Note: Descriptions are shown in the official language in which they were submitted.
W095i31480 ~ ~ CA 02372569 2002-03-08 ~ pC'1'/CA95l00293
HETERODIMER POLYPEPTIDE IMMUNOGEN
CARRIER COMP08ITION AHD~3~SETHOD
Field of the Invention
The present invention relates in general to a
composition and methods of use of a°polypeptide carrier
complex which. caw have two different bioactive moieties
attached in a known st.oichiometry and molecular
i0 orientation. It, relates more spec~.fically to a synthetic
immunogenic polypeptide complex which can present two
different types of antigens in a pre-defined., precise
stoichiometry and molecular .orientation.
References Chad
Abbas, A.IC. , et 81. , ' In CELLULAR AND MOLECULP.R I!~.MUNOLOGY,
W. B. Saunders Company, Philadelphia PA (1991).
Ausubel , F . M . , a t a1. , in CaR~rrr PROTOCOLS IN Mor~a~'yLAx
o~ ,~ John Wiley.and Sons, Inc., Media PA.
Benjamin, D.C., et al. Aan. Rev. Immunol. ,x;67
(1984). ' .
. Berzotsky, JA. Vaccine ,x:89-93 (1988). .
Bittle, J.L.,,et al: Natu=e (.London) ,x:30-33
(1982).
DiMarchi, R., et al., Science x:639-641 (1986).
Eisel, U., et al., EM80 J. 5:2495-2502.(i986).
Etlinger, H.M., et al.., Vacciye x:512-514 (1891).
Felix, A:M., et a3., Int. J. Reptide.Protein Res.
31:231-238 (1988).
Francs, et al. , Nature ,(London) 300.:168 (1987) .
Good, M.F., et al~. Ann. Rev. Lmmunol. 6:663-688
(1988) .
. Harlow, E. , et a.I . , in ArtTZSOD=gs : A LasoRATO9~ _MArrvAZ,,
Coid Spring Harbor Laboratory Press (1988).
Herzenberg, L.A., et al., J. Fxa. Med. x:1730-40
(1982).
Ho, P. C. , et al. , Eur. J. Itamunol. .0:477 (1990) .
. :~T~T~.3'~'E SHE"E~'
WO 95131480 . ~ ~ 02372569 2002-o3-oa ~ pC'1'/CA95100293
2
HOChull, E. , 111 GENETIC ENGINEERING, PRINCIPALS AND
PRACTICE, VoL. 12 (J. Stelow Ed. ) Plenum, NY, pp. 87-98
(1990).
Hodges, R.S., et al., Peptide Res. 1:19-30 (1988).
Hodges, R.S., et al., Peptide Res. 3_:123-137 (1990).
Hodges, R.S., et al., Peptide Res. 3_:123-I37 (1993).
Hodges, R.S., et al.,~U.S. Patent No. 5,223,604,
issued June 1993.
Hopp, T.P:, et al., Proc. Natl. Acad. Sci. USA
78:-3824-3828 (1981). '
Hu, et al., Science 250:1400-1403 (1990).
Lerner, R:A. Nature (London) 299:592-596-(1982).
McInnes, C., et al., Biochemistry 32:13432-40 (1993).
Maniatis, T. , et al . , in Mor.ECVraR CLONING: A LABORATORY
MANUAL, Cold Spring Harbor Laboratory (1982).
Miller, J.F., et a1.,_ Nature fiLondon) 216:69-63
(1969):
Panina-Bordignon, P., et al., Eur. J. Immunol
X9_:2237-2242 (1989).
Porath, J., Protein Exp. and Purif. 3:263 (1992).
Satin, et al., Anal. Biochem.. X7,:147-157 (1981).
Sela, M. and R. Arnon: , in NEw DEVELOPMENTS WITH HUMAN ANn
VETINARY VACCINES, (Mizrahi, A. , et al . , Eds. ) (Liss, New
York), pp 315-323 (1980).
Skerra, A., et aZ., Biotechnolagy ,x:273 (1991).
Tam, J.P., Proc: Natl. Acad. Sci. USA $x:5409-13
(1988)
Tam, J.P., U.S. Patent No. 5,229,490,issued July
1993. .
30. Wong, W.Y., et al., Protein Sci. x:1308-18 (1992).
Background of the Invention
Vaccines can be constructed using either largely-
intact, native antigenic molecules or portions of
antigenic molecules containing the epitope of interest.
As discussed by Tam (1988, 1993); recent studies have
shown that synthetic peptides can induce antibodies
e~. no c-r~-ra tT~ ~ ~; r ~. i
WO 95131480 ~ CA 02372569 2002-03-08 ~ p~/CA95I00293
3
reactive with their respective sequences in the native
protein (Sela, et al:; Lerner): Antibodies immunoreactive
with peptide antigens are useful laboratory and diagnostic
reagents. Synthetic peptide antigens, conveniently
available through chemical synthesis, can be used for
producing immunogens and for passive immunoprophylaxis
(Sela, et al.; Lerner; Bittle, et al.; DiMarchi, et al.).
A conventional approach to preparing antibodies
immunoreactive with peptide antigens is conjugation of a
l0, peptide to a known protein or synthetic polymer carrier to
give a macromolecular structure to the imiaunogeric entity
(Sela, et al.; Lerner; Bittle, e.t a~.). Methods designed
to avoid the use of carrier by polymerizing synthetic
peptide antigens to give peptide polymers have also been
reported (DiMarchi, et a1.). Although such constructs are
effective in producing animal antibodies, they are
ambiguous in composition and structure. This is
particularly disadvantageous if the antibodies are to be
used for a human'vaccine.
Vaccines typically comprise an. antigen on a natural
y ' carrier such as a protein, a carbohydrate, a lipid or a
liposome. Such vaccines are. useful and have been employed
~'or many years. There are however a number of recognized
problems with them, some of which are related to the
carrier. Since the carriers are usually isolated from
natural sources,- they are often not of uniform quality:
Additionally, despite expensive and arduous purification
efforts, it is difficult, and often impossible, to,'provide
products completely free of natural contaminants. Such
contaminants may themselves be antigenic. They cause the
undesirable side reactions often associated with the use
of .vaccines; particularly fevers and tissue swelling.
Additionally, the concentration of antigen may vary. from
one batch to. another because the amounts of antigen that
react with the.carrier or that are observed on its surface
are not uniform.
SUB~T~'~UTE SHEET
WO 9~I31480 ~ 02372569 2002-03-08
PCT/CA95IU0293
4
Summary of the Invention
It is therefore one object of the present invention
to provide a polypeptide compound comprised of two
subunits that interact to form a coiled-coil heterodimer.
Each subunit is deri~tatized to include. a different
functional or bioactive moiety, and the moieties do' not
substantially interfere with the formation of a coiled-
coil heterodimer: The coiled coil heterodimer may be
stabilized by ionic interactions betweeW the subunits.
:10 Various bioactive moieties may be linked or
incorporated into the subunits. The. moieties may be other
polypeptides (including antibodies and FAb_.fragments),
drugs, therapeutic. agents, radioactive substances, nucleic
acids,~glycoproteins, lipoproteins, carbohydrates, fatty
acids, or other biologically-active substances. These
substances may be linked'directly to amino acid residues
of the carrier polypeptides, or. they. may be linked through
a spacer,~such as 2-8 amino acids (e..g., poly-glycine), a
carbon chain.or the like. . '
' In particular,.the moieties may be antigens (.e.g., a
T-cell antigen on one.subunit and .a B-cell antigen on the
other subunit). In one embodiment, one subunit .is
derivatized with a T-cell antigen.comprised of a peptide
having a sequence represented by SEQ ID.NO:10, SEQ ID
N0:11, SEQ ID N0:12, SEQ ID N0:13 or SEQ ID N0:14. An
exemplary B-cell antigen has the sequence represented as
SEQ ID NQ:18. .
- The subunit and its bioactive.moiety may.'be a single
polypeptide Ehain, e.g., a fusion polypeptide.having,two
domains'which may be separated by a spacer. In one
embodiment, the single polypeptide chain has an amino acid
sequence that includes a sequence present in SEQ ID N0:28.
In another embodiment, the single polypeptide~chasn has an
amino acid sequence that includes a sequence present in
SEQ ID N0:30.
It is a related object of the invention to provide a
heterodimer polypeptide immunogen comprised of two
SUBSTI"~UT~ SHIES i
WO 95131480 / ~ 02372569 2002-03-08 ~ pCZyCA95100293
subunits where each subunit is comprised of a core peptide
and an antigen. Each core peptide is comprised of =-
terminal and internal amino acid repeat sequences having
the form gabcdef. Positions a and d of each terminal and
5 internal amino acid repeat sequence are isoleucine,
leucine or valine, and positions a and g are aspartic acid
or glutamic acid in one core peptide, and. lysine, arginine
or histidine in the other core peptide.
Peptide antigens are attached to the core peptides
through covalent linkages to amino acids at position b, c
or f of the internal repeats..The two subunits are
arranged in a stable a-helical coiled-coil configuration
having a 1:1 stoichiometry, and the peptide antigens are
disposed toward outer surfaces of the conf igura,tion.
The terminal repeat sequences of the each core
peptide can include a glutamic acid at position b, a~
lysine at position f and a lactam bridge formed between
positions b and f. The internal repeat sequences can
include an amino acid coupling residue at position f, and
this coupling residue can be a cysteine residue.
In a preferred embodiment, the core peptides have
sequences represented by SEQ ID NO:~. (EE) and SEQ ID N0:2
(KK), and the antigens have sequences represented by SEQ
ID N0:12 (T-cell antigen) and SEQ ID N0:18 (B-cell
antigen).
It is another object of the invention to provide a
pair of subunits for use as an a-helical coiled-coil
heterodimer antigen carrier. Each of the subunits
contains two terminal amino acid repeat peptide sequences
having the.form gabcdef, where b is glutamic acid, f is
lysine, and b and f are linked by a lactam~bridge, and at
least one internal-amino acid repeat sequence having the
form gabcdef, where position b, c or f is a cysteine
residue . The cysteine residue can be covalently.attached
to an antigen. Positions a and d of each terminal and
internal amino acid repeat sequence are isoleucine,
leucine or valine, positions a and g of one subunit are
SUSS'~'iT~J i C Si-iirCT
WO 95/31480 ~ .~ 02372569 2002-03-08 '~ pCTlCA95/00293
6
aspartic acid or glutamic acid, positions a and g of the
other subunit are lysine, argin'ine or histidine.
Two exemplary subunits capable of forming cz-helical
coiled-coils have sequences represented by SEQ ID N0:1
(EE) and S,EQ ID N0:2 (KK).
It is yet another object of the invention to provide
a method of preparing a polypeptide compound. The method
includes forming two peptide subunits that interact to
form a coiled-coil heterodimer. Each subunit is
derivatized to include a bioactive moiety, each subunit .
carries a different bioactive moiety and the bioactive
moieties do not substantially interfere with. the formation
of a coiled-coil heterodimer. The~polypeptide subunits
are mixed in a benign medium in a ratio of about 1:i under
Z5 conditions that promote formation of said coiled-coil
heterodimers. The coiled-coil heterodimer may be
stabilized by ionic interactions.
The bioactive moieties may be as described above,
e.g., antigens. In one embodiment, one subunit contains a
T-cell'antigen and the other subunit contains a 8-cell
antigen. The T-cell antigen can have.a~~equence
represented by SEQ ID NO:lO, SEQ ID NO:il,. SEQ ID N0:12,
SEQ ID N0:13 or SEQ ID N0:14.
It is a related object of the invention to provide a
method of preparing a polypeptide immunogen composition,
where two core peptides are formed, each of which contains
two terminal amino acid repeat sequences having the form
gabcdef and at least one internal amino acid repeat
sequence having the form gabcdef. Positions a and d of
'each teriainal and internal amino acid repeat sequence are
isoleucine, leucine or valine, positions a and g of each
terminal and internal amino acid repeat sequence are
aspartic acid or glutamic acid in one core peptide, and
lysine, arginine or histidine in the other core peptide.
~ Peptide antigens may be attached through covalent
linkages to amino acids at positions b, c or f of the core
peptides, and the derivatized peptides are mixed in a
C 1 1 Q CT! T! ' ST.= C :-! .L'~ 1=-r
WO 95/31.480 . CA 02372569 2002-03-08 ~ PC"TICA95100293
7
benign medium in.a ratio. of. about 1:1 under conditions
that promote formation of coiled-coil heterodimers.
A further embodiment of the present invention
includes two subunits capable of forming an a-helical
coiled-coil heterodimer dimer composition, as described
above, where the antigen on the first subunit is replaced
by a moiety capable of binding to a target cell, for .
example, a tumor, cell, and the antigen on the second
subunit is replaced by a_cytotoxic moiety, for example, a
radioactive compound. ~ .
The first.subunit is administered to a-subject and.
allowed to bind to a target cell. Following a selected
time interval, the second subunit is administered and
allowed ~o form a heterodimer with the first subunit. The
subunits are preferably~administered at doses. effective to
significantly inhibit or kill the target cell while having-
a minimal cytotoxic effect on non-target cells and causing:.
minimal side-effects .in the subject.
These and other objects and features of the invention
2o will~be more fully appreciated whey the following detailed
description of the invention is read in conjunction with
the accompanying drawings. ~ .
Brief Deseriptioa of the-Fiqures
Figures 1a-c show ayschematic representation of the
~'synthesis~and assembly of an immunogen:ic formulation
disclosed. in the speci~ication.~ Figure 1a showswa
schematic of two core polypeptides, each comprised of 5
heptads. Figure ib shows the core polypeptides after they
~haue been derivatized withantigenic peptides. Figure is
shows a schematic of an immunogenic complex of the present
invention, corngri$ed of two antigen-decorated core
polypeptides in a heterodimeric configuration.
Figure 2a shows helical wheel representations of
terminal heptads of two exemplary.core ,polypept.ides in a
parallel a-helical heterodimer configuration. Figure 2b
shows helical wheel representations of terminal heptads of
SUBS~'~'f l u'I"~ ."'.~~~~T
WO 95/31480 ~ ~ 02372569 2002-03-08 ' ~ p~~Cpgg~00293
8
two exemplary care polypeptides in an antiparallel a-
helical heterodimer configuration.
Figures 3a-a show a schematic representations of
adjacent heptads of two core polypeptides in a parallel
configuration comparing the stabilizing/destabilizing
'effects of charged residues at the a and g positions in
homodimers vs. heterodimers.. Figure 3a shows a homodimer
stabilized by ogpositely-charged residues at the a and g
positions of a heptad. Figure 3b shows a heterodimer.
/O destabilized by oppasitely-charged residues at the a and g
positions of a heptad. Figure 3c shows a homodimer
destabilized by positively-charged residues~at the. s and g.
positions of_a heptad. Figure 3d..shows a heterodimer
stabilized by like-charged residues at the a and g
positions of a heptad.~ Figure 3e shows a homodimer
destabilized by negatively-charged,residu~s at the a and g
positions of a heptad.
Figures 4a--c show a schematic of some possible
distributions of heptads, bearing either positive or
negative charges at their a and g positions, within
peptides designed to form coiled-coil, heterodimers.
Figure 4a shows~a schematic of a~heterodimer comprised of
core,polypeptides having alternating positively- and'
negatively-charged successive heptads. Figure 4b shows a
schematic of a heterodimer comprised,of core polypeptides,
one. of Which has predominantly positively-charged heptads,
and the other of which has predominantly negatively-
charged heptads.w Figure 4c shows a schematic of a
heterodimer comprised of core polypeptides, one of which
has, all positively-charged heptads, and the other of which
has all negatively-charged heptads. .
