Note: Descriptions are shown in the official language in which they were submitted.
CA 022167~ 1997-09-29
W096/30405 PCT~S96~0349a
PEPTIDES WITH GROW'TH PROMOTION PRO~K~ S
F;~l~ Qf Th~ Tnv~n t; nn
This invention relate~ to peptides which
elicit antibodies which ~nh~nce the activity of
porcine somatotropin and promote growth of warm-
blooded An; m~ 18.
~ch~ ~ ~ Of Th~ Tnvent; nn
Porcine somatotropin (pST) is a 191 amino
acid long pituitary hormone which has diverse
biological activities (Bibliography entry 1). Thîs
hormone exists as a single chain polypeptide which is
arranged in four anti-parallel a-helices (2). Each a-
helix contains 3.6 amino acids per turn. This
periodicity places every third or fourth residue on
the ~ame face of the helix.
Administration of pST to farm ~n; -18
increases muscle growth, feed efficiency and decreases
fat accumulation (3,4). Endogenous amounts of pST are
small; therefore, efforts have focused on the
preparation of exogenous pST for use in large-scale
agriculture. The widespread use of exogenous pST has
been hampered by difficulties in formulation and
~m;~; stration. Long-term implants re~uire the
stabilization of the pST molecule and control of the
release of pST over time. Alternatively, daily or
frequent periodic injections of pST involve labor
costs which may render the benefits of pST
uneconomical.
Some efforts to reduce these difficulties
have involved reducing the amount of pST needed
through the addition of a component which potentiates
CA 022167~ 1997-09-29
W096/30405 PCT~S96/03490
-- 2
the activity of pST.
Monoclonal and polyclonal antibodies
complexed with somatotropin have been reported to
further enh~n~e the biological activity of
somatotropin in vivo (5-9). Although the mechanism of
antibody-mediated growth ~nh~ncement is not totally
clear, altered ph~r~-cokinetics and/or bio-
distribution of somatotropin has been proposed in part
to explain the growth ~nh~ncement ph~nomenon (10). It
has also been suggested that the ~nh~ncement is due to
the selective modulation of b;n~;ng to subclasses of
somatotropin receptors by the antibodies (5,6,11,12).
Imp ov~ -nt of somatotropin b;n~;ng to hepatic
somatogenic receptors was also reported to be a
possible explanation (13). It is noteworthy that
these antibodies by themselves do not potentiate
growth in the absence of exogenous pST.
One attempt to increase ~n; ~'1 growth
; ologically has produced a monoclonal antibody
designated PS-7.6, which consistently increases the
biological activity of pST in a hypophysectomized
(hypox) rat assay (10), even though in the absence of
pST it does not by itself increase growth. However,
passive ; - ;zation of farm ~n; -18 with this growth-
~nh~ncing monoclonal antibody is not optimum
economically because of the cost of monoclonal
antibody and the labor required for the multiple
dosing of antibodies.
Thus, there is a need for a new approach to
growth potentiation which will reduce the difficultie~
of the current approaches. One such approach involves
the design of peptides which mimic the region of pST
which binds to PS-7.6 monoclonal antibody. The active
immunization of ~n; -18 with peptide fragments
correspo~;ng to the epitope recognized by PS-7.6
CA 022167~ 1997-09-29
WO 9~/30 ~C~t', PCT/US96/03490
monoclonal antibody should generate antibodies
functionally similar to PS-7.6 monoclonal antibody to
~ce the endogenous pST activities. Thus, the
identification of the epitope recognized by PS-7.6
monoclonal antibody is critical for the design of
peptide vaccines that ~nh~nce ~n; 1 growth
performance. Most peptides possess the primary, but
lack the secondary conformation of the native protein.
Short peptides typically exist in solution as rapidly
interconverting structures (random coils). A
conformation analogous to the desired region of pST
will be present in only a ~mall fraction of the
peptides at a given time. Thus, there is a need to
design peptides having a defined secon~-y
conformation.
~Um~Y Of Th~ Tnv~nt;on
Accordingly, it is an object of this
invention to design peptides which have well-defined
secondary structure, preferably a helical
conformation, which mimic the correspon~;ng region of
pST.
It is a further object of this invention to
design peptides which compete with pST for b;n~;ng to
PS-7.6 monoclonal antibody.
It is yet another object of this invention
to develop compositions which utilize these peptides
for the promotion of growth of warm-blooded ~n; =ls
These objects of the invention are
accomplished through a peptide which contain~ therein
the sequence of amino acids Xaa-Xaa-Leu-Xaa-Xaa-Ile-
Xaa-Xaa-Xaa-Leu-Xaa-Xaa-Val-Xaa-Xaa (SEQ ID N0:1),
wherein the sequence differs from the native sequence
of pST. The invention also include~ peptides wherein
CA 022167~ 1997-09-29
W096/30405 PCT~S96/03490
one or more of the first or second leucine or the
valine of SEQ ID NO:1 is replaced by normal (straight
chain) leucine (Nle). The invention is also directed
to a peptide in which the location of the essential
amino acids is shifted by three amino acids,
representing almost one turn along the helix, which
contains therein the sequence of amino acids Xaa-Xaa-
Xaa-Xaa-Xaa-Leu-Xaa-Xaa-Xaa-Ile-Xaa-Xaa-Leu-Xaa-Xaa-
Xaa-Val (SEQ ID NO:18) and wherein the sequence
differs from the native sequence of pST. The peptides
of SEQ ID NO:18 are further modified by limiting the
third amino acid residue to isoleucine, generating the
sequence of amino acids: Xaa-Xaa-Ile-Xaa-Xaa-Leu-Xaa-
Xaa-Xaa-Ile-Xaa-Xaa-Leu-Xaa-Xaa-Xaa-Val (SEQ ID NO:19)
and wherein the sequence differs from the native
sequence of pST.
These peptides preferably contain serine as
a promoter of helical conformation as the amino acid
; ~;ately amino-te~m;nAl to the first leucine of SEQ
ID NO:1 and as the amino acid ; -~;ately amino-
te ;n~l to the first isoleucine of SEQ ID NO:19.
The peptides of this invention are used with
a phA -ceutically acceptable adjuvant, diluent or
carrier to formulate compositions in methods for
promoting the growth of a warm-blooded An;m~l.
Br;ef Descr~p~; nn Of Th~ F; ~ne8
Figure 1 depicts the trypsin digeRts (lane
1), fractions #9 (lane 2) and #9-5 (lane 3) which are
incubated with PS-7.6 monoclonal antibody,
;mm~noprecipitated by magnetized goat anti-mouse IgG
antibody, subjected to SDS-PAGE, electroblotted onto a
PVDF membrane, probed with rabbit anti-pST polyclonal
antibody followed by iodinated donkey anti-rabbit IgG
CA 022167~ 1997-09-29
WO 96130405 PCT~US96/03490
antibody, and finally exposed to an x-ray film and
autoradiography.
Figure 2 depicts the peptides pST(70-95),
pST(64-95) and pST(54-95) (Figure 2A), and pST(75-95)
and pST(75-90) (Figure 2B), which are synthesized and
tested for their inhibitory effects on the interaction
between PS-7.6 monoclonal antibody and 125I-pST in a
competitive RIA. Cold intact pST is also tested and
serves as the positive control.
Figure 3 depicts the results of a
radio; s~say which measures the competition for
b; n~'; ng to PS-7.6 monoclonal antibody of radiolabelled
pST compared with: (1) the pGH(75-95) peptide, (2) a
pGH(80-90) peptide, and (3) a pGH(81-90) peptide.
Figure 4 depicts the amino acids of the
pST(75-95) fragment, of which amino acid residues 75-
90 are sequentially substituted by ~l~n;n~, These
analogs are tested by competition RIA. The IC50 of an
effective competition for each residue is averaged
from five separate experiments.
Figure 5 depicts a helical projection of
pST(75-95) of pST. The ~h~e~ circles are the amino
acidR critical for the recognition by PS-7.6
monoclonal antibody.
Figure 6 depicts the results of a
radio; ~ns~say which measures the competition for
b; n~; ng to PS-7.6 monoclonal antibody of radiolabelled
pST (referred to as "pGH" in Figure 5) compared with:
(1) a peptide correspQn~'; ng to the native sequence of
amino acids 75-95 of pST (referred to as "pGH(75-95)"
in Figure 5), (2) the peptide of SEQ ID N0:2 (referred
to as "peptide-Xn), and (3) a modified form of
peptide-X where the amino-te~; n~ 1 amino group i8
modified by the addition of an acetyl group (referred
to as "capped peptide-X").
CA 022167~ 1997-09-29
W096/30405 PCT~S96/03490
Figure 7 depicts the results of a
radioim~no~say which measures the competition for
bin~;ng to PS-7.6 monoclonal antibody of radiolabelled
pST compared with: (1) the pGH(75-95) peptide, and
(2) a peptide of SEQ ID NO:21 not within the scope of
this invention (referred to as "peptide-Y" in Figure
6).
Figure 8 depicts the results of a
radioi~ o~say which measures the competition for
b;n~;n~ to PS-7.6 monoclonal antibody of radiolabelled
pST compared with: (1) the pGH(75-95) peptide, (2)
peptide-X, (3) peptide-Y, and (4) peptide-X wherein
the first leucine iB replaced by nor~-l leucine (Nle),
generating SEQ ID NO:13 (referred to as "Nle80" in
Figure 7), (5) peptide-X wherein the second leucine is
replaced by normal leucine (Nle), generating SEQ ID
NO:14 (referred to as "Nle87n), and (6) peptide-X
wherein the valine is replaced by normal leucine
(Nle), generating SEQ ID NO:15 (referred to as
"Nle90").
Figure 9 depicts the effect on the growth of
hypophysectomized rats receiving either no treatment
(negative control, first bar); treatment with pST
alone (positive control, second bar); treatment with
pST plus swine antibodies before and after
;~-lln;zation with peptide-X (SEQ ID NO:2) from three
different pigs (three groups of rats, third through
eighth bars).
