
pharmaceutical agents using biologically active peptides. In this
invention, pharmacologically active compounds are prepared by a
process comprising: a) selecting at least one peptide that
modulates the activity of a protein of interest; and b) preparing a
pharmacologic agent comprising an Fc domain covalently linked to at
least one amino acid of the selected peptide. Linkage to the
vehicle increases the half-life of the peptide, which otherwise
would be quickly degraded in vivo. The preferred vehicle is an Fc
domain. The peptide is preferably selected by phage display, E.
coli display, ribosome display, RNA-peptide screening, or
chemical-peptide screening.
Inventors: |
Feige, Ulrich; (Newbury
Park, CA) ; Liu, Chuan-Fa; (Longmont, CO) ;
Cheetham, Janet C.; (Montecito, CA) ; Boone, Thomas
Charles; (Newbury Park, CA) |
Correspondence
Address: |
AMGEN INCORPORATED
MAIL STOP 27-4-A
ONE AMGEN CENTER DRIVE
THOUSAND OAKS
CA
91320-1799
US
|
Assignee: |
Amgen Inc.
|
Family ID: |
26802505 |
Appl. No.: |
10/645761 |
Filed: |
August 18, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10645761 |
Aug 18, 2003 |
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09428082 |
Oct 22, 1999 |
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6660843 |
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60105371 |
Oct 23, 1998 |
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Current U.S.
Class: |
424/178.1 ;
530/391.1 |
Current CPC
Class: |
C07K 14/525 20130101;
A61P 31/18 20180101; C07K 2319/30 20130101; A61P 7/04 20180101;
A61P 31/04 20180101; C12N 9/6491 20130101; A61K 38/00 20130101;
A61P 29/00 20180101; A61P 3/04 20180101; C07K 2319/00 20130101;
C07K 14/545 20130101; A61P 19/00 20180101; A61P 11/06 20180101;
A61P 37/02 20180101; A61P 7/08 20180101; A61P 35/00 20180101; C07K
14/8146 20130101; A61P 9/00 20180101; A61P 19/02 20180101; A61P
43/00 20180101; C07K 14/52 20130101; A61P 7/02 20180101; C07K
14/524 20130101; C07K 14/505 20130101; A61P 7/06 20180101; A61P
31/12 20180101; A61P 35/02 20180101; A61P 7/00 20180101 |
Class at
Publication: |
424/178.1 ;
530/391.1 |
International
Class: |
A61K 039/395; C07K
016/46 |
Claims
What is claimed is:
1. A composition of matter of the formula
(X.sup.1).sub.a-F.sup.1-(X.sup.2- ).sub.b and multimers thereof,
wherein: F.sup.1 is an Fc domain; X.sup.1 and X.sup.2 are each
independently selected from -(L.sup.1).sub.c-P.sup.1- ,
-(L.sup.1).sub.c-P.sup.1-(L.sup.2).sub.d-P.sup.2,
(L.sup.1).sub.c-P.sup.1-(L.sup.2).sub.d-P.sup.2-(L.sup.3).sub.e-P.sup.1,
and
-(L.sup.1).sub.c-P-(L.sup.2).sub.d-P.sup.2-(L.sup.1).sub.e-P.sup.1-(L-
.sup.4).sub.f-P.sup.4 P.sup.1, P.sup.2, P.sup.1, and P.sup.4 are
each independently sequences of pharmacologically active peptides;
L.sup.1, L.sup.2, L.sup.3, and L.sup.4 are each independently
linkers; and a, b, c, d, e, and f are each independently 0 or 1,
provided that at least one of a and b is 1.
2. The composition of matter of claim 1 of the formulae
X.sup.1-F.sup.1 or F.sup.1-X.sup.2.
3. The composition of matter of claim 1 of the formula
F.sup.1-(L.sup.1).sub.c-P.sup.1.
4. The composition of matter of claim 1 of the formula
F.sup.1-(L.sup.1).sub.c-P.sup.1-(L.sup.2).sub.d-P.sup.2.
5. The composition of matter of claim 1 wherein F.sup.1 is an IgG
Fc domain.
6. The composition of matter of claim 1 wherein F.sup.1 is an IgG1
Fc domain.
7. The composition of matter of claim 1 wherein F.sup.1 comprises
the sequence of SEQ ID NO: 2.
8. The composition of matter of claim 1 wherein X.sup.1 and X.sup.2
comprise an IL-1 antagonist peptide sequence.
9. The composition of matter of claim 8 wherein the IL-1 antagonist
peptide sequence is selected from SEQ ID NOS: 212, 907, 908, 909,
910, 917, and 979.
10. The composition of matter of claim 8 wherein the IL-1
antagonist peptide sequence is selected from SEQ ID NOS: 213 to
271, 671 to 906, 911 to 916, and 918 to 1023.
11. The composition of matter of claim 8 wherein F.sup.1 comprises
the sequence of SEQ ID NO: 2.
12. The composition of matter of claim 1 wherein X.sup.1 and
X.sup.2 comprise an EPO-mimetic peptide sequence.
13. The composition of matter of claim 12 wherein the EPO-mimetic
peptide sequence is selected from Table 5.
14. The composition of matter of claim 12 wherein F.sup.1 comprises
the sequence of SEQ ID NO: 2.
15. The composition of matter of claim 12 comprising a sequence
selected from SEQ ID NOS: 83, 84, 85, 124, 419, 420, 421, and
461.
16. The composition of matter of claim 12 comprising a sequence
selected from SEQ ID NOS: 339 and 340.
17. The composition of matter of claim 12 comprising a sequence
selected from SEQ ID NOS: 20 and 22.
18. The composition of matter of claim 3 wherein P.sup.1 is a
TPO-mimetic peptide sequence.
19. The composition of matter of claim 18 wherein P.sup.1 is a
TPO-mimetic peptide sequence selected from Table 6.
20. The composition of matter of claim 18 wherein F.sup.1 comprises
the sequence of SEQ ID NO: 2.
21. The composition of matter of claim 18 having a sequence
selected from SEQ ID NOS: 6 and 12.
22. A DNA encoding a composition of matter of any of claims 1 to
21.
23. An expression vector comprising the DNA of claim 22.
24. A host cell comprising the expression vector of claim 23.
25. The cell of claim 24, wherein the cell is an E. coli cell.
26. A process for preparing a pharmacologically active compound,
which comprises a) selecting at least one randomized peptide that
modulates the activity of a protein of interest; and b) preparing a
pharmacologic agent comprising at least one Fc domain covalently
linked to at least one amino acid sequence of the selected peptide
or peptides.
27. The process of claim 26, wherein the peptide is selected in a
process comprising screening of a phage display library, an E. coli
display library, a ribosomal library, or a chemical peptide
library.
28. The process of claim 26, wherein the preparation of the
pharmacologic agent is carried out by: a) preparing a gene
construct comprising a nucleic acid sequence encoding the selected
peptide and a nucleic acid sequence encoding an Fc domain; and b)
expressing the gene construct.
29. The process of claim 26, wherein the gene construct is
expressed in an E. coli cell.
30. The process of claim 26, wherein the protein of interest is a
cell surface receptor.
31. The process of claim 26, wherein the protein of interest has a
linear epitope.
32. The process of claim 26, wherein the protein of interest is a
cytokine receptor.
33. The process of claim 26, wherein the peptide is an EPO-mimetic
peptide.
34. The process of claim 26, wherein the peptide is a TPO-mimetic
peptide.
35. The process of claim 26, wherein the peptide is an IL-1
antagonist peptide.
36. The process of claim 26, wherein the peptide is an MMP
inhibitor peptide or a VEGF antagonist peptide.
37. The process of claim 26, wherein the peptide is a
TNF-antagonist peptide.
38. The process of claim 26, wherein the peptide is a CTLA4-mimetic
peptide.
39. The process of claim 26, wherein the peptide is selected from
Tables 4 to 20.
40. The process of claim 26, wherein the selection of the peptide
is carried out by a process comprising: a) preparing a gene
construct comprising a nucleic acid sequence encoding a first
selected peptide and a nucleic acid sequence encoding an Fc domain;
b) conducting a polymerase chain reaction using the gene construct
and mutagenic primers, wherein i) a first mutagenic primer
comprises a nucleic acid sequence complementary to a sequence at or
near the 5' end of a coding strand of the gene construct, and ii) a
second mutagenic primer comprises a nucleic acid sequence
complementary to the 3' end of the noncoding strand of the gene
construct.
41. The process of claim 26, wherein the compound is
derivatized.
42. The process of claim 26, wherein the derivatized compound
comprises a cyclic portion, a cross-linking site, a non-peptidyl
linkage, an N-terminal replacement, a C-terminal replacement, or a
modified amino acid moiety.
43. The process of claim 26 wherein the Fc domain is an IgG Fc
domain.
44. The process of claim 26, wherein the vehicle is an IgG1 Fc
domain.
45. The process of claim 26, wherein the vehicle comprises the
sequence of SEQ ID NO: 2.
46. The process of claim 26, wherein the compound prepared is of
the formula (X.sup.1).sub.a-F.sup.1-(X.sup.2).sub.b and multimers
thereof, wherein: F.sup.1 is an Fc domain; X.sup.1 and X.sup.2 are
each independently selected from -(L.sup.1).sub.c-P.sup.1,
-(L.sup.1).sub.c-P.sup.1-(L.sup.2).sub.d-P.sup.2,
-(L.sup.1).sub.c-P.sup.-
1-(L.sup.2).sub.d-P.sup.2(L.sup.3).sub.e-P.sup.3, and
-(L.sup.1).sub.c-P.sup.1-(L.sup.2).sub.d-P.sup.2-(L.sup.3).sub.e-P.sup.3--
(L.sup.4).sub.f-P.sup.4 P.sup.1, P.sup.2, P.sup.3, and P.sup.4 are
each independently sequences of pharmacologically active peptides;
L.sup.1, L.sup.2, L.sup.3, and L.sup.4 are each independently
linkers; and a, b, c, d, e, and f are each independently 0 or 1,
provided that at least one of a and b is 1.
47. The process of claim 46, wherein the compound prepared is of
the formulae X.sup.1-F.sup.1 or F.sup.1-X.sup.2.
48. The process of claim 46, wherein the compound prepared is of
the formulae F.sup.1-(L.sup.1).sub.c-P.sup.1 or
F.sup.1-(L.sup.1).sub.c-P.sup- .1-(L.sup.2).sub.d-P.sup.2.
49. The process of claim 46, wherein F.sup.1 is an IgG Fc
domain.
50. The process of claim 46, wherein F.sup.1 is an IgG1 Fc
domain.
51. The process of claim 46, wherein F.sup.1 comprises the sequence
of SEQ ID NO: 2.
Description
BACKGROUND OF THE INVENTION
[0001] Recombinant proteins are an emerging class of therapeutic
agents. Such recombinant therapeutics have engendered advances in
protein formulation and chemical modification. Such modifications
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.
[0002] One useful modification is combination with the "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.
1TABLE 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. CD30-L anaplastic lymphoma; T-
5,480,981 cell leukemia Murine Fc.gamma.2a IL-10 anti-inflammatory;
Zheng et al. (1995), J. Immunol. transplant rejection 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, TNF receptor inflammation, U.S. Pat. No.
5,808,029, IgM, or IgE autoimmune 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.gamma.1 Med. 174: 561-9
[0003] A much different approach to development of therapeutic
agents is peptide library screening. The interaction of a protein
ligand with its receptor often takes place at a relatively large
interface. However, as demonstrated for human growth hormone and
its receptor, only a few key residues at the interface contribute
to most of the binding energy. Clackson et al. (1995), Science 267:
383-6. The bulk of the protein ligand merely displays the binding
epitopes in the right topology or serves functions unrelated to
binding. Thus, molecules of only "peptide" length (2 to 40 amino
acids) can bind to the receptor protein of a given large protein
ligand. Such peptides may mimic the bioactivity of the large
protein ligand ("peptide agonists") or, through competitive
binding, inhibit the bioactivity of the large protein ligand
("peptide antagonists").
[0004] Phage display peptide libraries have emerged as a powerful
method in identifying such peptide agonists and antagonists. See,
for example, Scott et al. (1990), Science 249: 386; Devlin et al.
(1990), Science 249: 404; U.S. Pat. No. 5,223,409, issued Jun. 29,
1993; U.S. Pat. No. 5,733,731, issued Mar. 31, 1998; U.S. Pat. No.
5,498,530, issued Mar. 12, 1996; U.S. Pat. No. 5,432,018, issued
Jul. 11, 1995; U.S. Pat. No. 5,338,665, issued Aug. 16, 1994; U.S.
Pat. No. 5,922,545, issued Jul. 13, 1999; WO 96/40987, published
Dec. 19, 1996; and WO 98/15833, published Apr. 16, 1998 (each of
which is incorporated by reference). In such libraries, random
peptide sequences are displayed by fusion with coat proteins of
filamentous phage. Typically, the displayed peptides are
affinity-eluted against an antibody-immobilized extracellular
domain of a receptor. The retained phages may be enriched by
successive rounds of affinity purification and repropagation. The
best binding peptides may be sequenced to identify key residues
within one or more structurally related families of peptides. See,
e.g., Cwirla et al. (1997), Science 276: 1696-9, in which two
distinct families were identified. The peptide sequences may also
suggest which residues may be safely replaced by alanine scanning
or by mutagenesis at the DNA level. Mutagenesis libraries may be
created and screened to further optimize the sequence of the best
binders. Lowman (1997), Ann. Rev. Biophys. Biomol. Struct. 26:
401-24.
[0005] 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 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.
[0006] Other methods compete with phage display in peptide
research. A peptide library can be fused to the carboxyl terminus
of the lac repressor and expressed in E. coli. Another E.
coli-based method allows display on the cell's outer membrane by
fusion with a peptidoglycan-associated lipoprotein (PAL).
Hereinafter, these and related methods are collectively referred to
as "E. coli display." In another method, translation of random RNA
is halted prior to ribosome release, resulting in a library of
polypeptides with their associated RNA still attached. Hereinafter,
this and related methods are collectively referred to as "ribosome
display." Other methods employ chemical linkage of peptides to RNA;
see, for example, Roberts & Szostak (1997), Proc. Natl. Acad.
Sci. USA, 94:12297-303. Hereinafter, this and related methods are
collectively referred to as "RNA-peptide screening." Chemically
derived peptide libraries have been developed in which peptides are
immobilized on stable, non-biological materials, such as
polyethylene rods or solvent-permeable resins. Another chemically
derived peptide library uses photolithography to scan peptides
immobilized on glass slides. Hereinafter, these and related methods
are collectively referred to as "chemical-peptide screening."
Chemical-peptide screening may be advantageous in that it allows
use of D-amino acids and other unnatural analogues, as well as
non-peptide elements. Both biological and chemical methods are
reviewed in Wells & Lowman (1992), Curr. Opin. Biotechnol. 3:
355-62.
[0007] Conceptually, one may discover peptide mimetics of any
protein using phage display and the other methods mentioned above.
These methods have been used for epitope mapping, for
identification of critical amino acids in protein-protein
interactions, and as leads for the discovery of new therapeutic
agents. E.g., Cortese et al. (1996), Curr. Opin. Biotech. 7:
616-21. Peptide libraries are now being used most often in
immunological studies, such as epitope mapping. Kreeger (1996), The
Scientist 10(13): 19-20.
[0008] Of particular interest here is use of peptide libraries and
other techniques in the discovery of pharmacologically active
peptides. A number of such peptides identified in the art are
summarized in Table 2. The peptides are described in the listed
publications, each of which is hereby incorporated by reference.
The pharmacologic activity of the peptides is described, and in
many instances is followed by a shorthand term 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 monclonal antibody.
2TABLE 2 Pharmacologically active peptides Binding partner/ Form of
protein of Pharmacologic peptide interest.sup.a activity Reference
intrapeptide EPO receptor EPO-mimetic Wrighton et al. (1996),
disulfide- Science 273: 458-63; bonded U.S. 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 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 Paukovits et
al. (1984), linked dimer hematopoiesis Hoppe-Seylers Z.
("G-CSF-mimetic") Physiol. Chem. 365: 303-11; Laerum et al. (1988),
Exp. Hemat. 16: 274-80 alkylene- G-CSF-mimetic Bhatnagar et al.
(1996), 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
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(1984), Int. J. Immunopharmacol, 6: 141-6. intrapeptide CTLA4 MAb
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Takasaki et al. (1997), Nature Biotech. 15: 1266-70; WO 98/53842,
published Dec. 3, 1998 linear TNF-.alpha. receptor TNF-.alpha.
antagonist Chirinos-Rojas ( ), J. Imm., 5621-5626. intrapeptide C3b
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antiphospholipid Natl. Acad. Sci. USA 96: glycoprotein-i syndrome
(APS), 5164-8 (.beta.2GPI) thromboembolic antibodies phenomena,
thrombocytopenia, and recurrent fetal loss linear T Cell Receptor
diabetes WO 96/11214, published beta chain Apr. 18, 1996 .sup.aThe
protein listed in this column may be bound by the associated
peptide (e.g., EPO receptor, IL-1 receptor) or mimicked by the
associated peptide. The references listed for each clarify whether
the molecule is bound by or mimicked by the peptides. .sup.bFTS is
a thymic hormone mimicked by the molecule of this invention rather
than a receptor bound by the molecule of this invention.
[0009] Peptides identified by peptide library screening have been
regarded as "leads" in development of therapeutic agents rather
than as therapeutic agents themselves. Like other proteins and
peptides, they would be rapidly removed in vivo either by renal
filtration, cellular clearance mechanisms in the
reticuloendothelial system, or proteolytic degradation. Francis
(1992), Focus on Growth Factors 3: 4-11. As a result, the art
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. The art would
benefit from a process by which such peptides could more readily
yield therapeutic agents.
SUMMARY OF THE INVENTION
[0010] The present invention concerns a process by which the in
vivo half-life of one or more biologically active peptides is
increased by fusion with a vehicle. In this invention,
pharmacologically active compounds are prepared by a process
comprising:
[0011] a) selecting at least one peptide that modulates the
activity of a protein of interest; and
[0012] b) preparing a pharmacologic agent comprising at least one
vehicle covalently linked to at least one amino acid sequence of
the selected peptide.
[0013] The preferred vehicle is an Fc domain. The peptides screened
in step (a) are preferably expressed in a phage display library.
The vehicle and the peptide may be linked through the N- or
C-terminus of the peptide or the vehicle, as described further
below. Derivatives of the above compounds (described below) are
also encompassed by this invention.
[0014] The compounds of this invention may be prepared by standard
synthetic methods, recombinant DNA techniques, or any other methods
of preparing peptides and fusion proteins. Compounds of this
invention that encompass non-peptide portions may be synthesized by
standard organic chemistry reactions, in addition to standard
peptide chemistry reactions when applicable.
[0015] The primary use contemplated is as therapeutic or
prophylactic agents. The vehicle-linked peptide may have activity
comparable to--or even greater than--the natural ligand mimicked by
the peptide. In addition, certain natural ligand-based therapeutic
agents might induce antibodies against the patient's own endogenous
ligand; the vehicle-linked peptide avoids this pitfall by having
little or typically no sequence identity with the natural
ligand.
[0016] Although mostly contemplated as therapeutic agents,
compounds of this invention may also be useful in screening for
such agents. For example, one could use an Fc-peptide (e.g., Fc-SH2
domain peptide) in an assay employing anti-Fc coated plates. The
vehicle, especially Fc, may make insoluble peptides soluble and
thus useful in a number of assays.
[0017] The compounds of this invention may be used for therapeutic
or prophylactic purposes by formulating them with appropriate
pharmaceutical carrier materials and administering an effective
amount to a patient, such as a human (or other mammal) in need
thereof. Other related aspects are also included in the instant
invention.
[0018] Numerous additional aspects and advantages of the present
invention will become apparent upon consideration of the figures
and detailed description of the invention.
BRIEF DESCRIPTION OF THE FIGURES
[0019] FIG. 1 shows a schematic representation of an exemplary
process of the invention. In this preferred process, the vehicle is
an Fc domain, which is linked to the peptide covalently by
expression from a DNA construct encoding both the Fc domain and the
peptide. As noted in FIG. 1, the Fc domains spontaneously form a
dimer in this process.
[0020] FIG. 2 shows exemplary Fc dimers that may be derived from an
IgG1 antibody. "Fc" in the figure represents any of the Fc variants
within the meaning of "Fc domain" herein. "X.sup.1" and "X.sup.2"
represent peptides or linker-peptide combinations as defined
hereinafter. The specific dimers are as follows:
[0021] A, D: Single disulfide-bonded dimers. IgG1 antibodies
typically have two disulfide bonds at the hinge region between the
constant and variable domains. The Fc domain in FIGS. 2A and 2D may
be formed by truncation between the two disulfide bond sites or by
substitution of a cysteinyl residue with an unreactive residue
(e.g., alanyl). In FIG. 2A, the Fc domain is linked at the amino
terminus of the peptides; in 2D, at the carboxyl terminus.
[0022] B, E: Doubly disulfide-bonded dimers. This Fc domain may be
formed by truncation of the parent antibody to retain both
cysteinyl residues in the Fc domain chains or by expression from a
construct including a sequence encoding such an Fc domain. In FIG.
2B, the Fc domain is linked at the amino terminus of the peptides;
in 2E, at the carboxyl terminus.
[0023] C, F: Noncovalent dimers. This Fc domain may be formed by
elimination of the cysteinyl residues by either truncation or
substitution. One may desire to eliminate the cysteinyl residues to
avoid impurities formed by reaction of the cysteinyl residue with
cysteinyl residues of other proteins present in the host cell. The
noncovalent bonding or the Fc domains is sufficient to hold
together the dimer.
[0024] Other dimers may be formed by using Fc domains derived from
different types of antibodies (e.g., IgG2, IgM).
[0025] FIG. 3 shows the structure of preferred compounds of the
invention that feature tandem repeats of the pharmacologically
active peptide. FIG. 3A shows a single chain molecule and may also
represent the DNA construct for the molecule. FIG. 3B shows a dimer
in which the linker-peptide portion is present on only one chain of
the dimer. FIG. 3C shows a dimer having the peptide portion on both
chains. The dimer of FIG. 3C will form spontaneously in certain
host cells upon expression of a DNA construct encoding the single
chain shown in FIG. 3A. In other host cells, the cells could be
placed in conditions favoring formation of dimers or the dimers can
be formed in vitro.
[0026] FIG. 4 shows exemplary nucleic acid and amino acid sequences
(SEQ ID NOS: 1 and 2, respectively) of human IgG1 Fc that may be
used in this invention.
[0027] FIG. 5 shows a synthetic scheme for the preparation of
PEGylated peptide 19 (SEQ ID NO: 3).
[0028] FIG. 6 shows a synthetic scheme for the preparation of
PEGylated peptide 20 (SEQ ID NO: 4).
[0029] FIG. 7 shows the nucleotide and amino acid sequences (SEQ ID
NOS: 5 and 6, respectively) of the molecule identified as "Fc-TMP"
in Example 2 hereinafter.
[0030] FIG. 8 shows the nucleotide and amino acid sequences (SEQ.
ID. NOS: 7 and 8, respectively) of the molecule identified as
"Fc-TMP-TMP" in Example 2 hereinafter.
[0031] FIG. 9 shows the nucleotide and amino acid sequences (SEQ.
ID. NOS: 9 and 10, respectively) of the molecule identified as
"TMP-TMP-Fc" in Example 2 hereinafter.
[0032] FIG. 10 shows the nucleotide and amino acid sequences (SEQ.
ID. NOS: 11 and 12, respectively) of the molecule identified as
"TMP-Fc" in Example 2 hereinafter.
[0033] FIG. 11 shows the number of platelets generated in vivo in
normal female BDF1 mice treated with one 100 .mu.g/kg bolus
injection of various compounds, with the terms defined as
follows.
