Note: Descriptions are shown in the official language in which they were submitted.
W093/25576 2 1 ~ 7 3 6 7 PCT~US93/05325
PEPTIDES HAVING PLATELET-DERIVED GROWTH FACTOR
(PDGF) ACTIVITY
Field of the Invention
This invention is in the field of biologically
active peptides. These peptides may also be incorporated
into other peptides or proteins to yield hybrid, or
chimeric, multifunctional molecules. More particularly,
it concerns peptides which substantially correspond in
sequence to fragments of platelet-derived growth factor
("PDGF") and their preparation and activity as mitogens
and as chemotactic agents for fibroblasts, vascular
smooth muscle cells and other cells. These activities
lead to uses for these peptides as wound healing agents,
as agents for treatment of vascular diseases in addition
to uses in analytical methods for determ; n~ ng PDGF in
samples.
Background Materials
PDGF is a 32-kD protein heterodimer composed of
A and B polypeptide chains linked by disulfide bonds. It
is stored in the ~-granules of platelets and released
when platelets are activated by blood clotting and
contact with sites of injury. It stimulates specific
target cells by binding to cell-surface receptors,
thereby mediating a cascade of events that leads to DNA
synthesis and cell proliferation. PDGF is a strong
mitogen for fibroblasts, smooth muscle cells, and glial
cells. In addition, it is a potent chemotactic factor
for neutrophils, monocytes, fibroblasts, and smooth
W093/25576 PCT/US93/05325
~ 3~ -2-
muscle cells. Thus, PDGF plays an important role in the
migration of inflammatory cells and connective tissue
cells to sites of inflammation and injury and in the
repair or restructuring of injured tissues.
More especially, PDGF appears to play a part in
atherosclerosis. A degenerative disease of the arteries,
this condition is characterized by deposition of fatty
substances in, and the fibrous thickening of, intima,
resulting in the narrowing of the vessel passages and
ultimately their hardening and loss of elasticity. The
earliest lesion of atherosclerosis is a ubiquitous fatty
streak commonly found in children. This grossly flat,
lipid-rich lesion consists of macrophages and some smooth
muscle. The fibrous plaque is representative of the
various forms of advanced atherosclerosis and is made up
of increased internal smooth muscle cells surrounded by
connective tissue matrix and containing variable amounts
of intracellular and extracellular lipid. In the lumen
of the artery, this lesion is generally covered by a
dense, fibrous cap of smooth muscle and connective
tissue.
Important events in the development of lesions
of atherosclerosis involve mainly the injury to
endothelial cells -- the initiating event in
atherogenesis -- and advance lesions of atherosclerosis.
Injured endothelial cells produce several growth factors
in culture, including a mitogen resembling platelet-
derived growth factor (PDGF). Platelets contain PDGF,
which may be of particular importance in atherogenesis
because it is both chemotactic and mitogenic and thus can
induce both smooth muscle cell migration and
proliferation. It has been demonstrated that if
platelets are absent from sites of endothelial injury or
if platelet interaction can be pharmacologically
prevented, then the intimal proliferative lesions that
W093/25576 2 1 ~ 7 3 6 7 PCT/US93/05325
usually accompany such injury will not occur. Smooth
muscle cells are found in both fatty streaks and fibrous
plaques. Their proliferation is the key event that
determines how extensive fibrous plaques will become.
They can form enormous amounts of connective tissue
matrix and can accumulate lipid. They contain receptors
for PDGF and can respond to a number of chemotactic
factors. Thus, the event leading to the development of
lesions of atherosclerosis can be represented as:
Injury to endothelium
Release of PDGF
Migration of smooth muscle cells
from the media to intima
Smooth muscle cell proliferation
Plaque
PDGF plays an important role in wound healing.
Although autologous cellular grafts and reconstructed
skin are beneficial in certain instances, such costly
specialized and time-consuming procedures are not
applicable for all clinical situations. Enhancement of
wound healing using pharmaceutical agents can be of great
benefit.
The cellular events leading to the repopulation
of cells and restoration of a wounded area are controlled
in large part by specific peptide factors, which regulate
the migration and proliferation of cells involved in the
repair process. These factors have a variety of cellular
sources, including many cell types that are an integral
part of the cellular machinery of wound repair.
Activated monocytes (macrophages) have been shown to
release a PDGF-like activity and to express the C-sis
W O 93/25576 P~r/US93/05325
~ ~ ~ 6~l -4-
gene and to secrete a PDGF-like activity as well.
Pierce, G.F., at al., J. Exp. Med., 167, 974 (1988),
reported that PDGF and recombinant C-sis gene homodimeric
proteins augment in vivo incisional wound healing in
rats. PDGF increases collagen formation, DNA content,
and protein levels.
The following review articles describe the
native PDGF material and its activity (Deuel, Thomas F.,
Current Topics in Cellular Requlation, 26:51 (1985);
Deuel, Thomas F. et al., J. Clin. Invest. 74:669 (1984)).
It has been reported by Senior, R.M. et al., J.
Cell Biol. 100:351 (1985)-that separate dom~;n~ exist in
PDGF for fibroblast chemotactic and mitogenic activity
and for monocyte and fibroblast chemotactic activity.
This raises the possibility of selectively modifying the
biological activities of PDGF. Senior et al.
demonstrated that human leukocyte elastase (HLE)
abolishes the chemotactic activity of PDGF for
fibroblasts but has no effect on the chemotactic activity
of monocytes or its mitogenic activity for 3T3 cells or
its capacity to bind to 3T3 cells. Cathepsin G has no
effect on the chemotactic or mitogenic activities of
PDGF, suggesting that the active site of PDGF for
chemotaxis is different for different cell types and that
different sites on PDGF are involved in its chemotactic
activity and mitogenic activity for fibroblasts.
Observations of these investigators also suggest that the
cleavage produced by HLE is located at or near the N- and
C-terminal ends of either the A or B ch~' n~ since HLE
preferentially cleaves at Val-X bonds and, to a lesser
extent, at Ala-X bonds. On the other hand, cathepsin G
cleaves peptide bonds adjacent to the carboxyl group of
phenylalanine, leucine, tyrosine, isoleucine, and
methionine residues. The sensitivity of PDGF
fibroblasts' chemotactic activity to HLE but not the
W093/25576 h 13 7 ~ 6 ~ PCT/US93/05325
cathepsin G suggests that the active site for chemotactic
activity is located in a small region(s) of the PDGF
molecule. Reduction and alkylation of PDGF results in
loss of its mitogenic activity but has no effect on its
chemotactic activity, thus suggesting that sulfhydryl
groups of cysteine do not affect chemotaxis but do affect
mitogenesis. This also indicates that dimers (homo or
hetero) or disulfide loop structures are required for
mitogenesis. Because p28~-siS, the transforming protein
of the simian sarcoma virus, which is 92~ homologous to
the A chain of PDGF, has specific mitogenic activity
identical with that of PDGF, it is likely that the A
chain carries the active site for the mitogenic activity
of PDGF. The B chain may contain the active site for
chemotactic activity or the A chain may have the active
site for both the mitogenic activity and the chemotactic
activity.
Previous studies involving the receptors of
PDGF have been based on the premise that a single cell-
surface receptor binds all three isoforms of PDGF (AA,
BB, and AB). However, Hart et al., Science 240:1529
(1988) recently reported that two populations of PDGF
receptor exist and can be distinguished by their ligand-
binding specificity. The B receptor binds only the BB
dimer, whereas the A/B receptor binds AA, BB, and AB
dimers. They also reported that human dermal fibroblasts
appear to express seven times more B receptor than A/B
receptor. Their observations indicate that B receptor is
responsible for most PDGF receptor phosphorylation.
Studies of Escobedo et al., Science 240:1532 (1988) and
Nature 335:85 (1988) suggest that a common PDGF receptor
is activated by homodimeric A and B forms of PDGF. They
observed that both forms of PDGF bind to the transfected
receptor, stimulate the receptor ~yrosine kinase
actlvity, and elicit a mitogenic response of Balb/c 3T3
W093/25576 ~ 3~ 1 -6- PCT/US93/05325
cells. Their data indicate that the tyrosine kinase and
mitogenic responses of the Balb/c 3T3 cells to AA and BB
forms of PDGF can be attributed to a single type of
receptor and show that the AA form, like the BB form, is
a true mitogen.
