Note: Descriptions are shown in the official language in which they were submitted.
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VACCINE
The present invention relates to protein derivatives of a protein known as
prostase, a prostate-specific serine protease, to methods for their
purification and
manufacture, and also to pharmaceutical compositions containing such
derivatives,
and to their use in medicine. In particular such derivatives find utility in
cancer
vaccine therapy, particularly prostate cancer vaccine therapy and diagnostic
agents for
prostate tumours.
In particular the derivatives of the invention include fusion proteins
1o comprising prostase linked to an immunological or an expression enhancer
fusion
partner.
The present invention also provides methods for purifying the prostase
derivatives and for formulating vaccines for immunotherapeutically treating
prostate
cancer patients and prostase-expressing tumours other than prostate tumours,
prostatic
hyperplasia, and prostate intraepithelilial neoplasia (PIN).
Prostate cancer is the most common cancer among males, with an estimated
incidence of 30% in men over the age of S0. Overwhelming clinical evidence
shows
that human prostate cancer has the propency to metastasise to bone, and the
disease
appears to progress inevitably from androgen dependent to androgen refractory
status,
leading to increased patient mortality (Abbas F., Scardino P. "The Natural
History of
Clinical Prostate Carcinoma." In Cancer (1997); 80:827-833). This prevalent
disease
is currently the second leading cause of cancer death among men in the US.
Despite considerable research into therapies for the disease, prostate cancer
remains difficult to treat. Currently, treatment is based on surgery and/or
radiation
therapy, but these methods are ineffective in a significant percentage of
cases
(Frydenberg M., Stricker P., Kaye K. "Prostate Cancer Diagnosis and Management
"The Lancet (1997); 349:1681-1687). Several tumour-associated antigens are
already
known. Many of these antigens may be interesting targets for immunotherapy,
but are
either not fully tumour-specific or are closely related to normal proteins,
and hence
3o bear with them the risk of organ-specific auto-immunity, once targeted by a
potent
immune response. When an auto-immune response to non-crucial organs can be
tolerated, auto-immunity to heart, intestine and other crucial organs could
lead to
unacceptable safety profiles. Some previously identified prostate specific
proteins like
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prostate specific antigen (PSA) and prostatic acid phosphatase (PAP), prostate-
specific membrane antigen (PSMA) and prostate stem cell antigen (PSCA) have
limited therapeutic potential and moreover are not always correlated with the
presence
of prostate cancer or with the level of metastasis (Pound C., Partin A.,
Eisenberg M. et
al. "Natural History of Progression after PSA Elevation following Radical
Prostatectomy." In Jama (1999); 281:1591-1597) (Bostwick D., Pacelli A., Blute
M.
et al. "Prostate Specific Membrane Antigen Expression in Prostatic
Intraepithelial
Neoplasia and Adenocarcinoma." In Cancer ( 1998); 82:2256-2261 ).
The existence of tumour rejection mechanisms has been recognised since
1o several decades. Tumour antigens, though encoded by the genome of the
organism and thus theoretically not recognized by the immune system through
the
immune tolerance phenomenon, can occasionally induce immune responses
detectable in cancer patients. This is evidenced by antibodies or T cell
responses
to antigens expressed by the tumour (Xue BH., Zhang Y., Sosman J. et al.
"Induction of Human Cytotoxic T-Lymphocytes Specific for Prostate-Specific
Antigen." In Prostate (1997); 30(2):73-78). When relatively weak anti-tumour
effects can be observed through the administration of antibodies recognizing
cell
surface markers of tumour cells, induction of strong T cell responses to
antigens
expressed by tumour cells can lead to complete regression of established
tumours
in animal models (mainly murine).
It is now recognised that the expression of tumour antigens by a cell is not
sufficient for induction of an immune response to these antigens. Initiation
of a
tumour rejection response requires a series of immune amplification phenomena
dependent on the intervention of antigen presenting cells, responsible for
delivery
of a series of activation signals.
Prostase is a prostate-specific serine protease (trypsin-like), 254 amino acid-
long, with a conserved serine protease catalytic triad H-D-S and a amino-
terminal pre-
propeptide sequence, indicating a potential secretory function (P. Nelson, Lu
Gan, C.
Ferguson, P. Moss, R. Gelinas, L. Hood & K. Wand, "Molecular cloning and
3o characterisation of prostase, an androgen-regulated serine protease with
prostate
restricted expression, In Proc. Natl. Acad. Sci. USA ( 1999) 96, 3114-3119). A
putative glycosylation site has been described. The predicted structure is
very similar
to other known serine proteases, showing that the mature polypeptide folds
into a
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single domain. The mature protein is 224 amino acids-long, with one A2 epitope
shown to be naturally processed.
Prostase nucleotide sequence and deduced polypeptide sequence and
homologs are disclosed in Ferguson, et al. (Proc. Natl. Acad. Sci. USA 1999,
96,
3114-3119) and in International Patent Applications No. WO 98/12302 (and also
the
corresponding granted patent US 5,955,306), WO 98/20117 (and also the
corresponding granted patents US 5,840,871 and US 5,786,148) (prostate-
specific
kallikrein) and WO 00/04149 (P703P).
The present invention provides prostase protein fusions based on prostase
to protein and fragments and homologues thereof ("derivatives"). Such
derivatives are
suitable for use in therapeutic vaccine formulations which are suitable for
the
treatment of a prostate tumours.
Prostase fragments of the invention will be of at least about 10 consecutive
amino acids, preferably about 20, more preferably about 50, more preferably
about
15 100, more preferably about 150 contiguous amino acids selected from the
amino acid
sequences as shown in SEQ ID N°7 or SEQ ID N°8. More
particularly fragments will
retain some functional property, preferably a immunological activity, of the
larger
molecule set forth in SEQ ID N°7 or SEQ ID N°8, and are useful
in the methods
described herein (e.g. in vaccine compositions, in diagnostics, etc.). In
particular the
2o fragments will be able to generate an immune response, when suitable
attached to a
carrier, that will recognise the protein of SEQ ID N°7 or SEQ ID
N°8.
Prostase homologues will generally share substantial sequence similarity, and
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,
25 yet more preferably at least 95% identity, most preferably at least 97-99%
identity, to
that of SEQ ID N0:7 or SEQ ID N°8 over the entire length of SEQ ID N0:7
or SEQ
ID N°8. Such polypeptides include those comprising the amino acid of
SEQ ID N0:7
or SEQ ID N°8.
The prostase antigen derivative or fragments and homologues thereof may in a
3o preferred embodiment carry a mutation in the active site of the protein, to
reduce
substantially or preferably eliminate its protease biological activity.
Preferred
mutations involve replacing the Histidine and Aspartate catalytic residues of
the
serine protease. In a preferred embodiment, prostase contains a Histidine-
Alanine
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mutation at residue 71 of prostase sequence (Ferguson, et al. (Proc. Natl.
Acad. Sci.
USA 1999, 96, 3114-3119) which corresponds to amino acid 43 of P703PDE5
sequence (SEQ ID N°8). The mutated protein preferably has a significant
decrease in
the catalytic efficiency (expressed in enzymatic specific activity) of the
protein as
compared to the non-mutated one. Preferably the reduction in the catalytic
efficiency
is at least by a factor of 103, more preferably at least by a factor of 1 O6.
The protein
which has undergone a histidine alanine mutation is hereafrer referred to as *
(star).
In one embodiment, the present invention relates to fusions proteins,
comprising the tumour-associated prostase or fragment or homologues thereof
and a
1o heterologous protein or part of a protein acting as a fusion partner. The
protein and the
fusion partner may be chemically conjugated, but are preferably expressed as
recombinant fusion proteins in a heterologous expression system.
In a preferred embodiment of the invention there is provided a prostase fusion
protein or fragment or homologues thereof linked to an immunological fusion
partner
15 that may assist in providing T helper epitopes. Thus the fusion partner may
act
through a bystander helper effect linked to secretion of activation signals by
a large
number of T cells specific to the foreign protein or peptide, thereby
enhancing the
induction of immunity to the prostase component as compared to the non-fused
protein. Preferably the heterologous partner is selected to be recognizable by
T cells
2o in a majority of humans.
In another embodiment, the invention provides a prostase protein or fragment
or homologues thereof linked to a fusion partner that acts as an expression
enhancer.
Thus the fusion partner may assist in aiding in the expression of prostase in
a
heterologous system, allowing increased levels to be produced in an expression
25 system as compared to the native recombinant protein.
Preferably the fusion partner will be both an immunological fusion partner
and an expression enhancer partner. Accordingly, the present invention in the
embodiment provides fusion proteins comprising the tumour-specific prostase or
a
fragment thereof linked to a fusion partner. Preferably the fusion partner is
acting
3o both as an immunological fusion partner and as an expression enhancer
partner.