De~ ed Descr;pt;~n Of The Tnve~tion
This invention comprises the design of
peptides which mimic a helical region of pST. These
peptides possess minimal sequence homology to pST in
order that the peptides can retain a helical
CA 022167~ 1997-09-29
WO ~f/~0 ~' PCT/US96/03490
formation. These peptide mimics elicit antibodies
which can recognize native pST.
T ln; zation of a target An; ~1 species with
a peptide cont~;n;ng the epitope recognized by PS-7.6
- o~lonal antibody (raised against native pST)
produces antibodies functionally similar to PS-7.6
monoclonal antibody and promotes the growth of the
An; --1,
Active immunization of animals with the
epitope recognized by PS-7.6 monoclonal antibody is a
practical alternative to the passive A~n; n; ~tration of
PS-7.6 monoclonal antibody. This idea is supported by
a recent report of Pell et al (14) who ~ - ntrated
that ;~lln;zation of lambs with somatotropin fragment
(residues 135-154), a different region associated with
the ~nhAncement, increased the percentage of lean
muscle growth.
In this invention, the epitope of pST which
binds to PS-7.6 - ~clonal antibody is mapped by a
limited tryptic digestion of pST coupled with reverse-
phase liquid chromatographic separation and
sprecipitation. The critical amino acids of the
epitope which interact with PS-7.6 monoclonal antibody
are then identified by an; Inologic analysis of a
series of sequential alanine analogs of the epitope.
The peptide which contains the epitope recognized by
the monoclonal antibody designated PS-7.6, which is
raised against recombinant pST, is shown to enhAnce
the growth-promoting activity of pST in a
hypophysectomized rat -'el and to promote growth in
pigs.
A pST fragment correspo~; ng to amino acid
residues 70-95 (which contains the second helix of pST
(2)) is separated by reverse-phase high performance
liquid chromatography and also; sprecipitated by
CA 022167~ 1997-09-29
W096/30405 PCT~S96/03490
PS-7.6 monoclonal antibody. This fragment i8 found in
a radioimmunoassay (RIA) to compete with radiolabelled
pST for the bi n~; ng to PS-7.6 monoclonal antibody in a
dose-dependent fashion. However, sera from pigs
immunized with pST(70-95) do not raise significant
levels of anti-pST antibodies. This result suggests
that the conformation of pST(70-95) does not match
that of the true epitope or the peptide is not
suf~iciently ; ogenic.
Several peptides covering this potential
epitope region of pST(70-95) are synthesized and
assayed by competitive RIA. The results suggest that
pST(75-95) is the optimal sequence r~o~n;zed by PS-
7.6 monoclonal antibody. In contrast, pST(85-95)
binds very weakly and non- e~ od~cibly to PS-7.6
monoclonal antibody, while pST(81-95) is completely
devoid of b;~;ng. The peptide pGH(80-90) binds
almost as strongly as pGH(75-95), while pGH(81-90)
binds very weakly to PS-7.6 monoclonal antibody.
Furth~ re, if the peptide is too small, such as
pST(80-95), even though b;n~;ng is good, aggregation
is substantial due to the hydrophobic character of the
individual amino acid residues. To reduce the
ay~,eydtion problem and improve solubility, the
peptide should be longer.
The design of peptides in the epitopic
region pST(75-95) involves a number of factors. Most
peptides possess the primary, but lack the secondary
conformation of the native protein. Short peptides
typically exist in solution as rapidly interconverting
structures (r~ coils). A conformation analogous
to the desired region of pST will be present in only a
small fraction of the peptides at a given time.
The goal of the design is to stabilize the
helical conformation and thus to ~nh~nce the affinity
CA 022167~ 1997-09-29
w096/30405 PCT~S96/03490
of the antibody for the protein. This affinity
requires structural complementarity between the
b; n~; ~g site of the antibody and the epitope on the
protein.
Amino acid residues which contact the
antibody b; n~; n~ site are relatively intolerant of
~ub~titution. Replacement of leucine by isoleucine is
~ufficient to abolish peptide b;n~;ng to the antibody.
Non-critical residues can be replaced by residues
which promote or stabilize a desired structure, in
this case an a-helix, which allows the peptide to
better mimic the native protein conformation.
Among the ~trategies which can be employed
to stabilize ~-helices are: (1) interhelical
hydrophobic association (15): (2) side-chain charge-
charge interactions (16); (3) side-chain co~alent
linkages by a disulfide (17) or lactam bond (18); (4)
incorporating amino acid residues with high helical
propensity (19); (5) charge-helix dipole stabilization
(20): (6) end-capping of peptides (21) and metal-ion
side chain ~tabilization (22).
The criticality of the first leucine is
demonstrated in Example 1 below, where the first
leucine of a peptide (SEQ ID N0:2) is replaced by
isoleucine to produce the peptide of SEQ ID N0:21. In
contrast, non-contact residues may be replaced freely
and still bind effecti~ely. Thus, after identifying
the essential antibody contact residues, the rem~; n; ng
residues of the peptide are replaced 80 as to achieve
a stable helical conformation.
Se~uential alanine ~ubstitution of each
residue of pST(75-90) re~eals that the residues of
Leu80, Ile83 and Leu37, plus either Leu'6 or Val90, are
critical for b;n~;ng to PS-7.6 monoclonal antibody (as
depicted in the helical projection of Figure 5).
-
CA 022167~ 1997-09-29
W09~'30l~ PCT~S96/03490
-- 10 --
Other residues are replaced by alanine without
substantially altering the bin~;ng affinity. The
region of amino acids 75-95 comprises the second helix
of pST and the repeating pattern of i and i+3 (i+7) of
the critical amino acids appears consistent with PS-
7.6 monoclonal antibody b;n~;ng to a hydrophobic side
of the helix. This enables peptides to be synthesized
utilizing both the i and the i+3 (almost one helical
turn) patterns. The sequence and the helical
structure of the epitope recognized by PS-7.6
monoclonal antibody provide the basis for designing
effective peptide vaccines to enh~nce the growth
performance of ~n; ~ 18.
The pST and peptides are generated as
follows. Recombinant pST is produced by American
Cyanamid Co., Pearl River, NY. Peptides are
synthesized on a solid-phase automatic peptide
synthesizer (9600, Milligen/Biosearch, Bedford, MA).
All reagents are purchased from Peninsula
Laboratories, Inc., Belmont, CA. After being cleaved
from the resin support, all peptides are purified by
preparative reverse-phase high performance liguid
chromatography (HPLC) on a C18 coll-~n (R~; n; n
Instrument Corp., Woburn, MA). The purity of these
peptides is always greater than 97% as determined by
analytical HPLC, FAB mass spectrometry, and amino acid
composition analysis. Alternatively, the peptide~ of
this invention are also constructed by other
technigues known in the art, such as solution-phase
chemical synthesis or recombinant expression of a DNA
nucleotide se~uence in an appropriate host.
Recombinant expression may also be in the form of a
fusion of the peptide to a carrier, such as a maltose-
b; n~; ng protein or peptide.
Mapping of the antibody-recognizing sites
CA 022167~ 1997-09-29
W096t3040S PCT~S96/03490
has been appro~he~ conventionally by the enzymatic
~ dige tion of the antigen (23,24) or the concurrent
synthesis of multiple peptides on solid supports (25).
Initial attempts with multiple peptide synthesis were
not ~uccessful and, therefore, the proteolytic
degradation of pST is employed subsequently to
complete the epitope mapping of PS-7.6 monoclonal
antibody in this study. In the beg;nn;n~, pST is
treated at 37~C with various proteases, including
chymotrypsin, trypsin and endoproteinase Glu-C. They
all produce small pST fragments (m.w. c 4 kDa). The
rationale for selecting these small fragments is the
convenience of solid pha~e synthesis of future peptide
vaccine candidates. However, PS-7.6 monoclonal
antibody fails to re~ogn;ze any of these small
fr~m~nts by RIA and Western Analysis. In addition,
short peptides are generally conformationally flexible
and produce a variety of inter-converting
configurations in solution. As a result, resulting
anti-peptide antibodies frequently interact with the
correspon~; ng native protein with a very low
efficiency.
Results from the initial experiments of
enzymatic degradation indicate that the epitope
recognized by PS-7.6 monoclonal antibody is very
sensitive to protease at 37~C. It is possible that
the se~n~y/tertiary structure of pST might be
slightly unfolded at this temperature, thus exposing
the critical epitope region to the protea~e for a
total degradation. In order to limit the degree of
enzymatic digestion to protect the intact PS-7.6
epitope, especially the loops and turns (26), the
reaction temperature is restricted at 25~C for three
days.
Specifically, recombinant pST (100 mg) is
CA 022167~ 1997-09-29
W096/30405 PCT~S96/03490
subjected to limited tryptic digestion by incubation
with 1 ml bo~ine pancreatic trypsin cross-linked to
agarose (50 units enzymatic activity per ml of packed
gel, Sigma Co., St. Louis, MO) in 200 ml phosphate
buffer, pH 9.5, at 25~C for three days. The tryptic
fragments are separated by preparative HPLC (C18
column, 21.4 mm x 25 cm, flow rate = 24 ml/min., W at
235 nm., Dynamax-300A, R~;n;n) with a linear gradient
of 0-100% of B buffer in 30 minutes (A buffer = 0.1%
TFA in water; B buffer = 0.1% TFA in acetonitrile).
All fractions are collected and analyzed by RIA to
identify the acti~e cnmron~nts. A predominant and
stable tryptic fragment which is ab~ent in the 37~C
digestion is produced and designated as fraction #9.
This fraction is eluted at a retention time of 18.89
minutes and comprises more than 60% yield of the
initial pST digestion. It is later found to compete
with l25I-pST for the b;n~;ng to PS-7.6 monoclonal
antibody in RIA, suggesting that the fraction #9
contain~ the PS-7.6 epitope. The fraction #9 is
further reduced by a 10 molar excess of dithiothreitol
in 0.1 M ~ - ;um bicarbonate solution at pH 9.5 and
again fractionated by HPLC. One fraction, designated
#9-5 and eluted at a retention time of 18.43 minute~,
continues to exhibit an ability to compete with l25I-pST
for PS-7.6 monoclonal antibody b; n~; ng.
