Note: Descriptions are shown in the official language in which they were submitted.
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SYN'fHETIC SOMATOSTATIN IMMUNOGEN
FOR GROWTH PROMOTION IN FARM ANIMALS
FIEhD OF THE INVENTION
S This invention relates to a peptide composition that
is useful as an immunoge:n for growth promotion in farm
animals. The immunogenic peptides of the subject composition
contain helper T cell ep.itopes (Th) which comprise multiple
class II MHC binding motifs and have somatostatin at either
the C- or N- terminus. 'the peptides, optionally, contain an
invasin domain which act:; as a general immune stimulator. The
helper T cell epitopes and the invasin domain enable the
immune response against l~he somatostatin self-peptide.
BACKGROUND OF THE INVENTION
1S Recent understanding of neuro-endocrine and hormonal
factors involved in growth, together with rapid advances in
biotechnology, have provided potential avenues for the
improvement of growth rage in a variety of farm animal
species. A method for animal growth promotion that modifies
the regulatory effects of growth hormones through
immunoneutralization is ~~ valuable alternative to the present
methods which use antibiotics, anabolic steroids, and growth
hormones as growth promoters. This application of antibiotics
to animal husbandry is believed to be a driving force for the
2S selection of antibiotic resistance in certain pathogenic
bacterial species and has resulted in adverse consequences in
the control of human infectious disease (Witte, Science, 1998;
279:996). The application of anabolic steroids promotes
growth in farm animals, but is unpopular among consumers.
Steroid use is constrained in Europe both by legislation and
by public opinion (Buttery and Dawson, Proc Nutr Soc, 1990;
49:459). Recombinant DNA technology has enabled the production
of metabolic hormones in large quantity for direct
administration to animals. Such application can promote
3S growth. It is, however, just one of the steps that need to be
taken if the somatotrophic hormones are to be used as a means
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of improving animal performance.
The principal somatotrophic hormones are:
somatotropin (growth hormone, GH), somatomedin-C (insulin-like
growth factor 1, IGF-1), somatocrinin (GH releasing factor,
GRF) and somatostatin (G1~ release inhibiting factor). The
major potential applicat:LOns for the somatotrophic hormones
are in growth and lactation, however the hormonal control of
these phenomena relies upon a complex interaction between many
different hormones (Spenc:er, Livest Prod Sci, 1985, 12:31).
Thus, simple application of a single hormone may not be
sufficient to enhance productivity.
It appears that: GH plays two distinct roles. It has
a positive effect in stimulating an increase in muscle protein
synthesis (most, if not a.ll, of which is mediated by IGF-1)
and a potent catabolic effect by its ability to breakdown
fats. The overall effects are an increase in the lean content
of the carcass and a decrease in carcass fat, often an
increase in growth, and universally an improvement in food
conversion efficiency (Buttery and Dawson, Proc Nutr Soc,
1990; 49:459; and, Spencer, Reprod Nutr Develop, 1987;
27 (2B) :581) .
The effects on growth of GH administration are not
reliable for its use in practice; probably because
administration of exogenous hormones ignores the sensitive
interrelationship between various hormones and ignores the
possible effect of elevation of plasma levels on receptor
populations. Effective application of GH requires frequent
injections so as to provide a continuous physiologically
effective serum level of :hormone. This results in increased
risk of upsetting these balanced relationships and causing
adverse effects.
Thus, although pure preparations of growth hormones
can be made in large quantities by recombinant DNA technology,
this technology does not provide the modulated mechanism for
delivery that is needed for the desired effect on growth.
Moreover, the use of recombinant growth hormone in food
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animals, most recently in dairy cattle, has resulted in
widespread consumer opposition and regulatory obstacles. The
use of growth hormone to promote lean deposition in ruminants
and other farm animals has not received regulatory approval
within the European Economic Community. The regulatory and
political situation is similar for the application of IGF-1 or
GRF to promote growth in farm animal species.
As an alternative to increasing the levels of growth
stimulating hormones it may prove equally, or even more,
effective to remove endogenous growth inhibitors. The major
inhibitor of total somatic growth is somatostatin.
Somatostatin is a cyclic peptide of fourteen amino acids and
its structure is conserved across species. It is synthesized
as a ninety-two amino acid prosomatostatin molecule from which
six peptides including somatostatin itself and a twenty-eight
amino acid form of somatc:~tatin are known to be derived
(Reichlin, J hab Clin Meci, ~-987; 109:320). Somatostatin
inhibits the release of rnany gastro-intestinal hormones as
well as inhibits release of GH, insulin, thyroid hormones,
thereby affecting both the ability of the animal to absorb
nutrients and its subseauent ability to direct these nutrients
into tissue growth.
The use of a somatostatin antagonist has been found
to stimulate growth in .rat=s (Spencer et al., Life Sci,1985,
37:27), but this kind of treatment also suffers from the
drawbacks of GH, IGF-1 and GRF in that it requires daily
injections. A practical alternative is the use of the immune
response to induce immunc~neutralization of somatostatin. The
use of the immune system to promote growth may be more
acceptable to consumers a.nd regulatory agencies than direct
administration of hormones or synthetic steroids. The
immunoneutralization of somatostatin by vaccine was first
explored in sheep by Spencer et al. (Livest prod Sci, 1983,
10:469) .
In a preliminary study using twin St. Kilda lambs,
active immunization against somatostatin resulted in the
treated lambs growing at 176°s of the rate of the control lambs
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(Spencer et al., Anim Prod,1981, 32:376). Subsequent studies
have been unable to reproduce this figure, but an improvement
of 15-20% in growth rate is more usual. The somatostatin
molecule is the same in all farm animal species, and it has
S now been shown that active immunization against somatostatin
can stimulate growth in commercially important breeds of sheep
(Spencer et al., Livest Prod Sci, 1983, 10:25; Laarveld et
al., Can J Anim Sci, 1986, 66:77), cattle (Lawrence et al., J
Anim Sci, 1986, 63: (Supply 215), pigs and chickens (Spencer!
et al., Dom Anim Endocr, 1986, 3:55). A summary of successful
examples demonstrating e_Efective immunization against
somatostatin for farm an_Lmals is shown in Table 1.
In addition to stimulating growth rate and leading
to a 20% reduction in rearing time (Spencer, Reprod Nutr
Develop, 1987; 27(2B):581), active immunization against
somatostatin also has a beneficial effect on food conversion
efficiency. In addition t;o the saving on food by virtue of
more rapid growth, the animals actually utilize their food
more efficiently during t=he growing period (Spencer et al.,
Livest Prod Sci, 1983, 10:969), at least partly as a result of
changes in gut motility (Fadlalla et al., J Anim Sci, 1985,
61:234; Faichney et al., Can J Anirn Sci, 1985, 64(Suppl) 93).
The treatment does not have any marked effect on carcass
composition (Spencer et al., 1983, ibid.) but there are
indications that, when killed at equal weights, treated
animals may be leaner. Taken all experimental data together,
active immunization appears to be a powerful, safe, and
effective tool to enhance growth (Spencer, Dom Anim Endocr,
1986, 3:55).
Several immunogenic forms of somatostatin have been
designed and tested as reported in the literature. For
example, somatostatin has been conjugated with protein
carriers to enhance immunopotency. However, protein carriers
are too expensive for economical use in farm animals.
Further, effective immunization with somatostatin depends on
the conjugation site between somatostatin and the carrier.
Most if not all of the so:matostatin protein carrier conjugates
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were prepared by glutaraldehyde coupling, employing cross
linkage between the lysine residues present on somatostatin
and the carrier protein. The two lysines on somatostatin
available for coupling reside within a 12-mer functional loop
thus may result in significant loss of the native somatostatin
structure and reduction in crossreactivity to somatostatin
when such conjugates are used as vaccines.
Moreover, protein linkage to somatostatin is
problematic because the majority of immune responses are
directed to the carrier :rather than to somatostatin (the mass
of the carrier molecule(a) is much greater than that of
somatostatin) and immunization with hapten carrier conjugates
frequently leads to carrier-induced immune suppression (Schutz
et al., J Immunol, 1985, 135:2319). Accordingly, an immune
enhancer that is suitable for live stock use, inexpensive and
capable of stimulating an early and strong immune response to
somatostatin has been sou<~ht. This immune enhancer should
avoid carrier-induced suppression.