Accordingly, in a preferred form of the invention, the fusion partner is the
non-
structural protein from influenzae virus, NS 1 (hemagglutinin) or fragment
thereof.
Typically the N-terminal 81 amino acids are utilised, although different
fragments
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may be used provided they include T-helper epitopes (C. Hackett, D. Horowitz,
M.
Wysocka & S. Dillon, 1992, J. Gen. Virology, 73, 1339-1343). When NS1 is the
immunological fusion partner it has the additional advantage in that it allows
higher
expression yields to be achieved. In particular, such fusions are expressed at
higher
yields than the native recombinant prostase proteins.
In preferred embodiments, the prostase moiety within the fusion is selected
from the group comprising SEQ ID NO: 5 (Millenium WO 98/12302), SEQ ID NO: 6
(Incyte WO 98/20117), SEQ ID NO: 7 (PNAS (1999) 96, 3114-3119), and SEQ ID
NO: 8 (Corixa WO 00/04149, P703PDE5 sequence). Yet in a most preferred
1 o embodiment, the fusion protein comprises the N-terminal 81 amino acids of
NS 1 non
structural protein fused to the 5 to 226 carboxy-terminal amino acids from
mutated
prostase, as set forth in SEQ ID NO: 1 or SEQ ID N°3.
The proteins of the present invention are expressed in an appropriate host
cell,
and preferably in E. coli. In a preferred embodiment the proteins are
expressed with
an affinity tag, such as for example, a histidine tail comprising between S to
9 and
preferably six histidine residues, most preferably at least 4 histidine
residues. These
are advantageous in aiding purification through for example ion metal affinity
chromatography (IMAC).
The present invention also provides a nucleic acid encoding the proteins of
the
2o present invention. Such sequences can be inserted into a suitable
expression vector
and used for DNA/RNA vaccination or expressed in a suitable host. In
additional
embodiments, genetic constructs comprising one or more of the polynucleotides
of the
invention are introduced into cells in vivo. This may he achievPrl »cina anv
of a
variety or well-known approaches. One of the preferred methods for in vivo
delivery
of one or more nucleic acid sequences involves the use of an expression vector
such
as a recombinant live viral or bacterial microorganism. Suitable viral
expression
vectors are for example poxviruses (e.g; vaccinia, fowlpox, canarypox),
alphaviruses
(Sindbis virus, Semliki Forest Virus, Venezuelian Equine Encephalitis Virus),
adenoviruses, adeno-associated virus, picornaviruses (poliovirus, rhinovirus),
and
3o herpesviruses (varicella zoster virus, etc). Other preferred methods for in
vivo delivery
of one or more nucleic acid sequences involves the use of a bacterial
expression
vector, such as Listeria, Salmonella , Shigella and BCG. Inoculation and in
vivo
infection with this live vector will lead to in vivo expression of the antigen
and
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induction of immune responses. These viruses and bacteria can be virulent, or
attenuated in various ways in order to obtain live vaccines. Such live
vaccines also
form pan of the invention.
A DNA sequence encoding the proteins of the present invention can be
synthesized using standard DNA synthesis techniques, such as by enzymatic
ligation
as described by D.M. Roberts et al. in Biochemistry 1985, 24, 5090-5098, by
chemical synthesis, by in vitro enzymatic polymerization, or by PCR technology
utilising for example a heat stable polymerise, or by a combination of these
techniques. Enzymatic polymerisation of DNA may be carried out in vitro using
a
1 o DNA polymerise such as DNA polymerise I (Klenow fragment) in an
appropriate
buffer containing the nucleoside triphosphates dATP, dCTP, dGTP and dTTP as
required at a temperature of 10°-37°C, generally in a volume of
501 or less.
Enzymatic ligation of DNA fragments may be carried out using a DNA ligase such
as
T4 DNA ligase in an appropriate buffer, such as 0.05M Tris (pH 7.4), 0.01 M
MgCl2,
O.O1M dithiothreitol, 1mM spermidine, 1mM ATP and O.lmg/ml bovine serum
albumin, at a temperature of 4°C to ambient, generally in a volume of
50m1 or less.
The chemical synthesis of the DNA polymer or fragments may be carried out by
conventional phosphotriester, phosphite or phosphoramidite chemistry, using
solid
phase techniques such as those described in 'Chemical and Enzymatic Synthesis
of
2o Gene Fragments - A Laboratory Manual' (ed. H.G. Gassen and A. Lang), Verlag
Chemie, Weinheim (1982), or in other scientific publications, for example M.J.
Gait,
H.W.D. Matthes, M. Singh, B.S. Sproat, and R.C. Titmas, Nucleic Acids
Research,
1982, 10, 6243; B.S. Sproat, and W. Bannwarth, Tetrahedron Letters, 1983, 24,
5771;
M.D. Matteucci and M.H. Caruthers, Tetrahedron Letters, 1980, 21, 719; M.D.
Matteucci and M.H. Caruthers, Journal of the American Chemical Society, 1981,
103,
3185; S.P. Adams et al., loumal of the American Chemical Society, 1983, 105,
661;
N.D. Sinha, J. Biernat, J. McMannus, and H. Koester, Nucleic Acids Research,
1984,
12, 4539; and H.W.D. Matthes et al., EMBO Journal, 1984, 3, 801.
In a further embodiment of the invention is provided a method of producing a
3o protein as described herein. The process of the invention may be performed
by
conventional recombinant techniques such as described in Maniatis et al.,
Molecular
Cloning - A Laboratory Manual; Cold Spring Harbor, 1982-1989.
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In particular, the process of the invention may preferably comprise the steps
of:
i) preparing a replicable or integrating expression vector capable, in a
host cell, of expressing a DNA polymer comprising a nucleotide
sequence that encodes the protein or an immunogenic derivative
thereof;
ii) transforming a host cell with said vector;
ii) culturing said transformed host cell under conditions permitting
expression of said DNA polymer to produce said protein; and
iv) recovering said protein.
The term 'transforming' is used herein to mean the introduction of foreign
DNA into a host cell. This can be achieved for example by transformation,
transfection or infection with an appropriate plasmid or viral vector using
e.g.
conventional techniques as described in Genetic Engineering; Eds. S.M.
Kingsman
and A.J. Kingsman; Blackwell Scientific Publications; Oxford, England, 1988.
The
term 'transformed' or 'transformant' will hereafrer apply to the resulting
host cell
containing and expressing the foreign gene of interest. Preferably recombinant
antigens of the invention are expressed in unicellular hosts, most preferably
in
bacterial systems, most preferably in E. coli.
2o Preferably the recombinant strategy includes cloning a gene construct
encoding a NS 1 fusion protein, the gene construct comprising from 5' to 3' a
DNA
sequence encoding NSl joined to a DNA sequence encoding the protein of
interest,
mto an expression vector to form a DNA fragment encoding a NS 1- carboxyl-
terminal
P703P fusion protein. An affinity polyhistidine tail may be engineered at the
carboxy-
terminus of the fusion protein allowing for simplified purification through
affinity
chromatography.
The expression vectors are novel and also form part of the invention.
The replicable expression vectors may be prepared in accordance with the
invention, by cleaving a vector compatible with the host cell to provide a
linear DNA
3o segment having an intact replicon, and combining said linear segment with
one or
more DNA molecules which, together with said linear segment encode the desired
product, such as the DNA polymer encoding the protein of the invention, or
derivative
thereof, under ligating conditions.
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Thus, the hybrid DNA may be pre-formed or formed during the construction
of the vector, as desired.
The choice of vector will be determined in part by the host cell, which may be
prokaryotic or eukaryotic but are preferably E. coli, yeast or CHO cells.
Suitable
s vectors include plasmids, bacteriophages, cosmids and recombinant viruses.
Expression and cloning vectors preferably contain a selectable marker such
that only
the host cells expressing the marker will survive under selective conditions.
Selection
genes include but are not limited to the one encoding protein that confer a
resistance
to ampicillin, tetracyclin or kanamycin. Expression vectors also contain
control
sequences which are compatible with the designated host. For example,
expression
control sequences for E. coli, and more generally for prokaryotes, include
promoters
and ribosome binding sites. Promoter sequences may be naturally occurring,
such as
the ~3-lactamase (penicillinase) (Weissman 1981, In Interferon 3 (ed. L.
Gresser),
lactose (lac) (Chang et al. Nature, 1977, 198: 1056) and tryptophan (trp)
(Goeddel et
15 al. Nucl. Acids Res. 1980, 8, 4057) and lambda-derived PL promoter system.
In
addition, synthetic promoters which do not occur in nature also function as
bacterial
promoters. This is the case for example for the tac synthetic hybrid promoter
which is
derived from sequences of the trp and lac promoters (De Boer et al., Proc.