A combination of ; oprecipitation and
Western blotting experiments is carried out to analyze
these samples. The trypsin digests, fractions #9 and
#9-5, are incubated with PS-7.6 monoclonal antibody
for 60 minutes at 37~C. I~~gnetic beads coated with
goat anti-mouse IgG (Advanced Magnetics Inc.,
Cambridge, MA) are added to the mixture and incubated
for 30 minutes at room temperature with gentle,
constant ~h~k;ng. The beads are washed several times
CA 022l67~ l997-09-29
WO 96/30405 PCT/US96/~3490
-- 13 -
and collected by a ~gret. The protein~ are separated
~ from the beads by boiling in SDS-PAGE 8ample buffer
and ~ub~ected to reduced Tri~-tricine polyacrylamide
gel electrophoresi~ on a 16% gel (PAGE, Novex, San
Diego, CA). The separated protein components are
electroblotted from the gel onto a polyvinylidene
difluoride microporous (PVDF) membrane (Applied
Biosystems, Foster City, CA) in a 10 mM 3-
[cyclohexyl r- ;no] -l-propane~ulfonic acid (CAPS) buffer
at pH ll contA;n;ng 20% methanol. The membrane is
treated with 5% nonfat milk followed by sequential
expo~ure~ to rabbit anti-pST polyclonal antibody and
0 iodinated donkey anti-rabbit IgG antibody (Amersham
Corp., Arlington Heights, IL). The PVDF membrane in
finally expo~ed to an x-ray film for the development
of autoradiography. To determine the amino acid
~equence of the tryptic fragment~, the m~mhrane blot~
are ~tA;ne~ briefly with a solution contA;n;ng 0.1%
Coomassie, 40% methanol and 10% acetic acid and the
visible bands are exci~ed. These bands are arranged
in a single layer in the upper cartridge block of a
gas-phase sequenator (Applied Biosystems) and the
phenylthiohydantoin-derived amino acids are dete~m;neA
by the procedure of ~AnkApiller ~ Hood (27). The
results are seen in Figure 1.
It i~ clear that the predominant component
of pST after a limited digestion with trypsin is the
15 kDa material with minor cont~;n~nts of 10 kDa, 4
kDa and 2.5 kDa (lane 1). However, the~e minor
components are enriched and become pr~m;n~nt in
fraction #9 following HPLC fractionation (lane 2).
Finally, the 2.5 kDa fragment appears to be the only
cl _- ~nt present in the fraction #9-5, sugge~ting
that the epitope recognized by PS-7.6 monoclonal
antibody re~ides within this fraction (lane 3). The
=
CA 022167~ 1997-09-29
W096l30405 PCT~S96/03490
- 14 -
2.5 kDa fragment i8 then seguenced and found to
correspond to amino acids 70-95 of pST. The chemical
structure and ; ologic properties of this fragment
are confirmed by a solid phase peptide synthesis (28),
because the synthesized peptide is capable of
competing with 12sI-pST in a dose dependent fashion for
the b;n~;ng to PS-7. 6 monoclonal antibody in RIA
(Figure 2). The competition by pST (70-95) is
d~ Gximately 100-fold less active than that by cold
pST.
In order to further characterize the preci~e
epitope, three additional peptides are synthesized.
Peptides pST (54-95) and pST(64-95) are produced by
ext~n~; ng the amino acid residues of the N-terminu~,
while pST (75-90) is generated by truncating the
residues at both ends. These peptides are tested in
RIA and exhibit a similar competition effect as
c~ _-~ed to the tryptic pST (70-95) fragment,
suggesting that the residues between 75 and 90 contain
the epitope (Figure 2). The competition of these
peptides is substantially lower than that of a control
pST, probably due to the conformational flexibility
inherent in the short peptides and the involv - t of
other discontinuous residues in the antibody
recognition.
A separate competition RIA establishes the
criticality of the Leu80 residue. As depicted in
Figure 3, the peptide pGH(80-90) binds almo~t as
strongly as pGH(75-95) ~ while pGH(81-90) binds very
weakly to PS-7. 6 monoclonal antibody.
The conformational flexibility of peptide
antigens limits their practical utility as antigens. t
However, the limitation can be overcome by restricting
the conformational flexibility of the peptides to
mimic the secondary structure of the native protein
CA 022167~ 1997-09-29
w096J3040s PCT~S96/03490
.
- 15 -
anti~en. It is also known that not every residue in
~ an epitope is neceQsarily in contact with the antibody
b;n~;ng sites (29). Because the seco~ry structure
~ of the PS-7.6 monoclonal antibody epitope is
considered important, the amino acid side ~h~;n~ of
the pST(75-95) fragment that interact with PS-7.6
monoclonal antibody are det~_ ;n~ by sequentially
replacing each residue with ~1 ~n; ne. The ~equential
substitution with alanine, which ha_ a high propensity
to form a helical conformation in peptides or proteins
(30), is less likely to change the accessible backbone
conformation of pST(75-95).
This approach, called ~l~n;ne-8c~nn;ng
mutagenesis, ha~ been very useful for identifying the
critical amino acids of human somatotropin which
interact with its receptor (31). Al ~n;ne is ascumed
to leave the seco~y structure ~nch~nged, while
eliminating one potential site of interaction with the
antibody. If the peptide contA;n;ng a substituted
alanine still binds to the antibody, the replaced
amino acid iQ considered to be a non-contact residue.
Therefore, a ~eries of ~l~n;ne-substituted
analogs of pST(75-90) is Qynthesized by the solid-
phase method and their ability to compete for PS-7.6
monoclonal antibody b;n~;ng with pST is evaluated by
RIA. Side ch~;nQ of residues are considered critical
if the ~l~n;n~ replacement results in a significant
increase in IC50 to achieve an equivalent competition.
F;n~;ngs in Figure 4, which are ~ -~ized from five
separate experiments, indicate that Leu80 (see also
Figure 3), Ile83 and Leu37, which all are hydrophobic
amino acids, are critical to PS-7.6 - o~lonal
antibody recognition, and that either Leu76 or Val90 is
al_o critical. Although the bar value for position 89
3S i8 greater than that for position 90 in Figure 4, when
CA 022l67~ l997-09-29
W096/30405 PCT~S96/03490
p values are computed using Fisher'~ combined p-value
test, the p value for position 90 is statistically
significant (p=0.00162), while the p value for
position 89 is not statistically significant
(p=0.16048). Note that Figure 4 does not include the
data from a sixth experiment, where the results are
entirely inconsistent with the other five experiments.
Based on the tertiary structure of pST (2)
and the projection of the second helix, these five
critical residues presented by the ~ e~ circles in
Figure 5 are located on the same face of the helix,
representing a hydrophobic ribbon to interact with PS-
7.6 monoclonal antibody.
In s~ary, the epitope of growth-~nh~cing
antibody PS-7.6 monoclonal antibody is mapped by a
limited tryptic digestion and the critical amino acids
on the epitope are identified by the sequential
alanine substitution. The a-helical projection of pST
shows that the critical amino acids are a hydrophobic
ribbon located on the same face of the second helix.
The characterization of the sequence and secondary
structure of the epitope provide the basis for
designing effective peptide antigens that mimic the
native conformation of the correspQ~;ng epitope of
pST.
Based on the identification of critical
amino acids in the epitope, a series of peptides is
designed. In one embodiment of the invention, the
peptides are selected from sequences cont~;n;ng the
formula:
Xaa-Xaa-Leu-Xaa-Xaa-Ile-Xaa-Xaa-Xaa-Leu-Xaa-
Xaa-Val-Xaa-Xaa (SEQ ID NO:1)
wherein the sequence differs from the native sequence
of pST. Examples of such peptides include:
Tyr-Ser-Leu-Asp-Asp-Ile-Ile-Arg-Arg-Leu-Asp-
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WO 96130405 PCT/US96/03490
Asp-Val-Ile-Arg-Arg-Ile (SEQ ID N0:2),
Tyr-Ser-Leu-Asp-Arg-Ile-Ile-Arg-Asp-Leu-Asp-
Arg-Val-Ile-Arg-Asp-Ile (SEQ ID N0:3),
Tyr-Ser-Leu-Arg-Arg-Ile-Ile-Arg-Arg-Leu-Arg-
Arg-Val-Ile-Arg-Arg-Ile (SEQ ID N0:4),
Tyr-Ser-Leu-Asp-Asp-Ile-Ile-ARp-Asp-Leu-Asp-
Asp-Val-Ile-Asp-Asp-Ile (SEQ ID N0:5),
Tyr-Ser-Leu-Anp-Anp-Ile-Ala-Arg-Arg-Leu-Asp-
Asp-Val-Ala-Arg-Arg-Leu (SEQ ID N0:6),
Tyr-Ser-Leu-Lys-Ala-Ile-Ala-Glu-Ala-Leu-Lys-
Ala-Val-Ala-Glu-Ala (SEQ ID N0:7),
Tyr-Ser-Leu-Ly~-Glu-Ile-Glu-Lys-Leu-Leu-Lyn-
Glu-Val-Leu-Glu-Ly~-Leu (SEQ ID N0:8),
Tyr-Ser-Leu-Asp-Asp-Ile-Ala-Arg-Arg-Leu-Asp-
A~p-Val-Ala-Arg-Arg-Ala (SEQ ID N0:9),
Tyr-Ser-Leu-Lys-Ile-Ile-Ile-Glu-Ile-Leu-Lys-
Ile-Val-Ile-Glu-Ile (SEQ ID N0:10),
Tyr-Ser-Leu-Lys-Glu-Ile-Glu-Lys-Leu-Leu-Lys-
Glu-Val-Leu-Glu-Lys-Leu (SEQ ID N0:11), and
Tyr-Ser-Leu-Lys-Aib-Ile-Aib-Glu-Aib-Leu-Lys-
Aib-Val-Aib-Glu-Aib (SEQ ID N0:12), where
Aib i~ 2-aminoisobutyric acid.
