U.S. patent application number 10/000903 was filed with the patent office on 2002-12-05 for recombinant papillomavirus vaccine and method for production and treatment.
This patent application is currently assigned to SmithKline Beecham Biologicals. Invention is credited to Bruck, Claudine, Delisse, Anne-Marie Eva Fernande, Gerard, Catherine Marie Ghislaine, Lombardo-Bencheikh, Angela, Silva, Teresa Cabezon.
Application Number | 20020182221 10/000903 |
Document ID | / |
Family ID | 10817971 |
Filed Date | 2002-12-05 |
United States Patent
Application |
20020182221 |
Kind Code |
A1 |
Bruck, Claudine ; et
al. |
December 5, 2002 |
Recombinant papillomavirus vaccine and method for production and
treatment
Abstract
The present invention relates to fusions proteins, comprising a
protein or part of a protein that provides T helper epitopes and an
antigen from a human-papilloma virus. In particular the invention
relates to fusion proteins comprising an E6 or E7 protein from HPV
strain 16 or 18 linked to protein D from Haemophilus influenza B.
The invention also provides vaccine compositions that are useful in
the treatment or prophylaxis of human papilloma induced tumors.
Inventors: |
Bruck, Claudine; (Rixensart,
BE) ; Silva, Teresa Cabezon; (Lenkebeek, BE) ;
Delisse, Anne-Marie Eva Fernande; (Gosselies, BE) ;
Gerard, Catherine Marie Ghislaine; (Rhode Saint Genese,
BE) ; Lombardo-Bencheikh, Angela; (Wavre,
BE) |
Correspondence
Address: |
GLAXOSMITHKLINE
Corporate Intellectual Property - UW2220
P.O. Box 1539
King of Prussia
PA
19406-0939
US
|
Assignee: |
SmithKline Beecham
Biologicals
|
Family ID: |
10817971 |
Appl. No.: |
10/000903 |
Filed: |
November 1, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10000903 |
Nov 1, 2001 |
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09485885 |
Feb 18, 2000 |
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6342224 |
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09485885 |
Feb 18, 2000 |
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PCT/EP98/05285 |
Aug 17, 1998 |
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Current U.S.
Class: |
424/186.1 ;
424/185.1; 424/192.1; 424/204.1; 435/252.3; 435/320.1; 435/325;
435/69.3; 435/69.7; 530/350; 536/23.4; 536/23.72 |
Current CPC
Class: |
A61K 39/00 20130101;
C07K 14/005 20130101; C12N 2710/20022 20130101; A61P 31/12
20180101; C12N 2710/20034 20130101; A61P 35/00 20180101; A61P 37/00
20180101; C07K 2319/00 20130101 |
Class at
Publication: |
424/186.1 ;
530/350; 536/23.72; 424/204.1; 424/192.1; 424/185.1; 435/252.3;
435/320.1; 435/325; 435/69.3; 435/69.7; 536/23.4 |
International
Class: |
A61K 039/12; C07H
021/04; C07K 014/08; C12N 015/09; C12P 021/04; A61K 039/00; C12N
001/20; C12N 015/00; C12N 015/63; C12N 015/70; C12N 015/74; C12N
005/00; C12N 005/02; C07K 001/00; C07K 014/00; C07K 017/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 22, 1997 |
GB |
9717953.5 |
Claims
1. An E6 or E7 protein or E6/E7 fusion protein from HPV linked to
an immunological fusion partner.
2. A protein as claimed in claim 1 wherein the fusion partner is
selected from the group; protein D or a fragment thereof from
Heamophilius influenzae B, lipoprotein D or fragment thereof from
Heamophilius influenzae B, NS1 or fragment thereof from Influenzae
Virus, and LYTA or fragment thereof from Streptococcus
Pneumoniae.
3. A protein as claimed in claim 1 or 2 wherein the E6 or E7
proteins are derived from HPV16 or HPV18.
4. A protein as claimed in claim 1, 2 or 3 wherein the E7 protein
is mutated.
5. A protein as claimed in claim 1, 2 or 3 wherein the E6 protein
is mutated.
6. A protein as claimed in any of claims 1 to 5 additionally
comprising a hisitidine tag of at least 4 hisitidine residues.
7. A fusion protein comprising a heterologous protein, a hisitidine
tag and a C-LYTA tag.
8. A DNA sequence encoding a protein as claimed herein.
9. A vaccine containing a protein as claimed in any of claims 1 to
7 and a pharmaceutically acceptable diluent or excipient.
10. A vaccine as claimed in claim 9 additionally comprising an
adjuvant.
11. A vaccine as claimed in claim 9 or 10 wherein the protein is
presented in an oil in water emulsion vehicle.
12. A vaccine as claimed in claim 10 or 11 wherein the adjuvant
comprises 3D-MPL or QS21 or both.
13. A vaccine as claimed herein comprising an additional HPV
antigen.
14. A vaccine as claimed herein for use in medicine.
15. Use of a protein as claimed herein for the manufacture of a
vaccine for immunotherapeutically treating a patient suffering from
HPV induced tumour lesions (benign or malignant).