Figure_5 shows a map of plasmid pRLD-E.
Figure 6 shows the polylinker region (promoter, MCS
and insert) of plasmid..pRLD-E.
' Figure 7 shows a.map of plasmid pRLD-K.
Figure 8 shows the polylinker region of pRLD-K.
Figure 9 shows a map~of plas~aid pHIL-S1/E.
aUB~: f ~'i'~ ~i-i~~~
WO 95!31480 . CA 02372569 2002-03-08 ~ ~ PCTlCA95/00293
9
rFigure l0 shows the polylinker region of plasmid
pHIL-S1/E.
Figure !l shows.a map of plasmid,pHIL-S1/K. y
Figure i2 shows the polylinker region of plasmid
pHIL-Sl/K: '
Figure l3 shows the nucleotide and translated amino
acid sequences of a fragment containing sequences encoding
PAK pili-C1 cloned in the poly~.inker region of pHIL-S1/E.
Figure 14 shows the nucleotide and translated amino
acid sequences of a fragment containing sequences encoding
MVF-C1 (measles virus F protein) cloned in the polylinker
region of pHIL-S1/E.
Detailed Description of the IaveatioW
I. Definitions
The tenas "peptide" and "polypeptide", used
interchangeably, designate a chain of amino acid based
polyamides.~ The chain_can vary:in length anywhere from 2
amino acids to 100 or more amino acids. Chains longer
than approximately 100 amino acids~are typically termed
"proteins". Further, the term "heterodimer polypepti.de"'.
refers to two associated non-identical polypeptide~chains.
The term "derivatized", in the context of a:
polypeptide subunit "derivatized" to include a bioactive
moiety, is~understood to refer to a polypeptide subunit
having one or more functional or bioactive moieties
covalently attached to one or more amino acid residues
forming the subunit, where the moiety may be (i) coupled
to one or more amino acid residues in the subunit either
~30 before or after polypeptide subunit synthesis,. or (ii)-
form an elongation of the peptide subunit, e.g., at_the
subunit's N-terminus. Further, the functional or
bioactive moiety~may be attached to the.polypeptide
subunit directly, or through a linker or spacer, e.g.; a
poly-glycine spacer. _ .
St JE3S'1"~ ~ t jTF SHEET
WO 95!31480 ~ 02372569 2002-03-08 ~ p~pA95100293
Unless otherwise indicated, the sequence for peptides
and polypeptides is given in, the order from the amino
terminus to the carboxyl terminus.
The term "epitope" as used herein,~designates the .
5 structural component of a molecule that is responsible for
specific interactions with~corresponding antibody
(immunoglobulin~ molecules elicited by the same or related
antigen. More generally, the term refers to a peptide
having the same or similar imaiunoreactive properties, such
10 as specific antibody binding affinity, as the antigenic
protein or peptide used to generate the antibody-.
Therefore, an epitope that is formed by a_specific peptide
sequence generally refers to any peptide which is reactive
with antibodies. directed against the specific sequence.
The term "antigen" as used herein,.means a molecule
which''is used to induce production of antibodies'. The
~- w term is.alternatively used to denote a molecule which is
reactive with a specific antibody.
The tens "H-cell antigen" as used herein, means a
.2.0 molecule which is used toinduce production of antibodies.
The term is alternatively used. to denote a molecule that
is reactive with-a specific B-lymphocyte clone, or that
elicits a B-lymphocyte-mediated iiamunogenic response in a
subject or test animal.
The term "T-cell antigen" as used herein, denotes a
molecule that is reactive with a specific T-lymphocyte
clone; or a-molecule that elicits a T-lymphocyte-mediated
immunogenic response in a subject or test animal.
The term"immunogen" as used herein, describes an
entity that induces antibody production in a host~animal.
In some instances the;antigen and the immunogen are the
same entity,.while in other instances the two entities are
different.
All amino acid residues identified herein. are in the
natural or L-configuration unless otherwise specified. In
keeping with standard peptide nomenclature, abbreviations
~.._. . ,~.
~ I,~~'~T: 'T"_; .~. ..
.:.L.i 3~~ ... l ~ . ~..~ ~ :_ ::: ~--''. . ~.T
WO 95131480 , . ~ 02372569 2002-03-08 ~ /~ p~ICA95100293 ,
I1
for amino acid residues are standard 3-letter and/or 1
letter codes commonly used in the art. -
The term '!benign medium" as used herein, describes a
physiologically-compatible aqueous solution typically
having a pH of. between about 6 and about 8 and a salt
concentration of between about 50 mM and about 500 mM.
Preferably, the salt concentration is between about 100 mM
and about 200 mM.. An exemplary benign laedium, designated
as_buffer A, has the following composition: 50 mM
potassium phosphate, 100 mM KCl, pH~7. Equally effective
benign media may be made by substituting, for example,
_sodium phosphate for potassium phosphate and/or NaCl for
KCl.
II. General Overyiew of the Invention
In one aspect, the invention is,a synthetic vaccine ~-
fonaulation having two subunits., each subunit being
comprised of a core golypeptide (CP) and one or more -
antigen molecules (Ag). ~ .
. The core polypeptides are~tWO~~non-identical
polypeptide chains, typically about~2l to about 70
residues in length, having an amino.acid sequence
compatible with their formation into two-stranded cx-
helical heterodimeric coiled-coils in a benign medium:
They are designated hesein~as CP1 (core polypeptide i~,
and CP2 (core polypeptide 2). In benign aqueous, medium
the isolated core polypept.ides are random coils. When CP1
and CP2 are.mixed together., preferably in equal -
quantities, they interact to form a two-stranded a-helical
heterodimeric coiled-coil carrier, designated~as CP1-CP2.
Peptides ~.n an a-helical coiled-coil conformation
interact~with one another in a characteristic manner that
is determined by the primary sequence of~each peptide.
The tertiary structure of~an a-helix is such that 7 amino
acid residues in the primary sequence correspond to
approximately 2 turns of the a-helix. Accordingly, a
primary. amino acid sequence giving rise to an a-helical
~t .l~S' ,TiT~.3TE SHEET'
WO 95131480 ~ ~ 02372569 2002-03-08
PCTlCA95/00293
12
conformation may be broken down into units of 7 residues
each, termed heptads. The core polypeptides are comprised
of a series of heptads in.tandem. When the sequence 'of a
heptad is repeated in a particular core polypeptide, the
heptad may be referred to as a "heptad repeat'., or simply
..repeat" .
As is detailed below, specific types of amino acid
residues at defined positions in each heptad act to
stabilize the two'-stranded a-helical coiled-coil
~10 heterodimeric structure.
CP1 and CP2 mau be independently derivatized, or
decorated, with different antigens (Agl and Ag2) through
amino acid coupling residues. The coupling residues are
. placed at locations.in the sequences of CP1 and CP2 so as
i5' to be positioned at the.outward, or hydrophilic aspects of
an a-helical coiled-coil structure. Antigen-derivatized
carriers are designated.as [Agl];=CPl or CP2-[Ag2]~, where i
and j refer to the number cf antigens attached, to a single
core polypeptide. Antigens are selected such that.when
20 they are derivatized to core polypeptides, they,.do not
block the formation. an a-helical heterodimeric coiled-coil
.structure. [Agi]; CPl.and CP2-[Ag2]~ may be purified prior
to. their assembly into a final immunogenic .structure.
CP.1 and CP2 may also contain residues that can be
25 reacted.(either intra- or inter-helically) to stabilize
the a-helical or coiled-coil nature of the polypeptides.
one example of a stabilizing. modification is the
incorporation of lactam bridges in the first and last
(terminal) -repeats of core peptides.
30 . A complete antigenic structure can be made by mixing
[Agl];-CP1 and CP2-jAg2]3. The decorated core~polypeptides
self-assemble to form an antigen-derivatized~a-helical
coiled-coil structure, denoted as [Agl]; CP1~CP2 -[Ag2]~.
This structure can be used as an immunogen for the
35 production of antibodies or in a'vaccine formulation.'
A~diagram of the general steps outlined above is
shown in Figure 1 for core polypeptides containing 5
SUBS i i"~"L1TE 5i-'~~~'~1' . .. . .
WO 95131480 . ~ 02372569 2002-03-08 ~~ pCTlCA95100293
13 '
heptad repeats (indicated as boxes with varying degrees of
shading), one antigen-binding residue per core
polypeptide, and lactam-bridge modification sites an the
terminal repeats. Part A of Figure 1 shows a schematic of
CP1 and CP2 .after the peptides.had been synthesized under
reaction conditions, detailed in Example 4, to induce the
formation of lactam bridges. Part-B shows a schematic of
CP1 and CP2 after the modified core peptides had been
derivatized with antigens, as detailed for instance in
Example 5. Part C is a schematic of the entire
heterodimeric immunogenic complex, showy for simplicity as
a linear (as opposed to a coiled-coil) structure, after,
mixing the individual decorated peptides as described, for
instance, in Example 6.
In another aspect; the invention includes polypeptide
complexes comprised of two core polypeptides (as described
above), each of which has different bioactive moiety °-
attached to it. The bioactive moieties attached to the
core polypeptides are not necessarily antigenic,-but
typically serve a therapeutic or targeting function. The
individual core polypeptides derivatized with~the
bioactive moiety.may be administered,together, in a
coiled-coil configuration as described below, or they may
be administered separately and allowed to foria coiled-coli
heterodimers in the animal or subject to which they are
administered.
III. Features of Corg Polvpentides
The two core polypeptides (CP1 and CP2) are of
similar, if not identical size, each~typically ranging
from about 21 to about 70 residues (3..to 10 heptads) in
length. ' .,
The peptides may be'synthesized by .a variety of
methods known to those skilled in the art. For example,
an AHI Model 430A peptide synthesizer may be used with
conventional t-Hoc chemistry as described previously by
Hodges, .et al., (1988), and in Example 1.
-~e ~~~;';1TUTE S1~"'q~E
' CA 02372569 2002-03-08
W 0 95131480 pCT/CA95100293
I4
. y Subsequent to synthesis, the peptides are purified by
any of a number of methods known to those skilled in the
art; for example using reversed-phase high performance
liquid chromatography (RPC) and a "SYNCHROPAIC"~~RP-P
column, as detailed in Example 1.
The composition,and purity of the peptides can be
verified by several methods, including amino acid
composition mass analysis 'on a Beckman model 6300 amino
acid analyzer~and molecular weight.analysis using time of
flight mass spectroscopy on a "BIOION-20"i~Nordic, as
detailed in Example 1:..
A. Coiled-Coil Formation
The dimerization of CPi and CP2~occurs due to the
presence of a repeated heptad motif. of conserved amino
acid residues. The individual positions in each heptad
are designated by the letters a through g for Cpl, and a'
through g' for CP~,~as shown'in Figures 2a and 2b. The
positions (e.g., a~, g') of CP2 are sometimes referred to
without the~(~) symbol in general discussions of heptad
positions in core heterodimers, below. ..
An appropriate.heptad motif,~or repeat, directs the
CP1 and CP2 polypeptides to assemble into a heterodimeric
a-helical coiled-coil structure under permissible
conditions, presented in part D, below. The individual a-
helical peptides contact one another along their
respective hydrophobia faces, defined as the a and d
positions of each~heptad.
CPi.and CP2 may assemble into a heterodimer coiled-
~30 coil helix.(coiled-coil heterodimer) in either parallel or
antiparallel configurations. In a parallel configuration,
. the two core polypeptide helixes are aligned such that
they have the. same orientation (amino-terminal to
carboxyl-terminal). In an antiparallel configuration, the
helixes'are arranged such that the~amino-terminal end of
one helix is aligned with the carboxyl-terminal end of the
other helix, and vice vez~sa.
* Trademark ~~ 3~~"~.i~:~.~.~ ~~,.~C~T
WO 95131480 ' - ~ 02372569 2002-03-08 ~~ p~~CA951()0293
Diagrams of the relative orientations of the a-g
positions of two interacting a-helices are shown in
._ Figures 2a and 2b. Figure 2a shows an end-on schematic of
the first two turns (one heptad) of two exemplary core
5 polypeptides, EE and KK (SEQ ID NO:l and SEQ ID NO:2)
arranged in_a parallel configuration.- Figure 2b shows an,
end-on schematic of the same core polypeptides arranged in
an antiparallel configuration.
Core polypeptides designed in accord with the
l0 guidance~presented~herein typically show a slight
preference .for assembling in a parallel orientation vs. an
antiparallel orientation. Generally, hoiaever, the
. orientation (parallel vs. antiparallel) in which the two
core polypeptides form an a-helical coiled coil. is not
15 necessarily relevant to their function as carriers for
bringing together moieties attached to the core
polypeptides. .-
y ' In Figures 2a and 2b, amino acids are circled and
indicated by the one-letter code, and consecutive amino
acid positions. are numbered and joined by lines. with arrow
heads indicating the N-terminal to C-terminal direction.
- Interactions between the two helixes are indicated by
arrows. Wide arrows crossing between the helixes depict
hydrophobic interactions between the a and d positions of
adjacent helixes..
Ionic:interactions between the a and g-positions of
adjacent,helixes are indicated as curving arrows above and
below the nexus of the helixes. ~ Position a of peptide EE~
(SEQ ID N0:1) is a Gln.in the first and last heptad, and a
Glu in the internal heptads. The (bottom) curving arrow
depicting. ionic interactions with this position is drawn
with a dashed line to.indicate that~ionic interactions are
present between internal heptads of the helixes, but not
between the'first and last, cr'terminal, heptads.
_ ' Lactam bridges are indicated as a right-angle line
between the f and b positions within each helix.
~ = .,-..._. ........ .- -~r
WO 95131480 ~ 02372569 2002-03-08
rcrica9s~ooz93
16
8. Hydrophobic Interactions in Coiled-Coil
Vita 'litv
The hydrophobic interactions between the helixes are
due to hydrophobic residues at the a and .depositions of
the core polypeptides. Residues at these positions,v
effective~to maintain the helixes in contact,v include
leucine, isoleucine, valine, phenylalanine, methionine,
tryptophan, tyrosine, alanine.and derivatives of any of
the above. Other residues, including alanine, cysteine,
serine, threonine, asparagine and glutamine may also
occupya or d positions in some heptads, so long as others
are occupied by hydrophobic residues:
Appropriate selection of the specific residues to
occupy the a and d positions is an important aspect of the
present invention. If the hydrophobic interactions are
strong, as is the case, for example, between helixes
containing Ile at one of the positions and Leu at the
other gosition, a significant fraction of the helixes will..
form as homodimers at pH 7, even if like-charged residues
2.0 are present at. the a and g positions~to discourage
homodimer formation .(see part C.., .below). If, on the
other hand, residues at the a and d positions are selected
such that the hydrophobic interactions are~toy weak (for
example, Ala at both positions), the helixes'may not form
coiled-coil~dimers at all. Preferably, residue'pairs are
selected that promote the fonaation >_ 95% heterodimers at
pH 7. The. degree of heterodimer vs. homodimer formation
may be measured as described, for instance., in Example 3.
An exemplary pair, of. residues at the a and d positions,
3o that results in hydrophobic interactions conducive to __>95%
heterodimer formation at pH 7, comprises Leu at one of the
positions and Calmat the other.positipn. These residues
are present at the a and d,positions of exemplary core
polypeptides EE (SEQ ID N0:1) and KiC (SEQ,ID N0:2).