An important far_tor affecting immunogenicity of a
synthetic peptide for an somatostatin immunogen is its
presentation to the immure system by T helper cell epitopes.
Formerly, those were provided by a carrier protein with the
concomitant disadvantage°_> discussed above. These may also be
supplied as hybrid polypeptides by recombinant DNA expression
systems (Riggs, US 4,812,554; US 4,563,924; and Xu et al.,
Science in China (Series B), 1994; 37:1234). These may also
be more simply and less expensively supplied by a synthetic
peptide comprising the target hapten B cell site and T-helper
epitopes (Th) appropriate for the host. Such peptides react
with helper T-cell receptors and the class II MHC molecules,
in addition to antibody binding sites (Babbitt et al., Nature,
1985, 317:359) and thus stimulate a tightly site-specific
antibody response to the target antibody binding site (target
site). A wholly synthetic peptide immunogen for somatostatin
would enjoy the following advantages over carrier conjugates
and recombinant polypeptides: the product is chemically
defined for easy quality control, it is stable, no elaborate
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downstream processing is needed, no elaborate production plant
is required, and the engendered immune response is site-
specific so that undesirable responses such as epitopic
suppression are avoided.
S Immunogenicity of synthetic somatostatin immunogens
can be optimized by (1) combining somatostatin with selected
promiscuous Th sites to which the majority of a population are
responsive; (2) combining samatostatin with an enlarged
repertoire of Th through combinatorial chemistry and thereby
accommodate to the variable immune responsiveness of a
population, and (3) the stabilization of a desirable
conformational. feature o:E somatostatin by cyclic constraint.
Epitopes termed promiscuous Th evoke efficient T
cell help and can be combined with B cell epitopes that by
themselves are poorly imrnunogenic to provide potent
immunogens. Well-designed promiscuous Th/B cell epitope
chimeric peptides are capable of eliciting Th responses and
resultant antibody responses in most members of a genetically
diverse population expre:>sing diverse MHC haplotypes.
Promiscuous Th can be provided by specific sequences derived
from potent immunogens including measles virus F protein and
hepatitis B virus surface antigen. Many known promiscuous Th
have been shown to be effective in potentiating a poorly
immunogenic peptide corresponding to the decapeptide hormone
LHRH (US 5, 759, 551 ) .
Potent Th epitopes range in size from approximately
15-30 amino acid residue; in length, often share common
structural features, and may contain specific landmark
sequences. For example, a common feature is amphipathic
helices, which are alpha-helical structures with hydrophobic
amino acid residues dominating one face of the helix and with
charged and polar residues dominating the surrounding faces
(Cease et al., Proc Natl Acad Sci USA, 1987; 84: 4249-4253).
Th epitopes frequently contain additional primary amino acid
patterns such as a Gly or charged residue followed by two to
three hydrophobic residues, followed in turn by a charged or
polar residue. This pattern defines what are called Rothbard
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sequences. Also, Th epitopes often obey the 1, 4, S, 8 rule,
where a positively charged residue is followed by hydrophobic
residues at the fourth, :E.ifth and eighth positions after the
charged residue. Since all of these structures are composed
of common hydrophobic, charged and polar amino acids, each
structure can exist simu:~Ltaneously within a single Th epitope
(Partidos et al., J Gen ~~irol, 1991; 72:1293). Most, if not
all, of the promiscuous T cell epitopes fit at least one of
the periodicities described above. These features may be
incorporated into the den>igns of idealized artificial Th
sites, including combinatorial Th epitopes. In regard to the
design of combinatorial fh sites, lists of variable positions
and preferred amino acids are available for MHC-binding motifs
(Meister et al., Vaccine, 1. 1995; 13:581-591); and, a method
for producing combinatorial Th has been disclosed for library
peptides termed structured synthetic antigen library or SSAL
(Wang et al., WO 95/11998). Thus, the 1,4,5,8 rule can be
applied together with combinatorial MHC-binding motifs in the
assignment of positions for the invariant and degenerate sites
of an SSAL and for the selection of residues for these sites,
so as to vastly enlarge the range of immune responsiveness to
an artificial Th (WO 95/119981.
Peptide immunogens are generally more flexible than
proteins and tend not to retain any preferred structure.
Therefore it is useful t:o stabilize a peptide immunogen by the
introduction of cyclic constraints. A correctly cyclized
peptide immunogen can mimic and preserve the conformation of
the targeted epitope and thereby evoke antibodies with cross-
reactivities on that site on the authentic molecule (Moore,
Chapter 2 in Synthetic Peptides A User's guide, ed Grant, WH
Freeman and Company: New '.fork, 1992, pp 63-67).
Peptide immunogens that have been designed with the
peptide technologies and peptide design elements discussed
above, i.e., design of promiscuous potent Th epitopes, Th SSAL
combinatorial peptides, and cyclic constraint, are the basis
for effective synthetic somatostatin immunogens. Such
peptides are preferred for. their presentation of the
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somatostatin by optimized positioning and cyclization, and for
broadly reactive Th responsiveness. Hence, it has been found
that peptides containing particular structural arrangements of
a Th epitope alone or linked to a general immune enhancer,
e.g., an invasin domain (US 5,759,551) and somatostatin in its
intact form where the functional site within the 12 mer loop
structure is not disturbed (as target antigen), are effective
in stimulating the production of antibodies against
somatostatin.
SUMMARY OF THE INVENTION
The present invention relates to an immunogenic
peptide composition comprising synthetic peptides, which are
capable of inducing antibodies against somatostatin that lead
to the suppression of sornatostatin levels, promote growth and
improve food conversion efficiency in farm animals. In
particular, peptides of t:his invention have a Th epitope
linked to a carboxyl- or amino- terminal somatostatin (SEQ ID
N0:1) or a peptide analog of somatostatin. Optionally, the
peptides have an invasin domain (SEQ ID N0:2) as a general
immune stimulator. These peptides are effective as
immunogens, capable of increasing serum growth hormone level
in immunized hosts to promote daily weight gain in farm
animals.
Another aspect of this invention provides an
antigenic composition comprising an immunologically effective
amount of a peptide composition in accordance with this
invention and one or more pharmaceutically acceptable vaccine
formulations and instructions for dosage such that
immunotherapeutic antibodies directed against the targeted
somatostatin site are generated. Such peptide compositions
are useful for growth promotion in farm animals.
A further aspect of the invention relates to a
method for increasing circulating somatotropic hormone levels
in a mammal by administering one or more of the subject
peptides to the mammal fc>r a time and under conditions
sufficient to induce functional antibodies directed against
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said somatostatin.
Yet another aspect of the invention relates to an
immunogenic synthetic peptide of about 30 to about 90 amino
acids which contains a helper T cell (Th) epitope,
somatostatin (SEQ ID NO:1) or a peptide analog of
somatostatin, spacers to separate the immunogenic domains and
optionally general immunostimulatory sites, for example, an
invasin domain (SEQ ID N0:2). These three immunogenic domain
elements of the peptide and spacer can be covalently joined in
any order provided that either the immunoreactivity of the
peptide hapten is subst.a:ntially preserved or that
~immunoreactivity to the somatostatin self-peptide can be
generated.
DETAILED DE:>CRIPTION OF THE INVENTION
This invention .is directed to a novel peptide
composition for the generation of high titer polycional
antibodies with specificity for somatostatin. The high site-
specificity of the peptide composition minimizes the
generation of antibodies that are directed to irrelevant sites
on carrier proteins. Therefore, the invention is further
directed to an effective method for the growth promotion in
farm animals.
Somatostatin (:>EQ ID NO:1) is a short cyclized
peptide hormone which, by itself is non-immunogenic, more so
for being a self-antigen. This short peptide can be
immunopotentiated by chemical coupling to a carrier protein,
for example, keyhole limpet hemocyanin (KLH) or by fusion to a
carrier polypeptide through recombinant DNA expression, for
example, hepatitis B surface antigen. Major deficiencies of
such "somatostatin-carrier" vaccines is that the largest
portion of antibodies generated by the combinations are the
non-functional antibodies directed against the carrier protein
or polypeptide and the potential for epitopic suppression.
The immunogens of the present invention are wholly synthetic
peptides which minimize the generation of irrelevant
antibodies to elicit an immune response mare focused to
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somatostatin. However, because somatostatin is a non-
immunogenic T cell-dependent antigen, it is completely
dependent on extrinsic Th epitopes for immunogenicity. These
are provided for the peptides of the invention as covalently
linked promiscuous Th epi.topes. The immunogens of the
invention are all of site-specific immunoreactivity to provide
for effective growth promotion in livestock.