16. Use of a protein as claimed herein for the manufacture of a
vaccine to prevent HPV viral infection.
17. A vector containing a DNA sequence of claim 8.
18. A vector containing a DNA sequence as claimed in claim 8 and a
DNA sequence encoding thioredoxin.
19. A host transformed with a DNA sequence of claim 8.
20. A host transformed with a vector of claim 17 or 18.
21. A host as claimed in claim 19 additionally transformed with a
DNA sequence encoding thioredoxin.
22. A process for the production of a protein as claimed herein
comprising transforming a host cell with a DNA sequence of claim 6,
expressing said sequence and isolating the desired product.
23. A process for the production of a vaccine as claimed herein,
comprising admixing a protein as claimed herein with a suitable
adjuvant, diluent or other pharmaceutically acceptable excipient.
Description
[0001] The present invention relates to fusions proteins,
comprising a protein or part of a protein that provides T helper
epitopes and an antigen from a human-papilloma virus that find
utility in the treatment or prophylaxis of human papilloma induced
tumours. In particular the invention relates to fusion proteins
comprising an E6 or E7 protein from HPV strain 16 or 18 linked to
protein D from Heamophilius influenza B.
[0002] Papillomaviruses are small naked DNA tumour viruses (7.9
kilobases, double strand), which are highly species-specific. Over
70 individual human papillomavirus (HPV) genotypes have been
described. Papillomaviruses are classified on the basis of species
of origin (human, bovine etc.) and of the degree of genetic
relatedness with other papillomaviruses from the same species. HPVs
are generally specific for the skin or mucosal surfaces and have
been broadly classified into "low" and "high" risk on the basis of
rare and common, respectively, detection in abnormal or tumour
tissue. Low risk HPVs usually cause benign lesions (warts or
papillomas) that persist for several months or years. High risk
HPVs are associated with cancer. The strongest positive association
between an HPV virus and human cancer is that which exist between
HPV 16 and 18 and cervical carcinoma. More than ten other HPV types
have also been found in cervical carcinomas including HPV 31 and
HPV 33 although at less frequency.
[0003] Genital HPV infection in young sexually active women is
common and most individuals either clear the infection, or if
lesions develop, these regress. Only a subset of infected
individuals has lesions which progress to high grade
intraephithelial neoplasia and only a fraction of these progress
further to invasive carcinoma.
[0004] The molecular events leading to HPV infection have not been
clearly established. The lack of an adequate in vitro system to
propagate human papillomaviruses has hampered the progress to a
best information about the viral cycle.
[0005] Today, the different types of HPVs have been isolated and
characterised with the help of cloning systems in bacteria and more
recently by PCR amplification. The molecular organisation of the
HPV genomes has been defined on a comparative basis with that of
the well characterised bovine papillomavirus type 1 (BPV 1).
[0006] Although minor variations do occur, all HPVs genomes
described have at least seven early genes, E1 to E7 and two late
genes L1 and L2. In addition, an upstream regulatory region harbors
the regulatory sequences which appears to control most
transcriptional events of the HPV genome.
[0007] E1 and E2 genes are involved in viral replication and
transcriptional control, respectively and tend to be disrupted by
viral integration. E6 and E7 are involved in viral transformation.
E5 has also been implicated in this process.
[0008] In the HPVs involved in cervical carcinoma such as HPV 16
and 18, the oncogenic process starts after integration of viral
DNA. The integration results in the inactivation of genes coding
for the capsid proteins L1 and L2 and loss of E2 repressor function
leads to deregulation of the E6/E7 open reading frame installing
continuously overexpression of the two early proteins E6 and E7
that will lead to gradually loss of the normal cellular
differentiation and the development of the carcinoma. E6 and E7
overcome normal cell cycle by inactivating major tumor suppressor
proteins, p53 and pRB, the retinoblastoma gene product,
respectively.
[0009] Carcinoma of the cervix is common in women and develops
through a pre-cancerous intermediate stage to the invasive
carcinoma which frequently leads to death. The intermediate stages
of the disease is known as cervical intraepithelial neoplasia and
is graded I to III in terms of increasing severity (CIN I-III).
[0010] Clinically, HPV infection of the female anogenital tract
manifests as cervical flat condylomas, the hallmark of which is the
koilocytosis affecting predominantly the superficial and
intermediate cells of the cervical squamous epithelium.
[0011] Koilocytes which are the consequence of a cytopathic effect
of the virus, appear as multinucleated cells with a perinuclear
clear haloe. The epithelium is thickened with abnormal
keratinisation responsible for the warty appearance of the
lesion.
[0012] Such flat condylomas when positive for the HPV 16 or 18
serotypes, are high-risk factors for the evolution toward cervical
intraepithelial neoplasia (CIN) and carcinoma in situ (CIS) which
are themselves regarded as precursor lesions of invasive cervix
carcinoma.
[0013] The natural history of oncogenic HPV infection presents 3
consecutive phases, namely:
[0014] (1) a latent infection phase,
[0015] (2) a phase of intranuclear viral replication with product
of complete virions, which corresponds to the occurrence of
koilocytes. At this stage, the HPV is producing its full range of
proteins including E2, E5, E6, E7, L1 and L2.