Natl Acad
Sci. USA 1983, 80, 21-26). These systems are particularly suitable with E.
coli.
2o Yeast compatible vectors also carry markers that allow the selection of
successful transformants by conferring prototrophy to auxotrophic mutants or
resistance to heavy metals on wild-type strains. Control sequences for yeast
vectors
include promoters for glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg.
1968, 7,
149), PHOS gene encoding acid phosphatase, CUP1 gene, ARG3 gene, GAL genes
'S promoters and synthetic promoter sequences.. Other control elements useful
in yeast
expression are terminators and leader sequences. The leader sequence is
particularly
useful since it typically encodes a signal peptide comprised of hydrophobic
amino
acids which direct the secretion of the protein from the cell. Suitable signal
sequences
can be encoded by genes for secreted yeast proteins such as the yeast
invertase gene
3o and the a-factor gene, acid phosphatase, killer toxin, the a-mating factor
gene and
recently the heterologous inulinase signal sequence derived from INDIA gene of
Kluyveromyces marxianus.. Suitable vectors have been developed for expression
in
Pichia pastoris and Saccharomvces cerevisiae.
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A variety of P. pastoris expression vectors are available based on various
inducible or constitutive promoters ( Cereghino and Cregg, FEMS Microbiol.
Rev.
2000,24:45-66). For the production of cytosolic and secreted proteins,the most
commonly used P. pastoris vectors contain the very strong and tightly
regulated
alcohol oxidase (AOX1) promoter. The vectors also contain the P. pastoris
histidinol
dehydrogenase (HIS4) gene for selection in his4 hosts. Secretion of foreign
protein
require the presence of a signal sequence and the S. cerevisiae prepro alpha
mating
factor leader sequence has been widly and successfully used in Pichia
expression
system. Expression vectors are integrated into the P. pastoris genome to
maximize the
1o stability of expression strains. As in S.cerevisiae, cleavage of a
P.pastoris expression
vector within a sequence shared by the host genome (AOX 1 or HIS4) stimulates
homologous recombination events that efficiently target integration of the
vector to
that genomic locus. In general, a recombinant strain that contains multiple
integrated
copies of an expression cassette can yield more heterologous protein than
single-copy
strain. The most effective way to obtain high copy number transformants
requires the
transformation of Pichia recipient strain by the sphaeroplast technique (Cregg
et all
1985, Mol.Cell.Biol. 5: 3376-3385) .
The preparation of the replicable expression vector may be carried out
conventionally with appropriate enzymes for restriction, polymerisation and
ligation
of the DNA, by procedures described in, for example, Maniatis et al. cited
above.
The recombinant host cell is prepared, in accordance with the invention, by
transforming a host cell with a replicable expression vector of the invention
under
transforming conditions. Suitable transforming conditions are conventional and
are
described in, for example, Maniatis et al. cited above, or "DNA Cloning" Vol.
II,
D.M. Glover ed., IRL Press Ltd, 1985.
The choice of transforming conditions depends upon the choice of the host cell
to be transformed. For example, in vivo transformation using a live viral
vector as the
transforming agent for the polynucleotides of the invention is described
above.
Bacterial transformation of a host such as E. coli may be done by direct
uptake of the
3o polynucleotides (which may be expression vectors containing the desired
sequence)
after the host has been treated with a solution of CaCh (Cohen et al., Proc.
Nat. Acad.
Sci., 1973, 69, 2110) or with a solution comprising a mixture of rubidium
chloride
(RbCI), MnCl2, potassium acetate and glycerol, and then with 3-[N-morpholino]-
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propane-sulphonic acid, RbC 1 and glycerol. Transformation of lower eukaryotic
organisms such as yeast cells in culture by direct uptake may be carried out
by using
the method of Hinnen et al (Proc. Natl. Acad. Sci. 1978, 75 : 1929-1933).
Mammalian
cells in culture may be transformed using the calcium phosphate co-
precipitation of
s the vector DNA onto the cells (Graham & Van der Eb, Virology 1978, 52, 546).
Other
methods for introduction of polynucleotides into mammalian cells include
dextran
mediated transfection, polybrene mediated transfection, protoplast fusion,
electroporation, encapsulation of the polynucleotide(s) into liposomes, and
direct
micro-injection of the polynucleotides into nuclei.
1o The invention also extends to a host cell transformed with a nucleic acid
encoding the protein of the invention or a replicable expression vector of the
invention.
Culturing the transformed host cell under conditions permitting expression of
the DNA polymer is carried out conventionally, as described in, for example,
1 s Maniatis et al. and "DNA Cloning" cited above. Thus, preferably the cell
is supplied
with nutrient and cultured at a temperature below 50°C, preferably
between 25°C and
35°C, most preferably at 30°C. The incubation time may vary from
a few minutes to a
few hours, according to the proportion of the polypeptide in the bacterial
cell, as
assessed by SDS-PAGE or Western blot.
20 The product may be recovered by conventional methods according to the host
cell and according to the localisation of the expression product (in~acellular
or
secreted into the culture medium or into the cell periplasm). Thus, where the
host cell
is bacterial, such as E. coli it may, for example, be lysed physically,
chemically or
enzymatically and the protein product isolated from the resulting lysate.
Where the
2s host cell is mammalian, the product may generally be isolated from the
nutrient
medium or from cell free extracts. Where the host cell is a yeast such as
Saccharomyces cerevisiae or Pichia pastoris, the product may generally be
isolated
from from lysed cells or from the culture medium, and then further purified
using
conventional techniques. The specificity of the expression system may be
assessed by
3o western blot using an antibody directed against the polypeptide of
interest.
Conventional protein isolation techniques include selective precipitation,
adsorption chromatography, and affinity chromatography including a monoclonal
antibody affinity column. When the proteins of the present invention are
expressed
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with a histidine tail (His tag), they can easily be purified by affinity
chromatography
using an ion metal affinity chromatography column (IMAC) column.
In a preferred embodiment of the invention the proteins of the present
invention is provided with an affinity tag, such as a polyhistidine tail. In
such cases
the protein after the blocking step is preferably subjected to affinity
chromatography.
For those proteins with a polyhistidine tail, immobilised metal ion affinity
chromatography (IMAC) may be performed. The metal ion, may be any suitable ion
for example zinc, nickel, iron, magnesium or copper, but is preferably zinc or
nickel.
Preferably the IMAC buffer contains detergent, preferably a non-ionic
detergent such
as Tween 80, or a zwitterionic detergent such as Empigen BB, as this may
result in
lower levels of endotoxin in the final product.
Further chromatographic steps include for example a Q-Sepharose step that
may be operated either before of after the IMAC column. Preferably the pH is
in the
range of 7.5 to 10, more preferably from 7.5 to 9.5, optimally between 8 and
9, ideally
is 8.5.
The proteins of the present invention are provided either soluble in a liquid
form or in a lyophilised form, which is the preferred form. It is generally
expected
that each human dose will comprise 1 to 1000 pg of protein, and preferably 30-
300
l~g~
2o The present invention also provides pharmaceutical composition comprising a
protein of the present invention in a pharmaceutically acceptable excipient.
A preferred vaccine composition comprises at least NS1-P703P* (SEQ ID
I~T°1) or
NS1-P703P (SEQ ID N°3). Said protein has, preferably, blocked thiol
groups and is
highly purified, e.g. has less than 5% host cell contamination. Such vaccine
may
25 optionally contain one or more other tumour-associated antigen and
derivatives. For
example, suitable other associated antigen include PAP-1, PSA (prostate
specific
antigen), PSMA (prostate-specific membrane antigen), PSCA (Prostate Stem Cell
Antigen), STEAP.
Vaccine preparation is generally described in Vaccine Design ("The subunit
3o and adjuvant approach" (eds. Powell M.F. & Newman M.J). (1995) Plenum Press
New Fork). Encapsulation within liposomes is described by Fullerton, US Patent
4,235,877.
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The proteins of the present invention are preferably adjuvanted in the vaccine
formulation of the invention. Suitable adjuvants are commercially available
such 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 derivatized polysaccharides; polyphosphazenes; biodegradable
microspheres; monophosphoryl lipid A and quit A. Cytokines, such as GM-CSF or
t0 interleukin-2, -7, or -12, may also be used as adjuvants.
In the formulations of the invention it is preferred that the adjuvant
composition induces an immune response predominantly of the THl type. High
levels
of Thl-type cvtokines (e.g., IFN-y, TNFa, IL-2 and IL-12) tend to favour the
induction of cell mediated immune responses to an administered antigen. 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 Coffman,
Ann.
Rev. Immunol. 7:145-173, 1989.
