Note: Descriptions are shown in the official language in which they were submitted.
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Novel Compounds
The present invention relates to polynucleotides, herein referred to as
CASB6411
polynucleotides, polypeptides encoded thereby (referred to herein as CASB6411
polypeptides), recombinant materials and methods for their production. In
another aspect,
the invention relates to methods for using such polypeptides and
polynucleotides, including
the treatment of cancer and autoimmune diseases and other related conditions.
In a further
aspect, the invention relates to methods for identifying agonists and
antagonists/inhibitors
using the materials provided by the invention, and treating conditions
associated with
l0 CASB6411 polypeptide imbalance with the identified compounds. In a still
further
aspect, the invention relates to diagnostic assays for detecting diseases
associated with
inappropriate CASB6411 polypeptide activity or levels.
Polypeptides and polynucleotides of the present invention are believed to be
important
immunogens for specific prophylactic or therapeutic immunization against
tumours, because
they are specifically expressed or highly over-expressed in tumours compared
to normal
cells and can thus be targeted by antigen-specific immune mechanisms leading
to the
destruction of the tumour cell. They can also be used to diagnose the
occurrence of tumour
cells. Furthermore, their inappropriate expression in certain circumstances
can cause an
2o induction of autoimmune, inappropriate immune responses, which could be
corrected
through appropriate vaccination using the same polypeptides or
polynucleotides. In this
respect the most important biological activities to our purpose are the
antigenic and
immunogenic activities of the polypeptide of the present invention. A
polypeptide of the
present invention may also exhibit at least one other biological activity of a
CASB6411
polypeptide, which could qualify it as a target for therapeutic or
prophylactic intervention
different from that linked to the immune response.
In a first aspect, the present invention relates to CASB6411 polypeptides.
Such peptides
include isolated polypeptides comprising an amino acid sequence which has at
least 70%
identity, preferably at least 80% identity, more preferably at least 90%
identity, yet more
preferably at least 95% identity, most preferably at least 97-99% identity, to
that of SEQ
D7 N0:2 OR 4 over the entire length of SEQ ID N0:2 OR 4. Such polypeptides
include
those comprising the amino acid of SEQ ID N0:2 OR 4.
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Further peptides of the present invention include isolated polypeptides in
which the
amino acid sequence has at least 70% identity, preferably at least 80%
identity, more
preferably at least 90% identity, yet more preferably at least 95% identity,
most
preferably at least 97-99% identity, to the amino acid sequence of SEQ ID N0:2
OR 4
over the entire length of SEQ ID N0:2 OR 4. Such polypeptides include the
polypeptide
of SEQ ID NO:2 OR 4.
Further peptides of the present invention include isolated polypeptides
encoded by a
polynucleotide comprising the sequence contained in SEQ ID NO:l.
The invention also provides an immunogenic fragment of a CASB6411 polypeptide,
that is
a contiguous portion of the CASB6411 polypeptide which has the same or similar
immunogenic properties to the polypeptide comprising the amino acid seqeunce
of SEQ ID
N0:2 OR 4. That is to say, the fragment (if necessary when coupled to a
carrier) is capable
of raising an immune response which recognises the CASB6411 polypeptide. Such
an
immunogenic fragment may include, for example, the CASB6411 polypeptide
lacking an N-
terminal leader sequence, a transmembrane domain or a C-terminal anchor
domain. In a
preferred aspect the immunogenic fragment of CASB6411 according to the
invention
2o comprises substantially all of the extracellular domain of a polypeptide
which has at least
70% identity, preferably at least 80% identity, more preferably at least 90%
identity, yet
more preferably at least 95% identity, most preferably at least 97-99%
identity, to that of
SEQ ID N0:2 OR 4 over the entire length of SEQ ID N0:2 OR 4. Preferably an
immunogenic fragment according to the invention comprises at least one
epitope.
Peptide fragments incorporating an epitope of CASB6411 typically will comprise
at least
7, preferably 9 or 10 contiguous amino acids from SEQ ID N0:2 OR 4. Preferred
epitopes are shown in SEQ ID N0:9 to SEQ ID N0:72.
Peptides that incorporate these epitopes form a preferred aspect of the
present invention.
Mimotopes which have the same characteristics as these epitopes, and
immunogens
comprising such mimotopes which generate an immune response which cross-react
with
2
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an epitope in the context of the CASB6411 molecule, also form part of the
present
invention.
The present invention, therefore, includes isolated peptides encompassing
these epitopes
themselves, and any mimotope thereof. The meaning of mimotope is defined as an
entity
which is sufficiently similar to the native CASB6411 epitope so as to be
capable of being
recognised by antibodies which recognise the native molecule; (Gheysen, H.M.,
et al.,
1986, Synthetic peptides as antigens. Wiley, Chichester, Ciba foundation
symposium
119, p130-149; Gheysen, H.M., 1986, Molecular Immunology, 23,7, 709-715); or
are
l0 capable of raising antibodies, when coupled to a suitable carrier, which
antibodies cross-
react with the native molecule.
Peptide mimotopes of the above-identified epitopes may be designed for a
particular
purpose by addition, deletion or substitution of elected amino acids. Thus,
the peptides of
the present invention may be modified for the purposes of ease of conjugation
to a protein
carrier. For example, it may be desirable for some chemical conjugation
methods to
include a terminal cysteine to the epitope. In addition it may be desirable
for peptides
conjugated to a protein Garner to include a hydrophobic terminus distal from
the
conjugated terminus of the peptide, such that the free unconjugated end of the
peptide
2o remains associated with the surface of the carrier protein. This reduces
the
conformational degrees of freedom of the peptide, and thus increases the
probability that
the peptide is presented in a conformation which most closely resembles that
of the
peptide as found in the context of the whole molecule. For example, the
peptides may be
altered to have an N-terminal cysteine and a C-terminal hydrophobic amidated
tail.
Alternatively, the addition or substitution of a D-stereoisomer form of one or
more ofthe
amino acids may be performed to create a beneficial derivative, for example to
enhance
stability of the peptide. Those skilled in the art will realise that such
modified peptides, or
mimotopes, could be a wholly or partly non-peptide mimotope wherein the
constituent
residues are not necessarily confined to the 20 naturally occurring amino
acids. In
addition, these may be cyclised by techniques known in the art to constrain
the peptide
into a conformation that closely resembles its shape when the peptide sequence
is in the
context of the whole molecule. A preferred method of cyclising a peptide
comprises the
addition of a pair of cysteine residues to allow the formation of a disulphide
bridge.
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Further, those skilled in the art will realise that mimotopes or immunogens of
the present
invention may be larger than the above-identified epitopes, and as such may
comprise the
sequences disclosed herein. Accordingly, the mimotopes of the present
invention may
consist of addition of N and/or C terminal extensions of a number of other
natural
residues at one or both ends. The peptide mimotopes may also be retro
sequences of the
natural sequences, in that the sequence orientation is reversed; or
alternatively the
sequences may be entirely or at least in part comprised of D-stereo isomer
amino acids
(inverso sequences). Also, the peptide sequences may be retro-inverso in
character, in
that the sequence orientation is reversed and the amino acids are of the D-
stereoisomer
form. Such retro or retro-inverso peptides have the advantage of being non-
self, and as
such may overcome problems of self tolerance in the immune system.
Alternatively, peptide mimotopes may be identified using antibodies which are
capable
themselves of binding to the epitopes of the present invention using
techniques such as
phage display technology (EP 0 552 267 B1). This technique, generates a large
number of
peptide sequences which mimic the structure of the native peptides and are,
therefore,
capable of binding to anti-native peptide antibodies, but may not necessarily
themselves
share significant sequence homology to the native peptide. This approach may
have
significant advantages by allowing the possibility of identifying a peptide
with enhanced
immunogenic properties, or may overcome any potential self antigen tolerance
problems
which may be associated with the use of the native peptide sequence.
Additionally this
technique allows the identification of a recognition pattern for each native-
peptide in
terms of its shared chemical properties amongst recognised mimotope sequences.
The covalent coupling of the peptide to the immunogenic carrier can be carried
out in a
manner well known in the art. Thus, for example, for direct covalent coupling
it is
possible to utilise a carbodiimide, glutaraldehyde or (N-[y-
maleimidobutyryloxy]
succinimide ester, utilising common commercially available heterobifunctional
linkers
such as CDAP and SPDP (using manufacturers instructions). After the coupling
reaction,
the immunogen can easily be isolated and purified by means of a dialysis
method, a gel
filtration method, a fractionation method etc.
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The types of Garners used in the immunogens of the present invention will be
readily
known to the man skilled in the art. The function of the carrier is to provide
cytokine help
in order to help induce an immune response against the peptide. A non-
exhaustive list of
carriers which may be used in the present invention include: Keyhole limpet
Haemocyanin (KLH), serum albumins such as bovine serum albumin (BSA),
inactivated
bacterial toxins such as tetanus or diptheria toxins (TT and DT), or
recombinant
fragments thereof (for example, Domain 1 of Fragment C of TT, or the
translocation
domain of DT), or the purified protein derivative of tuberculin (PPD).
Alternatively the
mimotopes or epitopes may be directly conjugated to liposome carriers, which
may
l0 additionally comprise immunogens capable of providing T-cell help.
Preferably the ratio
of mimotopes to carrier is in the order of 1:1 to 20:1, and preferably each
carrier should
carry between 3-15 peptides.
In an embodiment of the invention a preferred carrier is Protein D from
Haemophilus
influenzae (EP 0 594 610 Bl). Protein D is an IgD-binding protein from
Haemophilus
influenzae and has been patented by Forsgren (WO 91/18926, granted EP 0 594
610 B1).
In some circumstances, for example in recombinant immunogen expression systems
it
may be desirable to use fragments of protein D, for example Protein D 1/3Ta
(comprising
the N-terminal 100-110 amino acids of protein D (GB 9717953.5)).
Another preferred method of presenting the peptides of the present invention
is in the
context of a recombinant fusion molecule. For example, EP 0 421 635 B
describes the use
of chimaeric hepadnavirus core antigen particles to present foreign peptide
sequences in a
virus-like particle. As such, immunogens of the present invention may comprise
peptides
presented in chimaeric particles consisting of hepatitis B core antigen.
Additionally, the
recombinant fusion proteins may comprise the mimotopes of the present
invention and a
carrier protein, such as NS 1 of the influenza virus. For any recombinantly
expressed
protein which forms part of the present invention, the nucleic acid which
encodes said
immunogen also forms an aspect of the present invention.
Peptides used in the present invention can be readily synthesised by solid
phase
procedures well known in the art. Suitable syntheses may be performed by
utilising "T-
boc" or "F-moc" procedures. Cyclic peptides can be synthesised by the solid
phase
CA 02386028 2002-03-28
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procedure employing the well-known "F-moc" procedure and polyamide resin in
the fully
automated apparatus. Alternatively, those skilled in the art will know the
necessary
laboratory procedures to perform the process manually. Techniques and
procedures for
solid phase synthesis are described in 'Solid Phase Peptide Synthesis: A
Practical
Approach' by E. Atherton and R.C. Sheppard, published by IRL at Oxford
University
Press (1989). Alternatively, the peptides may be produced by recombinant
methods,
including expressing nucleic acid molecules encoding the mimotopes in a
bacterial or
mammalian cell line, followed by purification of the expressed mimotope.
Techniques for
recombinant expression of peptides and proteins are known in the art, and are
described
to in Maniatis, T., Fritsch, E.F. and Sambrook et al., Molecular cloning, a
laboratory
manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New
York
(1989).
The polypeptides or immunogenic fragment of the invention may be in the form
of the
"mature" protein or may be a part of a larger protein such as a precursor or a
fusion
protein. It is often advantageous to include an additional amino acid sequence
which
contains secretory or leader sequences, pro-sequences, sequences which aid in
purification such as multiple histidine residues, or an additional sequence
for stability
during recombinant production. Furthermore, addition of exogenous polypeptide
or lipid
tail or polynucleotide sequences to increase the immunogenic potential of the
final
molecule is also considered.
In one aspect, the invention relates to genetically engineered soluble fusion
proteins
comprising a polypeptide of the present invention, or a fragment thereof, and
various
portions of the constant regions of heavy or light chains of immunoglobulins
of various
subclasses (IgG, IgM, IgA, IgE). Preferred as an immunoglobulin is the
constant part of
the heavy chain of human IgG, particularly IgGl, where fusion takes place at
the hinge
region. In a particular embodiment, the Fc part can be removed simply by
incorporation
of a cleavage sequence which can be cleaved with blood clotting factor Xa.
Furthermore,
3o this invention relates to processes for the preparation of these fusion
proteins by genetic
engineering, and to the use thereof for drug screening, diagnosis and therapy.
A further
aspect of the invention also relates to polynucleotides encoding such fusion
proteins.
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Examples of fusion protein technology can be found in International Patent
Application
Nos. W094/29458 and W094/22914.
The proteins may be chemically conjugated, or expressed as recombinant fusion
proteins
allowing increased levels to be produced in an expression system as compared
to non-
fused protein. The fusion partner may assist in providing T helper epitopes
(immunological fusion partner), preferably T helper epitopes recognised by
humans, or
assist in expressing the protein (expression enhancer) at higher yields than
the native
recombinant protein. Preferably the fusion partner will be both an
immunological fusion
to partner and expression enhancing partner.
Fusion partners include protein D from Haemophilus influenza B and the non-
structural
protein from influenzae virus, NS 1 (hemagglutinin). Another immunological
fusion
partner is the protein known as LYTA. Preferably the C terminal portion of the
molecule
is used. Lyta is derived from Streptococcus pneumoniae which synthesize an N-
acetyl-L-
alanine amidase, amidase LYTA, (coded by the lytA gene {Gene, 43 (1986) page
265-
272} an autolysin that specifically degrades certain bonds in the
peptidoglycan backbone.
The C-terminal domain of the LYTA protein is responsible for the affinity to
the choline
or to some choline analogues such as DEAF. This property has been exploited
for the
development of E.coli C-LYTA expressing plasmids useful for expression of
fusion
proteins. Purification of hybrid proteins containing the C-LYTA fragment at
its amino
terminus has been described {Biotechnology: 10, (1992) page 795-798}. It is
possible to
use the repeat portion of the Lyta molecule found in the C terminal end
starting at residue
178, for example residues 188 - 305.
The present invention also includes variants of the aforementioned
polypeptides, that is
polypeptides that vary from the referents by conservative amino acid
substitutions, whereby
a residue is substituted by another with like characteristics. Typical such
substitutions are
among Ala, Val, Leu and Ile; among Ser and Thr; among the acidic residues Asp
and Glu;
among Asn and Gln; and among the basic residues Lys and Arg; or aromatic
residues Phe
and Tyr. Particularly preferred are variants in which several, 5-10, 1-5, 1-3,
1-2 or 1 amino
acids are substituted, deleted, or added in any combination.
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Polypeptides of the present invention can be prepared in any suitable manner.
Such
polypeptides include isolated naturally occurring polypeptides, recombinantly
produced
polypeptides, synthetically produced polypeptides, or polypeptides produced by
a
combination of these methods. Means for preparing such polypeptides are well
understood
in the art.
In a further aspect, the present invention relates to CASB6411
polynucleotides. Such
polynucleotides include isolated polynucleotides comprising a nucleotide
sequence encoding
a polypeptide which has at least 70% identity, preferably at least 80%
identity, more
l0 preferably at least 90% identity, yet more preferably at least 95%
identity, to the amino
acid sequence of SEQ ID N0:2 OR 4, over the entire length of SEQ ID N0:2 OR 4.
In this
regard, polypeptides which have at least 97% identity are highly preferred,
whilst those with
at least 98-99% identity are more highly preferred, and those with at least
99% identity are
most highly preferred. Such polynucleotides include a polynucleotide
comprising the
15 nucleotide sequence contained in SEQ ID NO:1 or 3 encoding the polypeptide
of SEQ m
N0:2 OR 4 respectively.
Further polynucleotides of the present invention include isolated
polynucleotides comprising
a nucleotide sequence that has at least 70% identity, preferably at least 80%
identity, more
20 preferably at least 90% identity, yet more preferably at least 95%
identity, to a nucleotide
sequence encoding a polypeptide of SEQ ID N0:2 OR 4, over the entire coding
region. In
this regard, polynucleotides which have at least 97% identity are highly
preferred, whilst
those with at least 98-99% identity are more highly preferred, and those with
at least 99%
identity are most highly preferred.
Further polynucleotides of the present invention include isolated
polynucleotides
comprising a nucleotide sequence which has at least 70% identity, preferably
at least 80%
identity, more preferably at least 90% identity, yet more preferably at least
95% identity,
to SEQ ID NO:1 or 3 or to the coding sequence of SEQ ID NO:1 or 3 over the
entire
length of SEQ ID NO:1 or 3 or over the entire length of the coding sequence of
SEQ m
NO:1 or 3 respectively. In this regard, polynucleotides which have at least
97% identity are
highly preferred, whilst those with at least 98-99% identiy are more highly
preferred, and
those with at least 99% identity are most highly preferred. Such
polynucleotides include a
8
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polynucleotide comprising the polynucleotide of SEQ ID NO:1 or 3 as well as
the
polynucleotide of SEQ ID NO:1 or 3 or the coding region of SEQ ID NO:1 or 3.
Said
polynucleotide can be inserted in a suitable plasmid or recombinant
microrganism vector
and used for immunization ( see for example Wolff et. al., Science 247:1465-
1468 (1990);
Corr et. al., J. Exp. Med. 184:1555-1560 (1996); Doe et. al., Proc. Natl.
Acad. Sci. 93:8578-
8583 (1996)).
The invention also provides polynucleotides which are complementary to all the
above
described polynucleotides.
The invention also provides a fragment of a CASB6411 polynucleotide which when
administered to a subject has the same immunogenic properties as the
polynucleotide of
SEQ ID NO:1 or 3.
The invention also provides a polynucleotide encoding an immunological
fragment of a
CASB6411 polypeptide as hereinbefore defined.
The fragments have a level of immunogenic activity of at least about SO%,
preferably at
least about 70% and more preferably at least about 90% of the level of
immunogenic
2o activity of a polypeptide sequence set forth in SEQ ID N0:2 OR 4 or a
polypeptide
sequence encoded by a polynucleotide sequence set forth in SEQ ID NO: 1.
The polypeptide fragments according to the invention preferably comprise at
least about
5, 10, 15, 20, 25, 50, or 100 contiguous amino acids, or more, including all
intermediate
lengths, of a polypeptide composition set forth herein, such as those set
forth in SEQ ID
N0:2 OR 4, or those encoded by a polynucleotide sequence set forth in a
sequence of
SEQ ID NO:1.
The nucleotide sequence of SEQ ID NO:1 is a cDNA sequence which comprises a
3o polypeptide encoding sequence (nucleotide 349 to 1761) encoding a
polypeptide of 460
amino acids, the polypeptide of SEQ ID N0:2. The nucleotide sequence encoding
the
polypeptide of SEQ ID N0:2 may be identical to the polypeptide encoding
sequence
contained in SEQ ID NO:1 or it may be a sequence other than the one contained
in SEQ
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ID NO:1, which, as a result of the redundancy (degeneracy) of the genetic
code, also
encodes the polypeptide of SEQ ID N0:2. The polypeptide of the SEQ ID N0:2 is
structurally related to other proteins of the LAK-4p family, having homology
and/or
structural similarity with Homo sapiens LAK-4p (GenBank accession BAA24179).
SEQ ID N0:3 is a cDNA sequence which is an alternative isoform of CASB6411 SEQ
ID
NO:1, and probably the result of alternative exon splicing. SEQ ID N0:3
comprises a
polypeptide encoding sequence (nucleotide 382 to 844) encoding a polypeptide
of 154
aminoacids, the polypeptide of SEQ ID N0:4. SEQ )D N0:4 is a truncated form of
SEQ ID
1o N0:2 polypeptide. The nucleotide sequence encoding the polypeptide of SEQ
ID N0:4
may be identical to the polypeptide encoding sequence contained in SEQ ID N0:3
or it
may be a sequence other than the one contained in SEQ ID N0:3, which, as a
result of the
redundancy (degeneracy) of the genetic code, also encodes the polypeptide of
SEQ ID
N0:4.
Preferred polypeptides and polynucleotides of the present invention are
expected to have,
inter alia, similar biological functions/properties to their homologous
polypeptides and
polynucleotides. Furthermore, preferred polypeptides, immunological fragments
and
polynucleotides of the present invention have at least one activity of either
SEQ ID NO:1 or
3 or SEQ 117 N0:2 OR 4, as appropriate.
The present invention also relates to partial or other incomplete
polynucleotide and
polypeptide sequences which were first identified prior to the determination
of the
corresponding full length sequences of SEQ ID NO:1 or 3 and SEQ ID N0:2 OR 4.
Accordingly, in a fiuther aspect, the present invention provides for an
isolated
polynucleotide which:
(a) comprises a nucleotide sequence which has at least 70% identity,
preferably at least
80% identity, more preferably at least 90% identity, yet more preferably at
least 95%
identity, even more preferably at least 97-99% identity to SEQ ID N0:5 and 7
over the
entire length of SEQ ID N0:5 and 7, respectively;
(b) has a nucleotide sequence which has at least 70% identity, preferably at
least 80%
identity, more preferably at least 90% identity, yet more preferably at least
95% identity,
CA 02386028 2002-03-28
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even more preferably at least 97-99% identity, to SEQ ID NO: l or 3 over the
entire
length of SEQ ID NO:S and 7;
(c) the polynucleotide of SEQ ID NO:S and 7; or
(d) a nucleotide sequence encoding a polypeptide which has at least 70%
identity,
preferably at least 80% identity, more preferably at least 90% identity, yet
more
preferably at least 95% identity, even more preferably at least 97-99%
identity, to the
amino acid sequence of SEQ ID N0:6 and 8, over the entire length of SEQ D7
N0:6 and 8 ,
respectively,;
as well as the polynucleotide of SEQ ID NO:S and 7.
to
The present invention further provides for a polypeptide which:
(a) comprises an amino acid sequence which has at least 70% identity,
preferably at least
80% identity, more preferably at least 90% identity, yet more preferably at
least 95%
identity, most preferably at least 97-99% identity, to that of SEQ ID N0:6 OR
8 over the
15 entire length of SEQ >D N0:6 and 8;
(b) has an amino acid sequence which is at least 70% identity, preferably at
least 80%
identity, more preferably at least 90% identity, yet more preferably at least
95% identity,
most preferably at least 97-99% identity, to the amino acid sequence of SEQ >D
N0:6 OR
8 over the entire length of SEQ ID N0:6 and 8;
2o (c) comprises the amino acid of SEQ m N0:6 and 8; and
(d) is the polypeptide of SEQ ID N0:6 and 8;
as well as polypeptides encoded by a polynucleotide comprising the sequence
contained
in SEQ m NO:S and 7.