In addition, one or more of the leucines or
~aline of SEQ ID N0:1 can be replaced by no ~1
leucine (Nle), an follows:
Tyr-Ser-Nle-Asp-Asp-Ile-Ile-Arg-Arg-Leu-Asp-
Asp-Val-Ile-Arg-Arg-Ile (SEQ ID N0:13),
Tyr-Ser-Leu-Asp-Asp-Ile-Ile-Arg-Arg-Nle-Asp-
Asp-Val-Ile-Arg-Arg-Ile (SEQ ID N0:14),
Tyr-Ser-Leu-Asp-Asp-Ile-Ile-Arg-Arg-Leu-Asp-
Anp-Nle-Ile-Arg-Arg-Ile (SEQ ID N0:15), and
Tyr-Ser-Nle-Asp-Asp-Ile-Ile-Arg-Arg-Nle-Asp-
A~p-Val-Ile-Arg-Arg-Ile (SEQ ID N0:16).
Fur~h~ -re, a cysteine may be ~ to
either or both ends of the peptiden of SEQ ID N0:1.
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-- 18 --
An example of such a peptide is:
Cy~-Tyr-Ser-Leu-Asp-Asp-Ile-Ile-Arg-Arg-Leu-
Asp-Asp-Val-Ile-Arg-Arg-Ile (SEQ ID N0:17).
In another .e ~o~; ~nt of the invention, the
location of the essential amino acid residues is
shifted by three amino acids, representing almost one
turn along the helix. These peptides are selected
from sequences cont~; n; ng the formula:
Xaa-Xaa-Xaa-Xaa-Xaa-Leu-Xaa-Xaa-Xaa-Ile-Xaa-
Xaa-Leu-Xaa-Xaa-Xaa-Val (SEQ ID NO:18)
wherein the sequence differs from the native sequence
of pST. In particular, these peptides include an
isoleucine as the third residue and are selected from
sequences cont~; n; ng the formula:
Xaa-Xaa-Ile-Xaa-Xaa-Leu-Xaa-Xaa-Xaa-Ile-Xaa-
Xaa-Leu-Xaa-Xaa-Xaa-Val (SEQ ID NO:l9)
wherein the seguence differs from the native sequence
of pST. An example of such a peptide i8:
Tyr-Ser-Ile-Asp-Asp-Leu-Ile-Arg-Arg-Ile-Asp-
Asp-Leu-Ile-Arg-Arg-Val (SEQ ID NO:20).
TheQe peptides preferably contain serine as
a promoter of helical conformation as the amino acid
; ~~;ately amino-t~m;n~l to the first leucine of SEQ
ID N0:1 and as the amino acid ; -~;ately amino-
t~r~;n~l the first isoleucine of SEQ ID N0:19.
The amino acid sequences of these peptides
are described using the convention that the first
essential residue is toward the amino-terminus and the
last essential residue is toward the carboxy-terminus.
The invention also encompasses identical sequenceQ in
opposite orientations where the first essential
residue is toward the carboxy-te~m;nl~Q and the last
essential residue is toward the amino-te~;nnQ. Both
orientations have the same helical projection on the
side chain (see Figure 5 ) and are functionally
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WO 96130405 PCT~US96/03490
equivalent.
- other peptides within the scope of the
invention are generated readily by uRing the methods
and criteria described herein.
The peptide~ of this invention are tested in
various experiments, as described in detail in the
Examples below.
Fir~t, radioimmunoassay~ are performed to
determine the extent to which various peptides (both
within and outside the scope of this invention)
compete with radiolabelled pST for b;n~;ng to PS-7.6
- ~clonal antibody. As detailed in Example 1 and
shown in Figure 6, the peptide of SEQ ID NO:2
(referred to as "peptide-X") inhibits the interaction
of ~S-7.6 ~no~lonal antibody and radiolabelled pST in
a dose dependent manner more strongly than a peptide
corre8p~n~; ng to the native sequence of amino acids
75-95 of pST. A modified form of peptide-X, where the
amino-teTm;nA1 amino group i8 replaced by an acetyl
group (referred to as "capped peptide-X") does not
inhibit the interaction. In experiments not shown in
Figure 6, the peptide~ of SEQ ID NOS:8-11 inhibit the
interaction as well as peptide-X, while the peptides
of SEQ ID NOS:3 and 12 inhibit the interaction, but at
a lower rate than peptide-X.
As detailed in Example l and shown in Figure
7, a peptide not within the scope of the invention and
having the sequence Tyr-Ser-Ile-Asp-Asp-Ile-Ile-Arg-
Arg-Ile-Asp-Asp-Ile-Arg-Arg-Ile (SEQ ID NO:21;
referred to as "peptide-Y") is much less effective in
inhibiting the interaction than the pGH(75-95)
peptide. Thus, the substitution of isoleucines for
the first leucine and the valine in the peptide of SEQ
ID NO:2 abolishes the b;n~;~g to the antibody.
As detailed in Example l and shown in Figure
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- 20 -
8, peptide-X and three variants thereof (SEQ ID
NOS:13-15) where the leucines or valine are replaced
by normal leucine (Nle) are more effective than
pGH(75-95) in inhibiting the interaction, whereas
peptide-Y is again much less effective. In an
experiment not shown in Figure 8, the peptide of SEQ
ID NO:16 inhibits the interaction as well as peptide-
X.
Next, the biological activity of these
peptides is tested in hypophysectomized (hypox) rats.
Hypox-rats are growth-deficient as a result of
surgical ~ vdl of their pituitary glands. Hypox-rats
serve as a useful model for studying the effect of
somatotropin on growth (32).
As described in Example 2 and shown in
Figure 9, peptide-X (SEQ ID NO:2) is conjugated to
ovalbumin and emulsified in Complete Freund's
Adjuvant. Three pigs are ; ;zed with peptide-X and
receive two booster doses with Incomplete Freund's
Adjuvant. Blood samples are taken from thege ~n;m~l 8
and control sera are al~o harvested from the same pigs
prior to the first injection. Antibodies are
purified, mixed with pST and injected into hypox rats
for five consecutive days. Rats receiving pST alone
grow in a statistically signficant manner. Rats
receiving pST plu8 antibodies from pigs #1 or #2 after
; ni zation have significantly increased growth.
Although the effect of the antibodies from pig #3 does
not reach a statistically significant level (p=0.08),
there is visible growth ~nh~ncement. In contrast,
pre-; ;zation antibodies are not effective. As a
result of the hypox rat experiments, ; ;zation
trials are conducted with pigs.
Peptides may be administered alone or, more
preferably, linked to a macromolecule which serves to
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- 21 --
~nh~nce the production of antibodies in ~ vo. For
~ example, the peptide may be conjugated to a protein
such as keyhole limpet haemocyanin (RLH). Other
macromolecules within the scope of this invention
include those known in the art such as hl -n and
bovine 6erum albumins, ovalbumin, myoglobins, ~-
galactosidase, penicillanase, heat shock protein and
bacterial toxoids. Synthetic molecules such as multi-
poly-DL-alanyl-poly-L-lysine and poly-~-lysine are
also suitable.
The peptides may be A~m; n; stered by
conventional routes such as subcutaneous injection,
intramuscular injection and intravenous flow, as well
as trans~e ~l and oral administration. It is
preferred to administer the peptides (or their
conjugates) in association with a ph~ ceutically
acceptable adjuvant, diluent or carrier. It is
particularly preferred to use a dosage regimen where
an initial ~m;n; stration of the peptides is followed
by one or more booster ~; n; stration of the ~ame
peptides at regular time intervals.
As detailed in Examples 3-5, the
; ;zation of pigs with the peptides of this
invention results in the desirable results of an
increase in feed efficiency, and an increased lean and
decreased fat.
In Example 3, Table 1, finishing pigs
receiving the peptide of SEQ ID NO:2 have a
statistically significant increase in feed efficiency
over the course of the experiment when compared to
pigs receiving o~albumin only. In Example 3, Table 2,
the aame pigs receiving the peptide display improved
carcass characteristics, with a statistically
significant increase in lean and decrease in fat when
compared to pigs receiving ovalbumin only. It is
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- 22 -
interesting to note that the pigs receiving the
peptide do not have a significant increase in most
organ weights. In contrast, pST is known to increase
organ weights.
When the experiment of Example 3 iB repeated
in Example 5 with another group of finishing pigs
receiving either the peptide of SEQ ID NO:2 or the
peptide of SEQ ID NO:17 ("Cys-Peptide"), both groups
of pigs have a statistically significant decrease in
undesirable leaf fat when compared to pigs receiving
ovalbumin only, while the pigs receiving the peptide
of SEQ ID NO:2 have a statistically significant
decrease in semit~n~;nosis (see Tables 5 and 6).
In Example 4, Tables 3 and 4, growing pigs
receiving the peptide have a statistically significant
increase in lean over the course of the experiment
when compared to pigs receiving ovalbumin only. This
is particularly significant during the treatment
stages, as compared to the finishing period where
treatment i~ ~topped.
In order that this invention may be better
understood, the following examples are set forth. The
examples are for the purposes of illustration only and
are not to be construed as limiting the scope of the
invention.