[0016] (3) a phase of viral integration into the cellular genome,
which triggers the onset of malignant transformation, and
corresponds to CIN II and CIN III/CIS with progressive
disappearance of koilocytes. At this stage, the expression of E2 is
down-regulated, the expression of E6 and E7 is enhanced. Between
CIN II/III and CIN III/Cervix carcinoma the viral DNA changes from
being episomal in the basal cells to integration of E6 and E7 genes
only (tumoral cells). 85% of all cervix carcinomas are squamos cell
carcinomas most predominantly related to the HPV16 serotype. 10%
and 5% are adenocarcinomas and adenosquamos cell carcinomas
respectively, and both types are predominantly related to HPV 18
serotype. Nevertheless other oncogenic HPV's exist.
[0017] International Patent Application No. WO 96/19496 discloses
variants of human papilloma virus E6 and E7 proteins, particularly
fusion proteins of E6/E7 with a deletion in both the E6 and E7
proteins. These deletion fusion proteins are said to be
immunogenic.
[0018] The present invention provides compositions comprising
either an E6 or E7 or an E6/E7 fusion protein linked to an
immunological fusion partner having T cell epitopes.
[0019] In a preferred form of the invention, the immunological
fusion partner is derived from protein D of Heamophilus influenza
B. Preferably the protein D derivative comprises approximately the
first {fraction (1/3)} of the protein, in particular approximately
the first N-terminal 100-110 amino acids. The protein D may be
lipidated (Lipo Protein D). Other immunological fusion partners
include the non-structural protein from influenzae virus, NS1
(hemagglutinin). Typically the N terminal 81 amino acids are
utilised, although different fragments may be used provided they
include T-helper epitopes.
[0020] In another embodiment the 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 gen {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}. As used herein a preferred embodiment
utilises the repeat portion of the Lyta molecule found in the C
terminal end starting at residue 178. A particularly preferred form
incorporates residues 188-305.
[0021] Accordingly, the present invention in preferred embodiment
provides fusion proteins comprising Protein D-E6 from HPV 16,
Protein D-E7 from HPV 16 Protein D-E7 from HPV 18, Protein D-E6
from HPV 18, and Protein D E6 E7 from both HPV 16 and 18. The
protein D part preferably comprises the first {fraction (1/3)} of
protein D. It will be appreciated that other E6 and E7 proteins may
be utilised from other HPV subtypes.
[0022] The proteins of the present invention preferably are
expressed in E. coli. In a preferred embodiment the proteins are
expressed with a Histidine tail comprising between 5 to 9 and
preferably six Histidine residues. These are advantageous in aiding
purification.
[0023] The protein E7 may in a preferred embodiment carry a
mutation to reduce the binding for the rb site (retinoblastoma gene
product) and hence eliminate any potential transforming capacity.
Preferred mutations for HPV 16 E7 involve replacing Cys.sub.24 with
Glycine, or Glutamic acid.sub.26 with Glutamine. In a preferred
embodiment the E7 protein contains both these mutations.
[0024] Preferred mutations for the HPV 18 E.sub.7 involve replacing
Cys.sub.27 with Glycine and/or Glutamic acid.sub.26 with Glutamine.
Again preferably both mutations are present.
[0025] Single or double mutations may also be introduced p53 region
of E.sub.6 to eliminate any potential transforming ability.
[0026] In a further embodiment of the invention there is provided
and E6 E7 fusion protein from HPV linked to an immunological fusion
partner. A preferred Immunological fusion partner is Protein D,
more preferable the first {fraction (1/3)} of protein D.
[0027] The present invention also provides a DNA encoding the
proteins of the present invention. Such sequences can be inserted
into a suitable expression vector and expressed in a suitable
host.
[0028] 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 polymerase, or by a
combination of these techniques.
[0029] Enzymatic polymerisation of DNA may be carried out in vitro
using a DNA polymerase such as DNA polymerase I (Klenow fragment)
in an appropriate buffer containing the nucleoside triphosphates
dATP, dCTP, dGTP and dTTP as required at a temperature of
10.degree.-37.degree. C., generally in a volume of 50 .mu.l 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), 0.01M MgCl.sub.2, 0.01M dithiothreitol, 1
mM spermidine, 1 mM ATP and 0.1 mg/ml bovine serum albumin, at a
temperature of 4.degree. C. to ambient, generally in a volume of 50
ml 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 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.,
Journal 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.
[0030] 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.
[0031] In particular, the process may comprise the steps of:
[0032] 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;
[0033] ii) transforming a host cell with said vector;
[0034] iii) culturing said transformed host cell under conditions
permitting expression of said DNA polymer to produce said protein;
and
[0035] iv) recovering said protein.
[0036] 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
hereafter apply to the resulting host cell containing and
expressing the foreign gene of interest.