25 Polynucleotides of the present invention may be obtained, using standard
cloning and
screening techniques, from a cDNA library derived from mRNA in cells of human,
colorectal tumours, stomach tumors and normal stomach, normal pancreas and
pancreas
tumors, ovarian tumors, lung tumors, and normal prostate, (for example
Sambrook et al.,
Molecular Cloning: A Laboratory Manual, 2"d Ed., Cold Spring harbor Laboratory
Press,
3o Cold Spring harbor, N.Y. (1989)). Polynucleotides of the invention can also
be obtained
from natural sources such as genomic DNA libraries or can be synthesized using
well
known and commercially available techniques.
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When polynucleotides of the present invention are used for the recombinant
production of
polypeptides of the present invention, the polynucleotide may include the
coding sequence
for the mature polypeptide, by itself; or the coding sequence for the mature
polypeptide in
reading frame with other coding sequences, such as those encoding a leader or
secretory
sequence, a pre-, or pro- or prepro- protein sequence, or other fusion peptide
portions. For
example, a marker sequence which facilitates purification of the fused
polypeptide can be
encoded. In certain preferred embodiments of this aspect of the invention, the
marker
sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen,
Inc.) and
described in Gentz et al., Proc Natl Acad Sci USA (1989) 86:821-824, or is an
HA tag. The
to polynucleotide may also contain non-coding S' and 3' sequences, such as
transcribed, non-
translated sequences, splicing and polyadenylation signals, ribosome binding
sites and
sequences that stabilize mRNA.
Further embodiments of the present invention include polynucleotides encoding
polypeptide
variants which comprise the amino acid sequence of SEQ ID N0:2 OR 4 and in
which
several, for instance from 5 to 10, 1 to 5, 1 to 3, 1 to 2 or 1, amino acid
residues are
substituted, deleted or added, in any combination.
Polynucleotides which are identical or sufficiently identical to a nucleotide
sequence
2o contained in SEQ >D NO:1 or 3, may be used as hybridization probes for cDNA
and
genomic DNA or as primers for a nucleic acid amplification (PCR) reaction, to
isolate full
length cDNAs and genomic clones encoding polypeptides of the present invention
and to
isolate cDNA and genomic clones of other genes (including genes encoding
paralogs from
human sources and orthologs and paralogs from species other than human) that
have a high
sequence similarity to SEQ ID NO:1 or 3. Typically these nucleotide sequences
are 70%
identical, preferably 80% identical, more preferably 90% identical, most
preferably 95%
identical to that of the referent. The probes or primers will generally
comprise at least 1 S
nucleotides, preferably, at least 30 nucleotides and may have at least 50
nucleotides.
Particularly preferred probes will have between 30 and 50 nucleotides.
Particularly
preferred primers will have between 20 and 25 nucleotides. In particular,
polypeptides or
polynucleotides derived from sequences from homologous animal origin could be
used as
immunogens to obtain a cross-reactive immune response to the human gene.
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A polynucleotide encoding a polypeptide of the present invention, including
homologs from
species other than human, may be obtained by a process which comprises the
steps of
screening an appropriate library under stringent hybridization conditions with
a labeled
probe having the sequence of SEQ ID NO: 1 or 3 or a fragment thereof; and
isolating full-
y length cDNA and genomic clones containing said polynucleotide sequence. Such
hybridization techniques are well known to the skilled artisan. Preferred
stringent
hybridization conditions include overnight incubation at 42oC in a solution
comprising: 50%
formamide, SxSSC (150mM NaCI, lSmM trisodium citrate), 50 mM sodium phosphate
(pH7.6), Sx Denhardt's solution, 10 % dextran sulfate, and 20 microgram/ml
denatured,
l0 sheared salmon sperm DNA; followed by washing the filters in O.lx SSC at
about 65°C.
Thus the present invention also includes polynucleotides obtainable by
screening an
appropriate library under stingent hybridization conditions with a labeled
probe having the
sequence of SEQ ID NO:1 or 3 or a fragment thereof.
15 The skilled artisan will appreciate that, in many cases, an isolated cDNA
sequence will be
incomplete, in that the region coding for the polypeptide is short at the 5'
end of the
cDNA.
There are several methods available and well known to those skilled in the art
to obtain
20 full-length cDNAs, or extend short cDNAs, for example those based on the
method of
Rapid Amplification of cDNA ends (RACE) (see, for example, Frohman et al.,
PNAS
USA 85, 8998-9002, 1988). Recent modifications of the technique, exemplified
by the
MarathonTM technology (Clontech Laboratories Inc.) for example, have
significantly
simplified the search for longer cDNAs. In the MarathonTM technology, cDNAs
have
25 been prepared from mRNA extracted from a chosen tissue and an'adaptor'
sequence
ligated onto each end. Nucleic acid amplification (PCR) is then carned out to
amplify the
'missing' S' end of the cDNA using a combination of gene specific and adaptor
specific
oligonucleotide primers. The PCR reaction is then repeated using 'nested'
primers, that is,
primers designed to anneal within the amplified product (typically an adaptor
specific
3o primer that anneals further 3' in the adaptor sequence and a gene specific
primer that
anneals further 5' in the known gene sequence). The products of this reaction
can then be
analysed by DNA sequencing and a full-length cDNA constructed either by
joining the
13
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WO 01/23417 PCT/EP00/09500
product directly to the existing cDNA to give a complete sequence, or carrying
out a
separate full-length PCR using the new sequence information for the design of
the 5'
pnmer.
Recombinant polypeptides of the present invention may be prepared by processes
well
known in the art from genetically engineered host cells comprising expression
systems.
Accordingly, in a further aspect, the present invention relates to an
expression system which
comprises a polynucleotide of the present invention, to host cells which are
genetically
engineered with such expression sytems and to the production of polypeptides
of the
invention by recombinant techniques. Cell-free translation systems can also be
employed to
produce such proteins using RNAs derived from the DNA constructs of the
present
invention.
For recombinant production, host cells can be genetically engineered to
incorporate
expression systems or portions thereof for polynucleotides of the present
invention.
Introduction of polynucleotides into host cells can be effected by methods
described in many
standard laboratory manuals, such as Davis et al., Basic Methods in Molecular
Biology
(1986) and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed.,
Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). Preferred
such methods
2o include, for instance, calcium phosphate transfection, DEAF-dextran
mediated transfection,
transvection, microinjection, cationic lipid-mediated transfection,
electroporation,
transduction, scrape loading, ballistic introduction or infection.
Preferably the proteins of the invention are coexpressed with thioredoxin in
trans (TIT).
Coexpression of thioredoxin in trans versus in cis is preferred to keep
antigen free of
thioredoxin without the need for protease. Thioredoxin coexpression eases the
solubilisation of the proteins of the invention. Thioredoxin coexpression has
also a
significant impact on protein purification yield, on purified-protein
solubility and quality.
Representative examples of appropriate hosts include bacterial cells, such as
Streptococci,
Staphylococci, E. coli, Streptomyces and Bacillus subtilis cells; fungal
cells, such as yeast
cells and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera
Sf~ cells;
14
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WO 01/23417 PCT/EP00/09500
animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes
melanoma
cells; and plant cells.
A great variety of expression systems can be used, for instance, chromosomal,
episomal
and virus-derived systems, e.g., vectors derived from bacterial plasmids, from
bacteriophage, from transposons, from yeast episomes, from insertion elements,
from
yeast chromosomal elements, from viruses such as baculoviruses, papova
viruses, such as
SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses
and
retroviruses, and vectors derived from combinations thereof, such as those
derived from
1o plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
The
expression systems may contain control regions that regulate as well as
engender
expression. Generally, any system or vector which is able to maintain,
propagate or
express a polynucleotide to produce a polypeptide in a host may be used. The
appropriate
nucleotide sequence may be inserted into an expression system by any of a
variety of
15 well-known and routine techniques, such as, for example, those set forth in
Sambrook et
al., Molecular Cloning, A Laboratory Manual (supra). Appropriate secretion
signals may
be incorporated into the desired polypeptide to allow secretion of the
translated protein
into the lumen of the endoplasmic reticulum, the periplasmic space or the
extracellular
environment. These signals may be endogenous to the polypeptide or they may be
2o heterologous signals.
The expression system may also be a recombinant live microorganism, such as a
virus or
bacterium. The gene of interest can be inserted into the genome of a live
recombinant
virus or bacterium. Inoculation and in vivo infection with this live vector
will lead to in
25 vivo expression of the antigen and induction of immune responses.
Therefore, in certain embodiments, polynucleotides encoding immunogenic
polypeptides
of the present invention are introduced into suitable mammalian host cells for
expression
using any of a number of known viral-based systems. In one illustrative
embodiment,
3o retroviruses provide a convenient and effective platform for gene delivery
systems. A
selected nucleotide sequence encoding a polypeptide of the present invention
can be
inserted into a vector and packaged in retroviral particles using techniques
known in the
art. The recombinant virus can then be isolated and delivered to a subject. A
number of
CA 02386028 2002-03-28
WO 01/23417 PCT/EP00/09500
illustrative retroviral systems have been described (e.g., U.S. Pat. No.
5,219,740; Miller
and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene
Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993)
Proc.
Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur.
Opin.
Genet. Develop. 3:102-109.
In addition, a number of illustrative adenovirus-based systems have also been
described.
Unlike retroviruses which integrate into the host genome, adenoviruses persist
extrachromosomally thus minimizing the risks associated with insertional
mutagenesis
to (Haj-Ahmad and Graham (1986) J. Virol. 57:267-274; Bett et al. (1993) J.
Virol.
67:5911-5921; Mittereder et al. (1994) Human Gene Therapy 5:717-729; Seth et
al.
(1994) J. Virol. 68:933-940; Barr et al. (1994) Gene Therapy 1:51-58; Berkner,
K. L.
(1988) BioTechniques 6:616-629; and Rich et al. (1993) Human Gene Therapy
4:461-
476).
Various adeno-associated virus (AAV) vector systems have also been developed
for
polynucleotide delivery. AAV vectors can be readily constructed using
techniques well
known in the art. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941;
International
Publication Nos. WO 92/01070 and WO 93/03769; Lebkowski et al. (1988) Molec.
Cell.
2o Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor
Laboratory
Press); Carter, B. J. (1992) Current Opinion in Biotechnology 3:533-539;
Muzyczka, N.
(1992) Current Topics in Microbiol. and Immunol. 158:97-129; Kotin, R. M.
(1994)
Human Gene Therapy 5:793-801; Shelling and Smith (1994) Gene Therapy 1:165-
169;
and Zhou et al. (1994) J. Exp. Med. 179:1867-1875.
Additional viral vectors useful for delivering the nucleic acid molecules
encoding
polypeptides of the present invention by gene transfer include those derived
from the pox
family of viruses, such as vaccinia virus and avian poxvirus. By way of
example, vaccinia
virus recombinants expressing the novel molecules can be constructed as
follows. The
DNA encoding a polypeptide is first inserted into an appropriate vector so
that it is
3o adjacent to a vaccinia promoter and flanking vaccinia DNA sequences, such
as the
sequence encoding thymidine kinase (TK). This vector is then used to transfect
cells
16
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WO 01/23417 PCT/EP00/09500
which are simultaneously infected with vaccinia. Homologous recombination
serves to
insert the vaccinia promoter plus the gene encoding the polypeptide of
interest into the
viral genome. The resulting TK(-) recombinant can be selected by
culturing the cells
in the presence of 5-bromodeoxyuridine and picking viral plaques resistant
thereto.
A vaccinia-based infection/transfection system can be conveniently used to
provide for
inducible, transient expression or coexpression of one or more polypeptides
described
herein in host cells of an organism. In this particular system, cells are
first infected in
vitro with a vaccinia virus recombinant that encodes the bacteriophage T7 RNA
l0 polymerase. This polymerase displays exquisite specificity in that it only
transcribes
templates bearing T7 promoters. Following infection, cells are transfected
with the
polynucleotide or polynucleotides of interest, driven by a T7 promoter. The
polymerase
expressed in the cytoplasm from the vaccinia virus recombinant transcribes the
transfected DNA into RNA which is then translated into polypeptide by the host
15 translational machinery. The method provides for high level, transient,
cytoplasmic
production of large quantities of RNA and its translation products. See, e.g.,
Elroy-Stein
and Moss, Proc. Natl. Acad. Sci. USA (1990) 87:6743-6747; Fuerst et al. Proc.
Natl.
Acad. Sci. USA (1986) 83:8122-8126.
2o Alternatively, avipoxviruses, such as the fowlpox and canarypox viruses,
can also be used
to deliver the coding sequences of interest. Recombinant avipox viruses,
expressing
immunogens from mammalian pathogens, are known to confer protective immunity
when
administered to non-avian species. The use of an Avipox vector is particularly
desirable
in human and other mammalian species since members of the Avipox genus can
only
25 productively replicate in susceptible avian species and therefore are not
infective in
mammalian cells. Methods for producing recombinant Avipoxviruses are known in
the
art and employ genetic recombination, as described above with respect to the
production
of vaccinia viruses. See, e.g., WO 91/12882; WO 89/03429; and WO 92/03545.
3o Any of a number of alphavirus vectors can also be used for delivery of
polynucleotide
compositions of the present invention, such as those vectors described in U.S.
Patent Nos.
17
CA 02386028 2002-03-28
WO 01/23417 PCT/EP00/09500
5,843,723; 6,015,686; 6,008,035 and 6,015,694. Certain vectors based on
Venezuelan
Equine Encephalitis (VEE) can also be used, illustrative examples of which can
be found
in U.S. Patent Nos. 5,505,947 and 5,643,576.
Moreover, molecular conjugate vectors, such as the adenovirus chimeric vectors
described in Michael et al. J. Biol. Chem. (1993) 268:6866-6869 and Wagner et
al. Proc.
Natl. Acad. Sci. USA (1992) 89:6099-6103, can also be used for gene delivery
under the
W vention.
to Additional illustrative information on these and other known viral-based
delivery systems
can be found, for example, in Fisher-Hoch et al., Proc. Natl. Acad. Sci. USA
86:317-321,
1989; Flexner et al., Ann. N. Y. Acad. Sci. 569:86-103, 1989; Flexner et al.,
Vaccine
8:17-21, 1990; U.S. Patent Nos. 4,603,112, 4,769,330, and 5,017,487; WO
89/01973;
U.S. Patent No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner,
Biotechniques 6:616-627, 1988; Rosenfeld et al., Science 252:431-434, 1991;
Kolls et al.,
Proc. Natl. Acad. Sci. USA 91:215-219, 1994; Kass-Eisler et al., Proc. Natl.
Acad. Sci.
USA 90:11498-11502, 1993; Guzman et al., Circulation 88:2838-2848, 1993; and
Guzman et al., Cir. Res. 73:1202-1207, 1993.
2o The recombinant live microorganisms described above can be virulent, or
attenuated in
various ways in order to obtain live vaccines. Such live vaccines also form
part of the
invention.
In certain embodiments, a polynucleotide may be integrated into the genome of
a target
cell. This integration may be in the specific location and orientation via
homologous
recombination (gene replacement) or it may be integrated in a random, non-
specific
location (gene augmentation). In yet further embodiments, the polynucleotide
may be
stably maintained in the cell as a separate, episomal segment of DNA. Such
3o polynucleotide segments or "episomes" encode sequences sufficient to permit
maintenance and replication independent of or in synchronization with the host
cell cycle.
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WO 01/23417 PCT/EP00/09500
The manner in which the expression construct is delivered to a cell and where
in the cell
the polynucleotide remains is dependent on the type of expression construct
employed.
In another embodiment of the invention, a polynucleotide is
administered/delivered as
"naked" DNA, for example as described in Ulmer et al., Science 259:1745-1749,
1993
and reviewed by Cohen, Science 259:1691-1692, 1993. The uptake of naked DNA
may
be increased by coating the DNA onto biodegradable beads, which are
efficiently
transported into the cells.
l0 In still another embodiment, a composition of the present invention can be
delivered via a
particle bombardment approach, many of which have been described. In one
illustrative
example, gas-driven particle acceleration can be achieved with devices such as
those
manufactured by Powderject Pharmaceuticals PLC (Oxford, UK) and Powderject
Vaccines Inc. (Madison, WI), some examples of which are described in U.S.
Patent Nos.
15 5,846,796; 6,010,478; 5,865,796; 5,584,807; and EP Patent No. 0500 799.
This approach
offers a needle-free delivery approach wherein a dry powder formulation of
microscopic
particles, such as polynucleotide or polypeptide particles, are accelerated to
high speed
within a helium gas jet generated by a hand held device, propelling the
particles into a
target tissue of interest.
In a related embodiment, other devices and methods that may be useful for gas-
driven
needle-less injection of compositions of the present invention include those
provided by
Bioject, Inc. (Portland, OR), some examples of which are described in U.S.
Patent Nos.
4,790,824; 5,064,413; 5,312,335; 5,383,851; 5,399,163; 5,520,639 and
5,993,412.
Polypeptides of the present invention can be recovered and purified from
recombinant cell
cultures by well-known methods including ammonium sulfate or ethanol
precipitation, acid
extraction, anion or cation exchange chromatography, phosphocellulose
chromatography,
hydrophobic interaction chromatography, affinity chromatography,
hydroxylapatite
chromatography and lectin chromatography. Most preferably, ion metal affinity
chromatography (INIAC) is employed for purification. Well known techniques for
19
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WO 01/23417 PCT/EP00/09500
refolding proteins may be employed to regenerate active conformation when the
polypeptide
is denatured during intracellular synthesis, isolation and or purification.
Another important aspect of the invention relates to a method for inducing ,
re-inforcing
or modulating an immunological response in a mammal which comprises
inoculating the
mammal with a fragment or the entire polypeptide or polynucleotide of the
invention,
adequate to produce antibody and/or T cell immune response for prophylaxis or
for
therapeutic treatment of cancer and autoimmune disease and related conditions.
Yet
another aspect of the invention relates to a method of inducing, re-inforcing
or
l0 modulating immunological response in a mammal which comprises, delivering a
polypeptide of the present invention via a vector or cell directing expression
of the
polynucleotide and coding for the polypeptide in vivo in order to induce such
an
immunological response to produce immune responses for prophylaxis or
treatment of
said mammal from diseases.
A further aspect of the invention relates to an immunological/vaccine
formulation
(composition) which, when introduced into a mammalian host, induces, re-
inforces or
modulates an immunological response in that mammal to a polypeptide of the
present
invention wherein the composition comprises a polypeptide or polynucleotide of
the
invention or an immunological fragment thereof as herein before defined.The
vaccine
formulation may further comprise a suitable carrier. Since a polypeptide may
be broken
down in the stomach, it is preferably administered parenterally (for instance,
subcutaneous, intramuscular, intravenous, or intradermal injection).
Formulations
suitable for parenteral administration include aqueous and non-aqueous sterile
injection
solutions which may contain anti-oxidants, buffers, bacteriostats and solutes
which render
the formulation isotonic with the blood of the recipient; and aqueous and non-
aqueous
sterile suspensions which may include suspending agents or thickening agents.
The
formulations may be presented in unit-dose or multi-dose containers, for
example, sealed
ampoules and vials and may be stored in a freeze-dried condition requiring
only the
3o addition of the sterile liquid carrier immediately prior to use.
A further aspect of the invention relates to the in vitro induction of immune
responses to a
fragment or the entire polypeptide or polynucleotide of the present invention
or a
CA 02386028 2002-03-28
WO 01/23417 PCT/EP00/09500
molecule comprising the polypeptide or polynucleotide of the present
invention, using
cells from the immune system of a mammal, and reinfusing these activated
immune cells
of the mammal for the treatment of disease. Activation of the cells from the
immune
system is achieved by in vitro incubation with the entire polypeptide or
polynucleotide of
the present invention or a molecule comprising the polypeptide or
polynucleotide of the
present invention in the presence or absence of various immunomodulator
molecules.
A further aspect of the invention relates to the immunization of a mammal by
administration of antigen presenting cells modified by in vitro loading with
part or the
entire polypeptide of the present invention or a molecule comprising the
polypeptide of
to the present invention and administered in vivo in an immunogenic way.
Alternatively,
antigen presenting cells can be transfected in vitro with a vector containing
a fragment or
the entire polynucleotide of the present invention or a molecule comprising
the
polynucleotide of the present invention, such as to express the corresponding
polypeptide,
and administered in vivo in an immunogenic way.
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WO 01/23417 PCT/EP00/09500
According to another embodiment, the pharmaceutical compositions described
herein will
comprise one or more immunostimulants in addition to the immunogenic
polynucleotide,
polypeptide, antibody, T-cell and/or antigen presenting cell (APC)
compositions of this
invention. An immunostimulant refers to essentially any substance that
enhances or
potentiates an immune response (antibody and/or cell-mediated) to an exogenous
antigen.
One preferred type of immunostimulant comprises an adjuvant. Many adjuvants
contain
a substance designed to protect the antigen from rapid catabolism, such as
aluminum
hydroxide or mineral oil, and a stimulator of immune responses, such as lipid
A,
Bortadella pertussis or Mycobacterium tuberculosis derived proteins. Certain
adjuvants
to are commercially available as, for example, Freund's Incomplete Adjuvant
and Complete
Adjuvant (Difco Laboratories, Detroit, MI); Merck Adjuvant 65 (Merck and
Company,
Inc., Rahway, NJ); AS-2 (SmithKline Beecham, Philadelphia, PA); aluminum salts
such
as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron
or zinc;
an insoluble suspension of acylated tyrosine; acylated sugars; cationically or
anionically
15 derivatized polysaccharides; polyphosphazenes; biodegradable microspheres;
monophosphoryl lipid A and quil A. Cytokines, such as GM-CSF, interleukin-2, -
7, -12,
and other like growth factors, may also be used as adjuvants.
Within certain embodiments of the invention, the adjuvant composition is
preferably one
2o that induces an immune response predominantly of the Thl type. High levels
of Thl-
type cytokines (e.g., IFN-~y, TNFa,, IL-2 and IL-12) tend to favor the
induction of cell
mediated immune responses to an administered antigen. In contrast, high levels
of Th2-
type cytokines (e.g., IL-4, IL-5, IL-6 and IL-10) tend to favor the induction
of humoral
immune responses. Following application of a vaccine as provided herein, a
patient will
25 support an immune response that includes Thl- and Th2-type responses.
Within a
preferred embodiment, in which a response is predominantly Thl-type, the level
of Thl-
type cytokines will increase to a greater extent than the level of Th2-type
cytokines. The
levels of these cytokines may be readily assessed using standard assays. For a
review of
the families of cytokines, see Mosmann and Coffinan, Ann. Rev. Immunol. 7:145-
173,
30 1989.
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WO 01/23417 PCT/EP00/09500
Certain preferred adjuvants for eliciting a predominantly Thl-type response
include, for
example, a combination of monophosphoryl lipid A, preferably 3-de-O-acylated
monophosphoryl lipid A, together with an aluminum salt. MPL~ adjuvants are
available
from Corixa Corporation (Seattle, WA; see, for example, US Patent Nos.
4,436,727;
4,877,61 l; 4,866,034 and 4,912,094). CpG-containing oligonucleotides (in
which the
CpG dinucleotide is unmethylated) also induce a predominantly Thl response.