Accordingly, suitable adjuvants for use in eliciting a predominantly Thl-type
response include, for example a combination of monophosphoryl lipid A,
preferably
3-de-O-acylated monophosphoryl lipid A (3D-MPL) together with an aluminium
salt.
Other known adjuvants which preferentially induce a THl type immune response
include CpG containing oligonucleotides. The oligonucleotides are
characterised in
that the CpG dinucleotide is unmethylated. Such oligonucleotides are well
known
and are described in, for example WO 96/02555. Immunostimulatory DNA sequences
are also described, for example, by Sato et al., Science 273:352, 1996.
Another
preferred adjuvant is a saponin, preferably QS21 (Aquila Biopharmaceuticals
Inc.,
Framingham, MA), which may be used alone or in combination with other
adjuvants.
For example, an enhanced system involves the combination of a monophosphoryl
lipid A and saponin derivative, such as the combination of QS21 and 3D-MPL 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
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formulations comprise an oil-in-water emulsion and tocopherol. A particularly
potent
adjuvant formulation involving QS21, 3D-MPL and tocopherol in an oil-in-water
emulsion is described in WO 95/17210.
A particularly potent adjuvant formulation involving QS21 3D-MPL &
tocopherol in an oil in water emulsion is described in WO 95/17210 and is a
preferred
formulation.
Other preferred adjuvants include Montanide ISA 720 (Seppic, France), SAF
(Chiron, California, United States), ISCOMS (CSL), MF-59 (Chiron), Detox
(Ribi,
Hamilton, MT), RC-529 (Corixa, Hamilton, MT) and other aminoalkyl
glucosaminide
l0 4-phosphates (AGPs).
Other preferred adjuvants include adjuvant molecules of the general formula
(I):
HO(CHZCHZO)~-A-R
Wherein, n is 1-50, A is a bond or -C(O)-, R is C,_SO alkyl or Phenyl C,.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 C,_SO,
preferably C4-
CZO alkyl and most preferably C,2 alkyl, and A is a bond. The concentration of
the
polyoxyethylene ethers 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, poIyoxyethylene-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 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
3o combination is preferably with CpG as described in the pending UK patent
application GB 9820956.2.
Accordingly in one embodiment of the present invention there is provided a
vaccine comprising a protein of the present invention, more preferably a NS 1-
P703P*
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adjuvanted with a monophosphoryl lipid A or derivative thereof, QS21 and
tocopherol
in an oil in water emulsion.
Preferably the vaccine additionally comprises a saponin, more preferably
QS21. Another particular suitable adjuvant formulation including CpG and a
saponin
is described in WO 00/09159 and is a preferred formulation. Most preferably
the
saponin in that particular formulation is QS21. Preferably the formulation
additionally
comprises an oil in water emulsion and tocopherol.
Any of a variety of delivery vehicles may be employed within pharmaceutical
compositions and vaccines to facilitate production of an antigen-specific
immune
to response that targets tumour cells. Delivery vehicles include antigen-
presenting cells
(APCs), 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-tumour effects per se
and/or to
be immunologically compatible with the receiver (i.e., matched HLA haplotype).
APCs may generally be isolated from any of a variety of biological fluids and
organs,
including tumour and peri-tumoural tissues, and may be autologous, allogeneic,
syngeneic or xenogeneic cells.
Certain preferred embodiments of the present invention use dendritic cells or
2o progenitors 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
antitumour 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 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
3o present invention. As an alternative to 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).
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Dendritic cells and progenitors may be obtained from peripheral blood, bone
marrow, tumour-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,
lipopolysaccharide LPS, flt3 ligand and/or other compounds) that induce
t0 differentiation, maturation and proliferation of dendritic 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 4-1BB).
APCs may generally be transfected with a polynucleotide encoding prostase
tumour protein (or derivative thereof) such that the prostase tumour
polypeptide, or an
immunogenic portion thereof, is expressed on the cell surface. Such
transfection may
take place ex vivo, and a composition or vaccine 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 prostase tumour polvpeptide, DNA (naked or
within
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a plasmid vector) or RNA; or with antigen-expressing recombinant bacterium or
viruses (e.g., vaccinia, fowlpox, adenovirus or lentivirus vectors).
Vaccines and pharmaceutical compositions may be presented in unit-dose or
multi-dose containers, such as sealed ampoules or vials. Such containers are
preferably hermetically sealed to preserve sterility of the formulation until
use. In
general, formulations may be stored as suspensions, solutions or emulsions in
oily or
aqueous vehicles. Alternatively, a vaccine or pharmaceutical composition may
be
stored in a freeze-dried condition requiring only the addition of a sterile
liquid carrier
immediately prior to use.
The present invention also provides a method for producing a vaccine
formulation comprising mixing a protein of the present invention together with
a
pharmaceutically acceptable excipient, such as 3D-MPL.
Another aspect of the invention is the use of a protein or nucleic acid as
claimed herein for the manufacture of a vaccine for immunotherapeutically
treating a
patient suffering from prostate cancer or other prostase-associated tumours.
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FIGURES LEGENDS
Fi re 1: Design of the fusion protein NS1- p703* -His expressed in E. coli
Fi re 2: Primary structure of the fusion protein NS1- p703* -His expressed in
E. coli
(SEQ ID N° 1 ).
Fi re 3: Coding sequence of NS 1,_8,-P703P*-His (SEQ ID N°2).
Fi re 4: Cloning strategy to produce NS1-P703P*-His in E. coli
Fi re 5: Plasmid map of RIT 14952
Fi re 6: E. coli ITS 1-P703P*-His fermentation process
Fi a 7: E. coli NS 1-P703P*-His purification process
Fi re 8: Immunogenicity of NS 1-P703P* adjuvanted with SBAS2
Fi re 9: Primary structure of the fusion protein NS 1- p703 -His expressed in
E. coli
(SEQ ID N°3).
Figure 10: Coding sequence ofNSl,_8,-P703P-His (SEQ ID N°4).
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The invention will be further described by reference to the following
examples:
EXAMPLE I:
Preparation of the recombinant E. coli strain expressing the fusion protein
NS1-
P7I1'iP*_'i_u;c
1. - Protein design
The expression strategy followed for this candidate included the design of the
most appropriate primary structure for the recombinant protein that could have
the
best expectation for both, good level of expression and easy purification
process.
Although the chance that a recombinant protein could keep its protease
biological activity when formulated for vaccination is really very low, the
mutation of
the active side was made in order to reduce substantially or preferably
eliminate its
proteolytical biological activity. Accordingly, the His residue at position 43
of SEQ
ID N° 8, has been mutated into an Ala residue.
The design of the fusion protein NS 1- p703 * -His to be expressed in E. coli
is
2o described in figure 1. This fusion contains the N-terminal (81 amino acid)
of non
structural protein of Influenzae virus, followed by the non processed amino
acid
sequence of prostate antigen (amino acids 5-X226 of p703pde5 sequence
described in
SEQ ID N°8 containing the mutation His-~Ala of the 43 residue of the
protease active
site followed by the His tail. The Histidine tail was added to prostase to
enable
versatile purification of the fusion and processed protein. The length of the
fusion is
313 aminoacids.
The primary structure of the resulting protein has the sequence described in
figure 2. The coding sequence corresponding to the above protein is
illustrated in
figure 3 and was subsequently placed under the control of ~.pL promoter in a
E. coli
3o expression plasmid.
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2. - The E. Coli expression system
For the production of NS 1 the DNA encoding the 81 amino-terminal residues
of NS 1 (non-structural protein from influenzae virus) has been cloned into
the
expression vector pMG 81. This plasmid utilises signals from lambda phage DNA
to
drive the transcription and translation of inserted foreign genes. The vector
contains
the lambda PL promoter PL, operator OL and two utilisation sites (NutL and
NutR) to
relieve transcriptional polarity effects when N protein is provided (Gross et
al., 1985.
Mol. & Cell. Biol. 5:1015). Vectors containing the PL promoter, are introduced
into
an E. coli lysogenic host to stabilise the plasmid DNA. Lysogenic host strains
contain
replication-defective lambda phage DNA integrated into the genome (Shatzman et
al.,
1983; In Experimental Manipulation of Gene Expression. Inouya (ed) pp 1-14.
Academic Press I~~. The lambda phage DNA directs the synthesis of the cI
repressor
protein which binds to the OL repressor of the vector and prevents binding of
RNA
polymerase to the PL promoter and thereby transcription of the inserted gene.
The cI
gene of the expression strain AR58 contains a temperature sensitive mutation
so that
PL directed transcription can be regulated by temperature shift, i.e. an
increase in
culture temperature inactivates the repressor and synthesis of the foreign
protein is
initiated. This expression system allows controlled synthesis of foreign
proteins
2o especially of those that may be toxic to the cell (Shimataka & Rosenberg,
1981.