~1~8~TETLJ-~'~ ~HEE'Z'
WO 95131480 ~ 02372569 2002-03-08
PCTJCA9S/00293
17
C. Ionic Interactions in Coiled-coil Stab~.litv
Dimeric coiled-coil conformations of a-helixes can be
stabilized by ionic interactions between residues at the a
and g positions of adjacent helixes, as is illustrated in .
Figure 3. If each helix of a dimer has a positively-
charged~residue at one position, for exaiaple, e, and a
negatively-charged residue at the other position, for
example, g, homodimer formation is favored (Fig. 3A;
compare with heterodimer in Fig..3B)., However, if each
helix has like-charged residues at both positions, then
two oppositely-charged helixes will. tend to associate into
heterodimers (Fig. 3D),- a5 opposed to forming homodimers
(Fig. 3C, 3E). ,
The conformation of polypeptides, such as CPl and
CP2, in solution can be~determined from CD spectra of the
solution. These data provide information as to the
conformation of the. individual peptides themselves (random
coil vs. a-helical), as well information as to the
relative amounts of heterodimer vs. homodimer complexes
of,.for example, CP1 and CP2. Example 2 details one
method of measuring CD sgectra. ,Example 3 details how a .
CD spectra measurements can be used to assess the
conformation of peptides in solution.
In the. diagram shown in: Figure 2, ionic interactions
between the two helixes arise from negatively-charged
(Glu).residues at the s and g positions on CP1 (EE;v SEQ ID
NO~.1), and positively-charged (Lys) residues.at the a and
g positions on CP2 (RK; SEQ ID N0:,2).However, the
terminal heptads of peptide EE (SEQ ID NO:1) have
uncharged residues (Gln) at the a position, as opposed to
the charged Glu.at.that position in internal repeats.
Accordingly, ionic interactions inv~lving the a position
of EE will occur at internal, and not terminal, repeats.
Negatively-charged residues can be aspartic acid,
glutamic acid or derivatives thereof. Positively-charged
residues can be lysine, arginine, histidine, or
derivatives thereof.
.r.
CA 02372569 2002-03-08
WO 95131480 PCT/CA9510U293
18
Ionic interactions between other positions in a
heptad_may also exert significant influences on helix
stability. For, example, position a in EE carrier peptide
(SEQ ID NO:1) terminal repeats is a Gln, as opposed to a
Glu, because Glu residues at both.positions would tend to
destabilize an a-helical conformation through ionic
repulsions (see Figs. 2a and 2b). Certain destabilizing
effects, however, may be overcome by introducing.
stabilizing covalent modifications, such~as lactam bridge
formation discussed below in part E.
D. Conditions Favorable for Coiled-coil Formation
Core polypeptides comprised of repeating heptads and
designed according to the guidance presented in parts A
through C, above, will readily form coiled-coil
heterodimers in a benign medium, defined above in part I.
The degree of a-helical coiled-coil heterodimer formation
can be determined from CD spectra, as described, for
instance, in Example 3.
Coiled-coil heterodimers may form under conditions.
outside the pH and salt range given f.or.a benign medium,
but some of the molecular interactions and relative
stability of heterodimers vs. homodimers may differ from
characteristics detailed above. For examples ionic
interactians between the a and g positions that tend to
stabilize heterodimers may break down at low or high pH
values due to the protonation of, for example, GIu side
chains at acidic pH, or the deprotonation.of,.forexample,
Lys side.chains at basic pH.
. Aforementioned effects of low and high pH values on
coiled-coil heterodimer formation maybe overcome,
however, by increasing salt concentration. Increasing the
salt concentration can neutralize the stabilizing ionic
attractions or suppress the destabilizing ionic
35, repulsions. Certain salts have greater efficacy at
neutralizing the ionic interactions. For example, in the
case of the KK peptide (SEQ ID N0:2), a 1M or greater
,..,..
ct.1~3~?~~~~~' ~ ~H~~ ~
WO 95!31480 ~ CA 02372569 2002-03-08 ~ p~~CA~gI00Z93
19 ,
.concentration of ClOa anions is required induce maximal a-
helical structure (as determined by CD measurements
performed as detailed in Example 2), whereas a 3M or
greater concentration of C1' ions is required .for the same
effect. The effects of high salt on coiled-coil formation
at low and high pH also show that interhelical'ionicw
attractions are not essential for~helix~formation, but
rather, control whether.a coiled-coil tends to form as a
heterodimer vs. a~homodimer.
E. Heutad~ Variation in Core Polvneuti
Parts A,~ H and C, above, present guidelines as.to
which amino acid residues may be included, and which amino
acid residues are.preferable, at specific positions in . .
heptads of core polypeptides that w~ill.typically result ~in
those peptides forming a-helical coiled-coil structures in
a benign medium. This part describes some examples of how
heptads with sequences which are in compliance~with the
guidelines presented in parts A.through C, above, can be
arranged within the core polypeptides.
Core polypeptides of the present invention may each
contain~.from three to a plurality of hegtads. The
sequences of each of those heptads may all be the same, or
.they may differ. .In particular, the sequences of the
first and last heptads, or terminal repeats, may differ
fxom the sequences of the interior yr intermediate heptads
or repeats. Furthermore, the sequences of the internal
repeats ~aay differ from one another depending on, for
example, whether or not the repeats incorporate amino acid
coupling residues. ' -
For example, peptide EE (SEQ ID NO:i)'has a total of
5 heptad repeats. The two terminal repeats have the
sequence represented by SEQ_ID~N0:3, and the three
intermediate repeats have sequences represented by SEQ.ID
N0:.4 and SEQ ID NO:S. The sequence represented by SEQ ID
N0:5,,present in~the central repeat, differs from the
_ ,.. ~~T
SL.jE3'r''~ .~_".~ "~ ,.:.~"'~
..,:
CA 02372569 2002-03-08
WO 95131480 PCTICA95100293
internal repeat sequence (SEQ ID No:4) by the presence of
a cysteine coupling residue. Peptide KK (SEQ ID N0:2)
also has a total of 5 heptad repeats, and the repeats are
arranged in-a manner analogous to those of peptide EE.
5 The two terminal repeats. of .KK have the sequence
represented by SEQ ID N0:6, and the three intermediate
repeats have sequences represented by SEQ ID N0:7 and SEQ
- ID N0:8; with the sequence represented by SEQ fD N0:8
including a cysteine coupling residue.
l0 The terminal repeats of both the EE and KK peptides
incorporate residues designed to form lactam bridges. to
stabilize an a-helical conformation. The. central internal
repeats of both peptides contain amino acid coupling
residues (cysteines), and are termed "peptide conjugation
15 internal repeats". ~.
Many other variations in heptad.arrangement are
possible. For example, it.may be desirable to design a
core polypeptide with a~different amino acid coupling
residue on each intermediate repeat, i.n order to couple
20 different compounds at defined positions on one core
polypeptide. This strategy is discussed in more detail in
part G, below. Alternatively, one may want to place a
unique coupling residue on one of the repeats on one or
both core peptides to anchor them to a resin or another
. 25 polypeptide.
Because the salient interactions between. two core
polypeptides in an a-helical coiled-coil heterodimer pair
are between adjacent, "complimentary" heptads in each
peptide;. the primary sequence of heptads Within a core
polypeptide can vary, so long as the residues.within each
heptad interact:favorably with residues in the
complimentary heptad of the second core polypeptide.
It follows, then, that adjacent heptads may vary in
sequence such that, for example, the net charge on the
core polypeptiiies can be altered without affecting the
ability of the polypeptides o form a-helical heterodimer
coiled'-coils. This relationship is illustrated in Figure
"l., 3~~ ~ i'~ ~~~
~t.1~ST~ ' "
CA 02372569 2002-03-08
WO 95!31480 'PCT/CA95100293
21
4. The figure shows three examples of CP dimes pairs.
Each core polypeptide, has 5.heptads. The + or'- symbols
in each heptad. each represent two charges (one'at'the a
position and one at the g position). Note that adjacent
complimentary heptads have opposite charges. For the
purpose of this example, it is assumed that positions
other than a and g in each heptad sum to a net charge of
zero. It can be appreciated that CP1 and CP2 forming the
dimes in Figure 4A have net charges of +2 and -2,
respectively, due to an excess of one positively-charged
heptad, and one negatively-charged heptad, respectively.
Similarly, CPi and CP2 in Figure 4B have net charges of +6
and -s, respectively,, and CP1 and CP2 in Figure 4C have
net charges of +10 and -10, respectively.. Other
25 -variations an this theme are, of course,~possible without
departing from the spirit of the invention. .
Peptides EE (SEQ ID NO:1) and KK (SEQ ID N0:2) arev
similar to the case schematized 'in Figure 4C, in that the
a andvq positions of all heptads comprising peptide EE ,
have a.net negative charge, whereas, the a and g positions
of all heptads comprising peptide KK have a net positive
charge.
F. Covalent Modification of Core Polypepaides.
~ The core polypeptide sequences may also include
residues designed to stabilize the c~-helical conformation
of each core polypeptide in a~coiled-COil~dimer. For
examp3e, peptides EE and KK have gTutamic acid. and lysine
residues. at the b and f -positions, respectively, of, the
terminal repeats. These residues can react under the '
appropriate conditions, detailed in Example 4, to form a
lactam bridge, as schematized in Figure L: Lactam bridges
at these positions stabilize an a-helical conformation.
G. Bioactive Moiety.Couu~4ina to Core Polvoegt.ide~
Another aspect of the invention includes the
incorporation of amino-acid coupling residues at positions
St,~!'.~~'.i-r-'-%"~'"~ ~';"~~~T
'CA 02372569 2002-03-08 .,
WO 95/3!480 PCT/CA95100293
22
b, c and/or f of one or more heptads. These positions lie
along the outward~face of a coiled-coil heterodimer. Each
heptad may contain up to three coupling: residues.
.Various embodiments ara possible. For example, the
amino acid coupling residues may be incorporated in the
internal repeat. sequences, but not in the terminal repeat.
sequences. Additionally, coupling residues may be
simultaneously incorporated at all three positions, at two
of the three.posi'tions, or 'at only one position (for .
I0 example; f) in each heptad. I~t an exemplary embodiment
(EE,~KK peptides; SEQ ID N0:1 and 2)., the coupling
residues are cysteines placed at the f position of the
central.heptad of each core polypeptide.
Preferred coupling groups are the thiol groups of
cysteine residues, which are easily modified by standard.
methods: .Example 5 details how the cysteine.thiol groups
. present in the peptide~conjugation internal repeats of
peptides EE (SEQ ID N0:1) and~KK (SEQ ID N0:2) can be used
to attach antigenic peptides at those positions.
Other useful coupling groups include the thioester of
methionine; the imida2olyl group of histidine, the
guanidinyl group of arginine, the phenolic group of
tyrosine and the indolyl group of tryptophan,. These
coupling groups can be derivatized in a manner similar to
that detailed in Example 5, using reaction conditions
knoww to those skilled in the azt. ,
As was mentioned~in part E, above, it may be
desirable to incorporate. a different amino acid. coupling
residue in different heptads comprising,a core
polypeptide, allowing the attachment of different antigens
on a single core polypeptide in defined locations. The
cone polypeptide can be sequentially decorated with the
different antigens by carrying out a series of coupling
reactions. A single antigen is coupled to the core-
polypeptide in a given reaction step. In cases where the
antigen is~a peptide,of less than approximately 40 amino
acids, it is typically desirable to add a spacer between
ct iF~~'"~~T'~.l'~'E 5i1~'~~~.'~'
WO 95131480 cA 02372569 2002-03-08 ~~ p~/CA95/00~93
23
the antigenic peptide and the core polypeptide. The
spacer may comprise, for example, 2 to 5 amino acids. Two
exemplary spacers (one for the TT2 peptide, SEQ ID N0:12-,
and the:other for the PAK peptider SEQ ID N0:18) are
detailed in Example 5. . '
Iri a~ preferred embodiment of the invention, the .
bioactive moieties are peptide antigens linked through
amino acid spacers to the coupling. residues.
In another aspect of the invention, bioactive
moieties, such. as antigens, may be coupled to the core
poJ.ypeptides not via the amino acid residues at positions
b, c and/or f, but rather, directly in or. at either end. of
the core polypeptide (e.g., at the ~1-terminal or C-
terminal end). Such coupling may be carried out using
either synthetic or recombinant approaches: In a . ..
recombinant approach, polynucleotide sequences encoding
the core polypeptides. and the bioactive moieties (e.g., .
antigenic peptides) are engineered into suitable.
expression plasmids using methods known to-those skilled
2o in the art (e.g., Maniatis, et al., Ausubel, et al.). In
one embodiment, fusion peptides, containing.a core ,
polypeptide or a core polypeptide in tandem with a
. bioactive moiety may then be,produced by inducing
expression of the plasmids in a suitable express;i,on ~syste~m
and purifying the expressed fusion protein.
The expression plasmid typically contain the
following elements: an origin of replication (ori), a
selection marker (e. g., ampicil.lin; Amp-R), a,promoter
(e..g.,- lac promoter/operator; 1ac p/o), a multiple clloning
3o~ site (NiCS) and a aranscription term.iriator. The plasmid
may contain a number of other elements,~such as signal
peptide sequences .(e . g. , ompA) , f.1 ori, a f lag or.. of f inity
sequence to facilitate purification.of the recombinant
protein (e:g., a His tail) and the.like.
Figures 5-Z2 illustrate maps (Figs. 5, 7, 9 and 1l)
and the poly3inke-r regions (Figs. 6, 8, 10 and. l2) of four
exemplary..plasmids suitable for generating. recombinant
S~~'v.i f i"i tJ 1 IW'.~l"'1
~CA 02372569 2002-03-08
WO 95131480 ~ PCTICA95100293
24
polypeptides useful in the.~practice of the present
invention. Fig. 5 shows a map of plasmid pRLD-E, an E.
cold expression plasmid modified from pASK40 (Skerra, et
a1.) by changing the polylinker sites to correspond to the
polylinker of pHIL-Sl and PIC9 (both pHIL-S1 and PIC9 are
. commercially available from Invitrogen, San Diego, CA).
Fig. 6, illustrating the polylinker region (promoter, MCS
and insert) of pRLD-E, shows. that pRLD-E contains
polynucleotide sequences (SEQ ID N0:19) encoding the~E-
coil peptide (SEQ ID N0:20), comprised of five repeats of
the EE internal repeat (SEQ ID N0:3) in tandem with a 5-
' residue His tail. w
Fig. 7 shows a map of pTasmid pRLD~K, Which is
identical to pRLD-E with the exception that ~it contains , .
polyriucleotide sequences (SEQ ID_N0:2I) encoding the K-
coil peptide, (SEQ ID N0:2~2) , comprised of five repeats of
the KK internal.repeat (SEQ ID N0:7) in tandem with a 5-,
residue His tail (Fig. 8).
Fig. 9 shows ~a map of plasmid pHIL-S1/E; a yeast
(e.g., P~chia pastoris) expression plasmid constructed by
cloning an EcoRI/BamHI.fragment containing a signal
cleavage site, a.sequence~encoding a poly-glycine spacer
('8 glycines),.a sequence (SEQ ID N0:19) encoding the E-
coil peptide (SEQ ID N0:20) another poly-Gly spacer and a
His tail (Fig: 10) into the EcoRIJBgIII sites of pHIL-S1
(Invitrogen). Fig. 11 shows a map of plasmid PHIL--S1/K,
Which is identical to pHIL-Sl/E with the exception that
the insert contains nucleotide sequences (SEQ ID No:21)
eneoding~the K-coil peptide (SEQ ID N0:22) instead of the
E-coil peptide (Fig. i2).