[0034] PEG-MGDF: 20 kD average molecular weight PEG attached by
reductive amination to the N-terminal amino group of amino acids
1-163 of native human TPO, which is expressed in E. coli (so that
it is not glycosylated);
[0035] TMP: the TPO-mimetic peptide having the amino acid sequence
IEGPTLRQWLAARA (SEQ ID NO: 13); TMP-TMP: the TPO-mimetic peptide
having the amino acid sequence
IEGPTLRQWLAARA-GGGGGGGG-IEGPTLRQWLAARA (SEQ ID NO: 14);
[0036] PEG-TMP-TMP: the peptide of SEQ ID NO: 14, wherein the PEG
group is a 5 kD average molecular weight PEG attached as shown in
FIG. 6;
[0037] Fc-TMP-TMP: the compound of SEQ ID NO: 8 (FIG. 8) dimerized
with an identical second monomer (i.e., Cys residues 7 and 10 are
bound to the corresponding Cys residues in the second monomer to
form a dimer, as shown in FIG. 2); and
[0038] TMP-TMP-Fc is the compound of SEQ ID NO: 10 (FIG. 9)
dimerized in the same way as TMP-TMP-Fc except that the Fc domain
is attached at the C-terminal end rather than the N-terminal end of
the TMP-TMP peptide.
[0039] FIG. 12 shows the number of platelets generated in vivo in
normal BDF1 mice treated with various compounds delivered via
implanted osmotic pumps over a 7-day period. The compounds are as
defined for FIG. 7.
[0040] FIG. 13 shows the nucleotide and amino acid sequences (SEQ.
ID. NOS: 15 and 16, respectively) of the molecule identified as
"Fc-EMP" in Example 3 hereinafter.
[0041] FIG. 14 shows the nucleotide and amino acid sequences (SEQ
ID NOS: 17 and 18, respectively) of the molecule identified as
"EMP-Fc" in Example 3 hereinafter.
[0042] FIG. 15 shows the nucleotide and amino acid sequences (SEQ
ID NOS:19 and 20, respectively) of the molecule identified as
"EMP-EMP-Fc" in Example 3 hereinafter.
[0043] FIG. 16 shows the nucleotide and amino acid sequences (SEQ
ID NOS: 21 and 22, respectively) of the molecule identified as
"Fc-EMP-EMP" in Example 3 hereinafter.
[0044] FIGS. 17A and 17B show the DNA sequence (SEQ ID NO: 23)
inserted into pCFM1656 between the unique AatII (position #4364 in
pCFM1656) and SacII (position #4585 in pCFM1656) restriction sites
to form expression plasmid pAMG21 (ATCC accession no. 98113).
[0045] FIG. 18A shows the hemoglobin, red blood cells, and
hematocrit generated in vivo in normal female BDF1 mice treated
with one 100 .mu.g/kg bolus injection of various compounds. FIG.
18B shows the same results with mice treated with 100 .mu.g/kg per
day delivered the same dose by 7-day micro-osmotic pump with the
EMPs delivered at 100 .mu.g/kg, rhEPO at 30 U/mouse. (In both
experiments, neutrophils, lymphocytes, and platelets were
unaffected.) In these figures, the terms are defined as
follows.
[0046] Fc-EMP: the compound of SEQ ID NO: 16 (FIG. 13) dimerized
with an identical second monomer (i.e., Cys residues 7 and 10 are
bound to the corresponding Cys residues in the second monomer to
form a dimer, as shown in FIG. 2);
[0047] EMP-Fc: the compound of SEQ ID NO: 18 (FIG. 14) dimerized in
the same way as Fc-EMP except that the Fc domain is attached at the
C-terminal end rather than the N-terminal end of the EMP
peptide.
[0048] "EMP-EMP-Fc" refers to a tandem repeat of the same peptide
(SEQ ID NO: 20) attached to the same Fc domain by the carboxyl
terminus of the peptides. "Fc-EMP-EMP" refers to the same tandem
repeat of the peptide but with the same Fc domain attached at the
amino terminus of the tandem repeat. All molecules are expressed in
E. coli and so are not glycosylated.
[0049] FIGS. 19A and 19B show the nucleotide and amino acid
sequences (SEQ ID NOS: 1055 and 1056) of the Fc-TNF-.alpha.
inhibitor fusion molecule described in Example 4 hereinafter.
[0050] FIGS. 20A and 20B show the nucleotide and amino acid
sequences (SEQ ID NOS: 1057 and 1058) of the TNF-.alpha.
inhibitor-Fc fusion molecule described in Example 4
hereinafter.
[0051] FIGS. 21A and 21B show the nucleotide and amino acid
sequences (SEQ ID NOS: 1059 and 1060) of the Fc-IL-1 antagonist
fusion molecule described in Example 5 hereinafter.
[0052] FIGS. 22A and 22B show the nucleotide and amino acid
sequences (SEQ ID NOS: 1061 and 1062) of the IL-1 antagonist-Fc
fusion molecule described in Example 5 hereinafter.
[0053] FIGS. 23A, 23B, and 23C show the nucleotide and amino acid
sequences (SEQ ID NOS: 1063 and 1064) of the Fc-VEGF antagonist
fusion molecule described in Example 6 hereinafter.
[0054] FIGS. 24A and 24B show the nucleotide and amino acid
sequences (SEQ ID NOS: 1065 and 1066) of the VEGF antagonist-Fc
fusion molecule described in Example 6 hereinafter.
[0055] FIGS. 25A and 25B show the nucleotide and amino acid
sequences (SEQ ID NOS: 1067 and 1068) of the Fc-MMP inhibitor
fusion molecule described in Example 7 hereinafter.
[0056] FIGS. 26A and 26B show the nucleotide and amino acid
sequences (SEQ ID NOS: 1069 and 1070) of the MMP inhibitor-Fc
fusion molecule described in Example 7 hereinafter.
DETAILED DESCRIPTION OF THE INVENTION
[0057] Definition of Terms
[0058] The terms used throughout this specification are defined as
follows, unless otherwise limited in specific instances.
[0059] 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.
[0060] 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 (which is
preferred) as well as a linear polymer (e.g., polyethylene glycol
(PEG), polylysine, dextran, etc.); a branched-chain polymer (see,
for example, U.S. Pat. No. 4,289,872 to Denkenwalter et al., issued
Sep. 15, 1981; U.S. Pat. No. 5,229,490 to Tam, issued Jul. 20,
1993; WO 93/21259 by Frechet et al., published Oct. 28, 1993); a
lipid; a cholesterol group (such as a steroid); a carbohydrate or
oligosaccharide; or any natural or synthetic protein, polypeptide
or peptide that binds to a salvage receptor. Vehicles are further
described hereinafter.
[0061] 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 preferably of human origin and may be any of the
immunoglobulins, although IgG1 and IgG2 are preferred. Native Fc's
are made up of monomeric polypeptides that may be linked into
dimeric or multimeric forms by covalent (i.e., disulfide bonds) and
non-covalent association. The number of intermolecular disulfide
bonds between monomeric subunits of native Fc molecules ranges from
1 to 4 depending on class (e.g., IgG, IgA, IgE) or subclass (e.g.,
IgG1, IgG2, IgG3, IgA1, IgGA2). One example of a native Fc is a
disulfide-bonded dimer resulting from papain digestion of an IgG
(see Ellison et al. (1982), Nucleic Acids Res. 10: 4071-9). The
term "native Fc" as used herein is generic to the monomeric,
dimeric, and multimeric forms.
[0062] 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 Sep. 25, 1997) and WO 96/32478 describe exemplary Fc
variants, as well as interaction with the salvage receptor, and are
hereby incorporated by reference. Thus, the term "Fc variant"
comprises a molecule or sequence that is humanized from a non-human
native Fc. Furthermore, a native Fc comprises sites that may be
removed because they provide structural features or biological
activity that are not required for the fusion molecules of the
present invention. Thus, the term "Fc variant" comprises a molecule
or sequence that lacks one or more native Fc sites or residues that
affect or are involved in (1) disulfide bond formation, (2)
incompatibility with a selected host cell (3) N-terminal
heterogeneity upon expression in a selected host cell, (4)
glycosylation, (5) interaction with complement, (6) binding to an
Fc receptor other than a salvage receptor, or (7)
antibody-dependent cellular cytotoxicity (ADCC). Fc variants are
described in further detail hereinafter.
[0063] The term "Fc domain" encompasses native Fc and Fc variant
molecules and sequences as defined above. As with Fc variants and
native Fc's, the term "Fc domain" includes molecules in monomeric
or multimeric form, whether digested from whole antibody or
produced by other means.
[0064] 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.
[0065] The term "dimer" as applied to Fc domains or molecules
comprising Fc domains refers to molecules having two polypeptide
chains associated covalently or non-covalently. Thus, exemplary
dimers within the scope of this invention are as shown in FIG.
2.
[0066] The terms "derivatizing" and "derivative" or "derivatized"
comprise processes and resulting compounds respectively in which
(1) the compound has a cyclic portion; for example, cross-linking
between cysteinyl residues within the compound; (2) the compound is
cross-linked or has a cross-linking site; for example, the compound
has a cysteinyl residue and thus forms cross-linked dimers in
culture or in vivo; (3) one or more peptidyl linkage is replaced by
a non-peptidyl linkage; (4) the N-terminus is replaced by
--NRR.sup.1, NRC(O)R.sup.1, --NRC(O)OR.sup.1,
--NRS(O).sub.2R.sup.1, --NHC(O)NHR, a succinimide group, or
substituted or unsubstituted benzyloxycarbonyl-NH--, wherein R and
R.sup.1 and the ring substituents are as defined hereinafter; (5)
the C-terminus is replaced by --C(O)R.sup.2 or --NR.sup.3R.sup.4
wherein R.sup.2, R.sup.3 and R.sup.4 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.
[0067] The term "peptide" refers to molecules of 2 to 40 amino
acids, with molecules of 3 to 20 amino acids preferred and those of
6 to 15 amino acids most preferred. Exemplary peptides may be
randomly generated by any of the methods cited above, carried in a
peptide library (e.g., a phage display library), or derived by
digestion of proteins.
[0068] 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, RNA-peptide screening,
chemical screening, and the like.
[0069] 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.
[0070] 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. Thus, the term
"EPO-mimetic peptide" comprises any peptides that can be identified
or derived as described in Wrighton et al. (1996), Science 273:
458-63, Naranda et al. (1999), Proc. Natl. Acad. Sci. USA 96:
7569-74, or any other reference in Table 2 identified as having
EPO-mimetic subject matter. Those of ordinary skill in the art
appreciate that each of these references enables one to select
different peptides than actually disclosed therein by following the
disclosed procedures with different peptide libraries.
[0071] The term "TPO-mimetic peptide" comprises peptides that can
be identified or derived as described in Cwirla et al. (1997),
Science 276: 1696-9, U.S. Pat. Nos. 5,869,451 and 5,932,946 and any
other reference in Table 2 identifed as having TPO-mimetic subject
matter, as well as the U.S. patent application, "Thrombopoietic
Compounds," filed on even date herewith and 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.
[0072] 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.
[0073] 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.
[0074] 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.
[0075] 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.
[0076] 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.
[0077] 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.
[0078] Additionally, physiologically acceptable salts of the
compounds of this invention are also encompassed herein. By
"physiologically acceptable salts" is meant any salts that are
known or later discovered to be pharmaceutically acceptable. Some
specific examples are: acetate; trifluoroacetate; hydrohalides,
such as hydrochloride and hydrobromide; sulfate; citrate; tartrate;
glycolate; and oxalate.
[0079] Structure of Compounds
[0080] In General. In the compositions of matter prepared in
accordance with this invention, the peptide may be attached to the
vehicle through the peptide's N-terminus or C-terminus. Thus, the
vehicle-peptide molecules of this invention may be described by the
following formula I:
(X.sup.1).sub.a-F.sup.1-(X.sup.2).sub.b I
[0081] wherein:
[0082] F.sup.1 is a vehicle (preferably an Fc domain);
[0083] X.sup.1 and X.sup.2 are each independently selected from
-(L.sup.1).sub.c-P.sup.1,
-(L.sup.1).sub.c-P.sup.1-(L.sup.1).sub.d-P.sup.- 2,
-(L.sup.1).sub.c-P.sup.1-(L.sup.2).sub.d-P.sup.2-(L.sup.3).sub.e-P.sup.-
3, and
-(L.sup.1).sub.c-P.sup.1-(L.sup.2).sub.d-P.sup.2-(L.sup.3).sub.e-P.-
sup.3-(L.sup.4).sub.f-P.sup.4
[0084] P.sup.1, P.sup.2, P.sup.3, and P.sup.4 are each
independently sequences of pharmacologically active peptides;
[0085] L.sup.1, L.sup.2, L.sup.3, and L.sup.4 are each
independently linkers; and
[0086] a, b, c, d, e, and f are each independently 0 or 1, provided
that at least one of a and b is 1.
[0087] Thus, compound I comprises preferred compounds of the
formulae
X.sup.1-F.sup.1 II
[0088] and multimers thereof wherein F.sup.1 is an Fc domain and is
attached at the C-terminus of X.sup.1;
F.sup.1-X.sup.2 III
[0089] and multimers thereof wherein F.sup.1 is an Fc domain and is
attached at the N-terminus of X.sup.2;
F.sup.1-(L.sup.1).sub.c-P.sup.1 IV
[0090] and multimers thereof wherein F.sup.1 is an Fc domain and is
attached at the N-terminus of -(L.sup.1).sub.c-P.sup.1; and
F-(L.sup.1).sub.c-P.sup.1-(L.sup.2).sub.d-P.sup.2 V
[0091] and multimers thereof wherein F.sup.1 is an Fc domain and is
attached at the N-terminus of -L.sup.1-P.sup.1-L.sup.2-P.sup.2.
[0092] 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-.alpha., and TGF-.beta.. 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.
[0093] Phage display, in particular, is useful in generating
peptides for use in the present invention. It has been stated that
affinity selection from libraries of random peptides can be used to
identify peptide ligands for any site of any gene product. Dedman
et al. (1993), J. Biol. Chem. 268: 23025-30. Phage display is
particularly well suited for identifying peptides that bind to such
proteins of interest as cell surface receptors or any proteins
having linear epitopes. Wilson et al. (1998), Can. T. Microbiol.
44: 313-29; Kay et al. (1998), Drug Disc. Today 3: 370-8. Such
proteins are extensively reviewed in Herz et al. (1997), T.
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.
[0094] A particularly preferred group of peptides are those that
bind to cytokine receptors. Cytokines have recently been classified
according to their receptor code. See Inglot (1997), Archivum
Immunologiae et Therapiae Experimentalis 45: 353-7, which is hereby
incorporated by reference. Among these receptors, most preferred
are the CKRs (family I in Table 3). The receptor classification
appears in Table 3.
3TABLE 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
.gamma.Cr cytokines IL-9, IL-13, IL- (CKR) 15 2. IL-3, IL-5, GM- 2.
shared GP CSF 140 .beta.R 3. IL-6, IL-11, IL- 3. 3.shared 12, LIF,
OSM, RP 130 CNTF, leptin (OB) 4. G-CSF, EPO, 4. "single TPO, PRL,
GH chain" R 5. IL-17, HVS-IL- 5. other R.sup.c 17 II. IL-10 ligands
IL-10, BCRF-1, II. IL-10 R HSV-IL-10 III. Interferons 1.
IFN-.alpha.l, .alpha.2, .alpha.4, III. Interferon R 1. IFNAR m, t,
IFN-.beta..sup.d 2. IFN-.gamma. 2. IFNGR IV. IL-1 ligands 1.
IL-1.alpha., IL-1.beta., IL- IV. IL-1R 1Ra V. TNF ligands 1.
TNF-.alpha., TNF-.beta. V. NGF/TNF R.sup.e (LT), FAS1, CD40 L,
CD30L, CD27 L VI. Chemokines 1. .alpha. chemokines: VI. Chemokine R
1. CXCR IL-8, GRO .alpha., .beta., .gamma., IF-10, PF-4, SDF-1 2.
.beta. chemokines: 2. CCR MIP1.alpha., MIP1.beta., MCP-1, 2, 3, 4,
RANTES, eotaxin 3. .gamma. chemokines: 3. CR lymphotactin 4.
DARC.sup.f VII. Growth 1.1 SCF, M-CSF, VII. RKF 1. TK factors
PDGF-AA, AB, sub-family BB, FLT-3L, 1.1 IgTK VEGF, SSV- III R PDGF
1.2 FGF.alpha., FGF.beta. 1.2 IgTK 1.3 EGF, TGF-.alpha., IV R
VV-F19 (EGF- 1.3 Cysteine- like) rich TK-I 1.4 IGF-I, IGF-II, 1.4
Cysteine Insulin rich TK-II 1.5 NGF, BDNF, 1.5 Cysteine NT-3,
NT-4.sup.g knot TK V 2. TGF-.beta.1, .beta.2, .beta.3 2. STK
subfamily.sup.h .sup.cIL-17R belongs to the CKR family but is not
assigned to any of the 4 indicated subjamilies. .sup.dOther 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. .sup.eTNF receptors use multiple, distinct
intracellular molecules for signal transduction including "death
domain" of FAS R and 55 kDa TNF-.alpha.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 G protein-coupled,
seven transmembrane (7TM, serpentine) domain receptors. .sup.fThe
Duffy blood group antigen (DARC) is an erythrocyte receptor that
can bind several different chemokines. It belongs to the
immunoglobulin superfamily but characteristics of its signal
transduction events remain unclear. .sup.gThe neurotrophic
cytokines can associate with NGF/TNF receptors also. .sup.hSTKS may
encompass many other TGF-.beta.-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.
[0095] Exemplary peptides for this invention appear in Tables 4
through 20 below. These peptides may be prepared by methods
disclosed in the art. 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 "A" 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 --NH.sub.2. For derivatives in which amino acid
residues are substituted by moieties other than amino acid
residues, the substitutions are denoted by .sigma., which signifies
any of the moieties described in Bhatnagar et al. (1996), J. Med.
Chem. 39: 3814-9 and Cuthbertson et al. (1997), T. Med. Chem. 40:
2876-82, which are incorporated by reference. The J substituent and
the Z substituents (Z.sub.5, Z.sub.6, . . . Z.sub.40) 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 X.sub.2 through X.sub.11 and the
integer "n" are as defined in WO 96/40772, which is incorporated by
reference. The substituents ".PSI.," ".THETA.," and "+" are as
defined in Sparks et al. (1996), Proc. Natl. Acad. Sci. 93: 1540-4,
which is hereby incorporated by reference. X.sub.4, X.sub.5,
X.sub.6, and X.sub.7 are as defined in U.S. Pat. No. 5,773,569,
which is hereby incorporated by reference, except that: for
integrin-binding peptides, X.sub.1, X.sub.2, X.sub.3, X.sub.4,
X.sub.5, X.sub.6, X.sub.7, and X.sub.8 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, X.sup.1, X.sup.1',
X.sup.1", X.sub.2, X.sub.3, X.sub.4, X.sub.5, X.sub.6 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. AA.sub.1, AA.sub.2, AB.sub.1, AB.sub.2,
and AC are as defined in International application WO 98/53842,
published Dec. 3, 1998, which is incorporated by reference.
X.sup.1, X.sup.2, X.sup.3, and X.sup.4 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.