Such
oligonucleotides are well known and are described, for example, in WO
96/02555, WO
99/33488 and U.S. Patent Nos. 6,008,200 and 5,856,462. Immunostimulatory DNA
sequences are also described, for example, by Sato et al., Science 273:352,
1996.
to Another preferred adjuvant comprises a saponin, such as Quil A, or
derivatives thereof,
including QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham, MA);
Escin;
Digitonin; or Gypsophila or Chenopodium quinoa saponins . Other preferred
formulations include more than one saponin in the adjuvant combinations of the
present
invention, for example combinations of at least two of the following group
comprising
QS21, QS7, Quil A, ~3-escin, or digitonin.
Alternatively the saponin formulations may be combined with vaccine vehicles
composed
of chitosan or other polycationic polymers, polylactide and polylactide-co-
glycolide
particles, poly-N-acetyl glucosamine-based polymer matrix, particles composed
of
polysaccharides or chemically modified polysaccharides, liposomes and lipid-
based
particles, particles composed of glycerol monoesters, etc. The saponins may
also be
formulated in the presence of cholesterol to form particulate structures such
as liposomes
or ISCOMs. Furthermore, the saponins may be formulated together with a
polyoxyethylene ether or ester, in either a non-particulate solution or
suspension, or in a
particulate structure such as a paucilamelar liposome or ISCOM. The saponins
may also
be formulated with excipients such as CarbopolR to increase viscosity, or may
be
formulated in a dry powder form with a powder excipient such as lactose.
In one preferred embodiment, the adjuvant system includes the combination of a
monophosphoryl lipid A and a saponin derivative, such as the combination of
QS21 and
3D-MPL~ adjuvant, as described in WO 94/00153, or a less reactogenic
composition
where the QS21 is quenched with cholesterol, as described in WO 96/33739.
Other
preferred formulations comprise an oil-in-water emulsion and tocopherol.
Another
23
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WO 01/23417 PCT/EP00/09500
particularly preferred adjuvant formulation employing QS21, 3D-MPL~ adjuvant
and
tocopherol in an oil-in-water emulsion is described in WO 95/17210.
Another enhanced adjuvant system involves the combination of a CpG-containing
oligonucleotide and a saponin derivative particularly the combination of CpG
and QS21
as disclosed in WO 00/09159. Preferably the formulation additionally comprises
an oil in
water emulsion and tocopherol.
Additional illustrative adjuvants for use in the pharmaceutical compositions
of the
to invention include Montanide ISA 720 (Seppic, France), SAF (Chiron,
California, United
States), ISCOMS (CSL), MF-59 (Chiron), the SBAS series of adjuvants (e.g.,
SBAS-2 or
SBAS-4, available from SmithKline Beecham, Rixensart, Belgium), Detox
(Enhanzyn~
(Corixa, Hamilton, MT), RC-529 (Corixa, Hamilton, MT) and other aminoalkyl
glucosaminide 4-phosphates (AGPs), such as those described in pending U.S.
Patent
15 Application Serial Nos. 08/853,826 and 09/074,720, the disclosures of which
are
incorporated herein by reference in their entireties, and polyoxyethylene
ether adjuvants
such as those described in WO 99/52549A1.
Other preferred adjuvants include adjuvant molecules of the general formula
(I):
HO(CHZCHzO)"-A-R
2o Wherein, n is 1-50, A is a bond or-C(O)-, R is C~_so alkyl or Phenyl C1-so
alkyl.
One embodiment of the present invention consists of a vaccine formulation
comprising a
polyoxyethylene ether of general formula (I), wherein n is between 1 and 50,
preferably
4-24, most preferably 9; the R component is C1-so, preferably C4-Czo alkyl and
most
preferably CIZ alkyl, and A is a bond. The concentration of the
polyoxyethylene ethers
25 should be in the range 0.1-20%, preferably from 0.1-10%, and most
preferably in the
range 0.1-1%. Preferred polyoxyethylene ethers are selected from the following
group:
polyoxyethylene-9-lauryl ether, polyoxyethylene-9-steoryl ether,
polyoxyethylene-8
steoryl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl
ether, and
polyoxyethylene-23-lauryl ether. Polyoxyethylene ethers such as
polyoxyethylene lauryl
24
CA 02386028 2002-03-28
WO 01/23417 PCT/EP00/09500
ether are described in the Merck index (12'h edition: entry 7717). These
adjuvant
molecules are described in WO 99/52549.
The polyoxyethylene ether according to the general formula (I) above may, if
desired, be
combined with another adjuvant. For example, a preferred adjuvant combination
is
preferably with CpG as described in the pending UK patent application GB
9820956.2.
Preferably a carrier is also present in the vaccine composition according to
the invention.
l0 The carrier may be an oil in water emulsion, or an aluminium salt, such as
aluminium
phosphate or aluminium hydroxide.
A preferred oil-in-water emulsion comprises a metabolisible oil, such as
squalene, alpha
tocopherol and Tween 80. In a particularly preferred aspect the antigens in
the vaccine
composition according to the invention are combined with QS21 and 3D-MPL in
such an
emulsion. Additionally the oil in water emulsion may contain span 85 and/or
lecithin
and/or tricaprylin.
Typically for human administration QS21 and 3D-MPL will be present in a
vaccine in the
range of lp,g - 200pg, such as 10-100~,g, preferably 10~g - SO~g per dose.
Typically the
oil in water will comprise from 2 to 10% squalene, from 2 to 10% alpha
tocopherol and
from 0.3 to 3% tween 80. Preferably the ratio of squalene: alpha tocopherol is
equal to
or less than 1 as this provides a more stable emulsion. Span 85 may also be
present at a
level of 1%. In some cases it may be advantageous that the vaccines of the
present
invention will further contain a stabiliser.
Non-toxic oil in water emulsions preferably contain a non-toxic oil, e.g.
squalane or
squalene, an emulsifier, e.g. Tween 80, in an aqueous carrier. The aqueous
Garner may
be, for example, phosphate buffered saline.
25
CA 02386028 2002-03-28
WO 01/23417 PCT/EP00/09500
A particularly potent adjuvant formulation involving QS21, 3D-MPL and
tocopherol in
an oil in water emulsion is described in WO 95/17210.
The present invention also provides a polyvalent vaccine composition
comprising a vaccine
formulation of the invention in combination with other antigens, in particular
antigens useful
for treating cancers, autoimmune diseases and related conditions. Such a
polyvalent vaccine
composition may include a TH-1 inducing adjuvant as hereinbefore described.
According to another embodiment of this invention, an immunogenic composition
1o described herein is delivered to a host via antigen presenting cells
(ADCs), such as
dendritic cells, macrophages, B cells, monocytes and other cells that may be
engineered
to be efficient APCs. Such cells may, but need not, be genetically modified to
increase
the capacity for presenting the antigen, to improve activation and/or
maintenance of the T
cell response, to have anti-tumor effects per se and/or to be immunologically
compatible
15 with the receiver (i.e., matched HLA haplotype). APCs may generally be
isolated from
any of a variety of biological fluids and organs, including tumor and
peritumoral tissues,
and may be autologous, allogeneic, syngeneic or xenogeneic cells.
Certain preferred embodiments of the present invention use dendritic cells or
progenitors
2o thereof as antigen-presenting cells. Dendritic cells are highly potent APCs
(Banchereau
and Steinman, Nature 392:245-251, 1998) and have been shown to be effective as
a
physiological adjuvant for eliciting prophylactic or therapeutic antitumor
immunity (see
Timmerman and Levy, Ann. Rev. Med. 50:507-529, 1999). In general, dendritic
cells
may be identified based on their typical shape (stellate in situ, with marked
cytoplasmic
25 processes (dendrites) visible in vitro), their ability to take up, process
and present
antigens with high efficiency and their ability to activate naive T cell
responses.
Dendritic cells may, of course, be engineered to express specific cell-surface
receptors or
ligands that are not commonly found on dendritic cells in vivo or ex vivo, and
such
modified dendritic cells are contemplated by the present invention. As an
alternative to
3o dendritic cells, secreted vesicles antigen-loaded dendritic cells (called
exosomes) may be
used within a vaccine (see Zitvogel et al., Nature Med. 4:594-600, 1998).
26
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WO 01/23417 PCT/EP00/09500
Dendritic cells and progenitors may be obtained from peripheral blood, bone
marrow,
tumor-infiltrating cells, peritumoral tissues-infiltrating cells, lymph nodes,
spleen, skin,
umbilical cord blood or any other suitable tissue or fluid. For example,
dendritic cells
may be differentiated ex vivo by adding a combination of cytokines such as GM-
CSF, IL-
4, IL-13 and/or TNFa to cultures of monocytes harvested from peripheral blood.
Alternatively, CD34 positive cells harvested from peripheral blood, umbilical
cord blood
or bone marrow may be differentiated into dendritic cells by adding to the
culture
medium combinations of GM-CSF, IL-3, TNFa, CD40 ligand, LPS, flt3 ligand
and/or
other compounds) that induce differentiation, maturation and proliferation of
dendritic
to cells.
Dendritic cells are conveniently categorized as "immature" and "mature" cells,
which
allows a simple way to discriminate between two well characterized phenotypes.
However, this nomenclature should not be construed to exclude all possible
intermediate
stages of differentiation. Immature dendritic cells are characterized as APC
with a high
capacity for antigen uptake and processing, which correlates with the high
expression of
Fcy receptor and mannose receptor. The mature phenotype is typically
characterized by a
lower expression of these markers, but a high expression of cell surface
molecules
responsible for T cell activation such as class I and class II MHC, adhesion
molecules
(e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80, CD86 and
4-
2o 1BB).
APCs may generally be transfected with a polynucleotide of the invention (or
portion or
other variant thereof) such that the encoded polypeptide, or an immunogenic
portion
thereof, is expressed on the cell surface. Such transfection may take place ex
vivo, arid a
pharmaceutical composition comprising such transfected cells may then be used
for
therapeutic purposes, as described herein. Alternatively, a gene delivery
vehicle that
targets a dendritic or other antigen presenting cell may be administered to a
patient,
resulting in transfection that occurs in vivo. In vivo and ex vivo
transfection of dendritic
cells, for example, may generally be performed using any methods known in the
art, such
as those described in WO 97/24447, or the gene gun approach described by Mahvi
et al.,
Immunology and cell Biology 75:456-460, 1997. Antigen loading of dendritic
cells may
be achieved by incubating dendritic cells or progenitor cells with the tumor
polypeptide,
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WO 01/23417 PCT/EP00/09500
DNA (naked or within a plasmid vector) or RNA; or with antigen-expressing
recombinant bacterium or viruses (e.g., vaccinia, fowlpox, adenovirus or
lentivirus
vectors). Prior to loading, the polypeptide may be covalently conjugated to an
immunological partner that provides T cell help (e.g., a carrier molecule).
Alternatively,
a dendritic cell may be pulsed with a non-conjugated immunological partner,
separately
or in the presence of the polypeptide.
While any suitable carrier known to those of ordinary skill in the art may be
employed in
the pharmaceutical compositions of this invention, the type of carrier will
typically vary
to depending on the mode of administration. Compositions of the present
invention may be
formulated for any appropriate manner of administration, including for
example, topical,
oral, nasal, mucosal, intravenous, intracranial, intraperitoneal, subcutaneous
and
intramuscular administration.
Carriers for use within such pharmaceutical compositions are biocompatible,
and may
15 also be biodegradable. In certain embodiments, the formulation preferably
provides a
relatively constant level of active component release. In other embodiments,
however, a
more rapid rate of release immediately upon administration may be desired. The
formulation of such compositions is well within the level of ordinary skill in
the art using
known techniques. Illustrative Garners useful in this regard include
microparticles of
20 poly(lactide-co-glycolide), polyacrylate, latex, starch, cellulose, dextran
and the like.
Other illustrative delayed-release Garners include supramolecular biovectors,
which
comprise a non-liquid hydrophilic core (e.g., a cross-linked polysaccharide or
oligosaccharide) and, optionally, an external layer comprising an amphiphilic
compound,
such as a phospholipid (see e.g., U.S. Patent No. 5,151,254 and PCT
applications WO
25 94/20078, WO/94/23701 and WO 96/06638). The amount of active compound
contained
within a sustained release formulation depends upon the site of implantation,
the rate and
expected duration of release and the nature of the condition to be treated or
prevented.
In another illustrative embodiment, biodegradable microspheres (e.g.,
polylactate
3o polyglycolate) are employed as carriers for the compositions of this
invention. Suitable
biodegradable microspheres are disclosed, for example, in U.S. Patent Nos.
4,897,268;
5,075,109; 5,928,647; 5,811,128; 5,820,883; 5,853,763; 5,814,344, 5,407,609
and
28
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WO 01/23417 PCT/EP00/09500
5,942,252. Modified hepatitis B core protein Garner systems. such as described
in
WG/99 40934, and references cited therein, will also be useful for many
applications.
Another illustrative carrier/delivery system employs a carrier comprising
particulate-
protein complexes, such as those described in U.S. Patent No. 5,928,647, which
are
capable of inducing a class I-restricted cytotoxic T lymphocyte responses in a
host.
The pharmaceutical compositions of the invention will often further comprise
one or
more buffers (e.g., neutral buffered saline or phosphate buffered saline),
carbohydrates
(e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins,
polypeptides or amino
acids such as glycine, antioxidants, bacteriostats, chelating agents such as
EDTA or
1o glutathione, adjuvants (e.g., aluminum hydroxide), solutes that render the
formulation
isotonic, hypotonic or weakly hypertonic with the blood of a recipient,
suspending agents,
thickening agents and/or preservatives. Alternatively, compositions of the
present
invention may be formulated as a lyophilizate.
The pharmaceutical compositions described herein may be presented in unit-dose
or
multi-dose containers, such as sealed ampoules or vials. Such containers are
typically
sealed in such a way to preserve the sterility and stability of the
formulation until use. In
general, formulations may be stored as suspensions, solutions or emulsions in
oily or
aqueous vehicles. Alternatively, a pharmaceutical composition may be stored in
a freeze-
dried condition requiring only the addition of a sterile liquid Garner
immediately prior to
use.
The development of suitable dosing and treatment regimens for using the
particular
compositions described herein in a variety of treatment regimens, including
e.g., oral,
parenteral, intravenous, intranasal, and intramuscular administration and
formulation, is
well known in the art, some of which are briefly discussed below for general
purposes of
illustration.
In certain applications, the pharmaceutical compositions disclosed herein may
be
delivered via oral administration to an animal. As such, these compositions
may be
formulated with an inert diluent or with an assimilable edible carrier, or
they may be
29
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WO 01/23417 PCT/EP00/09500
enclosed in hard- or soft-shell gelatin capsule, or they may be compressed
into tablets, or
they may be incorporated directly with the food of the diet.
The active compounds may even be incorporated with excipients and used in the
form of
ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions,
syrups, wafers,
and the like (see, for example, Mathiowitz et al., Nature 1997 Mar
27;386(6623):410-4;
Hwang et al., Crit Rev Ther Drug Carrier Syst 1998;15(3):243-84; U. S. Patent
5,641,515; U. S. Patent 5,580,579 and U. S. Patent 5,792,451). Tablets,
troches, pills,
capsules and the like may also contain any of a variety of additional
components, for
1o example, a binder, such as gum tragacanth, acacia, cornstarch, or gelatin;
excipients, such
as dicalcium phosphate; a disintegrating agent, such as corn starch, potato
starch, alginic
acid and the like; a lubricant, such as magnesium stearate; and a sweetening
agent, such
as sucrose, lactose or saccharin may be added or a flavoring agent, such as
peppermint,
oil of wintergreen, or cherry flavoring. When the dosage unit form is a
capsule, it may
15 contain, in addition to materials of the above type, a liquid Garner.
Various other
materials may be present as coatings or to otherwise modify the physical form
of the
dosage unit. For instance, tablets, pills, or capsules may be coated with
shellac, sugar, or
both. Of course, any material used in preparing any dosage unit form should be
pharmaceutically pure and substantially non-toxic in the amounts employed. In
addition,
20 the active compounds may be incorporated into sustained-release preparation
and
formulations.
Typically, these formulations will contain at least about 0.1 % of the active
compound or
more, although the percentage of the active ingredients) may, of course, be
varied and
25 may conveniently be between about 1 or 2% and about 60% or 70% or more of
the
weight or volume of the total formulation. Naturally, the amount of active
compounds)
in each therapeutically useful composition may be prepared is such a way that
a suitable
dosage will be obtained in any given unit dose of the compound. Factors such
as
solubility, bioavailability, biological half life, route of administration,
product shelf life,
30 as vc~ell as other pharmacological considerations will be contemplated by
one skilled in
the art of preparing such pharmaceutical formulations, and as such, a variety
of dosages
and treatment regimens may be desirable.
CA 02386028 2002-03-28
WO 01/23417 PCT/EP00/09500
For oral administration the compositions of the present invention may
alternatively be
incorporated with one or more excipients in the form of a mouthwash,
dentifrice, buccal
tablet, oral spray, or sublingual orally-administered formulation.
Alternatively, the active
ingredient may be incorporated into an oral solution such as one containing
sodium
borate, glycerin and potassium bicarbonate, or dispersed in a dentifrice, or
added in a
therapeutically-effective amount to a composition that may include water,
binders,
abrasives, flavoring agents, foaming agents, and humectants. Alternatively the
compositions may be fashioned into a tablet or solution form that may be
placed under
1o the tongue or otherwise dissolved in the mouth.
In certain circumstances it will be desirable to deliver the pharmaceutical
compositions
disclosed herein parenterally, intravenously, intramuscularly, or even
intraperitoneally.
Such approaches are well known to the skilled artisan, some of which are
further
described, for example, in U. S. Patent 5,543,158; U. S. Patent 5,641,515 and
U. S. Patent
5,399,363. In certain embodiments, solutions of the active compounds as free
base or
pharmacologically acceptable salts may be prepared in water suitably mixed
with a
surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared
in
glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under
ordinary
conditions of storage and use, these preparations generally will contain a
preservative to
prevent the growth of microorganisms.
Illustrative pharmaceutical forms suitable for injectable use include sterile
aqueous
solutions or dispersions and sterile powders for the extemporaneous
preparation of sterile
injectable solutions or dispersions (for example, see U. S. Patent 5,466,468).
In all cases
the form must be sterile and must be fluid to the extent that easy
syringability exists. It
must be stable under the conditions of manufacture and storage and must be
preserved
against the contaminating action of microorganisms, such as bacteria and
fungi. The
earner can be a solvent or dispersion medium containing, for example, water,
ethanol,
polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and
the like),
suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be
maintained, for
31
CA 02386028 2002-03-28
WO 01/23417 PCT/EP00/09500
example, by the use of a coating, such as lecithin, by the maintenance of the
required
particle size in the case of dispersion and/or by the use of surfactants. The
prevention of
the action of microorganisms can be facilitated by various antibacterial and
antifungal
agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal,
and the
like. In many cases, it will be preferable to include isotonic agents, for
example, sugars
or sodium chloride. Prolonged absorption of the injectable compositions can be
brought
about by the use in the compositions of agents delaying absorption, for
example,
aluminum monostearate and gelatin.
to In one embodiment, for parenteral administration in an aqueous solution,
the solution
should be suitably buffered if necessary and the liquid diluent first rendered
isotonic with
sufficient saline or glucose. These particular aqueous solutions are
especially suitable for
intravenous, intramuscular, subcutaneous and intraperitoneal administration.
In this
connection, a sterile aqueous medium that can be employed will be known to
those of
i5 skill in the art in light of the present disclosure. For example, one
dosage may be
dissolved in 1 ml of isotonic NaCI solution and either added to 1000 ml of
hypodermoclysis fluid or injected at the proposed site of infusion, (see for
example,
"Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-
1580).
Some variation in dosage will necessarily occur depending on the condition of
the subject
2o being treated. Moreover, for human administration, preparations will of
course
preferably meet sterility, pyrogenicity, and the general safety and purity
standards as
required by FDA Office of Biologics standards.
In another embodiment of the invention, the compositions disclosed herein may
be
25 formulated in a neutral or salt form. Illustrative pharmaceutically-
acceptable salts include
the acid addition salts (formed with the free amino groups of the protein) and
which are
formed with inorganic acids such as, for example, hydrochloric or phosphoric
acids, or
such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts
formed with the
free carboxyl groups can also be derived from inorganic bases such as, for
example,
3o sodium, potassium, ammonium, calcium, or ferric hydroxides, and such
organic bases as
isopropylamine, trimethylamine, histidine, procaine and the like. Upon
formulation,
32
CA 02386028 2002-03-28
WO 01/23417 PCT/EP00/09500
solutions will be administered in a manner compatible with the dosage
formulation and in
such amount as is therapeutically effective.
The carriers can further comprise any and all solvents, dispersion media,
vehicles,
coatings, diluents, antibacterial and antifungal agents, isotonic and
absorption delaying
agents, buffers, carrier solutions, suspensions, colloids, and the like. The
use of such
media and agents for pharmaceutical active substances is well known in the
art. Except
insofar as any conventional media or agent is incompatible with the active
ingredient, its
use in the therapeutic compositions is contemplated. Supplementary active
ingredients
to can also be incorporated into the compositions. The phrase
"pharmaceutically-acceptable" refers to molecular entities and compositions
that do not
produce an allergic or similar untoward reaction when administered to a human.
In certain embodiments, the pharmaceutical compositions may be delivered by
intranasal
15 sprays, inhalation, and/or other aerosol delivery vehicles. Methods for
delivering genes,
nucleic acids, and peptide compositions directly to the lungs via nasal
aerosol sprays has
been described, e.g., in U. S. Patent 5,756,353 and U. S. Patent 5,804,212.
Likewise, the
delivery of drugs using intranasal microparticle resins (Takenaga et al., J
Controlled
Release 1998 Mar 2;52(1-2):81-7) and lysophosphatidyl-glycerol compounds (U.
S.
2o Patent 5,725,871) are also well-known in the pharmaceutical arts. Likewise,
illustrative
transmucosal drug delivery in the form of a polytetrafluoroetheylene support
matrix is
described in U. S. Patent 5,780,045.
In certain embodiments, liposomes, nanocapsules, microparticles, lipid
particles, vesicles,
25 and the like, are used for the introduction of the compositions of the
present invention
into suitable host cells/organisms. In particular, the compositions of the
present invention
may be formulated for delivery either encapsulated in a lipid particle, a
liposome, a
vesicle, a nanosphere, or a nanoparticle or the like. Alternatively,
compositions of the
present invention can be bound, either covalently or non-covalently, to the
surface of
3o such carrier vehicles.
33
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WO 01/23417 PCT/EP00/09500
The formation and use of liposome and liposome-like preparations as potential
drug
carriers is generally known to those of skill in the art (see for example,
Lasic, Trends
Biotechnol 1998 Ju1;16(7):307-21; Takakura, Nippon Rinsho 1998 Mar;56(3):691-
5;
Chandran et al., Indian J Exp Biol. 1997 Aug;35(8):801-9; Margalit, Crit Rev
Ther Drug
Carrier Syst. 1995;12(2-3):233-61; U.S. Patent 5,567,434; U.S. Patent
5,552,157; U.S.