Figs. l3 and l4 show nucleotide andytranslated amino
acid sequence of exemplary fusion construct insert
fragments for making recombinant polypegtides suitable for
use in vaccine compositions and methods of the pre ent
- 35 invention. The fragment in Fig. 13 (SEQ ID~NO:23)
contains a nucleotide sequence (SEQ ID N0:25) encoding a
'PAK antigen (PAK 128-144; SEQ ID N0:18, S~Q ID N0:26) .
CA 02372569 2002-03-08
WO 95!31480 PCTICA95I00293
cloned upstream of the sequence encoding 8 glycines in the
polylinker region of pHIL-S1/E. The fusion polypeptide
produced from such a fragment, in combination with a
correspondiiig decorated peptide, may be particularly:
5 ~ useful as a vaccine composition against Pseudor~onas
aeruginosa, and may be evaluated for such use~using, for
example, the protocol in Example 8. .
The fragment in Fig. 14.(SEQ ID N0:27) contains a
nucleotide sequence (SEQ ID N0:29) encoding an MVF T .y
10 antigen (mea~sLes virus F protein.; region 288-302; SEQ ID
N0:15, SEQ ID N0:30) cloned upstream of the sequence
encoding 8 glycines in the polylinker region of PHIL-S1/E.
Expression pla-smids such as those described above may
be transformed into, suitable host cells,~:such as bacteria
15 or yeast; and induced to produce recombinant polypeptides,
which may then be purified using methods known to those
skilled in the art and employed for uses such as~are
detailed herein. Fusion polypeptides containing a poly-
iiis tail., such as those described above, may be
20 conveniently purified by means ofimmobilized metal ion
affinity chromatography (IMAC; Hochuli; Porath):
The pHIL and pPIC -derived vectors are especially
suitable f.or high level expression of recombinant
polypeptides. They employ a methanol-regulated alcohol
25 oxidase (AOX) promoter which-is particularly useful in
Pichia pastoris host cells (for example, the AOX promoter
is used in pHIL and pPIC vectors included in the Pichia
expression kit, available from Invitrogen, San Diego; CA).
The plasmids are used to transform Pichia pastoris (strain
GS115; Invitrogen) spheroplasts, and the transformed cells
used~to produce recombinant polypeptide according to the
manufacturer's 'instructions. .
The pRLD-derived vectors may also be employed for
expression of recombinant polypeptides of the present
invention. The plasmids are used to transform E. coli
cells (e. g., JM83 cells}, the cells are induced.with
isopropyl-/3-thiogalactopyranoside (IPTG), the outer
.r..-. .rr . ! Yr 1°'~~'
WO 95131480 ~ 02372569 2002-os-oa "~ p~pA9510~293
26
membrane is broken, and the periplasmic membrane proteins
are isolated and passed over an Ni'" IMAC column (Hochuli;
Porath) for purification.
Recombinant proteins purified as described above may
be further purified and/or modified using methods .known to
those skilled in the art (e.g., as were,used for
synthetically-produced peptides described herein) prior to
the use of such proteins in the practice of the present .
invention.
l0 A variety of bioactive moieties may be expressed in
tandem with a carriex polypeptide, such, that.they form a
single polypeptide chain, to form a decorated peptide.
They include antigens, such as exemplified in the
constructs shown in Figs. 13 and 14, far use as vaccine
compositions as well as other polypeptides, such as cloned
antibodies. The antibodies may, be directed, for example,
against a pathogen (P. aeruginose) or against an antigen:
expressed on a tissue to be targeted by 'specific drugs
(e: g., a tumor tissue). Cloned human antibodies directed
against pathogens may be particularly useful, since they
typically do not generate an immune response when used in
humans.
The specific moieties selected will depend on the
application,.and can be readily determined by one of
ordinary skill in the art following the guidance herein.
Among the suitable applications for the present invention,
are a delivery system for use in binding assays (e.g., one
subunit contains an antibody, and the other contains a
detection moiety, such as alkaline phosphatase.(AP) or ~-
galactosidase)_, a delivery system for a vaccine
composition, and an affinity protein purification. system
(e.g., with one subunit..derivatized to a column and the
other containing an antibody directed against a desired
polypeptide).
Exemplary carrier molecules CP1 and CP2 employed in
the recombinant methods described 'above are the E=coil
peptide (SEQ ID N0:20) and the K-coil peptide (SEQ ID
W0 95131480 ~ ~ ~ ~ 02372569 2002-03-08 ~ pC'f/CA95/00293
N0:22~) . They differ from EE (SEQ ID NO: I) and KK (SEQ ID
NO:) peptides, respectively, in that the E-coin and K-coil
peptides are comprised exclusively of "internal" repeats,
rather than containing the ''terminal" repeats at their
ends, but have characteristics (conditions favoring coil-
coil formation, etc.) comparable to those of the EE and KK
peptides.-
H. Generating Antigen-decorated Heterodimers
The individual antigen-decorated core peptides may be .
purified as detailed in Example 1, precipitated and
lyophilized, by standard methods. Antigen-decorated
heterodi:mers may be generated by,mixing purified [Agl);-CP1
with purified CP2-[Ag2]~1, as described in Example 6 for the
[PAK~1-KK ([PAK]-KK) and EE-[TT2]1 (EE-[TT21) decorated
core .polypeptides. ~ - .
The peptides are individually resuspended in a benign
medium, for example buffer-A, at a concentration,of .
between about 0.25 mM and O.S mM. Approximately equal
amounts of each peptide suspended in so3.ut'iori are combined
and allowed to react for between'5 and IO minutes at room
temperature. The fraction of peptides in a coiled-coil
vs.'a random orientation.is assayed using a C~D ~.
measurement, as detailed in Example 2. Typically, over
% of the total protein is in an a-helical heterodimeric
coiled-coil~conformation.
Alternatively, equal portions of lyophilized mixtures
of the peptides can:be mixed and resuspended in .benign
medium.
IV. Advantages for Vaccine Development .
Important features of the present invention related
to vaccine development include (i) two or more different
types of antigens, comprised of a plurality of individual
35' antigenic polypeptides,.can be incorporated into one
immunogenic macromolecule of well-defined structure, (ii)
the components are synthesized and purified to homogeneity
, ..~, r-' .....,.
CA 02372569 2002-03-08'
WO 95!31480 " PCTICA95/00293
28
prior to their assembly, allowing for :control over the
composition at each step of synthesis, and enabling the
production of a pure, well-defined product and (iii) a
high~concentration of antigens can be achieved in a
relatively small volume..
These features are advantageous in the design of
effective and reproducible vaccines.
An effective vaccine must elicit a strong. immune
response. To elicit an immune response that affords
potent and prolonged protection, it is desirable. to
stimulate both B- and T-lymphocytes (B- and T-cells;
Benjamin, e't a1.). B=cells respond to circulating
antigens that bind to specific 'immunoglobulin (Ig)
receptors on their surface, whereas T-cells are stimulated
by binding to antigens that had been internalized,
processed and appropriately presented by antigen
presenting cells (ADCs). APCs present foreign antigens on
their surface as antigen fragments bound to the major
histocompatibility complex (MIiC), for recognition by.T-
cells bearing the appropriate T-cell receptor complex
(Abbas, et a1.).
B-cell and T-cell epitopes,are typically not '
identical, even though both may be derived from the same
immunogenic molecule (Benjamin, et al.). Effective T-cell
antigens are, usually amphipathic helixes, presumably
because the hydrophobic face interacts well with a groove
in the I~iC type IZ and the hydrophilic face is exposed to
the extracellular medium for interaction With the T-cell
receptor. (Berzofsky) .
~ The strongest immune responses are mounted when a B-
cell functions as an APC. Thisybrings,B- and T-cells in
close proximity and increases the effectiveness of
cytokines, released by both cell types, that.stimulate the
cells to proliferate and generate "memory" cells.
A B-cell. displaying the appropriate Ig antibody binds
a B-cell antigen o.n a foreign antigenic molecule,
internalizes the molecule, processes it, and displays a T-
r., ., ...-. .-.---,--v ts~ C i-.i1 ~ E="~'
WO 95/31480 . ~ 02372569 2002-03-08 ~ p~~CA95100293
29
antigen fragment in association with an MHC type II for
binding by an appropriate helper T-cell.
Native antigen molecules typically contain both B-
and T-cell antigens, and are thus capable of eliciting a
strong immune response. There are several disadvantages,
however, to using intact proteins as antigens ,
particularly in human vaccines. They include (i) the
chance of generating antibodies against a part of an
antigenic molecule that is variable among closely-related
.strains of the pathogen (thus reducing the effectiveness
of the vaccine) , and (~.i) the ohance of generating
antibodies to an epitope that is similar to one in an
endogenous,protein, thus increasing the risk of developing
an autoiminune response. Furthermore, obtaining intact
~ protein in large quantities and of sufficient purity for
use in humans is difficult. The purification~of crude
antigens isolated~from the pathogenic organism is tedious,
costly'and carries with it.the risk of infection for
individuals involved in production. Large-scale culture
of bacteria or yeast to harvest and purify recombinant
proteins in the amounts required for.vaccination of a
v large number of individuals is impractical (Good).
The present invention offers an alternate to thewse
of intact antigenic molecules for eliciting a strong
immune response. According to one method of- the
invention, a synthetic polypeptide comprising., for
example, a B-cell epitope is.derivatized to CPl, and a
synthetic polypeptide bearing, for example, a T-cell
epitope is-derivatized to CP2. The decorated polypeptides
are purified and mixed to form a-stable heterodimer
coiled-coil structure-decorated on its outer surface by
e.pitopes of interest. .
A vaccine formulation made according to the present
invention may thus. incorporate well-characterized,
effective H-cell antigen peptides.together with proven and
effective T-cell antigen peptides coupled to a single
molecule: Such a formulation is highly reproducible
-.v s ! "1 t~_'~'~,'~~r~~ ~T
CA 02372569 2002-03-08
WO 9513.1480 ~ PCTICA95100293
because the antigens arewpresent in a pre-defined
molecular orientation and stoichiometry that-~is
essentially invariant from batch to batch.
Among the advantages of the present invention are
5 that the exact structure- is known,~there are no v
contaminants which may themselves be anti.genic~, produce
tissue irritation; or other undesirable reactions, the
exact amount and orientation of the antigen is known, the
antigen is symmetrically-distributed on the carrier; the
l0 components- can be purified independently to homogeneity
prior to final assembly and the carrier can:be utilized as
a base for more than one antigen, se that multivalent
vaccines can be produced. Unlike previous systems using
natural carriers such as keyhole limpet hemocyanin,
i5 tetanus toxoid and bovine serum albumin, the carriers of
this invention are fully-defined chemical entities to
which the antigens are derivatized in known orientations
and stoichiometries.
. The present invention addresses the above-identified
20 shortcomings of current immunogenic formulations and
vaccines, and furthermore pro~tides a-general method of
assembling and presenting~two different bioactive moieties
in a well-defined special orientation and stoichiometry.
25 V. Selection of Peptide An~iaens
Tn a preferred embodiment of the invention, the
substances linked~to the core molecules are antigenic'
peptides, for the construction of antigenic~formulations
to be used in antibody production or vaccine development.
- , 30 In an exemplary embodiment, the invention inchides a 8-
cell antigen linked to one core polypeptide (e. g., CP1,),
and a T-cell antigen linked to the other core polypeptide
(e: g., CP2).
A. B-cell Anti ens
Effective vaccines result in the production of
antibodies by 8-cells in the vaccinated individual which
~t i:~~ a ' ~ ~.~~ C6-9~'~'i"
WO 95!31480 CA 02372569 2002-03-08 ~ p~~CA95/00293 . .
3Z
are directed against epitopes~of~the pathogen,.. Some
epitopes are more antigenic than others, and readily
stimulate the production of potent antibodies effective to
inactivate the pathogen. The identification of
particularly antigenic B-cell epitopes; and epitopes that
will significantly inh;ibit.the pathogen, depends on the
resources available. The~techniques for such an
identification; however; are w,e:Cl-known to those skilled
in the art. Several examples are~listed~below.
10, If the DNA encoding a particularly antigenic protein
of the pathogen has been cloned.,- it may be 'possible to use
one of a number of computer programs to identify regions
of isolated sequences that are likely to encode protein
antigenic determinants (for example, Hopp, et al.;
'ANTIGEN," Intelligenetics, Mountain View CA).
If sera from infected individuals.are available, one
can screen the sera, either individually or in..a mixture,
using an ELISA assay such as is described in the Materials
and Methods section of the present invention, to identify
reactive proteins or peptides.
If an animal-model for a disease or affliction
exists, one can screen antibodies generated. against
defined.proteins or peptides of the pathogen for the
antibodies' ability to neutralize the infectivity.of a.
virulent mixture of pathogen administered to the model
animal.
Effective antigens may also be~identified as regions
of pathogen proteins that are involved in specific host=
pathogen interactions in the disease cycle. This is true
particularly in cases whew a cellular model for the
disease or, affliction exists., as in.the case for example,
for~Pseudomonas aeruginosa infection. As demonstrated, by
Hodges, et al. (1993.), peptides derived from exoenzyme S
(.Exo S), a bacterial toxin having ADP ribosyl transf erase
activity which ,is present on the.surface of P.. aeruginosa
cells, and antibodies directed against these peptides, are
effective to block the attachment of P, aeruginosa and
~,.~-r
.~..T1~~ ~ . ~ ,.
i~lV.: ~ . ' ..
~ 02372569 2002-03-08
WO 95!31480 PCTICA95/00293
32
other micro-organisms to tracheal epithelial cells (TECs)
and buccal.epithelial (BECs). The Exo S peptide antigen
includes the sequence represented by 5EQ ID N0:9.
Otheryexamples of peptides that are effective B-cell
antigens and that can be used in vaccine formulations.
designed to. protect against the respective organisms
include the~MVF peptide from measles F protein (residues
288-302; S~Q ID NO:I5; SEQ ID N0:30), the HBV peptide (a
hepatitis T antigen; SEQ ID N0:16), the CSP peptide from
P. vivax CSP protein, residues 317-336 (SEQ ID N0:17), and
the PAK Peptide (P. aeruginosa strain K pilin antigen,
residues 128-144; SEQ ID, NO:-18, SEQ ID N0:26).
Although some B-lymphocytes have been found to
interact directly with certain antigens, the majority of-
B-cells, and all memory B cells, have been found to
require cooperation with T-cells before they can
differentiate towards antibody secretion: A brief summary
of.T-cells and T-cell antigens, as it relates to aspects
of the present invention, is_presenbed below.