4TABLE 4 IL-1 antagonist peptide sequences Sequence/structure SEQ
ID NO: Z.sub.11Z.sub.7Z.sub.8QZ.sub- .5Y.sub.4Z.sub.9Z.sub.10 212
XXQZ.sub.5YZ.sub.6XX 907 Z.sub.7XQZ.sub.5YZ.sub.6XX 908
Z.sub.7Z.sub.8QZ.sub.5YZ.sub.6Z.sub- .9Z.sub.10 909
Z.sub.11Z.sub.7Z.sub.8QZ.sub.5YZ.sub.6Z.sub.9Z.sub.1- 0 910
Z.sub.12Z.sub.13Z.sub.14Z.sub.15Z.sub.16Z.sub.17Z.sub.18Z.sub-
.19Z.sub.20Z.sub.21Z.sub.22Z.sub.11Z.sub.7Z.sub.8QZ.sub.5YZ.sub.6Z.sub.9Z.-
sub.10L 917
Z.sub.23NZ.sub.24Z.sub.39Z.sub.25Z.sub.26Z.sub.27Z.sub.-
28Z.sub.29Z.sub.30Z.sub.40 979 TANVSSFEWTPYYWQPYALPL 213
SWTDYGYWQPYALPISGL 214 ETPFTWEESNAYYWQPYALPL 215
ENTYSPNWADSMYWQPYALPL 216 SVGEDHNFWTSEYWQPYALPL 217
DGYDRWRQSGERYWQPYALPL 218 FEWTPGYWQPY 219 FEWTPGYWQHY 220
FEWTPGWYQJY 221 AcFEWTPGWYQJY 222 FEWTPGWpYQJY 223 FAWTPGYWQJY 224
FEWAPGYWQJY 225 FEWVPGYWQJY 226 FEWTPGYWQJY 227 AcFEWTPGYWQJY 228
FEWTPaWYQJY 229 FEWTPSarWYQJY 230 FEWTPGYYQPY 231 FEWTPGWWQPY 232
FEWTPNYWQPY 233 FEWTPvYWQJY 234 FEWTPecGYWQJY 235 FEWIPAibYWQJY 236
FEWTSarGYWQJY 237 FEWTPGYWQPY 238 FEWTPGYWQHY 239 FEWTPGWYQJY 240
AcFEWTPGWYQJY 241 FEWTPGW-pY-QJY 242 FAWTPGYWQJY 243 FEWAPGYWQJY
244 FEWVPGYWQJY 245 FEWTPGYWQJY 246 AcFEWTPGYWQJY 247 FEWTPAWYQJY
248 FEWTPSarWYQJY 249 FEWTPGYYQPY 250 FEWTPGWWQPY 251 FEWTPNYWQPY
252 FEWTPVYWQJY 253 FEWTPecGYWQJY 254 FEWTPAibYWQJY 255
FEWTSarGYWQJY 256 FEWTPGYWQPYALPL 257 1NapEWTPGYYQJY 258
YEWTPGYYQJY 259 FEWVPGYYQJY 260 FEWTPSYYQJY 261 FEWTPNYYQJY 262
TKPR 263 RKSSK 264 RKQDK 265 NRKQDK 266 RKQDKR 267 ENRKQDKRF 268
VTKFYF 269 VTKFY 270 VTDFY 271 SHLYWQPYSVQ 671 TLVYWQPYSLQT 672
RGDYWQPYSVQS 673 VHVYWQPYSVQT 674 RLVYWQPYSVQT 675 SRVWFQPYSLQS 676
NMVYWQPYSIQT 677 SVVFWQPYSVQT 678 TFVYWQPYALPL 679 TLVYWQPYSIQR 680
RLVYWQPYSVQR 681 SPVFWQPYSIQI 682 WIEWWQPYSVQS 683 SLIYWQPYSLQM 684
TRLYWQPYSVQR 685 RCDYWQPYSVQT 686 MRVFWQPYSVQN 687 KIVYWQPYSVQT 688
RHLYWQPYSVQR 689 ALVWWQPYSEQI 690 SRVWFQPYSLQS 691 WEQPYALPLE 692
QLVWWQPYSVQR 693 DLRYWQPYSVQV 694 ELVWWQPYSLQL 695 DLVWWQPYSVQW 696
NGNYWQPYSFQV 697 ELVYWQPYSIQR 698 ELMYWQPYSVQE 699 NLLYWQPYSMQD 700
GYEWYQPYSVQR 701 SRVWYQPYSVQR 702 LSEQYQPYSVQR 703 GGGWWQPYSVQR 704
VGRWYQPYSVQR 705 VHVYWQPYSVQR 706 QARWYQPYSVQR 707 VHVYWQPYSVQT 708
RSVYWQPYSVQR 709 TRVWFQPYSVQR 710 GRIWFQPYSVQR 711 GRVWFQPYSVQR 712
ARTWYQPYSVQR 713 ARVWWQPYSVQM 714 RLMFYQPYSVQR 715 ESMWYQPYSVQR 716
HFGWWQPYSVHM 717 ARFWWQPYSVQR 718 RLVYWQ PYAPIY 719 RLVYWQ PYSYQT
720 RLVYWQ PYSLPI 721 RLVYWQ PYSVQA 722 SRVWYQ PYAKGL 723 SRVWYQ
PYAQGL 724 SRVWYQ PYAMPL 725 SRVWYQ PYSVQA 726 SRVWYQ PYSLGL 727
SRVWYQ PYAREL 728 SRVWYQ PYSRQP 729 SRVWYQ PYFVQP 730 EYEWYQ PYALPL
731 IPEYWQ PYALPL 732 SRIWWQ PYALPL 733 DPLFWQ PYALPL 734 SRQWVQ
PYALPL 735 IRSWWQ PYALPL 736 RGYWQ PYALPL 737 RLLWVQ PYALPL 738
EYRWFQ PYALPL 739 DAYWVQ PYALPL 740 WSGYFQ PYALPL 741 NIEFWQ PYALPL
742 TRDWVQ PYALPL 743 DSSWYQ PYALPL 744 IGNWYQ PYALPL 745 NLRWDQ
PYALPL 746 LPEFWQ PYALPL 747 DSYWWQ PYALPL 748 RSQYYQ PYALPL 749
ARFWLQ PYALPL 750 NSYFWQ PYALPL 751 RFMYWQPYSVQR 752 AHLFWQPYSVQR
753 WWQPYALPL 754 YYQPYALPL 755 YFQPYALGL 756 YWYQPYALPL 757
RWWQPYATPL 758 GWYQPYALGF 759 YWYQPYALGL 760 IWYQPYAMPL 761
SNMQPYQRLS 762 TFVYWQPY AVGLPAAETACN 763 TFVYWQPY SVQMTITGKVTM 764
TFVYWQPY SSHXXVPXGFPL 765 TFVYWQPY YGNPQWAIHVRH 766 TFVYWQPY
VLLELPEGAVRA 767 TFVYWQPY VDYVWPIPIAQV 768 GWYQPYVDGWR 769
RWEQPYVKDGWS 770 EWYQPYALGWAR 771 GWWQPYARGL 772 LFEQPYAKALGL 773
GWEQPYARGLAG 774 AWVQPYATPLDE 775 MWYQPYSSQPAE 776 GWTQPYSQQGEV 777
DWFQPYSIQSDE 778 PWIQPYARGFG 779 RPLYWQPYSVQV 780 TLIYWQPYSVQI 781
RFDYWQPYSDQT 782 WHQFVQPYALPL 783 EWDS VYWQPYSVQ TLLR 784 WEQN
VYWQPYSVQ SFAD 785 SDV VYWQPYSVQ SLEM 786 YYDG VYWQPYSVQ VMPA 787
SDIWYQ PYALPL 788 QRIWWQ PYALPL 789 SRIWWQ PYALPL 790 RSLYWQ PYALPL
791 TIIWEQ PYALPL 792 WETWYQ PYALPL 793 SYDWEQ PYALPL 794 SRIWCQ
PYALPL 795 EIMFWQ PYALPL 796 DYVWQQ PYALPL 797 MDLLVQ WYQPYALPL 798
GSKVIL WYQPYALPL 799 RQGANI WYQPYALPL 800 GGGDEP WYQPYALPL 801
SQLERT WYQPYALPL 802 ETWVRE WYQPYALPL 803 KKGSTQ WYQPYALPL 804
LQARMN WYQPYALPL 805 EPRSQK WYQPYALPL 806 VKQKWR WYQPYALPL 807
LRRHDV WYQPYALPL 808 RSTASI WYQPYALPL 809 ESKEDQ WYQPYALPL 810
EGLTMK WYQPYALPL 811 EGSREG WYQPYALPL 812 VIEWWQ PYALPL 813 VWYWEQ
PYALPL 814 ASEWWQ PYALPL 815 FYEWWQ PYALPL 816 EGWWVQ PYALPL 817
WGEWLQ PYALPL 818 DYVWEQ PYALPL 819 AHTWWQ PYALPL 820 FIEWFQ PYALPL
821 WLAWEQ PYALPL 822 VMEWWQ PYALPL 823 ERMWQ PYALPL 824 NXXWXX
PYALPL 825 WGNWYQ PYALPL 826 TLYWEQ PYALPL 827 VWRWEQ PYALPL 828
LLWTQ PYALPL 829 SRIWXX PYALPL 830 SDIWYQ PYALPL 831 WGYYXX PYALPL
832 TSGWYQ PYALPL 833 VHPYXX PYALPL 834 EHSYFQ PYALPL 835 XXIWYQ
PYALPL 836 AQLHSQ PYALPL 837 WANWFQ PYALPL 838 SRLYSQ PYALPL 839
GVTFSQ PYALPL 840 SIVWSQ PYALPL 841 SRDLVQ PYALPL 842 HWGH
VYWQPYSVQ DDLG 843 SWHS VYWQPYSVQ SVPE 844 WRDS VYWQPYSVQ PESA 845
TWDA VYWQPYSVQ KWLD 846 TPPW VYWQPYSVQ SLDP 847 YWSS VYWQPYSVQ SVHS
848 YWY QPY ALGL 849 YWY QPY ALPL 850 EWI QPY ATGL 851 NWE QPY AKPL
852 AFY QPY ALPL 853 FLY QPY ALPL 854 VCK QPY LEWC 855
ETPFTWEESNAYYWQPYALPL 856 QGWLTWQDSVDMYWQPYALPL 857
FSEAGYTWPENTYWQPYALPL 858 TESPGGLDWAKIYWQPYALPL 859
DGYDRWRQSGERYWQPYALPL 860 TANVSSFEWTPGYWQPYALPL 861 SVGEDHNFWTSE
YWQPYALPL 862 MNDQTSEVSTFP YWQPYALPL 863 SWSEAFEQPRNL YWQPYALPL 864
QYAEPSALNDWG YWQPYALPL 865 NGDWATADWSNY YWQPYALPL 866 THDEHI
YWQPYALPL 867 MLEKTYTTWTPG YWQPYALPL 868 WSDPLTRDADL YWQPYALPL 869
SDAFTTQDSQAM YWQPYALPL 870 GDDAAWRTDSLT YWQPYALPL 871 AIIRQLYRWSEM
YWQPYALPL 872 ENTYSPNWADSM YWQPYALPL 873 MNDQTSEVSTFP YWQPYALPL 874
SVGEDHNFWTSE YWQPYALPL 875 QTPFTWEESNAY YWQPYALPL 876 ENPFTWQESNAY
YWQPYALPL 877 VTPFTWEDSNVF YWQPYALPL 878 QIPFTWEQSNAY YWQPYALPL 879
QAPLTWQESAAY YWQPYALPL 880 EPTFTWEESKAT YWQPYALPL 881 TTTLTWEESNAY
YWQPYALPL 882 ESPLTWEESSAL YWQPYALPL 883 ETPLTWEESNAY YWQPYALPL 884
EATFTWAESNAY YWQPYALPL 885 EALFTWKESTAY YWQPYALPL 886 STP-TWEESNAY
YWQPYALPL 887 ETPFTWEESNAY YWQPYALPL 888 KAPFTWEESQAY YWQPYALPL 889
STSFTWEESNAY YWQPYALPL 890 DSTFTWEESNAY YWQPYALPL 891 YIPFTWEESNAY
YWQPYALPL 892 QTAFTWEESNAY YWQPYALPL 893 ETLFTWEESNAT YWQPYALPL 894
VSSFTWEESNAY YWQPYALPL 895 QPYALPL 896 Py-1-NapPYQJYALPL 897
TANVSSFEWTPG YWQPYALPL 898 FEWTPGYWQPYALPL 899 FEWTPGYWQJYALPL 900
FEWTPGYYQJYALPL 901 ETPFTWEESNAYYWQPYALPL 902 FTWEESNAYYWQJYALPL
903 ADVL YWQPYA PVTLWV 904 GDVAE YWQPYA LPLTSL 905 SWTDYG YWQPYA
LPISGL 906 FEWTPGYWQPYALPL 911 FEWTPGYWQJYALPL 912 FEWTPGWYQPYALPL
913 FEWTPGWYQJYALPL 914 FEWTPGYYQPYALPL 915 FEWTPGYYQJYALPL 916
TANVSSFEWTPGYWQPYALPL 918 SWTDYGYWQPYALPISGL 919
ETPFTWEESNAYYWQPYALPL 920 ENTYSPNWADSMYWQPYALPL 921
SVGEDHNFWTSEYWQPYALPL 922 DGYDRWRQSGERYWQPYALPL 923 FEWTPGYWQPYALPL
924 FEWTPGYWQPY 925 FEWTPGYWQJY 926 EWTPGYWQPY 927 FEWTPGWYQJY 928
AEWTPGYWQJY 929 FAWTPGYWQJY 930 FEATPGYWQJY 931 FEWAPGYWQJY 932
FEWTAGYWQJY 933 FEWTPAYWQJY 934 FEWTPGAWQJY 935 FEWTPGYAQJY 936
FEWTPGYWQJA 937 FEWTGGYWQJY 938 FEWTPGYWQJY 939 FEWTJGYWQJY 940
FEWTPecGYWQJY 941 FEWTPAibYWQJY 942 FEWTPSarWYQJY 943 FEWTSarGYWQJY
944 FEWTPNYWQJY 945 FEWTPVYWQJY 946 FEWTVPYWQJY 947 AcFEWTPGWYQJY
948 AcFEWTPGYWQJY 949 INap-EWTPGYYQJY 950 YEWTPGYYQJY 951
FEWVPGYYQJY 952 FEWTPGYYQJY 953 FEWTPsYYQJY 954 FEWTPnYYQJY 955
SHLY-Nap-QPYSVQM 956 TLVY-Nap-QPYSLQT 957 RGDY-Nap-QPYSVQS 958
NMVY-Nap-QPYSIQT 959 VYWQPYSVQ 960 VY-Nap-QPYSVQ 961 TFVYWQJYALPL
962 FEWTPGYYQJ-Bpa 963 XaaFEWTPGYYQJ-Bpa 964 FEWTPGY-Bpa-QJY 965
AcFEWTPGY-Bpa-QJY 966 FEWTPG-Bpa-YQJY 967 AcFEWTPG-Bpa-YQJY 968
AcFE-Bpa-TPGYYQJY 969 AcFE-Bpa-TPGYYQJY 970 Bpa-EWTPGYYQJY 971
AcBpa-EWTPGYYQJY 972 VYWQPYSVQ 973 RLVYWQPYSVQR 974
RLVY-Nap-QPYSVQR 975 RLDYWQPYSVQR 976 RLVWFQPYSVQR 977 RLVYWQPYSIQR
978 DNSSWYDSFLL 980 DNTAWYESFLA 981 DNTAWYENFLL 982 PARE
DNTAWYDSFLI WC 983 TSEY DNTTWYEKFLA SQ 984 SQIP DNTAWYQSFLL HG 985
SPFI DNTAWYENFLL TY 986 EQIY DNTAWYDHFLL SY 987 TPFI DNTAWYENFLL TY
988 TYTY DNTAWYERFLM SY 989 TMTQ DNTAWYENFLL SY 990 TI DNTAWYANLVQ
TYPQ 991 TI DNTAWYERFLA QYPD 992 HI DNTAWYENFLL TYTP 993 SQ
DNTAWYENFLL SYKA 994 QI DNTAWYERFLL QYNA 995 NQ DNTAWYESFLL QYNT
996 TI DNTAWYENFLL NHNL 997 HY DNTAWYERFLQ QGWH 998
ETPFTWEESNAYYWQPYALPL 999 YIPFTWEESNAYYWQPYALPL 1000
DGYDRWRQSGERYWQPYALPL 1001 pY-INap-pY-QJYALPL 1002
TANVSSFEWTPGYWQPYALPL 1003 FEWTPGYWQJYALPL 1004 FEWTPGYWQPYALPLSD
1005 FEWTPGYYQJYALPL 1006 FEWTPGYWQJY 1007 AcFEWTPGYWQJY 1008
AcFEWTPGWYQJY 1009 AcFEWTPGYYQJY 1010 AcFEWTPaYWQJY 1011
AcFEWTPaWYQJY 1012 AcFEWTPaYYQJY 1013 FEWTPGYYQJYALPL 1014
FEWTPGYWQJYALPL 1015 FEWTPGWYQJYALPL 1016 TANVSSFEWTPGYWQPYALPL
1017 AcFEWTPGYWQJY 1018 AcFEWTPGWYQJY 1019 AcFEWTPGYYQJY 1020
AcFEWTPAYWQJY 1021 AcFEWTPAWYQJY 1022 AcFEWTPAYYQJY 1023
[0096]
5TABLE 5 EPO-mimetic peptide sequences SEQ Sequence/structure ID
NO: YXCXXGPXTWXCXP 83 YXCXXGPXTWXCXP-YXCXXGPXTWXCXP 84
YXCXXGPXTWXCXP-.LAMBDA.-YXCXXGPXTWXCXP 85 1 86 86
GGTYSCHFGPLTWVCKPQGG 87 GGDYHCRMGPLTWVCKPLGG 88
GGVYACRMGPITWVCSPLGG 89 VGNYMCHFGPITWVCRPGGG 90
GGLYLCRFGPVTWDCGYKGG 91 GGTYSCHFGPLTWVCKPQGG- 92
GGTYSCHFGPLTWVCKPQGG GGTYSCHFGPLTWVCKPQGG-.LAMBDA.- 93
GGTYSCHFGPLTWVCKPQGG GGTYSCHFGPLTWVCKPQGGSSK 94
GGTYSCHFGPLTWVCKPQGGSSK- 95 GGTYSCHFGPLTWVCKPQGGSSK
GGTYSCHFGPLTWVCKPQGGSSK-.LAMBDA.- 96 GGTYSCHFGPLTWVCKPQGGSSK 2 97
97 GGTYSCHFGPLTWVCKPQGGSSK(-.LA- MBDA.-biotin) 98
CX.sub.4X.sub.5GPX.sub.6TWX.sub.7C 421 GGTYSCHGPLTWVCKPQGG 422
VGNYMAHMGPITWVGRPGG 423 GGPHHVYACRMGPLTWIC 424 GGTYSCHFGPLTWVCKPQ
425 GGLYACHMGPMTWVCQPLRG 426 TIAQYICYMGPETWECRPSPKA 427
YSCHFGPLTWVCK 428 YCHFGPLTWVC 429 X.sub.3X.sub.4X.sub.5GP-
X.sub.6TWX.sub.7X.sub.8 124 YX.sub.2X.sub.3X.sub.4X.sub.5GPX.sub.6-
TWX.sub.7X.sub.8 461 X.sub.1YX.sub.2X.sub.3X.sub.4X.sub.5GPX.sub.6-
TWX.sub.7X.sub.8X.sub.9X.sub.10X.sub.11 419
X.sub.1YX.sub.2CX.sub.4X.sub.5GPX.sub.6TWX.sub.7CX.sub.9X.sub.10X.sub.11
420 GGLYLCRFGPVTWDCGYKGG 1024 GGTYSCHFGPLTWVCKPQGG 1025
GGDYHCRMGPLTWVCKPLGG 1026 VGNYMCHFGPITWVCRPGGG 1029
GGVYACRMGPITWVCSPLGG 1030 VGNYMAHMGPITWVCRPGG 1035
GGTYSCHFGPLTWVCKPQ 1036 GGLYACHMGPMTWVCQPLRG 1037
TIAQYICYMGPETWECRPSPKA 1038 YSCHFGPLTWVCK 1039 YCHFGPLTWVC 1040
SCHFGPLTWVCK 1041
(AX.sub.2).sub.nX.sub.3X.sub.4X.sub.5GPX.sub.6TWX.sub.7X.sub.8
1042
[0097]
6TABLE 6 TPO-mimetic peptide sequences SEQ Sequence/structure ID
NO: IEGPTLRQWLAARA 13 IEGPTLRQWLAAKA 24 IEGPTLREWLAARA 25
IEGPTLRQWLAARA-.LAMBDA.-IEGPTLRQWLAARA 26 IEGPTLRQWLAAKA-.LAMBDA.--
IEGPTLRQWLAAKA 27 3 28 IEGPTLRQWLAARA-.LAMBDA.-K(Br-
Ac)-.LAMBDA.-IEGPTLRQWLAARA 29
IEGPTLRQWLAARA-.LAMBDA.-K(PEG)-.LAMB- DA.-IEGPTLRQWLAARA 30 4 31 31
5 32 32 VRDQIXXXL 33 TLREWL 34 GRVRDQVAGW 35 GRVKDQIAQL 36
GVRDQVSWAL 37 ESVREQVMKY 38 SVRSQISASL 39 GVRETVYRHM 40 GVREVIVMHML
41 GRVRDQIWAAL 42 AGVRDQILIWL 43 GRVRDQIMLSL 44 GRVRDQI(X).sub.3L
45 CTLRQWLQGC 46 CTLQEFLEGC 47 CTRTEWLHGC 48 CTLREWLHGGFC 49
CTLREWVFAGLC 50 CTLRQWLILLGMC 51 CTLAEFLASGVEQC 52 CSLQEFLSHGGYVC
53 CTLREFLDPTTAVC 54 CTLKEWLVSHEVWC 55 CTLREWL(X).sub.2-6C 56-60
REGPTLRQWM 61 EGPTLRQWLA 62 ERGPFWAKAC 63 REGPRCVMWM 64
CGTEGPTLSTWLDC 65 CEQDGPTLLEWLKC 66 CELVGPSLMSWLTC 67
CLTGPFVTQWLYEC 68 CRAGPTLLEWLTLC 69 CADGPTLREWISFC 70
C(X).sub.1-2EGPTLREWL(X).sub.1-2C 71-74 GGCTLREWLHGGFCGG 75
GGCADGPTLREWISFCGG 76 GNADGPTLRQWLEGRRPKN 77 LAIEGPTLRQWLHGNGRDT 78
HGRVGPTLREWKTQVATKK 79 TIKGPTLRQWLKSREHTS so ISDGPTLKEWLSVTRGAS 81
SIEGPTLREWLTSRTPHS 82
[0098]
7TABLE 7 G-CSF-mimetic peptide sequences SEQ Sequence/structure ID
NO: EEDCK 99 6 99 99 EED.sigma.K 100 7 100 100 pGluED.sigma.K 101 8
101 101 PicSD.sigma.K 102 9 102 102 EEDCK-.LAMBDA.-EEDCK 103
EEDXK-.LAMBDA.-EEDXK 104
[0099]
8TABLE 8 TNF-antagonist peptide sequences SEQ Sequence/structure ID
NO: YCFTASENHCY 106 YCFTNSENHCY 107 YCFTRSENHGY 108 FCASENHCY 109
YCASENHCY 110 FCNSENHCY 111 FCNSENRCY 112 FCNSVENRCY 113
YCSQSVSNDCF 114 FCVSNDRCY 115 YCRKELGQVCY 116 YCKEPGQCY 117
YCRKEMGCY 118 FCRKEMGCY 119 YCWSQNLCY 120 YCELSQYLCY 121 YCWSQNYCY
122 YCWSQYLCY 123 DFLPHYKNTSLGHRP 1085 10 NR
[0100]
9TABLE 9 Integrin-binding peptide sequences Sequence/structure SEQ
ID NO: RX.sub.1ETX.sub.2WX.sub.3 441 RX.sub.1ETX.sub.2WX.sub.3 442
RGDGX 443 CRGDGXC 444 CX.sub.1X.sub.2RLDX.sub.3X.sub.4C 445
CARRLDAPC 446 CPSRLDSPC 447 X.sub.1X.sub.2X.sub.3RGDX.sub-
.4X.sub.5X.sub.6 448 CX.sub.2CRGDCX.sub.5C 449 CDCRGDCFC 450
CDGRGDCLC 451 CLCRGDCIC 452
X.sub.1X.sub.2DDX.sub.4X.sub.5X.sub.7X.sub.8 453
X.sub.1X.sub.2X.sub.3DDX.sub.4X.sub.5X.sub.6X.sub.7X.sub.8 454
CWDDGWLC 455 CWDDLWWLC 456 CWDDGLMC 457 CWDDGWMC 458 CSWDDGWLC 459
CPDDLWWLC 460 NGR NR GSL NR RGD NR CGRECPRLCQSSC 1071 CNGRCVSGCAGRC
1072 CLSGSLSC 1073 RGD NR NGR NR GSL NR NGRAHA 1074 CNGRC 1075
CDCRGDCFC 1076 CGSLVRC 1077 DLXXL 1043 RTDLDSLRTYTL 1044 RTDLDSLRTY
1053 RTDLDSLRT 1054 RTDLDSLR 1078 GDLDLLKLRLTL 1079 GDLHSLRQLLSR
1080 RDDLHMLRLQLW 1081 SSDLHALKKRYG 1082 RGDLKQLSELTW 1083
RGDLAALSAPPV 1084
[0101]
10TABLE 10 Selectin antagonist peptide sequences Sequence/structure
SEQ ID NO: DITWDQLWDLMK 147 DITWDELWKIMN 148 DYTWFELWDMMQ 149
QITWAQLWNMMK 150 DMTWHDLWTLMS 151 DYSWHDLWEMMS 152 EITWDQLWEVMN 153
HVSWEQLWDIMN 154 HITWDQLWRIMT 155 RNMSWLELWEHMK 156
AEWTWDQLWHVMNPAESQ 157 HRAEWLALWEQMSP 158 KKEDWLALWRIMSV 159
ITWDQLWDLMK 160 DITWDQLWDLMK 161 DITWDQLWDLMK 162 DITWDQLWDLMK 163
CQNRYTDLVAIQNKNE 462 AENWADNEPNNKRNNED 463 RKNNKTWTWVGTKKALTNE 464
KKALTNEAENWAD 465 CQXRYTDLVAIQNKXE 466 RKXNXXWTWVGTXKXLTEE 467
AENWADGEPNNKXNXED 468 CXXXYTXLVAIQNKXE 469 RKXXXXWXWVGTXKXLTXE 470
AXNWXXXEPNNXXXED 471 XKXKTXEAXNWXX 472
[0102]
11TABLE 11 Antipathogenic peptide sequences Sequence/structure SEQ
ID NO: GFFALIPKIISSPLFKTLLSAVGSAL- SSSGGQQ 503
GFFALIPKIISSPLFKTLLSAVGSALSSSGGQE 504 GFFALIPKIISSPLFKTLLSAV 505
GFFALIPKIISSPLFKTLLSAV 506 KGFFALIPKIISSPLFKTLLSAV 507
KKGFFALIPKIISSPLFKTLLSAV 508 KKGFFALIPKIISSPLFKTLLSAV 509
GFFALIPKIIS 510 GIGAVLKVLTTGLPALISWIKRKRQQ 511
GIGAVLKVLTTGLPALISWIKRKRQQ 512 GIGAVLKVLTTGLPALISWIKRKRQQ 513
GIGAVLKVLTTGLPALISWIKR 514 AVLKVLTTGLPALISWIKR 515 KLLLLLKLLLLK 516
KLLLKLLLKLLK 517 KLLLKLKLKLLK 518 KKLLKLKLKLKK 519 KLLLKLLLKLLK 520
KLLLKLKLKLLK 521 KLLLLK 522 KLLLKLLK 523 KLLLKLKLKLLK 524
KLLLKLKLKLLK 525 KLLLKLKLKLLK 526 KAAAKAAAKAAK 527 KVVVKVVVKVVK 528
KVVVKVKVKVVK 529 KVVVKVKVKVK 530 KVVVKVKVKVVK 531 KLILKL 532 KVLHLL
533 LKLRLL 534 KPLHLL 535 KLILKLVR 536 KVFHLLHL 537 HKFRILKL 538
KPFHILHL 539 KIIIKIKIKIIK 540 KIIIKIKIKIIK 541 KIIIKIKIKIIK 542
KIPIKIKIKIPK 543 KIPIKIKIKIVK 544 RIIIRIRIRIIR 545 RIIIRIRIRIIR 546
RIIIRIRIRIIR 547 RIVIRIRIRLIR 548 RIIVRIRLRIIR 549 RIGIRLRVRIIR 550
KIVIRIRIRLIR 551 RIAVKWRLRFIK 552 KIGWKLRVRIIR 553 KKIGWLIIRVRR 554
RIVIRIRIRLIRIR 555 RIIVRIRLRIIRVR 556 RIGIRLRVRIIRRV 557
KIVIRIRARLIRIRIR 558 RIIVKIRLRIIKKIRL 559 KIGIKARVRIIRVKII 560
RIIVHIRLRIIHHIRL 561 HIGIKAHVRIIRVHII 562 RIYVKIHLRYIKKIRL 563
KIGHKARVHIIRYKII 564 RIYVKPHPRYIKKIRL 565 KPGHKARPHIIRYKII 566
KIVIRIRIRLIRIRIRKIV 567 RIIVKIRLRIIKKIRLIKK 568 KIGWKLRVRIIRVKIGRLR
569 KIVIRIRIRLIRIRIRKIVKVKRIR 570 RFAVKIRLRIIKKIRLIKKIRKRVIK 571
KAGWKLRVRIIRVKIGRLRKIGWKKRVRIK 572 RIYVKPHPRYIKKIRL 573
KPGHKARPHIIRYKII 574 KIVIRIRIRLIRIRIRKIV 575 RIIVKIRLRIIKKIRLIKK
576 RIYVSKISIYIKKIRL 577 KIVIFTRIRLTSIRIRSIV 578 KPIHKARPTIIRYKMI
579 cyclicCKGFFALIPKIISSPLFKTLLSAVC 580 CKKGFFALIPKIISSPLFKTLLSAVC
581 CKKKGFFALIPKIISSPLFKTLLSAVC 582 CyclicCRIVIRIRIRLIRIRC 583
CyclicCKPGHKARPHIIRYKIIC 584 CyclicCRFAVKIRLRIIKKIRLIKKIRKAVIKC 585
KLLLKLLL KLLKC 586 KLLLKLLLKLLK 587 KLLLKLKLKLLKC 588 KLLLKLLLKLLK
589
[0103]
12TABLE 12 VIP-mimetic peptide sequences SEQ Sequence/structure ID
NO: HSDAVFYDNYTR LRKQMAVKKYLN SILN 590 Nle HSDAVFYDNYTR
LRKQMAVKKYLN SILN 591 X.sub.1X.sub.1'X.sub.1"X.sub.2 592 X.sub.3 S
X.sub.4 LN 593 11 594 KKYL 595 NSILN 596 KKYL 597 KKYA 598 AVKKYL
599 NSILN 600 KKYV 601 SILauN 602 KKYLNle 603 NSYLN 604 NSIYN 605
KKYLPPNSILN 606 LauKKYL 607 CapKKYL 608 KYL NR KKYNle 609 VKKYL 610
LNSILN 611 YLNSILN 612 KKYLN 613 KKYLNS 614 KKYLNSI 615 KKYLNSIL
616 KKYL 617 KKYDA 618 AVKKYL 619 NSILN 620 KKYV 621 SILauN 622
NSYLN 623 NSIYN 624 KKYLNle 625 KKYLPPNSILN 626 KKYL 627 KKYDA 628
AVKKYL 629 NSILN 630 KKYV 631 SILauN 632 LauKKYL 633 CapKKYL 634
KYL NR KYL NR KKYNle 635 VKKYL 636 LNSILN 637 YLNSILN 638 KKYLNle
639 KKYLN 640 KKYLNS 641 KKYLNSI 642 KKYLNSIL 643 KKKYLD 644
cyclicCKKYLC 645 12 646 KKYA 647 WWTDTGLW 648 WWTDDGLW 649
WWDTRGLWVWTI 650 FWGNDGIWLESG 651 DWDQFGLWRGAA 652 RWDDNGLWVVVL 653
SGMWSHYGIWMG 654 GGRWDQAGLWVA 655 KLWSEQGIWMGE 656 CWSMHGLWLC 657
GCWDNTGIWVPC 658 DWDTRGLWVY 659 SLWDENGAWI 660 KWDDRGLWMH 661
QAWNERGLWT 662 QWDTRGLWVA 663 WNVHGIWQE 664 SWDTRGLWVE 665
DWDTRGLWVA 666 SWGRDGLWIE 667 EWTDNGLWAL 668 SWDEKGLWSA 669
SWDSSGLWMD 670
[0104]
13TABLE 13 Mdm/hdm antagonist peptide sequences Sequence/structure
SEQ ID NO: TFSDLW 130 QETFSDLWKLLP 131 QPTFSDLWKLLP 132