Patent 5,565,213; U.S. Patent 5,738,868 and U.S. Patent 5,795,587, each
specifically
incorporated herein by reference in its entirety).
Liposomes have been used successfully with a number of cell types that are
normally
1o difficult to transfect by other procedures, including T cell suspensions,
primary
hepatocyte cultures and PC 12 cells (Renneisen et al., J Biol Chem. 1990 Sep
25;265(27):16337-42; Muller et al., DNA Cell Biol. 1990 Apr;9(3):221-9). In
addition,
liposomes are free of the DNA length constraints that are typical of viral-
based delivery
systems. Liposomes have been used effectively to introduce genes, various
drugs,
15 radiotherapeutic agents, enzymes, viruses, transcription factors,
allosteric effectors and
the like, into a variety of cultured cell lines and animals. Furthermore, he
use of
liposomes does not appear to be associated with autoimmune responses or
unacceptable
toxicity after systemic delivery.
20 In certain embodiments, liposomes are formed from phospholipids that are
dispersed in
an aqueous medium and spontaneously form multilamellar concentric bilayer
vesicles
(also termed multilamellar vesicles (MLVs).
Alternatively, in other embodiments, the invention provides for
pharmaceutically
25 acceptable nanocapsule formulations of the compositions of the present
invention.
Nanocapsules can generally entrap compounds in a stable and reproducible way
(see, for
example, Quintanar-Guerrero et al., Drug Dev Ind Pharm. 1998 Dec;24(12):1113-
28).
To avoid side effects due to intracellular polymeric overloading, such
ultrafme particles
(sized around 0.1 p,m) may be designed using polymers able to be degraded in
vivo. Such
30 particles can be made as described, for example, by Couvreur et al., Crit
Rev Ther Drug
Carrier Syst. 1988;5(1):1-20; zur Muhlen et al., Eur J Pharm Biopharm. 1998
34
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WO 01/23417 PCT/EP00/09500
Mar;45(2):149-55; Zambaux et al. J Controlled Release. 1998 Jan 2;50(1-3):31-
40; and
U. S. Patent 5,145,684.
This invention also relates to the use of polynucleotides, in the form of
primers derived from
the polynucleotides of the present invention, and of polypeptides, in the form
of antibodies
or reagents specific for the polypeptide of the present invention, as
diagnostic reagents.
The identification of genetic or biochemical markers in blood or tissues that
will enable the
detection of very early changes along the carcinogenesis pathway will help in
determining
l0 the best treatment for the patient. Surrogate tumour markers, such as
polynucleotide
expression, can be used to diagnose different forms and states of cancer. The
identification
of expression levels of the polynucleotides of the invention will be useful in
both the
staging of the cancerous disorder and grading the nature of the cancerous
tissue. The staging
process monitors the advancement of the cancer and is determined on the
presence or
15 absence of malignant tissue in the areas biopsied. The polynucleotides of
the invention can
help to perfect the staging process by identifying markers for the aggresivity
of a cancer, for
example the presence in different areas of the body. The grading of the cancer
describes
how closely a tumour resembles normal tissue of its same type and is assessed
by its cell
morphology and other markers of differentiation. The polynucleotides of the
invention can
20 be useful in determining the tumour grade as they can help in the
determination of the
differentiation status of the cells of a tumour.
The diagnostic assays offer a process for diagnosing or determining a
susceptibility to
cancers, autoimmune disease and related conditions through diagnosis by
methods
25 comprising determining from a sample derived from a subject an abnormally
decreased or
increased level of polypeptide or mRNA. This method of diagnosis is known as
differential expression. The expression of a particular gene is compared
between a
diseased tissue and a normal tissue. A difference between the polynucleotide-
related
gene, mRNA, or protein in the two tissues is compared, for example in
molecular weight,
30 amino acid or nucleotide sequence, or relative abundance, indicates a
change in the gene,
or a gene which regulates it, in the tissue of the human that was suspected of
being
diseased.
CA 02386028 2002-03-28
WO 01/23417 PCT/EP00/09500
Decreased or increased expression can be measured at the RNA level. PolyA RNA
is
first isolated from the two tissues and the detection of mRNA encoded by a
gene
corresponding to a differentially expressed polynucleotide of the invention
can be
detected by, for example, in situ hybridization in tissue sections, reverse
trascriptase-
PCR, using Northern blots containing poly A+ mRNA, or any other direct or
inderect
RNA detection method. An increased or decreased expression of a given RNA in a
diseased tissue compared to a normal tissue suggests that the transcript
and/or the expressed
protein has a role in the disease. Thus detection of a higher or lower level
of mRNA
to corresponding to SEQ m NO 1 or 3 relative to normal level is indicative of
the presence
of cancer in the patient.
mRNA expression levels in a sample can be determined by generation of a
library of
expressed sequence tags (ESTs) from the sample. The relative representation of
ESTs in
the library can be used to assess the relative representation of the gene
transcript in the
starting sample. The EST analysis of the test can then be compared to the EST
analysis
of a reference sample to determine the relative expression levels of the
polynucleotide of
interest.
2o Other mRNA analyses can be carned out using serial analysis of gene
expression (SAGE)
methodology (Velculescu et. Al. Science (1995) 270:484) , differential display
methodology (For example, US 5,776,683) or hybridization analysis which relies
on the
specificity of nucleotide interactions.
Alternatively, the comparison could be made at the protein level. The protein
sizes in the
two tissues may be compared using antibodies to detect polypeptides in Western
blots of
protein extracts from the two tissues. Expression levels and subcellular
localization may
also be detected immunologically using antibodies to the corresponding
protein. Further
assay techniques that can be used to determine levels of a protein, such as a
polypeptide of
3o the present invention, in a sample derived from a host are well-known to
those of skill in the
art. A raised or decreased level of polypeptide expression in the diseased
tissue compared
with the same protein expression level in the normal tissue indicates that the
expressed
protein may be involved in the disease.
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WO 01/23417 PCT/EP00/09500
In the assays of the present invention, the diagnosis can be determined by
detection of gene
product expression levels encoded by at least one sequence set forth in SEQ ID
NOS: 1 or 3.
A comparison of the mRNA or protein levels in a diseased versus normal tissue
may also be
used to follow the progression or remission of a disease.
A large number of polynucleotide sequences in a sample can be assayed using
polynucleotide arrays. These can be used to examine differential expression of
genes and to
determine gene function. For example, arrays of the polynucleotide sequences
SEQ ID NO:
l0 1 or 3 can be used to determine if any of the polynucleotides are
differentially expressed
between a normal and cancer cell. In one embodiment of the invention, an array
of
oligonucleotides probes comprising the SEQ ID NO: 1 or 3 nucleotide sequence
or
fragments thereof can be constructed to conduct efficient screening of e.g.,
genetic
mutations. Array technology methods are well known and have general
applicability and
is can be used to address a variety of questions in molecular genetics
including gene
expression, genetic linkage, and genetic variability (see for example: M.Chee
et al., Science,
Vol 274, pp 610-613 (1996)).
"Diagnosis" as used herein includes determination of a subject's
susceptibility to a
20 disease, determination as to whether a subject presently has the disease,
and also the
prognosis of a subject affected by the disease.
The present invention, further relates to a diagnostic kit for performing a
diagnostic assay
which comprises:
25 (a) a polynucleotide of the present invention, preferably the nucleotide
sequence of SEQ
ID NO: 1 or 3, or a fragment thereof ;
(b) a nucleotide sequence complementary to that of (a);
(c) a polypeptide of the present invention, preferably the polypeptide of SEQ
ID NO: 2 or
4, or a fragment thereof; or
30 (d) an antibody to a polypeptide of the present invention, preferably to
the polypeptide of
SEQ ID N0:2 OR 4.
37
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WO 01/23417 PCT/EP00/09500
The nucleotide sequences of the present invention are also valuable for
chromosomal
localisation. The sequence is specifically targeted to, and can hybridize
with, a particular
location on an individual human c'nromosome. The mapping of relevant sequences
to
chromosomes according to the present invention is an important first step in
correlating
those sequences with gene associated disease. Once a sequence has been mapped
to a
precise chromosomal location, the physical position of the sequence on the
chromosome can
be correlated with genetic map data. Such data are found in, for example, V.
McKusick,
Mendelian Inheritance in Man (available on-line through Johns Hopkins
University Welch
Medical Library). The relationship between genes and diseases that have been
mapped to
l0 the same chromosomal region are then identified through linkage analysis
(coinheritance of
physically adjacent genes).The differences in the cDNA or genomic sequence
between
affected and unaffected individuals can also be determined.
The polypeptides of the invention or their fragments or analogs thereof, or
cells expressing
them, can also be used as immunogens to produce antibodies immunospecific for
polypeptides of the present invention. The term "immunospecific" means that
the antibodies
have substantially greater affinity for the polypeptides of the invention than
their affinity for
other related polypeptides in the prior art.
In a further aspect the invention provides an antibody immunospecific for a
polypeptide
according to the invention or an immunological fragment thereof as
hereinbefore defined.
Preferably the antibody is a monoclonal antibody
Antibodies generated against polypeptides of the present invention may be
obtained by
administering the polypeptides or epitope-bearing fragments, analogs or cells
to an animal,
preferably a non-human animal, using routine protocols. For preparation of
monoclonal
antibodies, any technique which provides antibodies produced by continuous
cell line
cultures can be used. Examples include the hybridoma technique (Kohler, G. and
Milstein,
C., Nature (1975) 256:495-497), the trioma technique, the human B-cell
hybridoma
technique (Kozbor et al., Immunology Today (1983) 4:72) and the EBV-hybridoma
technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, 77-96, Alan
R. Liss,
Inc., 1985).
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CA 02386028 2002-03-28
WO 01/23417 PCT/EP00/09500
Techniques for the production of single chain antibodies, such as those
described in U.S.
Patent No. 4,946,778, can also be adapted to produce single chain antibodies
to polypeptides
of this invention. Also, transgenic mice, or other organisms, including other
mammals, may
be used to express humanized antibodies.
The above-described antibodies may be employed to isolate or to identify
clones expressing
the polypeptide or to purify the polypeptides by affinity chromatography.
The antibody of the invention may also be employed to prevent or treat cancer,
particularly
ovarian and colon cancer, autoimmune disease and related conditions.
1o
Another aspect of the invention relates to a method for inducing or modulating
an
immunological response in a mammal which comprises inoculating the mammal with
a
polypeptide of the present invention, adequate to produce antibody and/or T
cell immune
response to protect or ameliorate the symptoms or progression of the disease.
Yet
15 another aspect of the invention relates to a method of inducing or
modulating
immunological response in a mammal which comprises, delivering a polypeptide
of the
present invention via a vector directing expression of the polynucleotide and
coding for
the polypeptide in vivo in order to induce such an immunological response to
produce
antibody to protect said animal from diseases.
It will be appreciated that the present invention therefore provides a method
of treating
abnormal conditions such as, for instance, cancer and autoimmune diseases, in
particular,
ovarian and colon cancer, related to either a presence of, an excess of, or an
under-
expression of, CASB6411 polypeptide activity.
The present invention further provides for a method of screening compounds to
identify
those which stimulate or which inhibit the function of the CASB6411
polypeptide. In
general, agonists or antagonists may be employed for therapeutic and
prophylactic purposes
for such diseases as hereinbefore mentioned. Compounds may be identified from
a variety
3o of sources, for example, cells, cell-free preparations, chemical libraries,
and natural product
mixtures. Such agonists, antagonists or inhibitors so-identified may be
natural or modified
substrates, ligands, receptors, enzymes, etc., as the case may be, of the
polypeptide; or may
be structural or functional mimetics thereof (see Coligan et al., Current
Protocols in
39
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Immunology 1(2):Chapter 5 (1991)). Screening methods will be known to those
skilled in
the art. Further screening methods may be found in for example D. Bennett et
al., J Mol
Recognition, 8:52-58 (1995); and K. Johanson et al., J Biol Chem, 270(16):9459-
9471
(1995) and references therein.
Thus the invention provides a method for screening to identify compounds which
stimulate
or which inhibit the function of the polypeptide of the invention which
comprises a method
selected from the group consisting of
(a) measuring the binding of a candidate compound to the polypeptide (or to
the cells or
membranes bearing the polypeptide) or a fusion protein thereof by means of a
label
directly or indirectly associated with the candidate compound;
(b) measuring the binding of a candidate compound to the polypeptide (or to
the cells or
membranes bearing the polypeptide) or a fusion protein thereof in the presense
of a
labeled competitior;
(c) testing whether the candidate compound results in a signal generated by
activation or
inhibition of the polypeptide, using detection systems appropriate to the
cells or cell
membranes bearing the polypeptide;
(d) mixing a candidate compound with a solution containing a polypeptide of
claim l, to
form a mixture, measuring activity of the polypeptide in the mixture, and
comparing the
2o activity of the mixture to a standard; or
(e) detecting the effect of a candidate compound on the production of mRNA
encoding
said polypeptide and said polypeptide in cells, using for instance, an ELISA
assay.
The polypeptide of the invention may be used to identify membrane bound or
soluble
receptors, if any, through standard receptor binding techniques known in the
art. Well
known screening methods may also be used to identify agonists and antagonists
of the
polypeptide of the invention which compete with the binding of the polypeptide
of the
invention to its receptors, if any.
Thus, in another aspect, the present invention relates to a screening kit for
identifying
agonists, antagonists, ligands, receptors, substrates, enzymes, etc. for
polypeptides of the
present invention; or compounds which decrease or enhance the production of
such
polypeptides, which comprises:
CA 02386028 2002-03-28
WO 01/23417 PCT/EP00/09500
(a) a polypeptide of the present invention;
(b) a recombinant cell expressing a polypeptide of the present invention;
(c) a cell membrane expressing a polypeptide of the present invention; or
(d) antibody to a polypeptide of the present invention;
which polypeptide is preferably that of SEQ ID N0:2 OR 4.
It will be readily appreciated by the skilled artisan that a polypeptide of
the present
invention may also be used in a method for the structure-based design of an
agonist,
antagonist or inhibitor of the polypeptide, by:
to (a) determining in the first instance the three-dimensional structure of
the polypeptide;
(b) deducing the three-dimensional structure for the likely reactive or
binding sites) of
an agonist, antagonist or inhibitor;
(c) synthesing candidate compounds that are predicted to bind to or react with
the
deduced binding or reactive site; and
15 (d) testing whether the candidate compounds are indeed agonists,
antagonists or
inhibitors.
Gene therapy may also be employed to effect the endogenous production of
CASB6411
polypeptide by the relevant cells in the subject. For an overview of gene
therapy, see
2o Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic
Approaches,
(and references cited therein) in Human Molecular Genetics, T Strachan and A P
Read,
BIOS Scientific Publishers Ltd (1996).
Vaccine preparation is generally described in Pharmaceutical Biotechnology,
Vo1.61
25 Vaccine Design - the subunit and adjuvant approach, edited by Powell and
Newman,
Plenurn Press, 1995. New Trends and Developments in Vaccines, edited by Voller
et al.,
University Park Press, Baltimore, Maryland, U.S.A. 1978. Encapsulation within
liposomes is described, for example, by Fullerton, U.S. Patent 4,235,877.
Conjugation of
proteins to macromolecules is disclosed, for example, by Likhite, U.S. Patent
4,372,945
30 and by Armor et al., U.S. Patent 4,474,757.
The amount of protein in each vaccine dose is selected as an amount which
induces an
immunoprotective response without significant, adverse side effects in typical
vaccinees.
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Such amount will vary depending upon which specific immunogen is employed.
Generally, it is expected that each dose will comprise 1-1000p.g of protein,
preferably
2-lUOp,g, most preferably 4-40p,g. An optimal amount for a particular vaccine
can be
ascertained by standard studies involving observation of antibody titres and
other
responses in subjects. Following an initial vaccination, subjects may receive
a boost in
about 4 weeks.
"Isolated" means altered "by the hand of man" from the natural state. If an
"isolated"
composition or substance occurs in nature, it has been changed or removed from
its
1o original environment, or both. For example, a polynucleotide or a
polypeptide naturally
present in a living animal is not "isolated," but the same polynucleotide or
polypeptide
separated from the coexisting materials of its natural state is "isolated", as
the term is
employed herein.
15 "Polynucleotide" generally refers to any polyribonucleotide or
polydeoxribonucleotide,
which may be unmodified RNA or DNA or modified RNA or DNA including single and
double stranded regions.
"Variant" refers to a polynucleotide or polypeptide that differs from a
reference
20 polynucleotide or polypeptide, but retains essential properties. A typical
variant of a
polynucleotide differs in nucleotide sequence from another, reference
polynucleotide.
Changes in the nucleotide sequence of the variant may or may not alter the
amino acid
sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide
changes
may result in amino acid substitutions, additions, deletions, fusions and
truncations in the
25 polypeptide encoded by the reference sequence, as discussed below. A
typical variant of
a polypeptide differs in amino acid sequence from another, reference
polypeptide.
Generally, differences are limited so that the sequences of the reference
polypeptide and
the variant are closely similar overall and, in many regions, identical. A
variant and
reference polypeptide may differ in amino acid sequence by one or more
substitutions,
30 additions, deletions in any combination. A substituted or inserted amino
acid residue
may or may not be one encoded by the genetic code. A variant of a
polynucleotide or
polypeptide may be a naturally occurring such as an allelic variant, or it may
be a variant
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that is not known to occur naturally. Non-naturally occurring variants of
polynucleotides
and polypeptides may be made by mutagenesis techniques or by direct synthesis.
"Identity," as known in the art, is a relationship between two or more
polypeptide sequences
or two or more polynucleotide sequences, as determined by comparing the
sequences. In the
art, "identity" also means the degree of sequence relatedness between
polypeptide or
polynucleotide sequences, as the case may be, as determined by the match
between
strings of such sequences. "Identity" and "similarity" can be readily
calculated by known
methods, including but not limited to those described in (Computational
Molecular
1o Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988;
Biocomputing:
Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York,
1993;
Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G.,
eds.,
Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von
Heinje,
G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and
Devereux,
J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D.,
SIAM J.
Applied Math., 48: 1073 (1988). Preferred methods to determine identity are
designed to
give the largest match between the sequences tested. Methods to determine
identity and
similarity are codified in publicly available computer programs. Preferred
computer
program methods to determine identity and similarity between two sequences
include, but
2o are not limited to, the GCG program package (Devereux, J., et al., Nucleic
Acids
Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S.F. et al.,
J.
Molec. Biol. 215: 403-410 (1990). The BLAST X program is publicly available
from
NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH
Bethesda,
MD 20894; Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990). The well
known Smith
Waterman algorithm may also be used to determine identity.
The preferred algorithm used is FASTA. The preferred parameters for
polypeptide or
polynuleotide sequence comparison using this algorithm include the following:
Gap Penalty:l2
3o Gap extension penalty: 4
Word size: 2, max 6
43
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Preferred parameters for polypeptide sequence comparison with other methods
include
the following:
1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)
Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad.
Sci.
USA. 89:10915-10919 (1992)
Gap Penalty: 12
Gap Length Penalty: 4
A program useful with these parameters is publicly available as the "gap"
program from
l0 Genetics Computer Group, Madison WI. The aforementioned parameters are the
default
parameters for polypeptide comparisons (along with no penalty for end gaps).
Preferred parameters for polynucleotide comparison include the following:
1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)
Comparison matrix: matches = +10, mismatch = 0
Gap Penalty: 50
Gap Length Penalty: 3
A program useful with these parameters is publicly available as the "gap"
program from
Genetics Computer Group, Madison WI. The aforementioned parameters are the
default
parameters for polynucleotide comparisons.
By way of example, a polynucleotide sequence of the present invention may be
identical
to the reference sequence of SEQ ID NO:1, that is be 100% identical, or it may
include
up to a certain integer number of nucleotide alterations as compared to the
reference
sequence. Such alterations are selected from the group consisting of at least
one
nucleotide deletion, substitution, including transition and transversion, or
insertion, and
wherein said alterations may occur at the 5' or 3' terminal positions of the
reference
nucleotide sequence or anywhere between those terminal positions, interspersed
either
individually among the nucleotides in the reference sequence or in one or more
contiguous groups within the reference sequence. The number of nucleotide
alterations is
determined by multiplying the total number of nucleotides in SEQ ID NO:1 by
the
44
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WO 01/23417 PCT/EP00/09500
numerical percent of the respective percent identity(divided by 100) and
subtracting that
product from said total number of nucleotides in SEQ ID NO:1, or:
nn ~ xn ' (xn' Y)
wherein nn is the number of nucleotide alterations, xn is the total number of
nucleotides
in SEQ 117 NO:I, and y is, for instance, 0.70 for 70%, 0.80 for 80%, 0.85 for
85%, 0.90
for 90%, 0.95 for 95%,etc., and wherein any non-integer product of xn and y is
rounded
down to the nearest integer prior to subtracting it from xn. Alterations of a
polynucleotide sequence encoding the polypeptide of SEQ ID N0:2 OR 4 may
create
nonsense, missense or frameshift mutations in this coding sequence and thereby
alter the
l0 polypeptide encoded by the polynucleotide following such alterations.
Similarly, a polypeptide sequence of the present invention may be identical to
the
reference sequence of SEQ ID N0:2 OR 4, that is be 100% identical, or it may
include up
to a certain integer number of amino acid alterations as compared to the
reference
sequence such that the % identity is less than 100%. Such alterations are
selected from
the group consisting of at least one amino acid deletion, substitution,
including
conservative and non-conservative substitution, or insertion, and wherein said
alterations
may occur at the amino- or carboxy-terminal positions of the reference
polypeptide
sequence or anywhere between those terminal positions, interspersed either
individually
among the amino acids in the reference sequence or in one or more contiguous
groups
within the reference sequence. The number of amino acid alterations for a
given
identity is determined by multiplying the total number of amino acids in SEQ
ID N0:2
OR 4 by the numerical percent of the respective percent identity(divided by
100) and then
subtracting that product from said total number of amino acids in SEQ ID N0:2
OR-4, or:
na~xa ' (xa ~ y),
wherein na is the number of amino acid alterations, xa is the total number of
amino acids
in SEQ ID N0:2 OR 4, and y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85
for 85%
etc., and wherein any non-integer product of xa and y is rounded down to the
nearest
integer prior to subtracting it from xa.
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"Homolog" is a generic term used in the art to indicate a polynucleotide or
polypeptide
sequence possessing a high degree of sequence relatedness to a subject
sequence. Such
relatedness may be quntified by determining the degree of identity and/or
similarity between
the sequences being compared as hereinbefore described. Falling within this
generic term
are the terms "ortholog", meaning a polynucleotide or polypeptide that is the
functional
equivalent of a polynucleotide or polypeptide in another species and "paralog"
meaning a
functionally similar sequence when considered within the same species.
to Examples
Example 1
Subtractive cDNA cloning of colon tumour-associated antigen (TAA) candidates.