P!y~ e 1
Cn~?etitive Radioi m"~ln~assay
Radio; lno~says are performed to determine
the extent to which various peptides compete with
radiolabelled pST for b;n~;~g to PS-7.6 monoclonal
antibody. For any given concentration of peptide, the
lower the amount of pST which binds to the antibody in
the assay, the greater the competition of the peptide
to the antibody. Peptides which conformationally
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- 23 -
mimic native pST ~hould diQplace pST from PS-7.6
~ monoclonal antibody more efficiently. The
radio; O~Qays are performed aQ followR.
oble microtiter wellQ are coated with
PS-7.6 monoclonal antibody at 1 ~g/well and the
r~m~;n;n~ microtiter well b;n~;n~ sites are blocked
with 2% BSA. Iodinated pST (lZ~I-pST, specific activity
= 180-250 ~Ci/~g, New England Nuclear/DuPont, Co.,
BoRton, M~) iQ added together with competitive agents
at various dilution~. After a 60 minute incubation,
all wells are washed thoroughly to ~_~ve the unbound
5I-pST and the re~idual radioactivity, reflecting the
amounts of l25I-pST which remain associated with PS-7.6
monoclonal antibody in each well, iQ counted
individually in a gamma counter.
In a first experiment depicted in Figure 6,
a radio; ~ay iR performed which mea~ure~ the
competition for b;n~;n~ to PS-7.6 monoclonal antibody
of radiolabelled pST (referred to aQ "pGH" in Figure
6) compared with: (1) a peptide corresp~n~;ng to the
nati~e sequence of amino acids 75-95 of pST (referred
to a~ "pGH(75-95) n in Figure 6), (2) the peptide of
SEQ ID N0:2 (referred to as "peptide-X"), and (3) a
modified form of peptide-X where the amino-te ;n~l
amino group is replaced by an acetyl group (referred
to a~ "capped peptide-Xn).
The results indicate that addition of
pGH(75-95) to the wells inhibits the interaction of
PS-7.6 monoclonal antibody and radioactive pST in a
3~ dose dependent manner. Peptide-X iQ alQo ~hown to
inhibit the interaction in a ~imilar, though somewhat
Qtronger, fashion, whereas capped peptide-X fails to
do 80. Without being bound by theory, the failure of
capped peptide-X in the competition assay may be due
to the inter-peptide aggregation in the test solution.
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In a second experiment depicted in Figure 7,
a radioimmunoassay i8 performed which measures the
competition for b;n~;ng to PS-7.6 monoclonal antibody
of radiolabelled pST compared with: (1) the pGH(75-
95) peptide, and (2) a peptide of SEQ ID N0:21 not
within the scope of this invention (referred to as
"peptide-Y" in Figure 7).
The results indicate that addition of
pGH(75-95) to the wells inhibits the interaction of
PS-7.6 monoclonal antibody and radioacti~e pST in a
dose dependent -nne~. In contrast, peptide-Y is
shown to be much less effective.
In a third experiment depicted in Figure 8,
a radio; o~say is performed which measures the
competition for b;n~;ng to PS-7.6 monoclonal antibody
of radiolabelled pST ro~r~ed with: (1) the pGH(75-
95) peptide, (2) peptide-X, (3) peptide-Y, (4)
peptide-X wherein the first leucine is replaced by
normal leucine (Nle), generating SEQ ID NO:13
(referred to as "Nle80" in Figure 8), (5) peptide-X
wherein the second leucine is replaced by normal
leucine (Nle), generating SEQ ID NO:14 (referred to as
"Nle87"), and (6) peptide-X wherein the ~aline is
replaced by normal leucine (Nle), generating SEQ ID
NO:15 (referred to as "Nle90").
The results indicate that addition of
pGH(75-95) to the wells inhibits the interaction of
PS-7.6 monoclonal antibody and radioactive pST in a
dose dependent ~-nner. Several peptides within the
scope of this invention, including peptide-X, Nle80,
Nle87 and Nle90, are found to be more effecti~e than
pGH(75-95) in this competition assay. However,
peptide-Y is again shown to be much less effective.
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WO 96130405 PCT/US96/03490
E~~le 2
~em~nt Of pST Act;v;ty Tn ~ypox ~t8 W; th
SW;n~ Ant~h~A;es To A Pept;~
In this example, swine are injected with a
peptide. Serum samples cont~;n;ng ant;hoA;es raised
against the peptide are injected together with pST
into hypox rats. The ant;~oA;es potentiate the
ability of pST to promote the growth of the hypox
rats. The experiment is conducted as follows.
Peptide-X (SEQ ID NO:2) is conjugated to
ovalbumin and e_ulsified in Complete Freund' 8
Adjuvant. Three pigs are ; ;zed with peptide-X and
receive booster doses with Incomplete Freund's
Adjuvant four and eight weeks thereafter. Blood
samples are taken from these ~n ; - 18 one week after
the last boosting. Control sera are also harvested
from the same pigs prior to the first injection.
Antibodies are purified by ; - ;um sulfate
precipitation, m; Ye~ with pST to provide a dose of 1
mg antibodies and 5 ~g pST, and injected into hypox
rats for five consecutive days. Three separate groups
of rats each receives ant;hoA;es from one different
pig; a fourth group of rats receives no injections,
while a fifth group of rats receives only pST. The
net weight gains of the rats of day 5 are depicted in
Figure 9.
Treatment with pST alone stimulates growth
in hypox rats in a statistically signficant ~~nner (*,
pcO.05). Rats receiving pST plus antibodies from pigs
#1 or #2 after immunization have significantly
increased growth. Although the effect of the
antibodies from pig #3 does not reach a statistically
significant level (p=0.08), there is visible growth
~nh~cement. In contrast, pre-; ;zation antibodies
are not effective.
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Ex~mple 3
n; zat; ~n of F; n; ~h; n~ ~; gs With A Peptide
This example sets forth the effects of
immunization of finishing pigs (final growth phase
from 70-80 kg and above) with a peptide of this
invention on growth rate, feed efficiency and carcass
composition.
The peptide (Tyr-Ser-Leu-Asp-Asp-Ile-Ile-
Arg-Arg-Leu-Asp-Asp-Val-Ile-Arg-Arg-Ile; SEQ ID NO:2)
is synthesized and conjugated to ovalbumin. For
comparison, on ovalbumin antigen is generated by
conjugating ovalbumin to itself.
Mixed-breed barrows (a new commercial DeRalb
term;nAl cross genetic line) weighing 70-80 kg are
purchased from Gold Rist Pork, Hillsborough, NC. Pigs
are the target ~n; 1 for the peptides and at least 25
pigs per treatment group are needed to obtain
statistical significance between treatment8. An;m~l 8
are individually identified by numbered eartags and
maintained in numbered pens. The pigs receive a diet
which is medicated with chlortetracycline at 200 g/ton
and are fed for about one week after the pigs are
delivered. The pigs receive a Swine ST ration (20%
crude protein) for the rr~ e~ of the experiment.
Feed and water are available ad libitl-m.
An; ~18 are assigned to each of the two
treatments in a rAn~ ;zed complete block design. The
An;m~l 8 are blocked by initial body weight. The
following treatments are ~m;n; stered:
GrQUp An~;g~n n N~mher Pen~
A Ovalbumin/ovalbumin 25 5
B Peptide/ovalbumin 25 5
The pigs are rAn~mly assigned to treatment groups A
or B within each of the blocks. Each block of 15 pigs
start treatment when their average body weights are
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WO 96130405 PCT/US96/03490
about 75 kg.
The pigs are allowed at least a 1-week
pretreatment period in the pen~ before the first
; ;zation injection. The flow of feed is adjusted
to pro~ide unlimited access with a minimum of wastage.
Pigs are ; ;zed with 0.5 mg of
peptide/ovalbumin conjugate totally emulsified with
Complete Freund's Adjuvant using a subcutaneous (SC)
injection in the neck area ju~t behind the ear~. All
pigs receive one booster injection after a 4-week
interval using the same quantity of conjugate, but
with Incomplete Freund'~ Adjuvant. Control pigs
receive the ovalbumin conjugate without the peptide.
Pigs are observed daily. Any ~ick or
injured An; ~18 receive appropriate veterinary care.
An; -18 that do not recover within a few days or that
are isolated to recover (e.g. lameness that is
aggravated by penmate~) are -ved from the
experiment.
Body weights and feed con~umption are
determined weekly and more often if necessary as the
pigs approach the slaughter weight.
Each pen of pigs is sacrificed when the mean
live weight is 115+2 kg. Carcass measurements include
chilled weight, backfat thickness, rib eye area,
carcass length and weights of heart, liver, spleen,
kidney, leaf fat and semit~n~;nous muscle.
The data are analyzed using the analysis of
variance procedures for a rAn~ ;zed complete block
design. The experimental unit for the evaluation of
weight gain and carcass measurement~ is the individual
pig. Feed consumption and feed efficiency data use
the pen as the experimental unit. Mean comparisons
are made using Fisher's Protected LSD.
The experiment proceeds on the following
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- 2 8
srhe~le. On Day 1, all pigs are weighed and
treatment (including the initial immunization for
Group B) starts for each block when the average body
weight reaches 75 kg.
Booster injections are :~m;n; stered 4 and 8
weeks after the initial immunization. Pigs are
weighed and feed consumption iB dete~m;ne~ on a weekly
basis. The pigs are then sacrificed at mean pen
weights of 115i2 kg.
The results of this experiment are shown in
Tables 1 and 2.