B. T-cell Antigens -
In many cases, pathogen epitopes that,. due to their
accessibility, structural invariance among different
pathogenic.strains, or unique role in the life cycle of
the pathogen, would be. well-suited for targeting by a
vaccine, are not particularly antigenic. The antigenicity
of these epitopes can be increased by coupling them to a~
highly immunogenic carrier protein, such as tetanus
toxoid.' Unfortunately, this strategy has not been
uniformly successful in clinical trials (Etlinger, et
a1.). One reason may be that the carrier proteins were
themselves used in previous vaccinations of the
individuals, either as carriers for other vaccines or as
vaccines themselves (e. g. tetanus toxoid), and have
resulted in epitopic suppression. Epitopic suppression
occurs when pre-immunization with a carrier protein can
inhibit the subsequent antibody response to new epitopes
SUB '~a''i :'TINE SHEET
CA 02372569 2002-03-08
WO 95/3148f~ PCTlCA95/00293 w
33
attached to the carrier protein (Herzenberg, et al.). By
using peptides derived from antigenic carrier proteins,''as
opposed to the intact carrier proteins, epitopic
suppression can be made advantageous. It appears that
certain such peptides, termed "helper" peptides (Francis,
et al.), are recognized by previously-primed htelper T-
cells, but not by cells responsible for suppression (H-
cells and suppressor.T-cells).
Some helper peptides, in combination with B-antigens,
l0 elicit.immune responses that are genetically restricted to
only one or: a few alleles Qf class II I~:C. This
phenomenon of I~iC "restriction" arises from the fact that
T-cells do not recognize the native protein,,but a
processed for:a of protein antigen. Therresulting
~15 fragments must presented on the surface of. cells bearing
the sane haplotype as the T-cells themselves, but not one
cells bearing different haplotypes. Recent. data show that
some T-antigenic peptides are permissive in their
interaction with a wide range of M~iC haplotypes (Fio, et
20 al.). In particular, peptides derived from tetanus toxoid
ale typically very effective at stimulating T-cells. .
These peptides include the TTO peptide (.tetanus
toxoid residues 88-99; SEQ ID N0:10), TT Peptide. (also
referred to as TT12, tetanus toxoid residues 580-599; SEQ
25 ID NO:11), TT2 peptide (also referred to as P2; tetanus
toxoid residues 830-846;. SEQ ID.N0:12), TTl peptide (also-
referred o as TT21; tetanus toxoid residues 916-932,~ SEQ
ID N0:13), and TT3 peptide (a2so referred to as X30;
tetanus t~oxoid residues 94'7-96.7; SEQ ID N0:14).
30. ~ According to a method of the present invention, an
effective vaccine formulation can be constructed utilizing
w an appropriate B-cell antigen in combination with an
antigenic T-cell antigen capable of interacting with a
wide range of MfiC haplotypes. One such~exemplary
35 formulation is identified in section VI, below.
SUBSTITUTE S~"'~L~T
1 CA 02372569 2002-03-08
WO 9513!A80 PCTlCA9510a293
34.
VI. Exemplary Carrier/antigen Combination
Anexemplary vaccine composition of the present
invention contains the PAK peptide (SEQ ID N0:18; B-cell
antigen) coupled to the KK (SEQ ID N0:2) core peptide, and-
a tetanus toxoid peptide (TT2, SEQ ID NO: I2; T-cell
antigen) coupled to the EE (SEQ ID NO:1) core peptide.
The PAK peptide (SEQ ID N0:18) has been previously
identified as an effective B-cell antigen (along:, et al.,
1992). The epitope formed by this peptide is recognized
by Pseudomonas,aeruginosa strain K-specific monoclonal
antibody PK93H; which blocks pilus-mediated adherence to
buccal and tracheal epithelial cells (along, et.al,., 1992).
The TT2 peptide (SEQ ID N0:12) was chosen as a T-cell
antigen based on Work by Panina-Bordignon, et al. (1989). ,
These authors showed that the TT2 peptide, as well as the
TT3 peptide (SEQ,ID N0:14.), are universally immunogenic,
since they are recognized by all primed (human) donors,
irrespective of their I~iC ,haplotypes.
The EE (SEQ-ID NO:.1) and KK (SEQ ID.NO:2) core
peptides are exemplary CPl and CP2'core polypeptides.
Both peptides contain ~Fal residues at their a positions,
and Leu residues at their d positions, ensuring
hydrophobic interactions effective to stabilize coiled-
coil heterodimers, but not strong enough to overcome the
electrostatic repulsion between homodiiners.
The a and g positions of EE internal repeats.(SEQ ID .
N0:4, SEQ ID N0:5) contain Glu residues, whereas the a and
g positions of both~terminal (SEQ ID N0:6) a~td internal
repeats (SEQ ID N0:7, SEQ ID NO:8) of KK contain-Lysw
residues. The. opposite charges at corresponding positions
within complimentary heptads of~EE and KK s.tabilize: a-
helical coiled-coil heterodimers, as was described in
section III, parts C and D above, and illustrated in Figs.
3a-a and 4.
In an analogous manner; the charged groups at the a
and g positions discourage the formation of, and
destabilize homodimers. According to an aspect.of the
SUBST~'~'a'E a~-IE~ET
WO 95131480 ~ , ~ 02372569 2002-03-08 ~ 'p~'/CA95/00293
present invention, this destabilization is strong enough
to overcome the hydrophobic interactions present between
appropriately-chosen residues at~the a.and d positions,
which favor the formation of both heterodimers and
5 homodimers.
The terminal repeats of both peptides contain Glu at
the b positipns, and Lys.at the f positions, which can .
form intra-helical lactam bridges. The lactam bridges caw
be formed, for instance; under the reaction conditions
10 detailed in Example 4. The bridges, schematized in
Figures 2a and 2b as straight lines forming a right angle
and connecting positions b and f within each a-helix,
stabilize an a-helical conformation when formed under the
appropriate conditions, detailed in Example 4.:.
15 The geptide conjugation internal repeats of both:
peptides (SEQ ID N0:5 (EEy, and SEQ ID N0:8 (KK)) contain
Cys at the f position. ; The thiol -groups of these .
cysteines are,used.to couple antigenic peptides to the
core polypeptides using, for instance, the protocol
~.20 detailed in Example 5. A B-cell antigenic peptide, the
PAK strain pilin antigen peptide (SEQ ID N0:18~) i's coupled
to the Cys residues of. the internal repeats in the KIC
peptide,(SEQ ID N0:2),. while the tetanus toxoid,derived
TT2 peptide (SEQ ID N0:12) is coupled to the Cys residues
25 of the internal repeats in the EE.peptide (SEQ ID NO:1).
Another set of exemplary vaccines includes, the
recombinantly-produced. fusion peptides described above.
For example, the polypeptide encoded as shown in.Fig. f3,
.contains the PAK antigen~coupled to the E-coil carrier
30 peptide.. The fusion protein nay be expressed and purified
as described above, and used as. an antigen-decorated core
peptide {as described above) in conjunction with a
complimentary (e.g.,~K-coil or KK-based) antigen-decorated
(*e.g., T-antigen) core peptide to-make.a vaccine
35 composition. The conditions for mixing the decorated core
peptides are as were used,.above. ~ .
suss-rrr~~ sy=E-r
. CA 02372569 2002-03-08 ._
WO 95/31480 PCTlCA95100293
36
VII. Antibodies and Immunizations.
A. Antibodies
In another aspect, the invention includes the
production of specific antibodies directed against
pol.ypeptide formulations of the present invention. To
prepare antibodies, a host.animal, such as~a rabbit'; is
immunized with a polypeptide formulation of the present
invention. The host serum or plasma is collected
following an appropriate time interval, and this serum is
1o tested for antibodies specific against the antigen. The
gamma globulin fraction of the IgG antibodies of immunized
animals can be:pbtained, for example, by use of saturated
ammonium sulfate or DEAF ~'SEPHADEX"~~; or other techniques
known to those skilled in tha art forproducing polyclonal
antibodies.
Alternatively, an antigenic formulation of the
present invention may be used for producing monoclonal'
antibodies. Here the spleen or lymphocytes from an
immunized animal are removed and.izmnortalized or used to
2o prepare hybridomas by methods known to those skilled in
the art. ~ . .'
Example 7 describes the production of mouse
antibodies which are sgecific against~the PAK antigenic
peptide (SEQ ID N0:18) in the [PAK]-KK-EE-[TT2),synthetic
vaccine formulation.
8. Vaccines and Neutralizing Antibod'~es. .
'Vaccines can be prepaied using immunogenic~
. polypeptides synthesized by the method of the present
invention. One way to identify potential antigens which
may be useful as vaccines is by screening for antigens
which result in neutralizing antibodies. The protocols
for achieving this are well-known in the art. Briefly, a
.potentially-antigenic formulation is used to prepare
antibodies in a suitabl.e~animal, for example a rabbit.
Antibodies or antibody-containing serum are then isolated
from the animal and incubated- with a virulent mixture of
. * Trademark
. r-rr y'~ ~ ' ,r","G'1'
.... ..-,~T. ."=W- w
CA 02372569 2002-03-08
WO 95131480 ~ PCTICA95/00293
37
the pathogen against which the antibodies were designed.
The pathogenicity of the mixture is then evaluated in an
appropriate assay system, for example a model animal or
susceptible cell culture, and compared with the (positive
~5 control) pathogenicity of a pathogenic mixture incubated
only with adjuvant or carrier. Neutralizing antibodies
will significantly diminish the infective potential of the
pathogenic mixture. An antigenic polypeptide that:
produces good~neutrali2ing antibodies is considered to be
an effective immunogenic polypeptide.
Vaccines containing immunogenic polypeptides as
active ingredients are typically prepared as injectable
either as solutions or suspensions. Further, the
immunogenic polypeptides may be prepared in a solid or
lyophilized state that is suitable for resuspension, prior
to injection, in an aqueous. form. The immunogenic
polypeptides may also be emulsified or encapsulated in
liposomes. The polypeptides are frequently nixed with
pharmaceutically acceptable excipients that are compatible
with the polypeptides. Such excipients include, but are
not limited to, the following and combinations of the
following: saline, water,, sugars (such as dextrose and
sorbitol), glycerol, alcohols (such as ethanol), and
others known in the art: Further, vaccine preparations
may contain minor amounts of other auxiliary substances
such as wetting agents, emulsifying agents (e: g.,
detergents), and pH buffering agents. In addition, a
number of adjuvants are available which may enhance the
effectiveness of vaccine preparations. Examples of such
adjuvants include, but are not limited to, the following:
the group of related compounds including N-acetyl-muranyl-
L-threonyl-D-isoglutamine,and N-acetyl=nor-muranyl-L-
alanyl-D-isoglutamine, and aluminum hydroxide.
The polypepticles are commonly formulated into
vaccines in neutral or salt forms. Pharmaceutically
acceptable organic and inorganic salts are well known in ,
the art.
~e wt~"T't"~'L)~~'~ ~~iE~~
-' . .~ 02372569 2002-03-08 ~.
WO 95131480 . PCTICA95100293
38
Other possible formulations include oral and
suppository formulations. Oral formulations commonly
employ excipients (e. g., pharmaceutical grade ugars;
. saccharine, cellulose, and the like) and usually contain
within l0-98% immunogenic polypeptide. Oral compositions
take the form of pills, capsules, tablets, solutions,
suspensions, powders, etc., and mad be formulated to allow
sustained or~long-term. release. Suppository formulations
use traditional binders and carriers and typically contain
10. between 0.1% and 10% of the immunvgenic.polypeptide.
An example of a vaccine~is a composition including
the [PAK]-KK-EE-[TT2] polypeptide immunogen described in
section VI, above. This immunogen is used as a vaccine
against infection by microorganisms which have surface
proteins which are antigenically cross-reactive with
antibodies produced against the epitope formed by the
sequence SEQ.ID N0:.18, as described in Example 8.
In view.of the above information, multivalent
vaccines against a variety of antigens can be generated.
The vaccines of the present invention are . .
adminis Bred is dosages compatible with the method of
formulation, and in such amounts that will be
pharmacologically effective for prophylactic or
therapeutic treatments. The quantity of immunogen
administered depends on the sub-je.ct being treated, the
capacity of the treatment subj.ect's ,immune system for
antibody synthesis, and the desired level of protection.
The~amounts to be administered are routinely determined by
the administering health care professional.
~ The vaccines of the present invention can be
administered in single or multiple.doses. Dosage regimens
are also determined relative to the treatment subject's
needs and tolerances.
W0 95131480 ~ ~ 02372569 2002-03-08 ~ pCT/CA95I00293
39
VIII.. Utility
Compositions made according to the methods of the
present invention can be used in a number of ways.
Several examples are described below.
A polypeptide designed according to one aspect of the
present invention can be used as a general immunocarrier,
derivatized with antigenic substances or polypeptides of
choice. The general immunocarrier can be used to produce
antibodies in rabbits, or antibodies in mice casing well-
known methodologies. Further, the iinmunocarrier can be
used in vaccine formulations.
As an examgle, a general immunocarrier can be
synthesized with an antigen that is cross-reactive with
antibodies effective to inhibit P. aeruginosa infection in
animals. In this embodiment of the invention, one subunit
would be derivatized.with a B-cell antigen, such as PAK.
peptide (SEQ ID_NO:18),- and the other subunit would be.
derivatized with a T-cell antigen, such as a tetanus
toxoid peptide TT2 (SEQ ID N0:12). An immunocarrier .
designed in this manner can be used as part of a vaccine
formulation to protect against P. aeruginosa in animals.
In a related embodiment of the invention, an immunocarrier
can be designed with an antigen that is cross-reactive
with antibodies effective to inhibit a P. aeruginosa
25~ infection in humans, and can be used as part of a vaccine
formulation to protect against P. aerugiaosa infection, or
to ameliorate an existing P. aeruginosa infection in
humans.
Compositions synthesized according to the present
invention can also be used as part. of a vaccine and/or
antibody development kit. Core polypeptides included in
such a kit can be sold with the coupling residues already
activated, such that all that is required to generate
[Agl~;-CP1 and CP2-[Ag2]~ is:the addition of activated CP1
to a solution containing Agi and the addition of activated
CP2 to a solution containing Ag2.
SUB,:~~'f i'~i.~ i ~ St-~SET
CA 02372569 2002-03-08
WO 95!31480 ' ~ PCTlCA95l00293
Alternatively, the kit can be sold with nan-activated
core polypeptides, with appropriate instructions for
carrying out coupling reactions, and (optionally)
including required coupling reagants: A kit formulated in
5 this manner can also include~an exemplary T-cell helper
. peptide capable of interacting with a wide range of ~iC
haplotypes. The T-cell helper peptide can be already
coupled to one of the core polypeptides,. or it can be
included as a.separate reagent. The later case provides
~10 the option of using a T-cell antigen of the user's own
choosing. ~_
Compositions made according to one aspect of the
present invention,can be.usecl to develop potentially
therapeutic antibodies. Antibodies can be developed, for
15 example, by (i) following the guidance set forth in the
present specification, cr (ii) using a kit developed in
accordance with the present invention, as described in the
above paragraph. Therapeutic antibodies can he produced
in any appropriate animal by techniques well known~in,the
2o art,~for example the methods. detailed in Example 7. Such
antibodies can be used to.treat~diseases or afflictions
for which they weredeveloped, or for diagnosing such
diseases and afflictions.
Polypeptides .designed -according to an aspect of .the
25 present invention can also be used as a "molecular glue",
that .can bring together two different bioactive moieties
linked to CPi and CP2. This strategy can find
applications in vjvo, both intra-cellularly and
extracellularly. as well as in vitro, in cell-free
30 extracts, homogenate, or. general reaction~mixtures where
it is desired-to bring rote-close apposition two
polypeptides or other substances.
