Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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COMPOUNDS RELATED TO PAP-1
The present invention relates to polynucleotides, herein referred to as CASB47
polynucleotides, polypeptides encoded thereby (referred to herein as CASB47
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
1 o CASB47 polypeptide imbalance with the identified compounds. In a still
fiuther aspect,
the invention relates to diagnostic assays for detecting diseases associated
with inappropriate
CASB47 polypeptide activity or levels.
Polypeptides and polynucleotides of the present invention are believed to be
important
t 5 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
CASB47
25 polypeptide, which could qualify it as a target for therapeutic or
prophylactic intervention
different from that linked to the immune response.
Functional genomics relies heavily on high-throughput DNA sequencing
technologies and
the various tools of bioinformatics to identify gene sequences of potential
interest from the
3o many molecular biology databases now available. cDNA libraries enriched for
genes of
relevance to a particular tissue or physiological situation can be constructed
using recently
developed subtractive cloning strategies. Furthermore, cDNAs found in
libraries of certain
tissues and not others can be identified using appropriate electronic
screening methods.
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High throughput genome- or gene-based biology allows new approaches to the
identification
and cloning of target genes for useful immune responses for the prevention and
vaccine
therapy of diseases such as cancer and autoimmunity.
In a first aspect, the present invention relates to CASB47 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
to ID N0:2 over the entire length of SEQ ID N0:2. Such polypeptides include
those
comprising the amino acid of SEQ ID N0:2.
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
15 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
over the
entire length of SEQ ID N0:2. Such polypeptides include the polypeptide of SEQ
ID
N0:2.
2o Further peptides of the present invention include isolated polypeptides
encoded by a
polynucleotide comprising the sequence contained in SEQ ID NO:1.
The invention also provides an immunogenic fiagrnent of a CASB47 polypeptide,
that is a
contiguous portion of the CASB47 polypeptide which has the same or similar
immunogenic
25 properties to the polypeptide comprising the amino acid seqeunce of SEQ ID
N0:2. That is
to say, the fragment (if necessary when coupled to a carrier) is capable of
raising an immune
response which recognises the CASB47 polypeptide. Such an immunogenic fragment
may
include, for example, the CASB47 polypeptide lacking an N-terminal leader
sequence, a
transmembrane domain or a C-terminal anchor domain. In a preferred aspect the
3o immunogenic fi~agment of CASB47 according to the invention 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
2
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95% identity, most preferably at least 97-99% identity, to that of SEQ ID N0:2
over the
entire length of SEQ ID N0:2
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
t o 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
15 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,
2o 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.
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
partner and expression enhancing partner.
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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 DEAE. 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.
t 5 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
2o and Tyr. Particularly preferred are variants in which several, S-10, 1-5, 1-
3, 1-2 or 1 amino
acids are substituted, deleted, or added in any combination.
Polypeptides of the present invention can be prepared in any suitable manner.
Such
polypeptides include isolated naturally occurring polypeptides, recombinantly
produced
25 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 CASB47 polynucleotides.
Such
3o polynucleotides include isolated polynucleotides comprising 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 9S% identity,
to the amino
acid sequence of SEQ ID N0:2, over the entire length of SEQ ID N0:2. In this
regard,
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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
nucleotide
sequence contained in SEQ ID NO:1 encoding the polypeptide of SEQ ID N0:2.
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
preferably at least 90% identity, yet more preferably at least 95% identity,
to a nucleotide
sequence encoding a polypeptide of SEQ ID N0:2, over the entire coding region.
In this
t o regard, poiynucleotides 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
15 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 over the entire length of SEQ ID NO: l . 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
2o preferred. Such polynucleotides include a polynucleotide comprising the
polynucleotide of
SEQ ID NO:1 as well as the polynucleotide of SEQ ID NO:1. 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. 184:155-1560 (1996); Doe et. al., Proc. Natl. Acad. Sci. 93:8578-
8583 (1996)).
25 The invention also provides polynucleotides which are complementary to all
the above
described polynucleotides.
The invention also provides a &~agznent of a CASB47 polynucleotide which when
administered to a subject has the same immunogenic properties as the
polynucleotide of
3o SEQ ID NO:1.
The invention also provides a polynucleotide encoding an immunological
fragment of a
CASB47 polypeptide as hereinbefore defined.
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The nucleotide sequence of SEQ ID NO:l shows homology with mouse PAP-I
(GenBank
accession D78255). The nucleotide seqlence of SEQ ID NO:1 is a cDNA sequence
and
comprises a polypeptide encoding sequence (nucleotide 25 to 690) encoding a
polypeptide
of 221 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: l or it may be a sequence other than the one contained
in SEQ
ID NO: I, 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
t o structurally related to other proteins of a novel family, having homology
and/or structural
similarity with mouse PAP-1 (accession D1011981).
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
SEQ ID N0:2, as appropriate.
The present invention also relates to partial polynucleotide and polypeptide
sequences which
2o were first identified prior to the determination of the corresponding full
length sequences of
SEQ ID NO:1 and SEQ ID N0:2.
Accordingly, in a further 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 N0:3 over
the entire
length of SEQ ID N0:3;
(b) has a nucleotide sequence which has at least 70% identity, preferably at
least 80%
3o 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 NO:1 over the entire
length of
SEQ ID N0:3;
(c) the polynucleotide of SEQ ID N0:3; or
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(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 N0:4, over the entire length of SEQ ID N0:4;
as well as the polynucleotide of SEQ ID N0:3.
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%
t o identity, most preferably at least 97-99% identity, to that of SEQ ID N0:2
over the entire
length of SEQ ID N0:4;
(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 ID
N0:2
t 5 over the entire length of SEQ ID N0:4;
(c) comprises the amino acid of SEQ ID N0:4; and
(d) is the polypeptide of SEQ ID N0:4;
as well as polypeptides encoded by a polynucleotide comprising the sequence
contained
in SEQ ID N0:3.