Subtractive cDNA libraries are produced using standard technologies. Briefly,
total
15 RNA is extracted from frozen (-70°C) tumour and matched normal colon
samples using the
TriPure reagent and protocol (Boehringer). Target RNA is prepared by pooling
total RNA
from three tumour samples (30 ~.g each). Driver RNA is prepared by pooling
total RNA
from three matched normal colon samples (10 ug each) and total RNA from seven
normal
tissues other than colon(brain, heart, kidney, liver, bladder, skin, spleen;
10 ~.g each). Total
2o RNA from non-colon normal tissues is purchased from InVitrogen.
Messenger RNA is purified from total RNA using oligo-dT magnetic bead
technology (Dynal) and quantified by spectrofluorimetry (BioRad).
Target and driver mRNA are reverse transcribed into cDNA using one of two
strategies: 1) Target sequences for PCR oligonucleotides are introduced onto
the ends of the
25 newly synthesised cDNA during reverse transcription using the template
switching
capability of reverse transcriptase (ClonTech SMART PCR cDNA synthesis kit).
2)
Alternatively, the target and driver mRNA are reverse transcribed into cDNA
using an
oligo-dT primer and converted to double-strand cDNA; the cDNA is cleaved with
RsaI and
linkers for PCR amplification are ligated onto the extremities of the cDNA
fragments.
3o In both cases, target and driver cDNA are amplified by long range PCR
(ClonTech
SMART PCR Synthesis Kit and Advantage PCR Polymerise Mix) and used as starting
material for subtractive cloning. For amplification, cycling conditions and
optimisation of
the number of PCR cycles are as described in the Advantage PCR protocol.
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Two subtractive cloning strategies are used: ClonTech PCR SELECT (see
ClonTech kit protocol and N. Gurskaya et al. 1996. Analytical Biochemistry:
240, 90) and
cRDA(M. Hubank and D. Schatz. 1994. Nucleic Acids Research: 22, 5640) . When
the PCR
SELECT protocol is used, the primary PCR SELECT subtraction products are
submitted to
a supplementary round of cRDA subtraction. When the cRDA protocol is used, two
consecutive cycles of cRDA subtraction are performed. In each case the
products of both
cycles of subtraction are cloned into pCR-TOPO (InVitrogen) and transformed
into E. coli
to produce a subtracted cDNA plasmid library.
to An alternative strategy is also followed: subtraction of normal colon
sequences and
sequences from non-colon normal tissues are subtracted in separate
hybridizations. In this
case, target and driver RNA are assembled for the first subtraction as above
with the
exception that non-colon RNA is left out of the driver pool and amounts of
normal colon are
increased to 10 p.g. Preparation of target and driver cDNA and subtractive
hybridization are
15 performed as described above. A second subtraction is then performed on the
products of the
first subtraction, but the driver is now composed of a pool of normal colon
and normal non-
colon mRNA from the seven normal tissues.
Example 2
2o Differential Screening of cDNA arrays.
Identification of tumour-associated genes in the subtracted cDNA library is
accomplished by
differential screening.
Total bacterial DNA is extracted from 100 ~1 over-night cultures. Bacteria are
lysed with
25 guanidium isothiocyantate and the bacterial DNA is affinity purified using
magnetic glass
(Boehringer). Plasmid inserts are recovered from the bacterial DNA by
Advantage PCR
amplification (Clontech). The PCR products are dotted onto two nylon membranes
to
produce high density cDNA arrays using the Biomek 96 HDRT tool (Beekman). The
spotted cDNA is covalently linked to the membrane by LTV irradiation. The
first membrane
30 is hybridised with a mixed cDNA probe prepared from the tumour of a single
patient. The
second membrane is hybridised with an equivalent amount of mixed cDNA probe
prepared
from normal colon of the same patient. The probe cDNA is prepared by PCR
amplification
as described above and is labelled using the AlkPhos Direct System (Amersham).
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Hybridisation conditions and stringency washes are as described in the AlkPhos
Direct kit.
Hybridized probe is detected by chemiluminescence. Hybridisation intensities
for each
cDNA fragment on both blots are measured by film densitometry or direct
measurement
(BioRad Fluor-S Max). The ratio of the tumour to normal hybridisation
intensities (T/I~ is
calculated for each gene to evaluate the degree of over-expression in the
tumour. Genes
which are significantly over-expressed in colon tumours are followed-up.
Significance is
arbitrarily defined as one standard deviation of the T/N frequency
distribution. Differential
screening experiments are repeated using RNA from multiple patient donors
(>18) to
estimate the frequency of over-expressing tumours in the patient population.
l0 In addition, the DNA arrays are hybridised with mixed cDNA probes from
normal tissues
other than colon (see list above) to determine the level of expression of the
candidate gene in
these tissues.
Example 3
Real-time RT-PCR analysis
Real-time RT-PCR (LT. Gibson. 1996. Genome Research: 6,996) is used to compare
mRNA transcript abundance of the candidate antigen in matched tumour and
normal
colon tissues from multiple patients. In addition, mRNA levels of the
candidate gene in a
panel of normal tissues are also evaluated by this approach.
Total RNA from normal and tumour colon is extracted from snap frozen biopsies
using
TriPure reagent (Boehringer). Total RNA from normal tissues is purchased from
InVitrogen or is extracted from snap frozen biopsies using TriPure reagent.
Poly-A+
mRNA is purified from total RNA after DNAase treatment using oligo-dT magnetic
beads (Dynal). Quantification of the mRNA is performed by spectrofluorimetry
(VersaFluor, BioRad) using SybrII dye (Molecular Probes). Primers for real-
time PCR
amplification are designed with the Perkin-Elmer Primer Express software using
default
options for TaqMan amplification conditions.
3o Real-time reactions are assembled according to standard PCR protocols using
2 ng of
purified mRNA for each reaction. SybrI dye (Molecular Probes) is added at a
final
dilution of 1/75000 for real-time detection. Amplification (40 cycles) and
real-time
detection is performed in a Perkin-Elmer Biosystems PE7700 system using
conventional
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instrument settings. Ct values are calculated using the PE7700 Sequence
Detector
software. Several Ct values are obtained for each samples : for the patient
samples, the
tumour Ct (CtT) and the matched normal colon Ct (CtI~ values on the candidate
TAA,
and for the panel of normal tissue samples, a CtXY for each normal tissue XY.
An
another Ct (CtA) is also calculated on Actin gene, as an internal reference,
for all of the
samples.
As the efficiency of PCR amplification under the prevailing experimental
conditions is
close to the theoretical amplification efficiency, 2~~tNrr/XY-cw> value is an
estimate of the
to relative TAA transcript level of the sample, standardised with respect to
Actin transcript
level. A value of 1 thus suggests the candidate antigen and Actin have the
same
expression level.
Real-time PCR reactions were performed on tumour colon and adjacent normal
colon
from biopsies of 18 patients. Results are shown in figure 1. 36 normal tissue
samples,
representing 28 different tissues (see table 2), were also tested by the same
procedure.
Results are shown in figure 2.
TAA transcript levels are calculated as described above. The proportion of
patients over-
2o expressing the candidate antigen, as well as the average transcript over-
expression versus
normal tissues is also calculated from this data set.
Overall results are shown in Table 1
Table 1 : CASB6411 Real-time PCR expression results
of patients with a CASB6411 transcript level 83%
higher in tumour colon than
adjacent normal colon (positive patients)
of positive patients with a CASB6411 transcript 60%
level at least 3 fold higher
CASB6411 transcript than adjacent normal colon
of positive patients with a CASB6411 transcript 40%
level at least 10 fold higher in
tumour colon than adjacent normal colon
Average transcript over-expression fold in positive40
patients
of patients with a CASB6411 transcript level 100%
higher in tumour colon than
normal tissue median
of patients with a mRNA level at least 3 fold 100%
higher in tumour colon than
normal tissue median
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of patients with a mRNA level at least 10 fold 40%
higher in tumour colon than
normal tissue median
Normal tissues where CASB6411 transcript expressionLung,
is equivalent than tumour
transcript level in tumours prostate
Table 1 clearly suggest CASB6411 transcript is over-expressed in colorectal
tumours
compared to adjacent normal colon and to most of the above mentioned normal
tissues.
More than 80% of the patients strongly over-express CASB6411 transcript in
tumour, as
compared to adjacent normal colon. Average over-expression fold is at least of
40, with
40 % of patients having overexpressing at least 10 fold. More over, all of the
patients
over-express CASB6411 transcript in colorectal tumors, as compared to other
normal
tissues, 40 % of them overexpressing it at least 10 fold.
l0
Table 2 : listing of normal tissues used for CASB7439 transcript expression
analysis.
Tissue Abbreviation Categorie
Adrenal gland Ad_GI non dispensable
Aorta Ao non dispensable
Bladder BI non dispensable
Bone marrow Bo_Ma non dispensable
Brain Bra non dispensable
Cervix Ce non dispensable
Colon Co non dispensable
Fallopian tubeFa_Tu non dispensable
Heart He non dispensable
Ileon II non dispensable
Kidney Ki non dispensable
Liver Li non dispensable
Lung Lu non dispensable
Lymph node Ly No non dispensable
Oesophagus Oe non dispensable
Parathyroid Pa_Thy non dispensable
gland
Rectum Re non dispensable
Skin Sk non dispensable
Skeletal muscleSk_Mu non dispensable
Small intestineSm_In non dispensable
Spleen Sp non dispensable
Stomach St non dispensable
Thyroid gland Thy non dispensable
Trachea Tra non dispensable
Ovary Ov dispensable
Placenta PI dispensable
Prostate Pr dispensable
Testis Te dispensable
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Example 4
DNA microarrays
DNA micro-arrays are used to examine mRNA expression profiles of large
collections of
genes in multiple samples. This information is used to complement the data
obtained by
real-time PCR and provides an independent measure of gene expression levels in
tumors
and normal tissues.
Examples of current technologies for production of DNA micro-arrays include 1
) The
Affymetrix "GeneChip" arrays in which oligonucleotides are synthetized on the
surface
to of the chip by solid phase chemical synthesis using a photolithographic
process 2) DNA
spotting technology in which small volumes of a DNA solution are robotically
deposited
and then immobilized onto the surface of a solid phase (e.g. glass). In both
instances, the
chips are hybridized with cDNA or cRNA which has been extracted from the
tissue of
interest (e.g. normal tissue, tumour etc...) and labeled with radioactivity or
with a
15 fluorescent reporter molecule. The labeled material is hybridized to the
chip and the
amount of probe bound to each sequence on the chip is determined using a
specialized
scanner. The experiment can be set-up with a single fluorescent reporter (or
radioactivity)
or, alternatively, can be performed using two fluorescent reporters. In this
latter case,
each of the two samples is labeled with one of the reporter molecules. The two
labeled
20 samples are then hybridized competitively to the sequences on the DNA chip.
The ratio
of the two fluorescent signals is determined for each sequence on the chip.
This ratio is
used to calculate the relative abundance of the transcript in the two samples.
Detailed
protocols are available from a number of sources including "DNA Microarrays: A
practical approach. Schena M. Oxford University Press 1999" and the World Wide
Web
25 (http://cmgm.Stanford.edu/pbrown/protocols/index.html),
http://arrayit.com/DNA-
Microarray-Protocolsn and specialized distributors (e.g. Affymetrix).
Example 5
3o Northern-Southern blot analysis
Limited amounts of mixed tumour and matched normal colon cDNA are amplified by
Advantage PCR (see above). Messenger RNA from multiple normal tissues is also
amplified using the same procedure. The amplified cDNA (1 fig) is
electrophoresed on a
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1.2% agarose gel and transferred onto a nylon membrane. The membrane is
hybridised
(AlkPhos Direct System) with a probe prepared using a fragment of the
candidate TAA
cDNA. Northern-Southern analysis provides information on transcript size,
presence of
splice variants and transcript abundance in tumour and normal tissues.
Example 6
Northern Blot Analysis
Northern blots are produced according to standard protocols using 1 pg of poly
A+
mRNA. Radioactive probes are prepared using the Ready-to-Go system
(Pharmacia).
Example 7
In silico detection of the full length cDNA sequence
EST sequence databases are screened with experimentally obtained cDNA sequence
fragments, using the Blast algorithm (Altschul, S.F., Gish, W., Miller, W.,
Myers, E.W.
& Lipman, D.J. (1990) "Basic local alignment search tool." J. Mol. Biol.
215:403-41).
The aim is to search for overlapping or longer identical EST sequences.
Matched EST
sequences are then assembled together, using the SeqMan software of the
Lasergene
package (DNASTAR). The consensus sequence of the resulting assembly is an EST-
derived longer cDNA. This EST-derived cDNA is further analysed using the
GeneMark
software to find a potential open reading frame (ORF). The translated sequence
of the
ORF is compared with protein databases, using the Blast algorithm, to find
homologues.
If any, the homologous protein sequences are further used to complete the cDNA
prediction by searching for genomic contig homologies using the Wise2
algorithm,
leading to a genome-derived, virtual cDNA sequence. This virtual cDNA in
finally
assembled with EST-derived cDNA, and the new consensus cDNA undergoes a final
check against ESTs to confirm the Wise2 prediction, and correct potential
sequencing
errors and frameshi$s. The virtual cDNA is considered as a virtual full length
cDNA
once a full ORF (from start to stop codons), with clear protein homologies and
coding
potential.
The SEQ ID NO: l has been obtained using this in silico cloning method of the
full length
cDNA sequence of CASB6411, which has a putative open reading frame of 460
amino
acids (SEQ ID N0:2), and a potential isoform generated by alternative splicing
(SEQ ID
N0:3), encoding a truncated protein (SEQ ID N0:4).
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Virtual full-length cDNA sequence is experimentally checked as described
below.
Example 8
Experimental Identification of the full length cDNA sequence
Colon tumour cDNA libraries are constructed using the Lambda Zap II system
(Stratagene) from 5 p.g of polyA+ mRNA. The supplied protocol is followed
except that
SuperscriptII (Life Technologies) is used for the reverse transcription step.
Oligo dT-
primed and random-primed libraries are constructed. About 1.5 x106 independent
phages
are plated for each screening of the library. Phage plaques are transferred
onto nylon
1o filters and hybridised using a cDNA probe labelled with AlkPhos Direct.
Positive phages
are detected by chemiluminescence. Positive phage are excised from the agar
plat, eluted
in SOOp.I SM buffer and confirmed by gene-specific PCR. Eluted phages are
converted to
single strand M13 bacteriophage by in vivo excision. The bacteriophage is then
converted
to double strand plasmid DNA by infection of E. coli. Infected bacteria are
plated and
submitted to a second round of screening with the cDNA probe. Plasmid DNA is
purified
from positive bacterial clones and sequenced on both strands.
When the full length gene cannot be obtained directly from the cDNA library,
missing
sequence is isolated using RACE technology (Marathon Kit, ClonTech.). This
approach
2o relies on reverse transcribing mRNA into double strand cDNA, ligating
linkers onto the
ends of the cDNA and amplifying the desired extremity of the cDNA using a gene-
specific primer and one of the linker oligonucleotides. Marathon PCR products
are cloned
into a plasmid (pCRII-TOPO, InVitrogen) and sequenced.
Example 9.
EST profiles
A complementary approach to experimental antigen tissue expression
characterization is
to explore the human EST database. ESTs ('Expressed Sequence Tags ) are small
fragments of cDNA made from a collection of mRNA extracted from a particular
tissue
or cell line. Such database currently provides a massive amount of human ESTs
(106)
from several hundreds of cDNA tissue libraries, including tumoral tissues from
various
types and states of disease. By means of informatics tools (Blast), a
comparison search of
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the CASB6411 sequence is performed in order to have further insight into
tissue
expression.
EST distribution of CASB6411
EST GenBank Accession number EST cDNA tissue library
C00562 Human adult (K.Okubo)
C05837 Human pancreatic islet
AA172076 Stratagene ovarian cancer (#937219)
AA172244 Stratagene ovarian cancer (#937219)
AA612697 NCI_CGAP_Co10
AA371314 Prostate gland I
AI264367 NCI_CGAP_Co8
AI270207 NCI_CGAP_Co14
AI278830 NCI_CGAP_Co8
AI281230 NCI_CGAP_Co8
AI283827 NCI_CGAP_Co8
AI285194 NCI_CGAP_Co8
AI285227 NCI_CGAP_Co8
AI346622 NCI_CGAP_Co8
AI362363 NCI_CGAP_Gas4
AI473464 NCI_CGAP_Gas4
AI697014 NCI_CGAP_Pan1
AI799626 NCI_CGAP_Gas4
AI830044 NCI_CGAP_Lu19
AI921465 NCI_CGAP_Gas4
AI925050 NCI_CGAP_Gas4
AW029127 NCI_CGAP Gas4
AW365013 DT0057
AW452356 NCI_CGAP_Sub5
AW469177 NCI_CGAP_Gas4
AW469181 NCI_CGAP Gas4
AW582253 ST0212
AW810203 ST0125
AW810268 ST0125
AW810418 ST0125
AW814058 ST0198
--
AW869793 SN0075
SEQ ID NO:1 perfectly aligns with 32 ESTs : 13 are from 2 stomach cDNA
libraries, 9
are from 3 tumor colon libraries, 2 are from one tumor ovary library, one is
from one
tumor pancreas library, one is from one lung tumor library, one is from one
mixed tumor
library, one is from one normal prostate library, one is from one normal
stomach library,
one is from normal pancreas library, 2 are from libraries of unlrnown type.
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This clearly suggests, as expected, that CASB6411 is over-expressed in tumor
tissues,
with an emphasis in colorectal and stomach tumor tissues, as compared to
normal
tissues.
Example 10
10.1 Expression and purification of tumour-specific antigens
Expression in microbial hosts, or alternatively in vitro
transcription/translation, is used to
produce the antigen of the invention for vaccine purposes and to produce
protein
l0 fragments or whole protein for rapid purification and generation of
antibodies needed for
characterization of the naturally expressed protein by immunohistochemistry or
for
follow-up of purification.
Recombinant proteins may be expressed in two microbial hosts, E. coli and in
yeast
(such as Saccharomyces cerevisiae or Pichia pastoris). This allows the
selection of the
15 expression system with the best features for this particular antigen
production. In
general, the recombinant antigen will be expressed in E. coli and the reagent
protein
expressed in yeast.
The expression strategy first involves the design of the primary structure of
the
recombinant antigen. In general an expression fusion partner (EFP) is placed
at the N
20 terminal extremity to improve levels of expression that could also include
a region useful
for modulating the immunogenic properties of the antigen, an immune fusion
partner
(IFP). In addition, an affinity fusion partner (AFP) useful for facilitating
further
purification is included at the C-terminal end.
When the recombinant strains are available, the recombinant product is
characterized by
25 the evaluation of the level of expression and the prediction of further
solubility of the
protein by analysis of the behavior in the crude extract.
After growth on appropriate culture medium and induction of the recombinant
protein
expression, total extracts are analyzed by SDS-PAGE. The recombinant proteins
are
visualized in stained gels and identified by Western blot analysis using
specific
3o antibodies.
A comparative evaluation of the different versions of the expressed antigen
will allow the
selection of the most promising candidate that is to be used for further
purification and
immunological evaluation.
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The purification scheme follows a classical approach based on the presence of
an His
affinity tail in the recombinant protein. In a typical experiment the
disrupted cells are
filtered and the acellular extracts loaded onto an Ion Metal Affinity
Chromatography
(IMAC; Ni++NTA from Qiagen) that will specifically retain the recombinant
protein.
The retained proteins are eluted by 0-500 mM Imidazole gradient (possibly in
presence of
a detergent) in a phosphate buffer.
10.2 Antibody production and immunohistochemistry
to Small amounts of relatively purified protein can be used to generate
immunological
tools in order to
a) detect the expression by immunohistochemistry in normal or cancer tissue
sections;
b) detect the expression, and to follow the protein during the purification
process
(ELISA/ Western Blot); or
c) characterise/ quantify the purified protein (ELISA).
10.2.1 Polyclonal antibodies:
Immunization
Rabbits are immunised , intramuscularly (LM.) , 3 times at 3 weeks intervals
with
100~g of protein, formulated in the adjuvant 3D-MPL/QS21. Three weeks after
each
immunisation a blood sample is taken and the antibody titer estimated in the
serum by
ELISA using the protein as coating antigen following a standard protocol.
ELISA
96 well microplates (maxisorb Nunc) are coated with S~.g of protein overnight
at 4°C.
After lhour saturation at 37°C with PBS NCS 1%, serial dilution of the
rabbit sera is
added for 1H 30 at 37°C (starting at 1/10). After 3 washings in PBS
Tween, anti rabbit
biotinylated anti serum (Amersham ) is added (1/5000). Plates are washed and
peroxydase coupled streptavidin (1/5000) is added for 30 min at 37°C.
After washing,
3o SOpI TMB (BioRad) is added for 7 min and the reaction then stopped with
H2S04 0.2M.
The OD can be measured at 450 nm and midpoint dilutions calculated by
SoftmaxPro.
10.2.2 Monoclonal antibodies:
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Tmmnni~atinn
BALB/c mice are immunized 3 times at 3 week intervals with S p.g of purified
protein.
Bleedings are performed 14 days post II and 1 week post 3. The sera are tested
by Elisa
on purified protein used as coated antigen. Based on these results (midpoint
dilution >
5 10000 ) one mouse is selected for fusion
Fusion/ HATselection
Spleen cells are fused with the SP2/0 myeloma according to a standard protocol
using
PEG 40% and DMSO 5%. Cells are then seeded in 96 well plates 2.5 x104 - 105
to cells/well and resistant clones will be selected in HAT medium. The
supernatant of these
hybridomas will be tested for their content in specific antibodies and when
positive,
will be submitted to 2 cycles of limited dilution . After 2 rounds of
screening, 3
hybridomas will be chosen for ascitis production.
10.2.3 Immunohistochemistry
When antibodies are available, immuno staining is performed on normal or
cancer tissue
sections, in order to determine
0 the level of expression of the antigen of the invention in cancer relative
to normal
tissue or
0 the proportion of cancer of a certain type expressing the antigen
0 if other cancer types also express the antigen
0 the proportion of cells expressing the antigen in a cancer tissue
Tissue sample preparation
After dissection, the tissue sample is mounted on a cork disk in OCT compound
and
rapidly frozen in isopentane previously super cooled in liquid nitrogen (-
160°C). The
block will then be conserved at -70°C until use. 7-l0um sections will
be realised in a
cryostat chamber (-20, -30°C).
Staining
Tissue sections are dried for 5 min at room Temperature (RT), fixed in acetone
for
lOmin at RT, dried again, and saturated with PBS 0.5% BSA 5% serum. After 30
min at
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RT either a direct or indirect staining is performed using antigen specific
antibodies. A
direct staining leads to a better specificity but a less intense staining
whilst an indirect
staining leads to a more intense but less specific staining.