Nature 292:128).
3. - The E. Coli strain AR58:
The AR58 lysogenic E. coli strain used for the production of the NS1-P703P*-
His protein is a derivative of the standard NIH E.coli K12 strain N99 (F- su-
galK2,
lacZ- thr-). It contains a defective lysogenic lambda phage (galE::TN10, I Kil-
cI857
DH 1 ). The Kil- phenotype prevents the shut off of host macromolecular
synthesis.
The cI857 mutation confers a temperature sensitive lesion to the cI repressor.
The
3o DH 1 deletion removes the lambda phage right operon and the hosts bio,
uvr3, and
chlA loci. The AR58 strain was generated by transduction of N99 with a P
lambda
phage stock previously grown on an SA500 derivative (galE::TN10, 1 Kil- cI857
DH 1 ). The introduction of the defective lysogen into N99 was selected with
tetracycline by virtue of the presence of a TN10 transposon coding for
tetracyclin
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resistance in the adjacent galE gene. N99 and SA500 are E.coli K12 strains
derived
from Dr. Martin Rosenberg's laboratory at the National Institutes of Health.
4. - Construction of the vector designed to express the recombinant protein
NS1-
P703P*-His
The starting materials were:
1) A cDNA plasmid received from CORIX.A p703pde5 (WO 00/04149),
where the putative signal sequence and a piece of the pro-peptide of P703P is
missing
to (see fig 1) and containing the coding sequence for prostase antigen;
2) Vector pRIT14901 containing the long version of the PL promoter; and
3) Plasmid PMG81 containing the 8laa of NS, coding region from Influenzae
virus.
The cloning strategy outlined in figure 4 included the following steps:
a) PCR amplification of the p703 sequence with NcoI and SpeI restriction sites
The template for the PCR reaction was the cDNA plasmid received from
CORIXA, the oligonucleotide sense can139 : 5'GCG CCC ATG GTT GGG
GAG GAC TGC AGC CCG 3', and the oligonucleotide antisense can 134
5'GGG ACT AGT ACT GGC CTG GAC GGT TTT CTC 3';
b) Insertion of the amplified sequences into the commercial vector Litmus 28
(biolabs), leading to the intermediate plasmid pRIT 14949;
c) Directed His -j Ala mutagenesis of residue 43 of the p703 sequence
contained in
the plasmid pRIT 14949, using the sense oligonucleotide can140 : 5'CTG TCA
GCC GCA GCG TGT TTC CAG 3'and the antisense oligonucleotide can141 : 5'
CTG GAA ACA CGC TGC GGC TGA CAG 3', leading to the obtention of
plasmid pRIT 14950;
d) Isolation of the NcoI - SpeI fragment from the plasmid pRIT 14950;
3o e) From pMG81 plasmid, purification of NS 1 fragment (8laa) afrer digestion
of the
restriction sites BamHI - NcoI;
f) Ligation of both fragments were ligated to the expression plasmid pRIT
14901
(pr PL long);
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g) Selection and characterisation of the E. coli AR58 strain transformants
containing
the plasmid pRIT14952 (see figure 5) expressing the NS1- p703 mutated -His
fusion protein
The recombinant strain thus produces the NS1-P703P* His-tailed fusion
protein of 313 amino acid residues long (see Figure 2), with the amino acids
sequence
described in ID No 1 and the coding sequence is described in ID No2.
1 o EXAMPLE II:
Preparation of the recombinant NS1-P703P*-3-His fusion protein
1. - Growth and induction of bacterial strain B1225 - Expression of NS1-P703P*-
3-His
Cells of AR58 transformed with plasmid pRIT14952 (strain B1225) were
grown in a 2 L flask containing 400 ml FEC015AA medium supplemented with
kanamycin sulphate (100mg/L). After a 16h of incubation at 30°C and at
200 rpm, a
2o small sample was removed from this flask for microscopic examination.
50 ml of this pre-culture was transferred into a 20-L fermentor containing 8.7
L
of FEC012AF medium supplemented with kanamycin sulphate (50mg/L). The pH
was adjusted to and maintained at 6.8 by addition of NH40H (25 % v/v), and the
temperature was maintained at 30°C. The aeration rate was kept constant
at 20 Llmin
and the p02 was regulated at 20% of saturation by feedback control of the
agitation
speed. The head pressure was maintained at 0.5 bar.
This fed-batch fermentation process is based on glycerol as a carbon source.
The feed solution was added at an initial rate of 0.04m1/min, and increased
exponentially during the first 30 hours to limit the growth rate in order to
be able to
keep a minimum p02 level of 20%.
After 30 hours, the temperature of the fermentor was rapidly increased to
39.5°C in order to induce the intracellular expression of the antigen
NS 1-P703P*-His.
The feeding rate was maintained constant at 1.28 ml/min during the whole
induction
phase ( 18h).
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Samples of broth were taken during both growth and induction phases in order
to monitor bacterial growth and antigen expression. Microbiological
identification and
purity tests were also realised on these materials.
At the end of fermentation, the biomass reached an optical density of about
130,
corresponding to a dry cell weight of about 50g/L. The final volume was
approximately 10.5 L. The cells containing the antigen were directly separated
from
the culture medium by centrifugation at 5000g for 1 h at 4°C and the
pellet was stored
in plastic bags at -70°C.
to 2. - Extraction of the protein:
Recombinant NS1-P703P*-His protein, expressed in E. coli as inclusion
bodies, was purified from cell homogenate using different steps (see figure
6).
Briefly, frozen concentrated cells from fermentation harvest were thawed to
+4°C
before being resuspended in disruption buffer (phosphate 20 mM - NaCI 2M -
EDTA
5 mM pH 7.5) to a final optical density (0D650) of 120. Two passes through a
high
pressure homogeniser ( 1000 bars) disrupted the cells.
EXAMPLE III:
Purification of fusion Protein NS1-P703P*-His
a) Introduction
As said above, the recombinant protein, NS 1-P703P*-His is produced in E.
coli in the form of inclusion bodies. A major issue for the set-up of the
purification
method was the oxidation of the recombinant protein with itself or with host
cell
contaminants, likely through covalent binding with disulphide bonds. The
process as
developed aimed at reducing the massive oxidation phenomenon in order to have
a
highly purified product together with an acceptable global yield, while
preserving the
3o product ability to mount an effective immune response against the antigen
of interest.
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a) Description of the process
The broken cell suspension was treated on a Pallsep VMF (Vibrating
Membrane Filtration) system (Pall-Filtron) equipped with 0.45 um membrane. The
"pellet fraction" was first washed by diafiltration with 20 mM phosphate
buffer pH
7.5 containing 0.5% Empigen BB detergent. The washed material was then
solubilised in the same buffer containing 4M guanidine hydrochoride and 20 mM
glutathion. The product was recovered through 0.45 qm filter and the permeate
was
treated with 200 mM iodoacetamide to prevent oxidative re-coupling.
1o The carboxyamidated fraction was subjected to IMAC (Nickel-Chelating-
Sepharose FF, Pharmacia). The column was first equilibrated with 20 mM Tris
buffer
pH 7.5 containing 4M urea, 0.5% Tween 80, and 20 mM imidazole. After the
sample
loading, the column was washed with the same buffer. The protein was then
eluted in
the previous buffer with 400 mM Imidazole.
Before continuing the anion exchange chromatography, the conductance of the
IMAC-eluate was reduced to below 5 mS/cm with 20 mM Tris buffer pH 8.5
containing 4M urea and 0.5-1.0 % Tween 80. The packed bed support (Q-Sepharose
FF, Pharmacia) was equilibrated with the dilution buffer. After the sample
loading
and a washing step with the equilibration buffer, the protein was eluted with
the same
2o buffer containing 250 mM NaCI.
The Q-Sepharose FF-eluate was then diafiltered against the appropriate
storage buffer (20 mM Tris buffer pH 8.0) in a tangential flow filtration unit
equipped
with a 10 Kd cut-off membrane (Omega, Filton).
Ultrafiltration retentate containing NS1-P703P*-His was sterile filtered
through 0.22 qm membrane.
The global purification yield was very high: around 3-4 g of purified material
/
L of homogenate (D0120).
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EXAMPLE IV:
Vaccine preparation using NS1-P703P*-His protein
1. - Vaccine preparation using NS1-P703P*-His protein:
The vaccine used in these experiments is produced from a recombinant DNA,
encoding a NS1-P703P*-His, expressed in E. coli from the strain AR58, either
adjuvanted or not. As an adjuvant, the formulation comprises a mixture of 3 de
-O-
to acylated monophosphoryl lipid A (3D-MPL) and QS21 in an oil/water emulsion.
The
adjuvant system SBAS2 has been previously described WO 95/17210.