The nucleotide sequence of SEQ ID N0:3 and the peptide sequence encoded
thereby are
derived from EST (Expressed Sequence Tag) sequences. It is recognised by those
skilled
in the art that there will inevitably be some nucleotide sequence reading
errors in EST
sequences (see Adams, M.D. et al, Nature 377 (supp) 3, 1995). Accordingly, the
23 nucleotide sequence of SEQ ID N0:3 and the peptide sequence encoded
therefrom are
therefore subject to the same inherent limitations in sequence accuracy.
Furthermore, the
peptide sequence encoded by SEQ ID N0:3 comprises a region of identity or
close
homology and/or close structural similarity (for example a conservative amino
acid
difference) with the closest homologous or structurally similar protein.
3o
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
colon
cancer, (for example Sambrook et al., Molecular Cloning: A Laboratory Manual,
2"~ Ed.,
7
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Cold Spring harbor Laboratory Press, 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.
s 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
1 o 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
polynucleotide may also cantain non-coding 5' and 3' sequences, such as
transcribed, non-
t 5 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 and in which
several, for
20. 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
contained in SEQ ID NO:1, may be used as hybridization probes for cDNA and
genomic
25 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. Typically these nucleotide sequences are
70%
3o 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 15
nucleotides, preferably, at least 30 nucleotides and may have at least 50
nucleotides.
Particularly preferred probes will have between 30 and 50 nucleotides.
Particularly
8
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WO 99/49030 PCT/EP99/01892
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.
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 a fragment thereof; and isolating
full-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% fonmamide,
SxSSC
( 1 SOmM NaCI, lSmM trisodium citrate), SO mM sodium phosphate (pH7.6), Sx
Denhardt's
solution, 10 % dextran sulfate, and 20 microgram/ml denatured, sheared salmon
sperm
DNA; followed by washing the filters in O.lx SSC at about 65oC. Thus the
present
~ 5 invention also includes polynucleotides obtainable by screening an
appropriate library
under stingent hybridization conditions with a labeled pmbe having the
sequence of SEQ ID
NO: I or a fragment thereof.
The skilled artisan will appreciate that, in many cases, an isolated cDNA
sequence will be
2o 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
full-length cDNAs, or extend short cDNAs, for example those based on the
method of
2s 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 MarathonT"" technology, cDNAs
have
been prepared from mRNA extracted from a chosen tissue and an'adaptor'
sequence
30 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.
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WO 99/49030 PCT/EP99/01892
primers designed to anneal within the amplified product (typically an adaptor
specific
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
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'
primer.
Recombinant polypeptides of the present invention may be prepared by processes
well
t o 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
t 5 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 poiynucleotides of the present
invention.
2o 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
include, for instance, calcium phosphate transfection, DEAF-dextran mediated
transfection,
25 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 traps versus in cis is preferred to keep
antigen free of
3o 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.
CA 02323501 2000-09-18
WO 99!49030 PCT/EP99/01892
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
Sf7 cells;
animal cells such as CHO, COS, HeLa, C 127, 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
plasmid and bacteriophage genetic elements, such as cosmids and phagemids. The
expression systems may contain control regions that regulate as well as
engender
~ 5 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
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
2o 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
heterologous signals.
25 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 Iead to in
vivo expression of the antigen and induction of immune responses. Viruses and
bacteria
used for this purpose are for instance: poxviruses (e.g; vaccinia, fowlpox,
canarypox),
3o alphaviruses (Sindbis virus, Semliki Forest Virus, Venezuelian Equine
Encephalitis
Virus), adenoviruses, adeno-associated virus, picornaviruses (poliovirus,
rhinovirus),
herpesviruses (varicella zoster virus, etc), Listeria, Salmonella , Shigella,
BCG. These
CA 02323501 2000-09-18
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viruses and bacteria can be virulent, or attenuated in various ways in order
to obtain live
vaccines. Such live vaccines also form part of the invention.
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 canon exchange chromatography, phosphocellulose
chromatography,
hydrophobic interaction chromatography, affinity chromatography,
hydroxylapatite
chromatography and lectin chromatography. Most preferably, ion metal affinity
chromatography (IMAC) is employed for purification. Well known techniques for
t o 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-enforcing
or modulating an immunological response in a mamrnai 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-enforcing
or
modulating immunological response in a mammal which comprises, delivering a
2o 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 immunologicaUvaccine
formulation
(composition) which, when introduced into a mammalian host, induces, re-
enforces 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
3o 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
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WO 99/49030 PCT/EP99/01892
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 mufti-dose containers, for
example, sealed
ampoules and vials and may be stored in a freeze-dried condition requiring
only the
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
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
15 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
the present invention and administered in vivo in an immunogenic way.
Alternatively,
2o 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.
25 The vaccine formulation of the invention may also include adjuvant systems
for
enhancing the immunogenicity of the formulation. Preferably the adjuvant
system raises
preferentially a TH 1 type of response.
An immune response may be broadly distinguished into two extreme categories,
being a
3o humoral or cell mediated immune responses (traditionally characterised by
antibody and
cellular effector mechanisms of protection respectively). These categories of
response
have been termed TH 1-type responses (cell-mediated response), and TH2-type
immune
responses (humoral response).
13
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WO 99/49030 PCT/EP99/01892
Extreme THl-type immune responses may be characterised by the generation of
antigen
specific, haplotype restricted cytotoxic T lymphocytes, and natural killer
cell responses.
In mice TH1-type responses are often characterised by the generation of
antibodies of the
IgG2a subtype, whilst in the human these correspond to IgGl type antibodies.
TH2-type
immune responses are characterised by the generation of a broad range of
immunoglobulin isotypes including in mice IgGI, IgA, and IgM.
It can be considered that the driving force behind the development of these
two types of
t o immune responses are cytokines. High levels of TH 1-type cytokines tend to
favour the
induction of cell mediated immune responses to the given antigen, whilst high
levels of
TH2-type cytokines tend to favour the induction of humoral immune responses to
the
antigen.
15 The distinction of TH 1 and TH2-type immune responses is not absolute. In
reality an
individual will support an immune response which is described as being
predominantly
TH1 or predominantly TH2. However, it is often convenient to consider the
families of
cytokines in terms of that described in marine CD4 +ve T cell clones by
Mosmann and
Coffman (Mosmann, T.R. and Cof,~'man, R.L. (1989) THl and TH2 cells: different
2o patterns of lymphokine secretion lead to dif~''erentfunctional properties.