[0037] Preferably recombinant antigen of the invention are
expressed in E. coli. The expression strategy include fusion of E7,
E6 or E6/E7 fusion to the 1/3-N-terminal portion of protein D from
Haemophilus influenzae B, an immunological fusion partner providing
T cell helper epitopes. An affinity polyhistidine tail is
engineered at the carboxy terminus of the fusion protein allowing
for simplified purification. Such recombinant antigen is
overexpressed in E. coli as insoluble protein.
[0038] Preferably the proteins of the invention are coexpressed
with thioredoxin in trans (TIT). Coexpression of thioredoxin in
trans versus in cis is preferred to keep antigen free of
thioredoxin without the need for protease. Thioredoxin coexpression
eases the solubilisation of the proteins of the invention.
Thioredoxin coexpression has also a significant impact on protein
purification yield, on purified-protein solubility and quality.
[0039] The expression vectors are novel and also form part of the
invention.
[0040] 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 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.
[0041] Thus, the DNA polymer may be preformed or formed during the
construction of the vector, as desired.
[0042] The choice of vector will be determined in part by the host
cell, which may be prokaryotic or eukaryotic but preferably is E.
coli. Suitable vectors include plasmids, bacteriophages, cosmids
and recombinant viruses.
[0043] 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.
[0044] 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.
[0045] The choice of transforming conditions is determined by the
host cell. Thus, a bacterial host such as E. coli may be treated
with a solution of CaCl.sub.2 (Cohen et al., Proc. Nat. Acad. Sci.,
1973, 69, 2110) or with a solution comprising a mixture of RbCl,
MnCl.sub.2, potassium acetate and glycerol, and then with
3-[N-morpholino]-propane-su- lphonic acid, RbCl and glycerol.
Mammalian cells in culture may be transformed by calcium
co-precipitation of the vector DNA onto the cells. The invention
also extends to a host cell transformed with a replicable
expression vector of the invention.
[0046] Culturing the transformed host cell under conditions
permitting expression of the DNA polymer is carried out
conventionally, as described in, for example, Maniatis et al. and
"DNA Cloning" cited above. Thus, preferably the cell is supplied
with nutrient and cultured at a temperature below 50.degree. C.
[0047] The product is recovered by conventional methods according
to the host cell. Thus, where the host cell is bacterial, such as
E. coli it may be lysed physically, chemically or enzymatically and
the protein product isolated from the resulting lysate. Where the
host cell is mammalian, the product may generally be isolated from
the nutrient medium or from cell free extracts. Conventional
protein isolation techniques include selective precipitation,
adsorption chromatography, and affinity chromatography including a
monoclonal antibody affinity column.
[0048] When the proteins of the present invention are expressed
with a hisitidine tail (His tag). The proteins can easily be
purified by affinity chromatography using an ion metal affinity
chromatography column (IMAC) column.
[0049] A second chromatographic step, such as Q-sepharose may be
utilised either before or after the IMAC column to yield highly
purified protein. If the immunological fusion partner is C-LYTA,
then it is possible to exploit the affinity of CLYTA for choline
and/or DEAE to purify this product. Products containing both C-LYTA
and his tags can be easily and efficiently purified in a two step
process involving differential affinity chromatography. One step
involves the affinity of the His tag to IMAC columns, the other
involves the affinity of the C-terminal domain of LYTA for choline
or DEAE.
[0050] Proteins comprising both a C-LYTA and Hisitidine tag are new
and accordingly form one aspect of the invention. These may be
purified to high levels (greater than 80% preferably greater than
90%) by a simple two step differential affinity procedure.
[0051] The proteins of the present invention are provided
preferably at least 80% pure more preferably 90% pure as visualized
by SDS PAGE. The protein present a major single band when analysed
by SDS PAGE under reducing conditions, and western blot analysis
show less than 5% host cell protein contamination.
[0052] 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 Protein D-E6 from HPV 16 or
derivative thereof together with Protein D-E7 from HPV 16.
Alternatively the E6 and E7 may be presented in a single molecule,
preferably a Protein D E6/E7 fusion. Such vaccine may optionally
contain either or both E6 and E7 proteins from HPV 18, preferably
in the form of a Protein D-E6 or Protein D-E7 fusion protein or
Protein D E6/E7 fusion protein. The vaccines of the present
invention may contain other HPV antigens from HPV 16 or 18. In
particular, the vaccine may contain L1 or L2 antigen monomers.
Alternatively such L1 or L2 antigens may be presented together as a
virus like particle or the L1 alone protein may be presented as
virus like particle or caposmer structure. Such antigens, virus
like particles and capsomer are per se known. See for example
WO94/00152, WO94/20137, WO94/05792, & WO93/02184. Additional
early proteins may be included such as E2 or preferably E5 for
example The vaccine of the present invention may additionally
comprise antigens from other HPV strains, preferably from strains
HPV 6 11, HPV 31 or 33.
[0053] Vaccine preparation is generally described in Vaccine
Design--The subunit and adjuvant approach (Ed. Powell and Newman)
Pharmaceutical Biotechnology Vol. 6 Plenum Press 1995.
Encapsulation within liposomes is described by Fullerton, U.S. Pat.
No. 4,235,877.