. A "molecular glue"~ application, such as is presented
in the above paragraph, can be made largely irreversible,
35 by the incorporation of inter-helical coupling residues,
or by utilizing condiaions.under which the decorated core
.~'-..~~.JBS r ~~~%'rr s'~"I~ET
WO 95131480 CA 02372569 2002-03-08 ~ pCT/CA95I00293
41
polypeptides remain almost exclusively as a-helical
heterodimeric coiled-coils.
Alternatively, the "molecular, glue" could be made
reversible. For example, core polypeptides .can be
designed that wil_1 associate into cx-helical coiled-coil
dimers under one set of reaction conditions, but
dissociate into monomers under~a different set of
conditions. The different conditions can include changes
in variables such as pH and salt~concentration, the
effects of which on coiled-coil formation are outlined in
section III, part D: Conditions compatible with a
selected "molecular glue" applicatiow that enable
reversible coiled-coil formation in a selected application
can be detenained based on the guidance in the ..
I5 specification: .
A "molecularvglue" approach could be utilized to ..
bring into molecular proximity two substances or which-may
v not be in hand; but for which a ligand, preferably a high-
affinity Iigand or antibody~fragment, is known. According
to this aspect of the invention, ayligand (bioactive
moiety) for the first substance is coupled to CP1, and a
ligand (bioactive moiety) for the second substance is
coupled to CP2.. Such an application can be used
therapeutically for targeting endogenous beneficial
molecules~to appropriate targets.
Combinations and variations of the.applications
described above will be obvious to those skilled in the
art. For exampie,.a drug ar therapeutic agent can be
coupled.to one carrier polypeptide, and a,binding.site for
a cellular target can be coupled to the other carrier
polypeptide. The composition can be administered to
deliver the drug or therapeutic agent to the appropriate
site in the body.
Alternatively, one.of the derivatized core
polypeptides can be administered to a subject by itself,
allowed. to bind to a target; and a second polypeptide
derivatized with a therapeutic bioactive moiety can be
S~ic~"'~'i l ~.~-~ ~~-icE'~
CA 02372569 2002-03-08
WO 95131480 ' PCTICA95100293
42
administered to the subject at a later time, with the
understanding that the core polypeptides will interact to
form coiled-coil heterodimers, and will thus be effective
to deliver the therapeutic substance-to the target.
Such an, approach may be employed. to deliver a drug
specifically to a target site, such a tumor undergoing
chemotherapy, with reduced undesirable side-effects due to
drug accumulation at non-target tissues. Prior to drug
delivery, CPl is~ligated to a drug therapeutic and CP2 is
conjugated to a target recognition domain such as a
monoclonal antibody that recognizes a cancer ce3l. The
antibody-CP2 conjugate is delivered into the host first to
search for the target. The drug-CP1 conjugate: is
delivered later. The drug will be localized to the target
1~5 site as a result of preferential dimeri,zati~on of CP1 and
CP2 to form a coiled-coil heterodimer.
The use of the coiled-coil heterodimer as a delivery
vehicle offers several advantages over directly-targeted
therapeutics, such as drugs conjugated directly.to
antibodies. First, optimum conjugation chemistry can be
independently sought for the linking of the individual CP1
and CP2 peptides to.the respective bioactive entities
(antibody~and drug). Further; the chemistries used for
such ~ligations are simpler, since the ligation of a
pegtide to an. antibody, and a peptide to a drug, require
.only basic,-organic chemistry.techniques. 'In contrast, the
conjugation of a drug to a protein (such as an antibody)
could be significantly more complex, as conditions for
ligation are often. harsh and can damage larger proteins
such as antibodies.
Second,_methods of the present invention allow~the
targeting of multiple bioactive moieties (e.g.;.different
drugs) to the same target (e. g., organ or tumor) without
the-need to design and prepare a different drug/antibody
conjugate for each drug. Third, the effective dose of
drug at the target can be modulated by locally modulating
factors that affect the binding affinity of CP1 for CP2.
SUBS"E-iTUTE Si-1EET
WO 95!31480 ~ ~ ~ 02372569 2002-03-08 ~ pC'f1CA95J00293
43
The following examples illustrate, but in no way are
intended to limit the present invention. .
Materials and Metho s
Overview of ELISA Protocol
A purified antigenic polypeptide formulation is
immobilized on a solid support,'such as.a multiwell
polystyrene.plate. Sera to be tested are diluted and
added to the wells. After a period of time sufficient for
the binding of antibodies to the immobilized antigens, the
sera are washed out of the wells. A labelled reporter
antibody is added to each well along with.aw appropriate
substrate.: wells containing antibodies bound to the
immobilized antigen polypept~de are detected by a positive
signal.
. . ELISA Protocol
(adapted from worobec, E.A:, et al., J. EioZ. Chem.
2 0:938 (1985).)
Antigenic peptides (10 mg/Mlwin O.O1 M carbonate
buffer, pH 9.5.) are added to each. well (100 ~cl/well) of a
NUNC'~96-well polystyrene plate and left for 6.hours at
room~teingerature. The wells are washed 3 times with 250
- . 25 ~1~ of P8S pH 7 . 4 supplemented with 0. 02% (wt/vol) . BSA
(wash buffer) , and 250 ~Cl ~5% (wt/vol) B$A in PBS pH 7.4
are added to each well. The plates are incubated
overnight at 4°C to~block non-specific binding sites in
the wells. The wells are then washed three times with
30 wash buffer and 100 ~1 of primary mouse antibady is added
and allowed to incubate at room temperature for 2 hours.
The wells are~washed 3 times with 250 ~cl wash buffer: A
goat anti-mouse IgG (.H+L) immunoglobulin-horse radish v
peroxidase conjugate (Jackson Laboratories,~Bar Harbor,
35 ~ ME) in wash buffer is added (100 ~C1/well) and incubated
for 2 hours at room temgerature. The wells are washed 3
times with Wash buffer and 350 ~C1 of substrate olution .
are added to each well. The substrate solution consists-
* j~$Cv~iT~ ~~.. ,'r~'j' ~"~~~
Trademark
CA 02372569 2002-03-08
WO 95/31480 PCTlCA95/00293
44
of 1 mM 2,2~-azino-di-(3-ethylbenzthiazoline sulfonic
acid), '0.03% (vol/vol) hydrogen peroxide in l0 mM sodium
citrate buffer at pH 4.2. The reactiow is stopped by the
addition. of 250~~c1/well of 4 mM sodium azide. Absorbance
at 405 nm is determined using an EL-407 plate reader.
Example 1
Peptide Synthesis. Purification and Analysis
All peptides-were synthesized by solid-phase peptide
synthesis using a benzhydryl amine-hydro.chlaride resin .on
an Applied Biosystems (Foster City, CA) peptide'
synthesizer Model 430A with conventional N-t- .
butyloxycarbonyl (t-Boc) chemistry. as described pre iously
(Hodges, et al., 1988). The peptides were~cleaved from
the resin by reactiori with hydrofluoric acid (HF; 20 ml/g
resin) containing 10% anisole and 2% 1,2-ethanedithiol .for
1 hour.at -5°C tc 0°C. . ~ ~ ' .
The crude reduced peptides, were'purified by reversed-
phase high performance liquid chromatography (RPC) =and a
t~SYNCHROPAK'"ARP-P semi-preparative' C18 column (250 x 10 mm
inner diameter, 6.5 hem pa=ticle size, 300 ~ pore size;
SynChrom, Lafayette, INy with a linear AB gradient_of 0.5%
B/min and 2 ml./min, where solvent A is 0.05%
trifluoroacetic,acid (TFA) in water ahd solvent B is 0.05%
f5 TFA in acetonitrile.
The amino acid: composition and mass analysis were
consistent with theydesigned sequence. For amino acid
analysis., purified peptides were hydrolyzed in 6 N.HC1
containing~,0.1% phenol at 100°C for 24' hours or: 1 hour at
16f°C in evacuated sealed tubes. Amino acid analysis was
performed on a Beckman model 6300 amino acid analyzer
(Beckman, San Ramon, CA). The correct primary ion
molecular weights of the reduced peptides were confirmed
by plasma desorption time of flight mass spectroscopy on a
BIOION-2~0~ Nordic (Uppsala, Sweden) .
SUBSTfTUTE SHEET
* Trademark
W0 95131480 , ~ 02372569 2002-03-08 ~ p~~CA95100293 .
Examp a 2
Circular -Dichroism Measurements
Circular dichroism (CD) spectra were recorded at 20°C
on a Jasco J-500C spectropolarimeter (Jasco, Easton, MD)
.5 equipped with a Jasco DP-500N data processor and a Laude
(model RMS) water bath (Brinkmann instruments, Rexdale,
Ontario, Canada) for control of the temperature 'of the
cuvette. Constant N2 flushing was employed. The
instrument was routinely calibrated with an agueous
10, solution of recrystallized d-l0-(+)-camphorsulfonic acid
at 290 nm. . .
Molar ellipticity at 200 nm is reported as mean
residue . molar ellipticity. ( [ B ] no, deg~cm2~dmol'') and
calculated from the equation: ''
- [g]cbs x x/10 x 1 x
[8]obs is the ellipticity measured in degrees, mrw is the
mean residue molecular weight (molecular weight of the
20 peptide divided by the number of amino acid residues), c
is the peptide concentration in grams per_milliliter, and
1 is the optical path length of the cell in~centimeters.
CD spectra were the average of four scans obtained by
collecting data at 0.1-nm~intervals from 250 to 190 nm.
25 ' Peptide concentrations were detenained by amino acid
analysis. The pIi was measured at room temperature.
Example 3
Heterodimer vs. Homodimer Formation
30 Two peptides, EE (SEQ ,ID NO:1) and ICK (SEQ ID N0:2),
were synthesized as described in .Examples 1 and 4. CD
spectra .of peptide mixtures of different ratios of the
first subunit peptide (EE; SEQ ID NO:lj and the second
subunit peptide (KK; SEQ ID N0:2) were measured as
35 described in Example 2, to determine the degree of
heterodimerws, homodimer formation.
c~ ic~,cTiTU"1~~ SHEET
CA 02372569 2002-03-08
WO 95/31480 PCTICA95100293
46
The peptides were suspended in a solution containing
0.1 M KC1 and 50 mM potassium phosphate buffer, pH 7 at 20
°C (react'ion buffer).. The total peptide concentration
(sum of EE and KK concentrations) was 196 ACM for all w
measurements.
The data show that as the ratio of the peptides is.
changed from 0:100 to 50:50, the conformation of the
peptide mixture. is changed from a random coil structure to
an a-helical structure. An equimolar mixture of the EE
and KK peptides displays the double minima at~220 and 208
nm with -31, 000 deg~cm2~-dmol'~ of. mean residue ellipticity
at 220 nm, which corresponds to -100% a-helical structure
(Hodges.., et al., 1990), suggesting that the interhelical
ionic repulsions which destabilize the homo-stranded
coiled-coil provide adriving force for the formation of
the hetero-stranded coiled-coil.
These results indicate that the mixture of peptides
EE and KK ,forms a hetero-stranded coiled-coil.
Example 4 '
Creation of Lactam Bridges
The N- and C-terminal~heptads (terminal repeats) were
synthesized semi-automatically using a Labortec peptide
synthesizer (Bubendorf, Switzer.land). Double couplings
25. with ~ equivalents of 2-(1H-benzotriazol-yl)-1,1,3,3-
tetramethyluronium hexfluorophosphate (HBTU1, Z-
hydoxybenzotriazole (HOBt) and hoc amino acids.and 7.5
equivalents~of N=methylmorpholine (NMM) in N-
methylpyrrolidone (NMP) were utilized for each cycle.
During each cycle, the Boc~group was~removed with.50%
trifluoroacetic acid.(TFA) in methylene chloride (DCM).
~Cyclizations.involving the side chains of lysine and
glutaniic acid residues at the N- and C-termini of the
coiled coil~forming peptides were carriesi out on the resin
using a modified protocol of Felix and co-workers (Felix,
et al., 1988) . In-order to facilitate. the intramolecular
cyclizatian reaction and. avoid the undesired
SUBSTITUTE SHEET v
WO 9~I31480 '~ CA 02372569 2oo2-os-oa ~ PCTlCA95100293
47
intermolecular reaction, a lo~a substitution level (0.13
mmol per gram of resin) was employed. The E=amino-..group
of Lysines 35 and 7 and the y-carboxyl. group of glu'tamic
acids 31 and 3 for both peptides were protected with Fmoc
and OFm groups; respectively. This allowed for the
selective deprotection of these residues with 20%
piperidine prior~to the solid phase cyclization with 3
equivalents of HBTU, HOBt and 4.5 equivalents of NMM in
NMP. The synthesis of the C-terminal heptad of peptide
EE, showw in Figure 2, serves to outline the cyciization
procedure.
The intervening heptads 2-4-were prepared on a
Applied Biosystems 430A peptide sgnthesizer. All amino
acids were double coupled using Dicyclohexylcarbodi:imide
(DCC) generated symmetric anhydrides (5 equivalents) in~
dimethyl formamide (DMF) for the'f~.rst coupling step and
DCM foz the second coupling step:
A. Pre~aration.of BocLvs(Fmoc)-
. Resin lLabortec SP 640 Peptide~Synthes'zer;Z
Benzhydrylamine resin (3.0 g, 0.74 meq/g zesin, 2.2
meq) was washed with 30 mL each of DCM~, methanol (.MeOH),
DCM, 5% diisopropylethylamine (DIEA) in DCM (x 2) DCM, and
' ~p ( x 2 } . ~ BocLys ( Fntoc) ( 1. 24, g, 2 . 4 mmol ) ,, HBTU ( 0 . 91 g ,
2.4 mmol);'H08t (0.37 g, 2.4 mmol) were dissolved in NMP
(1~5 iaL) to which was added NMri (0.51 mL, 3.63 mmol) and
solution was preactivated for 5, minutes. This solution
was added to the swelled resin and allowed to sir for 5
minutes.. The resultant BocLys(Fmoc)-resin was washed with
NMP (2 x 1 min) and DCM (3 x 1 min).
B. Preparation of the C- and N-Terminal Hegtads
After deprotection (50% TFA in DCM, 1 x,20 min) and
neutralization (5% DIEA in DCM, 2 x 2.min) the resin was
washed with DCM (2 x 1 min) and.NMP (3 x 1 min). The next
amino acid and all following amino acids for.the C-
terminal heptad and subsequent amino acids of the N-
SL9~~"~i . ~~: ~ ~~-i~;:~T
CA 02372569 2002-03-08
W0 95/31480 PCTlCA95/00293
48
terminal heptad were double coupled according to the
following grotocol.
Boc amino acid (5 eq.), HBTU (5 eq.), HOBt (5 eq.)
were dissolved in NMP (l5 mL) to which was added NN~i (7.5
eq.) and the solution was .allowed to greactivate for 5
minutes. This~solution was added to the reaction vessel
and allowed to gently agitate for 30 minutes. One cyc~l.e
of the synthesis consisted of the following operations (10
mL of solvent per gram of resin: 1) 50% TFA in DCM (1 x 1
min); 2) 50% TFA in DCM (I x 20 min)'; 3) DCM (3 x 1 min);
4) 5% DIEA in DCM (2 x 2 mini) ; 5) DCM (1 x~ l min) ;~ 6) NMP
( 3 x 1 min) ;. 7 ) couple ( 3 0 min) ; 8 ) NMP ( 3 x 1 min) ; 9 )
couple (30 min); 10) NMP~(2 x l min); 11j DCM (3 x 1 min).