Annual Review of
Immunology, 7, p145-173). Traditionally, TH1-type responses are associated
with the
production of the INF-y and IL-2 cytokines by T-lymphocytes. Other cytokines
often
directly associated with the induction of TH1-type immune responses are not
produced by
T-cells, such as IL-12. In contrast, TH2- type responses are associated with
the secretion
2s of IL-4, IL-5, IL-6 and IL-13.
It is known that certain vaccine adjuvants are particularly suited to the
stimulation of
either TH 1 or TH2 - type cytokine responses. Traditionally the best
indicators of the
TH1:TH2 balance of the immune response after a vaccination or infection
includes direct
3o measurement of the production of TH1 or TH2 cytokines by T lymphocytes in
vitro after
restimulation with antigen, and/or the measurement of the IgG 1:IgG2a ratio of
antigen
specif c antibody responses.
14
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WO 99/49030 PCT/EP99/01892
Thus, a TH 1-type adjuvant is one which preferentially stimulates isolated T-
cell
populations to produce high levels of TH 1-type cytokines when re-stimulated
with
antigen in vitro, and promotes development of both CD8+ cytotoxic T
lymphocytes and
antigen specific immunoglobulin responses associated with TH1-type isotype.
Adjuvants which are capable of preferential stimulation of the TH 1 cell
response are
described in International Patent Application No. WO 94/00153 and WO 95/17209.
3 De-O-acylated monophosphoryl lipid A (3D-MPL) is one such adjuvant. This is
known
to from GB 2220211 (Ribi). Chemically it is a mixture of 3 De-O-acylated
monophosphoryl lipid A with 4, 5 or 6 acylated chains and is manufactured by
Ribi
Immunochem, Montana. A preferred form of 3 De-O-acylated monophosphoryl lipid
A
is disclosed in European Patent 0 689 454 B 1 (SmithKline Beecham Biologicals
SA).
t 5 Preferably, the particles of 3D-MPL are small enough to be sterile
filtered through a
0.22micron membrane (European Patent number 0 689 454).
3D-MPL will be present in the range of lOp,g - 100p.g preferably 25-SOp,g per
dose
wherein the antigen will typically be present in a range 2-SOp,g per dose.
2o Another preferred adjuvant comprises QS21, an Hplc purified non-toxic
fraction derived
from the bark of Quillaja Saponaria Molina. Optionally this may be admixed
with 3 De-
O-acylated monophosphoryl lipid A (3D-MPL), optionally together with an
carrier.
The method of production of QS21 is disclosed in US patent No. 5,057,540.
Non-reactogenic adjuvant formulations containing QS21 have been described
previously
(WO 96/33739). Such formulations comprising QS21 and cholesterol have been
shown
to be successful TH1 stimulating adjuvants when formulated together with an
antigen.
3o Further adjuvants which are preferential stimulators of TH1 cell response
include
immunomodulatory oligonucleotides, for example unmethylated CpG sequences as
disclosed in WO 96/02555.
CA 02323501 2000-09-18
WO 99/49030 PCT/EP99/01892
Combinations of different TH1 stimulating adjuvants, such as those mentioned
hereinabove, are also contemplated as providing an adjuvant which is a
preferential
stimulator of TH1 cell response. For example, QS21 can be formulated together
with 3D-
MPL. The ratio of QS21 : 3D-MPL will typically be in the order of 1 : 10 to 10
: 1;
preferably 1:5 to 5 : 1 and often substantially 1 : 1. The preferred range for
optimal
synergy is 2.5 : l.to 1 : 1 3D-MPL: QS21.
Preferably a carrier is also present in the vaccine composition according to
the invention.
The carrier may be an oil in water emulsion, or an aluminium salt, such as
aluminium
t 0 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
I5 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 lpg - 200pg, such as 10-100p.g, preferably l0ug - SOpg per dose.
Typically the
20 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.
3o A particularly potent adjuvant formulation involving QS21, 3D-MPL and
tocopherol in
an oil in water emulsion is described in WO 95/I7210.
16
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WO 99/49030 PCT/EP99101892
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.
This invention also relates to the use of polynucleotides of the present
invention as
diagnostic reagents. Detection of a mutated form of the gene characterised by
the
polynucleotide of SEQ ID NO: l which is associated with a dysfunction will
provide a
diagnostic tool that can add to, or define, a diagnosis of a disease, or
susceptibility to a
t o disease, which results finm under-expression, over-expression or altered
spatial or temporal
expression of the said gene. Individuals carrying mutations in the gene may be
detected at
the DNA level by a variety of techniques.
Nucleic acids for diagnosis may be obtained from a subject's cells, such as
from blood,
t 5 urine, saliva, tissue biopsy or autopsy material. The genomic DNA may be
used directly for
detection or may be amplified enzymatically by using PCR or other
amplification techniques
prior to analysis. RNA or cDNA may also be used in similar fashion. Deletions
and
insertions can be detected by a change in size of the amplified product in
comparison to the
normal genotype. Point mutations can be identified by hybridizing amplified
DNA to
?o labeled CASB47 nucleotide sequences. Perfectly matched sequences can be
distinguished
from mismatched duplexes by RNase digestion or by differences in melting
temperatures.
DNA sequence differences may also be detected by alterations in
electrophoretic mobility of
DNA fragments in gels, with or without denaturing agents, or by direct DNA
sequencing
(ee, e.g., Myers et al., Science {1985) 230:1242). Sequence changes at
specific locations
25 may also be revealed by nuclease protection assays, such as RNase and S 1
protection or the
chemical cleavage method (see Cotton et al., Proc Natl Acad Sci USA (1985) 85:
4397-
4401 ). In another embodiment, an array of oligonucleotides probes comprising
CASB47
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
3o applicability and 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)).