Another recent study, Khachigian, L.M. et al.,
J. Biol. Chem. 267, No. 3, 1660 (1992) described an 18
unit peptide corresponding to the 194-211 region of the A
chain of PDGF and its ability to bind cultured cells and
interact with other important growth factors. All of
this prior activity shows that PDGF is an important
material which plays a part in many biological processes~
While in some cases it might be possible to
obtain, make and/or use the native material to moderate
body functions and treat injury or disease or to use this
native material in assays, this is not really practical.
The native material exists in such small amounts that its
isolation on a commercial scale is not feasible. Also,
the native material, at 32 kD, is too large to synthesize
in any sort of reasonable yield. What is needed is
synthetic peptides which exhibit desired PDGF activity
but which are significantly shorter and which correspond
substantially to a region of the PDGF sequence.
Statement of the Invention
In accord with this invention, it has now been
found that peptide fragments of from about 6 to about 26
amino acids in length and having amino acid sequences
which have substantial homology to sequences found in the
A or B chain of PDGF as well as salts, dimers and
derivatives of such fragments can exhibit substantial
PDGF-mimicking biological activity.
Accordingly, in one aspect, the invention
provides novel peptides corresponding to a sequence
W093/25~76 ~ 1 3 7 ~ ~ 7 PCT/US93/05325
--7--
having substantial homology to one of the following PDGF
A chain sequences:
33-53, 38-46, 44-51, 48-60, 53-63, 55-68,
61-71, 101-i25, or 106-125. These sequences are shown in
Table 1.
In another aspect this invention provides novel
active peptides corresponding to or having substantial
homology to one of the following PDGF B-chain sequences:
25-42, 30-47, 36-50, 40-50, 45-52, 76-91,
80-95, 84-97, 89-101, 94-109, 98-111, 104-116, 110-122,
or 115-128. These sequences are shown in Table 2.
In another aspect this invention provides novel
active peptide dimers, peptide ACM derivatives and
oxidized peptides corresponding to or having substantial
homology to one of the PDGF A-chain sequences shown in
Table 3.
In yet another aspect this invention provides
novel active peptide dimers, peptide ACM derivatives and
oxidized peptides corresponding to or having substantial
homology to one of the PDGF B-chain sequences shown in
Table 4.
In another aspect, this invention provides
hybrid (i.e., chimeric) multifunctional molecules
incorporating the peptides of this invention into other
peptides and proteins.
In other aspects this invention provides the
salts of these peptides, the amide and acyl derivatives
of the carbonyl and amino end groups of these peptides
and their salts.
In addition, any of the active peptides of this
invention may be conjugated to peptide polymers and
proteins. These immobilizing peptides and proteins are
high molecular weight (5 kD to 80 kD) materials such as
bovine serum albumin (BSA), ovalbumin (OVA), poly(lysine)
and the like. The peptides can also be attached to
W093/25576 PCT/US93/05325
~ 36~ -8-
lipophillic moieties such as 12 to 24 carbon atom long
saturated and unsaturated hydrocarbon and fatty acid
residues.
In an additional aspect this invention concerns
labeled versions of these peptides, their salts and
derivatives and their application to label-dependent
assay methods such as radioimmunoassays (RIA),
fluoroassays, and enzyme-linked immunoabsorbent assays
(ELISA) for the determination or assaying of PDGF in
laboratory and clinical samples. In addition, non-
labeled materials can be used as standards and the like
in assay methods.
In further aspects, active peptides, salts,
derivatives and hybrids of this invention and
particularly those of the B-chain are used as mitogens,
and as chemotactic agents and the invention provides
pharmaceutical agents based on these materials and
provides for their use in wound healing, in the
alleviation of the effects of vascular disease or injury,
and in the modulation of calcium uptake into cells.
Detailed Description of the Invention
Brief Description of the Drawings
This invention will be described with reference
being made to the accompanying drawings wherein Figs. 1,
5 and 6 each show results of the competitive binding of
3T3 cells of three peptides of this invention; Figs. 3
and 4 graph the effect of a material of this invention
and PDGF on intracellular calcium influx; and Fig. 2 is a
graph depicting competitive binding of 3T3 cells by PDGF
itself as a standard for comparison.
W093/25576 2 1 3 7 3 ~ 7 PCT/US93/05325
g
Definitions
In this specification and claims, reference
will be made to certain terms which are defined as
follows.
As used herein:
~'Acyl" refers to an alkyl-containing carbonyl
group, e.g., R-C(=O)-, wherein R is an alkyl group having
from 1 to 8 carbon atoms, such as methyl, ethyl,
n-propyl, isopropyl, hexyl, octyl and the like. The acyl
group usually preferred in this invention is acetyl.
Acyl groups are used to block the term' n~ 1 amino group of
a peptide.
"Amide" refers to an amino-containing group
formed to block the terminal carboxyl group of a peptide.
The amide group has the structure
-C (=O) -N-RlR2,
where R1 and R2 are each hydrogen or a lower alkyl.
"Conservative substitution" refers to a
substitution in a peptide of an amino acid by another
amino acid which is similar in chemical and
hydrophobicity properties to the original.
"ELISA" refers to an enzyme-linked
immunosorbent assay which employs an antibody or antigen
bound to a solid phase and an enzyme-antigen or
enzyme-antibody conjugate to detect and quantify the
amount of antigen or antibody present in a sample. A
description of the ELISA technique is found in Chapter 22
of the 4th Edition of Basic and Clinical Immunology by
D.P. Sites et al, published by Lange Medical Publications
of Los Altos, California, in 1982, which is incorporated
herein by reference.
"EMIT" refers to an enzyme-multiplied
immunoassay technique which uses (1) an enzyme-labeled
hapten, (2) specific antibody to the hapten, (3)
pretreatment reagent, (4) buffered-enzyme substrate, and
W093/25S76 ~ PCT/US93/05325
6 -lo-
(5) standards to detect the amount of an unknown in a
sample. A description of the EMIT technique is found in
Enzyme Immunoassay, edited by E.T. Maggio, published in
1980 by CRC Press, Inc., Boca Raton, Florida,
particularly on pp. 141-150, 234-5, and 242-3. These
materials are incorporated by reference.
"Fluoroimmunoassay" refers to an antibody-based
assay in which the species to be measured binds to,
displaces or competes for binding with a material
labelled with a fluorescent species in an antibody-ligand
complex. In some embodiments of this assay, the complex
is separated and the presence or absence of fluorescent
species gives a measure of the amount of measured
species. In other embodiments, the complex has different
fluorescent properties than the uncomplexed fluorescent
species so that the formation of the complex can be
detected without separation of the complex. A description
of fluoroimmunoassay techniques is found in "A Review of
Fluoroimmunoassay and Immunofluorometric Assay", D.S.
Smith et al. (1981) Ann. Clin. Biochem. (1981) 18:253-274
which is incorporated herein by reference.
"Label" refers to a detectable group in a
molecule. Among the common labels are radioactive
species useful in radioimmunoassays, fluorescent species
useful in fluoroimmunoassays, and enzymatic species
useful in the ELISA and EMIT methods and the like.
"Lower alkyl" refers to a straight or branched
chain saturated hydrocarbon group having from 1 to 4
carbon atoms such as, for example, methyl, ethyl,
n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and
tert-butyl.
"Peptide" or "polypeptide" refers to relatively
low molecular weight compounds which yield two or more,
such as up to about 30 units of amino acid on hydrolysis.