QETFSDYWKLLP 133 QPTFSDYWKLLP 134 MPRFMDYWEGLN 135 VQNFIDYWTQQF 136
TGPAFTHYWATF 137 IDRAPTFRDHWFALV 138 PRPALVFADYWETLY 139
PAFSRFWSDLSAGAH 140 PAFSRFWSKLSAGAH 141 PXFXDYWXXL 142 QETFSDLWKLLP
143 QPTFSDLWKLLP 144 QETFSDYWKLLP 145 QPTFSDYWKLLP 146
[0105]
14TABLE 14 Calmodulin antagonist peptide sequences
Sequence/structure SEQ ID NO: SCVKWGKKEFCGS 164 SCWKYWGKECGS 165
SCYEWGKLRWCGS 166 SCLRWGKWSNCGS 167 SCWRWGKYQICGS 168 SCVSWGALKLCGS
169 SCIRWGQNTFCGS 170 SCWQWGNLKICGS 171 SCVRWGQLSICGS 172
LKKFNARRKLKGAILTTMLAK 173 RRWKKNFIAVSAANRFKK 174 RKWQKTGHAVRAIGRLSS
175 INLKALAALAKKIL 176 KIWSILAPLGTTLVKLVA 177 LKKLLKLLKKLLKL 178
LKWKKLLKLLKKLLKKLL 179 AEWPSLTEIKTLSHFSV 180 AEWPSPTRVISTTYFGS 181
AELAHWPPVKTVLRSFT 182 AEGSWLQLLNLMKQMNN 183 AEWPSLTEIK 184
[0106]
15TABLE 15 Mast cell antagonists/Mast cell protease inhibitor
peptide sequences Sequence/structure SEQ ID NO: SGSGVLKRPLPILPVTR
272 RWLSSRPLPPLPLPPRT 273 GSGSYDTLALPSLPLHPMSS 274
GSGSYDTRALPSLPLHPMSS 275 GSGSSGVTMYPKLPPHWSMA 276
GSGSSGVRMYPKLPPHWSMA 277 GSGSSSMRMVPTIPGSAKHG 278 RNR NR QT NR RQK
NR NRQ NR RQK NR RNRQKT 436 RNRQ 437 RNRQK 438 NRQKT 439 RQKT
440
[0107]
16TABLE 16 SH3 antagonist peptide sequences Sequence/structure SEQ
ID NO: RPLPPLP 282 RELPPLP 283 SPLPPLP 284 GPLPPLP 285 RPLPIPP 286
RPLPIPP 287 RRLPPTP 288 RQLPPTP 289 RPLPSRP 290 RPLPTRP 291 SRLPPLP
292 RALPSPP 293 RRLPRTP 294 RPVPPIT 295 ILAPPVP 296 RPLPMLP 297
RPLPILP 298 RPLPSLP 299 RPLPSLP 300 RPLPMIP 301 RPLPLIP 302 RPLPPTP
303 RSLPPLP 304 RPQPPPP 305 RQLPIPP 306 XXXRPLPPLPXP 307
XXXRPLPPIPXX 308 XXXRPLPPLPXX 309 RXXRPLPPLPXP 310 RXXRPLPPLPPP 311
PPPYPPPPIPXX 312 PPPYPPPPVPXX 313 LXXRPLPX.PSI.P 314
.PSI.PXXRPLPXLP 315 PPX.THETA.PPP.PSI.P 316 +PP.PSI.PXKPXWL 317
RPX.PSI.P.PSI.R+SXP 318 PPVPPRPXXTL 319 .PSI.P.PSI.LP.PSI.K 320
+.THETA.DXPLPXLP 321
[0108]
17TABLE 17 Somatostatin or cortistatin mimetic peptide sequences
Sequence/structure SEQ ID NO:
X.sup.1-X.sup.2-Asn-Phe-Phe-Trp-Lys-Thr-Phe-X.sup.3-Ser-X.sup.4 473
Asp Arg Met Pro Cys Arg Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys Lys
474 Met Pro Cys Arg Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys Lys 475
Cys Arg Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys Lys 476 Asp Arg Met
Pro Cys Arg Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys 477 Met Pro Cys
Arg Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys 478 Cys Arg Asn Phe Phe
Trp Lys Thr Phe Ser Ser Cys 479 Asp Arg Met Pro Cys Lys Asn Phe Phe
Trp Lys Thr Phe Ser Ser Cys 480 Met Pro Cys Lys Asn Phe Phe Trp Lys
Thr Phe Ser Ser Cys Lys 481 Cys Lys Asn Phe Phe Trp Lys Thr Phe Ser
Ser Cys Lys 482 Asp Arg Met Pro Cys Lys Asn Phe Phe Trp Lys Thr Phe
Ser Ser Cys 483 Met Pro Cys Lys Asn Phe Phe Trp Lys Thr Phe Ser Ser
Cys 484 Cys Lys Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys 485 Asp Arg
Met Pro Cys Arg Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys Lys 486 Met
Pro Cys Arg Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys Lys 487 Cys Arg
Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys Lys 488 Asp Arg Met Pro Cys
Arg Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys 489 Met Pro Cys Arg Asn
Phe Phe Trp Lys Thr Phe Thr Ser Cys 490 Cys Arg Asn Phe Phe Trp Lys
Thr Phe Thr Ser Cys 491 Asp Arg Met Pro Cys Lys Asn Phe Phe Trp Lys
Thr Phe Thr Ser Cys Lys 492 Met Pro Cys Lys Asn Phe Phe Trp Lys Thr
Phe Thr Ser Cys Lys 493 Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr Ser
Cys Lys 494 Asp Arg Met Pro Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr
Ser Cys 495 Met Pro Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys
496 Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys 497
[0109]
18TABLE 18 UKR antagonist peptide sequences Sequence/structure SEQ
ID NO: AEPMPHSLNFSQYLWYT 196 AEHTYSSLWDTYSPLAF 197
AELDLWMRHYPLSFSNR 198 AESSLWTRYAWPSMPSY 199 AEWHPGLSFGSYLWSKT 200
AEPALLNWSFFFNPGLH 201 AEWSFYNLHLPEPQTIF 202 AEPLDLWSLYSLPPLAM 203
AEPTLWQLYQFPLRLSG 204 AEISFSELMWLRSTPAF 205 AELSEADLWTTWFGMGS 206
AESSLWRIFSPSALMMS 207 AESLPTLTSILWGKESV 208 AETLFMDLWHDKHILLT 209
AEILNFPLWHEPLWSTE 210 AESQTGTLNTLFWNTLR 211 AEPVYQYELDSYLRSYY 430
AELDLSTFYDIQYLLRT 431 AEFFKLGPNGYVYLHSA 432 FKLXXXGYVYL 433
AESTYHHLSLGYMYTLN 434 YHXLXXGYMYT 435
[0110]
19TABLE 19 Macrophage and/or T-cell inhibiting peptide sequences
Sequence/structure SEQ ID 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
IIe-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
[0111]
20TABLE 20 Additional Exemplary Pharmacologically Active Peptides
SEQ ID Sequence/structure NO: Activity VEPNCDIHVMWEWECFERL 1027
VEGF-antagonist GERWCFDGPLTWVCGEES 1084 VEGF-antagonist
RGWVEICVADDNGMCVTEAQ 1085 VEGF-antagonist GWDECDVARMWEWECFAGV 1086
VEGF-antagonist GERWCFDGPRAWVCGWEI 501 VEGF-antagonist
EELWCFDGPRAWVCGYVK 502 VEGF-antagonist RGWVEICAADDYGRCLTEAQ 1031
VEGF-antagonist RGWVEICESDVWGRCL 1087 VEGF-antagonist
RGWVEICESDVWGRCL 1088 VEGF-antagonist GGNECDIARMWEWECFERL 1089
VEGF-antagonist RGWVEICAADDYGRCL 1090 VEGF-antagonist CTTHWGFTLC
1028 MMP inhibitor CLRSGXGC 1091 MMP inhibitor CXXHWGFXXC 1092 MMP
inhibitor CXPXC 1093 MMP inhibitor CRRHWGFEFC 1094 MMP inhibitor
STTHWGFTLS 1095 MMP inhibitor CSLHWGFWWC 1096 CTLA4-mimetic
GFVCSGIFAVGVGRC 125 CTLA4-mimetic APGVRLGCAVLGRYC 126 CTLA4-mimetic
LLGRMK 105 Antiviral (HBV) ICVVQDWGHHRCTAGHMANLTSHASAI 127 C3b
antagonist ICVVQDWGHHRCT 128 C3b antagonist CVVQDWGHHAC 129 C3b
antagonist STGGFDDVYDWARGVSSALTTTLVATR 185 Vinculin-binding
STGGFDDVYDWARRVSSALTTTLVATR 186 Vinculin-binding
SRGVNFSEWLYDMSAAMKEASNVFPSRRSR 187 Vinculin-binding
SSQNWDMEAGVEDLTAAMLGLLSTIHSSSR 188 Vinculin-binding
SSPSLYTQFLVNYESAATRIQDLLIASRPSR 189 Vinculin-binding
SSTGWVDLLGALQRAADATRTSIPPSLQNSR 190 Vinculin-binding
DVYTKKELIECARRVSEK 191 Vinculin-binding EKGSYYPGSGIAQFHIDYNNVS 192
C4BP-binding SGIAQFHIDYNNVSSAEGWHVN 193 C4BP-binding
LVTVEKGSYYPGSGIAQFHIDYNNVSSAEGWHVN 194 C4BP-binding SGIAQFHIDYNNVS
195 C4BP-binding LLGRMK 279 anti-HBV ALLGRMKG 280 anti-HBV LDPAFR
281 anti-HBV CXXRGDC 322 Inhibition of platelet aggregation RPLPPLP
323 Src antagonist PPVPPR 324 Src antagonist XFXDXWXXLXX 325
Anti-cancer (particularly for sarcomas) KACRRLFGPVDSEQLSRDCD 326
p16-mimetic RERWNFDFVTETPLEGDFAW 327 p16-mimetic
KRRQTSMTDFYHSKRRLIFS 328 p16-mimetic TSMTDFYHSKRRLIFSKRKP 329
p16-mimetic RRLIF 330 p16-mimetic
KRRQTSATDFYHSKRRLIFSRQIKIWFQNRRMKWKK 331 p16-mimetic
KRRLIFSKRQIKIWFQNRRMKWKK 332 p16-mimetic Asn Gln Gly Arg His Phe
Cys Gly Gly Ala Leu Ile His Ala 498 CAP37 mimetic/LPS Arg Phe Val
Met Thr Ala Ala Ser Cys Phe Gln binding Arg His Phe Cys Gly Gly Ala
Leu Ile His Ala Arg Phe Val 499 CAP37 mimetic/LPS Met Thr Ala Ala
Ser Cys binding Gly Thr Arg Cys Gln Val Ala Gly Trp Gly Ser Gln Arg
Ser 500 CAP37 mimetic/LPS Gly Gly Arg Leu Ser Arg Phe Pro Arg Phe
Val Asn Val binding WHWRHRIPLQLAAGR 1097 carbohydrate (GD1 alpha)
mimetic LKTPRV 1098 .beta.2GPI Ab binding NTLKTPRV 1099 .beta.2GPI
Ab binding NTLKTPRVGGC 1100 .beta.2GPI Ab binding KDKATF 1101
.beta.2GPI Ab binding KDKATFGCHD 1102 .beta.2GPI Ab binding
KDKATFGCHDGC 1103 .beta.2GPI Ab binding TLRVYK 1104 .beta.2GPI Ab
binding ATLRVYKGG 1105 .beta.2GPI Ab binding CATLRVYKGG 1106
.beta.2GPI Ab binding INLKALAALAKKIL 1107 Membrane- transporting
GWT NR Membrane- transporting GWTLNSAGYLLG 1108 Membrane-
transporting GWTLNSAGYLLGKINLKALAALAKKIL 1109 Membrane-
transporting
[0112] The present invention is also particularly useful with
peptides having activity in treatment of:
[0113] cancer, wherein the peptide is a VEGF-mimetic or a VEGF
receptor antagonist, a HER2 agonist or antagonist, a CD20
antagonist and the like;
[0114] asthma, wherein the protein of interest is a CKR3
antagonist, an IL-5 receptor antagonist, and the like;
[0115] thrombosis, wherein the protein of interest is a GPIIb
antagonist, a GPIIIa antagonist, and the like;
[0116] 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.
[0117] Vehicles. This invention requires the presence of at least
one vehicle (F.sup.1, F.sup.2) 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; e.g., Fc's at each
terminus or an Fc at a terminus and a PEG group at the other
terminus or a sidechain.
[0118] An Fc domain is the preferred vehicle. The Fc domain may be
fused to the N or C termini of the peptides or at both the N and C
termini. For the TPO-mimetic peptides, molecules having the Fc
domain fused to the N terminus of the peptide portion of the
molecule are more bioactive than other such fusions, so fusion to
the N terminus is preferred. As noted above, Fc variants are
suitable vehicles within the scope of this invention. A native Fc
may be extensively modified to form an Fc variant in accordance
with this invention, provided binding to the salvage receptor is
maintained; see, for example WO 97/34631 and WO 96/32478.
[0119] 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:
[0120] 1. Sites involved in disulfide bond formation are removed.
Such removal may avoid reaction with other cysteine-containing
proteins present in the host cell used to produce the molecules of
the invention. For this purpose, the cysteine-containing segment at
the N-terminus may be truncated or cysteine residues may be deleted
or substituted with other amino acids (e.g., alanyl, seryl). In
particular, one may truncate the N-terminal 20-amino acid segment
of SEQ ID NO: 2 or delete or substitute the cysteine residues at
positions 7 and 10 of SEQ ID NO: 2. Even when cysteine residues are
removed, the single chain Fc domains can still form a dimeric Fc
domain that is held together non-covalently.
[0121] 2. A native Fc is modified to make it more compatible with a
selected host cell. For example, one may remove the PA sequence
near the N-terminus of a typical native Fc, which may be recognized
by a digestive enzyme in E. coli such as proline iminopeptidase.
One may also add an N-terminal methionine residue, especially when
the molecule is expressed recombinantly in a bacterial cell such as
E. coli. The Fc domain of SEQ ID NO: 2 (FIG. 4) is one such Fc
variant.
[0122] 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.
[0123] 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).
[0124] 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.
[0125] 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.
[0126] 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.
[0127] 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.
[0128] Preferred Fc variants include the following. In SEQ ID NO: 2
(FIG. 4) the leucine at position 15 may be substituted with
glutamate; the glutamate at position 99, with alanine; and the
lysines at positions 101 and 103, with alanines. In addition, one
or more tyrosine residues can be replaced by phenyalanine
residues.
[0129] 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).
[0130] As noted above, polymer vehicles may also be used for
F.sup.1 and F.sup.2. Various means for attaching chemical moieties
useful as vehicles are currently available, see, e.g., Patent
Cooperation Treaty ("PCT") International Publication No. WO
96/11953, entitled "N-Terminally Chemically Modified Protein
Compositions and Methods," herein incorporated by reference in its
entirety. This PCT publication discloses, among other things, the
selective attachment of water soluble polymers to the N-terminus of
proteins.
[0131] A preferred polymer vehicle is polyethylene glycol (PEG).
The PEG group may be of any convenient molecular weight and may be
linear or branched. The average molecular weight of the PEG will
preferably range from about 2 kiloDalton ("kD") to about 100 kDa,
more preferably from about 5 kDa to about 50 kDa, most preferably
from about 5 kDa to about 10 kDa. The PEG groups will generally be
attached to the compounds of the invention via acylation or
reductive alkylation through a reactive group on the PEG moiety
(e.g., an aldehyde, amino, thiol, or ester group) to a reactive
group on the inventive compound (e.g., an aldehyde, amino, or ester
group).
[0132] A useful strategy for the PEGylation of synthetic peptides
consists of combining, through forming a conjugate linkage in
solution, a peptide and a PEG moiety, each bearing a special
functionality that is mutually reactive toward the other. The
peptides can be easily prepared with conventional solid phase
synthesis (see, for example, FIGS. 5 and 6 and the accompanying
text herein). The peptides are "preactivated" with an appropriate
functional group at a specific site. The precursors are purified
and fully characterized prior to reacting with the PEG moiety.
Ligation of the peptide with PEG usually takes place in aqueous
phase and can be easily monitored by reverse phase analytical HPLC.
The PEGylated peptides can be easily purified by preparative HPLC
and characterized by analytical HPLC, amino acid analysis and laser
desorption mass spectrometry.
[0133] 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 .alpha.1-6 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.
[0134] Linkers. Any "linker" group is optional. When present, its
chemical structure is not critical, since it serves primarily as a
spacer. The linker is preferably made up of amino acids linked
together by peptide bonds. Thus, in preferred embodiments, the
linker is made up of from 1 to 20 amino acids linked by peptide
bonds, wherein the amino acids are selected from the 20 naturally
occurring amino acids. Some of these amino acids may be
glycosylated, as is well understood by those in the art. In a more
preferred embodiment, the 1 to 20 amino acids are selected from
glycine, alanine, proline, asparagine, glutamine, and lysine. Even
more preferably, a linker is made up of a majority of amino acids
that are sterically unhindered, such as glycine and alanine. Thus,
preferred linkers are polyglycines (particularly (Gly).sub.4,
(Gly).sub.5), poly(Gly-Ala), and polyalanines. Other specific
examples of linkers are:
21 (Gly).sub.3Lys(Gly).sub.4; (SEQ ID NO: 333)
(Gly).sub.3AsnGlySer(Gly).sub.2; (SEQ ID NO: 334)
(Gly).sub.3Cys(Gly).sub.4; and (SEQ ID NO: 335) GlyProAsnGlyGly.
(SEQ ID NO: 336)
[0135] To explain the above nomenclature, for example,
(Gly).sub.3Lys(Gly).sub.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.
[0136] Non-peptide linkers are also possible. For example, alkyl
linkers such as --NH--(CH.sub.2).sub.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.,
C.sub.1-C.sub.6) lower acyl, halogen (e.g., Cl, Br), CN, NH.sub.2,
phenyl, etc. An exemplary non-peptide linker is a PEG linker,
13
[0137] 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.
[0138] Derivatives. The inventors also contemplate derivatizing the
peptide and/or vehicle portion 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:
[0139] 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.
[0140] 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. 14
[0141] 3.
[0142] 4. One or more peptidyl [--C(O)NR--] linkages (bonds) is
replaced by a non-peptidyl linkage. Exemplary non-peptidyl linkages
are --CH.sub.2-carbamate [--CH.sub.2--OC(O)NR--], phosphonate,
--CH.sub.2-sulfonamide [--CH.sub.2--S(O).sub.2NR--], urea
[--NHC(O)NH--], --CH.sub.2-secondary amine, and alkylated peptide
[--C(O)NR.sup.6-- wherein R.sup.6 is lower alkyl].
[0143] 5. The N-terminus is derivatized. Typically, the N-terminus
may be acylated or modified to a substituted amine. Exemplary
N-terminal derivative groups include --NRR.sup.1 (other than
--NH.sub.2), --NRC(O)R.sup.1, --NRC(O)OR.sup.1,
--NRS(O).sub.2R.sup.1, --NHC(O)NHR.sup.1, succinimide, or
benzyloxycarbonyl-NH-- (CBZ-NH--), wherein R and R' 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 C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy,
chloro, and bromo.
[0144] 6. 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--CH.sub.2--CH.sub.2--NH.sub.2).sub.2 to compounds of this
invention having any of SEQ ID NOS: 504 to 508 at the C-terminus.
Likewise, one may use methods described in the art to add
--NH.sub.2 to compounds of this invention having any of SEQ ID NOS:
924 to 955, 963 to 972, 1005 to 1013, or 1018 to 1023 at the
C-terminus. Exemplary C-terminal derivative groups include, for
example, --C(O)R.sup.2 wherein R.sup.2 is lower alkoxy or
--NR.sup.3R.sup.4 wherein R.sup.3 and R.sup.4 are independently
hydrogen or C.sub.1-C.sub.8 alkyl (preferably C.sub.1-C.sub.4
alkyl).