[0054] The proteins of the present invention are preferably
adjuvanted in the vaccine formulation of the invention. Suitable
adjuvants include an aluminium salt such as aluminium hydroxide gel
(alum) or aluminium phosphate, but may also be a salt of calcium,
iron or zinc, or may be an insoluble suspension of acylated
tyrosine, or acylated sugars, cationically or anionically
derivatised polysaccharides, or polyphosphazenes.
[0055] In the formulation of the inventions it is preferred that
the adjuvant composition induces a preferential TH 1 response.
Suitable adjuvant systems include, for example, a combination of
monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl
lipid A (3D-MPL) together with an aluminium salt.
[0056] An enhanced system involves the combination of a
monophosphoryl lipid A and a saponin derivative particularly the
combination of QS21 and 3D-MPL as disclosed in WO 94/00153, or a
less reactogenic composition where the QS21 is quenched with
cholesterol as disclosed in WO 96/33739.
[0057] 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.
[0058] Accordingly in one embodiment of the present invention there
is provided a vaccine comprising a protein D (or derivative
thereof)--E6 or protein D (or derivative thereof)--E7 adjuvanted
with a monophosphoryl lipid A or derivative thereof.
[0059] Preferably the vaccine additionally comprises a saponin,
more preferably QS21.
[0060] Preferably the formulation additional comprises an oil in
water emulsion and tocopherol. 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.
[0061] The invention will be further described by reference to the
following examples:
EXAMPLE I
Construction of an E. coli Strain Expressing Fusion
Protein-D1/3-E7-His (HPV16)
[0062] 1)--Construction of Expression Plasmid
[0063] a)--Plasmid pMG MCS prot D1/3 (=pRIT14589) is a derivative
of pMG81 (described in UK patent application n.degree. 951 3261.9
published as WO97/01640) in which the codons 4-81 of NS 1 coding
region from Influenza were replaced by the codons corresponding to
residues Ser 20.fwdarw.Thrl27 of mature protein D of Haemophilus
Influenzae strain 772, biotype 2 (H. Janson et al., 1991, Infection
and Immunity, Jan. p.119-125). The sequence of Prot-D1/3 is
followed by a multiple cloning site (11 residues) and a coding
region for a C-terminal histidine tail (6 His). This plasmid is
used to express the fusion protein D1/3-E7-His.
[0064] b)--HPV genomic E6 and E7 sequences type HPV 16 (See Dorf et
al., Virology 1985, 145, p. 181-185) were amplified from HPV 16
full length genome cloned in pBR322 (obtained from Deutsches
Krebsforschungszentrum (DKFZ), Referenzzentrum fur human pathogen
Papillomaviruses--D 69120--Heidelberg) and were subcloned into
pUC19 to give TCA 301 (=pRIT14462).
[0065] Construction of Plasmid TCA 308 (=pRIT14501): a Plasmid
Expressing the Fusion Protein-D1/3-E7-His
[0066] The nucleotides sequences corresponding to amino acids
1.fwdarw.98 of E7 protein are amplified from pRIT14462. During the
polymerase chain reaction, NcoI and Spel restriction sites were
generated at the 5' and 3' ends of the E7 sequences allowing
insertion into the same sites of plasmid pMGMCS Prot D1/3 to give
plasmid TCA308 (=pRIT14501). The insert was sequenced to verify
that no modification had been generated during the polymerase chain
reaction. The sequence for the fusion protein-D1/3-E7-His (HPV 16)
is described in FIG. 1.
[0067] 2)--Transformation of AR58 Strain
[0068] Plasmid pRIT14501 was introduced into E. coli AR58 (Mott et
al., 1985, Proc. Natl. Acad. Sci., 82:88) a defective .lambda.
lysogen containing a thermosensitive repressor of the .lambda. pL
promoter.
[0069] 3)--Growth and Induction of Bacterial Strain--Expression of
Prot -D1/3-E7-His
[0070] Cells of AR58 transformed with plasmid pRIT14501 were grown
in 100 ml of LB medium supplemented with 50 .mu.gr/ml of Kanamycin
at 30.degree. C. During the logarithmic phase of growth bacteria
were shifted to 39.degree. C. to inactivate the .lambda. repressor
and turn on the synthesis of protein D1/3-E7-His. The incubation at
39.degree. C. was continued for 4 hours. Bacteria were pelleted and
stored at -20.degree. C.
EXAMPLE II
Characterisation of fusion Protein D1/3-E7-His (HPV 16)
[0071] Frozen cells are thawed and resuspended in 10 ml of PBS
buffer. Cells are broken in a French pressure cell press SLM Aminco
at 20.000 psi (three passages). The extract is centrifuged at
16.000 g for 30 minutes at 4.degree. C.
[0072] After centrifugation of extracts described above, aliquots
of supernatant and pellet were analysed by SDS-polyacrylamide gel
electrophoresis and Western blotting. A major band of about 33 kDa,
localised in the pellet fraction, was visualised by Coomassie
stained gels and identified in Western blots by rabbit polyclonal
anti-protein-D and by Ni-NTA conjugate coupled to calf intestinal
alkaline phosphatase (Qiagen cat. n.degree. 34510) which detects
accessible histidine tail. The level of expression represents about
5% of total protein as shown on a Coomassie-stained
SDS-polyacrylamide gel.