W093/25~76 ~1 3 7 3 6 7 PCT/US93/0532
"Pharmaceutically acceptable salt" and "salt"
refer to salts that retain the desired antigenic activity
of the parent peptide. "Pharmaceutically acceptable
salt" refers to salts that are suitable for ingestion or
parenteral administration or the like in that they do not
impart any undesired toxicological effects. Examples of
such salts and ph~ ceutically acceptable salts include
(a) acid addition salts formed with inorganic acids, for
example hydrochloric acid, hydrobromic acid, sulfuric
acid, phosphoric acid, nitric acid and the like; and
salts formed with organic acids such as, for example,
acetic acid, oxalic acid, tartaric acid, succinic acid,
maleic acid, fumaric acid, gluconic acid, citric acid,
malic acid, ascorbic acid, benzoic acid, tannic acid,
alginic acid, polyglutamic acid, naphthalenesulfonic
acids, naphthalenedisulfonic acids, polygalacturonic
acid, and the like; (b) salts with monovalent and
polyvalent metal cations such as sodium, potassium, zinc,
calcium, barium, magnesium, aluminum, copper, cobalt,
nickel, cadmium, and the like; or with an organic cation
formed from N,N'-dibenzylethylenediamine or
ethylenediamine; and (c) combinations of (a) and (b),
e.g., a zinc tannate salt and the like.
"Radioimmunoassay" or "RIA" refers to an
antibody-based assay in which the species to be measured
binds to, displaces or competes for binding with a
radiolabeled material in an antibody-ligand complex. The
complex is separated and the presence or absence of
radioactivity gives a measure of the amount of measured
species.
"Substantially corresponding" o~ "substantial
homology" refers to the property of two amino acid
sequences being identical to one another or differing
from one another by no more than three amino acid unitsO
Sequences can differ by having a different amino acid at
c~ 12- PCT/US93/0532'
a given position or by having an extra amino acid or by
missing an amino acid. Preferably, the sequences have at
most two points of difference and more preferably have
one difference or are identical.
As used herein, the following abbreviations are
used for the amino acids described:
Alanine A
Arginine R
Asparagine N
Aspartic Acid D
Cysteine C
Glutamine Q
Glutamic Acid - E
Glycine G
Histidine H
Isoleucine
Leucine L
Lysine K
Methionine M
Phenylalanine F
Proline P
Serine S
Threonine T
Tryptophan W
Tyrosine Y
Valine V
These represent L-amino acids with the
exception of the achiral amino acid glycine. All peptide
sequences mentioned herein are written according to the
generally accepted convention whereby the N-terminal (or
amino-terminal) amino acid is on the left and C-terminal
(carboxyl-terminal) amino acid is on the right.
Description of Preferred Embodiments
In one aspect, this invention relates to
synthetic peptides and their salts; peptide dimers, and
derivatives which have PDGF properties and activity.
WO 93/25576 2 1 3 7 3 5 7 PCT/US93/05325
-13-
The synthetic peptides have about 6 to 26 amino
acids substantially corresponding to a 6 to 26 amlno acid
sequence of a PDGF chain.
The peptide can correspond to portions of the A
5 chain in which case they can have sequences substantially
corresponding to the peptides shown in Table 1.
The peptides correspond to portions of the B
chain in which case they can have sequences substantially
corresponding to the peptides shown in Table 2.
The peptides can also be in the form of dimers
and derivatives substantially corresponding to those A-
chain materials shown in Table 3.
The peptides can also be in the form of dimers
and derivatives substantially corresponding to those B-
chain materials shown in Table 4.
Any of these materials can be in the form of
pharmaceutically acceptable salts. Labeled forms can be
prepared, as well.
Any of these materials can also be incorporated
into hybrid, multifunctional molecules. This is done by
incorporating these peptide sequences into other peptides
or proteins such as hirudin, or the like. These
materials can be prepared by direct synthesis as
exemplified by Church, F.C. et al., J. Biol. Chem. (1991)
266:11975-11979. This article illustrates the formation
and hybrid activity of a hirudin sequence-containing
peptide.
W093/25576 PCT/US93/05325
~ ~3~ 3 - 14-
Table 1
38 46
V E V K R C T G C (SEQ ID NO:1)
44 51
T G C C N T S S (SEQ ID NO:2)
48 60
N T S S V KC Q P S RVH (SEQ ID NO:3)
53 63
KC Q P S RVHHRS(SEQ ID NO:4)
55 68
Q P S RVHHRSVKVAK (SEQ ID NO:5)
61 71
HRSVKVAKV E Y (SEQ ID NO:6)
66 78
VAKV E Y V RKKPKL (SEQ ID NO:7)
106 125
D T G RPR E S G KKRKRKRLKPT (SEQ ID NO:8)
33 53
I W P P C V E V KRCT G C C N T S S V K (SEQ ID NO:9)
101 125
D Y R E E D T G R P R E S G KK R K R K R L KPT
(SEQ ID NO:10)
106 125
D T G RPR E S G KKRK R K R L K P T (SEQ ID NO:11)
108 125
G R P R E S G KKRKRKR L K P T (SEQ ID NO:12)
W O 93/25S76 2 1 3 7 3 6 7 PC~r/US93/05325
-15-
Table 2
42
I S R R L I D R T N A N F L V W P P (SEQ ID NO:13)
30 47
I D R T N A N F L V W P P C V E V Q (SEQ ID NO:14)
36 50
N F L V W P P C V E V Q R C S (SEQ ID NO:15)
40 50
W P P C V E V Q R C S (SEQ ID NO:16)
45 52
E V Q R C S G C (SEQ ID NO:17)
76 91
E I V A K K P I F K K A T V T L (SEQ ID NO:18)
K K P I F K K A T V T L E D H L (SEQ ID NO:19)
84 97
F K K A T V T L E D H L A C (SEQ ID NO:20)
89 101
V T L E D H L A C K C E T (SEQ ID NO:21)
94 109
H L A C K C E T V A A A R P V T (SEQ ID NO:22)
98 111
K C E T V A A A R P V T R S (SEQ ID NO:23)
104 116
A A R P V T R S P G G S Q (SEQ ID NO:24)
110 122
R S P G G S Q E Q R A K T (SEQ ID NO:25)
115 128
S Q E Q R A K T P Q T R V T (SEQ ID NO:26)
W093/25576 PCT/US93/05325
Cl~3~ 3~ -16-
Table 3
38 46
V E V K R C T G C (SEQ ID NO:27)
1 l
Acm Acm
38 46
V E V K R C T G C (SEQ ID NO:28)
38 46
V E V K R C T G C (SEQ ID NO:29)
V E V K R C T G C (SEQ ID NO:29)
44 51
T G C C N T S S ~SEQ ID NO:30)
1 1
AcmAcm
44 51
T G C C N T S S (SEQ ID NO:31)
44 51
T G C C N T S S (SEQ ID NO:32)
T G C C N T S S (SEQ ID NO:32)
48 60
N T S S V K C Q P S R V H (SEQ ID NO:33)
Acm
48 60
(N T S S V K C Q P S R V H)2 (SEQ ID NO:34)
W093/25576 ~ 1~ 7 3 6 7 PCT/US93/05325
-17-
Table 4
36 - 50
N F L V W P P C V E V Q R C S (SEQ ID NO:35)
1 l
N F L V W P P C V E V Q R C S (SEQ ID NO:35)
W P P C V E V Q R C S (SEQ ID NO:36)
W P P C V E V Q R C S (SEQ ID NO:36)
52
E V Q R f s G C (SEQ ID NO:37)
E V Q R C S G C (SEQ ID NO:37)
94 109
H L A C K C E T V A A A R P V T (SEQ ID NO:38)
H L A C K C E T V A A A R P V T (SEQ ID NO:38)
98 111
(K C E T V A A A R P V T R S)2 (SEQ ID NO:39)
Acm peptide analogs of 35 and 36
Oxidized analogs of 35, 36, 37 and 38
Conjugates
The active peptides of this invention can
advantageously be coupled or conjugated to other moieties
to enhance cell attachment or to immobilize the peptidesO
These complexing and conjugating techniques are generally
well known in the art and can employ methods and reagents
of the art. Immobilization is usually brought about by
covalently attaching the peptide to a peptide polymer or
protein of 5 kD to 80 kD. BSA and OVA are two well known
proteins for this use as are the regular lysine polymers
which range in size from about 5 kD to about 30 kD. This
W093/25576 PCT/US93/OS325
~ 18-
immobilization locks the conformation of the peptide and
leads to higher activity.