Specific examples are provided in the present
invention as embodiments of the peptides of the invention.
These examples provide for the linkage of synthetic
immunostimulatory elements to the somatostatin peptide such
that potent somatostatin-reactive antibodies are generated, in
a genetically diverse host population. These antibodies, in
turn, lead to inhibition of the function of somatostatin, thus
resulting in an effective growth promotion for livestock.
For active immunization, the term "immunogen"
referred to herein relates to a peptide composition which is
capable of inducing antibcdies against somatostatin, leading
to inhibition or suppression of somatostatin levels in a
mammal. The peptide composition of the present invention
includes peptides which contain promiscuous helper T cell
epitopes (Th epitopes). The peptides are covalently attached
to the somatostatin peptide, with a spacer (e.g. Gly-Gly), so
as to be adjacent to either the N- or C-terminus of the target
somatostatin peptide, in order to evoke efficient antibody
responses. The immunogen may also comprise a generalized
immunostimulatory element, for example, a domain of an invasin
protein from the bacteria Yersinia spp (Brett et al., Eur J
.Immunol, 1993, 23: 1608-1619) (SEQ ID N0:2). The invasin
domain is attached through a spacer to a Th peptide.
The peptides of this invention can be represented by
the formulas:
H2N- (A) n- (somatostatin peptide) - (B) o- (Th) m-X
or
HzN- (A) n- (Th) m- (:B) o- ( somatostatin peptide) -X
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wherein
HZN is the N-terminal a-NHz of the peptide conjugate,
each A is independently an amino acid or a general
immunostimulatory sequence;
each B is chosen from the group consisting of amino
acids, -NHCH (X) CHZSCHZCO-~, -NHCH (X) CHZSCHZCO (s-N) Lys-,
-NHCH (X) CH2S-succinimidy:L (e-N) Lys-, and -NHCH (X) CH2S-
(succinimidyl)-;
each Th is independently a sequence of amino acids
that comprises a helper T cell epitope, or an immune enhancing
analog or segment thereof;
somatostatin peptide is somatostatin or a
crossreactive,and immuno.iogically functional analog thereof;
X is an amino acid a-COON or a-CONHz;
n is from i to .about 10;
m is from 1 to about 4; and
o is from C to ,shout 10.
The peptide imrnunogen of the present invention
comprises from about 20 t=o about 1C0 amino acid residues,
preferably from about 25 fo about 80 amino acid residues and
more preferably from about 25 to about 65 amino acid residues.
When A is an amino acid or a general
immunostimulatory element:, e.g., Inv, it can be covalently
linked to either the N-terminal of the peptide immunogen as
shown by the formulas, on to the C-terminal (not shown).
When A is an amino acid, it can be any non-naturally
occurring or any naturally occurring amino acid. Non-
naturally occurring amino acids include, but are not limited
to, t3-alanine, ornithine, norleucine, norvaline,
hydroxyproline, thyroxine~, y-amino butyric acid, homoserine,
citruiline and the like. Naturally-occurring amino acids
include alanine, arginine, asparagine, aspartic acid,
cysteine, glutamic acid, glutamine, glycine, histidine,
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isoleucine, leucine, lysine, methionine, phenylalanine,
proline, serine, threonine, tryptophan, tyrosine and valine.
Moreover, when m is greater than one, and two or more of the A
groups are amino acids, then each amino acid may be
independently the same or different.
When A is an invasin domain, it is an immune
stimulatory epitope from the invasin protein of a Yersinia
species. This immune stimulatory property results from the
capability of this invasin domain to interact with the 131
integrin molecules present on T cells, particularly activated
immune or memory T cells. The specific sequence for an
invasin domain found to .interact with the (31 integrins has
been described by Brett et al (Eur J Immunol, 1993). A
preferred embodiment of ~~he invasin domain (Inv) for linkage
to a promiscuous Th epitope has been previously described in
US 5,759,551 and is incorporated herein by reference. The
said Inv domain has the sequence:
Thr-Ala-Lys-Ser-Lys-Lys-Phe-Pro-Ser-Tyr-Thr-Ala-Thr-Tyr-Gln-Phe
(SEQ ID N0:2)
or is an immune stimulatory homologue thereof from the
corresponding region in another Yersinia species invasin
protein. Such homologues thus may contain substitutions,
deletions or insertions of amino acid residues to accommodate
strain to strain variation, provided that the homologues
retain immune stimulatory properties.
In one embodiment, n is 1 and A is oc-NH2. In
another embodiment, n is 9 and A is a-NHz, an invasin domain
(Inv), glycine and glycine, in that order.
B is a spacer and is an amino acid which can be
naturally occurring or the non-naturally occurring amino acids
as described above. Each B is independently the same or
different.. The amino acids of B can also provide a spacer,
e.g., Gly-Gly, between th.e promiscuous Th epitope and the
somatostatin peptide (e.g., SEQ ID NO:1) and crossreactive and
functional immunological analogs thereof. In addition to
physically separating the Th epitope from the B cell epitope,
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i.e., the somatostatin peptide and immunological analogs
thereof, the Gly-Gly spacer can disrupt any artifactual
secondary structures created by the joining of the Th epitope
with the somatostatin peptide and crossreactive and functional
immunological analogs thereof and thereby eliminate
interference between the Th and/or B cell responses. The
amino acids of B can also form a spacer which acts as a
flexible hinge that enhances separation of the Th and IgE
domains. Examples of sequences encoding flexible hinges are
found in the immunoglobulin heavy chain hinge region.
Flexible hinge sequences are often proline rich. One
particularly useful flexible hinge is provided by the sequence
Pro-Pro-Xaa-Pro-Xaa-Pro (SEQ ID N0:3), where Xaa is any amino
acid, and preferably aspa.rtic acid. The conformational
separation provided by the amino acids of B permits more
efficient interactions between the presented peptide immunogen
and the appropriate Th ce:lls and B cells and thus enhances the
immune responses to the Th epitope and the antibody-eliciting
epitope and their crossreactive and functional immunological
analogs thereof.
Th is a sequence of amino acids (natural or non-
natural amino acids) that: comprises a Th epitope. A Th
epitope can consist of 3 continuous or discontinuous epitope.
Hence not every amino acid of Th is necessarily part of the
epitope. Accordingly, Th. epitopes, including analogs and
segments of Th epitopes, are capable of enhancing or
stimulating an immune response to the somatostatin peptide and
immunological analogs thereof. Th epitopes that are
immunodominant and promiscuous are highly and broadly reactive
in animal and human populations with widely divergent MHC
types (Partidos et al., 1991; US 5,759,551). The Th domain of
the subject peptides has from about 10 to about 50 amino acids
and preferably from about 10 to about 30 amino acids. When
multiple Th epitopes are present (i.e., m ? 2), then each Th
epitope is independently the same or different. Th segments
are contiguous portions of a Th epitope that are sufficient to
enhance or stimulate an i~~nmune response to the somatostatin
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peptide (SEQ ID NO:1) and immunological analogs thereof.
Th epitopes of the present invention include those
derived from foreign pathogens including but not limited to,
as examples, hepatitis H. surface and core antigen helper T
cell epitopes (HBS Th and HBO Th), pertussis toxin helper T cell
epitopes (PT Th), tetanus toxin helper T cell epitopes (TT
Th), measles virus F protein helper T cell epitopes (MVF Th),
Chlamydia trachomatis major outer membrane protein helper T
cell epitopes (CT Th), diphtheria toxin helper T cell epitopes
(DT Th), Plasmodium falciparum circumsporozoite helper T cell
epitopes (PF Th), Schistosoma mansoni triose phosphate
isomerase helper T cell epitopes (SM Th), and Escherichia coli
TraT helper T cell epitopes (TraT Th). The pathogen-derived
Th selected here as representative examples of promiscuous Th
were listed as SEQ ID NOS:2-9 and 42-52 in US 5,759,551; as CT
Th P11 in Stagg et al., Immunology, 1993; 79;1-9; and as HBc
peptide 50-69 in Ferrari et al., J Clin Invest, 1991; 88: 214-
222; and incorporated herein by reference. Further, Th
epitopes include idealized artificial Th (e. g., SEQ ID NOS:14)
and artificial SSAL Th (~~.g., SEQ ID NOS:7,30,31). Peptides
comprising SSAL Th are produced simultaneously in a single
solid-phase peptide synthesis in tandem with somatostatin and
other sequences. Th sit~as also include functional
immunological analogs. Functional Th analogs include immune-
enhancing analogs, cross:reactive analogs and segments of any
of these Th epitopes. Functional Th analogs further include
conservative substitutions, additions, deletions and
insertions of from one to about 10 amino acid residues in the
Th epitope which do not f~ssentially modify the Th-stimulating
function of the Th epitope.