[0145] 7. 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.
[0146] 8. 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.
[0147] 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.
[0148] 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.
[0149] 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.
[0150] Carboxyl sidechain groups (aspartyl or glutamyl) may be
selectively modified by reaction with carbodiimides
(R.sup.1-N.dbd.C.dbd.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.
[0151] 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.
[0152] 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.
[0153] 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.
[0154] 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.
[0155] 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).
[0156] 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.
[0157] 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.
[0158] Methods of Making
[0159] The compounds of this invention largely may be made in
transformed host cells using recombinant DNA techniques. To do so,
a recombinant DNA molecule coding for the peptide is prepared.
Methods of preparing such DNA molecules are well known in the art.
For instance, sequences coding for the peptides could be excised
from DNA using suitable restriction enzymes. Alternatively, the DNA
molecule could be synthesized using chemical synthesis techniques,
such as the phosphoramidate method. Also, a combination of these
techniques could be used.
[0160] The invention also includes a vector capable of expressing
the peptides in an appropriate host. The vector comprises the DNA
molecule that codes for the peptides operatively linked to
appropriate expression control sequences. Methods of effecting this
operative linking, either before or after the DNA molecule is
inserted into the vector, are well known. Expression control
sequences include promoters, activators, enhancers, operators,
ribosomal binding sites, start signals, stop signals, cap signals,
polyadenylation signals, and other signals involved with the
control of transcription or translation.
[0161] The resulting vector having the DNA molecule thereon is used
to transform an appropriate host. This transformation may be
performed using methods well known in the art.
[0162] Any of a large number of available and well-known host cells
may be used in the practice of this invention. The selection of a
particular host is dependent upon a number of factors recognized by
the art. These include, for example, compatibility with the chosen
expression vector, toxicity of the peptides encoded by the DNA
molecule, rate of transformation, ease of recovery of the peptides,
expression characteristics, bio-safety and costs. A balance of
these factors must be struck with the understanding that not all
hosts may be equally effective for the expression of a particular
DNA sequence. Within these general guidelines, useful microbial
hosts include bacteria (such as E. coli sp.), yeast (such as
Saccharomyces sp.) and other fungi, insects, plants, mammalian
(including human) cells in culture, or other hosts known in the
art.
[0163] 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.
[0164] The compounds may also be made by synthetic methods. For
example, solid phase synthesis techniques may be used. Suitable
techniques are well known in the art, and include those described
in Merrifield (1973), Chem. Polypeptides, pp. 335-61 (Katsoyannis
and Panayotis eds.); Merrifield (1963), J. Am. Chem. Soc. 85: 2149;
Davis et al. (1985), Biochem. Intl. 10: 394-414; Stewart and Young
(1969), Solid Phase Peptide Synthesis; U.S. Pat. No. 3,941,763;
Finn et al. (1976), The Proteins (3rd ed.) 2: 105-253; and Erickson
et al. (1976), The Proteins (3rd ed.)2: 257-527. Solid phase
synthesis is the preferred technique of making individual peptides
since it is the most cost-effective method of making small
peptides.
[0165] Compounds that contain derivatized peptides or which contain
non-peptide groups may be synthesized by well-known organic
chemistry techniques.
[0166] Uses of the Compounds
[0167] In general. The compounds of this invention have
pharmacologic activity resulting from their ability to bind to
proteins of interest as agonists, mimetics or antagonists of the
native ligands of such proteins of interest. The utility of
specific compounds is shown in Table 2. The activity of these
compounds can be measured by assays known in the art. For the
TPO-mimetic and EPO-mimetic compounds, in vivo assays are further
described in the Examples section herein.
[0168] 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.
[0169] 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.
[0170] 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.
[0171] The conditions to be treated are generally those that
involve an existing megakaryocyte/platelet deficiency or an
expected megakaryocyte/platelet deficiency (e.g., because of
planned surgery or platelet donation). Such conditions will usually
be the result of a deficiency (temporary or permanent) of active
Mpl ligand in vivo. The generic term for platelet deficiency is
thrombocytopenia, and hence the methods and compositions of the
present invention are generally available for treating
thrombocytopenia in patients in need thereof. Thrombocytopenia
(platelet deficiencies) may be present for various reasons,
including chemotherapy and other therapy with a variety of drugs,
radiation therapy, surgery, accidental blood loss, and other
specific disease conditions. Exemplary specific disease conditions
that involve thrombocytopenia and may be treated in accordance with
this invention are: aplastic anemia, idiopathic thrombocy openia,
metastatic tumors which result in thrombocytopenia, systemic lupus
erythematosus, splenomegaly, Fanconi's syndrome, vitamin B12
deficiency, folic acid deficiency, May-Hegglin anomaly,
Wiskott-Aldrich syndrome, and paroxysmal nocturnal hemoglobinuria.
Also, certain treatments for AIDS result in thrombocytopenia (e.g.,
AZT). Certain wound healing disorders might also benefit from an
increase in platelet numbers.
[0172] With regard to anticipated platelet deficiencies, e.g., due
to future surgery, a compound of the present invention could be
administered several days to several hours prior to the need for
platelets. With regard to acute situations, e.g., accidental and
massive blood loss, a compound of this invention could be
administered along with blood or purified platelets.
[0173] The TPO-mimetic compounds of this invention may also be
useful in stimulating certain cell types other than megakaryocytes
if such cells are found to express Mpl receptor. Conditions
associated with such cells that express the Mpl receptor, which are
responsive to stimulation by the Mpl ligand, are also within the
scope of this invention.
[0174] 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.
[0175] 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.
[0176] 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.
[0177] 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 .mu.g-1 mg inventive compound per
10.sup.6 cells.
[0178] Pharmaceutical Compositions
[0179] In General. The present invention also provides methods of
using pharmaceutical compositions of the inventive compounds. Such
pharmaceutical compositions may be for administration for
injection, or for oral, pulmonary, nasal, transdermal or other
forms of administration. In general, the invention encompasses
pharmaceutical compositions comprising effective amounts of a
compound of the invention together with pharmaceutically acceptable
diluents, preservatives, solubilizers, emulsifiers, adjuvants
and/or carriers. Such compositions include diluents of various
buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic
strength; additives such as detergents and solubilizing agents
(e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic
acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl
alcohol) and bulking substances (e.g., lactose, mannitol);
incorporation of the material into particulate preparations of
polymeric compounds such as polylactic acid, polyglycolic acid,
etc. or into liposomes. Hyaluronic acid may also be used, and this
may have the effect of promoting sustained duration in the
circulation. Such compositions may influence the physical state,
stability, rate of in vivo release, and rate of in vivo clearance
of the present proteins and derivatives. See, e.g., Remington's
Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co.,
Easton, Pa. 18042) pages 1435-1712 which are herein incorporated by
reference. The compositions may be prepared in liquid form, or may
be in dried powder, such as lyophilized form. Implantable sustained
release formulations are also contemplated, as are transdermal
formulations.
[0180] Oral dosage forms. Contemplated for use herein are oral
solid dosage forms, which are described generally in Chapter 89 of
Remington's Pharmaceutical Sciences (1990), 18th Ed., Mack
Publishing Co. Easton Pa. 18042, which is herein incorporated by
reference. Solid dosage forms include tablets, capsules, pills,
troches or lozenges, cachets or pellets. Also, liposomal or
proteinoid encapsulation may be used to formulate the present
compositions (as, for example, proteinoid microspheres reported in
U.S. Pat. No. 4,925,673). Liposomal encapsulation may be used and
the liposomes may be derivatized with various polymers (e.g., U.S.
Pat. No. 5,013,556). A description of possible solid dosage forms
for the therapeutic is given in Chapter 10 of Marshall, K., Modern
Pharmaceutics (1979), edited by G. S. Banker and C. T. Rhodes,
herein incorporated by reference. In general, the formulation will
include the inventive compound, and inert ingredients which allow
for protection against the stomach environment, and release of the
biologically active material in the intestine.
[0181] Also specifically contemplated are oral dosage forms of the
above inventive compounds. If necessary, the compounds may be
chemically modified so that oral delivery is efficacious.
Generally, the chemical modification contemplated is the attachment
of at least one moiety to the compound molecule itself, where said
moiety permits (a) inhibition of proteolysis; and (b) uptake into
the blood stream from the stomach or intestine. Also desired is the
increase in overall stability of the compound and increase in
circulation time in the body. Moieties useful as covalently
attached vehicles in this invention may also be used for this
purpose.
[0182] Examples of such moieties include: PEG, copolymers of
ethylene glycol and propylene glycol, carboxymethyl cellulose,
dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline.
See, for example, Abuchowski and Davis, Soluble Polymer-Enzyme
Adducts, Enzymes as Drugs (1981), Hocenberg and Roberts, eds.,
Wiley-Interscience, New York, N.Y., pp 367-83; Newmark, et al.
(1982), J. Appl. Biochem. 4:185-9. Other polymers that could be
used are poly-1,3-dioxolane and poly-1,3,6-tioxocane. Preferred for
pharmaceutical usage, as indicated above, are PEG moieties.
[0183] For oral delivery dosage forms, it is also possible to use a
salt of a modified aliphatic amino acid, such as sodium
N-(8-[2-hydroxybenzoyl]amino) caprylate (SNAC), as a carrier to
enhance absorption of the therapeutic compounds of this invention.
The clinical efficacy of a heparin formulation using SNAC has been
demonstrated in a Phase II trial conducted by Emisphere
Technologies. See U.S. Pat. No. 5,792,451, "Oral drug delivery
composition and methods".
[0184] The compounds of this invention can be included in the
formulation as fine multiparticulates in the form of granules or
pellets of particle size about 1 mm. The formulation of the
material for capsule administration could also be as a powder,
lightly compressed plugs or even as tablets. The therapeutic could
be prepared by compression.
[0185] Colorants and flavoring agents may all be included. For
example, the protein (or derivative) may be formulated (such as by
liposome or microsphere encapsulation) and then further contained
within an edible product, such as a refrigerated beverage
containing colorants and flavoring agents.
[0186] One may dilute or increase the volume of the compound of the
invention with an inert material. These diluents could include
carbohydrates, especially mannitol, .alpha.-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.
[0187] Disintegrants may be included in the formulation of the
therapeutic into a solid dosage form. Materials used as
disintegrants include but are not limited to starch including the
commercial disintegrant based on starch, Explotab. Sodium starch
glycolate, Amberlite, sodium carboxymethylcellulose,
ultramylopectin, sodium alginate, gelatin, orange peel, acid
carboxymethyl cellulose, natural sponge and bentonite may all be
used. Another form of the disintegrants are the insoluble cationic
exchange resins. Powdered gums may be used as disintegrants and as
binders and these can include powdered gums such as agar, Karaya or
tragacanth. Alginic acid and its sodium salt are also useful as
disintegrants.
[0188] Binders may be used to hold the therapeutic agent together
to form a hard tablet and 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.
[0189] An antifrictional agent may be included in the formulation
of the therapeutic to prevent sticking during the formulation
process. Lubricants may be used as a layer between the therapeutic
and the die wall, and these can include but are not limited to;
stearic acid including its magnesium and calcium salts,
polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and
waxes. Soluble lubricants may also be used such as sodium lauryl
sulfate, magnesium lauryl sulfate, polyethylene glycol of various
molecular weights, Carbowax 4000 and 6000.
[0190] Glidants that might improve the flow properties of the drug
during formulation and to aid rearrangement during compression
might be added. The glidants may include starch, talc, pyrogenic
silica and hydrated silicoaluminate.
[0191] To aid dissolution of the compound of this invention into
the aqueous environment a surfactant might be added as a wetting
agent. Surfactants may include anionic detergents such as sodium
lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium
sulfonate. Cationic detergents might be used and could include
benzalkonium chloride or benzethonium chloride. The list of
potential nonionic detergents that could be included in the
formulation as surfactants are lauromacrogol 400, polyoxyl 40
stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60,
glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty
acid ester, methyl cellulose and carboxymethyl cellulose. These
surfactants could be present in the formulation of the protein or
derivative either alone or as a mixture in different ratios.
[0192] Additives may also be included in the formulation to enhance
uptake of the compound. Additives potentially having this property
are for instance the fatty acids oleic acid, linoleic acid and
linolenic acid.
[0193] Controlled release formulation may be desirable. The
compound of this invention could be incorporated into an inert
matrix which permits release by either diffusion or leaching
mechanisms e.g., gums. Slowly degenerating matrices may also be
incorporated into the formulation, e.g., alginates,
polysaccharides. Another form of a controlled release of the
compounds of this invention is by a method based on the Oros
therapeutic system (Alza Corp.), i.e., the drug is enclosed in a
semipermeable membrane which allows water to enter and push drug
out through a single small opening due to osmotic effects. Some
enteric coatings also have a delayed release effect.
[0194] Other coatings may be used for the formulation. These
include a variety of sugars which could be applied in a coating
pan. The therapeutic agent could also be given in a film coated
tablet and the materials used in this instance are divided into 2
groups. The first are the nonenteric materials and include methyl
cellulose, ethyl cellulose, hydroxyethyl cellulose,
methylhydroxy-ethyl cellulose, hydroxypropyl cellulose,
hydroxypropyl-methyl cellulose, sodium carboxy-methyl cellulose,
providone and the polyethylene glycols. The second group consists
of the enteric materials that are commonly esters of phthalic
acid.
[0195] A mix of materials might be used to provide the optimum film
coating. Film coating may be carried out in a pan coater or in a
fluidized bed or by compression coating.
[0196] Pulmonary delivery forms. Also contemplated herein is
pulmonary delivery of the present protein (or derivatives thereof).
The protein (or derivative) is delivered to the lungs of a mammal
while inhaling and traverses across the lung epithelial lining to
the blood stream. (Other reports of this include Adjei et al.,
Pharma. Res. (1990) 7: 565-9; Adjei et al. (1990), Internatl. J.
Pharmaceutics 63: 135-44 (leuprolide acetate); Braquet et al.
(1989), J. Cardiovasc. Pharmacol. 13 (suppl.5): s.143-146
(endothelin-1); Hubbard et al. (1989), Annals Int. Med. 3: 206-12
(.alpha.1-antitrypsin); Smith et al. (1989), J. Clin. Invest.
84:1145-6 (.alpha.1-proteinase); Oswein et al. (March 1990),
"Aerosolization of Proteins", Proc. Symp. Resp. Drug Delivery II,
Keystone, Colo. (recombinant human growth hormone); Debs et al.
(1988), J. Immunol. 140: 3482-8 (interferon-.gamma. and tumor
necrosis factor .alpha.) and Platz et al., U.S. Pat. No. 5,284,656
(granulocyte colony stimulating factor).
[0197] Contemplated for use in the practice of this invention are a
wide range of mechanical devices designed for pulmonary delivery of
therapeutic products, including but not limited to nebulizers,
metered dose inhalers, and powder inhalers, all of which are
familiar to those skilled in the art. Some specific examples of
commercially available devices suitable for the practice of this
invention are the Ultravent nebulizer, manufactured by
Mallinckrodt, Inc., St. Louis, Mo.; the Acorn II nebulizer,
manufactured by Marquest Medical Products, Englewood, Colo.; the
Ventolin metered dose inhaler, manufactured by Glaxo Inc., Research
Triangle Park, North Carolina; and the Spinhaler powder inhaler,
manufactured by Fisons Corp., Bedford, Mass.
[0198] All such devices require the use of formulations suitable
for the dispensing of the inventive compound. Typically, each
formulation is specific to the type of device employed and may
involve the use of an appropriate propellant material, in addition
to diluents, adjuvants and/or carriers useful in therapy.
[0199] The inventive compound should most advantageously be
prepared in particulate form with an average particle size of less
than 10 .mu.m (or microns), most preferably 0.5 to 5 .mu.m, for
most effective delivery to the distal lung.
[0200] Pharmaceutically acceptable carriers include carbohydrates
such as trehalose, mannitol, xylitol, sucrose, lactose, and
sorbitol. Other ingredients for use in formulations may include
DPPC, DOPE, DSPC and DOPC. Natural or synthetic surfactants may be
used. PEG may be used (even apart from its use in derivatizing the
protein or analog). Dextrans, such as cyclodextran, may be used.
Bile salts and other related enhancers may be used. Cellulose and
cellulose derivatives may be used. Amino acids may be used, such as
use in a buffer formulation.
[0201] Also, the use of liposomes, microcapsules or microspheres,
inclusion complexes, or other types of carriers is
contemplated.
[0202] 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.
[0203] 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.
[0204] 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.
[0205] Nasal delivery forms. Nasal delivery of the inventive
compound is also contemplated. Nasal delivery allows the passage of
the protein to the blood stream directly after administering the
therapeutic product to the nose, without the necessity for
deposition of the product in the lung. Formulations for nasal
delivery include those with dextran or cyclodextran. Delivery via
transport across other mucous membranes is also contemplated.
[0206] Dosages. The dosage regimen involved in a method for
treating the above-described conditions will be determined by the
attending physician, considering various factors which modify the
action of drugs, e.g. the age, condition, body weight, sex and diet
of the patient, the severity of any infection, time of
administration and other clinical factors. Generally, the daily
regimen should be in the range of 0.1-1000 micrograms of the
inventive compound per kilogram of body weight, preferably 0.1-150
micrograms per kilogram.
[0207] Specific Preferred Embodiments
[0208] The inventors have determined preferred peptide sequences
for molecules having many different kinds of activity. The
inventors have further determined preferred structures of these
preferred peptides combined with preferred linkers and vehicles.
Preferred structures for these preferred peptides listed in Table
21 below.
22TABLE 21 Preferred embodiments SEQ ID Sequence/structure NO:
Activity F.sup.1-(G).sub.5-IEGPTLRQWLAARA-(G).sub.8-IEGPTLRQWLAARA
337 TPO-mimetic
IEGPTLRQWLAARA-(G).sub.8-IEGPTLRQWLAARA-(G).sub.5-F.sup.1 338
TPO-mimetic F.sup.1-(G).sub.5-IEGPTLRQWLAARA 1032 TPO-mimetic
IEGPTLRQWLAARA-(G).sub.5-F.sup.1 1033 TPO-mimetic
F.sup.1-(G).sub.5-GGTYSCHFGPLTWVCKPQGG-(G).sub.4- 339 EPO-mimetic
GGTYSCHFGPLTWVCKPQGG GGTYSCHFGPLTWVCKPQGG-(G).sub.4- 340
EPO-mimetic GGTYSCHFGPLTWVCKPQGG-(G).sub.5-F.sup.1
GGTYSCHFGPLTWVCKPQGG-(G).sub.5-F.sup.1 1034 EPO-mimetic
F.sup.1-(G).sub.5-DFLPHYKNTSLGHRP 1045 TNF-.alpha. inhibitor
DFLPHYKNTSLGHRP-(G).sub.5-F.sup.1 1046 TNF-.alpha. inhibitor
F.sup.1-(G).sub.5-FEWTPGYWQPYALPL 1047 IL-1 R antagonist
FEWTPGYWQPYALPL-(G).sub.5-F.sup.1 1048 IL-1 R antagonist
F.sup.1-(G).sub.5-VEPNCDIHVMWEWECFERL 1049 VEGF-antagonist
VEPNCDIHVMWEWECFERL-(G).sub.5-F.sup.1 1050 VEGF-antagonist
F.sup.1-(G).sub.5-CTTHWGFTLC 1051 MMP inhibitor
CTTHWGFTLC-(G).sub.5-F.sup.1 1052 MMP inhibitor "F.sup.1" is an Fc
domain as defined previously herein.
WORKING EXAMPLES
[0209] The compounds described above may be prepared as described
below. These examples comprise preferred embodiments of the
invention and are illustrative rather than limiting.
Example 1
TPO-Mimetics
[0210] The following example uses peptides identified by the
numbers appearing in Table A hereinafter.
[0211] Preparation of peptide 19. Peptide 17b (12 mg) and
MeO-PEG-SH 5000 (30 mg, 2 equiv.) were dissolved in 1 ml aqueous
buffer (pH 8). The mixture was incubated at RT for about 30 minutes
and the reaction was checked by analytical HPLC, which showed a
>80% completion of the reaction. The pegylated material was
isolated by preparative HPLC.
[0212] Preparation of peptide 20. Peptide 18 (14 mg) and
MeO-PEG-maleimide (25 mg) were dissolved in about 1.5 ml aqueous
buffer (pH 8). The mixture was incubated at RT for about 30
minutes, at which time about 70% transformation was complete as
monitored with analytical HPLC by applying an aliquot of sample to
the HPLC column. The pegylated material was purified by preparative
HPLC.
[0213] Bioactivity assay. The TPO in vitro bioassay is a mitogenic
assay utilizing an IL-3 dependent clone of murine 32D cells that
have been transfected with human mpl receptor. This assay is
described in greater detail in WO 95/26746. Cells are maintained in
MEM medium containing 10% Fetal Clone II and 1 ng/ml mIL-3. Prior
to sample addition, cells are prepared by rinsing twice with growth
medium lacking mIL-3. An extended twelve point TPO standard curve
is prepared, ranging from 33 to 39 .mu.g/ml. Four dilutions,
estimated to fall within the linear portion of the standard curve,
(100 to 125 .mu.g/ml), are prepared for each sample and run in
triplicate. A volume of 100 .mu.l of each dilution of sample or
standard is added to appropriate wells of a 96 well microtiter
plate -containing 10,000 cells/well. After forty-four hours at
37.degree. C. and 10% CO.sub.2, MTS (a tetrazolium compound which
is bioreduced by cells to a formazan) is added to each well.
Approximately six hours later, the optical density is read on a
plate reader at 490 nm. A dose response curve (log TPO
concentration vs. O.D.--Background) is generated and linear
regression analysis of points which fall in the linear portion of
the standard curve is performed. Concentrations of unknown test
samples are determined using the resulting linear equation and a
correction for the dilution factor.
[0214] TMP tandem repeats with polyglycine linkers. Our design of
sequentially linked TMP repeats was based on the assumption that a
dimeric form of TMP was required for its effective interaction with
c-Mpl (the TPO receptor) and that depending on how they were wound
up against each other in the receptor context, the two TMP
molecules could be tethered together in the C- to N-terminus
configuration in a way that would not perturb the global dimeric
conformation. Clearly, the success of the design of tandem linked
repeats depends on proper selection of the length and composition
of the linker that joins the C- and N-termini of the two
sequentially aligned TMP monomers. Since no structural information
of the TMP bound to c-Mpl was available, a series of repeated
peptides with linkers composed of 0 to 10 and 14 glycine residues
(Table A) were synthesized. Glycine was chosen because of its
simplicity and flexibility, based on the rationale that a flexible
polyglycine peptide chain might allow for the free folding of the
two tethered TMP repeats into the required conformation, while
other amino acid sequences may adopt undesired secondary structures
whose rigidity might disrupt the correct packing of the repeated
peptide in the receptor context.
[0215] The resulting peptides are readily accessible by
conventional solid phase peptide synthesis methods (Merrifield
(1963), J. Amer. Chem. Soc. 85: 2149) with either Fmoc or t-Boc
chemistry. Unlike the synthesis of the C-terminally linked parallel
dimer which required the use of an orthogonally protected lysine
residue as the initial branch point to build the two peptide chains
in a pseudosymmetrical way (Cwirla et al. (1997), Science 276:
1696-9), the synthesis of these tandem repeats was a
straightforward, stepwise assembly of the continuous peptide chains
from the C- to N-terminus. Since dimerization of TMP had a more
dramatic effect on the proliferative activity than binding affinity
as shown for the C-terminal dimer (Cwirla et al. (1997)), the
synthetic peptides were tested directly for biological activity in
a TPO-dependent cell-proliferation assay using an IL-3 dependent
clone of murine 32D cells transfected with the full-length c-Mpl
(Palacios et al., Cell 41:727 (1985)). As the test results showed,
all the polyglycine linked tandem repeats demonstrated >1000
fold increases in potency as compared to the monomer, and were even
more potent than the C-terminal dimer in this cell proliferation
assay. The absolute activity of the C-terminal dimer in our assay
was lower than that of the native TPO protein, which is different
from the previously reported findings in which the C-terminal dimer
was found to be as active as the natural ligand (Cwirla et al.