EXAMPLE III
Protein -D1/3-E7-His (HPV 16) Purification
[0073] One liter culture of bacteria expressing
protein-D1/3-E7-His, is centrifuged at 11,300 g for 30 min at
4.degree. C. and cell pellet is kept at -80.degree. C. until
further treatment. After resuspension in 75 ml PBS buffer, E. coli
cells are broken in a French pressure cell press (SLM Aminco.RTM.)
at 20,000 psi. Lysed cells are pelleted by centrifugation at 17,000
g for 30 minutes. Pellet, containing the protein-D1/3-E7-His, is
washed once in 30 ml of 2M NaCI, 50 mM Phosphate pH 7.5, then twice
in 30 ml 50 mM Phosphate pH 7.5. Proteins are solubilised after 2
hours incubation of the pellet in 30 ml of 8 M urea, 50 mM
phosphate pH 7.5 at RT. Cells debris are eliminated by 15 min
centrifugation at 17,000 g, 4.degree. C. Protein purification is
carried out at RT.degree., 15 ml of solubilised protein are applied
onto a 5 ml Ni2+NTA (Qiagen) resin (Pharmacia column XK 16/20)
preequilibrated in 8M urea, 50 mM phosphate pH 7.5 at a flow rate
of 0.2 ml/min. The column is washed in the same buffer until the
absorbance at 280 nm reaches the base line. The protein is eluted
with a 0-600 mM Imidazole gradient in 8M urea, 50 mM phosphate pH
7.5. The flow rate of these two last steps is brought to 1 ml/min.
Eluted fractions are analysed by SDS polyacrylamide gel
electrophoresis and by Western blotting. ProtD1/3-E7-His,
visualised by Coomassie blue staining, by a polyclonal anti protein
D or by a monoclonal anti E7 antibody, appears as a major single
band at about 32 kDalton and is estimated as a 95% pure protein. No
E. coli contaminants, traced with a polyclonal anti E. coli
proteins antibody, are observed.
[0074] In order to eliminate urea, 9 ml of purified antigen, at
1.33 mg/ml (Bradford), is dialysed against 3 liters of PBS buffer
overnight at RT.degree. followed by a 4 hours dialysis against a
fresh PBS buffer. 80% of urea free protein is recovered as soluble
protein. To eliminate contaminating endotoxins, 6 ml of dialysed
protein are incubated with 1 ml of Affiprep polymixin gel (Biorad),
for 3 hours at 4.degree. C. under gentle stirring. A second
incubation with 500 .mu.l of Affiprep polymixin resin is performed
to minimise the endotoxin level to 8.8 EU/.mu.g protein. After
sterile filtration on a 0.22 .mu.m filter device (Millex 0.22 GV,
Millipore), prot-D1/3-E7-His at 0.665 mg/ml is assayed for
stability. SDS PAGE analysis showed no evolution of the protein
after 7 days incubation at -20.degree. C., 4.degree. C., RT.degree.
or 37.degree. C.
EXAMPLE IV
Construction of an E.coli strain expressing fusion
Protein-D1/3-E6-his/HPV- 16
[0075] 1. Construction of Expression Plasmid
[0076] a) Plasmid pMG MCS prot D1/3 (=pRIT14589) is a derivative of
pMG81 (described in WO97/01640 in which the codons 4-81 of NS1
coding region from Influenza were replaced by the codons
corresponding to residues Ser 20.fwdarw.Thr 127 of mature protein D
of Haemophilus Influenzae strain 772, biotype 2 (H. Janson et al.,
1991, Infection and Immunity, Jan. p.119-125). The sequence of
Prot-D1/3 is followed by a multiple cloning site (11 residues) and
a coding region for a C-terminal histidine tail (6 His). This
plasmid is used to express the fusion protein D1/3-E6-his.
[0077] b) HPV genomic E6 and E7 sequences type HPV 16 (Seedorf et
al., Virology 1985, 145, p.181-185) were amplified from HPV16 full
length genome cloned in pBR322 (obtained from Deutsches
Krebsforschungszentrum (DKFZ), Referenzzentrum fur human pathogen
Papillomaviruses--
[0078] c) D 69120--Heidelberg) and were subcloned into pUC19 to
give TCA 301 (=pRIT14462).
[0079] Construction of Plasmid TCA 307 (=pRIT14497): a Plasmid
Expressing the Fusion Protein-D1/3-E6-His/HPV16
[0080] The nucleotides sequences corresponding to amino acid.
[0081] 1.fwdarw.151 of E6 protein were amplified from pRIT 14462.
During the polymerase chain reaction, NcoI and Spel restriction
sites were generated at the 5' and 3' ends of the E6 sequences
allowing insertion into the same sites of plasmid pMGMCS Prot D1/3
to give plasmid TCA307 (=pRIT14497) (see FIG. 2). The insert was
sequenced to verify that no modification had been generated during
the polymerase chain reaction. The coding sequence for the fusion
protein-D1/3-E6-His is described in FIG. 3.