3D-MPL: is an immunostimulant derived from the lipopolysaccharide (LPS)
of the Gram-negative bacterium Salmonella minnesota. MPL has been deacylated
and
is lacking a phosphate group on the lipid A moiety. This chemical treatment
dramatically reduces toxicity while preserving the immunostimulant properties
(Ribi,
1986). Ribi Immunochemistry produces and supplies MPL to SB-Biologicals.
Experiments performed at Smith Kline Beecham Biologicals have shown that
3D-MPL combined with various vehicles strongly enhances both the humoral and a
2o TH 1 type of cellular immunity.
QS21: is a natural saponin molecule extracted from the bark of the South
American tree Quillaja saponaria Molina. A purification technique developed to
separate the individual saponines from the crude extracts of the bark,
permitted the
isolation of the particular saponin, QS21, which is a triterpene glycoside
demonstrating stronger adjuvant activity and lower toxicity as compared with
the
parent component. QS21 has been shown to activate MHC class I restricted CTLs
to
several subunit Ags, as well as to stimulate Ag specific lymphocytic
proliferation
(Kensil, 1992). Aquila (formally Cambridge Biotech Corporation) produces and
3o supplies QS21 to SB-Biologicals.
Experiments performed at SmithKline Beecham Biologicals have
demonstrated a clear synergistic effect of combinations of MPL and QS21 in the
induction of both humoral and TH 1 type cellular immune responses.
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The oiUwater emulsion is composed an organic phase made of of 2 oils
(a tocopherol and squalene), and an aqueous phase of PBS containing Tween 80
as
emulsifier. The emulsion comprised 5% squalene 5% tocopherol 0.4% Tween 80 and
had an average particle size of 180 nm and is known as SB62 (see WO 95/17210).
Experiments performed at SmithKline Beecham Biologicals have proven that
the adjunction of this O/W emulsion to 3D-MPL/QS21 (SBAS2) further increases
the
immunostimulant properties of the latter against various subunit antigens.
to 2. - Preparation of emulsion SB62 (2 fold concentrate):
Tween 80 is dissolved in phosphate buffered saline (PBS) to give a 2%
solution in the PBS. To provide 100 ml two fold concentrate emulsion Sg of DL
alpha tocopherol and Sml of squalene are vortexed to mix thoroughly. 90m1 of
PBS/Tween solution is added and mixed thoroughly. The resulting emulsion is
then
passed through a syringe and finally microfluidised by using an M110S
microfluidics
machine. The resulting oil droplets have a size of approximately 180 nm.
3. - Lyophilisation of NS1-P703P*-His:
In practice, all compounds are placed in solution and sterilisation is
achieved
by filtration on a 0.2pm membrane. Formulations were performed the day of
freeze-
drying.
The sequence of formulation was:
HZO + sucrose 15.75% + Tris 100 mM pH8 + Tween80 10%- 5 min + NS 1-P703p
The volumes of all compounds are adjusted to have in final:
250-SO-10 pg NS 1-P703P*-His, in Tris 10 mM, tween 80 0.2%, 3.15% sucrose.
The vial was overfilled with by1.25 x (reconstitution with 625 p1 diluant,
injection of
SOOpI).
Using the Lyovac GT6 lyophilisation apparatus purchased from Steris (Germany),
the
lyophilisation cycle was performed during 3 days as follows:
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+35
-28°C l2 h
6h
-32°C 6 h
4h
-52°C h 30 h
4. - Preparation of NS1-P703P*-His QS21/3D MPL oil in water (SBAS2)
formulation:
The adjuvant is formulated as a combination of MPL and QS21, in an
oil/water emulsion.
l0 1) Formulation composition (injection volume: 100p1); group 1 has received
P703P*-His (20 pg) formulated in a combination of MPL and QS21, in an
oil/water
emulsion. Group 2 has received NS1-P703P*-His (25 p.g) in a combination of MPL
and QS21, in an oil/water emulsion.
2) Components
Components Conc mg/ml Buffer
P703p-His 0.513 Po4 20mM pH 7.5
NS1-P703p-his carboxy0.846 Tris 20 mM Tween 80 0.2%
pH7.5
SB62 2 x PBS pH 6.8
MPL 8.175 H24
QS21 2 H2p
Thiomersal 0.2 H2p
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3) Formulations
The formulations were prepared extemporaneously on the day of injection.
The formulations containing 3D-MPL and QS21 in an oil/water emulsion
(SBAS2B formulations - Groups 2 and 3) were performed as follows: P703p (20pg)
(group 2) and NS1-P703P*-His (25~g) (group 3) were diluted in 10-fold
concentrated
PBS pH 6.8 and H20 before consecutive addition of SB62 (SOpI), MPL (20p.g),
QS21
(20ug) and 1 pg/ml thiomersal as preservative at S min intervals. All
incubations
were carried out at room temperature with agitation.
The non-adjuvanted formulations (Groups 4 and 5) were performed as follows:
P703p (20pg) (group 4) and NS1-P703P*-His (25ug) (group 5) were diluted in 1.5
M
NaCI and HZO before addition of 1 pg/ml thiomersal as preservative at 5 min
intervals. All incubations were carried out at room temperature with
agitation.
The final vaccine is obtained after reconstitution of the lyophilised NS 1-
P703P*-His preparation with the adjuvant or with PBS alone.
The adjuvant controls without antigen were prepared by replacing the protein
by
PBS.
2o EXAMPLE V:
Immuno~enicity using NS1-P703P*-His protein
1. - Immunogenicity of NS1-P703P*-His in mice
The aim of the experiment was to characterise the immune response induced
in mice by vaccination with the purified recombinant mutated NS1-p703*-His
molecule produced in E coli, in the presence or the absence of an adjuvant.
a) - Immunization protocol:
Groups of 10 immunocompetent Balb/c mice, 6 to 8 weeks old mice, were
vaccinated twice, intramuscularly, at 2 weeks interval with 25 ug of mutated
NS1-
P703 formulated or not in SB AS02B (50p1 SB62 / lOpg MPL / lOpg QS21).
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14 days after the second injection, blood was taken and the sera were tested
for the
presence of anti-P703 antibodies.
b) - Total IeG Antibody response:
The anti P703 antibody response has been assessed in the sera of the mice
14 days after the latest vaccination. This has been done by ELISA using NS1-
P703P* as coating antigen.
E coli extracts were used to check for the possible presence of antibodies
against
host contaminants.
c - Results:
The results show that 1) a higher immune response (IgGl) is induced by NS1-
P703P* as evidenced in the sera of mice injected with the NS 1-P703P* protein
alone
as compared to the sera of normal control mice; 2) high antibody titers are
found in
animals receiving the NS 1-P703P* molecule formulated in the AS02B adjuvant.
The isotypic profile of the NS1-p703p specific IgG response has also been
measured. As shown in Figure 8, IgG 1 were detected when mice received the NS
1-
P703P* protein alone, however the isotypic profile was pushed towards a TH1
response (more IgG2a) by the presence of the AS02 adjuvant.
EXAMPLE VI:
1. - NS1-P703P-His
In an analogous fashion NS 1-P703P-His was prepared. The amino acid and
DNA sequences are depicted in SEQUENCE ID Nos. 3 and 4
Briefly, the strategy to express a NS1-P703P-His fusion protein in E.coli
included the following steps:
a) As a starting materiel, the same starting material as described in Example
I
(amino acids 5226 of p703pde5 sequence described in SEQ ID N°8);
b) PCR amplification to flank the p703 unmutated sequence cloning restriction
saes;
c) Insertion in a PMG81 vector (promoter pL long) containing the NS 1 gene;
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d) Transformation of the recipient strain AR58 or AR120
e) Selection of recombinant strain.
The resulting protein can be purified in an analogous manner to the NS 1-
P703P*-His mutated protein. The primary structure of the resulting protein has
the
sequence described in figure 9. The coding sequence corresponding to the above
protein is illustrated in figure 10.
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REFERENCES:
- Abbas F., Scardino P. "The Natural History of Clinical Prostate Carcinoma."