(1997)). This might be due to differences in the conditions used in
the two assays. Nevertheless, the difference in activity between
tandem (C terminal of first monomer linked to N terminal of second
monomer) and C-terminal (C terminal of first monomer linked to C
terminal of second monomer; also referred to as parallel) dimers in
the same assay clearly demonstrated the superiority of tandem
repeat strategy over parallel peptide dimerization. It is
interesting to note that a wide range of length is tolerated by the
linker. The optimal linker between tandem peptides with the
selected TMP monomers apparently is composed of 8 glycines.
[0216] Other tandem repeats. Subsequent to this first series of TMP
tandem repeats, several other molecules were designed either with
different linkers or containing modifications within the monomer
itself.
[0217] The first of these molecules, peptide 13, has a linker
composed of GPNG, a sequence known to have a high propensity to
form a .beta.-turn-type secondary structure. Although still about
100-fold more potent than the monomer, this peptide was found to be
>10-fold less active than the equivalent GGGG-linked analog.
Thus, introduction of a relatively rigid .beta.-turn at the linker
region seemed to have caused a slight distortion of the optimal
agonist conformation in this short linker form.
[0218] The Trp9 in the TMP sequence is a highly conserved residue
among the active peptides isolated from random peptide libraries.
There is also a highly conserved Trp in the consensus sequences of
EPO mimetic peptides and this Trp residue was found to be involved
in the formation of a hydrophobic core between the two EMPs and
contributed to hydrophobic interactions with the EPO receptor.
Livnah et al. (1996), Science 273: 464-71). By analogy, the Trp9
residue in TMP might have a similar function in dimerization of the
peptide ligand, and as an attempt to modulate and estimate the
effects of noncovalent hydrophobic forces exerted by the two indole
rings, several analogs were made resulting from mutations at the
Trp. So in peptide 14, the Trp residue was replaced in each of the
two TMP monomers with a Cys, and an intramolecular disulfide bond
was formed between the two cysteines by oxidation which was
envisioned to mimic the hydrophobic interactions between the two
Trp residues in peptide dimerization. Peptide 15 is the reduced
form of peptide 14. In peptide 16, the two Trp residues were
replaced by Ala. As the assay data show, all three analogs were
inactive. These data further demonstrated that Trp is critical for
the activity of the TPO mimetic peptide, not just for dimer
formation.
[0219] The next two peptides (peptide 17a, and 18) each contain in
their 8-amino acid linker a Lys or Cys residue. These two compounds
are precursors to the two PEGylated peptides (peptide 19 and 20) in
which the side chain of the Lys or Cys is modified by a PEG moiety.
A PEG moiety was introduced at the middle of a relatively long
linker, so that the large PEG component (5 kDa) is far enough away
from the critical binding sites in the peptide molecule. PEG is a
known biocompatible polymer which is increasingly used as a
covalent modifier to improve the pharmacokinetic profiles of
peptide- and protein-based therapeutics.
[0220] A modular, solution-based method was devised for convenient
PEGylation of synthetic or recombinant peptides. The method is
based on the now well established chemoselective ligation strategy
which utilizes the specific reaction between a pair of mutually
reactive functionalities. So, for pegylated peptide 19, the lysine
side chain was preactivated with a bromoacetyl group to give
peptide 17b to accommodate reaction with a thiol-derivatized PEG.
To do that, an orthogonal protecting group, Dde, was employed for
the protection of the lysine .epsilon.-amine. Once the whole
peptide chain was assembled, the N-terminal amine was reprotected
with t-Boc. Dde was then removed to allow for the bromoacetylation.
This strategy gave a high quality crude peptide which was easily
purified using conventional reverse phase HPLC. Ligation of the
peptide with the thiol-modified PEG took place in aqueous buffer at
pH 8 and the reaction completed within 30 minutes. MALDI-MS
analysis of the purified, pegylated material revealed a
characteristic, bell-shaped spectrum with an increment of 44 Da
between the adjacent peaks. For PEG-peptide 20, a cysteine residue
was placed in the linker region and its side chain thiol group
would serve as an attachment site for a maleimide-containing
PEG.
[0221] Similar conditions were used for the pegylation of this
peptide. As the assay data revealed, these two pegylated peptides
had even higher in vitro bioactivity as compared to their
unpegylated counterparts.
[0222] Peptide 21 has in its 8-amino acid linker a potential
glycosylation motif, NGS. Since our exemplary tandem repeats are
made up of natural amino acids linked by peptide bonds, expression
of such a molecule in an appropriate eukaryotic cell system should
produce a glycopeptide with the carbohydrate moiety added on the
side chain carboxyamide of Asn. Glycosylation is a common
post-translational modification process which can have many
positive impacts on the biological activity of a given protein by
increasing its aqueous solubility and in vivo stability. As the
assay data show, incorporation of this glycosylation motif into the
linker maintained high bioactivity. The synthetic precursor of the
potential glycopeptide had in effect an activity comparable to that
of the -(G).sub.8-linked analog. Once glycosylated, this peptide is
expected to have the same order of activity as the pegylated
peptides, because of the similar chemophysical properties exhibited
by a PEG and a carbohydrate moiety.
[0223] The last peptide is a dimer of a tandem repeat. It was
prepared by oxidizing peptide 18, which formed an intermolecular
disulfide bond between the two cysteine residues located at the
linker. This peptide was designed to address the possibility that
TMP was active as a tetramer. The assay data showed that this
peptide was not more active than an average tandem repeat on an
adjusted molar basis, which indirectly supports the idea that the
active form of TMP is indeed a dimer, otherwise dimerization of a
tandem repeat would have a further impact on the bioactivity.
[0224] In order to confirm the in vitro data in animals, one
pegylated TMP tandem repeat (compound 20 in Table A) was delivered
subcutaneously to normal mice via osmotic pumps. Time and
dose-dependent increases were seen in platelet numbers for the
duration of treatment. Peak platelet levels over 4-fold baseline
were seen on day 8. A dose of 10 .mu.g/kg/day of the pegylated TMP
repeat produced a similar response to rHuMGDF (non-pegylated) at
100 .mu.g/kg/day delivered by the same route.
23TABLE A TPO-mimetic Peptides Peptide SEQ ID Relative No. Compound
NO: Potency TPO ++++ TMP monomer 13 + TMP C-C dimer +++-
TMP-(G).sub.n-TMP: 1 n = 0 341 ++++- 2 n = 1 342 ++++ 3 n = 2 343
++++ 4 n = 3 344 ++++ 5 n = 4 345 ++++ 6 n = 5 346 ++++ 7 n = 6 347
++++ 8 n = 7 348 ++++ 9 n = 8 349 ++++- 10 n = 9 350 ++++ 11 n = 10
351 ++++ 12 n = 14 352 ++++ 13 TMP-GPNG-TMP 353 +++ 14 15 354 - 15
IEGPTLRQWCLAARA-GGGGGGGG- 355 - IEGPTLRQCLAARA-(linear) 16
IEGPTLRQALAARA-GGGGGGGG- 356 - IEGPTLRQALAARA 17a TMP-GGGKGGGG-TMP
357 ++++ 17b TMP-GGGK(BrAc)GGGG-TMP 358 ND 18 TMNP-GGGCGGGG-TMP 359
++++ 19 TMP-GGGK(PEG)GGGG-TMP 360 +++++ 20 TMP-GGGC(PEG)GGGG-TMP
361 +++++ 21 TMP-GGGN*GSGG-TMP 362 ++++ 22 TMP-GGGCGGGG-TMP 363
.vertline. ++++ TMP-GGGCGGGG-TMP 363
[0225] Discussion. It is well accepted that MGDF acts in a way
similar to hGH, i.e., one molecule of the protein ligand binds two
molecules of the receptor for its activation. Wells et al.(1996),
Ann. Rev. Biochem. 65: 609-34. Now, this interaction is mimicked by
the action of a much smaller peptide, TMP. However, the present
studies suggest that this mimicry requires the concerted action of
two TMP molecules, as covalent dimerization of TMP in either a C--C
parallel or C--N sequential fashion increased the in vitro
biological potency of the original monomer by a factor of greater
than 10.sup.3. The relatively low biopotency of the monomer is
probably due to inefficient formation of the noncovalent dimer. A
preformed covalent repeat has the ability to eliminate the entropy
barrier for the formation of a noncovalent dimer which is
exclusively driven by weak, noncovalent interactions between two
molecules of the small, 14-residue peptide.
[0226] It is intriguing that this tandem repeat approach had a
similar effect on enhancing bioactivity as the reported C--C
dimerization is intriguing. These two strategies brought about two
very different molecular configurations. The C--C dimer is a
quasi-symmetrical molecule, while the tandem repeats have no such
symmetry in their linear structures. Despite this difference in
their primary structures, these two types of molecules appeared
able to fold effectively into a similar biologically active
conformation and cause the dimerization and activation of c-Mpl.
These experimental observations provide a number of insights into
how the two TMP molecules may interact with one another in binding
to c-Mpl. First, the two C-termini of the two bound TMP molecules
must be in relatively close proximity with each other, as suggested
by data on the C-terminal dimer. Second, the respective N- and
C-termini of the two TMP molecules in the receptor complex must
also be very closely aligned with each other, such that they can be
directly tethered together with a single peptide bond to realize
the near maximum activity-enhancing effect brought about by the
tandem repeat strategy. Insertion of one or more (up to 14) glycine
residues at the junction did not increase (or decrease)
significantly the activity any further. This may be due to the fact
that a flexible polyglycine peptide chain is able to loop out
easily from the junction without causing any significant changes in
the overall conformation. This flexibility seems to provide the
freedom of orientation for the TMP peptide chains to fold into the
required conformation in interacting with the receptor and validate
it as a site of modification. Indirect evidence supporting this
came from the study on peptide 13, in which a much more rigid
b-turn-forming sequence as the linker apparently forced a deviation
of the backbone alignment around the linker which might have
resulted in a slight distortion of the optimal conformation, thus
resulting in a moderate (10-fold) decrease in activity as compared
with the analogous compound with a 4-Gly linker. Third, Trp9 in TMP
plays a similar role as Trp13 in EMP, which is involved not only in
peptide:peptide interaction for the formation of dimers but also is
important for contributing hydrophobic forces in peptide:receptor
interaction. Results obtained with the W to C mutant analog,
peptide 14, suggest that a covalent disulfide linkage is not
sufficient to approximate the hydrophobic interactions provided by
the Trp pair and that, being a short linkage, it might bring the
two TMP monomers too close, therefore perturbing the overall
conformation of the optimal dimeric structure.
[0227] An analysis of the possible secondary structure of the TMP
peptide can provide further understanding on the interaction
between TMP and c-Mpl. This can be facilitated by making reference
to the reported structure of the EPO mimetic peptide. Livnah et al.
(1996), Science 273:464-75 The receptor-bound EMP has a b-hairpin
structure with a b-turn formed by the highly consensus
Gly-Pro-Leu-Thr at the center of its sequence. Instead of GPLT, TMP
has a highly selected GPTL sequence which is likely to form a
similar turn. However, this turn-like motif is located near the
N-terminal part in TMP. Secondary structure prediction using
Chau-Fasman method suggests that the C-terminal half of the peptide
has a tendency to adopt a helical conformation. Together with the
highly conserved Trp at position 9, this C-terminal helix may
contribute to the stabilization of the dimeric structure. It is
interesting to note that most of our tandem repeats are more potent
than the C-terminal parallel dimer. Tandem repeats seem to give the
molecule a better fit conformation than does the C--C parallel
dimerization. The seemingly asymmetric feature of a tandem repeat
might have brought it closer to the natural ligand which, as an
asymmetric molecule, uses two different sites to bind two identical
receptor molecules.
[0228] Introduction of a PEG moiety was envisaged to enhance the in
vivo activity of the modified peptide by providing it a protection
against proteolytic degradation and by slowing down its clearance
through renal filtration. It was unexpected that pegylation could
further increase the in vitro bioactivity of a tandem repeated TMP
peptide in the cell-based proliferation assay.
Example 2
Fc-TMP Fusions
[0229] TMPs (and EMPs as described in Example 3) were expressed in
either monomeric or dimeric form as either N-terminal or C-terminal
fusions to the Fc region of human IgG1. In all cases, the
expression construct utilized the luxPR promoter promoter in the
plasmid expression vector pAMG21.
[0230] Fc-TMP. A DNA sequence coding for the Fc region of human
IgG1 fused in-frame to a monomer of the TPO-mimetic peptide was
constructed using standard PCR technology. Templates for PCR
reactions were the pFc-A3 vector and a synthetic TMP gene. The
synthetic gene was constructed from the 3 overlapping
oligonucleotides (SEQ ID NOS: 364, 365, and 366, respectively)
shown below:
24 1842-97 AAA AAA GGA TCC TCG AGA TTA AGC ACG AGC AGC CAG CCA CTG
ACG CAG AGT CGG ACC 1842-98 AAA GGT GGA GGT GGT GGT ATC GAA GGT CCG
ACT CTG CGT 1842-99 CAG TGG CTG GCT GCT CGT GCT TAA TCT CGA GGA TCC
TTT TTT
[0231] These oligonucleotides were annealed to form the duplex
encoding an amino acid sequence (SEQ ID NOS: 367 and 368,
respectively) shown below:
25 AAAGGTGGAGGTGGTGGTATCGAAGGTCCGACTCTGCGTCAGTGGCTGGCTGCTCGTGCT 1
---------+---------+---------+---------+---------+---------+ 60
CCAGGCTGAGACGCAGTCACCGACCGACGAGCACGA a K G G G G G I E G P T L R Q
W L A A R A - TAATCTCGAGGATCCTTTTTT 61 ---------+---------+- 81
ATTAGAGCTCCTAGGAAAAAA a *
[0232] This duplex was amplified in a PCR reaction using 1842-98
and 1842-97 as the sense and antisense primers.
[0233] The Fc portion of the molecule was generated in a PCR
reaction with pFc-A3 using the primers shown below (SEQ ID NOS: 369
and 370):
26 1216-52 AAC ATA AGT ACC TGT AGG ATC G 1830-51 TTCGATACCA
CCACCTCCAC CTTTACCCGG AGACAGGGAG AGGCTCTTCTGC
[0234] The oligonucleotides 1830-51 and 1842-98 contain an overlap
of 24 nucleotides, allowing the two genes to be fused together in
the correct reading frame by combining the above PCR products in a
third reaction using the outside primers, 1216-52 and 1842-97.
[0235] The final PCR gene product (the full length fusion gene) was
digested with restriction endonucleases XbaI and BamHI, and then
ligated into the vector pAMG21 and transformed into competent E.
coli strain 2596 cells as described for EMP-Fc herein. Clones were
screened for the ability to produce the recombinant protein product
and to possess the gene fusion having the correct nucleotide
sequence. A single such clone was selected and designated Amgen
strain #3728.
[0236] The nucleotide and amino acid sequences (SEQ ID NOS: 5 and
6) of the fusion protein are shown in FIG. 7.
[0237] Fc-TMP-TMP. A DNA sequence coding for the Fc region of human
IgG1 fused in-frame to a dimer of the TPO-mimetic peptide was
constructed using standard PCR technology. Templates for PCR
reactions were the pFc-A3 vector and a synthetic TMP-TMP gene. The
synthetic gene was constructed from the 4 overlapping
oligonucleotides (SEQ ID NOS: 371 to 374, respectively) shown
below:
27 1830-52 AAA GGT GGA GGT GGT GGT ATC GAA GGT CCG ACT CTG CGT CAG
TGG CTG GCT GCT CGT GCT 1830-53 ACC TCC ACC ACC AGC ACG AGC AGC CAG
CCA CTG ACG CAG AGT CGG ACC 1830-54 GGT GGT GGA GGT GGC GGC GGA GGT
ATT GAG GGC CCA ACC CTT CGC CAA TGG CTT GCA GCA CGC GCA 1830-55 AAA
AAA AGG ATC CTC GAG ATT ATG CGC GTG CTG CAA GCC ATT GGC GAA GGG TTG
GGC CCT CAA TAC CTC CGC CGC C
[0238] The 4 oligonucleotides were annealed to form the duplex
encoding an amino acid sequence (SEQ ID NOS: 375 and 376,
respectively) shown below:
28 AAAGGTGGAGGTGGTGGTATCGAAGGTCCGACTCTGCGTCAGTGGCTGGCTGCTCGTGCT 1
---------+---------+---------+---------+---------+---------+ 60
CCAGGCTGAGACGCAGTCACCGACCGACGAGCACGA a K G G G G G I E G P T L R Q
W L A A R A -
GGTGGTGGAGGTGGCGGCGGAGGTATTGAGGGCCCAACCCTTCGCCAATGGCTTGCAGCA 61
---------+---------+---------+---------+---------+---------+ 120
CCACCACCTCCACCGCCGCCTCCATAACTCCCGGGTTGGGAAGCGGTTACCGAACGTCGT a G G
G G G G G G I E G P T L R Q W L A A - CGCGCA 121
---------------------------148 GCGCGTATTAGAGCTCCTAGGAAAAAAA a R A
*-
[0239] This duplex was amplified in a PCR reaction using 1830-52
and 1830-55 as the sense and antisense primers.
[0240] The Fc portion of the molecule was generated in a PCR
reaction with pFc-A3 using the primers 1216-52 and 1830-51 as
described above for Fc-TMP. The full length fusion gene was
obtained from a third PCR reaction using the outside primers
1216-52 and 1830-55.
[0241] The final PCR gene product (the full length fusion gene) was
digested with restriction endonucleases XbaI and BamHI, and then
ligated into the vector pAMG21 and transformed into competent E.
coli strain 2596 cells as described in example 1. Clones were
screened for the ability to produce the recombinant protein product
and to possess the gene fusion having the correct nucleotide
sequence. A single such clone was selected and designated Amgen
strain #3727.
[0242] The nucleotide and amino acid sequences (SEQ ID NOS: 7 and
8) of the fusion protein are shown in FIG. 8.
[0243] TMP-TMP-Fc. A DNA sequence coding for a tandem repeat of the
TPO-mimetic peptide fused in-frame to the Fc region of human IgG1
was constructed using standard PCR technology. Templates for PCR
reactions were the EMP-Fc plasmid from strain #3688 (see Example 3)
and a synthetic gene encoding the TMP dimer. The synthetic gene for
the tandem repeat was constructed from the 7 overlapping
oligonucleotides shown below (SEQ ID NOS: 377 to 383,
respectively):
29 1885-52 TTT TTT CAT ATG ATC GAA GGT CCG ACT CTG CGT CAG TGG
1885-53 AGC ACG AGC AGC CAG CCA CTG ACG CAG AGT CGG ACC TTC GAT CAT
ATG 1885-54 CTG GCT GCT CGT GCT GGT GGA GGC GGT GGG GAC AAA ACT CAC
ACA 1885-55 CTG GCT GCT CGT GCT GGC GGT GGT GGC GGA GGG GGT GGC ATT
GAG GGC CCA 1885-56 AAG CCA TTG GCG AAG GGT TGG GCC CTC AAT GCC ACC
CCC TCC GCC ACC ACC GCC 1885-57 ACC CTT CGC CAA TGG CTT GCA GCA CGC
GCA GGG GGA GGC GGT GGG GAC AAA ACT 1885-58 CCC ACC GCC TCC CCC TGC
GCG TGC TGC
[0244] These oligonucleotides were annealed to form the duplex
shown encoding an amino acid sequence shown below (SEQ ID NOS 384
and 385):
30 TTTTTTCATATGATCGAAGGTCCGACTCTGCGTCAGTGGCTGGCTGCTCGTGCTGGCGGT 1
---------+---------+---------+---------+---------+---------+ 60
GTATACTAGCTTCCAGGCTGAGACGCAGTCACCGACCGACGAGCACGACCGCCA a M I E G P
T L R Q W L A A R A G G -
GGTGGCGGAGGGGGTGGCATTGAGGGCCCAACCCTTCGCCAATGGCTGGCTGCTCGTGCT 61
---------+---------+---------+---------+---------+---------+ 120
CCACCGCCTCCCCCACCGTAACTCCCGGGTTGGGAAGCGGTTACCGAACGTCGTGCGCGT a G G
G G G G I E G P T L R Q W L A A R A -
GGTGGAGGCGGTGGGGACAAAACTCTGGCTGCTCGTGCTGGTGGAGGCGGTGGGGACA- AA 121
---------+---------+---------+---------+---------+---------- + 180
CCCCCTCCGCCACCC a G G G G G D K T L A A R A G G G G G D K -
ACTCACACA 181 --------- 189 a T H T -
[0245] This duplex was amplified in a PCR reaction using 1885-52
and 1885-58 as the sense and antisense primers.
[0246] The Fc portion of the molecule was generated in a PCR
reaction with DNA from the EMP-Fc fusion strain #3688 (see Example
3) using the primers 1885-54 and 1200-54. The full length fusion
gene was obtained from a third PCR reaction using the outside
primers 1885-52 and 1200-54.
[0247] The final PCR gene product (the full length fusion gene) was
digested with restriction endonucleases XbaI and BamHI, and then
ligated into the vector pAMG21 and transformed into competent E.
coli strain 2596 cells as described for Fc-EMP herein. Clones were
screened for the ability to produce the recombinant protein product
and to possess the gene fusion having the correct nucleotide
sequence. A single such clone was selected and designated Amgen
strain #3798.
[0248] The nucelotide and amino acid sequences (SEQ ID NOS: 9 and
10) of the fusion protein are shown in FIG. 9.
[0249] TMP-Fc. A DNA sequence coding for a monomer of the
TPO-mimetic peptide fused in-frame to the Fc region of human IgG1
was obtained fortuitously in the ligation in TMP-TMP-Fc, presumably
due to the ability of primer 1885-54 to anneal to 1885-53 as well
as to 1885-58. A single clone having the correct nucleotide
sequence for the TMP-Fc construct was selected and designated Amgen
strain #3788.
[0250] The nucleotide and amino acid sequences (SEQ ID NOS: 11 and
12) of the fusion protein are shown in FIG. 10.
[0251] Expression in E. coli. Cultures of each of the
pAMG21-Fc-fusion constructs in E. coli GM221 were grown at
37.degree. C. in Luria Broth medium containing 50 mg/ml kanamycin.
Induction of gene product expression from the luxPR promoter was
achieved following the addition of the synthetic autoinducer
N-(3-oxohexanoyl)-DL-homoserine lactone to the culture media to a
final concentration of 20 ng/ml. Cultures were incubated at
37.degree. C. for a further 3 hours. After 3 hours, the bacterial
cultures were examined by microscopy for the presence of inclusion
bodies and were then collected by centrifugation. Refractile
inclusion bodies were observed in induced cultures indicating that
the Fc-fusions were most likely produced in the insoluble fraction
in E. coli. Cell pellets were lysed directly by resuspension in
Laemmli sample buffer containing 10% b-mercaptoethanol and were
analyzed by SDS-PAGE. In each case, an intense coomassie-stained
band of the appropriate molecular weight was observed on an
SDS-PAGE gel.
[0252] pAMG21. The expression plasmid pAMG21 can be derived from
the Amgen expression vector pCFM1656 (ATCC #69576) which in turn be
derived from the Amgen expression vector system described in U.S.
Pat. No. 4,710,473. The pCFM1656 plasmid can be derived from the
described pCFM836 plasmid (U.S. Pat. No. 4,710,473) by:
[0253] (a) destroying the two endogenous NdeI restriction sites by
end filling with T4 polymerase enzyme followed by blunt end
ligation;
[0254] (b) replacing the DNA sequence between the unique AatII and
ClaI restriction sites containing the synthetic P.sub.L promoter
with a similar fragment obtained from pCFM636 (U.S. Pat. No.
4,710,473) containing the P.sub.L promoter (see SEQ ID NO: 386
below); and
[0255] (c) substituting the small DNA sequence between the unique
ClaI and KpnI restriction sites with the oligonucleotide having the
sequence of SEQ ID NO: 388.