10.3 Analysis of human cellular immune responses to the antigen of the
invention
The immunological relevance of the antigen of the invention can be assessed by
in vitro
priming of human T cells. All T cell lymphocyte lines and dendritic cells are
derived
from PBMCs (peripheral blood mononuclear cells) of healthy donors (preferred
HLA-A2
to subtype). An HLA-A2.1/Kb transgenic mouse model is also used for screening
of HLA-
A2.1 peptides.
Newly discovered antigen-specific CD8+ T cell lines are raised and maintained
by
weekly in vitro stimulation. The lytic activity and the 0-IFN production of
the CD8 lines
15 in response to the antigen or antigen derived-peptides is tested using
standard procedures.
Two strategies to raise the CD8+ T cell lines are used: a peptide-based
approach and a
whole gene-based approach. Both approaches require the full-length cDNA of the
newly
discovered antigen in the correct reading frame to be either cloned in an
appropriate
2o delivery system or to be used to predict the sequence of HLA binding
peptides.
Peptide-based approach
Briefly, transgenic mice are immunized with adjuvanted HLA-A2 peptides, those
unable
to induce a CD8 response (as defined by an efficient lysis of peptide-pulsed
autologous
25 spleen cells) will be further analyzed in the human system.
Human dendritic cells (cultured according to Romani et al.) will be pulsed
with peptides
and used to stimulate CD8-sorted T cells (by Facs). After several weekly
stimulations, the
CD8 lines will be first tested on peptide-pulsed autologous BLCL (EBV-B
transformed
cell lines). To verify the proper in vivo processing of the peptide, the CD8
lines will be
30 tested on cDNA-transfected tumour cells (HLA-A2 transfected LnCaP, Skov3 or
CAMA
tumour cells).
Whole gene-based approach
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CD8+ T cell lines will be primed and stimulated with either gene-gun
transfected
dendritic cells, retrovirally transduced B7.1-transfected fibroblasts,
recombinant pox
virus (Kim et al.) or adenovirus (Butterfield et al.) infected dendritic
cells. Virus infected
cells are very efficient to present antigenic peptides since the antigen is
expressed at high
level but can only be used once to avoid the over-growth of viral T cells
lines.
After alternated stimulations, the CD8+ lines are tested on cDNA-transfected
tumour
cells as indicated above. Peptide specificity and identity is determined to
confirm the
immunological validation.
CD4+ T-cell response
Similarly, the CD4+ T-cell immune response can also be assessed. Generation of
specific
CD4+ T-cells is made using dendritic cells loaded with recombinant purified
protein or
peptides to stimulate the T-cells.
Predicted epitopes (nonamers and decamers) binding HLA alleles
The HLA Class I binding peptide sequences are predicted either by the Parker's
algorithm (Parker, K. C., M. A. Bednarek, and J. E. Coligan. 1994. Scheme for
ranking
2o potential HLA-A2 binding peptides based on independent binding of
individual peptide
side-chains. J. Immunol. 152:163 and http://bimas.dcrt.nih.gov/molbio/hla
binds or the
Rammensee method (Rammensee, Friede, Stevanovic, MHC ligands and peptide
motifs:
1st listing, Immunogenetics 41, 178-228, 1995 ; Rammensee, Bachmann,
Stevanovic:
MHC ligands and peptide motifs. Landes Bioscience 1997, and
http://134.2.96.221/scripts/hlaserver.dll/home.htm). Peptides are then
screened in the
HLA-A2.1/Kb transgenic mice model (Vitiello et al.).
The HLA Class II binding peptide sequences are predicted using the Tepitope
algorithm,
with a score cut-off set to 6 (Sturniolo, Hammer at al., Nature Biotechnology.
1999.
17;555-561).
3o The following tables gather the Class I and II predicted epitope sequences
HLA-A1
:
decamers
Rank Start Subsequence Residue Parker's Score * ~~ SEQ ID
Position Listing
1 379 ITEGRKIMIR 112.500 SEQ ID N0:9
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HLA-A0201 : nonamers
~
nk Start PositionSubsequence Residue Parker's SEQ ID
Listing Score
*
1 191 ___LLMDFVFSL 27926.980 SEQ ID NO:10
~
2 292 FLLFFPSFT 2650.811 SEQ ID NO:11
3 285 QMMTFFIFL 2266.849 ~ SEQ ID N0:12
r
4 286 MMTFFIFLL 1329.564 SEQ ID N0:13
403 FLIEKLIKL 926.658 SEQ ID N0:14
r 147 r VLLIRNIFL 739.032 SEQ ID N0:15
6
7 240 LVWIGIFFC 450.023 SEQ ID N0:16
8 305 TLAITIWRL 368.501 SEQ ID N0:17
9 ~ 22 LIFCWDFTV 348.892 SEQ ID N0:18
~
203 FLGEFLRRI 343.941 SEQ ID N0:19
11 112 LLLPFVVSC 273.114 SEQ ID N0:20
12 66 LLTRFSAYM 176.513 SEQ ID N0:21
!
13 21 KLIFCWDFT 119.495 SEQ ID N0:22
HLA-A0201 : decamers
Rank Start Subsequence Residue Parker's Score * SEQ ID
Position Listing
1 190 LLLMDFVFSL 6811.458 SEQ ID N0:23
2 191 LLMDFVFSLV 3407.985 SEQ ID N0:24
3 285 QMMTFFIFLL 2893.757 SEQ ID N0:25
4 93 YLAEYNLEFL 2497.344 SEQ )D N0:26
5 21 KLIFCWDFTV 1411.906 SEQ ID N0:27
6 293 LLFFPSFTGV 831.216 SEQ ID N0:28
7 65 QLLTRFSAYM 384.175 SEQ ID N0:29
8 239 TLVWIGIFFC 364.502 SEQ ID N0:30
9 284 SQMMTFFIFL 318.231 SEQ ID N0:31
10 111 VLLLPFVVSC 273.114 SEQ ID N0:32
11 162 ILCYYWLNTV 271.948 SEQ ID N0:33
12 366 TLIVLIITYL 270.234 SEQ ID N0:34
13 146 YVLLIRNIFL 174.977 SEQ ID N0:35
14 195 FVFSLVNSFL 174.977 SEQ ID N0:36
112 LLLPFVVSCI 150.931 SEQ ID N0:37
16 355 LIGSVHFFFI 147.808 SEQ ID N0:38
17 138 YEMPRHEVYV 126.598 SEQ ID N0:39
18 66 LLTRFSAYMV 118.238 SEQ ID N0:40
HLA
A205
:
nonamers
Rank Start Subsequence Residue Parker's Score SEQ ID
Position Listing *
1 191 r LLMDFVFSL 514.080 SEQ ID NO:10
2 403 FLIEKLIKL 126.000 SEQ ID N0:14
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HLA
A205
:
decamers
Rank Start Subsequence Residue Parker's Score * SEQ ID
PositionListing
1 190 LLLMDFVFSL 171.360 SEQ ID N0:23
2 146 YVLLIRNIFL 126.000 SEQ ID N0:35
3 195 FVFSLVNSFL 126.000 SEQ ID N0:36
4 285 QMMTFFIFLL ~ 100.800 SEQ ID N0:25
HLA
A24
:
nonamers
Rank Start Subsequence ResidueParker's Score * SEQ ID
Position Listing
1 14 IYSGGITKL 220.000 SEQ ID N0:41
2 91 VYYLAEYNL 200.000 SEQ ID N0:42
3 165 YYWLNTVAL 200.000 SEQ ID N0:43
4 187 IYRLLLMDF 120.000 SEQ ID N0:44
HLA A24 : d ecamers
Rank Start PositionSubsequence ResidueParker's Score SEQ ID
Listing
1 145 VYVLLIRNIF 252.000 SEQ ID N0:45
2 164 CYYWLNTVAL 200.000 SEQ ID N0:46
3 92 YYLAEYNLEF 165.000 SEQ ID N0:47
HLA A3 : nonamers
Rank Start PositionSubsequence ResidueParler's Score SEQ ID
Listing *
1 258 MLFIMFYSK 900.000 SEQ ID N0:48
2 148 LLIRNIFLK 135.000 SEQ ID N0:49
3 221 GLQEFDIAR 108.000 SEQ ID N0:50
Estimate of Half Time of Disassociation of a Molecule Containing This
Subsequence.
HLA
A3
:
decamers
Rank Start PositionSubsequence ResidueParker's Score * _ SEQ ID
Listing
~
1 401 ~~ KMFLI 600.000 SEQ ID N0:51
EKLIK
2 257 IMLFIMFYSK 270.000 SEQ-ID N0:52
3 407 KLIKLQDMEK 180.000 SEQ ID N0:53
4 147 VLLIRNIFLK 135.000 SEQ ID N0:54
HLA
B7
:
nonamers
Rank Start Subsequence Residue Parker's Score SEQ ID
Position Listing *
1 140 MPRHEVYVL 800.000 SEQ ID N0:55
HLA B7 : decamers
Rank Start Position ~ Subsequence Residue Listing ~ Parker's Score * ~ SEQ ID
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1 140 ~ MPRHEVYVLL 800.000 SEQ ID N0:56
HLA
B4403
nonamers
Rank Start Subsequence Residue Parker's Score * SEQ ID
Position Listing
138 ~ YEMPRHEVY 480.000 SEQ ID N0:57
HLA
B4403
decamers
Rank Start Subsequence Residue Parker's Score * SEQ ID
Position Listing
1 179 WETLIGQDIY 120.000 SEQ ID N0:58
*: Estimate of half time of disassociation of a molecule containing this
subsequence.
HLA-DRB1*1501
:
nonamers
Rank Start Subsequence Residue Tepitope Score SEQ ID
Position Listing
1 363 VHFFFILTL 6.5 ~ SEQ ID N0:59
2 210 LRRI IGMQL 6.2 SEQ ID N0:60
HLA-DRB1*0301
:
nonamers
-
Rank Start Subsequence Residue Tepitope Score SEQ ID ~:
Position Listing
1 193 LLMDFVFSL 6.4 ~~ SEQ ID N0:61
HLA-DRB1*0703
:
nonamers
Rank Start Subsequence Residue Tepitope Score SEQ ID
Position Listing
~
y
1 216 MQLITSLGL 8.5 SEQ ID N0:62
2 197 FVFSLVNSF 8 SEQ ID N0:63
3 367 FILTLIVLI 8 SEQ ID N0:64
4 210 LRRIIGMQL 7.6 SEQ ID N0:60
5 381 WQITEGRKI 7.6 SEQ ID N0:65
6 148 VLLIRNIFL 7.3 SEQ )D N0:49
7 265 MFYSKNISL 7.3 SEQ ID N0:66
8 263 FIMFYSKNI 7.2 SEQ ID N0:67
9 305 VLCTLAITI 7.2 SEQ ID N0:17
10 284 WRASQMMTF 6.8 SEQ ID N0:68
11 365 FFFILTLIV 6.8 SEQ ID N0:69
12 133 FRLVERYEM 6.7 SEQ ID N0:70
13 366 FFILTLIVL 6.4 SEQ ID N0:
71
14 238 YAQTLVWI 6.4 SEQ ID N0:72
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SEQUENCE INFORMATION
SEQ ID NO:1
TGGGGAGGCAGA_AGGCAGACTGATCACTTGAGGCCAGGAGTTTGAGACCTCAT
GTCTAAAAAAAAAAAATTCTGTGAGGTGAGTTTTATTGTTATTCCCTCTCTACAG
ATATGGAAACTGAGGCTGAGAATCAGAACCATTCACAAGACAAAAATCCCCCAG
TTGGCAGATCCAGGGTTGCAAGCCAGGCCTGTGCAGCCCCAAAACCAGTGCTTG
TTTAACCACTGTGTGGTGACCACACCGCTCCAGGCCAACAGCTTGGGGCTAAGT
CTTCACGTTGCCTTTCACCATTAAATAATAGGGCTGCCCTTTGTTGAAGCCCTGC
ACTCCCAGTGACGGCCATAATAACCTTCAGGTGTTCTGCTTTCTGCCTTCTCTAG
Catggccaagtatttccggaacaacttcattaatccccacatttactccggagggatcaccaagctgatcttttgctgg
gacttcactg
tcactcatgaaaaagctgtgaagctaaaacagaagaatcttagcactgagataagggagaacctgtcagagctccgtca
ggagaa
ttccaagttgacgttcaatcagctgctgacccgcttctctgcctacatggtagcctgggttgtctctacaggagtggcc
atagcctgctg
tgcagccgtttattacctggctgagtacaacttagagttcctgaagacacacagtaaccctggggcggtgctgttactg
cctttcgttgt
gtcctgcattaatctggccgtgccatgcatctactccatgttcaggcttgtggagaggtacgagatgccacggcacgaa
gtctacgttc
tcctgatccgaaacatctttttgaaaatatcaatcattggcattctttgttactattggctcaacaccgtggccctgtc
tggtgaagagtg
ttgggaaaccctcattggccaggacatctaccggctccttctgatggattttgtgttctctttagtcaattccttcctg
ggggagtttctga
ggagaatcattgggatgcaactgatcacaagtcttggccttcaggagtttgacattgccaggaacgttctagaactgat
ctatgcaca
aactctggtgtggattggcatcttcttctgccccctgctgccctttatccaaatgattatgcttttcatcatgttctac
tccaaaaatatca
gcctgatgatgaatttccagcctccgagcaaagcctggcgggcctcacagatgatgactttcttcatcttcttgctctt
tttcccatcctt
caccggggtcttgtgcaccctggccatcaccatctggagattgaagccttcagctgactgtggcccttttcgaggtctg
cctctcttcat
tcactccatctacagctggatcgacaccctaagtacacggcctggctacctgtgggttgtttggatctatcggaacctc
attggaagtg
tgcacttctttttcatcctcaccctcattgtgctaatcatcacctatctttactggcagatcacagagggaaggaagat
tatgataaggc
tgctccatgagcagatcattaatgagggcaaagataaaatgttcctgatagaaaaattgatcaagctgcaggatatgga
gaagaaa
gcaaaccccagctcacttgttctggaaaggagagaggtggagcaacaaggctttttgcatttgggggaacatgatggca
gtcttgac
ttgcgatctagaagatcagttcaagaaggtaatccaagggcctgaTGACTCTTTTGGTAACCAGACACCAA
TCAAATAAGGGGAGGAGACGAAAATGGAATGATTTCTTCCATGCCACCTGTGCC
TTTAGGAACTGCCCAGAAGAAAATCCAAGGCTTTAGCCAGGAGCGGAAACTGAC
TACCATGTAATTATCAAAGTAAAATTGGGCATTCCATGCTATTTTTAATACCTGG
ATTGCTGATTTTTCAAGACAAAATACTTGGGGTTTTCCAATAAAGATTGTTGTAA
TATTGAAATGAGCCTACAAAAACCTAGGAAGAGATAACTAGGGAATAATGTATA
TTATCTTCAAGAAATGTGTGCAGGAATGATTGGTTCTTAGAAATCTCTCCTGCCA
GACTTCCCAGACCTGGCAAAGGTTTAGAAACTGTTGCTAAGAAAAGTGGTCCAT
CCTGAATAAACATGTAATACTCCAGCAGGGATATGAAGCCTCTGAATTGTAGAA
CCTGCATTTATTTGTGACTTTGAACTAAAGACATCCCCCATGTCCCAAAGGTGG
AATACAACCAGAGGTCTCATCTCTGAACTTTCTTGCGTACTGATTACATGAGTCT
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TTGGAGTCGGGGATGGAGGAGGTTCTGCCCCTGTGAGGTGTTATACATGACCAT
CAAAGTCCTACGTCAAGCT
SEQ ID N0:2
MAKYFRNNFINPHIYSGGITKLIFCWDFTVTHEKAVKLKQKNLSTEIRENLSELRQENSKLTFNQLLTRFSA
YMVAWVVSTGVAIACCAAVYYLAEYNLEFLKTHSNPGAVLLLPFVVSCINLAVPCIYSMFRLVERYEMPRHE
VYVLLIRNIFLKISIIGILCYYWLNTVALSGEECWETLIGQDIYRLLLMDFVFSLVNSFLGEFLRRIIGMQL
ITSLGLQEFDIARNVLELIYAQTLVWIGIFFCPLLPFIQMIMLFIMFYSKNISLMMNFQPPSKAWRASQMMT
FFIFLLFFPSFTGVLCTLAITIWRLKPSADCGPFRGLPLFIHSIYSWIDTLSTRPGYLWVVWIYRNLIGSVH
FFFILTLIVLIITYLYWQITEGRKIMIRLLHEQIINEGKDKMFLIEKLIKLQDMEKKANPSSLVLERREVEQ
QGFLHLGEHDGSLDLRSRRSVQEGNPRA
SEQ ID N0:3
TGGGGAGGCAGAAGGCAGACTGATCACTTGAGGCCAGGAGTTTGAGACCTCAT
GTCTAAAAAAAAAAAATTCTGTGAGGTGAGTTTTATTGTTATTCCCTCTCTACAG
ATATGGAAACTGAGGCTGAGAATCAGAACCATTCACAAGACAAAAATCCCCCAG
TTGGCAGATCCAGGGTTGCAAGCCAGGCCTGTGCAGCCCCAAAACCAGTGCTTG
TTTAACCACTGTGTGGTGACCACACCGCTCCAGGCCAACAGCTTGGGGCTAAGT
CTTCACGTTGCCTTTCACCATTAAATAATAGGGCTGCCCTTTGTTGAAGCCCTGC
ACTCCCAGTGACGGCCATAATAACCTTCAGGTGTTCTGCTTTCTGCCTTCTCTAG
Catggccaagtatttccggaacaacttcattaatccccacatttactccggagggatcaccaagctgatcttttgctgg
gacttcactg
tcactcatgaaaaagctgtgaagctaaaacagaagaatcttagcactgagataagggagaacctgtcagagctccgtca
ggagaa
ttccaagttgacgttcaatcagctgctgacccgcttctctgcctacatggtagcctgggttgtctctacaggagtggcc
atagcctgctg
tgcagccgtttattacctggctgagtacaacttagagttcctgaagacacacagtaaccctggggcggtgctgttactg
cctttcgttgt
gtcctgcattctggccgtgccatgcatctactccatgttcaggcttgtggagaggtacgagatgccacggcacgaagtc
tacgttctcc
tgatccgcaggggattgatgtagTTCTCAAGTATGGGATGTACAGATGGGCAGGCAGTGCAC
GCACAAAGGCTCCTGGGCTGAGGACGGGACTGAAATCATCCAGCGTTCCCCTTA
GTCAAGCTAAACATCTTTTTGAAAATATCAATCATTGGCATTCTTTGTTACTATT
GGCTCAACACCGTGGCCCTGTCTGGTGAAGAGTGTTGGGAAACCCTCATTGGCC
AGGACATCTACCGGCTCCTTCTGATGGATTTTGTGTTCTCTTTAGTCAATTCCTT
CCTGGGGGAGTTTCTGAGGAGAATCATTGGGATGCAACTGATCACAAGTCTTGG
CCTTCAGGAGTTTGACATTGCCAGGAACGTTCTAGAACTGATCTATGCACAAAC
TCTGGTGTGGATTGGCATCTTCTTCTGCCCCCTGCTGCCCTTTATCCAAATGATT
ATGCTTTTCATCATGTTCTACTCCAAAAATATCAGCCTGATGATGAATTTCCAGC
64
CA 02386028 2002-03-28
WO 01/23417 PCT/EP00/09500
CTCCGAGCAAAGCCTGGCGGGCCTCACAGATGATGACTTTCTTCATCTTCTTGC
TCTTTTTCCCATCCTTCACCGGGGTCTTGTGCACCCTGGCCATCACCATCTGGA
GATTGAAGCCTTCAGCTGACTGTGGCCCTTTTCGAGGTCTGCCTCTCTTCATTC
ACTCCATCTACAGCTGGATCGACACCCTAAGTACACGGCCTGGCTACCTGTGGG
TTGTTTGGATCTATCGGAACCTCATTGGAAGTGTGCACTTCTTTTTCATCCTCAC
CCTCATTGTGCTAATCATCACCTATCTTTACTGGCAGATCACAGAGGGAAGGAA
GATTATGATAAGGCTGCTCCATGAGCAGATCATTAATGAGGGCAAAGATAAAAT
GTTCCTGATAGAAAAATTGATCAAGCTGCAGGATATGGAGAAGAAAGCAAACCC
CAGCTCACTTGTTCTGGAAAGGAGAGAGGTGGAGCAACAAGGCTTTTTGCATTT
GGGGGAACATGATGGCAGTCTTGACTTGCGATCTAGAAGATCAGTTCAAGAAG
GTAATCCAAGGGCCTGATGACTCTTTTGGTAACCAGACACCAATCAAATAAGGG
GAGGAGACGAAAATGGAATGATTTCTTCCATGCCACCTGTGCCTTTAGGAACTG
CCCAGAAGAAAATCCAAGGCTTTAGCCAGGAGCGGAAACTGACTACCATGTAAT
TATCAAAGTAAAATTGGGCATTCCATGCTATTTTTAATACCTGGATTGCTGATTT
TTCAAGACAAAATACTTGGGGTTTTCCAATAAAGATTGTTGTAATATTGAAATGA
GCCTACAAAAACCTAGGAAGAGATAACTAGGGAATAATGTATATTATCTTCAAG
AAATGTGTGCAGGAATGATTGGTTCTTAGAAATCTCTCCTGCCAGACTTCCCAG
ACCTGGCAAAGGTTTAGAAACTGTTGCTAAGAAAAGTGGTCCATCCTGAATAAA
CATGTAATACTCCAGCAGGGATATGAAGCCTCTGAATTGTAGAACCTGCATTTA
TTTGTGACTTTGAACTAAAGACATCCCCCATGTCCCAAAGGTGGAATACAACCA
GAGGTCTCATCTCTGAACTTTCTTGCGTACTGATTACATGAGTCTTTGGAGTCG
GGGATGGAGGAGGTTCTGCCCCTGTGAGGTGTTATACATGACCATCAAAGTCCT
ACGTCAAGCT