The synthetic peptides of this invention, as
described by the formulaa
(A)n-(Th)m-(B)o-(somatostatin peptide)
or
(A) n- (somatostatin peptide) - (B) o- (Th) m,
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have the Th epitope covalently attached through spacer B to
either the N terminus or C terminus of the somatostatin
peptide and crossreactive and functional immunological analogs
thereof.
Crossreactive and functional immunological analogs
of the somatostatin peptide (e.g., SEQ ID N0:1) according to
the invention, may further comprise conservative
substitutions, additions, deletions, or insertions of from one
to about four amino acid residues provided that the peptide
analogs are capable of e:Liciting immune responses
crossreactive with the somatostatin peptides. The
conservative substitutions, additions, and insertions can be
accomplished with natura:L or non-natural amino acids as
defined herein.
Preferred peptide immunogens of this invention are
the peptides containing t:he somatostatin peptides or
crossreactive and functic;nal immunological analogs thereof; a
spacer (e.g., Gly-Gly); a Th epitope that is an HBS Th (SEQ ID
NO : 15 ) , HB~ Th ( SEQ I D NO : 4 ) , MVF Th ( SEQ I D NOS : 21, 2 9 ) , PT
Th
(SEQ ID N0:5), TT Th (SEQ ID N0:5); CT Th (SEQ ID N0:27), DT
Th (SEQ ID N0:28), an artificial Th (e.g., SEQ ID
NOS:7,14,30,31) or an analogue thereof; and, optionally, an
Inv domain (SEQ ID N0:2) or analog thereof.
Peptide compositions which contain cocktails of the subject peptide
immunogens with riwo or more of the Th epitopes may enhance immunoef ficacy in
a broader population and thus provide an improved immune
response to the somatostatin peptide.
The peptide immunogens of this invention can be made
by chemical synthesis methods which are well known to the
ordinarily skilled artisan. See, for example, Fields et al.,
Chapter 3 in Synthetic Peptides: A User's Guide, ed. Grant, W.
H. Freeman & Co., New York, NY, 1992, p. 77. Hence, peptides
can be synthesized using the automated Merrifield techniques
of solid phase synthesis 'with the a-NHZ protected by either t-
Boc or F-moc chemistry using side chain protected amino acids
on, for example, an Applied Biosystems Peptide Synthesizer
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Model 430A or 431. Preparation of peptide constructs
comprising SSALs far Th e;pitopes can be accomplished by
providing a mixture of alternative amino acids for coupling at
a given variable position.
After complete assembly of the desired peptide
immunogen, the resin is treated according to standard
procedures to cleave the peptide from the resin and deblock
the functional groups on the amino acid side chains. The free
peptide is purified by HPLC and characterized biochemically,
for example, by amino acid analysis or by sequencing.
Purification and characterization methods for peptides are
well known to one of ordinary skill in the art.
The subject immunogen may also be polymerized.
Polymerization can be accomplished for example by reaction
between glutaraldehyde and the -NH2 groups of the lysine
residues using routine methodology. By another method, the
synthetic
"A-Th-spacer-(somatostatin peptide)"
or
"(somatostatin peptide)-spacer-(Th)m-A"
immunogen can be polymerized or co-polymerized by utilization
of an additional cysteine added to the N-terminus of the
synthetic "A-Th-spacer-(somatostatin peptide) or
"(somatostatin peptide)-spacer-(Th)m-(A)n" immunogen. The
subject immunogen may also be prepared as a branched polymer
through synthesis of the desired peptide construct directly
onto a branched poly-lysyl core resin (Wang, et al., Science,
1991; 254:285-288).
Alternatively, the longer synthetic peptide
immunogens can be synthesized by well known recombinant DNA
techniques. Any standard manual on DNA technology provides
detailed protocols to produce the peptides of the invention.
To construct a gene encoding a peptide of this invention, the
amino acid sequence is reverse translated into a nucleic acid
sequence, and preferably using optimized codon usage for the
organism in which the gene will be expressed. Next, a
synthetic gene is made, typically by synthesizing overlapping
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oligonucleotides which encode the peptide and any regulatory
elements, if necessary. 'rhe synthetic gene is inserted in a
suitable cloning vector and recombinants are obtained and
characterized. The peptide is then expressed under suitable
conditions appropriate for the selected expression system and
host. The peptide is purified and characterized by standard
methods.
The efficacy of the peptide composition of the
present invention can be ~~stabiished by injecting an animal,
for example, rats, with an immunogenic composition comprising
peptides of the invention, e.g., SEQ ID NOS:8-13, 16-20, 22
followed by monitoring th~~ humoral immune response to the
somatostatin and crossrea~~ti.ve and functional immunological
homologues thereof, as detailed in the Examples.
Another aspect of this invention provides a peptide
composition comprising an immunologically effective amount of
one or more of the peptide immunogens of this invention in a
pharmaceutically acceptable delivery system. Accordingly, the
subject peptides can be formulated as a peptide composition
using adjuvants, pharmaceutically-acceptable carriers or other
ingredients routinely provided in peptide compositions.
Among the ingredients that can be used in this invention are
adjuvants or emulsifiers including alum, incomplete Freund's
adjuvant, liposyn, saponin, squalene, L121, emulsigen
monophosphyryl lipid A (MPL), QS21, ISA51, ISA35, ISA206 and
ISA 720 as well as the other efficacious adjuvants and
emulsifiers. The formulations include formulations for
immediate release and/or for sustained release, and induction
of systemic immunity, which may be accomplished by, for
example, immunogen entrapment by or coadministration with
microparticles. Such formulations are readily determined by
one of ordinary skill in the art. The present immunogens can
be administered by any convenient route including
subcutaneous, oral, intramuscular, or other parenteral or
enteral route. Similarly the immunogens can be administered
as a single dose or multiple doses. Immunization schedules
are readily determined by the ordinarily skilled artisan.
SUBSTtTUTE SHEET (RULE 25)
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The peptide composition of the instant invention
contain an effective amount of one or more of the peptide
immunogens of the present invention and a pharmaceutically
acceptable carrier. Such a composition in a suitable dosage
unit form generally contains about 0.5 ~g to about 1 mg of the
immunogen per kg body weight. When delivered in multiple
doses, it may be conveniently divided into an appropriate
amount per dosage unit form. For example, an initial dose,
e.g. 0.2-2.5 mg; preferably 1 mg, of immunogen represented as
a peptide composition of the present invention, is to be
administered by injection, preferably intramuscularly,
followed by repeat (booster) doses. Dosage will depend on the
age, weight and general health of the animal as is well known
in the vaccine and therapeutic arts.
The immune response to synthetic somatostatin
peptide immunogens can be improved by delivery through
entrapment in or on biodegradable microparticles of the type
described by O'Hagan et al. (Vaccine, 1991; 9: 768-771). The
immunogens can be encapsulated with or without an adjuvant in
biodegradable microparticles, to potentiate immune responses,
and to provide time-controlled release for sustained or
periodic responses, and for oral administration, (0'Hagan et
al, 1991; and, Eldridge et al., 1991; 28: 287-294).
Specific peptide immunogens and compositions are
provided in the following examples to illustrate the
invention. These example; are for purpose of illustration
only, and are not to be construed as limiting the scope of the
invention in any manner.