The peptide can be coupled to lypophillic
moieties of 6 to 20 carbons or so. These can be fatty
acid residues of 12 to 20 carbons such as palmitic, oleic
and linoleic acid and the like and alkyl amino acids
where the alkyl substituent is from about 6 to 12 carbons
in length and typically saturated and linear, such as
aminooctanoic acid. The preparation of amino acids
carrying these lypophillic groups is shown in Toth, I. et
al., In. Proc. of the 11th Amer. Peptide Symposium, J.E.
Rivier and G.R. Marshall (Eds.) ESCOM, Leiden 1990, pp.
1078-1079. A11 of these adducts are cumulatively
described as "conjugates."
General Preparative Techniques
Chemical Synthesis of the Peptide Sequence
The peptides may be synthesized by any
techniques that are known to those skilled in the peptide
art, such as may be found in Meienhofer, J., Hormonal
Proteins and Peptides, Vol. 2, p. 46, Academic Press, New
York, (1973) (for solid phase peptide synthesis) and
Schroder, E. et al., The Peptides, Vol. 1, Academic
Press, New York, (1965) (for classical solution
synthesis).
These methods comprise sequential addition of
amino acids or suitably protected amino acids to a
growing peptide chain. Generally, either the amino or
carboxyl group of the first amino acid is protected by a
suitable protecting group. The protected or derivatized
amino acid is contacted with the next amino acid in the
sequence having the complimentary (amino or carboxyl)
group suitably protected, under conditions suitable for
forming the amide linkage. The protecting group is then
W093/25576 2 1 3 7 3 6 7 PCT/US93/05325
-19 -
removed from this newly added amino acid residue and the
next amino acid is then added. After all the desired
amino acids have been linked in the proper sequence, any
r~m~;n;ng protecting groups (and any solid support) are
removed sequentially or concurrently to afford the final
peptide. ~lso, as is well known, it is possible to add
more than one amino acid at a time to a growing chain.
A preferred method of preparing compounds of
the present invention involves solid phase peptide
synthesis. In this method the alpha-amino function of
the amino acids is protected by an acid or base-sensitive
group. Suitable protecting groups are t-butyloxycarbonyl
(Boc), fluorenyl methoxy carbonyl (FMOC),
benzyloxycarbonyl (Z), and the like.
Side chain active sites are protected, as well,
to prevent undesired reactions or couplings.
Particularly preferred side chain protecting groups are,
for arginine: nitro, p-toluenesulfonyl,
4-methoxybenzenesulfonyl, Z, Boc, and adamantyloxy
carbonyl; for lysine: dichloro benzyloxyl carbonyl,
t-Boc; for aspartic acid and glutamic acid: o-benzyl,
t-butyl; for tyrosine: benzyl, o-bromobenzyloxycarbonyl,
2,6-dichlorobenzyl, isopropyl, cyclohexyl, cyclopentyl,
and acetyl; for serine and threonine: benzyl, t-butyl and
tetrahydropyranyl; for histidine: benzyl, p-
toluenesulfonyl and 2,4-dinitrophenyl; and for
tryptophan: formyl.
The carboxyl-terminal amino acid is attached to
a suitable solid support. Suitable supports are inert to
the reagents and reaction conditions of the reactions, as
well as insoluble in the media used. Suitable solid
supports include chloromethylpolystyrenedivinylbenzene
polymers and the like, especially
chloromethylpolystyrene-1~ divinylbenzene polymer. For
the special case where the carboxy-terminal amino acid of
W093/25576 PCT/US93/05325
~3~ -20-
the peptide becomes an amide [-C(=O)-NH2], a particularly
useful support is the benzhydrylaminopolystyrenedivinyl-
benzene polymer described by Vivaille, P. et al. (1971)
Helv. Chim. Acta. 54:2772. The attachment to the
chloro-methyl polystyrene-divinylbenzene type of resin is
made by means of the reaction of the alpha N-protected
amino acid, especially the Boc-amino acid, as its cesium,
- tetramethylammonium, 4,5-diazabicyclo[5.4.0]undec-5-ene,
or similar salt in ethanol, acetonitrile,
N,N-dimethylformamide (DMF), and the like, especially the
cesium salt in DMF, with the chloromethyl resins at an
elevated temperature, for example between about 40C and
60C, preferably about 50C, for from about 12 to 48
hours, preferably about 24 hours. The alpha N-Boc-amino
acid is attached to the benzhydrylamine resin by means of
an N,N'-dicyclohexylcarbodiimide (DCC)/
1-hydroxybenzotriazole (HBT) mediated coupling for from
about 2 to about 24 hours, preferably about 12 hours at a
temperature of between about 10C and 50C, preferably
25C in a solvent such as dichloromethane or DMF,
preferably dichloromethane.
The removal of the alpha N-protecting groups
may be performed in the presence of, for example, a
solution of trifluoroacetic acid in methylene chloride,
or other strong acid solution, preferably 50~
trifluoroacetic acid in dichloromethane at about ambient
temperature. Base-labile protecting groups may be
removed by treatment with a base such as piperidine in
DMF. Each protected amino acid is preferably introduced
in approximately 2.5 molar excess and coupling may be
carried out in dichloromethane and the like, especially
in dichloromethane at about ambient temperature. The
coupling agent is normally DCC in dichloromethane but may
be N,N'-diisopropylcarbodiimide or other carbodiimide
either alone or in the presence of HOBT,
W093/25576 2 1 3 7 3 6 7 PCT/US93/05325
N-hydroxysuccinimide, other N-hydroxyimideS or oximes.
Alternatively, protected amino acid active esters ~e.g.,
p-nitrophenyl, pentafluorophenyl and the like) or
symmetrical anhydrides may be used.
At the end of the solid phase synthesis, the
peptide is either carried through another deprotection
and neutralization cycle followed by acylation,
preferably acetylation with acetic anhydride to yield an
N-acetyl (N-Ac) blocked amino end group, or it may be
removed from the resin directly. If the carboxy
[-C(=O)-OH] terminal is to be blocked as the amide, the
peptide may be either synthesized on the
benzhydrylamino-polystyrene resin, which gives the amide
directly, or it may be removed from the resin by
ammonolysis with, for example, ammonia/methanol or
ammonia/ethanol, at a temperature of from about 0 to
about 50C, preferably about 25C for about 12 to about
48 hours, preferably about 18 hours. If a peptide with a
free amino-terminal and a carboxyl-ter~; n~ 1 is desired,
the peptide may be directly removed from the resin by
treatment with anhydrous liquid hydrogen fluoride in the
presence of a radical scavenger such as anisole. The
amino or carboxyl--blocked (protected) peptides, either on
the resin or removed from the resin by ammonolysis, are
similarly deprotected by treatment with anhydrous liquid
hydrogen fluoride. In cases where base-labile protection
of the alpha N function is used in conjunction with
t-butyl-based side chain protection, the final resin
removal and deprotection step may be performed with
trifluoroacetic acid.
Other means of removal of the (side chain)
protecting groups from the polypeptide are treatment with
hydrogen fluoride/pyridine complex, treatment with
tris(trifluoroacetyl)boron and trifluoroacetic acid, by
reduction with hydrogen and palladium on carbon or
W093/25576 PCT/US93/OS325
~ 3 - 22-
polyvinylpyrrolidone, or by reduction with sodium in
liquid ammonia or with liquid hydrogen fluoride plus
anisole at a temperature between about -10 and +10C,
preferably about 0C, for between about 15 minutes and 1
hour, preferably about 30 minutes. The latter treatment
(HF/anisole) may be used for simultaneous cleavage from
the resin and deprotection to yield free-C02H end groups
when a normal benzylester linkage has been used or to
form a CO-NH2 (amide) end groups when a benzhydrylamino
linkage has been used. For the amide term-n~l peptides
on the benzhydrylamine resins, the resin cleavage and
deprotection steps may be combined in a single step
utilizing liquid HF/anisole as described above. The
fully protected polypeptide can then be purified by
chromatographic steps.