17
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WO 99/49030 PCT/EP99/01892
The diagnostic assays offer a process for diagnosing or determining a
susceptibility to
cancers, autoimmune disease and related conditions through detection of
mutation in the
CASB47 nucleotide sequence by the methods described. In addition, such
diseases may be
diagnosed by methods comprising determining from a sample derived from a
subject an
abnormally decreased or increased level of polypeptide or mRNA. Decreased or
increased expression~can be measured at the RNA level using any of the methods
well
known in the art for the quantitation of polynucleotides. Assay techniques
that can be used
to determine levels of a protein, such as a polypeptide of the present
invention, in a sample
derived from a host are well-known to those of skill in the art.
to
Thus in another aspect, the present invention relates to a diagnostic kit for
performing a
diagnostic assay which comprises:
(a) a polynucleotide of the present invention, preferably the nucleotide
sequence of SEQ
ID NO: 1, or a fragment thereof ;
t 5 (b) a nucleotide sequence complementary to that of (a);
(c) a polypeptide of the present invention, preferably the polypeptide of SEQ
ID N0:2 or
a fragment thereof; or
(d) an antibody to a polypeptide of the present invention, preferably to the
polypeptide of
SEQ ID N0:2.
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 chromosome. 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
3o 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.
18
CA 02323501 2000-09-18
WO 99/49030 PCT/EP99/01892
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 polypepddes 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.
IO
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
~ 5 cultures can be used. Examples include the hybridoma technique (Kohler, G.
and Milstein,
C., Nature ( I 975) 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).
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
colon cancer, ovarian cancer, lung cancer and brain cancer, autoimmune disease
and related
3o conditions.
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
19
CA 02323501 2000-09-18
WO 99/49030 PCT/EP99/01892
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
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
t o abnormal conditions such as, for instance, cancer and autoimmune diseases,
in particular,
colon cancer, ovarian cancer, lung cancer and brain cancer, related to either
a presence of, an
excess of, or an under-expression of, CASB47 polypeptide activity.
The present invention further provides for a method of screening compounds to
identify
t 5 those which stimulate or which inhibit the function of the CASB47
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
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
2o substrates, ligands, receptors, enzymes, etc., as the case may be, of the
polypeptide; or may
be structural or functional mimedcs thereof (see Coligan et al., Current
Protocols in
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
25 (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
30 (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;
CA 02323501 2000-09-18
WO 99/49030 PCT/EP99/O1$92
(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 1, to
form a mixture, measuring activity of the polypeptide in the mixture, and
comparing the
activity of the mixture to a standard; or
t o (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
~ 5 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.
2o 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:
(a) a polypeptide of the present invention;
25 (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.
3o 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:
(a) determining in the first instance the three-dimensional structure of the
polypeptide;
21
CA 02323501 2000-09-18
WO 99/49030 PCT/EP99/01892
(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
(d) testing whether the candidate compounds are indeed agonists, antagonists
or
inhibitors.
Gene therapy may also be employed to effect the endogenous production of
CASB47
polypeptide by the relevant cells in the subject. For an overview of gene
therapy, see
t o 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
t 5 Vaccine Design - the subunit and adjuvant approach, edited by Powell and
Newman,
Plenurn Press, 1995. New Trends and Developments in Vaccines, edited by Volley
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
2o 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.
Such amount will vary depending upon which specific immunogen is employed.
25 Generally, it is expected that each dose will comprise 1-1000p,g of
protein, preferably
2-100p.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
original environment, or both. For example, a polynucleotide or a polypeptide
naturally
22
CA 02323501 2000-09-18
WO 99/49030 PCT/EP99/01892
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.
"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
poiynucleotide 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
t 5 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,
2o 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
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
3o strings of such sequences. "Identity" and "similarity" can be readily
calculated by known
methods, including but not limited to those described in (Computational
Molecular
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;
23
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WO 99/49030 PCT/EP99/01892
Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Grin, 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
are not limited to, the GCG program package (Devereux, J., et al., Nucleic
Acids
to Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S.F. et
al., J.
Molec. Biol. 21~: 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.
t5
The preferred algorithm used is FASTA. The preferred parameters for
polypeptide or
polynuleotide sequence comparison using this algorithm include the following:
Gap Penalty:12
Gap extension penalty: 4
2o Word size: 2, max 6
Preferred parameters for polypeptide sequence comparison with other methods
include
the following:
1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)
25 Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl.
Acad. Sci.
USA. 89:10915-10919 (1992)
Gap Penalty: 12
Gap Length Penalty: 4
3o 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 polypeptide comparisons (along with no penalty for end gaps).
24
CA 02323501 2000-09-18
WO 99/49030 PCT/EP99/01892
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.
to
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
15 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
2o determined by multiplying the total number of nucleotides in SEQ ID NO:1 by
the
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 5 xn - (xn ~ y),
wherein nn is the number of nucleotide alterations, xn is the total number of
nucleotides
25 in SEQ ID NO:1, 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 may create
nonsense,
missense or frameshift mutations in this coding sequence and thereby alter the
3o polypeptide encoded by the polynucleotide following such alterations.
CA 02323501 2000-09-18
WO 99/49030 PCf/EP99/01892
Similarly, a polypeptide sequence of the present invention may be identical to
the
reference sequence of SEQ ID N0:2, 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
I o 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 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:
na~a - (xa' Y)
15 wherein na is the number of amino acid alterations, xa is the total number
of amino acids
in SEQ ID N0:2, 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.
20 "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 detennining 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
25 equivalent of a polynucleotide or polypeptide in another species and
"paralog" meaning a
functionally similar sequence when considered within the same species.
26
CA 02323501 2000-09-18
WO 99/49030 PCT/EP99/01892
EXAMPLES
Example 1
A database screening method used to select novel genes that are differentially
expressed in cancers
1.1 Introduction
A complementary approach to experimental antigen discovery is to explore the
human
t o genome databases, particularly those of "Expressed Sequence Tags" (ESTs),
in search of
tumour-specific and tumour-associated antigens. ESTs are small fragments
(approximately 300 bp) of cDNA made from a collection of mRNA extracted from a
particular tissue or cell line. Such currently provide a massive amount of
ESTs ( 106) from
several hundreds of cDNA tissue libraries, including tumoural tissues from
various types
~ 5 and states of disease. By means of specifically designed informatics
tools, one can search
in this database for a subset of potential antigen candidates, provided that
artifacts are
carefully avoided. To allow a specific selection, the libraries, from both
healthy and
diseased tissues, have first to be selected on the basis of different quality
criteria (tissue
quality, library construction method, sequencing depth and quality, diversity
index,
2o frameshift). The EST sequences from these selected libraries are then
compared to
identify those genes specifically expressed, or significantly overexpressed,
in tumoural
tissues. Currently, the method is limited by the sequencing depth of these
libraries, i.e.
typically only about 10% of all the expressed genes of a particular tissue are
represented
by ESTs from a particular library. This limitation can be overcome by pooling
tissue
25 libraries.