~'v~MDT.F' l
TYPICAL METHODS TO SYNTHESIZE
SOMATOSTP,TIN PEPTIDE CONSTRUCTS
Peptides listed in Tables 2 and 3 were synthesized
individually by the Merrifield solid-phase synthesis technique
on Applied Biosystems automated peptide synthesizers (Models
430, 431 and 433A) using Fmoc chemistry. Preparation of
peptide constructs compr~_sing structured synthetic antigen
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libraries (SSALs), e.g., artificial Th site termed "1,4,9
PALINDROMIC" (SEQ ID N0:7), can be accomplished by providing a
mixture of the desired amino acids for chemical coupling at a
given position as specified in the design. After complete
assembly of the desired peptide, the resin was treated
according to standard procedure using trifluoroacetic acid to
cleave the peptide from l~he resin and deblock the protecting
groups on the amino acid side chains. For cyclic peptide, the
cleaved peptide was dissolved in 15% DMSO in water for 48 hrs
to facilitate intradisulfide bond formation between cysteines.
The cleaved, extracted and washed peptides were
purified by HPLC and chai:acterized by mass spectrometry and
reverse phase HPLC.
Peptides marked by "b" in the peptide code column
were synthesized as target. antigenic peptides in tandem with
Th sites as shown. Th si.tes used include, for example, the
HBs Th taken from hepatitis B virus (SEQ ID N0:15), and the
novel artificial Th site termed "1,4,9 PALINDROMIC" (SEQ ID
N0:7). Peptides marked !=>y "c" are variants of the "b"
constructs synthesized in tandem with the Inv domain
immunostimulatory peptide (SEQ ID N0:2). Peptides marked by
"d" are the reversal of the "b" constructs (e. g.,
somatostatin-Th) and peptides marked by "e" are the reversal
of the "c" constructs (e.g., somatostatin-Th-Inv). The "b",
"c", "d" and "e" constructs were synthesized with gly-gly
spacers for separation of the target antigenic site from the
Th site, and separation of the Th from the Inv
immunostimulatory site.
EXAMPLE 2
TYPICAL METHODS TO EVALUATE
IMMUNOGENICITY OF SOMATOSTATIN PEPTIDES
Somatostatin Peptide immunogens (e. g., SEQ ID NOS:B-
13,16-20,22 and 24 as shown in Tables 2 and 3) were evaluated
on groups of 4 or 5 rats as specified by the experimental
immunization protocol outlined below and by serological assays
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for determination of immunogenicity.
Standard Experimental Design:
Immunogens: (1) individual peptide immunogen; or
(2) mixtures comprising equal molar peptide
S immunogens as specified in each example.
Dose: 100 ~g in 0.5 mL per immunization unless
otherwise specified
Route: intramuscular unless otherwise specified
Adjuvants: (1) Freund's Complete Adjuvant (CFA)/
Incomplete Adjuvant (IFA); or
(2) 0.4o Alum (Aluminum hydroxide);
CFA/IFA groups received CFA week 0, IFA in
subsequent weeks. Alum groups received same formulations for
all doses
Dose Sched ule: 0, 2 and 9 weeks; 0, 3, and 6 weeks or
otherwise specified.
Bleed Sche dule: weeks 0, 3, 6 and 8 or otherwise specified
Species: Sprague-Dawley rats
Group Size : 4 or 5 rats/group
Assay: specific ELISAs for each immune serum's anti-peptide
activity, solid-phase substrate was the cyclized somatostatin
peptide EQ ID N0:1).
(S
Blood was collected and processed into serum, and
stored pri or to titering by ELISA with the target antigenic
peptides.
Anti-somatostatin antibody activities were
determined by ELISAs (enzyme-linked immunosorbent assays)
using 96-well flat bottam microtiter plates which were coated
with the cyclized somatostatin peptide (SEQ ID NO:1) as
immunosorbent. Aliquots (100 wL) of the peptide immunogen
solution at a concentration of 5 ~.g/mL were incubated for 1
hour at 37°C. The plates were blocked by another incubation at
37°C for 1 hour with a 3g gelatin/PBS solution. The blocked
plates were then dried and used for the assay. Aliquots (100
~L) of the test immune sera, starting with a 1:100 dilution in
a sample dilution buffer and ten-fold serial dilutions
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thereafter, were added to the peptide coated plates. The
plates were incubated fo:r 1 hour at 37°C.
The plates were washed six times with 0.05$
PBS/Tween~ buffer. 100 ~~L of horseradish peroxidase labeled
goat-anti-species specific antibody was added at appropriate
dilutions in conjugate dilution buffer (Phosphate buffer
containing 0.5M NaCl, and normal goat serum). The plates were
incubated for 1 hour at 37°C before being washed as above.
Aliquots (100 ~tL) of o-phenylenediamine substrate solution
were then added. The color was allowed to develop for 5-15
minutes before the enzymatic color reaction was stopped by the
addition of 50 ~L 2N HZSOa . The A99zr,n, of the contents of each
well was read in a plate reader. ELISA titers were calculated
based on linear regression analysis of the absorbances, with
cutoff A99zr~ set at 0.5. This cutoff value was rigorous as the
values for diluted norma.l_ control samples run with each assay
were less than 0.15.
Th peptide-based ELISAs. The Th peptide based
ELISAs were performed essentially the same as the somatostatin
ELISA described herein above except for the antigen coating
steps, where microtiter wells were coating for 1 hr at 37° with
the designated individual. Th peptide derived from its
corresponding somatostati.n vaccine construct (e. g., peptides
with SEQ ID NOS:14 and 1~~) at 5 ~g/mL.
EXAMPLE 3
IMMUNOGENICI'.CY STUDIES OF SOMATOSTATIN
ANTIGENIC PEPTIDES INCORPORATING
VARIOUS IMMUNOSTIMULATORY ELEMENTS
The somatostati.n peptide immunogens shown in Table 2
illustrate variations of the peptides of this invention
represented by the formulas:
(A) ~- (Th)m- (B) °- (Somatostatin peptide)
or
(A)n(Somatostatin peptide)-(B)o-(Th)m
wherein:
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A is an amino acid, aNH2, or Inv (SEQ ID N0:2);
when A is an amino acid or Inv it can be linked to
either the N-terminal or the C-terminal;
B is glycine;
Th is a helper T cell epitope derived from foreign
pathogens, a . g . , HBcso-s9Th ( SEQ ID N0: 4 ) , TTsis-s3iTh ( SEQ ID
N0: 5 ) , PTias-i7sTh ( SEQ I D NO: 6 ) , or and arti f icial Th a . g . ,
1,4,9 PALIDROMIC Th (SEQ ID N0:7);
n is 1, m is 1 and o is 2
These peptides were synthesized among others and
immune sera were generatEsd for immunogenicity evaluation.
Most of the peptides shown in Table 2, incorporating various
forms and orientations of Th epitopes, elicited high titer
somatostatin-specific antibodies in the immunized hosts. In
contrast, the somatostatin peptide (p1348a, SEQ ID N0:1)
lacking Th was devoid of immunogenicity. However, certain Th
are to be preferred over others. For example, in Table 2,
p2134b (SEQ ID N0:8) having HBc Th (SEQ ID N0:4), p2384b (SEQ
ID N0:20) having SynTh(1,2,3) (SEQ ID N0:14), an artificial Th
site, and p2138b (SEQ ID NO:10) having PT199-l~s Th (SEQ ID N0:6)
are more immunogenic than p2135b (SEQ ID N0:9) having TT Th
(SEQ ID N0:5) and all of these are preferable to p2136b (SEQ
ID N0:24) having CTAB Th (SEQ ID N0:23) which is scarcely
immunogenic at all. Also, for optimum immunogenicity, the
orientation of the Th sit:e to the somatostatin target site
must be specified. Compare the kinetics of the antibody
response for p2253b (SEQ ID N0:11) to p2255d (SEQ ID N0:12).
It is preferable to place 1,4,9 PALINDROMIC Th (SEQ ID N0:7)
on the C-terminus of soma.tostatin. From the comparison of
p1344b (SEQ ID N0:16) to p1349b (SEQ ID N0:22), the better
placement for MVE258-277 (SEQ ID N0:21) is on the N-terminus of
somatostatin. It is clear that the selection and arrangement
of each Th site must be specified for the preferred peptides
of the invention.