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Table 1
-
The effects of active immunization of finishing pigs
with a peptide on growth, feed consumption and feed
efficiency
Control T 1~; zed SEM
Item Ova/ova Peptide
No. pens/pigs 5/25 5/25
Body weight, kg
Initial 76.2 76.3 0.3
4 Weeks 99.2 98.8 0.8
Final 116.2 117.1 1.0
Days on Experiment51.2 51.0 1.3
Gain, g/day
1-4 Weeks 819 804 26.9
5 Weeks - End 743 805 27.5
1 Week - End 781 802 18.4
Feed intake, g/day
1-4 Weeks 3390 3250 59.7
5 Weeks - End 3316 3326 93.4
1 Week - End 3357 3285 54.5
Gain/Feed
1-4 Weeks .241 .247 .005
5 Week~ - End .222 .242 .009
1 Week - End .233 .244* .004
* Significantly different than control, Pc.05
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- 30 -
Table 2
The effects of active ; ;zation of finishing pigs with
a peptide on carcass measurements and organ weights
ItemControl Immunized SEM
Ova/ova Peptide
No. pens/pigs 5/25 5/25
Carcass weight, kg82.5 82.2 .8
Dressing percent 71.0 70.2 .3
Carcass length, cm82.6 83.0 .4
Organ weight, g
Heart 370 373 8.6
Liver 1804 1829 37.8
~idneys 375 384 8.7
Spleen 161 175 6.6
Leaf fat 1663 1389** 60.9
Semit~n~;nosus 394 434*** 8.2
Fat thickness, cm
1st rib 4.9 4.4* .14
Last rib 2.9 3.0 .10
Lumbar 2.6 2.3 .12
Backfat 3.5 3.2 .09
6th rib 3.6 3.1** .11
10th rib 3.7 3.2*** .11
Adjusted 10th rib 3.3 2.8*** .1
fat, cm
Loin eye area, cm244.2 44.6 .8
Adjusted loin eye 41.6 41.9 .8
area, cm
Calculated lean (33)
Kg 38.1 39.7** .4
% 56.1 57.6* .5
* Significantly different than control, Pc.05
** Significantly different than control, Pc.01
*** Significantly different than control, Pc.001
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Ex~le 4
~n;7~t.;on Of Grow;n~ P;g~ W;th A Pept~
The immunization experiment of Example 3 is
repeated using young growing pigs (approximately 15-20
kg) from the herd of American Cyanamid Company,
Princeton, NJ. Two booster injections are given at
four week intervals (during the growth phase to
approximately 50-60 kg). The ~n;~l~ are then
maint~;neA to sacrifice at a finishing weight of
approximately 115 kg. The re~ults of this experiment
are ~hown in Tables 3 and 4.
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Table 3
The effects of active ; ;zation of growing pigs with
a peptide-on growth, feed consumption and feed efficiency
Item ControlT~--n; zed SEM
Ova/ova Peptide
No. pens/pigs 6/27 6/26
Body weight, kg
Initial 18.5 18.8 .3
4 Weeks 39.8 40.7 .6
8 Weeks 56.3 59.1* .8
12 Weeks 72.9 76.3* 1.1
16 Weeks 97.6 100.7 1.4
Final 113.0 114.1 1.5
Days on Experiment135.0 131.2+ 1.4
Gain, g/day
1-4 Weeks 762.0 782.2 13.4
1-8 Weeks 675.4 718.9* 11.6
1-12 Weeks 647.8 683.7 12.3
1-16 Weeks 706.3 730.7 11.6
1 Week - End 704.0 728.5 11.6
8 Weeks - End 725.1 735.2 16.4
Lean tissue (34)326.6 343.6* 5.8
Feed Intake, g/day
1-4 Weeks 1702 1632 37.2
1-8 Weeks 1670 1631 52.1
1-12 Weeks 1843 1857 70.5
1-16 Weeks 2131 2187 72.0
1 Week - End 2352 2401 70.6
8 Weeks - End 2872 3061 89.2
Gain/Feed
1-4 Weeks .448 .480*.011
1-8 Weeks .404 .444*.013
1-12 Weeks .351 .371.010
1-16 Weeks .331 .337.011
Week 1 - End .298 .305.010
Week 8 - End .251 .242.009
Lean ti~sue (34).137 .144.005
+ Significantly different than control, Pc.10
* Significantly different than control, Pc.05
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Table 4
The effects of active immunization of growing pigs with
a peptide on carcass measurements and organ weights
Item Control T ;zed SEM
Ova/o~a Peptide
No. pen~/pigs 6/27 6/26
Carcass weight, kg79.8 80.6 1.1
Dressing percent71.1 71.0 .3
Carcass length, cm83.2 83.5 0.5
Organ weight, g
Heart 400 439* 10.9
Li~er 1796 1841 33.3
Ridney~ 387 385 8.5
Spleen 150 158 5.0
Leaf fat 1441 988*** 54.7
Semit~n~;no is 425 461* 12.1
Fat thickness, cm
1st rib 4.3 4.2 .16
Last rib 2.2 2.2 .09
Lumbar 2.2 2.0 .09
Backfat 2.9 2.8 .09
6th rib 2.1 2.0+ .07
10th rib 2.7 2.5* .08
Adjusted 10th 2.5 2.2* .07
rib fat, cm
Loin eye area, cm238.2 42.1** .8
Adjusted loin eye36.4 40.0*** .7
area, cmZ
Calculated lean (33)
Rg 39.9 41.1** .3
% 56.2 58.5*** .4
+ Significantly different than control, Pc.10
* Significantly different than control, P~.05
** Significantly different than control, P~.01
*** Significantly different than control, P~.001
CA 02216755 1997-09-29
W 096/30405 PCT~US96/03490
- 34 -
~le 5
;7at;~n of ~;n;~h;n~ Pi~s With ~ Pept;de
The ; ln; zation experiment of Example 3 is
repeated with the inclusion of a second peptide having
a cystine linkage at the amino-term;nl~ (SEQ ID
NO:17)(Cys-peptide), which is conjugated to ovalbumin
as follows: Ovalbumin (10 mg) is dissolved in an
aqueous solution (pH 8) and treated with iodoacetic
anhydride (16 mg; 20-fold excess) and stirred for 2
hours at ~hient temperature. The excess iodoacetic
anhydride-is separated by ultrafiltration and the
modified ovalbumin is reacted with the Cys-peptide for
four hours at ~hient temperature and dialyzed to
produce the final conjugate. The results of this
experiment are shown in Tables 5 and 6. Note that two
pigs receiving the conjugated Cys-peptide died during
the course of the study.
CA 022l6755 l997-09-29
W096l30405 PCT~S96103490
- 35 -
Table 5
The effect~ of active ; ;zation of finishing pigs with
peptide~ on growth, feed con~umption and feed
efficiency
Control Immunized T ; zed SEM
Item Ova/ova Peptide Cys-
Peptide
No. pen~/pigs 5/25 5/25 5/23
Body weight, kg
Initial 76.6 76.7 76.7 0.3
4 Week~ 100.8 102.2 102.2 0.9
Final 119.2 119.2 120.1 1.2
Day~ on Experiment49.8 49.0 48.4 1.2
Gain, g/day
1-4 Week~ 866 909 903 30.2
4 Weeks - End 845 811 896 28.7
1 Week - End 856 870 902 23.8
Feed intake, g/day
1-4 Weeks
4 Week~ - End 3180 3333 3222 118.7
1 Week - End 3430 3440 3630 116.2
3285 3381 3389 100.8
Gain/Feed
1-4 Weeks .273 .273 .281 .008
4 Week~ - End .247 .236 .247 .009
1 Week - End .261 .257 .266 .008
CA 022l67~ l997-09-29
W096/30405 PCT~S96103490
- 36 -
Table 6
The effects of active ; ;zation of finishing pigs with peptides on
carcass measurements and organ weights
ControlI =unized I =unized SEM
Ova/ova Peptide Cys-
Peptide
No. pens/pigs 5/25 5/25 5/23
Carcass weight, kg86.0 86.4 85.9 .9
Dressing percent 72.1 72.S 71.5 .3
Carcass length, cm82.6 82.7 83.5 .5
Organ weight, g
Heart 397 400 415 8.4
Liver 1838 1810 1885 38.9
Kidneys 397 388 411 9.3
Spleen 166 167 183 6.9
Leaf fat 1569 1291** 1313* 73.1
Semit~n~;nosis 457 492* 467 10.0
Fat thickness, cm
1st rib 4.5 4.3 4.5 .13
Last rib 2.6 2.6 2.6 .10
Lumbar 2.0 2.0 2.3 .09
Backfat 3.0 3.0 3.1 .09
6th rib 2.4 2.3 2.3 .09
10th rib 3.0 2.9 2.9 .13
Adjusted 10th rib fat, 2.6 2.5 2.4 .1
cm
Loin eye area, cm246.8 46.7 46.8 1.1
Adjusted loin eye area, 43.6 43.6 43.4 1.1
cm2
Calculated lean (33)
Rg 40.6 40.7 40.9 .5
% 58.2 58.5 58.6 .7
* Significantly different than control, Pc.05
** Significantly different than control, Pc.01
CA 02216755 1997-09-29
W 096/30405 PCT~US96/03~90
B;~l;ogr~hsy
1. Pell, J.M., and Bates, P.C., et al.,
Nutr;tion Res., Rev. 3., 163-192 (1990).
2. Abdel-Meguid, S.S., et al., Prod. Nat.
Acad. Sc;. USA, ~, 6434-6437 (1987).
3. McLaren, D.G. et al., J. Anim. Sci.,
68, 640-651 (1990).
4. Campbell, R.G., et al., J. An;m. SCi.,
69, 1522-1531 (1991).
5. Aston, R., et al., J. ~n~cr;
381-388 (1986).
6. Aston, R., et al., Molec~ Tmm~n~l ,
143-150 (1987).
7. Holder, A.T., et al., J ~n~ncr;nn
R9-R12 (1985).
8. Holder, A.T., et al., J. ~n~cr;nnl.
117, 85-90 (1988).
9. Wallis, M., et al., B;ot~hfm. Ri o~hy~.
Re8. ~mm7~n., 1~2~ 187-193 (1987).
10. Wang, B.S., et al., Molec. Tmm--n~l.,
29, 313-317 ~1992).
11. Ivanyi, J., Molec. Tm,~l~nnl, 19~ 1611-
1618 (1982).
12. Thomas, H.I., et al., B;o~hem. J., 243,
365-372 (1987).
13. Ma~sart, S., et al., J. ~n~:iocr; n~l ogy~
139, 383-393 (1993).
14. Pell, J.M., and Aston, R., J.
Fn~ncr;nol., 31, Rl-R4 (1991).
15. Ho, S.P., and DeGrado, W.F., J. Am.
Chem. Soc., 109, 6751 (1987).
16. Marqusee, S., and Baldwin, R.L., Pro~.
-t. Ac~. Sc;. USA, 84, 8898 (1987).