31 AatII 5' CTAATTCCGCTCTCACCTACCAAACAATGCCCCCCTGCAAAAAATA-
AATTCATAT- SEQ ID NO: 386 3'
TGCAGATTAAGGCGAGAGTGGATGGTTTGTTACGGGGG- GACGTTTTTTATTTAAGTATA-
-AAAAAACATACAGATAACCATCTGCGGTGAT- AAATTATCTCTGGCGGTGTTGACATAAA-
-TTTTTTGTATGTCTATTGGTAGACGCCACTATT- TAATAGAGACCGCCACAACTGTATTT-
-TACCACTGGCGGTGATACTGAGCACA- T 3': -ATGGTGACCGCCACTATGACTCGTGTAGC
5' ClaI 5' CGATTTGATTCTAGAAGGAGGAATAACATA-
TGGTTAACGCGTTGGAATTCGGTAC 3': SEQ ID NO: 387 3'
TAAACTAAGATCTTCCTCCTTATTGTATACCAATTGCGCAACCTTAAGC 5' ClaI KpnI
[0256] The expression plasmid pAMG21 can then be derived from
pCFM1656 by making a series of site-directed base changes by PCR
overlapping oligo mutagenesis and DNA sequence substitutions.
Starting with the BglII site (plasmid bp # 180) immediately 5' to
the plasmid replication promoter PcopB and proceeding toward the
plasmid replication genes, the base pair changes are as shown in
Table B below.
32TABLE B Base pair changes resulting in pAMG21 pAMG21 bp # bp in
pCFM1656 bp changed to in pAMG21 # 204 T/A C/G # 428 A/T G/C # 509
G/C A/T # 617 -- insert two G/C bp # 679 G/C T/A # 980 T/A C/G #
994 G/C A/T # 1004 A/T C/G # 1007 C/G T/A # 1028 A/T T/A # 1047 C/G
T/A # 1178 G/C T/A # 1466 G/C T/A # 2028 G/C bp deletion # 2187 C/G
T/A # 2480 A/T T/A # 2499-2502 AGTG GTCA TCAC CAGT # 2642 TCCGAGC 7
bp deletion AGGCTCG # 3435 G/C A/T # 3446 G/C A/T # 3643 A/T
T/A
[0257] The DNA sequence between the unique AatII (position #4364 in
pCFM1656) and SacII (position #4585 in pCFM1656) restriction sites
is substituted with the DNA sequence (SEQ ID NO: 23) shown in FIGS.
17A and 17B. During the ligation of the sticky ends of this
substitution DNA sequence, the outside AatII and SacII sites are
destroyed. There are unique AatII and SacII sites in the
substituted DNA.
[0258] GM221 (Amgen #2596). The Amgen host strain #2596 is an E.
coli K-12 strain derived from Amgen strain #393. It has been
modified to contain both the temperature sensitive lambda repressor
cI857s7 in the early ebg region and the lacIq repressor in the late
ebg region (68 minutes). The presence of these two repressor genes
allows the use of this host with a variety of expression systems,
however both of these repressors are irrelevant to the expression
from luxP.sub.R. The untransformed host has no antibiotic
resistances.
[0259] The ribosome binding site of the cI857s7 gene has been
modified to include an enhanced RBS. It has been inserted into the
ebg operon between nucleotide position 1170 and 1411 as numbered in
Genbank accession number M64441Gb_Ba with deletion of the
intervening ebg sequence. The sequence of the insert is shown below
with lower case letters representing the ebg sequences flanking the
insert shown below
33 (SEQ ID NO: 388) ttattttcgtGCGGCCGCACCATTATCACCGCCAGAGGT-
AAACTAGTCAACACGCACGGTGTTAGATATTTAT
CCCTTGCGGTGATAGATTGAGCACATCGATTTGATTCTAGAAGGAGGGATAATATATGAGCACAAAAAAGAAA
CCATTAACACAAGAGCAGCTTGAGGACGCACGTCGCCTTAAAGCAATTTATGAAAAA-
AAGAAAAATGAACTTG GCTTATCCCAGGAATCTGTCGCAGACAAGATGGGGATGGGG-
CAGTCAGGCGTTGGTGCTTTATTTAATGGCAT CAATGCATTAAATGCTTATAACGCC-
GCATTGCTTACAAAAATTCTCAAAGTTAGCGTTGAAGAATTTAGCCCT
TCAATCGCCAGAGAATCTACGAGATGTATGAAGCGGTTAGTATGCAGCCGTCACTTAGAAGTGAGTATGAGTA
CCCTGTTTTTTCTCATGTTCAGGCAGGGATGTTCTCACCTAAGCTTAGAACCTTTAC-
CAAAGGTGATGCGGAG AGATGGGTAAGCACAACCAAAAAAGCCAGTGATTCTGCATT-
CTGGCTTGAGGTTGAAGGTAATTCCATGACCG CACCAACAGGCTCCAAGCCAAGCTT-
TCCTGACGGAATGTTAATTCTCGTTGACCCTGAGCAGGCTGTTGAGCC
AGGTGATTTCTGCATAGCCAGACTTGGGGGTGATGAGTTTACCTTCAAGAAACTGATCAGGGATAGCGGTCAG
GTGTTTTTACAACCACTAAACCCACAGTACCCAATGATCCCATGCAATGAGAGTTGT-
TCCGTTGTGGGGAAAG TTATCGCTAGTCAGTGGCCTGAAGAGACGTTTGGCTGATAG-
ACTAGTGGATCCACTAGTgtttctgccc:
[0260] The construct was delivered to the chromosome using a
recombinant phage called MMebg-cI857s7enhanced RBS #4 into
F'tet/393. After recombination and resolution only the chromosomal
insert described above remains in the cell. It was renamed
F'tet/GM101. F'tet/GM101 was then modified by the delivery of a
lacI.sup.Q construct into the ebg operon between nucleotide
position 2493 and 2937 as numbered in the Genbank accession number
M64441Gb_Ba with the deletion of the intervening ebg sequence. The
sequence of the insert is shown below with the lower case letters
representing the ebg sequences flanking the insert (SEQ ID NO: 389)
shown below:
34 ggcggaaaccGACGTCCATCGAATGGTGCAAAACCTTTCGCGGTATGGCA
TGATAGCGCCCGGAAGAGAGTCAATTCAGGGTGGTGAATGTGAAACCAGT
AACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTT
CCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAA
GTCGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACA
ACAACTGGCGGGCAAACAGTCGCTCCTGATTGGCGTTGCCACCTCCAGTC
TGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCC
GATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGT
CGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTG
GGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAA
GCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGAC
ACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCG
TGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGC
CCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATA
TCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGA
GTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATC
GTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAAT
GCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACC
ACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTT
GCTGCAACTCTCTCAGGGCCAGCCGGTGAAGGGCAATCAGCTGTTGCCCG
TCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCC
TCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTC
CCGACTGGAAAGCGGACAGTAAGGTACCATAGGATCCaggcacagga
[0261] The construct was delivered to the chromosome using a
recombinant phage called AGebg-LacIQ#5 into F'tet/GM101. After
recombination and resolution only the chromosomal insert described
above remains in the cell. It was renamed F'tet/GM221. The F'tet
episome was cured from the strain using acridine orange at a
concentration of 25 .mu.g/ml in LB. The cured strain was identified
as tetracyline sensitive and was stored as GM221.
[0262] Expression. Cultures of pAMG21-Fc-TMP-TMP in E. coli GM221
in Luria Broth medium containing 50 .mu.g/ml kanamycin were
incubated at 37.degree. C. prior to induction. Induction of
Fc-TMP-TMP gene product expression from the luxPR promoter was
achieved following the addition of the synthetic autoinducer
N-(3-oxohexanoyl)-DL-homoserine lactone to the culture media to a
final concentration of 20 ng/ml and cultures were incubated at
37.degree. C. for a further 3 hours. After 3 hours, the bacterial
cultures were examined by microscopy for the presence of inclusion
bodies and were then collected by centrifugation. Refractile
inclusion bodies were observed in induced cultures indicating that
the Fc-TMP-TMP was most likely produced in the insoluble fraction
in E. coli. Cell pellets were lysed directly by resuspension in
Laemmli sample buffer containing 10% .cndot.-mercaptoethanol and
were analyzed by SDS-PAGE. An intense Coomassie stained band of
approximately 30 kDa was observed on an SDS-PAGE gel. The expected
gene product would be 269 amino acids in length and have an
expected molecular weight of about 29.5 kDa. Fermentation was also
carried out under standard batch conditions at the 10 L scale,
resulting in similar expression levels of the Fc-TMP-TMP to those
obtained at bench scale.
[0263] Purification of Fc-TMP-TMP. Cells are broken in water (1/10)
by high pressure homogenization (2 passes at 14,000 PSI) and
inclusion bodies are harvested by centrifugation (4200 RPM in J-6B
for 1 hour). Inclusion bodies are solubilized in 6M guanidine, 50
mM Tris, 8 mM DTT, pH 8.7 for 1 hour at a 1/10 ratio. The
solubilized mixture is diluted 20 times into 2M urea, 50 mM tris,
160 mM arginine, 3 mM cysteine, pH 8.5. The mixture is stirred
overnight in the cold and then concentrated about 10 fold by
ultafiltration. It is then diluted 3 fold with 10 mM Tris, 1.5M
urea, pH 9. The pH of this mixture is then adjusted to pH 5 with
acetic acid. The precipitate is removed by centrifugation and the
supernatant is loaded onto a SP-Sepharose Fast Flow column
equilibrated in 20 mM NaAc, 100 mM NaCl, pH 5(10 mg/ml protein
load, room temperature). The protein is eluted off using a 20
column volume gradient in the same buffer ranging from 100 mM NaCl
to 500 mM NaCl. The pool from the column is diluted 3 fold and
loaded onto a SP-Sepharose HP column in 20 mM NaAc, 150 mM NaCl, pH
5(10 mg/ml protein load, room temperature). The protein is eluted
off using a 20 column volume gradient in the same buffer ranging
from 150 mM NaCl to 400 mM NaCl. The peak is pooled and
filtered.
[0264] Characterization of Fc-TMP activity. The following is a
summary of in vivo data in mice with various compounds of this
invention.
[0265] Mice: Normal female BDF1 approximately 10-12 weeks of
age.
[0266] Bleed schedule: Ten mice per group treated on day 0, two
groups started 4 days apart for a total of 20 mice per group. Five
mice bled at each time point, mice were bled a minimum of three
times a week. Mice were anesthetized with isoflurane and a total
volume of 140-160 .mu.l of blood was obtained by puncture of the
orbital sinus. Blood was counted on a Technicon H1E blood analyzer
running software for murine blood. Parameters measured were white
blood cells, red blood cells, hematocrit, hemoglobin, platelets,
neutrophils.
[0267] Treatments: Mice were either injected subcutaneously for a
bolus treatment or implanted with 7-day micro-osmotic pumps for
continuous delivery. Subcutaneous injections were delivered in a
volume of 0.2 ml. Osmotic pumps were inserted into a subcutaneous
incision made in the skin between the scapulae of anesthetized
mice. Compounds were diluted in PBS with 0.1% BSA. All experiments
included one control group, labeled "carrier" that were treated
with this diluent only. The concentration of the test articles in
the pumps was adjusted so that the calibrated flow rate from the
pumps gave the treatment levels indicated in the graphs.
[0268] Compounds: A dose titration of the compound was delivered to
mice in 7 day micro-osmotic pumps. Mice were treated with various
compounds at a single dose of 100 .mu.g/kg in 7 day osmotic pumps.
Some of the same compounds were then given to mice as a single
bolus injection.
[0269] Activity test results: The results of the activity
experiments are shown in FIGS. 11 and 12. In dose response assays
using 7-day micro-osmotic pumps, the maximum effect was seen with
the compound of SEQ ID NO: 18 was at 100 .mu.g/kg/day; the 10
.mu.g/kg/day dose was about 50% maximally active and 1 .mu.g/kg/day
was the lowest dose at which activity could be seen in this assay
system. The compound at 10 .mu.g/kg/day dose was about equally
active as 100 .mu.g/kg/day unpegylated rHu-MGDF in the same
experiment.
Example 3
Fc-EMP Fusions
[0270] Fc-EMP. A DNA sequence coding for the Fc region of human
IgG1 fused in-frame to a monomer of the EPO-mimetic peptide was
constructed using standard PCR technology. Templates for PCR
reactions were a vector containing the Fc sequence (pFc-A3,
described in International application WO 97/23614, published Jul.
3, 1997) and a synthetic gene encoding EPO monomer. The synthetic
gene for the monomer was constructed from the 4 overlapping
oligonucleotides (SEQ ID NOS: 390 to 393, respectively) shown
below:
35 1798-2 TAT GAA AGG TGG AGG TGG TGG TGG AGG TAC TTA CTC TTG CCA
CTT CGG CCC GCT GAC TTG G 1798-3 CGG TTT GCA AAC CCA AGT CAG CGG
GCC GAA GTG GCA AGA GTA AGT ACC TCC ACC ACC ACC TCC ACC TTT CAT
1798-4 GTT TGC AAA CCG CAG GGT GGC GGC GGC GGC GGC GGT GGT ACC TAT
TCC TGT CAT TTT 1798-5 CCA GGT CAG CGG GCC AAA ATG ACA GGA ATA GGT
ACC ACC GCC GCC GCC GCC GCC ACC CTG
[0271] The 4 oligonucleotides were annealed to form the duplex
encoding an amino acid sequence (SEQ ID NOS: 394 and 395,
respectively) shown below:
36 TATGAAAGGTGGAGGTGGTGGTGGAGGTACTTACTCTTGCCACTTCGGCCCGCTGACTTG 1
---------+---------+---------+---------+---------+---------+ 60
TACTTTCCACCTCCACCACCACCTCCATGAATGAGAACGGTGAAGCCGGGCGACTGAAC b M K G
G G G G G G T Y S C H F G P L T W -
GGTTTGCAAACCGCAGGGTGGCGGCGGCGGCGGCGGTGGTACCTATTCCTGTCATTTT 61
---------+---------+---------+---------+---------+----------+------
-----+-- 133 CCAAACGTTTGGCGTCCCACCGCCGCCGCCGCCGCCACCATGGATAAGGACA-
GTAAAACCGGGCGACTGGACC b V C K P Q G G G G G G G G T Y S C H F -
[0272] This duplex was amplified in a PCR reaction using
37 1798-18 GCA GAA GAG CCT CTC CCT GTC TCC GGG TAA AGG TGG AGG TGG
TGG TGG AGG TAC TTA CTC T and 1798-19 CTA ATT GGA TCC ACG AGA TTA
ACC ACC CTG CGG TTT GCA A
[0273] as the sense and antisense primers (SEQ ID NOS: 396 and 397,
respectively).
[0274] The Fc portion of the molecule was generated in a PCR
reaction with pFc-A3 using the primers
38 1216-52 AAC ATA AGT ACC TGT AGG ATC G 1798-17 AGA GTA AGT ACC
TCC ACC ACC ACC TCC ACC TTT ACC CGG AGA CAG GGA GAG GCT CTT CTG
C
[0275] which are SEQ ID NOS: 398 and 399, respectively. The
oligonucleotides 1798-17 and 1798-18 contain an overlap of 61
nucleotides, allowing the two genes to be fused together in the
correct reading frame by combining the above PCR products in a
third reaction using the outside primers, 1216-52 and 1798-19.
[0276] The final PCR gene product (the full length fusion gene) was
digested with restriction endonucleases XbaI and BamHI, and then
ligated into the vector pAMG21 (described below), also digested
with XbaI and BamHI. Ligated DNA was transformed into competent
host cells of E. coli strain 2596 (GM221, described herein). Clones
were screened for the ability to produce the recombinant protein
product and to possess the gene fusion having the correct
nucleotide sequence. A single such clone was selected and
designated Amgen strain #3718.
[0277] The nucleotide and amino acid sequence of the resulting
fusion protein (SEQ ID NOS: 15 and 16) are shown in FIG. 13.
[0278] EMP-Fc. A DNA sequence coding for a monomer of the
EPO-mimetic peptide fused in-frame to the Fc region of human IgG1
was constructed using standard PCR technology. Templates for PCR
reactions were the pFC-A3a vector and a synthetic gene encoding EPO
monomer. The synthetic gene for the monomer was constructed from
the 4 overlapping oligonucleotides 1798-4 and 1798-5 (above) and
1798-6 and 1798-7 (SEQ ID NOS: 400 and 401, respectively) shown
below:
39 1798-6 GGC CCG CTG ACC TGG GTA TGT AAG CCA CAA GGG GGT GGG GGA
GGC GGG GGG TAA TCT CGA G 1798-7 GAT CCT CGA GAT TAC CCC CCG CCT
CCC CCA CCC CCT TGT GGC TTA CAT AC
[0279] The 4 oligonucleotides were annealed to form the duplex
encoding an amino acid sequence (SEQ ID NOS: 402 and 403,
respectively) shown below:
40 GTTTGCAAACCGCAGGGTGGCGGCGGCGGCGGCGGTGGTACCTATTCCTGTCATTTTGGC 1
---------+---------+---------+---------+---------+---------+ 60
GTCCCACCGCCGCCGCCGCCGCCACCATGGATAAGGACAGTAAAACCG A V C K P Q G G G
G G G G G T Y S C H F G -
CCGCTGACCTGGGTATGTAAGCCACAAGGGGGTGGGGGAGGCGGGGGGTAATCTCGAG 61
---------+---------+---------+---------+---------+---------+- 122
GGCGACTGGACCCATACATTCGGTGTTCCCCCACCCCCTCCGCCCCCCATTAGAGCTCCTAG A P
L T W V C K P Q G G G G G G G *
[0280] This duplex was amplified in a PCR reaction using
41 1798-21 TTA TTT CAT ATG AAA GGT GGT AAC TAT TCC TGT CAT TTT and
1798-22 TGG ACA TGT GTG AGT TTT GTC CCC CCC GCC TCC CCC ACC CCC
T
[0281] as the sense and antisense primers (SEQ ID NOS: 404 and 405,
respectively).
[0282] The Fc portion of the molecule was generated in a PCR
reaction with pFc-A3 using the primers
42 1798-23 AGG GGG TGG GGG AGG CGG GGG GGA CAA AAC TCA CAC ATG TCC
A and 1200-54 GTT ATT GCT CAG CGG TGG CA
[0283] which are SEQ ID NOS: 406 and 407, respectively. The
oligonucleotides 1798-22 and 1798-23 contain an overlap of 43
nucleotides allowing the two genes to be fused together in the
correct reading frame by combining the above PCR products in a
third reaction using the outside primers, 1787-21 and 1200-54.
[0284] The final PCR gene product (the full length fusion gene) was
digested with restriction endonucleases XbaI and BamHI, and then
ligated into the vector pAMG21 and transformed into competent E.
coli strain 2596 cells as described above. Clones were screened for
the ability to produce the recombinant protein product and to
possess the gene fusion having the correct nucleotide sequence. A
single such clone was selected and designated Amgen strain
#3688.
[0285] The nucleotide and amino acid sequences (SEQ ID NOS: 17 and
18) of the resulting fusion protein are shown in FIG. 14.
[0286] EMP-EMP-Fc. A DNA sequence coding for a dimer of the
EPO-mimetic peptide fused in-frame to the Fc region of human IgG1
was constructed using standard PCR technology. Templates for PCR
reactions were the EMP-Fc plasmid from strain #3688 above and a
synthetic gene encoding the EPO dimer. The synthetic gene for the
dimer was constructed from the 8 overlapping oligonucleotides (SEQ
ID NOS:408 to 415, respectively) shown below:
43 1869-23 TTT TTT ATC GAT TTG ATT CTA GAT TTG AGT TTT AAC TTT TAG
AAG GAG GAA TAA AAT ATG 1869-48 TAA AAG TTA AAA CTC AAA TCT AGA ATC
AAA TCG ATA AAA AA 1871-72 GGA GGT ACT TAC TCT TGC CAC TTC GGC CCG
CTG ACT TGG GTT TGC AAA CCG 1871-73 AGT CAG CGG GCC GAA GTG GCA AGA
GTA AGT ACC TCC CAT ATT TTA TTC CTC CTT C 1871-74 CAG GGT GGC GGC
GGC GGC GGC GGT GGT ACC TAT TCC TGT CAT TTT GGC CCG CTG ACC TGG
1871-75 AAA ATG ACA GGA ATA GGT ACC ACC GCC GCC GCC GCC GCC ACC CTG
CGG TTT GCA AAC CCA 1871-78 GTA TGT AAG CCA CAA GGG GGT GGG GGA GGC
GGG GGG GAC AAA ACT CAC ACA TGT CCA 1871-79 AGT TTT GTC CCC CCC GCC
TCC CCC ACC CCC TTG TGG CTT ACA TAC CCA GGT CAG CGG GCC
[0287] The 8 oligonucleotides were annealed to form the duplex
encoding an amino acid sequence (SEQ ID NOS: 416 and 417,
respectively) shown below:
44 TTTTTTATCGATTTGATTCTAGATTTGAGTTTTAACTTTTAGAAGGAGGAATAAAATATG 1
---------+---------+---------+---------+---------+---------+ 60
AAAAAATAGCTAAACTAAGATCTAAACTCAAAATTGAAAATCTTCCTCCTTATTTTATAC a M- -
GGAGGTACTTACTCTTGCCACTTCGGCCCGCTGACTTGGGTTTGCAAACCGCAGGGTGGC 61
---------+---------+---------+---------+---------+---------+ 120
CCTCCATGAATGAGAACGGTGAAGCCGGGCGACTGAACCCAAACGTTTGGCGTCCCACCG a G G
T Y S C H F G P L T W V C K P Q G G -
GGCGGCGGCGGCGGTGGTACCTATTCCTGTCATTTTGGCCCGCTGACCTGGGTATGTA- AG 121
---------+---------+---------+---------+---------+---------- + 180
CCGCCGCCGCCGCCACCATGGATAAGGACAGTAAAACCGGGCGACTGGACCCATACATT- C a G
G G G G G T Y S C H F G P L T W V C K -
CCACAAGGGGGTGGGGGAGGCGGGGGGGACAAAACTCACACATGTCCA 181
---------+---------+---------+---------+-------- 228
GGTGTTCCCCCACCCCCTCCGCCCCCCCTGTTTTGA a P Q G G G G G G G D K T H T
C P -
[0288] This duplex was amplified in a PCR reaction using 1869-23
and 1871-79 (shown above) as the sense and antisense primers.
[0289] The Fc portion of the molecule was generated in a PCR
reaction with strain 3688 DNA using the primers 1798-23 and 1200-54
(shown above).
[0290] The oligonucleotides 1871-79 and 1798-23 contain an overlap
of 31 nucleotides, allowing the two genes to be fused together in
the correct reading frame by combining the above PCR products in a
third reaction using the outside primers, 1869-23 and 1200-54.
[0291] The final PCR gene product (the full length fusion gene) was
digested with restriction endonucleases XbaI and BamHI, and then
ligated into the vector pAMG21 and transformed into competent E.
coli strain 2596 cells as described for Fc-EMP. Clones were
screened for ability to produce the recombinant protein product and
possession of the gene fusion having the correct nucleotide
sequence. A single such clone was selected and designated Amgen
strain #3813.
[0292] The nucleotide and amino acid sequences (SEQ ID NOS: 19 and
20, respectively) of the resulting fusion protein are shown in FIG.
15. There is a silent mutation at position 145 (A to G, shown in
boldface) such that the final construct has a different nucleotide
sequence than the oligonucleotide 1871-72 from which it was
derived.
[0293] Fc-EMP-EMP. A DNA sequence coding for the Fc region of human
IgG1 fused in-frame to a dimer of the EPO-mimetic peptide was
constructed using standard PCR technology. Templates for PCR
reactions were the plasmids from strains 3688 and 3813 above.