For the somatostatin peptides shown in Table 3,
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where somatostatin constructs comprising a promiscuous Th
epitope were already found to be immunogenic, attachment of
Inv to the "Th" construct:s, can improve the immunogenicity of
the somatostatin peptide. The comparisons of immunogenicities
for p1344b (SEQ ID N0:16) with p1343c (SEQ ID N0:17) and
p1346b (SEQ ID N0:18) with p1395c (SEQ TD N0:19) shows that
the addition of the Inv domain (SEQ ID N0:2) to the N-terminus
of the Th constructs improved immunogenicity in terms of
percentage of responding animals, in the intensity of the
somatostatin-specific antibody titer, and in the longevity of
that antibody~response (~~35 weeks). However, the comparison
of immunogenicities of p2134b (SEQ ID N0:8) with p2134c (SEQ
ID N0:25) and with p2134d. (SEQ ID N0:26) illustrates that in
combination with particular Th sites Inv may not be
immunostimulatory, and that a particular orientation on the N-
terminus is preferred over the C-terminus orientation for the
combination of Inv with the HBc Th site (SEQ ID N0:4). For
the example of p2135e (SE~~ ID N0:13), the combination of Inv
with TTsis-ssi Th (SEQ ID NO:S) on the C-terminus did result in
an effective immunogen. Thus, the addition of Inv and the
orientation of Th and Inv must be specified for the preferred
peptides of the invention.
For constructs having potent site-directed
immunogenicity for somatostatin, the antibody titers directed
at the immunostimulatory elements, a . g . , Inv-MVFzse-2~~ Th of
p1343c (SEQ ID N0:17) and KKK-HBsis-s2 Th-GG of p1346b (SEQ ID
N0:18) from Table 3, were <1 Loglo in comparison to those of >3
Loglo for somatostatin. Thus, the immune response generated by
the synthetic peptides of the present invention were directed
almost exclusively to the somatostatin target site.
L'YZ1MDT L' A
ADDITIONAL ANTIGENIC PEPTIDES OF THE INVENTION
Somatostatin peptide antigens illustrating
variations of the peptides of this invention represented by
the formulas:
(A) n- (Th) m- (B) o- ( somatostatin peptide)
SUBS?'iTUTE SHEET (RUL.E 26)
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or
(A)n-(Somatostatin peptide)-(B)o-(Th)m
wherein:
Th is a helper T cell epitope derived from any of
the foreign pathogens as shown in Table 4; or, an helper T
cell epitope from any of the artificial Th epitopes as shown
in Table 5;
A is an amino acid, aNH2. or an invasin domain (SEQ
ID N0:2);
when A is an amino acid or Inv it can be linked to
either the N-terminus or the C-terminus;
B is glycine;
n is l, m is 1 and o is 2
are synthesized and immune sera generated.
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Table 1
Immunization against Somatostatin for Growth Promotion
Species Growth as percent References
of controls
Sheep (twin) 17~'o Spencer et al., 1981
.
Sheep 12.'~ Spencer et al., 1983
Pig 1113 Spencer et al., 1983
Cattle llf3 Lawrence et al., 1986
Chicken 115 Spencer et al., 1986
Carrier protein-somatostatin conjugates were used as the
vaccines for immunization.
SUBS'fiTUTE SiiEET (RULE 26)
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CA 02329755 2000-12-11
WO 99/66950 PCTNS99/13923
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CA 02329755 2000-12-11
WO 99/66950 PCT/US99/13923
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; a z z a z ~z w ~n
2
D O O ~ w
H
a ~ 4Y p CxD L~ Q OD H x
D 10
H ,7 H ,"7H ,.'~H DH
H
~ N
m ' w . . H. z
c
a r~ ~r rvu' c" tr oo ~ ro
tr
~ n O ~ >n p
W A CJ! W ( H N
V1 W td
Cc, W v7 cn 3"
u: c4 fx
..
'
-- a
CT
N G
U
C W 0 N
O I~
U
Cn 'd
~C
H (() H H E
~ ~ L
s1 1~
H1
N yJ O
b +.~ a !.
o
, .1.' m a m ~ ~
E
m ~ ~ ', c~ 'r,
o
0 0 ~ ~ .d "
x ~ ~ a ~ ~
ro w
1 x H ~
G ro
1 ~ ~ I 1 ~C ..1
la
o~ -- ~ ~U' ~c, N a~7
~ vc
u7
1 no C H
OD N N
C (""
~ ('"
21
0 O N ' .~ 'rIO '.'I
I ~ O O ~Z .a
z z U ro N
z z m o
~
1 x . u .,.10 ~ ai
o n o o
Ur ~ I U] NH t~
H H H H a
'Cf C5 +~ ~ a~ ~
I fa
ar~ a' I ro mtr m o
tr~ rr
y U y ~ ~a~ ~ D
a~ N N a~ w
x 1: ~ ~~
In V7 ~
(n H
f/7
W _ W
G W W
r-i
O O O
U rn
_ N ~
U
M U .C7 U ' d ~ U U
i-1
a ~r c u7 N N
er M M M M
r-IM .1 H . -i .-i CJ'
CT'
N
fj,~ N N N N
roa a a a a
E., a a
a
SUBS1TTUTE SHEET (RULE 26)
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WO 99/66950 PCT/US99113923
- 29 -
Table 4
Amino Acid Sequences of Pathogen-derived Th Epitopes
Description of Th SEQ ID NO: Amino Acid Sequence
HBcso-ss Th 4 SDFFPSVRDLLDTASALYRE
TTsis-s3i Th 5 WVRDIIDDFTNESSQKT
PT199-176 Th 6 KKLRRLLYMIYMSGLAVRVHVSKEEQYY
DY
HBS19-32 Th 15 FFLLTRILTIPQSLD
MVF2se-277 Th 21 GILESRGIKARITHVDTESY
CTA$ios-iso Th 23 ALNIWDRFDVFSTLGATSGYLKGNS
CTP11 Th 27 TINKPKGYVGKE
DTI Th 28 DSETADNLEKTVAALSILPGHG
MVF1 Th 29 LSEIKGVIVHRLEGV
SUBSTITUTE SHEET (RULE 26)
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WO 99/66950 PCT/US99/13923
-30-
Table 5
Amino Acid Sequences of Artificial Th Epitopes
Description of Th SEQ ID NO: Amino Acid Sequence
Syn Th {1,2,4) 14 KKKIITITRIITIITTID
(1,4,9 PALINDROMIC) 7 ISEIKGVIVHKIEGI
Th
MT RT TRM TM
L L V
(1,4,9 PALINDROMIC) 30 ISEIKGVIVHKIEGI
simplified Th
T RT TR T
IS(1,9,9 31 ISISEIKGVIVHKIEGILF
PALINDROMIC)LF
simplified Th T RT TR T
SUBSTITUTE SHEET (RULE 26)
CA 02329755 2000-12-11
- 1 -
WO 99/66950 PCT/US99/13923
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: UNITED BIOMEDICAL INC.