Cancer 80:827-833 (1997)
- Bostwick D., Pacelli A., Blute M. et al. "Prostate Specific Membrane Antigen
"Expression in Prostatic Intraepithelial Neoplasia and Adenocarcinoma." Cancer
82:2256-2261 (1998)
- Frydenberg M., Stricker P., Kaye K. "Prostate Cancer Diagnosis and
1o Management." The Lancet 349:1681-1687 (1997)
- C. Hackett, D. Horowitz, M. Wysocka & S. Dillon, J. Gen. Virology, 73, 1339-
1343 ( 1992)
- Kensil C.R., Soltysik S., Patel U., et al. in: Channock R.M. , Ginsburg
H.S.,
Brown F., et al., (eds.), Vaccines 92, (Cold Spring Harbor Laboratory Press,
Cold
Spring Harbor, N.Y.), 36-40: (1992)
- Nelson P., Lu Gan, Ferguson C., Moss P., Gelinas R., Hood L. & Wand K.,
"Molecular cloning and characterisation of prostase, an androgen-regulated
serin
protease with prostate restricted expression", Proc. Natl. Acad. Sci. USA 96,
3114-3119 ( 1999)
2o - Pound C., Partin A., Eisenberg M. et al. "Natural History of Progression
after PSA
Elevation following Radical Prostatectomy." Jama 281:1591-1597 (1999)
- Ribi E., et al. in: Levine L., Bonventre P.F., Morello J., et al. (eds).,
American
Society for Microbiology, Washington DC, Microbiology 1986, 9-13; (1986)
- Xue BH., Zhang Y., Sosman J. et al. "Induction of Human Cytotoxic T-
Lymphocytes Specific for Prostate-Specific Antigen." Prostate 30(2):73-78
(1997)
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SEQUENCE LISTING
<110> Cabezon Teresa
Dillon Davin
<120> Prostase fusion
<130> B45222
<160> 8
<170> FastSEQ for Windows Version 3.0
<210> 1
<211> 312
<212> PRT
<213> Human
<400> 1
Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp
1 5 10 15
His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe
20 25 30
Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser
35 40 45
Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile
50 55 60
Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr
65 70 75 80
Met Val Gly Glu Asp Cys Ser Pro His Ser Gln Pro Trp Gln Ala Ala
85 90 95
Leu Val Met Glu Asn Glu Leu Phe Cys Ser Gly Val Leu Val His Pro
100 105 110
Gln Trp Val Leu Ser Ala Ala Ala Cys Phe Gln Asn Ser Tyr Thr Ile
115 120 125
Gly Leu Gly Leu His Ser Leu Glu Ala Asp Gln Glu Pro Gly Ser Gln
130 135 140
Met Val Glu Ala Ser Leu Ser Val Arg His Pro Glu Tyr Asn Arg Pro
145 150 155 160
Leu Leu Ala Asn Asp Leu Met Leu Ile Lys Leu Asp Glu Ser Val Ser
165 170 175
Glu Ser Asp Thr Ile Arg Ser Ile Ser Ile Ala Ser Gln Cys Pro Thr
180 185 190
Ala Gly Asn Ser Cys Leu Val Ser Gly Trp Gly Leu Leu Ala Asn Gly
195 200 205
Arg Met Pro Thr Val Leu Gln Cys Val Asn Val Ser Val Val Ser Glu
210 215 220
Glu Val Cys Ser Lys Leu Tyr Asp Pro Leu Tyr His Pro Ser Met Phe
225 230 235 240
1
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Cys Ala Gly Gly Gly Gln Asp Gln Lys Asp Ser Cys Asn Gly Asp Ser
245 250 255
Gly Gly Pro Leu Ile Cys Asn Gly Tyr Leu Gln Gly Leu Val Ser Phe
260 265 270
Gly Lys Ala Pro Cys Gly Gln Val Gly Val Pro Gly Val Tyr Thr Asn
275 280 285
Leu Cys Lys Phe Thr Glu Trp Ile Glu Lys Thr Val Gln Ala Ser Thr
290 295 300
Ser Gly His His His His His His
305 310
<210> 2
<211> 939
<212> DNA
<213> Human
<400> 2
atggatccaa acactgtgtc aagctttcag gtagattgct ttctttggca tgtccgcaaa
cgagttgcag accaagaact aggtgatgcc ccattccttg atcggcttcg ccgagatcag
120
aaatccctaa gaggaagggg cagcaccctc ggtctggaca tcgagacagc cacacgtgct
180
ggaaagcaga tagtggagcg gattctgaaa gaagaatccg atgaggcact taaaatgacc
240
atggttgggg aggactgcag cccgcactcg cagccctggc aggcggcact ggtcatggaa
300
aacgaattgt tctgctcggg cgtcctggtg catccgcagt gggtgctgtc agccgcagcg
360
tgtttccaga actcctacac catcgggctg ggcctgcaca gtcttgaggc cgaccaagag
420
ccagggagcc agatggtgga ggccagcctc tccgtacggc acccagagta caacagaccc
480
ttgctcgcta acgacctcat gctcatcaag ttggacgaat ccgtgtccga gtctgacacc
540
atccggagca tcagcattgc ttcgcagtgc cctaccgcgg ggaactcttg cctcgtttct
600
ggctggggtc tgctggcgaa cggcagaatg cctaccgtgc tgcagtgcgt gaacgtgtcg
660
gtggtgtctg aggaggtctg cagtaagctc tatgacccgc tgtaccaccc cagcatgttc
720
tgcgccggcg gagggcaaga ccagaaggac tcctgcaacg gtgactctgg ggggcccctg
780
atctgcaacg ggtacttgca gggccttgtg tctttcggaa aagccccgtg tggccaagtt
840
ggcgtgccag gtgtctacac caacctctgc aaattcactg agtggataga gaaaaccgtc
900
caggccagta ctagtggcca ccatcaccat caccattaa
939
<210> 3
<211> 312
2
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<212> PRT
<213> Human
<400> 3
Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp
1 5 10 15
His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe
20 25 30
Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser
35 40 45
Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile
50 55 60
Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr
65 70 75 80
Met Val Gly Glu Asp Cys Ser Pro His Ser Gln Pro Trp Gln Ala Ala
85 90 95
Leu Val Met Glu Asn Glu Leu Phe Cys Ser Gly Val Leu Val His Pro
100 105 110
Gln Trp Val Leu Ser Ala Ala His Cys Phe Gln Asn Ser Tyr Thr Ile
115 120 125
Gly Leu Gly Leu His Ser Leu Glu Ala Asp Gln Glu Pro Gly Ser Gln
130 135 140
Met Val Glu Ala Ser Leu Ser Val Arg His Pro Glu Tyr Asn Arg Pro
145 150 155 160
Leu Leu Ala Asn Asp Leu Met Leu Ile Lys Leu Asp Glu Ser Val Ser
165 170 175
Glu Ser Asp Thr Ile Arg Ser Ile Ser Ile Ala Ser Gln Cys Pro Thr
180 185 190
Ala Gly Asn Ser Cys Leu Val Ser Gly Trp Gly Leu Leu Ala Asn Gly
195 200 205
Arg Met Pro Thr Val Leu Gln Cys Val Asn Val Ser Val Val Ser Glu
210 215 220
Glu Val Cys Ser Lys Leu Tyr Asp Pro Leu Tyr His Pro Ser Met Phe
225 230 235 240
Cys Ala Gly Gly Gly Gln Asp Gln Lys Asp Ser Cys Asn Gly Asp Ser
245 250 255
Gly Gly Pro Leu Ile Cys Asn Gly Tyr Leu Gln Gly Leu Val Ser Phe
260 265 270
Gly Lys Ala Pro Cys Gly Gln Val Gly Val Pro Gly Val Tyr Thr Asn
275 280 285
Leu Cys Lys Phe Thr Glu Trp Ile Glu Lys Thr Val Gln Ala Ser Thr
290 295 300
Ser Gly His His His His His His
305 310
<210> 4
<211> 939
<212> DNA
<213> Human
<400> 4
3
SUBSTITUTE SHEET (RULE 26) RO/AU
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atggatccaa acactgtgtc aagctttcag gtagattgct ttctttggca tgtccgcaaa