17. Jack~30n, D.Y., et al., J. Am, ~h~--.
CA 022l67~ l997-09-29
W096/30405 PCT~S96/03490
- 38 -
~Q~~ , 9391 (l991).
18. Osapay, G., et al., J. Am, ~h~m. Soc.,
11~, 6966 (1992).
19. garle, I.L., et al., Prod. Nat. Acad.
Sci. USA, 84, 5087 (1987).
20. Shoemaker, R.R., et al., Prod. Nat.
Acad. Sc;. USA, 82, 2439 (1985).
21. Forwood, B., et al., Prod. Nat. Acad.
Sc;. USA, 90, 838 (1993).
22. ~n~el, T.M., et al., Sc;ence, ~1, 879
(1993).
23. Benjamin, D.C., and Wigle, W.D.,
Tmm~lnnrhem~, 8, 1087-1097 (1971).
24. Crumpton, M.J., and Wilkinson, J.M.,
B;och~. J., 94, 545 (1965).
25. Geysen, H.M., et al., J. Im~u~ol.
Me~h~ds, 102, 254-274 (1987).
26. Hara, ~., et al., Bioche~mistry, 17,
550-556 (1978).
27. ~nk;3piller, M.W., and Hood, ~.E.,
Methods in Enzy3~olo~y, 91, 486-493 (1983).
28. Merri~ield, R.B., J. ~m . ~h~m . SOC .,
85, 2149-2154 (1963).
29. Getzoff, E.D., et al., Adv. I r~ol.,
43, 1-98 (1988).
30. Chou, P.Y., and Fasman, G. D., ~31 .
Rev. B;ochem~, 47, 251-276 (1978).
31. C~ln~;n~h-3~, B.C., and Wells, J.C.,
Sc;~n~e, ~4~, 1081-1085 (1989).
32. Groesbeck, M.D., and Parlow, A. F.,
cr;n~logy, l~Q, 2582-2590 (1987).
33. Boggs, D.L., and Merkel, R.A-, ~iY~ -
An;m~l ~r~R8 ~v~ll3~t; ~n ~n~ S~l ect~on Ma~3~l (1979).
34. National Pork Producers Council,
Proce~3nres to ~v~ te M3rket ~ng Perform~n~e (1983).
CA 02216755 1997-09-29
W 096/30405 PCT~US96/03490
- 39 -
~QU~-N-~ LISTING
(1) ~RN~uAT- lN~ OK~ATION:
(i) APPLICANT: Buckwalter, Brian L.
Shieh, Hong-Ming
Wang, Bosco S.
(ii) TITL_ OF INv~llON: Peptides With Growth Pro ~ tion Properties
(iii) NUMBER OF ~yu~. - R.C: 21
(iv) Co~K~p~ ~KN. K ~n~ReS:
(A'~ AnD~R-eSRR: American Cyanamid Company
B STRBET: One Cy~ ~ Plaza
~C~ CITY: W~yne
D~ STATE: New Jersey
B CO~ ~Y: U.S.A.
~FJ ZIP: 07470-8426
(v) COMP~TER TR~n~T-R FORM:
(A) MBDIUM TYP_: Floppy disk
(B) COMP~TER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn RelQa~Q #1.0, VQrsion #1.25
(vi) ~UKK~ APPLICATION DATA:
(A) APPLICATION N~MBER: ~S
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) AllOK~._~/AG~NT lN~O~ ~TION:
(A) NAME: Gordon, Alan M.
(B) REGISTRATION N~MBER: 30,637
(C) R_~KK_N~/DOCRBT N~MBER 32,083-00
(ix) T_LECOMM~NICATION INFORMATION:
(A) TBLEPHONE: 201-831-3244
(B) TELEFAX: 201-831-3305
(2) INFORNATION FOR SBQ ID NO:l:
(i) ~yu~-N~ ~R~ CTR~ T-C TICS:
(A~ LBNGTH: 15 ~;no acids
(B TYPB: amino acid
(C I ST~N~ ~N KeS single
(DJ TOPOLOGY: linear
(ii) ~OTR~u~-~ TYPE: protein
(iii) nYr~ CAL: NO
(iv) ANTI-SENSE: NO
-
(xi) ~yu N~K DRSCRIPTION: SEQ ID NO:l:
Xaa Xaa LQu Xaa Xaa Ile Xaa Xaa Xaa ~eu Xaa Xaa Val Xaa Xaa
1 5 10 15
CA 022l6755 l997-09-29
W 096/30405 PCTAUS96/03490
- 40 -
(2) lN~ORrATION FOR SEQ ID NO:2:
Uu-N~ C~TR~ T .CTICS:
(A' LENGTH: 17 ~;no acids
(Bl TYPE: amino acid
(Cl STR~N~-K.. Kes single
(D TOPOLOGY: linear
(ii) r ~T~T~C~T~T~ TYPE: protein
(iii) ~Y~G~ CAL: NO
(i~) ANTI-SBNSE: NO
(xi) ~yl ~R DESCRIPTION: SEQ ID NO:2:
Tyr Ser Leu Asp Asp Ile Ile Arg Arg Leu Asp Asp Val Ile Arg Arg
1 5 10 15
Ile
(2) lN~Oh~ATION FOR SEQ ID NO:3:
(i) ~QU~N~ r~R~rTR~T,eTICS
'A) LENGTH: 17 amino acids
~B) TYPR: amino acid
C) STR~ ~eS: single
D) TOPOLOGY: linear
(ii) MOTRC~T~ TYPE: protein
(iii) nY~OLn~.lCAL: NO
(iv) ANTI-SENSE: NO
(xi) ~_yu~N.~_ DESCRIPTION: SEQ ID NO:3:
Tyr Ser Leu Asp Arg Ile Ile Arg Asp Leu A~p Arg Val Ile Arg Asp
1 5 10 15
Ile
(2) lN~K~ATION FOR SBQ ID NO:4:
(i) ~_QU~N~ ~T~TERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: ~no acid
(C) STR~N~ NK~S: single
(D) TOPOLOGY: linear
(ii) M~TRC~TT~ TYPE: protein
(iii) nY~O~n~llCAL: NO
(i~) ANTI-SENSE: NO
CA 02216755 1997-09-29
W 096/30405 PCTAUS96/03490
(Xi ) ~yU~N~ D_SCRIPTION: SEQ ID NO:4:
Tyr Ser Leu Arg Arg Ile Ile Arg Arg Leu Arg Arg Val Ile Arg Arg
1 5 10 15
Ile
(2) INFORMATION FOR SFQ ID NO:5:
( i ) ~ KQ v ~ ~R~rTR~T~Ics:
~A) LENGTH: 17 amino acids
B) TYPE: amino acid
C) ST~N~J~:IJNK~S single
~D) TOPOLOGY: linear
(ii) NOTRC~TR TYPE: protein
(iii) nYrO ~CAL: NO
(iv) ANTI-SENSF: NO
(Xi ) ~QU~N~ DESCRIPTION: SEQ ID NO:5:
Tyr Ser Leu Asp Asp Ile Ile A~p Asp Leu Asp Asp Val Ile Asp Asp
1 5 10 15
Ile
(2) INFORNATION FOR SEQ ID NO:6:
(i) ~Qu-~ ~ r~R~rTR~T~cTIcs:
(A) ~ENGTH: 17 A~i no acids
(B) TYPE: amino acid
(C) STR~N~JIC~NICCS E~ingle
(D) TOPOLOGY: linear
(ii) MOT~RC~TR TYPE: protein
(iii) nYrO~ CAL: NO
(iv) ANTI-SENSR: NO
(xi) S~UU~N~ DESCRIPTION: SEQ ID NO:6:
Tyr Ser ~eu Asp Asp Ile Ala Arg Arg Leu Asp Asp Val Ala Arg Arg
1 5 10 15
Leu
( 2 ) IN~ O~ATION FOR SEQ ID NO:7:
(i) ~yu~.~ R~rTRRTSTICS:
(A) LENGTH: 16 A~ no acids
(B) TYPE: A~;no acid
-
CA 022l6755 l997-09-29
W 096/30405 PCTAUS96/03490
- 42 -
(C) ST~Nl ~Kl l~ S single
(D) TOPOLOGY: linear
(ii) MOTRC~TR TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SK~U~NCK DESCRIPTION: SEQ ID NO:7:
Tyr Ser Leu Lys Ala Ile Ala Glu Ala Leu Ly8 Ala Val Ala Glu Ala
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:8:
KU~ R t~T~R~t~TR~TsTIcs
'A; L~NGTH: 17 amino acids
~Bl TYPE: amino acid
C~ STRAN~.~SS: single
~D TOPOLOGY: linear
(ii) ~C~TR TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) ~QUKW~ DESCRIPTION: SEQ ID NO:8:
Tyr Ser Leu Lys Glu Ile Glu Lys Leu Leu Lys Glu Val Leu Glu Ly~
1 5 10 15
Leu
(2) lN~vK~ATION FOR SEQ ID NO:9:
(i) ~uKN~ CHARACTERISTICS:
A) LENGTH: 17 amino acids
B) TYPE: amino acid
C) ST~2~NIlK~ ~NlCpS single
~D) TOPOLOGY: linear
(ii) I .Rc~T.R TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) ~U~._K DESCRIPTION: SEQ ID NO:9:
Tyr Ser Leu Asp ABP Ile Ala Arg Arg Leu Asp Asp Val Ala Arg Arg
l 5 10 15
CA 02216755 1997-09-29
W 096l30405 PCTAUS96/03490
- 43 -
Ala
(2) INFORNATION FOR SFQ ID NO:10:
(i) S~ R rU~ Ku~c~lcs:
(A) LBNGTH: 16 amino acidD
(B) TYPB: Am;no acid
(C) ST~~ K~Nlccs 8ingle
(D) TOPOLOGY: linear