C. I,~ysine-Glutamic Acid Side Chain Cyclizations
After addition of ~Boc-Ile, selective deprotectian of
the Fmoc group of lysine and OFm group of gluta~aic acid
was performed with 20% piperidine in DCM ~(1 x 20 min) and
the resin was subsequently washed with DCM (2 x 1 min) and
NMP (3 x 1 min): Cyclizations were performed using the
following protocol.
~iBTU ( 3 eq . ) ~HOBT ( 3 eq . ) and N'MM ( 4 . S eq . ) were
dissolved ~.n NMP to which was added 0.5 mL of
hcxafluoroisopropanol. The solution was added to the
reaction vessel and allowed to gently agitate far 8 hours.
The progress of the reaction-was monitored by quantitative
ninhydrin test (Sarin, et a3., 1981). Typically,. three
coupling were required to achieve coupling efficiency of
greater than 97%. The resin was acetylated'for l hour
c~tith 10 :equivalents of acetic anhydride irt 25 mL of 5%
DIEA in DCM~and washed with DCM, MeOH, DCM and'NMP (x 2).
The following steps were employed.for each cyclization: 1)
20% piperidine in DCM (1 x 1 min); 2) 20% piperidine in
D.CM (1 X 20 min) ; 3) DCM (2 x 1 min) ; 4) NMP (3 x 1 min) ;
5) couple (8 h); 6) NMP (2 x l min); 7) DCM (1 x l min);. .
8) 5% DIEA in DCM (1 x' 1 min); 9) DCM (1 x 1 min); NMP (2
Sl.lE3:.~ttttsi~ ~s..:.~T
WO 95/31480 ~ cA 02372569 2002-03-08 , ~ p~'/CA95J00293
49
x 1 min) 1i) couple (3 h); 12) repeat steps 6-10; 13)
couple (2 h).
Example 5 _.
Linking Peptide Antictens to Heterodimer Scaffold by
Alkylation of Thi.ol Groins
This example describes conjugation of the Na-terminal
iodacetylated PAK strain pilin antigen (SEQ ID N0:18) to
the KK carrier sequence (SEQ ID N0:2).
Prior to conjixgation, the Na-terminus of PAK antigen
(SEQ ID N0:18) was extended by the addition of norleucine,
an internal marker, and two glycine residues acting as
spacers, forming IAc-GG-Nle-PAK. Similarly, the Na-
terminus of the TT2 peptide (SEQ ID N0:12) was extended by
the addition of three glycines and a bromoacetyl group,
forming BrAc-GGG-TT2. These extensions serve to separate
the antigens from,the carrier polypeptides, tending. to
preserve their antigenicity: Exten ions like those
described above are generally recommended in, the synthesis
of immunogenic complexes of the present invention.
Conjugation to carrier peptide sulphydryl groups was
carried out at ambient temperature in 50 mM NH40Ac and 8 M
urea at pH 8. Bromo- or iodacetylated peptides were
dissolved in buffer (0.987 ~cM, ,2 ml) and carrier peptide
KK (SEQ ID N0:2) was added to a final concentration of
0.165 ACM (2 m1). The reaction mixture remained clear and
was allowed to react at ambient temperature for 22 hours;
at which time it was acidified by the careful addition of
TFA (pFi 2 ) , and lyophilized.
A. Con~uaate Purification and Identification
The reaction mixture (2 m1) was applied directly to a
Synchropalt~ RP-8 ,semi-prep column (250 mm x 10 mm I. D. ;
Synchrom Znc., Lafayette, IN). The conjugate was easily
separated from unreacted peptide using gradient~elution
(2% B/minute over 30 minutes; Solvent A: 0.05% TFA/H20;
Solvent B: 0.05% TFA/acetonitrile}. The isalated
~UBS'1"'iTUTE SHEET
* Trademark
W0 95131480 ~ 02372569 2002-03-08
PCT/CA95100293
conjugate was lyophilized and redissolved.in HPLC grade
water (200 ~.1) which was then applied to a Mono-S~strong
cation exchange column (Pharmacia, Uppsala, Sweden) for
further purification. The gradient employed during this
5 purification step was a 1% B/minute gradient (Solvent A: 5
mM NaH2P04/20% acetonitrile,~ pH 5, Solvent H: 5 mM
NaHZP04/20% acetonitrile, l M NaCl; pH 5). The isolated
conjugate was then desalted using a reversed-phase column
and a standard 2% B gradient (vide supra): In this way,
10 pure conjugate was obtained~which was shown through mass
spectrometric analysis to be the'desired product (MW calc:
7432.0, Found, 7432.4). . ~ .
Example 6
15 Generating Heterodimers by Mixins~
Con-iuaated Core-Anti4en Monomers
PAK (SEQ ID NO:iB). and TT2 (SEQ ID N0:12) peptides
were prepared and purified as described.in.Example 1. EE
(SEQ ID NO:i) and KK (SEQ.ID N0:2) peptides were~:prepared,
20 purified and modified as described in Examples l and 4.
The [PAK]-KK and EE-[TT2] peptide-carrier complexes were
prepared as described in~Example 5 and'purified as
detailed in Example 1:w Heterodimer compleXes were
generated by combining the [PAK]-KK complex with the EE-
25 [TT2] complex under the~following conditions:
-Purified, lyophilized, decorated [PAK]-RIB and EE-
[TT2] peptides were individually~resuspended in reaction
buffer, at a concentration of 0.25 - 0.5 mM. . 50 ~1 of
.each peptide solution were combined and allowed to.react
30 far 10 minutes~at room temperature.
Examp~,e 7
rune Lr,~rt ~-rw~nr.-t ~l-'1'G f nezeroaime~~:om~lex
35 ' Balb C mice (l0 animals) are immunized
intraperitoneaLly with a [PAK]-KK-EE-[TT2] heterodimer
conjugate mixture 'comprising 5 ~cg of the conjugate
SUBSTITUTE SHEET
* 'Trademark
WO 95131480 ~ ~ 02372569 2002-03-08 ~ ~~"j'/CA;95/00293
51
dissolved in 100 ~cl of a 1: 1 mixture of ,Adjuvax~ ADJ-20
(Alfa-Beta Technology, Worcester; MA) and 1O mM phosphate
buffered saline (PBS). The injections are,given
intraperitoneally at one abdominal site. The mice are'
boosted after 7,, 14, and,-2l days with~the same amount of
w conjugate in.AdjuvaX ADJ-20.
Control experimerits_are performed an 10 animals
immunized with 5 ~cg.of conjugate.[PAK]-KK prepared as
above. Immunizations are conducted in an identical
fashion to those described for the test' group above.
Sera. are tested for immun~areactivity using a standard
ELISA protocol, as described-in the Materials and Methods.
Titers are estimated from reactivity plate ELISA assays
using~either (i) purified- Pseudomonas eernginosa strain K
pili or (ii) N-linked synthetic PAK peptide (SEQ ID N0:18)
coupled to bovine serum albumin as the solid phase
reactive species.
Spleens from.three animals are pooled, and processed
to produce cells for~fusion with myeloma cells, as
described in Iiarlow, et a3. Hybridoma supernatants are
tested for presence of immunoreactive antibodies by ELISA
tests with the antigen. Supernatants from positive clones
are tested for immunoreactivity with purified Pseudomonas
derived antigens.
Example 8
A. ~ wStudy 1 .
Groups of Balb/c mice (5 to 10 animals per group) are
immunized with.either (i) a control formulafiion ([PAK]-KK)
or (ii) an antigenic coiled-coiled heterodimer formulation
([PAK]-KK-EE-[TT2]). Lnjections of 1, 5 or l0 ~Cg peptide
mixed withAdjuvax* in phosphate buffered sal~.ne are
administered to test animals intro-peritoneally (IP) at 0,
2,~~, and 6 weeks. Animals are exsanguinated weekly and
* Trademark SUBS i lTI:ITE SHEET
. ,CA 023725692002-03-08
WO 95!31480 ' PCTlCA95/00293
52
the serum is tested for antibody responses. Antibody
levels are assessed by direct ELLSAs employing (i)
purified Pseudomonas aeruginosa strain K pili and (ii) N-
3inked synthetic peptide coupled tci bovine serum albumin.
Following the procedures outlined above, titres of
<102 are possible for animals immunized with the control
peptide and tested with a peptide-SSA antigen. Even lower
titers are possible for control animals tested with a PAK
pilin antigen ELISA.
In contrast, immunization with the coiled-coil
heterodimer formu3.ation may result in high titres of
antibody against both the peptide-SSA conjugate and
purified PAK pili. Titers as high as 106 to 10$ (for both
the peptide and native antigen) are possible after 3 to 4
Z5 injections of a 5 ~cg/injection dose.
8. Study 2
Groups of 5 to 10 AB.Y/SnJ mice (--4 weeks of age) are
immunized with adjuvax in buffer, the control peptide and
the coiled-coiled fonaulation iw3 biweekly injections
(containing 5 ~g of peptide and adjuvax as an adjuvant)
intro-muscularly (IM). Two weeks after the last
immunization, the mice (- 12 weeks of age) are challenged
IP with, viable Pseudomonas aerugin~sa strain K at a dose
of 2 x 106 CFU (a challenge dose equal to 5 x LDso). The
mice are monitored over the next 60 hours to'determine the
level cf protection afforded by the vaccine formulations
against Pseudomanas aeruginosa infections.
Control animals (mice immunized with adjuvax or with
the~adjuvax and the control peptide formulation) may
succumb'to the Pseudomonas aeruginosa infection within I6
to 20 hours experiencing 100% mortality. Mice immunized
with the coiled-coiled peptide vaccine formulation may
survive the Pseudomonas aeruginosa challenge and
experience.less than 40% mortality. ,
SUBSTITUTE SHEET
WO 95!31480 ~ ~ 02372569 2002-03-08 ~ pC'IyCA95100293
w While the invention has been described with reference
to specific methods and embodiments, it will be
appreciated that various modifications and changes may. be
made without departing from the invention.
SUBSTtT'UTE SHEET .
CA 02372569 2002-03-08
WO 95131a80 PCTICA951oO293
54
SEQUENCE LISTING
{1) GENERAL INFORMATION:
(i) APPLICANT: S.P.I. Synthetic Peptides Incorporated
(ii) TITLE OF INVENTION: Heterodzmer Polypeptide Immunogen Carrier
Composition and Method
(iii).NUMBER OF SEQUENCES: 30.
(iv) CORRESPONDENCE ADDRESS: .
(A) ADDRESSEE: Dehlinger & Associates
(B) STREET: 350 Cambridge Avenue, Suite 250
{C)'CITY,: Palo Alto
(D) STATE: CA
(E) OOUNTRY: USA
(F) ZIP: 94306 -
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
( B ) ' COMPUTER: IBM FC cocapatible
(C) OPERAThNG SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release X1.0, t~ersion #1.25
(vi) CURRENT APPLICATION DATA: -'
(A) APPLICATION NUMBER:
(B) FILING DATE: 18-MAY-1995
(C) CLASSIFICATION: ~ ..
(vii) PRIOR APPLICATION DATA:
{A) APPIrICATION NUMBER: US 08/245,507
(B) FILING DATE: 18-MAY-1994
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Sholtz, Charles K. ,
(B) REGISTRATION NUMBER: 38,fi15
(C) REFERENCE/DOCKET NUMS~R: 8900-0009.41
(ix) TELECOMMUNICATION INFORMATION:
{A) TELEPFFONE: (415 y- 324-0880 .
. (B) TELEFAX: (415) 324=0960
. g~TI~.U'L'E SHEET'
SU
WO 95/31480 ~ 02372569 2oo2-os-oa ~ PCT/CA95I00293 .
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 amino acids
(B) TYPE: amino acid ..
(C) STRANDEDNESS: single .
(D) TOPOLOGY: unknown
(ii).MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
{iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: EE peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NOsl:
Glu Val Glu Ala Leu Gln Lys Glu Val Ser Ala Lau Glu Lys Glu Val
1 5 10 15
Ser Ala Leu Glu Cys Glu Val Ser Ala Leu Glu Lys Glu Val.Glu Ala
20 25 . 30
Leu Gln Lys
(2) INFORMATION FOR SE.Q ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 amine acids
~8) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown .
(ii). MOLECULE TYPE: peptide '
(iii) HYPOTHETICAL: NO '
(iv) ANTI-SENSES NO
(vi).ORIGINAL SOURCE:
SUBSI'tTUTE SHEET
CA 02372569 2002-03-08
WU 95131480 ~ PCT/CA95/00293
56
(C) INDIVIDUAL ISOLATE: KK peptide
(xij SEQUENCE DESCRIPTION: SEQ ID N0:2:,
Lys Val Glu Ala Leu Lys Lys Lys Val Ser Ala Leu Lys Glu Lys Val.
1 5 10 ~1 S
Ser Ala Leu Lys Cys Lys Val Ser Ala Leu Lys Glu Lys Val Glu Ala
20 25 30 -
Leu.Lys Lys
(2.) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(8) TYPE: ,amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide . '
(iii) HYPOTHETICAL: NO
(iv)'ANTI-SENSE: NO
(vi)~ ORIGINAL SOURCE: ~,
(C) INDIVIDUAL ISOLATE: EE terminal repeat
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
Glu Val Glu Ala Leu Glu Lys
(2) INFORMATION FOR SEQ ID N0:4:
(ij SEQUENCE CHARACTERISTICS: ..
(A) LENGTH: 7 amino acids
(8) TYPE: amino acid
(C) STRANDEDNESS:.single
(D) TOPOLOGY: unknown
t ( R ~Tr-rs ~-!-~ c a..nc c-r
CA 02372569 2002-03-08
WO 95131480 PCTICA9510(?293
57
(iiy MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: EE internal repeat
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Glu Val Ser Ala Leu Glu Lye.
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS: . ..
(A) LENGTH: 7 amino acids
(H) TYPE: amino acid ,
(C) STRANDEDNESS:_single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
( iii. ) HYPOTEiETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: EE conjugation internal repeat
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
Glu Val Ser Ala Leu Glu Cys
1 5
{2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE .CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(8) TYPE: amino acid
(C) STRANDEDNESS: single
... , . _., ..-..-...... ,--.- ..~, , , ~ ,~-~T
CA 02372569 2002-03-08
WO 95!31480 PCT/CA95/00293
58
( D ) TOPOLOGY : unknocln
(ii) MOLECULE TYPE: peptide
(iii} HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL. ISOLATE: KK terminal repeat
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Lys Val Glu Ala Leu Lys Lys
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single. .
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: KK internal repeat
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
Lys Val Ser Ala Leu Lys Glu
(2) INFORMATION FOR SEQ ID NO:8
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
SUBSTITUTE SHEET
WO 95131480 ~ ~ 02372569 2002-03-08 ~ pC'IyCA95/U0293
59
(8) TYPE: amino acid
(C) STRANDEDNESS: eingle
(Dy TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv).ANTI-SENSE: NO
(vi) ORIGINAL SOURCE: ~ '
(C) INDIVIDUAL ISOLATE: ItK conjugation internal repeat
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
Lys Val Ser Ala Leu Lye Cys
(2) INFORMATION FOR SEQ ID HO:9:
. . (f) SEQUENCE CIiARACTERISTICS: ~ . '
(A) LENGTFI: 12 amino acids
(B) TYPE: amino acid ' .
(C) STRANDEDNESS: single
( D ) TOP(SLOGY : ua3cao~m
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(ivj ANTI-SENSE: NO .