Salt Formation
The peptides can be obtained as salts, by
simple adjustment of the pH of the medium from which they
are finally recovered with acids or bases corresponding
to the desired counter ions.
Preparing Labeled Versions of the Peptides
Radiolabeled versions of the polypeptides can
be produced in several manners. For one, commercially
available 14carbon-labeled amino acids can be employed in
the synthesis of the polypeptides. Similarly 3H-amino
acids can be prepared by the magnesium oxide procedure of
Schwyzer et al. (1959) Helv. Clin. Acta, 42 2622. Except
for the precautions routinely associated with
radiochemicals, these processes can follow the usual
synthesis route. Alternatively, the finished polypeptide
or conjugate can be radiolabeled by tritium exchange.
W093/25~76 2 1 3 7 3 ~ 7 PCT/US93/0532~
-23-
Enzyme labels can be incorporated by using an
enzymic carrier for forming conjugates or by attaching an
enzymatically active group to the carrier or the peptide.
Synthesis of the Dimers
Parallel dimers are synthesized by using
acetamido methyl group (ACM) on appropriate cysteine
residues, thereby selectively forming disulfide bonds to
put the peptide ~h~l n~ in registered dimeric form. The
ACM group is resistant to HF and can be removed with
iodine in methanol, with simultaneous oxidation to a
disulfide bond.
Synthesis of Cyclic Derivatives
The cyclic disulfides can be formed by air
oxidation of a dilute solution of the corresponding
linear peptide in water. After HF cleavage and
extraction of the peptide off of the resin, the resulting
solution is adjusted to pH 8.1 by addition of
concentrated NH40H in water and then shaken slowly on a
shaker for 5-10 days. At the end of this time or during
it, an Ellman test can be performed to confirm the
completion of the disulfide bond formation. The peptide
solution is adjusted to pH 6.5 by addition of a weak acid
such as acetic acid. It is then passed through an acetic
acid-treated and water-washed resin column. After
washing the column with water, the peptide can be
extracted with aqueous acetic acid. This extract can be
concentrated such as by rotary evaporation at 35C at 1.0
mm Hg vacuum and reevaporated from water to remove most
of the acetic acid. The resulting residue can be
purified by preparative HPLC using a linear gradient of
acetonitrile-water with 0.1~ trifluoroacetic acid. The
purified product fractions can be combined, organics
evaporated off, and the aqueous phase lyophilized.
W O 93/25576 ~3~t PC~r/US93/05325
-24-
Assays for Biological Activity
All the peptides and peptide analogs of this
invention can be tested to determine their most
advantageous PDGF-mimicking activity. As already noted,
a major advantage of the peptides of this invention is
their ability to exhibit only one or only a portion of
the several activities ascribed to native PDGF. In a
preferred testing approach, they are first tested for
their ability to bind to PDGF receptors on the surface of
Balb/C 3TC fibroblasts by a competitive inhibition
radioimmunoassay (RIA) by the test of R.M. Senior et al.,
J. Cell Biol. 100:351 (1988). All the peptides are then
tested for their ability to stimulate mitogenesis and to
induce chemotaxis.
Biological assays on active peptides can be
carried out on cultured smooth muscle cells (SMC). Rat
aorta cells can be obtained from ATCC (CRL1476), or
alternatively, isolated and cultured by the procedure
described by J. Nakao et al., Atherosclerosis 43:143
(1982) and R. Ross, J. Cell Biol. 50:172 (1971). SMCs
can be isolated from media explants of thoracic aortas
from male Wistar rats 6 to 8 weeks old. In culture, the
cells begin to grow and migrate out of the explants after
10 to 14 days. Cells are then subcultured at a 1 to 2
split ratio when they become confluent and are maintained
in tissue culture media (mlnlm~l essential medium [MEM])
containing 10% fetal bovine serum (FBS).
Cell Binding Assay
Fibroblasts or smooth muscle cells are grown in
24-well tissue culture plates in a 1:1 mixture of
Dulbecco's modified Vogt's medium and Ham's F12 medium.
When the cells reach confluence (3-4 days), the plates
are washed with cold phosphate-buffered saline (PBS) and
then filled with 1 ml of binding serum (PBS + 1% human
W093/25576 PCT/US93/05325
h ~73~7
-25-
serum albumin) containing various concentrations of
synthetic peptide or PDGF standards (in triplicate).
After 3 h incubation at 4C, approximately 5 ng of 125I-
labelled PDGF is added to each well, and the plates are
incubated for an additional 3 h at 4C. After three
washes with binding medium, the cell monolayers are
solubilized with 1 ml of 1~ Triton X-100 for 20 min at
room temperature. The amount of radioactivity contained
in the lysates is determined on a Packard Gamma Counting
System and the results are expressed as mean counts per
well. Fibroblast growth factor (FGF) and epidermal
growth factor (EGF) are used as controls because they do
not compete with the binding of PDGF. J.S. Huang et al.,
J. Biol. Chem. 257:8130 (1982).
Assay for Mitoqenic Activity
The mitogenic activity of peptides is
determined according to the procedure described by G.R.
Grotendorst et al., J. Cell Physiol. 113:261 (1982).
Fibroblasts or smooth muscle cells form primary explant
cultures are trypsinized and resuspended in Dulbecco's
Modified Eagle's Medium (DMEM) with 10~ calf serum
prepared from platelet-free plasma. The cells are then
pelleted at a density of 6 x 104 cells/well in Costar
microtissue culture wells (6.4 mm diameter). After 24 h,
various dilutions of PDGF or peptides are added to the
wells and the cells are incubated for an additional 48 h.
The cells are released from the wells by trypsinization
and then counted using an electronic cell counter. One
unit of PDGF activity is defined as that amount of PDGF
which stimulates 50~ of the cells to divide in 48 h.
Assays are also performed to determine the
incorporation of 3H-thymidine. Cells are added to 96-
~ell microcultures plates at a concentration of 1 x 104
3S cells/well in 0.1 ml of Dulbecco's Modified Eagle's
W093/25576 ,e' PCT/US93/05325
26-
Medium (DMEM) containing 10% fetal calf serum (FCS).
Dilutions of PDGF or peptide are added in triplicate to
these wells in 0.1 ml of DMEM. The plates are incubated
for 48 h at 37C, with the cultures being labelled with
0.2 ~Ci 3H-thymidine during the last 18-24 h. The
cultures are then harvested onto glass fiber filters and
counted. Results are expressed as the percent increase
in 3H-thymidine incorporation by cells incubated with
PDGF or peptide as compared to the cells incubated with
media alone.
Assay for Chemotactic Activity
Chemotaxis assays are performed using
fibroblasts or smooth muscle cells in a 48-well
chemotaxis chamber (NeuroProbe, Cabin John, NJ) according
to manufacturer's instructions and as described by W.
Ealk et al., J. Immunol. Meth. 33:239 (1980). Various
dilutions of chemotactic solutions are added in
triplicate to bcttom wells of the chamber in 25 ~l of
Hank's Balanced Salt Solution (BSS) cont~1ning 2~ bovine
serum albumin plate and the gasket and top plate is
secured on the filter with thumb screws. Approximately 3
x 104 cells are added to wells in the top chamber in 50
~1 of Hank's BSS containing 2~ BSA. After incubation for
2 h at 37C, the apparatus is disassembled and the
nonmigrated cells removed from the top of the filter by
gentle scraping. The filter is then fixed with methanol,
mounted on a glass slide, and dried. The filter is then
stained with Diff-Quick and the number of cells migrating
through the filter are counted microscopically. Ten
microscope fields are counted per well and the results
are expressed as the mean number of migrated cells per
microscope field. Alternatively, the amount of stain is
measured by determining the optical density.
W O 93/25576 ~ L 3 7 3 5 7 PC~r/US93/05325
By these uses of these tests, or other similar
tests known in the art, one can determine the best
activity of the particular material of this invention and
thus its most advantageous use.
Utility and ~m; ni stration
The peptides, salts and derivatives of the
present invention find use as medically active agents and
as analytical agents.