After a careful screening using a set of defined criteria (novelty of the
gene, putative
expression pattern), the selected candidates can be further tested for
selective expression
in normal and tumoural tissues, for example by RT-PCR.
30 1.2 Method
The original EST database is reorganized by assembling all the fragments into
overlapping "genetic clusters". There are several well known algorithms which
can be
used to produce these assemblies. Each resulting assembly is thus a consensus
sequence
27
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WO 99/49030 PCT/EP99/01892
representing a fragment of, or a complete gene. This process reduces the total
amount of
information by one order of magnitude.
The method allows to select candidates by "customized differential expression"
by
ranking the number of ESTs by customizable tissue category.
The data are organized in a relational database comprising
- Table "ESTs": contains at least the EST names or Ids and the code of the
cDNA library
from which each EST was generated
- Table "Assemblies": contains at least for each assembly the list of EST
components
to - Table "Libraries": contains at least for each cDNA library its code,
tissue or cell line
type, disease state (normal, tumour or non-tumoural disease)
Links are made between these tables as shown in Figure 1.
The cDNA libraries are then classified in 5 categories, each subdivided in
several groups:
~5
- groups of cell lines (separated normal & cancer cell lines)
- groups of non-cancerous diseased tissues
- group of fetaUembryonic tissues
- gmups of normal tissues
20 - groups of tumoural tissues
- 1 group of unknown origin
An additional table is then added to the relational database, called "Groups
of libraries" as
shown in Figure 2.
The next step is the computing, for each assembly, of the number of ESTs
originating
from each group of libraries (for example using a Sybase Query Language
Query):
- For each assembly
3o - For each EST
- Check the corresponding library code
- Assign the corresponding group code
- Count the number of ESTs assigned to the same group code
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- Count the total number of ESTs
The result of this step is a table called "Results" containing one line per
assembly, and
one column per group code, and containing the final EST counts.
The table "Results" is used to compute several quantities for each assembly:
~ a "tumour-to-normal ratio" (TNR}:
(sum of EST T, EST F, EST P, EST O, EST Tes, EST Pl)/total number of ESTs
where (EST T) is the sum of all ESTs belonging to the groups that are tissue
or cell line
tumours, (EST F) is the sum of all ESTs belonging to groups of fetal tissues
or cell lines,
EST P is the total number of ESTs from normal prostate tissue, EST O is the
sum of
ESTs from normal ovarian tissues, EST Tes is the total number of ESTs from
normal
~ 5 testis tissues or cell lines, and EST Pl is the total number of ESTs from
placenta tissues.
Note that some normal tissues are included, namely "dispensable" tissues Like
testis,
ovary and prostate, which may often share expression patterns with tumours
(the so-
called "cancer-testis antigens" or CT-antigens), as well as placental, fetal
and embryonic
tissues.
~ Any other sum that is relevant to select candidates for a specific target
cancer or type
of cancers. As an example, one may sum the ESTs from the groups of libraries
from
tumour tissues or cell lines representative of breast tumours and from testis
and fetal
tissues. This may be relevant to detect the above-mentioned CT-antigens.
The resulting table is called "Customized results", and contains one line per
assembly and
one column per computed sum as well as any other relevant information, such as
the total
number or ESTs.
3o The "Customized results" table is then sorted according to the desired use.
A relevant
sorting is to use the TNR column as a primary sorting key, and the customized
sum as the
secondary sorting key.
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Each assembly over a defined threshold (for example: TNR > 0.8) is then
compared to a
sequence database of known genes or gene products using any sequence
comparison
algorithm (for example Blast) to screen for novelty of the gene. In a similar
way, a
sequence comparison can be performed using the original EST or assembly
database to
check for alternate splicing variants.
1.3 Results
Table 1: EST Distribution
1 o ID LibraryID Library Name
NCBI:405184 Soaves melanocyte
2NbHM
NCBI:5020I7 Soaves fetal lung
NbHLI9W
NCBI:922037 Soaves NbHTGBC
NCBI:932912 Soaves ovary tumor
NbHOT
NCBI:934101 Soaves ovary tumor
NbHOT
NCBI:934638 Soaves ovary tumor
NbHOT
NCBI:1040011 Soaves ovary tumor
NbHOT
NCBI:1122876 Soaves ovary tumor
NbHOT
NCBI:1171566 NCI_CGAP_Co4
NCBI:1180435 NCI_CGAP_Co3
NCB1:1073825 Soaves ovary tumor
NbHOT
NCBI:1073930 Soaves ovary tumor
NbHOT
NCBI:945746 Aorta endothelial
cells, TNF
NCBI:1431531 2 Soaves NFL T
GBC S1
NCBI:1451557 Soaves NbHTGBC
NCBI:1462735 Soaves NbHTGBC
NCBI:1463384 Soaves NbHTGBC
NCBI:1553758 Soaves NbHTGBC
NCBI:1558729 Soaves NbHTGBC
NCBI:1756330 2 Soaves NFL T
GBC S1
NCBI:1757672 2 Soaves NFL T
GBC S 1
NCBI:1850298 Soaves NhHMPu
S1
NCBI:1950621 Soaves fetal lung
NbHLI9W
NCBI:1992275 2 Soaves NFL T
GBC S1
NCBI:2020608 Soaves fetal liver
spleen 1N
NCBI:2050335 Soares placenta
8 to 9 weeks
NCBI:2050343 Soaves placenta
8 to 9 weeks
NCBI:2073110 6 NCI_CGAP_Lu5
NCBI:2075055 Soaves placenta
8 to 9 weeks
NCBI:2158404 1 NCI CGAP Bnn25
In summary, the EST distribution profile shows expression in ovary tumours,
colon
tumours, lung tumours and brain tumours.