[0082] 2. Transformation of AR58 Strain
[0083] Plasmid pRIT14497 was introduced into E. coli AR58 (Mott et
al., 1985, Proc. Natl. Acad. Sci., 82:88) a defective .lambda.
lysogen containing a thermosensitive repressor of the .lambda. pL
promoter.
[0084] 3. Growth and Induction of Bacterial Strain--Expression of
Prot-D1/3-E6-His
[0085] Cells of AR58 transformed with plasmid pRIT14497 were grown
in 100 ml of LB medium supplemented with 50 .mu.gr/ml of Kanamycin
at 30.degree. C. During the logarithmic phase of growth bacteria
were shifted to 39.degree. C. to inactivate the .lambda. repressor
and turn on the synthesis of protein D1/3-E6-his. The incubation at
39.degree. C. was continued for 4 hours. Bacteria were pelleted and
stored at -20C.
[0086] 4. Characterization of Fusion Protein D1/3-E6-his (HPV 16)
Preparation of Extracts
[0087] Frozen cells are thawed and resuspended in 10 ml of PBS
buffer. Cells are broken in a French pressure cell press SLM Aminco
at 20.000 psi (three passages). The extract is centrifuged at
16.000 g for 30 minutes at 4.degree. C.
[0088] Analysis on Coomassie-Stained SDS-Polyacrylamide Gels and
Western Blots
[0089] After centrifugation of extracts described above, aliquots
of supernatant and pellet were analysed by SDS-polyacrylamide gel
electrophoresis and Western blotting.
[0090] A major band of about 32 kDa, localized in the pellet
fraction, was visualised by Coomassie stained gels and identified
in Western blots by rabbit polyclonal anti-protein-D and by Ni-NTA
conjugate coupled to calf intestinal alkaline phosphatase (Qiagen
cat. n.degree. 34510) which detects accessible histidine tail. The
level of expression represents about 5% of total protein.
[0091] 5. Coexpression with Thioredoxin
[0092] In an analagons fashion to the expression of prot D 1/3 E7
His from HPV 18 (example XIII) an E.coli strain AR58 was
transformed with a plasmid encoding thioredoxin and protein D 1/3
E7 His (HPV 16).
EXAMPLE V
Purification of Prot D 1/3 E6 His (HPV 16)
[0093] HPV-16 ProtD1/3 E6 recombinant antigen was expressed in E.
coli (AR58). Expression strategy included fusion of E6 to the
1/3-N-terminal portion of protein D from Haemophilus influenzae, an
immunological fusion partner providing T cell helper epitopes. An
affinity polyhistidine tail was engineered at the carboxy terminus
of the fusion protein. The recombinant antigen was overexpressed in
E. coli as insoluble proteins.
[0094] Solubilisation of the antigen required denaturing agents. In
absence of denaturing agent, ProtD1/3-E6-His precipitated at
neutral pH. To circumvent the solubility problems, co-expression of
these proteins with Thioredoxin in Trans (TIT), a folding partner
was carried out.
[0095] Bacterial expressions are conducted in LB media in presence
of 0.05 mg/ml of kanamycin at 30.degree. C. plus 0.2 mg/ml of
Ampicillin when Thioredoxin is coexpressed. Recombinant protein
expression is thermally induced by transferring the cells to
42.degree. C., when cell optical density (OD 600 nm) of 0.4 is
reached. Protein expression is maintained for 4 hours. Purification
was carried out according to the following protocol.
1 Cell Culture Pellet 60 OD.sub.600 1 mM pefabloc, 2 M NaCl, PBS pH
7.4 (Buffer A) French Press Disruptor Three passes 20,000 psi
Centrifugation 17,000 g 30 min, 4.degree. C. Pellet Washes 2 M
NaCl, PBS pH 7.4 (Buffer B) x1 PBS pH 7.4 (Buffer C) x2
Centrifugation 17,000 g 30 min, 4.degree. C. Pellet Solubilisation
6 M Guanidine Chloride, 20 mM PO4, pH 7.0 (Buffer D) Overnight at
4.degree. C. Centrifugation 17,000 g 30 min, 4.degree. C.
Supernatant on IMAC Equilibration: 6 M Guanidine Chloride, 20 mM
PO4, pH 7.0 (Buffer D) Elution: Imidazole steps (0.025 M, 0.1 M,
0.5 M) in 8 M Urea, 20 mM PO4, pH 7.0 Affiprep Polymyxin 8 M Urea,
20 mM PO4, pH 7.0 (Buffer E) 2 h RT.degree. Dialysis 4 M Urea, 0.5
M Arginine, 150 mM NaCl, 10 mM PO4, pH 6.8 (Buffer I) 2 M Urea, 0.5
M Arginine, 150 mM NaCl, 10 mM PO4 pH 6.8, (Buffer J) 0 M Urea, 0.5
M Arginine, 150 mM NaCl, 10 mM PO4 pH 6.8 (Buffer K)
[0096] Cells are efficiently broken by high-pressure homogenisation
using a French pressure cell device. Antigen is extracted with high
concentration of protein denaturant. This first step breaks open
the bacterial cell wall and antigen is extracted from the bacterial
insoluble fraction. The following purification was carried out on 4
liter culture.