In medical applications they can be
administered locally or systemically to promote the
healing of wounds, burns and the like. They also can be
administered systemically to treat atherosclerosis or to
intervene in calcium uptake by cells.
For systemic administration one or a mixture of
two or more of these peptides may be administered
according to any convenient or effective methods for
introducing foreign substances into the blood stream of
m~mm~ls, such as by oral, rectal, nasal, buccal, vaginal,
or parenteral routes. The effective dosage level is, for
example, 0.01 to 100 mg/kg, preferably about 0.05 to 50
mg/kg. Doses of this size may be administered on a
regimen of 1 to 4 times per day.
Peptides here provided can be formulated into
pharmaceutical compositions by admixture with
pharmaceutically acceptable nontoxic carriers. As
mentioned above, such compositions may be prepared for
use for parenteral (subcutaneous, intramuscular or
intravenous) administration particularly in the form of
liquid solutions or suspensions; for use in vaginal or
rectal administration particularly in semisolid forms
such as creams and suppositories; for oral or buccal
administration particularly in the form of tablets or
capsules; or intranasally particularly in the form of
powders, nasal drops or aerosols.
L~ 36 PCT/US93/05325
-28-
The compounds may conveniently be administeredin unit dosage form and may be prepared by any of the
methods well known in the pharmaceutical art, for example
as described in Reminqton's Pharmaceutical Sciences, Mack
Publishing Company, Easton, Pa. 1975, incorporated by
reference. Formulations for parenteral administration
may contain as common excipients, sterile water or
saline, polyalkylene glycols such as polyethylene glycol,
oils of vegetable origin, hydrogenated naphthalenes and
the like. Formulations for vaginal or rectal
administration, e.g., suppositories, may contain as
excipients, for example, polyalkylene glycols, petroleum
jelly, cocoa butter, and the like. Formulations for
inhalation administration may be solid and contain as
excipients, for example, lactose, or may be aqueous or
oily solutions for administration in the form of nasal
drops. For buccal administration typical excipients
include sugars, calcium stearate, magnesium stearate,
pregelatinated starch, and the like.
For oral administration, a pharmaceutically
acceptable nontoxic composition can be formed by the
incorporation of any of the normally employed excipients,
oral dose extenders or carriers such as, for example,
pharmaceutical grades of mannitol, lactose, starch,
magnesium stearate, sodium saccharin, talcum, cellulose,
glucose, sucrose, magnesium, carbonate, and the like.
Such compositions can take the form of solutions,
suspensions, tablets, pills, capsules, powders,
sustained-release formulations, and the like. Such
compositions may contain 0.1-95~ active ingredient,
preferably 1-70~, with the r~m~in~er being carrier.
For local administration, the materials may be
formulated into lotions, salves and creams with carriers
such as water, mineral oil, salve base and the like.
W093/25576 2 1 ~ 7 ~ 6 ~ PCT/US93/05325
-29-
The peptides of this invention can be employed
as the sole active agent in a pharmaceutical composition
or can be used in combination with other active
ingredients.
In addition to these medical uses, the peptides
of this invention can also be employed as reagents and
standards in analytical schemes to detect the presence or
quantity of PDGF in samples. To this end they can be
used in labeled form in immunoassays such as fluoroim-
munoassays, radioimmunoassays, ELISA assays, EMIT assays
and the like.
Examples
The following examples serve to illustrate the
invention. They should not be construed as narrowing it,
or limiting its scope.
Example 1
Linear peptides of the invention are
synthesized by solid-phase techniques on a Beckman Model
990C automated peptide synthesizer using commercially
available t-BOC amino acid polystyrene resin and t-BOC
protected amino acids with the following side-chain
protecting groups: O-benzyl esters for aspartic acid and
glutamic acid; O-benzyl ethers for threonine and serine;
tosyl for arginine; DNP for histidine, p-methoxybenzyl or
acetamido methyl for cysteine; O-chlorobenzyloxycarbonyl
for lysine; and 2,6-dichlorobenzyl for tyrosine. All
couplings are performed using a 3-molar excess of t-BOC
amino acid and dicyclohexylcarbodiimide (DCC) over the
number of milliequivalents of amino acid on the resin.
In the cases of Asn and Gln, a 3-molar excess of t-BOC-
amino acid, DCC, and hydroxybenzotriazole (HOBT) is used.
All couplings are monitored by the ninhydrin test. 40
TFA-CH2Cl2 containing 10~ anisole and 0.1~ indole as
W093/25576 PCT/US93/OS325
~ 36~ - 30-
sc nger is used for BOC deprotection. The details of
the synthetic cycle are as follows:
SCHEDULE OF EVENTS FOR ASSEMBLING
A PEPTIDE ON A RESIN
Time
Step Reagent or Solvent(min)
1. CH2Cl2 x 3 1.5
10 2. 40~ TFA/CH2Cl2 prewash 5
3. 40~ TFA/CH2Cl2 30
4. CH2Cl2 x 6 1.5
5. 80~ Isopropanol/CH2Cl2 x 3 1.5
6. CH2Cl2 x 3 1.5
5~ Diisopropylethylamine/CH2Cl2 x 2 10
8. CH2Cl2 x 3 1.5
9. Coupling; 3-fold excess of t-BOC amino 120
in CH2Cl2:DMF (9:1; v/v); DCC/CH2C12
10. CH2Cl2 x 3 1.5
11. &0~ Isopropanol/CH2Cl2 x 3 1.5
After completion of the synthesis, the peptide
is cleaved from the resin using anhydrous hydrogen
fluoride in the presence of 10~ anisole as scavenger, at
4C for 1 hr. In the case of methionine, cysteine, and
tryptophan, 0.5~ dimethyl sulfide is used.
The DNP group of His is removed before HF
cleavage by treatment with 20-fold excess of thiophenol.
The peptides are separated from the various organic side-
products by extraction with ether and isolated from the
resin by extraction with various concentrations of
aqueous acetic acid depending on the solubilities of the
peptides. The solutions are diluted with water to about
5~ acetic acid concentration and lyophilized. The crude
peptides are then purified on Sephadex LH-20. Final
W093/25576 c~ 1 3 7 ~ 6 7 PCT/US93/05325
purification can be achieved on HPLC using 50 cm/20 mm
prep. column packed with Vydac 15-20 micron C18. Purity
of the peptides is checked by analytical HPLC and amino
acid analysis.
This process was used to prepare a peptide
corresponding to the 44-51 region of the PDGF "A"
chain--T-G-C-C-N-T-S-S (SEQ ID NO:2). This material was
tested to determine its ability to bind to receptors on
the 3T3 fibroblast cells. It bound competitively and
inhibited the binding of PDGF. The results of the test
are given in Fig. 1. The results of the PDGF binding are
shown in Fig. 2. This suggests that this peptide can
inhibit biological activity of PDGF since cell binding is
the initiating event for the induction of biological
activity. As such, this peptide can find application as
an antiatherosclerotic and antirestenosis agent. This
material was also tested for its ability to induce
chemotaxis and it showed chemotactic activity. The
results of this test are shown in Table 5. This suggests
that this peptide can also find application in wound
healing.
W O 93/25576 PC~r/US93/05325
~3~ 361 -32-
Table 5
CHEMOTACTIC ACTIVITY OF PEPTIDES
Concentration Chemotactic
Peptide (~g or ngtml) OD650 SD Activitya
Media 0.026 0.009
PDGFA 250 ~g 0.044 0.008 +
(108-125)
PDGFA 250 ~g 0.042 0.010 +
(101-125)
PDGFB 250 ~g 0.040 0.003 +
(45-52) Cyclic
PDGFA 250 ~g 0.043 0.011 +
(44-51)
PDGF 100 ng 0.057 0.007 +
50 ng 0.055 0.006 +
20 ng 0.059 0.010 +
10 ng 0.047 0.013
1 ng 0.021 0.012
0.5 ng 0.023 0.007
aPeptides were considered positive when their OD was sig-
nificantly higher than that of the media.