cgagttgcag accaagaact aggtgatgcc ccattccttg atcggcttcg ccgagatcag
120
aaatccctaa gaggaagggg cagcaccctc ggtctggaca tcgagacagc cacacgtgct
180
ggaaagcaga tagtggagcg gattctgaaa gaagaatccg atgaggcact taaaatgacc
240
atggttgggg aggactgcag cccgcactcg cagccctggc aggcggcact ggtcatggaa
300
aacgaattgt tctgctcggg cgtcctggtg catccgcagt gggtgctgtc agccgcacac
360
tgtttccaga actcctacac catcgggctg ggcctgcaca gtcttgaggc cgaccaagag
420
ccagggagcc agatggtgga ggccagcctc tccgtacggc acccagagta caacagaccc
480
ttgctcgcta acgacctcat gctcatcaag ttggacgaat ccgtgtccga gtctgacacc
540
atccggagca tcagcattgc ttcgcagtgc cctaccgcgg ggaactcttg cctcgtttct
600
ggctggggtc tgctggcgaa cggcagaatg cctaccgtgc tgcagtgcgt gaacgtgtcg
660
gtggtgtctg aggaggtctg cagtaagctc tatgacccgc tgtaccaccc cagcatgttc
720
tgcgccggcg gagggcaaga ccagaaggac tcctgcaacg gtgactctgg ggggcccctg
780
atctgcaacg ggtacttgca gggccttgtg tctttcggaa aagccccgtg tggccaagtt
840
ggcgtgccag gtgtctacac caacctctgc aaattcactg agtggataga gaaaaccgtc
900
caggccagta ctagtggcca ccatcaccat caccattaa
939
<210> 5
<211> 232
<212> PRT
<213> Human
<400> 5
Asn Ser Ala Arg Ala His Ser Gln Pro Trp Gln Ala Ala Leu Val Met
1 5 10 15
Glu Asn Glu Leu Phe Cys Ser Gly Val Leu Val His Pro Gln Trp Val
20 25 30
Leu Ser Ala Ala His Cys Phe Gln Lys Xaa Val Gln Ser Ser Tyr Thr
35 40 45
Ile Gly Leu Gly Leu His Ser Leu Glu Ala Asp Gln Glu Pro Gly Ser
50 55 60
Gln Met Val Glu Ala Ser Leu Ser Val Arg His Pro Glu Tyr Asn Arg
70 75 80
Pro Leu Leu Ala Asn Asp Leu Met Leu Ile Lys Leu Asp Glu Ser Val
85 90 95
Ser Glu Ser Asp Thr Ile Arg Ser Ile Ser Ile Ala Ser Gln Cys Pro
4
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100 105 110
Thr Ala Gly Asn Ser Cys Leu Val Ser Gly Trp Gly Leu Leu Ala Asn
115 120 125
Gly Arg Met Pro Thr Val Leu Gln Cys Val Asn Val Ser Val Val Ser
130 135 140
Glu Glu Val Cys Ser Lys Leu Tyr Asp Pro Leu Tyr His Pro Ser Met
145 150 155 160
Phe Cys Ala Gly Gly Gly Gln Asp Gln Lys Asp Ser Cys Asn Gly Asp
165 170 175
Ser Gly Gly Pro Leu Ile Cys Asn Gly Tyr Leu Gln Gly Leu Val Ser
180 185 190
Phe Gly Lys Ala Pro Cys Gly Gln Val Gly Val Pro Gly Val Tyr Thr
195 200 205
Asn Leu Cys Lys Phe Thr Glu Trp Ile Glu Lys Thr Val Pro Gly Gln
210 215 220
Leu Thr Leu Gly Thr Gly Asn Pro
225 230
<210> 6
<211> 248
<212> PRT
<213> Human
<400> 6
Met Trp Phe Leu Val Leu Cys Leu Ala Leu Ser Leu Gly Gly Thr Gly
1 5 10 15
Ala Ala Pro Pro Ile Gln Ser Arg Ile Val Gly Gly Trp Glu Cys Glu
20 25 30
Gln His Ser Gln Pro Trp Gln Ala Ala Leu Val Met Glu Asn Glu Leu
35 40 45
Phe Cys Ser Gly Val Leu Val His Pro Gln Trp Val Leu Ser Ala Ala
50 55 60
His Cys Phe Gln Asn Ser Tyr Thr Ile Gly Leu Gly Leu His Ser Leu
65 70 75 80
Glu Ala Asp Gln Glu Pro Gly Ser Gln Met Val Glu Ala Ser Leu Ser
85 90 95
Val Arg His Pro Glu Tyr Asn Arg Pro Leu Leu Ala Asn Asp Leu Met
100 105 110
Xaa Ile Lys Leu Asp Glu Ser Val Ser Glu Ser Asp Asn Ile Arg Xaa
115 120 125
Ile Ser Ile Xaa Ser Gln Cys Pro Thr Ala Gly Asn Phe Cys Leu Val
130 135 140
Ser Gly Trp Gly Leu Leu Ala Asn Gly Arg Met Pro Thr Val Leu Gln
145 150 155 160
Cys Val Asn Val Ser Val Val Ser Glu Glu Val Cys Ser Lys Leu Tyr
165 170 175
Asp Pro Leu Tyr His Pro Ser Met Phe Cys Ala Gly Gly Gly Gln Asp
180 185 190
Gln Lys Asp Ser Cys Asn Gly Asp Ser Gly Gly Pro Leu Ile Cys Asn
195 200 205
Gly Tyr Leu Gln Gly Leu Val Ser Phe Gly Lys Ala Pro Cys Gly Gln
210 215 220
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Val Gly Val Pro Gly Val Tyr Thr Asn Leu Cys Lys Phe Thr Glu Trp
225 230 235 240
Ile Glu Lys Thr Val Gln Ala Ser
245
<210> 7
<211> 254
<212> PRT
<213> Human
<400> 7
Met Ala Thr Ala Gly Asn Pro Trp Gly Trp Phe Leu Gly Tyr Leu Ile
1 5 10 15
Leu Gly Val Ala Gly Ser Leu Val Ser Gly Ser Cys Ser Gln Ile Ile
20 25 30
Asn Gly Glu Asp Cys Ser Pro His Ser Gln Pro Trp Gln Ala Ala Leu
35 40 45
Val Met Glu Asn Glu Leu Phe Cys Ser Gly Val Leu Val His Pro Gln
50 55 60
Trp Val Leu Ser Ala Ala His Cys Phe Gln Asn Ser Tyr Thr Ile Gly
65 70 75 80
Leu Gly Leu His Ser Leu Glu Ala Asp Gln Glu Pro Gly Ser Gln Met
85 90 95
Val Glu Ala Ser Leu Ser Val Arg His Pro Glu Tyr Asn Arg Pro Leu
100 105 110
Leu Ala Asn Asp Leu Met Leu Ile Lys Leu Asp Glu Ser Val Ser Glu
115 120 125
Ser Asp Thr Ile Arg Ser Ile Ser Ile Ala Ser Gln Cys Pro Thr Ala
130 135 140
Gly Asn Ser Cys Leu Val Ser Gly Trp Gly Leu Leu Ala Asn Gly Arg
145 150 155 160
Met Pro Thr Val Leu Gln Cys Val Asn Val Ser Val Val Ser Glu Glu
165 170 175
Val Cys Ser Lys Leu Tyr Asp Pro Leu Tyr His Pro Ser Met Phe Cys
180 185 190
Ala Gly Gly Gly His Asp Gln Lys Asp Ser Cys Asn Gly Asp Ser Gly
195 200 205
Gly Pro Leu Ile Cys Asn Gly Tyr Leu Gln Gly Leu Val Ser Phe Gly
210 215 220
Lys Ala Pro Cys Gly Gln Val Gly Val Pro Gly Val Tyr Thr Asn Leu
225 230 235 240
Cys Lys Phe Thr Glu Trp Ile Glu Lys Thr Val Gln Ala Ser
245 250
<210> 8
<211> 226
<212> PRT
<213> Human
<400> 8
Glu Phe His Cys Val Gly Glu Asp Cys Ser Pro His Ser Gln Pro Trp
1 5 10 15
6
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Gln Ala Ala Leu Val Met Glu Asn Glu Leu Phe Cys Ser Gly Val Leu
20 25 30
Val His Pro Gln Trp Val Leu Ser Ala Ala His Cys Phe Gln Asn Ser
35 40 45
Tyr Thr Ile Gly Leu Gly Leu His Ser Leu Glu Ala Asp Gln Glu Pro
50 55 60
Gly Ser Gln Met Val Glu Ala Ser Leu Ser Val Arg His Pro Glu Tyr
65 70 75 80
Asn Arg Pro Leu Leu Ala Asn Asp Leu Met Leu Ile Lys Leu Asp Glu
85 90 95
Ser Val Ser Glu Ser Asp Thr Ile Arg Ser Ile Ser Ile Ala Ser Gln
100 105 110
Cys Pro Thr Ala Gly Asn Ser Cys Leu Val Ser Gly Trp Gly Leu Leu
115 120 125
Ala Asn Gly Arg Met Pro Thr Val Leu Gln Cys Val Asn Val Ser Val
130 135 140
Val Ser Glu Glu Val Cys Ser Lys Leu Tyr Asp Pro Leu Tyr His Pro
145 150 155 160
Ser Met Phe Cys Ala Gly Gly Gly Gln Asp Gln Lys Asp Ser Cys Asn
165 170 175
Gly Asp Ser Gly Gly Pro Leu Ile Cys Asn Gly Tyr Leu Gln Gly Leu
180 185 190
Val Ser Phe Gly Lys Ala Pro Cys Gly Gln Val Gly Val Pro Gly Val
195 200 205
Tyr Thr Asn Leu Cys Lys Phe Thr Glu Trp Ile Glu Lys Thr Val Gln
210 215 220
Ala Ser
225
7
SUBSTITUTE SHEET (RULE 26) RO/AU