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1.4 References
Wan J. S., Sharp S. J., Poirier G. M.-C., Wagaman P. C., Chambers J., Pyati
J.. Hom Y.-
L., Galindo J. E., Huvar A., Peterson P. A., Jackson M. R., Erlander M. G.
(1996) Nature
Biotechnol. 14, 1685.
Pardoll D. M. ( 1996) Curr. Opin. Immunom. 8, 619.
Example 2
Qualitative RT-PCR amplification
to
Presence of mRNA transcripts in a panel of normal tissues and a small number
of tumour
samples is evaluated by non-quantitative RT-PCR.
Total RNA from 19 normal tissues and 3 tumour samples was purchased from
InVitrogen. mRNA is purified from total RNA after DNAse treatment using oligo-
dT
t 5 magnetic beads (Dynal). 200 ng of mRNA are reverse transcribed (Expand
reverse
transcriptase, Boehringer) in a 20 ~cl reaction and 2 pl of this reaction are
amplified by
PCR (AmpliTaq Gold, Perkin-Elmer) for 32 cycles (Perkin-Elmer 9600
thermocycler)
using standard protocols. Non-template controls (NTC) are always included.
Amplification products ( 10 wl) are visualised on ethidium bromide-stained
agarose gels.
2o Oligonucleotides for PCR amplification are designed by computer {LaserGene
PrimerSelect module). Specificity of the designed oligonucleotides is
evaluated in silico
by comparing their sequences to the sequences in the public databases using
the FASTA
algorithm. Transcripts of the housekeeping GAPDH gene are amplified under
identical
conditions on all tissue samples. GAPDH serves as a positive control and
provides a
25 visual reference of a highly expressed gene. Detection of CASB47 mRNA in 19
normal
tissues and 3 tumour samples by RT-PCR is shown in Figure 3.
Example 3
Real-time RT-PCR analysis
Real-time RT-PCR (U. Gibson. 1996. Genome Research: 6,996) is used to compare
mRNA transcript abundance of the candidate antigen in tumour and normal colon
tissues
from multiple patients. In addition, mRNA levels of the candidate gene are re-
evaluated
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WO 99/49030 PCT/EP99/01892
by this approach in a panel of normal tissues.
Total RNA is extracted from snap frozen colon tissue biopsies using TriPure
reagent
(Boehringer). Total RNA from normal tissues is from InVitrogen as above. 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
(BioRad)
using SybrII dye (Molecular Probes). Primers for amplification are designed
with the
Perkin-Elmer Primer Express software using default options for TaqMan
amplification
conditions.
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 PE 7700 system. Ct values are calculated using the
7700
~ 5 Sequence Detector software for the tumour (CtT) and normal (CtN) samples
of each
patient. The difference between Ct values (CtN-CtT) is a direct measure of the
difference
in transcript levels between the tumour and normal tissues. As Ct values are
log-linearly
related to copy number and that the efficiency of PCR amplification under the
prevailing
experimental conditions is close to the theoretical amplification efficiency,
2 ~~tN-ctT) iS an
2o estimate of the relative transcript levels in the two tissues (i.e. fold
mRNA over-
expression in tumor).The percentage of over-expressing patients and the
average level of
mRNA over-expression in the tumours of these patients is calculated from the
data set of
18 patients. In addition, Ct values obtained with 12 normal tissues are
provided for the
candidate antigen and beta-actin.
Table 2:
Patients over-expressing CASB47Average level of over-expression
in colon tumours in colon tumours
(/.) (fold)
59 3
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Table 3: Real-time RT-PCR Ct values for CASB47 and actin in 12 normal tissues.
Bla BraiBre Cer Hea KidLiv Lun Oes PlaRec Ute
CASBC47 40 40 27 27 29 34 36 37 30 29 40 27
~ Actin 14 ( ~ ~ ~ 16 ( 16 14 15 l6 15
~ 16 15 15 i 17
7
j
Legend. Bla: bladder, Bra: brain, Bre: breast, Cer: cervix, Hea: heart, Kid:
kidney, Liv:
liver, lun: lung, Oes: oesophagus, Pla: placenta, Rec: rectum, Ute: uterus.
Example 4
to Identification of the full length cDNA sequence
Colon tumour cDNA libraries are constructed using the Lambda Zap II system
(Stratagene) from 5 ~g of polyA+ mRNA. The supplied protocol is followed
except that
SuperscriptII (Life Technologies) is used for the reverse transcription step.
Oligo dT-
15 primed and random-primed libraries are constructed. About 1.5 x106
independent phage
are plated for each screening of the library. Phage plaques are transferred
onto nylon
filters and hybridised using a cDNA probe labelled with AIkPhos Direct.
Positive phage
are detected by chemiluminescence. Positive phage are excised from the agar
plat, eluted
in SOOpI SM buffer and co~rmed by gene-specific PCR. Eluted phage are
converted to
20 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.
25 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
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
3o into a plasmid (pCRII-TOPO, InVitrogen) and sequenced.
Example 5:
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5.1 Expression and purification of tumour-specific antigens
Expression in microbial hosts is used to produce the antigen of the invention
for vaccine
purposes and to produce protein fragments or whole protein for rapid
purification and
generation of antibodies needed for characterization of the naturally
expressed protein by
s 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
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
l o 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
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
~ 5 (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
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.
20 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
antibodies.
A comparative evaluation of the different versions of the expressed antigen
will allow the
25 selection of the most promising candidate that is to be used for further
purification and
immunological evaluation.
The purification work 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
30 (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. This step is optimally followed by an
Anion Exchange
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WO 99/49030 PCT/EP99/01892
resin step and a Size Exclusion chromatography step depending on the success
of the
Imac step and the nature of the contaminants.
~.2 Antibody production and immunohistochemistry
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
i o c) characterise/ quantify the purified protein (ELISA).