W093/25S76 Jl ~ 7 3 5 7 PCT/US93/05325
~ QJ ~
-33-
Table 6
COMPETITIVE INHIBITION FOR RECEPTOR BINDING
ON 3T3 CELLS BY ANALOGS OF PDGF B(45-52) CYCLIC PEPTIDE
Conc. Max.
(ng or Binding Inhibition
Peptide ~g/mL)
125I PDGF 100 0
PDGF BB 400 ng 27 73
200 ng 31 69
100 ng 38 62
PDGF B Ac-
(45-52)-NH2 200 ~g 39 61
100 ~g 49 51
50 ~g 75 25
25 ~g 95 5
12.5 ~g 100 0
PDGF B H2N
(45-52)-OH
(with free
amino and
carboxy end) 200 ~g 40 60
100 ~g 38 62
50 ~g 44 56
25 ~g 62 38
12.5 ~g 80 20
PDGF B Ac-(46-
52)-NH2 (Hepta-
peptide) 200 ~g 42 58
100 ~g 46 54
50 ~g 53 47
25 ~g 71 29
12.5 ~g 89 11
W O 93/25576 PC~r/US93/05325
,.~
~6 3 -34-
Table 7
COMP~'l'l'l'l~E INHIBITION FOR RECEPTOR BINDING
ON 3T3 CELLS BY PDGF PEPTIDES AND THEIR BSA CONJUGATES
Conc. Max.
(ng or Binding Inhibition
Peptide ~g/mL) (~
125I PDGF 5 NG 100 0
PDGF BB 100 ng 45 55
50 ng 54 46
25 ng 63 37
12.5 ng 72 28
PDGF A
(108-125) 1000 ~g 47 53
500 ~g 55 45
250 ~g 52 48
PDGF A (108- 400 ~g
125) BSA' (88 ~g)a 17 93
200 ~
(44 ~g)a 10 90
100 ~g
(22 ~g)a 14 86
PDGF A (44-51) 1000 ~g 43 57
500 ~g 61 39
250 ~g 76 24
25PDGF A (44-51) 400 ~g
-BSA (102 ~g)a 32 68
200 ~g
(51 ~g)a 39 61
100 ~g
(25.5 ~g)a 49 51
a Peptide concentration per milligram of conjugate.
W O 93/25576 2 1 3 7 3 ~ 7 PC~r/US93/05325
-35-
Example 2
The process and testing of Example 1 was
repeated this time to make and test a peptide
corresponding to the 108-125 region of the PDGF A
chain--G-R-P-R-E-S-G-K-K-R-K-R-K-R-L-K-P-T (SEQ ID
NO:12). This peptide was found to exhibit chemotactic
activity as shown in Table 5. This peptide was also
tested for its ability to induce intracellular calcium
influx and found to have activity of inducing
intracellular calcium uptake. The results of this test
are shown in Fig. 3. Induction of intracellular calcium
influx by PDGF is shown in Fig. 4. This suggests that
this peptide can stimulate cell proliferation and cell
growth and find application in wound healing.
Example 3
The process of Example 1 was repeated to
produce peptide corresponding to the 101-125 region of
the PDGA A-chain--D-Y-R-E-E-D-T-G-R-P-R-E-S-G-K-K-R-K-R-
K-R-L-K-P-T (SEQ ID N0:10). This peptide showed cell
binding and chemotactic activity. Results are shown in
Fig. 5 and Table 5.
Example 4
The cyclic disulfide E-V-Q-R-C-S-G-C (SEQ ID
NO: 17) was formed by air oxidation of a dilute solution
of B-chain 45-52 peptide in water. After HF cleavage of
the peptide off of the synthesis resin and extraction,
the resulting solution is adjusted to pH 8.1 by addition
of 50~ concentrated NH40H in water and then shaken slowly
on a shaker for 7 days. At the end of this time, an
Ellman test was performed to confirm the completion of
the disulfide bond formation. The peptide solution was
adjusted to pH 6.5 by addition of a few drops of acetic
W093/25~76 PCT/US93/05325
~3~6 -36-
acid. It was then passed through an acetic acid-treated
and water-washed Biorex 70 resin column. After washing
the column with water, the peptide was extracted into 50
aqueous acetic acid. This extract was concentrated by
rotary evaporation at 35C at 1.0 mm Hg vacuum and
reevaporated from water to remove most of the acetic
acid. The resulting residue was purified by preparative
HPLC using a linear gradient of acetonitrile-water with
0.1~ trifluoroacetic acid. The purified product
fractions were combined, organics are evaporated off, and
the aqueous phase lyophilized. This product was tested
for 3T3 receptor-binding activity using a competitive
binding assay and found to be very strongly active. The
results of this test are shown in Fig. 6.
This peptide being a very strong receptor
antagonist can inhibit biological activities of PDGF,
e.g., mitogenesis and chemotaxis and thereby can find
therapeutic use in atherosclerosis. It can also inhibit
intracellular calcium uptake and if properly targeted to
calcium-dependent cancer cells could intervene in the
course of this disease. This peptide when tested alone,
induced chemotaxis. The results are shown in Table 5.
The peptide was also treated to alter the ends
to give free amino and carboxy ends, and amidated carboxy
ends. These products were tested with the results given
in Table 6.
Example 5
The peptide dimer
(N T S S V K C Q P S R V H)2 (SEQ ID NO:3)
(A) 48 60
is formed. The monomer is formed as in Example 1, but
using an ACM-modified cysteine. The product is then
treated with iodine in methanol which removes ~he ACMs
and gives rise to the desired cysteine-linked dimer.
W093/2~576 ~31 3 7 3 fi 7 PCT/US93/05325
-37-
The dimer is recovered and purified.
Example 6
Conjugation of Peptides to Bovine Serum Albumin (BSA)
Peptides were conjugated to BSA by the method
of Atassi, M.Z. et al. Biochimia. Biophysica Acta,
670:300-302 (1981). BSA (100 mg) was dissolved in
0.025 M borate buffer (20 mL), pH 9.3. To this a
solution of succinic anhydride (540 mg) in dioxane (10
mL) was added in small aliquots over a period of 30 min
and the reaction mixture was stirred magnetically while
maintaining the pH at 9.3 by the addition of 3 M NaOH.
Following the last addition of succinic anhydride the
acylation reaction was allowed to continue for 3 hr. The
solution was then dialyzed against four changes of 0.01 M
triethylamine (Et3N), freeze-dried and finally dried in a
desiccator over P2O5.
Succinylated BSA (14 mg) was suspended in 1 mL
of anhydrous DMF and the suspension stirred magnetically
for 4 hr while shielding from direct light. p-Nitro-
phenol (6.5 mg) was added. The mixture was stirred for
15 min after which 5 mg of dicyclohexylcarbodiimide was
added. The reaction mixture was allowed to stir at room
temperature for 3 hr. To this was added 10 mg of peptide
and 100 ~L of Et3N. The reaction mixture was stirred at
room temperature for 24 hr while protected from direct
light. After this it was diluted with 3 mL of water and
dialyzed extensively against distilled water and
lyophilized.
The process was used to form several conjugates
which were found to be active. The results of these
tests are given in Table 7.
c~3 ~36 -38- PCI/US93/0532~
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: SRI INTERNATIONAL
(ii) TITLE OF INVENTION: PEPTIDES CORRESPONDING TO ACTIVE DOMAINS
OF PLATELET-DERIVED GROWTH FACTOR (PDGF)
(iii) NUMBER OF SEQUENCES: 39
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: SRI INTERNATIONAL
ATTN: INTELLECTUAL PROPERTY COUNSEL
(B) STREET: 333 Ravenswood Avenue
(C) CITY: Menlo Park
(D) STATE: California
(E) COUNTRY: USA
(F) ZIP: 94025
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 07/894,497
(8) FILING DATE: 05-JUN-1992
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: CLARK, JANET P.