SEQ ID N0:4
MAKYFP~NNFINPHIYSGGITKLIFCWDFTVTHEKAVKLKQKNLSTEIRENLSELRQE
NSKLTFNQLLTRFSAYMVAWWSTGVAIACCAAVYYLAEYNLEFLKTHSNPGAVLL
LPFWSCILAVPCIYSMFRLVERYEMPRHEVYVLLIRRGLM
SEQ ID N0:5
atcttttgctgggacttcactgtcactcatgaaaaagctgtgaagctaaaacagaagaatcttagcactgag
ataagggagaacctgtcagagctccgtcaggagaattccaagttgacgttcaatcagctgctgacccgcttc
tctgcctacatggtagcctgggttgtctctacaggagtggccatagcctgctgtgcagccgtttattacctg
gctgagtacaacttagagttcctgaagacacacagtaaccctggggcggtgctgttactgcctttcgttgtg
tcctgcattaatctggccgtgccatgcatctactccatgttcaggcttgtggagaggtacgagatgccacgg
CA 02386028 2002-03-28
WO 01/23417 PCT/EP00/09500
cacgaagtctacgttctcctgatccgaaacatctttttgaaaatatcaatcattggcattctttgttactat
tggctcaacaccgtggccctgtctggtgaagagtgttgggaaaccctcattggccaggacatctaccggctc
cttctgatggattttgtgttctctttagtcaattccttcctgggggagtttctgaggagaatcattgggatg
caactgatcacaagtcttggccttcaggagtttgacattgccaggaacgttctagaactgatctatgcacaa
actctggtgtggattggcatcttcttctgccccctgctgccctttatccaaatgattatgcttttcatcatg
ttctactccaaaaatatcagcctgatgatgaatttccagcctccgagcaaagcctggcgggcctcacagatg
atgactttcttcatcttcttgctctttttcccatctttcaccggggtcttgtgcaccctggccatcaccatc
tggagattgaagccttcagctgactgtggcccttttcgaggtctgcctctcttcattcactccatctacagc
tggatcgacaccctaagtacacggcctggctacctgtgggttgtttggatctatcggaacctcattggaagt
gtgcacttctttttcatcctcaccctcattgtgctgatcatcacctatctttactggcagatcacagaggga
aggaagattatgataaggctgctccatgagcagatcattaatgagggcaaagataaaatgttcctgatagaa
aaattgatcaagctgcaggatatggagaagaaagcaaaccccagctcacttgttctggaaaggagagaggtg
gagcaacaaggctttttgcatttgggggaacatgatggcagtcttgacttgcgatctagaagatcagttcaa
gaaggtaatccaagggcctgaTGACTCTTTTGGTAACCAGACACCAATCAAATAAGGGGAGGAGATGAAAAT
GGAATGATTTCTTCCATGCCACCTGTGCCTTTAGGAACTGCCCAGAAGAAAATCCAAGGCTTTAGCCAGGAG
CGGAAACTGACTACCATGTAATTATCAAAGTAAAATTGGGCATTCCATGCTATTTTTAATACCTGGATTGCT
GATTTTTCAAGACAAAATACTTGGGGTTTTCCAATAAAGATTGTTGTAATATTGAAATGAGCCTACAAAAAC
CTAGGAAGAGATAACTAGGGAATAATGTATATTATCTTCAAGAAATGTGTGCAGGAATGATTGGTTCTTAGA
AATCTCTCCTGCCAGACTTCCCAGACCTGGCAAAGGTTTAGAAACTGTTGCTAAGAAAAGTGGTCCATCCTG
AATAAACATGTAATACTCCAGCAGGGATATGAAGCCTCTGAATTGTAGAACCTGCATTTATTTGTGACTTTG
AACTAAAGACATCCCCCATGTCCCAAAGGTGGAATACAACCAGAGGTCTCATCTCTGAACTTTCTTGCGTAC
TGATTACATGAGTCTTTGGAGTCGGGGATGGAGGAGGTTCTGCCCCTGTGAGGTGTTATACATGACCATCAA
AGTCCTACGTCAAGCT
SEQ m N0:6
IFCWDFTVTHEKAVKLKQKNLSTEIRENLSELRQENSKLTFNQLLTRFSAYMVAWWSTGVAIACCAAVYYL
AEYNLEFLKTHSNPGAVLLLPFVVSCINLAVPCIYSMFRLVERYEMPRHEVYVLLIRNIFLKISIIGILCYY
WLNTVALSGEECWETLIGQDIYRLLLMDFVFSLVNSFLGEFLRRIIGMQLITSLGLQEFDIARNVLELIYAQ
TLVWIGIFFCPLLPFIQMIMLFIMFYSKNISLMMNFQPPSKAWRASQMMTFFIFLLFFPSFTGVLCTLAITI
WRLKPSADCGPFRGLPLFIHSIYSWIDTLSTRPGYLWVVWIYRNLIGSVHFFFILTLIVLIITYLYWQITEG
RKIMIRLLHEQIINEGKDKMFLIEKLIKLQDMEKKANPSSLVLERREVEQQGFLHLGEHDGSLDLRSRRSVQ
EGNPRA
SEQ ID N0:7
ctgatgatgaatttccagcctccgagcaaagcctggcgggcctcacagatgatgactttcttcatcttcttgctctttt
tcccatctttcac
cggggtcttgtgcaccctggccatcaccatctggagattgaagccttcagctgactgtggcccttttcgaggtctgcct
ctcttcattca
ctccatctacagctggatcgacaccctaagtacacggcctggctacctgtgggttgtttggatctatcggaacctcatt
ggaagtgtgc
66
CA 02386028 2002-03-28
WO 01/23417 PCT/EP00/09500
acttctttttcatcctcaccctcattgtgctgatcatcacctatctttactggcagatcacagagggaaggaagattat
gataaggctgc
tccatgagcagatcattaatgagggcaaagataaaatgttcctgatagaaaaattgatcaagctgcaggatatggagaa
gaaagc
aaaccccagctcacttgttctggaaaggagagaggtggagcaacaaggctttttgcatttgggggaacatgatggcagt
cttgactt
gcgatctagaagatcagttcaagaaggtaatccaagggcctgaTGACTCTTTTGGTAACCAGACACCAAT
CAAATAAGGGGAGGAGATGAAAATGGAATGATTTCTTCCAT'GCCACCTGTGCCT
TTAGGAACTGCCCAGAAGAAAATCCAAGGCTTTAGCCAGGAGCGGAAACTGACT
ACCATGTAATTATCAAAGTAAAATTGGGCATTCCATGCTATTTTTAATACCTGGA
TTGCTGATTTTTCAAGACAAAATACTTGGGGTTTTCCAATAAAGATTGTTGTAAT
ATTGAAATGAGCCTACAAAAACCTAGGAAGAGATAACTAGGGAATAATGTATAT
TATCTTCAAGAAATGTGTGCAGGAATGATTGGTTCTTAGAAATCTCTCCTGCCA
GACTTCCCAGACCTGGCAAAGGTTTAGAAACTGTTGCTAAGAAAAGTGGTCCAT
CCTGAATAAACATGTAATACTCCAGCAGGGATATGAAGCCTCTGAATTGTAGAA
CCTGCATTTATTTGTGACTTTGAACTAAAGACATCCCCCATGTCCCAAAGGTGG
AATACAACCAGAGGTCTCATCTCTGAACTTTCTTGCGTACTGATTACATGAGTCT
TTGGAGTCGGGGATGGAGGAGGTTCTGCCCCTGTGAGGTGTTATACATGACCAT
CAAAGTCCTACGTCAAGCT
SEQ ID N0:8
LMMNFQPPSKAWRASQMMTFFIFLLFFPSFTGVLCTLAITIWRLKPSADCGPFRGLPLFIHSIYSWIDTLST
RPGYLWVVWIYRNLIGSVHFFFILTLIVLIITYLYWQITEGRKIMIRLLHEQIINEGKDKMFLIEKLIKLQD
MEKKANPSSLVLERREVEQQGFLHLGEHDGSLDLRSRRSVQEGNPRA
SEQ ID N0:9
ITEGRKIMIR
SEQ ID NO:10
LLMDFVFSL
SEQ ID NO:11
FLLFFPSFT
SEQ ID N0:12
QMMTFFIFL
SEQ ID N0:13
MMTFFIFLL
SEQ ID N0:14
67
CA 02386028 2002-03-28
WO 01/23417 PCT/EP00/09500
FLIEKLIKL
SEQ ID NO:15
VLLIRNIFL
SEQ ID N0:16
LVWIGIFFC
SEQ ID N0:17
TLAITIWRL
SEQ ID N0:18
LIFCWDFTV
SEQ ID N0:19
FLGEFLRRI
SEQ ID N0:20
LLLPFVVSC
SEQ ID N0:21
LLTRFSAYM
SEQ ID N0:22
KLIFCWDFT
SEQ ID N0:23
LLLMDFVFSL
SEQ ID N0:24
LLMDFVFSLV
SEQ ID N0:25
QMMTFFIFLL
SEQ ID N0:26
YLAEYNLEFL
SEQ ID N0:27
KLIFCWDFTV
SEQ ID N0:28
LLFFPSFTGV
68
CA 02386028 2002-03-28
WO 01/23417 PCT/EP00/09500
SEQ ID N0:29
QLLTRFSAYM
SEQ ID N0:30
TLVWIGIFFC
SEQ ID N0:31
SQMMTFFIFL
SEQ ID N0:32
VLLLPFVVSC
SEQ ID N0:33
ILCYYWLNTV
SEQ ID N0:34
TLIVLIITYL
SEQ ID N0:35
YVLLIRNIFL
SEQ ID N0:36
FVFSLVNSFL
SEQ ID N0:37
LLLPFWSCI
SEQ ID N0:38
LIGSVHFFFI
SEQ ID N0:39
YEMPRHEVYV
SEQ ID N0:40
LLTRFSAYMV
SEQ ID N0:41
IYSGGITKL
SEQ ID N0:42
VYYLAEYNL
SEQ ID N0:43
69
CA 02386028 2002-03-28
WO 01/23417 PCT/EP00/09500
YYWLNTVAL
SEQ ID N0:44
IYRLLLMDF
SEQ ID N0:45
VYVLLIRNIF
SEQ ID N0:46
CYYWLNTVAL
SEQ ID N0:47
YYLAEYNLEF
SEQ ID N0:48
MLFIMFYSK
SEQ ID N0:49
LLIRNIFLK
SEQ ID N0:50
GLQEFDIAR
SEQ ID N0:51
KMFLIEKLIK
SEQ ID N0:52
IMLFIMFYSK
SEQ ID N0:53
KLIKLQDMEK
SEQ ID N0:54
VLLIRNIFLK
SEQ ID N0:55
MPRHEVYVL
SEQ ID N0:56
MPRHEVYVLL
SEQ ID N0:57
YEMPRHEVY
CA 02386028 2002-03-28
WO 01/23417 PCT/EP00/09500
SEQ ID N0:58
WETLIGQDIY
SEQ ID N0:59
VHFFFILTL
SEQ ID N0:60
LRRIIGMQL
SEQ ID N0:61
LLMDFVFSL
SEQ ID N0:62
MQLITSLGL
SEQ ID N0:63
FVFSLVNSF
SEQ ID N0:64
FILTLIVLI
SEQ ID N0:65
WQITEGRKI
SEQ ID N0:66
MFYSKNISL
SEQ ID N0:67
FIMFYSKNI
SEQ ID N0:68
WRASQMMTF
SEQ ID N0:69
FFFILTLIV
SEQ ID N0:70
FRLVERYEM
SEQ ID N0:71
FFILTLIVL
SEQ ID N0:72
71
Image
CA 02386028 2002-03-28
WO 01/23417 PCT/EP00/09500
SEQUENCE LISTING
<110> SmithKline Beecham Biologicals
<120> Novel compounds
<130> BC45263
<160> 72
<170> FastSEQ for Windows Version 3.0
<210> 1
<211> 2407
<212> DNA
<213> Artificial Sequence
<400> 1
tggggaggcagaaggcagactgatcacttgaggccaggagtttgagacctcatgtctaaa60
aaaaaaaaattctgtgaggtgagttttattgttattccctctctacagatatggaaactg120
aggctgagaatcagaaccattcacaagacaaaaatcccccagttggcagatccagggttg180
caagccaggcctgtgcagccccaaaaccagtgcttgtttaaccactgtgtggtgaccaca240
ccgctccaggccaacagcttggggctaagtcttcacgttgcctttcaccattaaataata300
gggctgccctttgttgaagccctgcactcccagtgacggccataataaccttcaggtgtt360
ctgctttctgccttctctagcatggccaagtatttccggaacaacttcattaatccccac420
atttactccggagggatcaccaagctgatcttttgctgggacttcactgtcactcatgaa480
aaagctgtgaagctaaaacagaagaatcttagcactgagataagggagaacctgtcagag540
ctccgtcaggagaattccaagttgacgttcaatcagctgctgacccgcttctctgcctac600
atggtagcctgggttgtctctacaggagtggccatagcctgctgtgcagccgtttattac660
ctggctgagtacaacttagagttcctgaagacacacagtaaccctggggcggtgctgtta720
ctgcctttcgttgtgtcctgcattaatctggccgtgccatgcatctactccatgttcagg780
cttgtggagaggtacgagatgccacggcacgaagtctacgttctcctgatccgaaacatc840
tttttgaaaatatcaatcattggcattctttgttactattggctcaacaccgtggccctg900
tctggtgaagagtgttgggaaaccctcattggccaggacatctaccggctccttctgatg960
gattttgtgttctctttagtcaattccttcctgggggagtttctgaggagaatcattggg1020
atgcaactgatcacaagtcttggccttcaggagtttgacattgccaggaacgttctagaa1080
ctgatctatgcacaaactctggtgtggattggcatcttcttctgccccctgctgcccttt1140
atccaaatgattatgcttttcatcatgttctactccaaaaatatcagcctgatgatgaat1200
ttccagcctccgagcaaagcctggcgggcctcacagatgatgactttcttcatcttcttg1260
ctctttttcccatccttcaccggggtcttgtgcaccctggccatcaccatctggagattg1320
aagccttcagctgactgtggcccttttcgaggtctgcctctcttcattcactccatctac1380
agctggatcgacaccctaagtacacggcctggctacctgtgggttgtttggatctatcgg1440
aacctcattggaagtgtgcacttctttttcatcctcaccctcattgtgctaatcatcacc1500
tatctttactggcagatcacagagggaaggaagattatgataaggctgctccatgagcag1560
atcattaatgagggcaaagataaaatgttcctgatagaaaaattgatcaagctgcaggat1620
atggagaagaaagcaaaccccagctcacttgttctggaaaggagagaggtggagcaacaa1680
ggctttttgcatttgggggaacatgatggcagtcttgacttgcgatctagaagatcagtt1740
caagaaggtaatccaagggcctgatgactcttttggtaaccagacaccaatcaaataagg1800
ggaggagacgaaaatggaatgatttcttccatgccacctgtgcctttaggaactgcccag1860
aagaaaatccaaggctttagccaggagcggaaactgactaccatgtaattatcaaagtaa1920
aattgggcattccatgctatttttaatacctggattgctgatttttcaagacaaaatact1980
tggggttttccaataaagattgttgtaatattgaaatgagcctacaaaaacctaggaaga2090
gataactagggaataatgtatattatcttcaagaaatgtgtgcaggaatgattggttctt2100
agaaatctctcctgccagacttcccagacctggcaaaggtttagaaactgttgctaagaa2160
aagtggtccatcctgaataaacatgtaatactccagcagggatatgaagcctctgaattg2220
tagaacctgcatttatttgtgactttgaactaaagacatcccccatgtcccaaaggtgga2280
atacaaccagaggtctcatctctgaactttcttgcgtactgattacatgagtctttggag2340
tcggggatggaggaggttctgcccctgtgaggtgttatacatgaccatcaaagtcctacg2400
tcaagct 2407
<210> 2
<211> 460
<212> PRT
<213> Artificial Sequence
CA 02386028 2002-03-28
WO 01/23417 PCT/EP00/09500
<400> 2
Met AlaLys TyrPheArgAsnAsnPheIleAsnProHisIleTyrSer
1 5 10 15
Gly G1yIle ThrLysLeuIlePheCysTrpAspPheThrValThrHis
20 25 30
Glu LysAla ValLysLeuLysGlnLysAsnLeuSerThrGluIleArg
35 40 45
Glu AsnLeu SerGluLeuArgGlnGluAsnSerLysLeuThrPheAsn
50 55 60
Gln LeuLeu ThrArgPheSerAlaTyrMetValAlaTrpValValSer
65 70 75 80
Thr GlyVal AlaIleAlaCysCysAlaAlaValTyrTyrLeuAlaGlu
85 90 95
Tyr AsnLeu GluPheLeuLysThrHisSerAsnProGlyAlaValLeu
100 105 110
Leu LeuPro PheValValSerCysIleAsnLeuAlaVa1ProCysIle
115 120 125
Tyr SerMet PheArgLeuValGluArgTyrGluMetProArgHisGlu
130 135 140
Val TyrVal LeuLeuIleArgAsnIlePheLeuLysIleSerIleIle
145 150 155 160
Gly IleLeu CysTyrTyrTrpLeuAsnThrValAlaLeuSerGlyGlu
165 170 175
Glu CysTrp GluThrLeuIleGlyGlnAspIleTyrArgLeuLeuLeu
180 185 190
Met AspPhe ValPheSerLeuValAsnSerPheLeuGlyGluPheLeu
195 200 205
Arg ArgIle IleGlyMetGlnLeuIleThrSerLeuGlyLeuGlnGlu
210 215 220
Phe AspIle AlaArgAsnValLeuGluLeuIleTyrAlaGlnThrLeu
225 230 235 240
Val TrpIle GlyIlePhePheCysProLeuLeuProPheIleGlnMet
245 250 255
Ile MetLeu PheIleMetPheTyrSerLysAsnIleSerLeuMetMet
260 265 270
Asn PheGln ProProSerLysAlaTrpArgAlaSerGlnMetMetThr
275 280 285
Phe PheIle PheLeuLeuPhePheProSerPheThrGlyValLeuCys
290 295 300
Thr LeuAla IleThrIleTrpArgLeuLysProSerAlaAspCysGly
305 310 315 320
Pro PheArg GlyLeuProLeuPheIleHisSerIleTyrSerTrpIle
325 330 335
Asp ThrLeu SerThrArgProGlyTyrLeuTrpValValTrpIleTyr
340 345 350
Arg AsnLeu IleGlySerValHisPhePhePheIleLeuThrLeuIle
355 360 365
Val LeuIle IleThrTyrLeuTyrTrpGlnIleThrGluGlyArgLys
370 375 380
Ile MetIle ArgLeuLeuHisGluGlnIleIleAsnGluGlyLysAsp
385 390 395 400
Lys MetPhe LeuIleGluLysLeuIleLysLeuGlnAspMetGluLys
405 410 415
Lys AlaAsn ProSerSerLeuValLeuGluArgArgGluValGluGln
420 425 430
Gln GlyPhe LeuHisLeuGlyGluHisAspGlySerLeuAspLeuArg
435 440 445
Ser ArgArg SerValGlnGluGlyAsnProArgAla
450 455 460
<210> 3
<211> 2521
<212> DNA
<213> Artificial
Sequence
2
CA 02386028 2002-03-28
WO 01/23417 PCT/EP00/09500
<400> 3
tggggaggcagaaggcagactgatcacttgaggccaggagtttgagacctcatgtctaaa60
aaaaaaaaattctgtgaggtgagttttattgttattccctctctacagatatggaaactg120
aggctgagaatcagaaccattcacaagacaaaaatcccccagttggcagatccagggttg180
caagccaggcctgtgcagccccaaaaccagtgcttgtttaaccactgtgtggtgaccaca240
ccgctccaggccaacagcttggggctaagtcttcacgttgcctttcaccattaaataata300
gggctgccctttgttgaagccctgcactcccagtgacggccataataaccttcaggtgtt360
ctgctttctgccttctctagcatggccaagtatttccggaacaacttcattaatccccac420
atttactccggagggatcaccaagctgatcttttgctgggacttcactgtcactcatgaa480
aaagctgtgaagctaaaacagaagaatcttagcactgagataagggagaacctgtcagag540
ctccgtcaggagaattccaagttgacgttcaatcagctgctgacccgcttctctgcctac600
atggtagcctgggttgtctctacaggagtggccatagcctgctgtgcagccgtttattac660
ctggctgagtacaacttagagttcctgaagacacacagtaaccctggggcggtgctgtta720
ctgcctttcgttgtgtcctgcattctggccgtgccatgcatctactccatgttcaggctt780
gtggagaggtacgagatgccacggcacgaagtctacgttctcctgatccgcaggggattg840
atgtagttctcaagtatgggatgtacagatgggcaggcagtgcacgcacaaaggctcctg900
ggctgaggacgggactgaaatcatccagcgttccccttagtcaagctaaacatctttttg960
aaaatatcaatcattggcattctttgttactattggctcaacaccgtggccctgtctggt1020
gaagagtgttgggaaaccctcattggccaggacatctaccggctccttctgatggatttt1080
gtgttctctttagtcaattccttcctgggggagtttctgaggagaatcattgggatgcaa1140
ctgatcacaagtcttggccttcaggagtttgacattgccaggaacgttctagaactgatc1200
tatgcacaaactctggtgtggattggcatcttcttctgccccctgctgccctttatccaa1260
atgattatgcttttcatcatgttctactccaaaaatatcagcctgatgatgaatttccag1320
cctccgagcaaagcctggcgggcctcacagatgatgactttcttcatcttcttgctcttt1380
ttcccatccttcaccggggtcttgtgcaccctggccatcaccatctggagattgaagcct1440
tcagctgactgtggcccttttcgaggtctgcctctcttcattcactccatctacagctgg1500
atcgacaccctaagtacacggcctggctacctgtgggttgtttggatctatcggaacctc1560
attggaagtgtgcacttctttttcatcctcaccctcattgtgctaatcatcacctatctt1620
tactggcagatcacagagggaaggaagattatgataaggctgctccatgagcagatcatt1680
aatgagggcaaagataaaatgttcctgatagaaaaattgatcaagctgcaggatatggag1740
aagaaagcaaaccccagctcacttgttctggaaaggagagaggtggagcaacaaggcttt1800
ttgcatttgggggaacatgatggcagtcttgacttgcgatctagaagatcagttcaagaa1860
ggtaatccaagggcctgatgactcttttggtaaccagacaccaatcaaataaggggagga1920
gacgaaaatggaatgatttcttccatgccacctgtgcctttaggaactgcccagaagaaa1980
atccaaggctttagccaggagcggaaactgactaccatgtaattatcaaagtaaaattgg2040
gcattccatgctatttttaatacctggattgctgatttttcaagacaaaatacttggggt2100
tttccaataaagattgttgtaatattgaaatgagcctacaaaaacctaggaagagataac2160
tagggaataatgtatattatcttcaagaaatgtgtgcaggaatgattggttcttagaaat2220
ctctcctgccagacttcccagacctggcaaaggtttagaaactgttgctaagaaaagtgg2280
tccatcctgaataaacatgtaatactccagcagggatatgaagcctctgaattgtagaac2340
ctgcatttatttgtgactttgaactaaagacatcccccatgtcccaaaggtggaatacaa2400
ccagaggtctcatctctgaactttcttgcgtactgattacatgagtctttggagtcgggg2960
atggaggaggttctgcccctgtgaggtgttatacatgaccatcaaagtcctacgtcaagc2520
t 2521
<210> 4
<211> 154
<212> PRT
<213> Artificial Sequence
<400> 4
Met Ala Lys Tyr Phe Arg Asn Asn Phe Ile Asn Pro His Ile Tyr Ser
1 5 10 15
Gly Gly Ile Thr Lys Leu Ile Phe Cys Trp Asp Phe Thr Val Thr His
20 25 30
Glu Lys Ala Val Lys Leu Lys Gln Lys Asn Leu Ser Thr Glu Ile Arg
35 40 45
Glu Asn Leu Ser Glu Leu Arg Gln Glu Asn Ser Lys Leu Thr Phe Asn
50 55 60
Gln Leu Leu Thr Arg Phe Ser Ala Tyr Met Val Ala Trp Val Val Ser
70 75 80