5.2.1 Polyclonal antibodies:
lmsnuni~atian
2- 3 Rabbits are immunized , intramuscularly (LM.) , 3 times at 3 weeks
intervals with
~ 5 100~,g of protein, formulated in the adjuvant 3D-MPL/QS21 . 3 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
20 96 well microplates (maxisorb Nunc) are coated with Spg 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,
25 501 TMB (BioRad) is added for 7 min and the reaction then stopped with
H2SO4 0.2M.
The OD can be measured at 450 nm and midpoint dilutions calculated by
SoftmaxPro.
5.2.2 Monoclonal antibodies:
Immunization
30 5 BALB/c mice are immunized 3 times at 3 week intervals with 5 ~,g of
purified protein.
Bleedings are performed 14 days post II and 1 week post 3. The sera is tested
by Elisa
on purified protein used as coated antigen. Based on these results (midpoint
dilution >
10000 ) one mouse is selected for fusion
CA 02323501 2000-09-18
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Fusio»l HATselection
Spleen cells are fused with the SP2/0 myeloma according to a standard protocol
using
PEG 40% and DMSO S%. Cells are then seeded in 96 well plates 2.5 x104 - 10'
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.
5.2.3 Immunohistochemistry
When antibodies are available, immuno staining is performed on normal or
cancer
tissue sections, in order to determine
~ the level of expression of the protein antigen of the invention in cancer
relative to
normal tissue or
~ the proportion of cancers of a certain type expressing the antigen
if other cancer types also express the antigen
the proportion of cells expressing the antigen in a cancer tissue
~ the cellular localisation of the antigen
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. 7-10~m sections will
be realized in a
cryostat chamber (-20, -30°C).
Sta- inin~
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
RT either a direct or indirect staining is performed using antigen specific
antibodies. A
3o 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.
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5.3 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
subtype). An HLA-A2.1/Kb transgenic mice 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 y-IFN production of
the CD8 lines
t o 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
15 delivery system or to be used to predict the sequence of HLA binding
peptides.
Peptide-based approach
The HLA-A2 binding peptide sequences are predicted by the Parker's algorithm.
Peptides
are then screened in the HLA-A2.1/Kb transgenic mice model (Vitiello et al.).
Briefly,
2o 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
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 stimulated CD8-sorted T cells (by Facs). After several weekly
stimulations,
25 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 tested on cDNA-transfected tumour cells (HLA-A2 transfected
LnCaP,
Skov3 or CAMA tumour cells).
3o Whole gene-based approach
CD8+ T cell lines will be primed and stimulated with either gene-gun
transfected
dendritic cells, retrovirally transduced B7.1-transfected fibroblastes,
recombinant pox
virus (Kim et al.) or adenovirus (Butterfield et al.) infected dentridic
cells. Virus infected
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cells are very effcient 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.
References
Vitiello et al. (L. Sherman), J. Exp. Med., J. Exp. Med, 1991, 173:1007-101.
to Romani et al., J. Exp. Med., 1994, 180:83-93.
Kim et al., J. Immunother., 1997, 20:276-286.
Butterfield et al., J. immunol., 1998, 161:5607-5613.
t 5 All publications, including but not limited to patents and patent
applications, cited in this
specification are herein incorporated by reference as if each individual
publication were
specifically and individually indicated to be incorporated by reference herein
as though
fully set forth.
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SEQUENCE LISTING
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CA 02323501 2000-09-18
WO 99/49030 PC'T/EP99/01892
<212> PRT
<213> homo sapiens
<400> 2
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1 5 10 15
Arg Pro Arg Glu Pro Pro Glu Gln Glu Leu Gln Arg Arg Arg Glu Gln
20 25 30
Lys Arg Arg Arg His Asp Ala Gln Gln Leu Gln Gln Leu Lys His Leu
35 40 45
Glu Ser Phe Tyr Glu Lys Pro Pro Pro Gly Leu Ile Lys Glu Asp Glu
50 55 60