[0294] The Fc portion of the molecule was generated in a PCR
reaction with strain 3688 DNA using the primers 1216-52 and 1798-17
(shown above). The EMP dimer portion of the molecule was the
product of a second PCR reaction with strain 3813 DNA using the
primers 1798-18 (also shown above) and SEQ ID NO: 418, shown
below:
[0295] 1798-20 CTA ATT GGA TCC TCG AGA TTA ACC CCC TTG TGG CTT
ACAT
[0296] The oligonucleotides 1798-17 and 1798-18 contain an overlap
of 61 nucleotides, allowing the two genes to be fused together in
the correct reading frame by combining the above PCR products in a
third reaction using the outside primers, 1216-52 and 1798-20.
[0297] The final PCR gene product (the full length fusion gene) was
digested with restriction endonucleases XbaI and BamHI, and then
ligated into the vector pAMG21 and transformed into competent E.
coli strain 2596 cells as described for Fc-EMP. Clones were
screened for the ability to produce the recombinant protein product
and to possess the gene fusion having the correct nucleotide
sequence. A single such clone was selected and designated Amgen
strain #3822.
[0298] The nucleotide and amino acid sequences (SEQ ID NOS: ______
and ______, respectively) of the fusion protein are shown in FIG.
16.
[0299] Characterization of Fc-EMP activity. Characterization was
carried out in vivo as follows.
[0300] Mice: Normal female BDF1 approximately 10-12 weeks of
age.
[0301] Bleed schedule: Ten mice per group treated on day 0, two
groups started 4 days apart for a total of 20 mice per group. Five
mice bled at each time point, mice were bled a maximum of three
times a week. Mice were anesthetized with isoflurane and a total
volume of 140-160 ml of blood was obtained by puncture of the
orbital sinus. Blood was counted on a Technicon H1E blood analyzer
running software for murine blood. Parameters measured were WBC,
RBC, HCT, HGB, PLT, NEUT, LYMPH.
[0302] Treatments: Mice were either injected subcutaneously for a
bolus treatment or implanted with 7 day micro-osmotic pumps for
continuous delivery. Subcutaneous injections were delivered in a
volume of 0.2 ml. Osmotic pumps were inserted into a subcutaneous
incision made in the skin between the scapulae of anesthetized
mice. Compounds were diluted in PBS with 0.1% BSA. All experiments
included one control group, labeled "carrier" that were treated
with this diluent only. The concentration of the test articles in
the pumps was adjusted so that the calibrated flow rate from the
pumps gave the treatment levels indicated in the graphs.
[0303] Experiments: Various Fc-conjugated EPO mimetic peptides
(EMPs) were delivered to mice as a single bolus injection at a dose
of 100 .mu.g/kg. Fc-EMPs were delivered to mice in 7-day
micro-osmotic pumps. The pumps were not replaced at the end of 7
days. Mice were bled until day 51 when HGB and HCT returned to
baseline levels.
Example 4
TNF-.alpha. Inhibitors
[0304] Fc-TNF-.alpha. inhibitors. A DNA sequence coding for the Fc
region of human IgG1 fused in-frame to a monomer of the TNF-.alpha.
inhibitory peptide was constructed using standard PCR technology.
The Fc and 5 glycine linker portion of the molecule was generated
in a PCR reaction with DNA from the Fc-EMP fusion strain #3718 (see
Example 3) using the sense primer 1216-52 and the antisense primer
2295-89 (SEQ ID NOS: 1112 and 1113, respectively). The nucleotides
encoding the TNF-.alpha. inhibitory peptide were provided by the
PCR primer 2295-89 shown below:
45 1216-52 AAC ATA AGT ACC TGT AGG ATC G 2295-89 CCG CGG ATC CAT
TAC GGA CGG TGA CCC AGA GAG GTG TTT TTG TAG TGC GGC AGG AAG TCA CCA
CCA CCT CCA CCT TTA CCC
[0305] The oligonucleotide 2295-89 overlaps the glycine linker and
Fc portion of the template by 22 nucleotides, with the PCR
resulting in the two genes being fused together in the correct
reading frame.
[0306] The PCR gene product (the full length fusion gene) was
digested with restriction endonucleases NdeI and BamHI, and then
ligated into the vector pAMG21 and transformed into competent E.
coli strain 2596 cells as described for EMP-Fc herein. Clones were
screened for the ability to produce the recombinant protein product
and to possess the gene fusion having the correct nucleotide
sequence. A single such clone was selected and designated Amgen
strain #4544.
[0307] The nucleotide and amino acid sequences (SEQ ID NOS: 1055
and 1056) of the fusion protein are shown in FIGS. 19A and 19B.
[0308] TNF-.alpha. inhibitor-Fc. A DNA sequence coding for a
TNF-.alpha. inhibitory peptide fused in-frame to the Fc region of
human IgG1 was constructed using standard PCR technology. The
template for the PCR reaction was a plasmid containing an unrelated
peptide fused via a five glycine linker to Fc. The nucleotides
encoding the TNF-.alpha. inhibitory peptide were provided by the
sense PCR primer 2295-88, with primer 1200-54 serving as the
antisense primer (SEQ ID NOS: 1117 and 407, respectively). The
primer sequences are shown below:
46 2295-88 GAA TAA CAT ATG GAC TTC CTG CCG CAC TAC AAA AAC ACC TCT
CTG GGT CAC CGT CCG GGT GGA GGC GGT GGG GAC AAA ACT 1200-54 GTT ATT
GCT CAG CGG TGG CA
[0309] The oligonucleotide 2295-88 overlaps the glycine linker and
Fc portion of the template by 24 nucleotides, with the PCR
resulting in the two genes being fused together in the correct
reading frame.
[0310] The PCR gene product (the full length fusion gene) was
digested with restriction endonucleases NdeI and BamHI, and then
ligated into the vector pAMG21 and transformed into competent E.
coli strain 2596 cells as described for EMP-Fc herein. Clones were
screened for the ability to produce the recombinant protein product
and to possess the gene fusion having the correct nucleotide
sequence. A single such clone was selected and designated Amgen
strain #4543.
[0311] The nucleotide and amino acid sequences (SEQ ID NOS: 1057
and 1058) of the fusion protein are shown in FIGS. 20A and 20B.
[0312] Expression in E. coli. Cultures of each of the
pAMG21-Fc-fusion constructs in E. coli GM221 were grown at
37.degree. C. in Luria Broth medium containing 50 mg/ml kanamycin.
Induction of gene product expression from the luxPR promoter was
achieved following the addition of the synthetic autoinducer
N-(3-oxohexanoyl)-DL-homoserine lactone to the culture media to a
final concentration of 20 ng/ml. Cultures were incubated at
37.degree. C. for a further 3 hours. After 3 hours, the bacterial
cultures were examined by microscopy for the presence of inclusion
bodies and were then collected by centrifugation. Refractile
inclusion bodies were observed in induced cultures indicating that
the Fc-fusions were most likely produced in the insoluble fraction
in E. coli. Cell pellets were lysed directly by resuspension in
Laemmli sample buffer containing 10% .beta.-mercaptoethanol and
were analyzed by SDS-PAGE. In each case, an intense
coomassie-stained band of the appropriate molecular weight was
observed on an SDS-PAGE gel.
[0313] Purification of Fc-peptide fusion proteins. Cells are broken
in water ({fraction (1/10)}) by high pressure homogenization (2
passes at 14,000 PSI) and inclusion bodies are harvested by
centrifugation (4200 RPM in J-6B for 1 hour). Inclusion bodies are
solubilized in 6M guanidine, 50 mM Tris, 8 mM DTT, pH 8.7 for 1
hour at a {fraction (1/10)} ratio. The solubilized mixture is
diluted 20 times into 2M urea, 50 mM tris, 160 mM arginine, 3 mM
cysteine, pH 8.5. The mixture is stirred overnight in the cold and
then concentrated about 10 fold by ultafiltration. It is then
diluted 3 fold with 10 mM Tris, 1.5M urea, pH 9. The pH of this
mixture is then adjusted to pH 5 with acetic acid. The precipitate
is removed by centrifugation and the supernatant is loaded onto a
SP-Sepharose Fast Flow column equilibrated in 20 mM NaAc, 100 mM
NaCl, pH 5 (10 mg/ml protein load, room temperature). The protein
is eluted from the column using a 20 column volume gradient in the
same buffer ranging from 100 mM NaCl to 500 mM NaCl. The pool from
the column is diluted 3 fold and loaded onto a SP-Sepharose HP
column in 20 mM NaAc, 150 mM NaCl, pH 5(10 mg/ml protein load, room
temperature). The protein is eluted using a 20 column volume
gradient in the same buffer ranging from 150 mM NaCl to 400 mM
NaCl. The peak is pooled and filtered.
[0314] Characterization of activity of Fc-TNF-.alpha. inhibitor and
TNF-.alpha. inhibitor-Fc. Binding of these peptide fusion proteins
to TNF-(x can be characterized by BIAcore by methods available to
one of ordinary skill in the art who is armed with the teachings of
the present specification.
Example 5
IL-1 Antagonists
[0315] Fc-IL-1 antagonist. A DNA sequence coding for the Fc region
of human IgG1 fused in-frame to a monomer of an IL-1 antagonist
peptide was constructed using standard PCR technology. The Fc and 5
glycine linker portion of the molecule was generated in a PCR
reaction with DNA from the Fc-EMP fusion strain #3718 (see Example
3) using the sense primer 1216-52 and the antisense primer 2269-70
(SEQ ID NOS: 1112 and 1118, respectively). The nucleotides encoding
the IL-1 antagonist peptide were provided by the PCR primer 2269-70
shown below:
47 1216-52 AAC ATA AGT ACC TGT AGG ATC G 2269-70 CCG CGG ATC CAT
TAC AGC GGC AGA GCG TAC GGC TGC CAG TAA CCC GGG GTC CAT TCG AAA CCA
CCA CCT CCA CCT TTA CCC
[0316] The oligonucleotide 2269-70 overlaps the glycine linker and
Fc portion of the template by 22 nucleotides, with the PCR
resulting in the two genes being fused together in the correct
reading frame.
[0317] The PCR gene product (the full length fusion gene) was
digested with restriction endonucleases NdeI and BamHI, and then
ligated into the vector pAMG21 and transformed into competent E.
coli strain 2596 cells as described for EMP-Fc herein. Clones were
screened for the ability to produce the recombinant protein product
and to possess the gene fusion having the correct nucleotide
sequence. A single such clone was selected and designated Amgen
strain #4506.
[0318] The nucleotide and amino acid sequences (SEQ ID NOS: 1059
and 1060) of the fusion protein are shown in FIGS. 21A and 21B.
[0319] IL-1 antagonist-Fc. A DNA sequence coding for an IL-1
antagonist peptide fused in-frame to the Fc region of human IgG1
was constructed using standard PCR technology. The template for the
PCR reaction was a plasmid containing an unrelated peptide fused
via a five glycine linker to Fc. The nucleotides encoding the IL-1
antagonist peptide were provided by the sense PCR primer 2269-69,
with primer 1200-54 serving as the antisense primer (SEQ ID NOS:
1119 and 407, respectively). The primer sequences are shown
below:
48 2269-69 GAA TAA CAT ATG TTC GAA TGG ACC CCG GGT TAC TGG CAG CCG
TAC GCT CTG CCG CTG GGT GGA GGC GGT GGG GAC AAA ACT 1200-54 GTT ATT
GCT CAG CGG TGG CA
[0320] The oligonucleotide 2269-69 overlaps the glycine linker and
Fc portion of the template by 24 nucleotides, with the PCR
resulting in the two genes 35 being fused together in the correct
reading frame.
[0321] The PCR gene product (the full length fusion gene) was
digested with restriction endonucleases NdeI and BamHI, and then
ligated into the vector pAMG21 and transformed into competent E.
coli strain 2596 cells as described for EMP-Fc herein. Clones were
screened for the ability to produce the recombinant protein product
and to possess the gene fusion having the correct nucleotide
sequence. A single such clone was selected and designated Amgen
strain #4505.
[0322] The nucleotide and amino acid sequences (SEQ ID NOS: 1061
and 1062) of the fusion protein are shown in FIGS. 22A and 22B.
Expression and purification were carried out as in previous
examples.
[0323] Characterization of Fc-IL-1 antagonist peptide and IL-1
antagonist peptide-Fc activity. IL-1 Receptor Binding competition
between IL-1.beta., IL-1RA and Fc-conjugated IL-1 peptide sequences
was carried out using the IGEN system. Reactions contained 0.4 nM
biotin-IL-1R+15 nM IL-1-TAG+3 uM competitor+20 ug/ml
streptavidin-conjugate beads, where competitors were IL-1RA,
Fc-IL-1 antagonist, IL-1 antagonist-Fc). Competition was assayed
over a range of competitor concentrations from 3 uM to 1.5 pM. The
results are shown in Table C below:
49TABLE C Results from IL-1 Receptor Binding Competition Assay
IL-1pep-Fc Fc-IL-1pep IL-1ra KI 281.5 59.58 1.405 EC50 530.0 112.2
2.645 95% Confidence Intervals EC50 280.2 to 1002 54.75 to 229.8
1.149 to 6.086 KI 148.9 to 532.5 29.08 to 122.1 0.6106 to 3.233
Goodness of Fit R.sup.2 0.9790 0.9687 0.9602
Example 6
VEGF-Antagonists
[0324] Fc-VEGF Antagonist. A DNA sequence coding for the Fc region
of human IgG1 fused in-frame to a monomer of the VEGF mimetic
peptide was constructed using standard PCR technology. The
templates for the PCR reaction were the pFc-A3 plasmid and a
synthetic VEGF mimetic peptide gene. The synthetic gene was
assembled by annealing the following two oligonucleotides primer
(SEQ ID NOS: 1120 and 1121, respectively):
50 2293-11 GTT GAA CCG AAC TGT GAC ATC CAT GTT ATG TGG GAA TGG GAA
TGT TTT GAA CGT CTG 2293-12 CAG ACG TTC AAA ACA TTC CCA TTC CCA CAT
AAC ATG GAT GTC ACA GTT CGG TTC AAC
[0325] The two oligonucleotides anneal to form the following duplex
encoding an amino acid sequence shown below (SEQ ID NOS 1122):
51 GTTGAACCGAACTGTGACATCCATGTTATGTGGGAATGGGAATGTTTTGAACGTCTG 1
---------+---------+---------+---------+---------+------- 57
CAACTTGGCTTGACACTGTAGGTACAATACACCCTTACCCTTACAAAACTTGCAGAC a V E P N
C D I H V M W E W E C F E R L -
[0326] This duplex was amplified in a PCR reaction using 2293-05
and 2293-06 as the sense and antisense primers (SEQ ID NOS. 1125
and 1126).
[0327] The Fc portion of the molecule was generated in a PCR
reaction with the pFc-A3 plasmid using the primers 2293-03 and
2293-04 as the sense and antisense primers (SEQ ID NOS. 1123 and
1124, respectively). The full length fusion gene was obtained from
a third PCR reaction using the outside primers 2293-03 and 2293-06.
These primers are shown below:
52 2293-03 ATT TGA TTC TAG AAG GAG GAA TAA CAT ATG GAC AAA ACT CAC
ACA TGT 2293-04 GTC ACA GTT CGG TTC AAC ACC ACC ACC ACC ACC TTT ACC
CGG AGA CAG GGA 2293-05 TCC CTG TCT CCG GGT AAA GGT GGT GGT GGT GGT
GTT GAA CCG AAC TGT GAC ATC 2293-06 CCG CGG ATC CTC GAG TTA CAG ACG
TTC AAA ACA TTC CCA
[0328] The PCR gene product (the full length fusion gene) was
digested with restriction endonucleases NdeI and BamHI, and then
ligated into the vector pAMG21 and transformed into competent E.
coli strain 2596 cells as described for EMP-Fc herein. Clones were
screened for the ability to produce the recombinant protein product
and to possess the gene fusion having the correct nucleotide
sequence. A single such clone was selected and designated Amgen
strain #4523.
[0329] The nucleotide and amino acid sequences (SEQ ID NOS: 1063
and 1064) of the fusion protein are shown in FIGS. 23A and 23B.
[0330] VEGF antagonist-Fc. A DNA sequence coding for a VEGF mimetic
peptide fused in-frame to the Fc region of human IgG1 was
constructed using standard PCR technology. The templates for the
PCR reaction were the pFc-A3 plasmid and the synthetic VEGF mimetic
peptide gene described above. The synthetic duplex was amplified in
a PCR reaction using 2293-07 and 2293-08 as the sense and antisense
primers (SEQ ID NOS. 1127 and 1128, respectively).
[0331] The Fc portion of the molecule was generated in a PCR
reaction with the pFc-A3 plasmid using the primers 2293-09 and
2293-10 as the sense and antisense primers (SEQ ID NOS. 1129 and
1130, respectively). The full length fusion gene was obtained from
a third PCR reaction using the outside primers 2293-07 and 2293-10.
These primers are shown below:
53 2293-07 ATT TGA TTC TAG AAG GAG GAA TAA CAT ATG GTT GAA CCG AAC
TGT GAC 2293-08 ACA TGT GTG AGT TTT GTC ACC ACC ACC ACC ACC CAG ACG
TTC AAA ACA TTC 2293-09 GAA TGT TTT GAA CGT CTG GGT GGT GGT GGT GGT
GAC AAA ACT CAC ACA TGT 2293-10 CCG CGG ATC CTC GAG TTA TTT ACC CGG
AGA CAG GGA GAG
[0332] The PCR gene product (the full length fusion gene) was
digested with restriction endonucleases NdeI and BamHI, and then
ligated into the vector pAMG21 and transformed into competent E.
coli strain 2596 cells as described for EMP-Fc herein. Clones were
screened for the ability to produce the recombinant protein product
and to possess the gene fusion having the correct nucleotide
sequence. A single such clone was selected and designated Amgen
strain #4524.
[0333] The nucleotide and amino acid sequences (SEQ ID NOS: 1065
and 1066) of the fusion protein are shown in FIGS. 24A and 24B.
Expression and purification were carried out as in previous
examples.
Example 7
MMP Inhibitors
[0334] Fc-MMP inhibitor. A DNA sequence coding for the Fc region of
human IgG1 fused in-frame to a monomer of an MMP inhibitory peptide
was constructed using standard PCR technology. The Fc and 5 glycine
linker portion of the molecule was generated in a PCR reaction with
DNA from the Fc-TNF-.alpha. inhibitor fusion strain #4544 (see
Example 4) using the sense primer 1216-52 and the antisense primer
2308-67 (SEQ ID NOS: 1112 and 1131, respectively). The nucleotides
encoding the MMP inhibitor peptide were provided by the PCR primer
2308-67 shown below:
54 1216-52 AAC ATA AGT ACC TGT AGG ATC G 2308-67 CCG CGG ATC CAT
TAG CAC AGG GTG AAA CCC CAG TGG GTG GTG CAA CCA CCA CCT CCA CCT TTA
CCC
[0335] The oligonucleotide 2308-67 overlaps the glycine linker and
Fc portion of the template by 22 nucleotides, with the PCR
resulting in the two genes being fused together in the correct
reading frame.
[0336] The PCR gene product (the full length fusion gene) was
digested with restriction endonucleases NdeI and BamHI, and then
ligated into the vector pAMG21 and transformed into competent E.
coli strain 2596 cells as described for EMP-Fc herein. Clones were
screened for the ability to produce the recombinant protein product
and to possess the gene fusion having the correct nucleotide
sequence. A single such clone was selected and designated Amgen
strain #4597.
[0337] The nucleotide and amino acid sequences (SEQ ID NOS: 1067
and 1068) of the fusion protein are shown in FIGS. 25A and 25B.
Expression and purification were carried out as in previous
examples.
[0338] MMP Inhibitor-Fc. A DNA sequence coding for an MMP
inhibitory peptide fused in-frame to the Fc region of human IgG1
was constructed using standard PCR technology. The Fc and 5 glycine
linker portion of the molecule was generated in a PCR reaction with
DNA from the Fc-TNF-.alpha. inhibitor fusion strain #4543 (see
Example 4). The nucleotides encoding the MMP inhibitory peptide
were provided by the sense PCR primer 2308-66, with primer 1200-54
serving as the antisense primer (SEQ ID NOS: 1132 and 407,
respectively). The primer sequences are shown below:
55 2308-66 GAA TAA CAT ATG TGC ACC ACC CAC TGG GGT TTC ACC CTG TGC
GGT GGA GGC GGT GGG GAC AAA 1200-54 GTT ATT GCT CAG CGG TGG CA
[0339] The oligonucleotide 2269-69 overlaps the glycine linker and
Fc portion of the template by 24 nucleotides, with the PCR
resulting in the two genes being fused together in the correct
reading frame.
[0340] The PCR gene product (the full length fusion gene) was
digested with restriction endonucleases NdeI and BamHI, and then
ligated into the vector pAMG21 and transformed into competent E.
coli strain 2596 cells as described for EMP-Fc herein. Clones were
screened for the ability to produce the recombinant protein product
and to possess the gene fusion having the correct nucleotide
sequence. A single such clone was selected and designated Amgen
strain #4598.
[0341] The nucleotide and amino acid sequences (SEQ ID NOS: 1069
and 1070) of the fusion protein are shown in FIGS. 26A and 26B.
[0342] The invention now being fully described, it will be apparent
to one of ordinary skill in the art that many changes and
modifications can be made thereto, without departing from the
spirit and scope of the invention as set forth herein.
Abbreviations
[0343] Abbreviations used throughout this specification are as
defined below, unless otherwise defined in specific
circumstances.
56 Ac acetyl (used to refer to acetylated residues) AcBpa
acetylated p-benzoyl-L-phenylalanine ADCC antibody-dependent
cellular cytotoxicity Aib aminoisobutyric acid bA beta-alanine Bpa
p-benzoyl-L-phenylalanine BrAc bromoacetyl (BrCH.sub.2C(O) BSA
Bovine serum albumin Bzl Benzyl Cap Caproic acid CTL Cytotoxic T
lymphocytes CTLA4 Cytotoxic T lymphocyte antigen 4 DARC Duffy blood
group antigen receptor DCC Dicylcohexylcarbodiimide Dde
1-(4,4-dimethyl-2,6-dioxo-cyclohexylidene)ethyl EMP
Erythropoietin-mimetic peptide ESI-MS Electron spray ionization
mass spectrometry EPO Erythropoietin Fmoc fluorenylmethoxycarbonyl
G-CSF Granulocyte colony stimulating factor GH Growth hormone HCT
hematocrit HGB hemoglobin hGH Human growth hormone HOBt
1-Hydroxybenzotriazole HPLC high performance liquid chromatography
IL interleukin IL-R interleukin receptor IL-1R interleukin-1
receptor IL-1ra interleukin-1 receptor antagonist Lau Lauric acid
LPS lipopolysaccharide LYMPH lymphocytes MALDI-MS Matrix-assisted
laser desorption ionization mass spectrometry Me methyl MeO methoxy
MHC major histocompatibility complex MMP matrix metalloproteinase
MMPI matrix metalloproteinase inhibitor 1-Nap 1-napthylalanine NEUT
neutrophils NGF nerve growth factor Nle norleucine NMP
N-methyl-2-pyrrolidinone PAGE polyacrylamide gel electrophoresis
PBS Phosphate-buffered saline Pbf
2,2,4,6,7-pendamethyldihydrobenzofuran-5-sulfonyl PCR polymerase
chain reaction Pec pipecolic acid PEG Poly(ethylene glycol) pGlu
pyroglutamic acid Pic picolinic acid PLT platelets pY
phosphotyrosine RBC red blood cells RBS ribosome binding site RT
room temperature (25.degree. C.) Sar sarcosine SDS sodium dodecyl
sulfate STK serine-threonine kinases t-Boc tert-Butoxycarbonyl tBu
tert-Butyl TGF tissue growth factor THF thymic humoral factor TK
tyrosine kinase TMP Thrombopoietin-mimetic peptide TNF Tissue
necrosis factor TPO Thrombopoietin TRAIL TNF-related
apoptosis-inducing ligand Trt trityl UK urokinase UKR urokinase
receptor VEGF vascular endothelial cell growth factor VIP
vasoactive intestinal peptide WBC white blood cells
[0344]
Sequence CWU 0
0
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