Thr Gly Val Ala Ile Ala Cys Cys Ala Ala Val Tyr Tyr Leu Ala Glu
85 90 95
Tyr Asn Leu Glu Phe Leu Lys Thr His Ser Asn Pro Gly Ala Val Leu
65 100 105 110
Leu Leu Pro Phe Val Val Ser Cys Ile Leu Ala Val Pro Cys Ile Tyr
3
CA 02386028 2002-03-28
WO 01/23417 PCT/EP00/09500
115 120 125
Ser Met Phe Arg Leu Val Glu Arg Tyr Glu Met Pro Arg His Glu Val
130 135 190
Tyr Val Leu Leu Ile Arg Arg Gly Leu Met
145 150
<210> 5
<211> 1960
<212> DNA
<213> Artificial Sequence
<400> 5
atcttttgctgggacttcactgtcactcatgaaaaagctgtgaagctaaaacagaagaat60
cttagcactgagataagggagaacctgtcagagctccgtcaggagaattccaagttgacg120
ttcaatcagctgctgacccgcttctctgcctacatggtagcctgggttgtctctacagga180
gtggccatagcctgctgtgcagccgtttattacctggctgagtacaacttagagttcctg240
aagacacacagtaaccctggggcggtgctgttactgcctttcgttgtgtcctgcattaat300
ctggccgtgccatgcatctactccatgttcaggcttgtggagaggtacgagatgccacgg360
cacgaagtctacgttctcctgatccgaaacatctttttgaaaatatcaatcattggcatt420
ctttgttactattggctcaacaccgtggccctgtctggtgaagagtgttgggaaaccctc480
attggccaggacatctaccggctccttctgatggattttgtgttctctttagtcaattcc540
ttcctgggggagtttctgaggagaatcattgggatgcaactgatcacaagtcttggcctt600
caggagtttgacattgccaggaacgttctagaactgatctatgcacaaactctggtgtgg660
attggcatcttcttctgccccctgctgccctttatccaaatgattatgcttttcatcatg720
ttctactccaaaaatatcagcctgatgatgaatttccagcctccgagcaaagcctggcgg780
gcctcacagatgatgactttcttcatcttcttgctctttttcccatctttcaccggggtc840
ttgtgcaccctggccatcaccatctggagattgaagccttcagctgactgtggccctttt900
cgaggtctgcctctcttcattcactccatctacagctggatcgacaccctaagtacacgg960
cctggctacctgtgggttgtttggatctatcggaacctcattggaagtgtgcacttcttt1020
ttcatcctcaccctcattgtgctgatcatcacctatctttactggcagatcacagaggga1080
aggaagattatgataaggctgctccatgagcagatcattaatgagggcaaagataaaatg1140
ttcctgatagaaaaattgatcaagctgcaggatatggagaagaaagcaaaccccagctca1200
cttgttctggaaaggagagaggtggagcaacaaggctttttgcatttgggggaacatgat1260
ggcagtcttgacttgcgatctagaagatcagttcaagaaggtaatccaagggcctgatga1320
ctcttttggtaaccagacaccaatcaaataaggggaggagatgaaaatggaatgatttct1380
tccatgccacctgtgcctttaggaactgcccagaagaaaatccaaggctttagccaggag1440
cggaaactgactaccatgtaattatcaaagtaaaattgggcattccatgctatttttaat1500
acctggattgctgatttttcaagacaaaatacttggggttttccaataaagattgttgta1560
atattgaaatgagcctacaaaaacctaggaagagataactagggaataatgtatattatc1620
ttcaagaaatgtgtgcaggaatgattggttcttagaaatctctcctgccagacttcccag1680
acctggcaaaggtttagaaactgttgctaagaaaagtggtccatcctgaataaacatgta1740
atactccagcagggatatgaagcctctgaattgtagaacctgcatttatttgtgactttg1800
aactaaagacatcccccatgtcccaaaggtggaatacaaccagaggtctcatctctgaac1860
tttcttgcgtactgattacatgagtctttggagtcggggatggaggaggttctgcccctg1920
tgaggtgttatacatgaccatcaaagtcctacgtcaagct 1960
<210> 6
<211> 438
<212> PRT
<213> artificalsequence
<400> 6
Ile Phe Cys Trp Asp Phe Thr Val Thr His Glu Lys Ala Val Lys Leu
1 5 10 15
Lys Gln Lys Asn Leu Ser Thr Glu Ile Arg Glu Asn Leu Ser Glu Leu
20 25 30
Arg Gln Glu Asn Ser Lys Leu Thr Phe Asn Gln Leu Leu Thr Arg Phe
35 40 45
Ser Ala Tyr Met Val Ala Trp Val Val Ser Thr Gly Val Ala Ile Ala
50 55 60
Cys Cys Ala Ala Val Tyr Tyr Leu Ala Glu Tyr Asn Leu G1u Phe Leu
70 75 80
Lys Thr His Ser Asn Pro Gly Ala Val Leu Leu Leu Pro Phe Val Val
85 90 95
65 Ser Cys Ile Asn Leu Ala Val Pro Cys Ile Tyr Ser Met Phe Arg Leu
100 105 110
4
CA 02386028 2002-03-28
WO 01/23417 PCT/EP00/09500
Va1 GluArgTyrGluMetProArgHisGluValTyrValLeuLeuIle
115 120 125
Arg AsnIlePheLeuLysIleSerIleIleGlyIleLeuCysTyrTyr
130 135 140
Trp LeuAsnThrValAlaLeuSerGlyGluGluCysTrpGluThrLeu
145 150 155 160
Ile GlyG1nAspIleTyrArgLeuLeuLeuMetAspPheValPheSer
165 170 175
Leu ValAsnSerPheLeuGlyGluPheLeuArgArgIleIleGlyMet
180 185 190
Gln LeuIleThrSerLeuGlyLeuGlnGluPheAspIleAlaArgAsn
195 200 205
Val LeuGluLeuIleTyrAlaGlnThrLeuValTrpIleGlyIlePhe
210 215 220
Phe CysProLeuLeuProPheIleGlnMetIleMetLeuPheIleMet
225 230 235 240
Phe TyrSerLysAsnIleSerLeuMetMetAsnPheGlnProProSer
245 250 255
Lys AlaTrpArgAlaSerGlnMetMetThrPhePheIlePheLeuLeu
260 265 270
Phe PheProSerPheThrGlyValLeuCysThrLeuAlaIleThrIle
275 280 285
Trp ArgLeuLysProSerAlaAspCysGlyProPheArgGlyLeuPro
290 295 300
Leu PheIleHisSerIleTyrSerTrpIleAspThrLeuSerThrArg
305 310 315 320
Pro GlyTyrLeuTrpValValTrpIleTyrArgAsnLeuIleGlySer
325 330 335
Val HisPhePhePheIleLeuThrLeuIleValLeuIleIleThrTyr
340 345 350
Leu TyrTrpGlnIleThrGluGlyArgLysIleMetIleArgLeuLeu
355 360 365
His GluGlnIleIleAsnGluGlyLysAspLysMetPheLeuIleGlu
370 375 380
Lys LeuIleLysLeuGlnAspMetGluLysLysAlaAsnProSerSer
385 390 395 400
Leu ValLeuGluArgArgGluValGluGlnGlnGlyPheLeuHisLeu
405 410 415
Gly GluHisAspGlySerLeuAspLeuArgSerArgArgSerValGln
420 425 430
Glu GlyAsnProArgAla
435
<210> 7
<211> 1219
<212> DNA
<213> Artificial Sequence
<400> 7
ctgatgatgaatttccagcctccgagcaaagcctggcgggcctcacagatgatgactttc60
ttcatcttcttgctctttttcccatctttcaccggggtcttgtgcaccctggccatcacc120
atctggagattgaagccttcagctgactgtggcccttttcgaggtctgcctctcttcatt180
cactccatctacagctggatcgacaccctaagtacacggcctggctacctgtgggttgtt240
tggatctatcggaacctcattggaagtgtgcacttctttttcatcctcaccctcattgtg300
ctgatcatcacctatctttactggcagatcacagagggaaggaagattatgataaggctg360
ctccatgagcagatcattaatgagggcaaagataaaatgttcctgatagaaaaattgatc420
aagctgcaggatatggagaagaaagcaaaccccagctcacttgttctggaaaggagagag480
gtggagcaacaaggctttttgcatttgggggaacatgatggcagtcttgacttgcgatct540
agaagatcagttcaagaaggtaatccaagggcctgatgactcttttggtaaccagacacc600
aatcaaataaggggaggagatgaaaatggaatgatttcttccatgccacctgtgccttta660
ggaactgcccagaagaaaatccaaggctttagccaggagcggaaactgactaccatgtaa720
ttatcaaagtaaaattgggcattccatgctatttttaatacctggattgctgatttttca780
agacaaaatacttggggttttccaataaagattgttgtaatattgaaatgagcctacaaa840
aacctaggaagagataactagggaataatgtatattatcttcaagaaatgtgtgcaggaa900
tgattggttcttagaaatctctcctgccagacttcccagacctggcaaaggtttagaaac960
tgttgctaagaaaagtggtccatcctgaataaacatgtaatactccagcagggatatgaa1020
5
CA 02386028 2002-03-28
WO 01/23417 PCT/EP00/09500
gcctctgaat tgtagaacct gcatttattt gtgactttga actaaagaca tcccccatgt 1080
cccaaaggtg gaatacaacc agaggtctca tctctgaact ttcttgcgta ctgattacat 1140
gagtctttgg agtcggggat ggaggaggtt ctgcccctgt gaggtgttat acatgaccat 1200
caaagtccta cgtcaagct 1219
<210> 8
<211> 191
<212> PRT
<213> Artificial Sequence
<400> 8
Leu MetMetAsnPheGlnProProSerLysAlaTrpArgAlaSerGln
1 5 10 15
Met MetThrPhePheIlePheLeuLeuPhePheProSerPheThrGly
20 25 30
Val LeuCysThrLeuAlaIleThrIleTrpArgLeuLysProSerAla
35 40 45
Asp CysGlyProPheArgGlyLeuProLeuPheIleHisSerIleTyr
50 55 60
Ser TrpIleAspThrLeuSerThrArgProGlyTyrLeuTrpValVal
65 70 75 80
Trp IleTyrArgAsnLeuIleGlySerValHisPhePhePheIleLeu
85 90 95
Thr LeuIleValLeuIleIleThrTyrLeuTyrTrpGlnIleThrGlu
100 105 110
Gly ArgLysIleMetIleArgLeuLeuHisGluGlnIleIleAsnGlu
115 120 125
Gly LysAspLysMetPheLeuIleGluLysLeuIleLysLeuGlnAsp
130 135 140
Met GluLysLysAlaAsnProSerSerLeuValLeuGluArgArgGlu
145 150 155 160
Val GluGlnGlnGlyPheLeuHisLeuGlyGluHisAspGlySerLeu
165 170 175
Asp LeuArgSerArgArgSerValGlnGluGlyAsnProArgAla
180 185 190
<210> 9
<211> 10
<212> PRT
<213> Artificial Sequence
<400> 9
Ile Thr Glu Gly Arg Lys Ile Met Ile Arg
1 5 10
50
<210> 10
<211> 9
<212> PRT
<213> Artificial Sequence
<400> 10
Leu Leu Met Asp Phe Val Phe Ser Leu
1 5
<210> 11
<211> 9
<212> PRT
<213> Artificial Sequence
<400> 11
Phe Leu Leu Phe Phe Pro Ser Phe Thr
1 5
<210> 12
<211> 9
<212> PRT
6
CA 02386028 2002-03-28
WO PCT/EP00/09500
01/23417
<213> Artificial Sequence
<400> 12
Gln Met Thr Phe Phe Ile Leu
Met Phe
1 5
<210> 13
<211> 9
<212> PRT
<213> Artificial Sequence
<400> 13
Met Met Phe Phe Ile Phe Leu
Thr Leu
1 5
<210> 14
<211> 9
<212> PRT
<213> Artificial Sequence
<400> 14
Phe Leu Glu Lys Leu Ile Leu
Ile Lys
1 5
<210> 15
<211> 9
<212> PRT
<213> Artificial Sequence
<900> 15
Val Leu Ile Arg Asn Ile Leu
Leu Phe
1 5
<210> 16
<211> 9
<212> PRT
<213> Artificial Sequence
<400> 16
40Leu Val Ile Gly Ile Phe Cys
Trp Phe
1 5
<210> 17
<211> 9
<212> PRT
<213> Artificial Sequence
<400> 17
Thr Leu Ile Thr Ile Trp Leu
Ala Arg
501 5
<210> 18
<211> 9
<212> PRT
<213> Artificial Sequence
<400> 18
Leu Ile Cys Trp Asp Phe Val
Phe Thr
1 5
<210> 19
<211> 9
<212> PRT
<213> Artificial Sequence
<400> 19
7
CA 02386028 2002-03-28
WO 01/23417 PCT/EP00/09500
Phe Leu Gly Glu Phe Leu Arg Arg Ile
1 5
<210> 20
<211> 9
<212> PRT
<213> ArtificialSequence
<400> 20
Leu Leu Pro Phe Val Cys
Leu Va1 Ser
1 5
<210> 21
<211> 9
<212> PRT
<213> ArtificialSequence
<400> 21
Leu Leu Arg Phe Ala Met
Thr Ser Tyr
1 5
<210> 22
<211> 9
<212> PRT
<213> ArtificialSequence
<400> 22
Lys Leu Phe Cys Asp Thr
Ile Trp Phe
1 5
<210> 23
<211> 10
<212> PRT
<213> ArtificialSequence
<400> 23
Leu Leu Met Asp Va1 Ser
Leu Phe Phe Leu
1 5 10
<210> 24
<211> 10
<212> PRT
<213> ArtificialSequence
<400> 24
Leu Leu Asp Phe Phe Leu
Met Val Ser Val
1 5 10
<210> 25
<211> to
<212> PRT
<213> ArtificialSequence
<400> 25
Gln Met Thr Phe Ile Leu
Met Phe Phe Leu
1 5 10
<210> 26
<211> 10
<212> PRT
<213> ArtificialSequence
<400> 26
Tyr Leu Glu Tyr Leu Phe
Ala Asn Glu Leu
1 5 10
8
CA 02386028 2002-03-28
WO 01/23417 PCT/EP00/09500
<210> 27
<211> 10
<212> PRT
<213> Artificial Sequence
<400> 27
Lys Leu Ile Phe Cys Trp Asp Phe Thr Val
1 5 10
<210> 28
<211> 10
<212> PRT
<213> Artificial Sequence
1$ <900> 28
Leu Leu Phe Phe Pro Ser Phe Thr Gly Val
1 5 10
<210> 29
<211> to
<212> PRT
<213> Artificial Sequence
<400> 29
G1n Leu Leu Thr Arg Phe Ser Ala Tyr Met
1 5 10
<210> 30
<211> 10
<212> PRT
<213> Artificial Sequence
<400> 30
Thr Leu Val Trp Ile Gly Ile Phe Phe Cys
1 5 10
<210> 31
<211> 10
<212> PRT
<213> Artificial Sequence
<400> 31
Ser Gln Met Met Thr Phe Phe Ile Phe Leu
1 5 10
<210> 32
<211> 10
<212> PRT
<213> Artificial Sequence
<400> 32
Val Leu Leu Leu Pro Phe Val Val Ser Cys
1 5 10
<210> 33
<211> 10
<212> PRT
<213> Artificial Sequence
<400> 33
Ile Leu Cys Tyr Tyr Trp Leu Asn Thr Val
1 5 10
<210> 34
<211> 10
<212> PRT
9
CA 02386028 2002-03-28
WO 01/23417 PCT/EP00/09500
<213> Artificial Sequence
<400> 34
Thr Leu Ile Val Leu Ile Ile Thr Tyr Leu
1 5 10
<210> 35
<211> 10
<212> PRT
<213> Artificial Sequence
<400> 35
Tyr Val Leu Leu Ile Arg Asn Ile Phe Leu
1 5 10
<210> 36
<211> 10
<212> PRT
<213> Artificial Sequence
<900> 36
Phe Val Phe Ser Leu Val Asn Ser Phe Leu
1 5 10
<210> 37
<211> 10
<212> PRT
<213> Artificial Sequence
<400> 3~
Leu Leu Leu Pro Phe Val Val Ser Cys Ile
1 5 10
<210> 38
<211> to
<212> PRT
<213> Artificial Sequence
<400> 38
Leu Ile Gly Ser Val His Phe Phe Phe Ile
1 5 10
<210> 39
<211> 10
<212> PRT
<213> Artificial Sequence
<400> 39
Tyr Glu Met Pro Arg His Glu Val Tyr Val
1 5 10
<210> 40
<211> 10
<212> PRT
<213> Artificial Sequence
<400> 40
Leu Leu Thr Arg Phe Ser Ala Tyr Met Val
1 5 10
<210> 91
<211> 9
<212> PRT
<213> Artificial Sequence
<400> 41
CA 02386028 2002-03-28
WO 01/23417 PCT/EP00/09500
Ile Tyr Ser Gly Gly Ile Thr Lys Leu
1 5
<210> 42
<211> 9
<212> PRT
<213> ArtificialSequence
<400> 42
Val Tyr Leu Ala Tyr Leu
Tyr Glu Asn
1 5
<210> 93
<211> 9
<212> PRT
<213> ArtificialSequence
<400> 43
Tyr Tyr Leu Asn Val Leu
Trp Thr Ala
1 5
<210> 44
<211> 9
<212> PRT
<213> ArtificialSequence
<400> 44
Ile Tyr Leu Leu Met Phe
Arg Leu Asp
1 5
<210> 45
<211> 10
<212> PRT
<213> ArtificialSequence
<400> 45
Val Tyr Leu Leu Arg Ile
Val Ile Asn Phe
1 5 10
<210> 46
<211> 10
<212> PRT
<213> ArtificialSequence
<400> 46
Cys Tyr Trp Leu Thr Ala
Tyr Asn Val Leu
1 5 10
<210> 47
<211> 10
<212> PRT
<213> ArtificialSequence
<400> 47
Tyr Tyr Ala Glu Asn Glu
Leu Tyr Leu Phe
1 5 10
<210> 48
<211> 9
<212> PRT
<213> ArtificialSequence
<400> 48
Met Leu Ile Met Tyr Lys
Phe Phe Ser
1 5
11
CA 02386028 2002-03-28
WO PCT/EP00/09500
01/23417
<210> 49
<211> 9
<212> PRT
<213> Artificial Sequence
<400> 49
Leu Leu Arg Asn Ile Phe Lys
Ile Leu
1 5
<210> 50
<211> 9
<212> PRT
<213> Artificial Sequence
<400> 50
Gly Leu Glu Phe Asp Ile Arg
Gln Ala
1 5
<210> 51
<211> 10
<212> PRT
<213> Artificial Sequence
<400> 51
25Lys Met Leu Ile Glu Lys IleLys
Phe Leu
1 5 10
<210> 52
<211> 10
<212> PRT
<213> Artificial Sequence
<400> 52
Ile Met Phe Ile Met Phe SerLys
Leu Tyr
351 5 10
<210> 53
<211> 10
<212> PRT
<213> Artificial Sequence
<400> 53
Lys Leu Lys Leu Gln Asp GluLys
Ile Met
1 5 10
<210> 54
<211> 10
<212> PRT
<213> Artificial Sequence
<400> 54
Val Leu Ile Arg Asn Ile LeuLys
Leu Phe
1 5 10
<210> 55
<211> 9
<212> PRT
<213> Artificial Sequence
<400> 55
Met Pro His Glu Val Tyr Leu
Arg Val
1 5
<210> 56
<211> 10
<212> PRT
12
CA 02386028 2002-03-28
WO 01/23417 PCT/EP00/09500
<213> Artificial Sequence
<400> 56
Met Pro Arg His Glu Val Tyr Val Leu Leu
$ 1 5 10
<210> 57
<211> 9
<212> PRT
<213> Artificial Sequence
<400> 57
Tyr Glu Met Pro Arg His Glu Val Tyr
1 5
1$
<210> 58
<211> 10
<212> PRT
<213> Artificial Sequence
<400> 58
Trp Glu Thr Leu Ile Gly Gln Asp Ile Tyr
1 5 10
2$ <210> 59
<211> 9
<212> PRT
<213> Artificial Sequence
<400> 59
Val His Phe Phe Phe Ile Leu Thr Leu
1 5
<210> 60
3$ <211> 9
<212> PRT
<213> Artificial Sequence
<400> 60
Leu Arg Arg Ile Ile Gly Met Gln Leu
1 5
<210> 61
<211> 9
4$ <212> PRT
<213> Artificial Sequence
<400> 61
Leu Leu Met Asp Phe Val Phe Ser Leu
$0 1 5
<210> 62
<211> 9
<212> PRT
$$ <213> Artificial Sequence
<400> 62
Met Gln Leu Ile Thr Ser Leu Gly Leu
1 5
<210> 63
<211> 9
<212> PRT
<213> Artificial Sequence
6$
<400> 63
13
CA 02386028 2002-03-28
WO 01/23417 PCT/EP00/09500
Phe Val Phe Ser Leu Val Asn Ser Phe
1 5
<210> 64
$ <211> 9
<212> PRT
<213> Artificial Sequence
<400> 64
Phe Ile Leu Thr Leu Ile Val Leu Ile
1 5
<210> 65
<211> 9
<212> PRT
<213> Artificial Sequence
<400> 65
Trp G1n Ile Thr Glu Gly Arg Lys Ile
1 5
<210> 66
<211> 9
<212> PRT
<213> Artificial Sequence
<400> 66
Met Phe Tyr Ser Lys Asn Ile Ser Leu
1 5
<210> 67
<211> 9
<212> PRT
<213> Artificial Sequence
<400> 67
Phe Ile Met Phe Tyr Ser Lys Asn Ile
1 5
<210> 68
<211> 9
<2i2> PRT
<213> Artificial Sequence
<400> 68
Trp Arg Ala Ser G1n Met Met Thr Phe
1 5
<210> 69
<211> 9
<212> PRT
<213> Artificial Sequence
<400> 69
Phe Phe Phe Ile Leu Thr Leu Ile Val
1 5
<210> 70
<211> 9
<212> PRT
<213> Artificial Sequence
<400> 70
Phe Arg Leu Val Glu Arg Tyr Glu Met
1 5
14
CA 02386028 2002-03-28
WO 01/23417 PCT/EP00/09500
<210> 71
<211> 9
<212> PRT
<213> Artificial Sequence
<400> 71
Phe Phe Ile Leu Thr Leu Ile Val Leu
1 5
<210> 72
<211> 9
<212> PRT
<213> Artificial Sequence
1j <400> 72
Tyr Ala Gln Thr Leu Val Trp Ile Ala
1 5
IS