(B) REGISTRATION NUMBER: 34,799
(C) REFERENCE/DOCKET NUMBER: PCT-2679
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (415) 859-2446
(B) TELEFAX: ~415) 859-3880
(C) TELEX: 334486
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Val Glu Val Lys Arg Cys Thr Gly Cys
1 5
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
WO93/25576 2137367 PCI/US93/05325
-39-
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Thr Gly Cys Cys Asn Thr Ser Ser
1 5
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Asn Thr Ser Ser Val Lys Cys Gln Pro Ser Arg Val His
1 5 10
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Lys Cys Gln Pro Ser Arg Val His His Arg Ser
1 5 10
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Gln Pro Ser Arg Val His His Arg Ser Val Lys Val Ala Lys
1 5 10
(2) INFORMATION FOR SEQ ID No:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
W O 93/25576 P~r/US93/05325
J. ~ 40-
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
His Arg Ser Val Lys Val Ala Lys Val Glu Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
~B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Val Ala Lys Val Glu Tyr Val Arg Lys Lys Pro Lys Leu
1 5 10
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Asp Thr Gly Arg Pro Arg Glu Ser Gly Lys Lys Arg Lys Arg Lys Arg
Leu Lys Pro Thr
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Ile Trp Pro Pro Cys Val Glu Val Lys Arg Cys Thr Gly Cys Cys Asn
1 5 10 15
Thr Ser Ser Val Lys
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
WO 93/25576 2 1 3 7 3 ~ 7 PCI`/US93/05325
--41--
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Asp Tyr Arg Glu Glu Asp Thr Gly Arg Pro Arg Glu Ser Gly Lys Lys
1 5 10 15
Arg Lys Arg Lys Arg Leu Lys Pro Thr
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll:
Asp Thr Gly Arg Pro Arg Glu Ser Gly Lys Lys Arg Lys Arg Lys Arg
1 5 10 15
Leu Lys Pro Thr
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Gly Arg Pro Arg Glu Ser Gly Lys Lys Arg Lys Arg Lys Arg Leu Lys
1 5 10 15
Pro Thr
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Ile Ser Arg Arg Leu Ile Asp Arg Thr Asn Ala Asn Phe Leu Val Trp
1 5 10 15
Pro Pro
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
WO 93/25576 PCr/US93/05325
2~3~ 3~ 42-
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
Ile Asp Arg Thr Asn Ala Asn Phe Leu Val Trp Pro Pro Cys Val Glu
l 5 10 15
Val Gln
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
Asn Phe Leu Val Trp Pro Pro Cys Val Glu Val Gln Arg Cys Ser
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
Trp Pro Pro Cys Val Glu Val Gln Arg Cys Ser
1 5 10
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Glu Val Gln Arg Cys Ser Gly Cys
1 5
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
- (D) TOPOLOGY: linear
WO 93/25576 2 ~ ~ 7 3 6' ~ PCI`/US93/05325
--43--
~ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
Glu Ile.Val Ala Lys Lys Pro Ile Phe Lys Lys Ala Thr Val Thr Leu
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
Lys Lys Pro Ile Phe Lys Lys Ala Thr Val Thr Leu Glu Asp His Leu
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
Phe Lys Lys Ala Thr Val Thr Leu Glu Asp His Leu Ala Cys
1 5 10
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
Val Thr Leu Glu Asp His Leu Ala Cys Lys Cys Glu Thr
1 5 10
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
W O 93/25576 PC~r/US93/05325
C~ 3~ -44-
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
His Leu Ala Cy5 Lys Cys Glu Thr Val Ala Ala Ala Arg Pro Val Thr
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
Lys Cys Glu Thr Val Ala Ala Ala Arg Pro Val Thr Arg Ser
1 5 10
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(D~ TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
Ala Ala Arg Pro Val Thr Arg Ser Pro Gly Gly Ser Gln
l 5 10
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
Arg Ser Pro Gly Gly Ser Gln Glu Gln Arg Ala Lys Thr
1 5 10
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
Ser Gln Glu Gln Arg Ala Lys Thr Pro Gln Thr Arg Val Thr
1 5 10
WO 93/25576 2 1 3 7 3 ~ 7 PCI/US93/OS325
--45--
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(D) OTHER INFORMATION: /note= "This position is
Acm-modified."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 9
(D) OTHER INFORMATION: /note= "This position is
Acm-modified."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
Val Glu Val Lys Arg Cys Thr Gly Cys
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: both
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
Val Glu Val Lys Arg Cys Thr Gly Cys
(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Disulfide-bond
(B) LOCATION: 6
(D) OTHER INFORMATION: /note= "This position has disulfide
bond to corresponding position on identical
sequence."
(ix) FEATURE:
(A) NAME/KEY: Disulfide-bond
(B) LOCATION: 9
(D) OTHER INFORMATION: /note= "This position has disulfide
bond to corresponding position of identical
sequence."
WO 93/25576 PCI/US93/05325
6~ -46-
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
Val Glu Val Lys Arg Cys Thr Gly Cys
1 5
(2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 3
(D) OTHER INFORMATION: /note= "This position is modified
with Acm."
tix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 4
(D) OTHER INFORMATION: /note= "This position is modified
with Acm."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
Thr Gly Cys Cys Asn Thr Ser Ser
1 5
(2) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: both
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
Thr Gly Cys Cys Asn Thr Ser Ser
1 5
(2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Disulfide-bond
(B) LOCATION: 3..4
(D) OTHER INFORMATION: /note= "This position has disulfide
bond to corresponding position of identical
sequence."
W O 93/25576 _47_ PC~r/US93/05325
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
Thr Gly Cys Cys Asn Thr Ser Ser
1 5
(2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 7
(D) OTHER INFORMATION: /note= "This position modified with
Acm.~l
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
Asn Thr Ser Ser Val Lys Cys Gln Pro Ser Arg Val His
(2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
Asn Thr Ser Ser Val Lys Cys Gln Pro Ser Arg Val His
1 5 10
(2) INFORMATION FOR SEQ ID NO:35:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/REY: Disulfide-bond
(B) LOCATION: 8
(D) OTHER INFORMATION: /note= "This position has disulfide
bond to corresponding position of identical
sequence."
(ix) FEATURE:
(A) NAME/REY: Disulfide-bond
(Bl LOCATION: 14
(D) OTHER INFORMATION: /note= "This position has disulfide
bond to corresponding position of identical
sequence."
WO 93/25576
PCI/US93/05325
37~ 3 -48-
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:
Asn Phe Leu Val Trp Pro Pro Cys Val Glu Val Gln Arg Cys Ser
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Disulfide-bond
(B) LOCATION: 4
(D) OTHER INFORMATION: /note= This position has disulfide
bond to corresponding position on identical
sequence.
(ix) FEATURE:
(A) NAME/KEY: Disulfide-bond
(B) LOCATION: 10
(D) OTHER INFORMATION: /note= "This position has disulfide
bond to corresponding position on identical
sequence."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:
Trp Pro Pro Cys Val Glu Val Gln Arg Cys Ser
1 5 10
(2) INFORMATION FOR SEQ ID NO:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/REY: Disulfide-bond
(B) LOCATION: 5
(D) OTHER INFORMATION: /note= "This position has disulfide
bond to corresponding position on identical
sequence."
(ix) FEATURE:
(A) NAME/KEY: Disulfide-bond
(B) LOCATION: 8
(D) OTHER INFORMATION: /note= "This position has disulfide
bond to corresponding position of identical
sequence."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:
Glu Val Gln Arg Cys Ser Gly Cys
1 5
W O 93/25576
~ ~ 7 3 6 PC~r/US93/05325
-49-
(2~ INFORMATION FOR SEQ ID NO:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Disulfide-bond
(B) LOCATION: 4
(D) OTHER INFORMATION: /note= "This position has disulfide
bond to corresponding position on identical
sequence."
(ix) FEATURE:
(A) NAME/REY: Disulfide-bond
(B) LOCATION: 6
(D) OTHER INFORMATION: /note= "This position has disulfide
bond to corresponding position on identical
sequence."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:
His Leu Ala Cys Lys Cys Glu Thr Val Ala Ala Ala Arg Pro Val Thr
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:
Lys Cys Glu Thr Val Ala Ala Ala Arg Pro Val Thr Arg Ser Lys Cys
1 5 10 15
Glu Thr Val Ala Ala Ala Arg Pro Val Thr Arg Ser