(ii) - RC~-~ TYPB: protein
(iii) AY~O.~lCAL: NO
(iv) ANTI-SENSE: NO
(xi) x~yu~ K L~ U~ ON: SEQ ID NO:10:
Tyr Ser ~eu Lys Ile Ile Ile Glu Ile Leu Lys Ile Val Ile Glu Ile
1 5 10 15
(2) INFORMATION FOR SBQ ID NO:11:
(i) XKyuL_,_K ~U~T.cTICS
A) LFNGTH: 17 amino acids
~B) TYPB: ~mino acid
C) ST~I ~KI ~ K.CS single
~D) TOPOLOGY: linear
(ii) MOT~RC~R TYPE: protein
(iii) ~-AL.LCAL: NO
(iv) ANTI-SFNSE: NO
(xi) ~Kyl ~ DESCRIPTION: SBQ ID NO:11:
Tyr Ser Leu Lys Glu Ile Glu Lys Leu Leu Lys Glu Val Leu Glu Lys
1 5 10 15
Leu
(2) INFORMATION FOR SBQ ID NO:12:
(i) ~Lyu~N~-K ~U~CTR~ T CTICS:
(A) L~NGTH: 16 amino acids
(B) TYPB: amino acid
(C) S'~ K~ '--KSS: single
(D) TOPOLOGY: linear
~ ~ TYPB: protein
(iii) AY~OlA~l~AL: NO
(i~) ~NTI-SBNSE: NO
CA 022167~ 1997-09-29
W O 96/30405 PCTrUS96/03490
- 44 -
(ix) FFAT~RB:
(A) NAML/KEY: Modi~ied-site
(B) LOCATION: 5
(D) OTHER INFORMATION: /label= Aib
/note= "Xaa at location 5 i8 Aibn
(ix) FRAT~RF
(A) NAM~/RFY: Modified-site
(B) LOCATION: 7
(D) OTHBR INFORMATION: /label= Aib
/note= nXaa at location 7 is Aib"
(ix) F AT~RF:
(A) NAMF/R_Y: Modi~ied-site
(B) LO QTION: 9
(D) OTHFR lN~OK~ATION: /label= Aib
/note= "Xaa at location 9 i8 Aib"
(ix) FFATURF:
(A) NAM~/REY: Modi~ied-site
(B) LO QTION: 12
(D) OTHER l~.~OKMATION: /label= Aib
/note= "Xaa at location 12 is Aib"
(ix) FFAT~RF:
(A) NAM_/R_Y: Modified-site
(B) LO QTION: 14
(D) OTHER INFORMATION: /label= Aib
/note= "Xaa at location 14 is Aib"
(ix) FFAT~R_:
(A) NAMF/RFY: Modified-site
(B) LO Q TION: 16
(D) OTHER lN~OK~ATION: /label= Aib
/note= "Xaa at location 16 is Aib"
(Xi ) XLQUL_._L DESCRIPTION: SFQ ID NO:12:
Tyr Ser Leu ~ys Xaa Ile Xaa Glu Xaa Leu Lys Xaa Val Xaa Glu Xaa
1 5 10 15
(2) lN~Oh~ATION FOR SFQ ID NO:13:
(i) SLYUL.._~ ~TFRISTICS:
(A' LFNGTH: 17 amino acids
(B~ TYPE: ~ino acid
(C. ST~N~ SS: single
(D) TOPOLOGY: linear
(ii) MnTRC~TR TYP_: protein
(iii) HYPOTHETICAL: NO
(i~) ANTI-S_NS_: NO
(ix) FFAT~R_:
(A) NAML/R_Y: Modi4ied-site
(B) ~OCATION: 3
CA 02216755 1997-09-29
WO 96130405 PCT/US96~03490
- 45 -
(D) OTHER lN~O~_TION: /label= Nle
/note= "Xaa at location 3 is Nle n
(xi) ~QU~N~ DESCRIPTION: SFQ ID NO:13:
~yr Ser Xaa Asp Asp Ile Ile Arg Arg Leu Asp Anp Val Ile Arg Arg
1 5 10 15
Ile
(2) lN~OkdATION FOR S~Q ID NO:14:
(i) ~yu~N~ CHARACT_RISTICS:
(A) ~ENGTH: 17 amino acids
(B) TYPE: amino acid
(C) S~R~ es: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) ~Y~O-~LlCAL: NO
(iv) ANTI-SENSE: NO
(iX) FEAT~RE
(A) NAME/KEY: Modi~ied-site
(B) LOCATION: 10
(D) OTHER lN~O~nATION: /label= Nle
/note= "Xaa at location 10 iB Nlen
(Xi) ~QU~N~ DESCRIPTION: SEQ ID NO:14:
Tyr Ser Leu Asp Asp Ile Ile Arg Arg Xaa Asp Asp Val Ile Arg Arg
1 5 10 15
Ile
(2) INFORMATION FOR SEQ ID NO:15:
(i) ~il:-~U~NI.;15 ~7~C'TRl2T~:TICS:
A~ LENGTH: 17 ~m; no acids
B TYPB: amino acid
~CJ STP~ CS: single
~D,~ TOPOLOGY: linear
(ii) MOTRC~ TYPE: protein
(iii) ~Y~O ~llCAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAMB/REY: -'; f; e~-site
(B) LOCATION: 13
(D) OTH~R LN~O~TION: /label= Nle
/note= "Xaa at location 13 is Nle n
CA 02216755 1997-09-29
W O 96t30405 PCTrUS96103490
- 4 6 -
(Xi ) 8_yu N~_ DESCRIPTION: SEQ ID NO:15:
Tyr Ser Leu Asp Asp Ile Ile Arg Arg Leu Asp Asp Xaa Ile Arg Arg
Ile
(2) INFOhMATION FOR SEQ ID NO:16:
(i) S_YU_NC_ ~U~rTERISTICS:
~A) L_NGTH: 17 amino acids
B) TYPE: amino acid
C) ST~N~ ~K~ Jr~K! S single
,D) TOPOLOGY: linear
( ii ) -.R~T.R TYPE: protein
(iii) ~lY~OIA~ NO
(iv) ANTI-SENSE: NO
(ix) FEAT~R_:
(A) NAME/REY: Modified-site
(B) LOCATION: 3
(D) OTHER l~.~uK~ATION: /label= Nle
/note= "Xaa at location 3 i~ Nle"
(ix) FEAT~RB:
(A) NAMB/REY: Modified-site
(B) LOCATION: 10
(D) OTHER l~OhnATION: /label= Nle
/note= "Xaa at location 10 i~ Nle"
(xi) ~yu~N~_ DESCRIPTION: SEQ ID NO:16:
Tyr Ser Xaa Asp Asp Ile Ile Arg Arg Xaa Asp Asp Val Ile Arg Arg
l 5 10 15
Ile
(2) INFORMATION FOR SEQ ID NO:17:
(i) ~_QU~N~ r~TR~TSTICS:
(A) LENGTH: 18 ~;no acids
(B) TYPE: ~ino acid
(C) STT~Nl~K~JNKcs single
(D) TOPO~OGY: linear
(ii) M~T~RC~-R TYPE: protein
(iii) ~Y~O~ lCAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQ~ENC_ nR~C~TPTION: SEQ ID NO:17:
CA 02216755 1997-09-29
W ~g6l30405 PCT~US96/03490
- 47 -
Cy~ Tyr Ser Leu Asp Asp Ile Ile Arg Arg L~u Asp Asp Val Ile Arg
1 5 10 15
Arg Ile
(2) INFORMATION FOR SEQ ID NO:18:
(i) XK5~UlsN~;ls ~T~rTERISTICS:
'A) LENGTH: 17 _mino acids
B) TYPE: ~ino acid
~C) ST~ N,J~, Kqs 13ingle
.D) TOPOLOGY: linear
(ii) MOtRC~TR TYPE: protein
(iii) ~Y~O~ CAL: NO
(iv) ANTI-SENSE: NO
(Xi) ~U~N~ DES~KI~lO_.: SEQ ID NO:18:
Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Ile Xaa Xaa Leu Xaa Xaa Xaa
1 5 10 15
Val
(2) INFORMATION FOR SEQ ID NO:l9:
(i) S~UU~N~-~ CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: ~m;no acid
(C) ST~N~ ~~. K. S single
(D) TOPOLOGY: linear
(ii) MOT-~Cu~T~ TYPE: protQin
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) ~UuKN~K DESCRIPTION: SEQ ID NO:l9:
Xaa Xaa Ile Xaa Xaa Leu Xaa Xaa Xaa Ile Xaa Xaa Leu Xaa Xaa Xaa
1 5 10 15
Val
-
(2) lN~OkdATION FOR SEQ ID NO:20:
(i) ~KyuL~ ~ ~TERISTICS:
~A) ~ENGTH: 17 _mino Acids
B) TYPE: ~;no acid
C) S~r~N~ ~K- ~ K~S single
~D) TOPOLOGY: linear
CA 022l6755 1997-09-29
W 096/30405 PCT~US96/03490
- 48 -
(ii) ~, ~RC~T-R TYPE: protein
(iii) HY~G~ lCAL: NO
(iv) ANTI-SENSE: NO
(Xi) ~U~N~ D_SCRIPTION: SEQ ID NO:20:
Tyr Ser Ile Asp A~p Leu Ile Arg Arg Ile Asp Asp Leu Ile Arg Arg
1 5 10 15
Val
(2) INFORMATION FOR S_Q ID NO:21:
(i) SEQ~ENC_ rU~T~rTF~TCTICS:
'A) ~ENGTH: 16 A~;no acids
B) TYPE: ~mino acid
C) ST~N~JK~JNK.C:S single
~D) TOPO~OGY: linear
(ii) MOTKC~TTK TYPE: protein
(iii) nY~ ~lCAL: NO
(i~) ANTI-SENSE: NO
(xi) ~yu~N~ DESCRIPTION: SEQ ID NO:21:
Tyr Ser Ile Asp Asp Ile Ile Arg Arg Ile Asp Asp Ile Arg Arg Ile
1 5 10 15