Thr Lys Pro Glu Asp Cys Ile Pro Asp Val Pro Gly Asn Glu His Ala
65 70 75 80
Arg Glu Phe Leu Ala His Ala Pro Thr Lys Gly Leu Trp Met Pro Leu
85 90 95
Gly Lys Glu Val Lys Val Met Gln Cys Trp Arg Cys Lys Arg Tyr Gly
100 105 110
His Arg Thr Gly Asp Lys Glu Cys Pro Phe Phe Ile Lys Gly Asn Gln
20 115 120 125
Lys Leu Glu Gln Phe Arg Val Ala His Glu Asp Pro Met Tyr Asp Ile
130 135 140
Ile Arg Asp Asn Lys Arg His Glu Lys Asp Val Arg Ile Gln Gln Leu
145 150 155 160
25 Lys Gln Leu Leu Glu Asp Ser Thr Ser Asp Glu Asp Arg Ser Ser Ser
165 170 175
Ser Ser Ser Glu Gly Lys Glu Lys His Lys Lys Lys Lys Lys Lys Glu
180 185 190
Lys His Lys Lys Arg Lys Lys Glu Lys Lys Lys Lys Lys Lys Arg Lys
30 1g5 200 205
His Lys Ser Ser Lys Ser Asn Glu Gly Ser Asp Ser Glu
210 215 220
<210> 3
35 <211> 505
<212> DNA
<213> homo sapiens
<400> 3
40 gcggccgcgt gaccgccgga gcaggagctg cagcgacgtc gggagcagaa gcgcggcgac 60
acgaccgcag agctgcagca gctcaagcac ctggagtcct tttacgaaaa acctcetcct 120
gggcttatca aggaagatga gactaagcca gaagattgca taccagatgt accaggcaat 180
2
CA 02323501 2000-09-18
WO 99/49030 PCT/EP99/01892
gaacacgcca gggaatttct ggctcatgca ccaactaaag gactttggat gccactgggg 240
aaagaagtca aagttatgcagtgttggcgttgcaaacgctatggtcaccgaacgggtgac300
aaagaatgcc ctttctttatcaaaggcaaccaaaagttagagcagttcagagtggcacat360
gaagatccca tgtatgacatcatacgagacaataaacgacatgaaaaggacgtaaggata420
cagcagttaa aacagttactggaggattctacctcagatgaagataggagcatccagtcc480
ctgagtaggt cccgtcgtgccgaac 505
<210> 4
<211> 164
<212> PRT
<213> homo sapiens
<400> 4
Pro Pro Glu Gln Glu Leu Gln Arg Arg Arg Glu Gln Lys Arg Gly Asp
1 5 10 15
Thr Thr Ala Glu Leu Gln Gln Leu Lys His Leu Glu Ser Phe Tyr Glu
25 30
Lys Pro Pro Pro Gly Leu Ile Lys Giu Asp Glu Thr Lys Pro Glu Asp
35 40 45
20 ~,s Ile Pro Asp Val Pro Gly Asn Glu His Ala Arg Glu Phe Leu Ala
50 55 60
His Ala Pro Thr Lys Gly Leu Trp Met Pro Leu Gly Lys Glu Val Lys
65 70 75 80
Val Met Gln Cys Trp Arg Cys Lys Arg Tyr Gly His Arg Thr Gly Asp
85 90 95
Lys Glu Cys Pro Phe Phe Ile Lys Gly Asn Gln Lys Leu Glu Gln Phe
100 105 110
Arg Val Ala His Glu Asp Pro Met Tyr Asp Ile Ile Arg Asp Asn Lys
115 120 125
Arg His Glu Lys Asp Val Arg Ile Gln Gln Leu Lys Gln Leu Leu Glu
130 135 140
Asp Ser Thr Ser Asp Glu Asp Arg Ser Ile Gln Ser Leu Ser Arg Ser
145 150 155 160
Arg Arg Ala Glu
3
CA 02323501 2000-09-18
WO 99/49030 PCT/EP99/01892
SEQUENCE INFORMATION
SEQ ID NO:1
CCGAGCGGCGCTGCGGGGGCTGACatgtcgtcccggcctgggcgcgaggacgtgggggctgcgggcgc
gcggcggccgcgtgagccgccggagcaggagctgcagcgacgtcgggagcagaagcggcggcgacacgacgcgcagcag
c
tgcagcagctcaagcacctggagtccttttacgaaaaacctcctcctgggcttatcaaggaagatgagactaagccaga
agattgcat
accagatgtaccaggcaatgaacacgccagggaatttctggctcatgcaccaactaaaggactttggatgccactgggg
aaagaag
tcaaagttatgcagtgttggcgttgcaaacgctatggtcaccgaacgggtgacaaagaatgccctttctttatcaaagg
caaccaaaa
gttagagcagttcagagtggcacatgaagatcccatgtatgacatcatacgagacaataaacgacatgaaaaggacgta
aggataca
gcagttaaaacagttactggaggattctacctcagatgaagataggagcagctccagttcctctgaaggtaaagagaaa
cacaagaa
~g~~~g~g~~gg~g~ggg~gaaaa
aacggaagcacaaatcttccaagtcaaatgagggttctgactcagagtgaCAAGGATGTGACTTGTTCAACAT
TCTCTTCTCAAACACTGACCAAGGAACAGAGGAAGATGCAGTCAGAGAAAGCA
GCAGGATAGAGACGCCGAGAGAGGAGTATATGTGGGTCACAGCAGTGAGCTC
~5 CCACCCGCCTTGCAGTGAAGATGTGACCCCAGGAGAGGGAGTGTCTCCTTCCA
GGTGCTAGCTCTGGACAGCAGCTGATTTTAGGCAGGAAAGTTTCTTCATCGTTG
TCCTCCCTGCTGGTCACATGAGTT'~ ACGATTCCTTTGAAGTGTCTCCCACAGGG
TGGCAGGACTGGGAGAATCTCTGAGGCGTGTCTTCCAGGCCCTCCCACAGCTT
GTGCCCTCCACAGTGTGGACTCAGGTCCCATAGACATCAGGCTGGAGTCTTCTC
2o TGTTGT
SEQ ID N0:2
MSSRPGREDVGAAGARRPREPPEQELQRRREQK;RRRHDAQQLQQLKHLESFYEKP
PPGLIKEDETKPEDCIPDVPGNEHAREFLAHAPTKGLWMPLGKEVKVMQCWRCKR
25 yG~TGDKECPFFIKGNQKLEQFRVAHEDPMYDIIRDNKRHEKDVRIQQLKQLLE
DSTSDEDRSSSSSSEGKEKHI~KICKKKEKHKKRKKEKKKKI~KRKHKSSKSNEGSDS
E.
SEQ ID N0:3
GCGGCCGCGTGACCGCCGGAGCAGGAGCTGCAGCGACGTCGGGAGCAGAAG
3o CGCGGCGACACGACCGCAGAGCTGCAGCAGCTCAAGCACCTGGAGTCCTTTT
ACGAAAAACCTCCTCCTGGGCTTATCAAGGAAGATGAGACTAAGCCAGAAGA
TTGCATACCAGATGTACCAGGCAATGAACACGCCAGGGAATTTCTGGCTCAT
GCACCAACTAAAGGACTTTGGATGCCACTGGGGAAAGAAGTCAAAGTTATGC
AGTGTTGGCGTTGCAAACgCTATGGTCACCGAACGGGTGACAAAGAATGCCC
35 T~C~ATCAAAGGCAACCAAAAGTTAGAGCAGTTCAGAGTGGCACATGAA
GATCCCATGTATGACATCATACGAGACAATAAACGACATGAAAAGGACGTAA
GGATACAGCAGTTAAAACAGTTACTGGAGGATTCTACCTCAGATGAAGATAG
GAGCATCCAGTCCCTGAGTAGGTCCCGTCGTGCCGAAC
SEQ ID N0:4
PPEQELQRRREQKRGDTTAELQQLKHLESFYEKPPPGLIKEDETKPEDCIPDVPGN
EHAREFLAHAPTKGLWMPLGKEVKVMQCWRCKRYGHRTGDKECPFFIKGNQK
LEQFRVAHEDPMYDIIRDNKRHEKDVRIQQLKQLLEDSTSDEDRSIQSLSRSRRAE