(ii) TITLE OF INVENTION: SYNTHETIC SOMATOSTATIN IMMUNOGEN FOR
GROWTH PROMOTION IN FARM ANIMALS
(iii) NUMBER OF SEQUENCES: 45
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Morgan & Finnegan
(B) STREET: 345 Park Avenue
(C) CITY: New York
(D) STATE: NY
( E ) COUNTRY : !;JSA
(F) ZIP: 10154-0054
(v) COMPUTER READA1BLE 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) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: TBA
(B) FILING DATE: 18-,JUNE-1999
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: MARIA C.H. LIN
(B) REGISTRATION NUMBER:29,323
(C) REFERENCE,/DOCKET NUMBER: 1151-9155PC1
(ix) TELECOMMUNICATION INFORMATTON:
(A) TELEPHONE: 212-758-4800
(B) TELEFAX: 2:12-751-6849
(2) INFORMATION FOR SEQ ID N0:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1~9 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: :Linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
Ala Gly Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr
1 5 10
Ser Cys
SUBS'T1TUTE SHEET (RULE 26)
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WO 99/66950 PCT/US99/13923
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: lfi amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Thr Ala Lys Ser Lys Lys Phe Pro Ser Tyr Thr Ala
1 5 10
Thr Tyr Gln Phe
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
Pro Pro Xaa Pro Xaa Pro
1 5
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp
1 5 10
Thr Ala Ser Ala Leu Tyr Arg Glu
I5 20
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
SUBSTITUTE SHEET (RULE 26)
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
Trp Val Arg Asp Ile Ile: Asp Asp Phe Thr Asn Glu
1 5 10
Ser Ser Gln Lys Thr
(2) INFORMATION FOR SEA! ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Lys Lys Leu Arg Arg Leu :Leu Tyr Met Ile Tyr Met
1 5 10
Ser Gly Leu Ala Val Arg Val His Val Ser Lys Glu
15 20
Glu Gln Tyr Tyr Asp Tyr
30
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1
(D) OTHER INFORMATION: /note= "Ile, Met or Leu"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 2
(D) OTHER INFORMATION: /note= "Ser or Thr"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 4
(D) OTHER INFORMATION: /note= "Ile or Arg"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 5
(D) OTHER INFORMATION: /note= "Lys or Thr"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 10
SUBSTITUTE SHEET (RULE 26)
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WO 99/66950
PCT/US99/13923
(D) OTHER INFORMATION: /note= "His or Thr"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 11
(D) OTHER INFORMATION: /note= "Lys or Arg"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 12
(D) OTHER INFORMATION: /note= "Ile, Met or Leu"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 19
(D) OTHER INFC>RMATION: /note= "Gly or Thr"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B} LOCATION: 15
(D) OTHER INFORMATION: /note= "Ile, Met or Val"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
Xaa Xaa Glu Xaa Xaa Gly Val Ile Val Xaa Xaa Xaa
1 5 10
Glu Xaa Xaa
(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE; peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp
1 5 10
Thr Ala Ser Ala Leu Tyr Arg Glu Gly Gly Ala Gly
15 20
Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys
30 35
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
SUBST'tTUTE SHEET (RULE 26)
CA 02329755 2000-12-11
WO 99/66950
PCT/US99/13923
Trp Val Arg Asp Ile Ile Asp Asp Phe Thr Asn Glu
1 5 10
Ser Ser Gln Lys Thr Gly Gly Ala Gly Cys Lys Asn
15 20
Phe Phe Trp Lys Thr Phe Thr Ser Cys
25 30
(2) INFORMATION FOR SEQ ID N0:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 46. amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:10:
Lys Lys Leu Arg Arg Leu Leu Tyr Met Ile Tyr Met
1 5 10
Ser Gly Leu Ala Val Arg Val His Val Ser Lys Glu
15 20
Glu Gln Tyr Tyr Asp Tyr Gly Gly Ala Gly Cys Lys
25 30 35
Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys
40 45
(2) INFORMATION FOR SEQ ID N0:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: :31 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: (peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1
(D) OTHER INF01RMATION: /note= "Ile, Met or Leu"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 2
(D) OTHER INFOIZMATION: /note= "Ser or Thr"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 4
(D) OTHER INFO1~MATION: /note= "Ile or Arg"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
( B ) LOCAT I ON : .'i
(D) OTHER INFORMATION: /note= "Lys or Thr"
SUBSTITUTE SHEET (RULE 26)
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WO 99/66950 PCT/US99/13923
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: LO
(D) OTHER INFORMATION: /note= "His or Thr"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 11
(D) OTHER INFORMATION: /note= "Lys or Arg"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 12
(D) OTHER INFORMATION: /note= "Ile, Met or Leu"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 7.4
(D) OTHER INFORMATION: /note= "Gly or Thr"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 15
(D) OTHER INFORMATION: /note= "Ile, Met or Val"
(xi) SEQUENCE DESCRI:fTION: SEQ ID NO:li:
Xaa Xaa Glu Xaa Xaa Gly ~'al Ile Val Xaa Xaa Xaa
1 5 10
Glu Xaa Xaa Gly Gly Ala Gly Cys Lys Asn Phe Phe
15 20
Trp Lys Thr Phe Thr Ser Cys
25 30
(2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 17
(D) OTHER INFORMATION: /note= "Ile, Met or Leu"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 18
(D) OTHER INFORMATION: /note= "Ser or Thr"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 21
(D) OTHER INFORMATION: /note= "Ile or Arg"
SUBSI~'1TUTE SHEET (RULE 26)
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WO 99/66950 PCT/US99/13923
(ix)FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 22
{D) OTHER INFORMATION: "Lys or Thr"
/note=
(ix)FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 26
(D) OTHER INFORMATION: "His or Thr"
/note=
{ix)FEATURE:
(A) NAME/KEY: Modified-site
{B) LOCATION: 27
(D) OTHER INFORMATION: "Lys or Arg"
/note=
(ix)FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: ~~8
{D) OTHER INFORMATION: "Ile,Met or
/note=' Leu"
(ix)FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 30
(D) OTHER INFORMATION: "Gly or Thr"
/note=
(ix)FEATURE:
{A) NAME/KEY: Modified-site
(B) LOCATION: 31
(D) OTHER INFORMATION: "Ile,Met or
/note= Val"
(xi)SEQUENCE DESCRIPTION: SEQ
ID N0:12:
AlaGly Cys Lys Asn Phe Phe Trp Phe hr
Lys Thr T
1 5 10
SerCys Gly Gly Xaa Xaa Glu Ile Val le
Xaa Xaa I
15 20
ValXaa Xaa Xaa Glu Xaa Xaa
25 30
(2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 57. amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCR7:PTION: SEQ ID N0:13:
Ala Gly Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr
1 5 10
Ser Cys Gly Gly Trp Val Arg Asp Ile Ile Asp Asp
15 20
Phe Thr Asn Glu Ser Ser Gln Lys Thr Gly Gly Thr
25 30 35
Ala Lys Ser Lys Lys Phe Pro Ser Tyr Thr Ala Thr
SUBSTITUTE SHEET (RULE 26)
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WO 99/66950 PCT/US99/113923
Tyr Gln Phe
40 45
(2) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY; linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
Lys Lys Lys Ile Ile Thr Ile Thr Arg Ile Ile Thr
1 5 10
Ile Ile Thr Thr Ile Asp
(2) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1.5 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: ;peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile Pro Gln
1 5 10
Ser Leu Asp
(2) INFORMATION FOR SEQ :ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: :Linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIJ?TION: SEQ ID N0:16:
Gly Ile Leu Glu Ser Arg Gly Ile Lys Ala Arg Ile
1 5 10
Thr His Val Asp Thr Glu Ser Tyr Gly Gly Ala Gly
15 20
Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys
30 35
(2) INFORMATION FOR SEQ ID N0:17:
SUBSTITUTE SHEET (RULE 26)
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
Thr Ala Lys Ser Lys Lys Phe Pro Ser Tyr Thr Ala
1 5 10
Thr Tyr Gln Phe Gly Ile Leu Glu Ser Arg Gly Ile
15 20
Lys Ala Arg Ile Thr His Val Asp Thr Glu Ser Tyr
25 30 35
Gly Gly
(2) INFORMATION FOR SEQ ID N0:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2C1 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:
Lys Lys Lys Phe Phe Leu Leu Thr Arg Ile Leu Thr
1 5 10
Ile Pro Gln Ser Leu Asp Gly Gly
15 20
{2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:19:
Thr Ala Lys Ser Lys Lys Phe Pro Ser Tyr Thr Ala
1 5 10
Thr Tyr Gln Phe Gly Gly Lys Lys Lys Phe Phe Leu
15 20
Leu Thr Arg Ile Leu Thr Ile Pro Gln Ser Leu Asp
25 30 35
Gly Gly
(2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
SUBSTITUTE SHEET (RULE 26)
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(A) LENGTH: ~!4 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCP.IPTION: SEQ ID N0:20:
Lys Lys Lys Ile Ile Thr Ile Thr Arg Ile Ile Thr
1 5 10
Ile Ile Thr Thr Ile Asp Gly Gly Ala Gly Cys Lys
15 20
Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys
25 30
(2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY': linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCR:CPTION: SEQ ID N0:21:
Gly Ile Leu Glu Ser Arg Gly Ile Lys Ala Arg Ile
1 5 10
Thr His Val Asp Thr Gl.u Ser Tyr
15 20
(2) INFORMATION FOR SEQ TD N0:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3E. amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
Ala Gly Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr
1 5 10
Ser Cys Gly Gly Gly Ile Leu Glu Ser Arg Gly Ile
15 20
Lys Ala Arg Ile Thr His Val Asp Thr Glu Ser Tyr
25 30 35
(2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
SUBST'1TUTE SHEET (RULE 26)
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WO 99/66950 PCT/US99/139Z3
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:
Ala Leu Asn Ile Trp Asp Arg Phe Asp Val Phe Ser
1 5 10
Thr Leu Gly Ala Thr Ser Gly Tyr Leu Lys Gly Asn
15 20
Ser
(2) INFORMATION FOR SEQ ID N0:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:
Ala Leu Asn Ile Trp Asp Arg Phe Asp Val Phe Ser
1 5 10
Thr Leu Gly Ala Thr Ser Gly Tyr Leu Lys Gly Asn
15 20
Ser Gly Gly Ala Gly Cys Lys Asn Phe Phe Trp Lys
25 30 35
Thr Phe Thr Ser Cys
(2) INFORMATION FOR SEQ ID N0:25:
(i) SEQUENCE CHARAC'.TERISTICS:
(A) LENGTH: 54 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:
Thr Ala Lys Ser Lys Lys Phe Pro Ser Tyr Thr Ala
1 5 10
Thr Tyr Gln Phe Gly Gly Ser Asp Phe Phe Pro Ser
15 20
Val Arg Asp Leu Leu Asp Thr Ala Ser Ala Leu Tyr
25 30 35
Arg Glu Gly Gly Ala Gly Cys Lys Asn Phe Phe Trp
40 45
Lys Thr Phe Thr Ser Cys
(2) INFORMATION FOR SEQ ID N0:26:
(i) SEQUENCE CHARACTERISTICS:
SUBSZITUTE SHEET (RULE 28)
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(A) LENGTH: 54 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:26:
Ala Gly Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr
1 5 10
Ser Cys Gly Gly Ser Asp Phe Phe Pro Ser Val Arg
15 20
Asp Leu Leu Asp Thr Ala Ser Ala Leu Tyr Arg Glu
25 30 35
Gly Gly Thr Ala Lys Ser Lys Lys Phe Pro Ser Tyr
40 45
Thr Ala Thr Tyr Gln Phe
(2) INFORMATION FOR SEQ ID N0:27:
(i) SEQUENCE CHARA(:TERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCR7:PTION: SEQ ID N0:27:
Thr Ile Asn Lys Pro Lys Gly Tyr Val Gly Lys Glu
1 5 10
(2) INFORMATION FOR SEQ ID N0:28:
(i) SEQUENCE CHARAC;TERISTICS:
(A) LENGTH: 22 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:
Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Val
1 5 10
Ala Ala Leu Ser Ile Leu Pro Gly His Gly
15 20
(2) INFORMATION FOR SEQ ID N0:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
SUBSTTTUTE SHEET (RULE 26)
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WO 99/66950 13 PCT/US99/139Z3
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCFtIPTION: SEQ ID N0:29:
Leu Ser Glu Ile Lys Gly Val Ile Val His Arg Leu
1 5 10
Glu Gly Val
(2) INFORMATION FOR SEQ ID N0:30:
{i) SEQUENCE CHARF,CTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 2
(D) OTHER INFORMATION: /note= "Ser or Thr"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 5
(D) OTHER INFOF;MATION: /note= "Lys or Arg"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(D) OTHER INFORMATION: /note= "Gly or Thr"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 10
(D) OTHER INFORMATION: /note "His or Thr"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 11
(D) OTHER INFORMATION: /note= "Lys or Arg"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 19
(D) OTHER INFORMATION: /note= "Gly or Thr"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:30:
Ile Xaa Glu Ile Xaa Xaa Val Ile Val Xaa Xaa Ile
1 5 10
Glu Xaa Ile
(2) INFORMATION FOR SEQ ID N0:31:
sues~rrru~ sHe~ (RUB zs~
CA 02329755 2000-12-11
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PCT/US99/13923
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 9
(D) OTHER INFORMATION: /note= "Ser or Thr"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 7
(D) OTHER INFORMATION: /note= "Lys or Arg"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 8
(D) OTHER INFORMATION: /note= "Gly or Thr"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 12
(D) OTHER INFORMATION: /note= "His or Thr"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 13
(D) OTHER INFORMATION: /note= "Lys or Arg"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1.6
(D) OTHER INFORMATION: /note= "Gly or Thr"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:31:
Ile Ser Ile Xaa Glu Ile Xaa Xaa Val Ile Val Xaa
1 5 10
Xaa Ile Glu Xaa Ile Leu Phe
(2) INFORMATION FOR SEQ ID N0:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:32
Lys Lys Gln Tyr .Ile Lys Ala Asn Ser Lys Phe Ile
1 5 10
SUBSITrUTE SHEET (RULE 26)
CA 02329755 2000-12-11
- 15 -
WO 99/66950 PCT/US99/13923
Gly Ile Thr Glu Leu
(2) INFORMATION FOR SEQ ID N0:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:33:
Lys Lys Phe Asn Asn Phe Thr Val Ser Phe Trp Leu
1 5 10
Arg Val Pro Lys Val Ser Ala Ser His Leu
15 20
(2) INFORMATION FOR SEQ ID N0:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2'7 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCR:OPTION: SEQ ID N0:34:
Tyr Asp Pro Asn Tyr Leu Arg Thr Asp Ser Asp Lys
1 5 10
Asp Arg Phe Leu Gln Thr Met Val Lys Leu Phe Asn
15 20
Arg Ile Lys
(2) INFORMATION FOR SEQ ID N0:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2~1 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCR7:PTION: SEQ ID N0:35:
Gly Ala Tyr Ala Arg Cys Pro Asn Gly Thr Arg Ala
1 5 10
Leu Thr Val Ala Glu Leu Arg Gly Asn Ala Glu Leu
15 20
(2) INFORMATION FOR SEQ ID N0:36:
SUBS'~1TUTE SHEET (RULE 26)
CA 02329755 2000-12-11
WO 99/66950 16 PCT/US99/13923
(i) SEQUENCE CHARPvCTERISTICS:
(A) LENGTH: 2:1 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:36:
Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala
1 5 10
Tyr Arg Pro Pro Asn Ala Pro Ile Leu
15 20
(2) INFORMATION FOR SEQ ID N0:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:37:
Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys
1 5 10
Trp Gly Glu Leu Met Thr Leu Ala
15 20
(2) INFORMATION FOR SEQ ID N0:38:
(i) SEQUENCE CHARA(:TERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCR7CPTION: SEQ ID N0:38:
Arg Ala Gly Arg Ala Ile Leu His Ile Pro Thr Arg
1 5 10
Ile Arg Gln Gly Leu Glu Arg
{2) INFORMATION FOR SEQ ID N0:39:
(i) SEQUENCE CHARAC;TERISTICS:
(A) LENGTH: 27. amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:39:
sues sHE~ cRU~ 2sZ
CA 02329755 2000-12-11
WO 99/66950 17
PCT/US99/13923
Ala Val Ala Glu Gly Thr Asp Arg Val Ile Glu Val
1 5 10
Leu Gln Arg Ala Gly Arg Ala Ile Leu
15 20
(2) INFORMATION FOR SEQ ID N0:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:40:
Glu Glu Ile Val Ala Gln Ser Ile Ala Leu Ser Ser
1 5 10
Leu Met Val Ala Gln Ala Ile Pro Leu Val Gly Glu
15 20
Leu Val Asp Ile Gly Phe Ala Ala Thr Asn Phe Val
25 30 35
Glu Ser Cys
(2) INFORMATION FOR SEQ ID N0:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:41:
Asp Ile Glu Lys Lys Ile Ala Lys Met Glu Lys Ala
1 5 10
Ser Ser Val Phe Asn Val Val Asn Ser
15 20
(2) INFORMATION FOR SEQ ID N0:42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCP.IPTION: SEQ ID N0:42:
Lys Trp Phe Lys Thr Asn. Ala Pro Asn Gly Val Asp
1 5 10
SU8:5TITUTE SHEET (RULE 26)
CA 02329755 2000-12-11
- 18 -
WO 99/66950 PCT/US99/13923
Glu Lys Ile Arg Ile
(2) INFORMATION FOR SEA! ID N0:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:43:
Gly Leu Gln Gly Lys Ile Ala Asp Ala Val Lys Ala
1 5 10
Lys Gly
(2) INFORMATION FOR SEQ ID N0:44:
(i) SEQUENCE CHAFLACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:44:
Gly Leu Ala Ala Gly Leu Val Gly Met Ala Ala Asp
1 5 10
Ala Met Val Glu Asp Val Asn
(2) INFORMATION FOR SEQ ID N0:45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:45:
Ser Thr Glu Thr Gly Asn Gln His His Tyr Gln Thr
1 5 10
Arg Val Val Ser Asn Ala Asn Lys
15 20
SUBSTITUTE SHEET (RULE 26)