(vi) ORIGINAL SOURCE:
(C) INDIVrDUAL ISOLATE: 8 antigen= Exo S geptide
(xi) SEQUENCE DESCRIPTIONS SEQ ID N0:9:
Cys Ala Thr Thr Ala Thr Gly P.ro Asw Gly.Ser Cys
1. . 5 1O
(2) INFORMATION FOR SEQ ID .N0:10:. ' '
SUBSTITUTE SHEET
CA 02372569 2002-03-08
WO 95131480 PCTlCA95l00293
6Q
(iy SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: T antigen, TTO peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Leu Gln Thr Met Vai Lya Leu Phe Asa Arg Ile Lys
1 . 5. 10
(2) INFORMATION FOR SEQ ID NOtll:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(8) TYPE: 'amino acid
(C) STRANDEDNESS: single
(D). TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide .
(iii) HYPOTHETICAL: NO
(iv),ANTI-SENSE: NO
( vi ) ORIGZI~tAL SOURCE
(C) INDIVIDUAL ISOLATE: T antigen, .TT peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Asn Ser.Val Asp Asp Ala Leu Ile Asn Ser Thr Lys Ile Tyr Ser Tyr
1 5 10 15
SUBSTITUTE SHEET
WO 95131480 . ~ 02372569 2002-03-08 ~ p~lCpgS/00293
' 61
Phe Pro 5er Val
(2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B') TYPE: amino acid ,
(C) STRANDEDNESS: single .
(D~) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: T antigen, TT2 peptide
(xi) SEQUENCE DESCRIPTION: SEQ ZD N0:12:
Gln Tyr Ile Lys Ala Aen Ser Lys phe~Ile Gly.Ils Thr Glu Leu Lys
1 5 10 15
Lya
(2) INFORMATION FOR SEQ~ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(8) TYPE: amino acid.
(C) STRANDEDNESSs single.
(D) TOPOLOGY: unknown
. (ii) MOLECULE TYPE: peptide
( iii ) FIYPOTIiETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORLGINAL SOURCE:
SUBS'T~TUTE SHEET
CA 02372569 2002-03-08' ~ p~pA95/00293
WO 95131480
62
(C) INDIVIDUAL ISOLATE: T antigen, TT1 peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
Pro Gly I1e Asn Gly Lys Ala.Ile His Leu Val Asn Asn Glu Ser Ser
1 5 10 . 15
Glu
(2) INFORMATION FOR SEQ ID NO:14.:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(8) TYPE: amino acid
(G~ STRANDEDNESS: single .
(D) TOPOLOGY: unknown .
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSEi NC
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: T antigen, TT3 peptide
a
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
Phe Aan Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lya Val~Ser_
Z 5 10 25
Ala Ser His Lau Glu,
(2) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS: .
(A) LENGTH: 15 amino acids.
(8) TYPE: amino acid
(C) STRANDEDNESS:'single
(D) TOPOLOGY: unknown
~~.:~~.-rf
Sl.lE3~-. . .~ i ;~ ,...
WO 95131480 ~ CA 02372569 2002-03-08 ~ PCTICA95100293
63
(ii) MOLECULE TYPE: peptide .
(.iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) .ORIGINAL SOURCE:
(C) INDIVIDUAL'.ISOLATE: T antigen, MVF gegtide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
Leu Ser Glu Ile Lys Gly Val IlewVal His Arg Leu Glu~Gly Val
1 ' S 10 15
(2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(Aj LENGTH: ~.5 amino acids
.(8) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: uaknosan
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv).ANTI-SENSE: NO .
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL, ISOLATE: T antigen, HBV peptide
(xi) SEQUENCE DESCRIPTIONx SEQ ID NOal6:
Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile Pro Gln Ser Leu Asp
1 5 10 15'
(2) INFORMP.TION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid ' ,
. ~(C) STRANDEDNESS: single
SUBS"~'iTU ~ E SHEET
~CA 02372569 2002-03-08 ... .
WO 95/31480 ' PCTICA95100293
64
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO ~ ,
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE: "
(C) INDIVIDUAL ISOLATE': B antigen, CSP peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:17: ,
Thr Cys Gly Val Gly Val Arg Val Arg Ser Arg Val Asn. Ala Ala Asn
1 5 10 . 15
Lys Lys Pro Glu
20 ' .
(2) INFORMATION FOR SEQ ID N0:18:
(~i) SEQUENCE CHARACTERISTICS: .
(A) LENGTH: 17 amino acids
' (8) TYPE: amino acid
(C) .STRANDEDNESS: single ,
(D)"TOPOLOGY: unknown
(ii).MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO .
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: PAK~geptide . .'
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:
Lys Cys Thr Ser Asg Gln Asp Glu Gln Phe Ile Pro Lys Gly Cys Ser
1 5 10 15
LYs
SUBS'F'IT~ ~ E SHS~T
WO 95/31480 , ~ ' ~ 02372569 2002-03-08 ~ pCTlCA95I00293
(2} INFORMATION FOR SEQ ID N0:19:
(i} SEQUENCE CHARACTERISTICS:
(A} LENGTH: 105 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D} TOPOLOGY: both
(ii) MOLECULE.TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv).ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C)-INDIVIDUAL ISOLATE: E-coil sequence
(ix) FEATURE:
(A) NAME/KEY: CDS
(8) LOCATION: 1..105.
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:19:
GAG GTA TCC GCT TTA GAG AAA GAA GTT TCT GCT CTC GAA AAA GAG GTC 48
Glu Val Ser Ala Leu Glu Lys Glu Val Ser Ala Leu Gnu Lys Glu Val
1 5 10 15
AGT GCT CTG GAA AAA GAG GTG TCA GCC TTG GAA AAG GAA GTA TCA GCA 96
Ser Ala Leu Glu~Lys Glu Va1 Ser Ala Leu Glu Lys Glu Vai Ser Ala
20 25 30
CTT GAG AAG 105
Leu Glu Lys
(2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 amino acids
(8) TYPE: amino 'acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
SUBSTITI.J~T~ SHEET
CA 02372569 2002-03-08
WO 95131480 PCTICA95100293
66
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:20:
Glu Val Ser Ala Leu Glu Lys Glu Val Ser Ala Leu Glu Lye Glu Val
1 ~ 5 10 I5
Ser Ala Leu Glu Lys Glu Val Ser Ala Leu Glu Lys Glu Val Ser Ala
20 25 30
Leu Glu Lys
(2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: lOS base pairs
(8) TYPE: nucleic acid
(C) STRANDEDNESS: double
(Dj TOPOLOGY:. both
(ii) MOLECULE TYPE: DNA
{iii) ~HYPOTHETICAL:,NO
{ib) ANTI-SENSE: NO '
(vi) ORIGINAL SOURCE:
{C) INDIVIDUAL ISOLATE: K-coil sequence
(ix) FEATURE:
(Aj NAME/KEY: CDS
(B) LOCATION: 1..10 5
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:
AAG GTA TCC GCT TTA AAA GAG AAA GTT TCT GCT' CTG AAA GAA AAG GTC 48
Lys Val Ser Ala Leu Lys Glu Lys Val Ser Ala Leu Lys Glu Lys Val
S 10 15
AGT GCT CTG AAG GAG AAG GTG TCA GCC TTG AAG GAA AAG GTT TCA GCA 96
Ser Ala Leu Lys Glu,Lys Val Ser Ala Leu Lys Glu Lya Val Ser Ala
20 25 30
~ ~
~ 02372569 2002-03-08 pCT/CA95100293' ..
WO 95131480 ~
67 .
CTT AAA GAG ' ~ 105
Leu Lye Glu
(2) TNFORMATION.FOR SEQ ZD N0:22:
(i) SEQUENCE CHARACTERISTICS:
_ (A) LENGTH: 35 amino acids ,
(B) TYPE: amino acid
(D) TOPOLOGY:.linear .
(ii) MOZ,ECULE TYPE: protein ,
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
Lys Val Ser Ala Leu Lys Glu Lys Val Ser Ala Leu Lys G1u Lys Val _
.
1 5 . 15 ~ ..
Ser Ala Leu-Lys.Glu Lys Val Ser Ala:Leu Lye Glu Lys Val Ser Ala
25 30.
Leu Lys Glu
(2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 231 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(Dj TOPOLOGY: both
(ii) MOLECULE TYPE: DNA
(iii).HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi)_ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: fragment in Fig. l3
(ix) FEATURE:
'
(A) NAME/KEY: CDS
C I 1l~ c~Tl--T 'T-- .., I , .~, ._
WO 95131480 ~ 02372569 2002-03-08
PCT/CA.95100293
68.
(B) LOCATIONe 1..219
(xi) SEQUENCE DESCRIPTION: SEQ ID.N0:23:.
CGA GAA TTC AAG TGT ACT TCT GAC CAA GAC GAG CAA TTC ATC CCT AAG 48
Arg G1u Phe Lys Cys Thr Ser Asp Gln Aep Glu Gln Phe Ile .Pro Lys
1 5 . .10 15
GGT TGT TCC AAA TTC GGA GGA GGT GGA GGT GGT GGT GGC GAG GTA TCC 96
Gly Cys Ser Lys Phe Gly Gly Gly Gly Gly Gly Gly Gly Glu Va1 Ser
20' 25 30
GCT TTA GAG AAA GAA GTT TCT GCT CTC GAA AAA GAG GTC AGT GCT CTG 144
Ala Leu Glu Lys Glu Val Se; Ala Leu Glu Lys.Glu Val Ser Ala Leu
35 ,40 ~ 45
GAA AAA GAG GTG TCA GCC TTG GAA AAG GAA GTA TCA GCA CTT GAG AAG' 192
GIu i,ys Glu Val Ser Ala Leu Glu Z.ys Glu Val Ser Ala Leu Glu Lys
50 55 60 w
GGC GGT GGA GGA CAT CAC CAC CAT .CAC TAATAAGGAT CC 231
Gly Gly Gly Gly His Hie His .His Flis "
65 70
(2) INFORMATION'FOR SEQ ID N0:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 73 amino acids
' (B) TYPE: a~iao acid
(D). TOPOLOGY: linear
. (ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:
Arg Glu Phe Lys Cys Thr Ser Asp Gln Asp G1u Gln Phe Ile Pro Lys
1 5 10 15
Gly Cys Ser Lys Phe Gly Gly Gly Gly Gly Gly Gly Gly Glu Val~Ser
' 20 25 30
Ala Leu Glu Lys Glu Val Ser Ala Leu Glu.Lys G1u Va1 Ser Ala Leu
35 40 . 45
-~~.~ ~~3CTPT1 1~y-r t-. t a r-.--.-
WO 95!31480 ~ CA 02372569 2002-03-08 ~ ~'~~CA95/00293
Glu Lys Glu Val Ser Ala Leu G1u Lys Glu Va1 Ser Ala Leu G1u Lys
50 55 60
Gly Gly Gly Gly His His His His His
65 70
(2),.INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(Ay LENGTH: 51 base pairs
(8) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: both
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: PAK antigen
(ix) FEATURE:
(A) NAME/KEY: CDS
(8) LOCATION: 1..51
(xi) SEQUENCE DESCRIPTION:.SEQ ID N0:25:
AAG TGT ACT TCT GAC CAA GAC GAG CAA TTC ATC CCT AAG GGT TGT TCC 4$
Lye Cys Thr Ser Asp Gln Asp Glu Gln Phe Ile Pro Lys Gly Cys Ser
1 5 10 15
AAA 51
Lys
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(8) TYPE: amino acid
SUBSTiTU"i-E SHEET
WO 95131480 ~ ~ ~ 02372569 2002-03-08 ~ . ~ . p~pp95100293
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE; protein ,
(xi)- SEQUENCE.DESCRIPTIONr SEQ ID N0:26:
Lys Cys Thr Ser Asp Gln Asp Glu Gln Phe~Ile Pro Lys Gly Cys Ser
1 5 10 15
Lys
(2) INFORMATION FOR SEQ ID N0:27:
(i) SEQUENCE CHARACTERISTICS:
(A~ LENGTH:.228 base pairs
(8) TYPE: nucleic acid .
(C) STRANDEDNESS: double
- (D) TOPOLOGY:'both
(ii)~MOLECULE TYPE: DNA
(iii) FIYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL.ISOLATE: fragment in Fig. 14
(ix) FEATURE:
tA) N~I~Y: CDS .
(8) LOCATION: 1..216
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:27:
CGA GAA TTC TTG TCT GAG ATC AAG GGA GTA ATC GTC CAC AGA CTT GAA 48
Arg.Glu Phe Leu Ser Glu Ile Lye Gly Val Ile Val His Arg Leu Glu
1 5 10 15
GGT GTC AAA TTC GGA GGA GGT GGA GGT GGT GGT GGC GAG GTA TCC GCT 96
Gly Val Lys Phe Gly Gly Gly Gly Gly Cly.Gly Gly Glu Val Ser Ala
20 25 30
... ,
WO 95131480 ~ ~ 02372569 2002-03-08 ~ PCTICA95100293
71
TTA GAG AAA GAA GTT TCT GCT CTC GAA AAA GAG GTC AGT GCT CTG GAA 144
Leu Glu Lys Glu Val Ser Ala Leu Glu Lys Glu Val Ser Ala Leu Glu
35 40 45
AAA GAG GTG TCA GCC TTG GAA AAG GAA GTA TCA GCA CTT GAG AAG GGC 192
Lye Glu Val Ser Ala Leu Glu Lys Glu Va1 Ser Ala Leu Glu I:ys Gly
50 55 60
GGT GGA GGA CAT CAC CAC CAT CAC TAATAAGGAT CC 228
Gly Gly Gly His His His His His
65 y p
(2) INFORMATION FOR SEQ ID N0:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 72 amino acids
(8j TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:
Arg Glu Phe Leu ser Glu_ Ile Lys Gly Val Ile Val His Arg Leu Glu
1 5 10 15
Gly Val Lys.Phe Gly Gly Gly Gly Gly Gly Gly Gly Glu Val Ser Ala
20 ~ 25 30
Leu Glu Lys G1u Val.Ser Ala Leu Glu Lys Glu Val Ser Ala Leu Glu
35 40 45
Lys Glu Val Ser Ala Leu Glu Lys Glu Val Ser Ala Leu Glu Lys Gly
50 . 56 60
Gly.Gly Gly His His His His His
65 70
(2) INFORMATION FOR SEQ ID N0:29:
(i) SEQUENCE~CHARACTERISTICS:
SUBSTITUTE SHEET
WO 95131480 ~ ~ 02372569 2002-03-08 ~ pCTICA95/00293
72
(A) LENGTH: 45 base pairs
(H) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: both
(ii) MOLECULE TYPE:. DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE': MVF antigen
(ix) FEATURE:
(A) NAME/KEY: CDS
(8) LOCATION: 1..45
w (xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:
TTG TCT GAG ATC AAG GGA GTA ATC GTC CAC AGA CTT GAA GGT GTC 45
Leu Ser Glu Ile Lys Gly Val Ile Val His Arg Leu Glu Gly Val
1 ~ 5 ZO ~ 15
(2) INFORMATION FOR SEQ ID N0:30:
(i) SEQUENCE CHARACTERISTICS:.
(A) LENGTH: 15 amino acids ,
(H) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi} SEQUENCE DESCRIPTION.: S.EQ ID N0:30:
Leu Ser Glu Ile Lys Gly Val Ile Val His Arg Leu Glu Gly Val
1 5 10 15
v _ _.r