[0097] Buffers
[0098] A. PBS/2M NaCl/1 mM Pefabloc
[0099] B. PBS/2MNaCl
[0100] C. PBS: 137 mM NaCl, 2.7 mM KCl, 8.1 mM NaH2PO4, 1.47 mM
KH2PO4 pH 7.4.
[0101] D. 6 M Guanidium Chloride, 20 mM PO4 (NaH2PO4 (2H2O)/K2HPO4
(3H2O)) pH 7.0
[0102] Starting material is 10 flasks of 400 ml culture each.
[0103] Cell paste is suspended to 60 OD.sub.600 in Buffer A (240 ml
of Buffer A in this case), prior cell lysis by three passes through
a French press disrupter (20,000 psi). Lysed cells are pelleted 30
min at 15,000 g at 4.degree. C. Bacterial cell pellet containing
the recombinant protein is washed once in 240 ml Buffer B, then
twice in 240 ml Buffer C.
[0104] Prot D E6-His (TIT) is solubilised by 240 ml Buffer D
overnight at 4.degree. C. on a rotating wheel. Cell debris are
pelleted 30 min at 15,000 g at 4.degree. C. Supernatant (230 ml) is
stored at -20.degree. C. The material is then subjected to IMAC
chromatography.
[0105] The chelating ligand NTA (nitrilo-tri-acetic-acid) is
attached to an Agarose support (Qiagen). NTA ligand is charged with
nickel metal ion with which it interacts through 4 of the 6
coordination sites of the nickel. The two remaining coordination
sites of nickel interact strongly with histidine residues of the
6.times. His-tagged protein. Elution is achieved by competition
with Imidazole which bind to the Ni-NTA and displace the tagged
antigen.
[0106] Ni-NTA Agarose Qiagen (catalogue number: 30 250) was
used.
[0107] Solutions
2 D: 6 M Guanidium Chloride, 20 mM PO4 (NaH.sub.2PO4
(2H.sub.2O)/K.sub.2HPO4 (3H.sub.2O)), pH 7.0 E: 8 M Urea, 20 mM PO4
(NaH.sub.2PO4 (2H.sub.2O)/K.sub.2HPO4 (3H.sub.2O)), pH 7.0 F: E +
0.025 M Imidazole G: E + 0.1 M Imidazole H: E + 0.5 M Imidazole
[0108] 0.5 M NaOH
[0109] Deionized water
[0110] 0.02% NaN.sub.3
[0111] Purification
[0112] a) The resin (15 ml resin/230 ml sample) is packed and
equilibrated in 10 column volumes (C.V.) of Buffer D at 15 cm
h.sup.-1.
[0113] b) Supernatant from solubilised fraction is injected onto
the column at 15 cm h.sup.-1.
[0114] c) Column is washed at 15 cm h.sup.-1 with buffer D until OD
280 nm returns to the baseline.
[0115] d) Column is washed with 2 CV of Buffer E at 15 cm h.sup.-1.
The wash fraction is recovered.
[0116] e) Column is first eluted with 5 CV of Buffer F. Elimination
of 25 kD major contaminant.
[0117] f) Column is then eluted with 2 CV of Buffer G.
[0118] g) Column is finally eluted with 3 CV of Buffer H. Elution
of the antigen.
[0119] Antigen positive fractions are pooled (30 ml).
[0120] Endotoxin is removed by affiprep chromatography.
[0121] Affi-Prep.RTM. Polymyxin support consists of USP Grade
Polymyxin B coupled to the Affi-Prep.RTM. Matrix. Due to its high
affinity to the lipid A moiety of endotoxins, polymixin B binds
endotoxin molecules with high capacity and selectivity.
[0122] Solutions
[0123] E: 8M Urea, 20 mM PO4 (NaH.sub.2PO4 (2H.sub.2O)/K.sub.2HPO4
(3H.sub.2O)), pH 7.0 (apyrogenic).
[0124] 0.5 M Na OH
[0125] Deionized apyrogenic water
[0126] Procedure
[0127] 1) Affi-Prep.RTM. Polymyxin resin is washed in 10 volumes of
0.1 M NaOH, followed by 10 volumes of pyrogen free water.
[0128] 2) Resin is equilibrated in 10 volumes of Buffer E.
[0129] 3) 15 ml (half-pool) of IMAC-eluted sample is incubated with
3 ml of Affi-Prep.RTM. Polymyxin resin in a batch mode.
[0130] 4) Incubation is pursued 4 hours at Room Temperature or O/N
at 4.degree. C. on a rotating wheel.
[0131] 5) Sample is centrifuged 10 min at 2000 g (Beckman
GS-6R).
[0132] 6) Supernatant containing the antigen is collected and
submitted to endotoxins and protein assays.
[0133] 7) Resin is discarded.
[0134] Small molecules diffuse through a semi-permeable membrane
while large molecules are retained. The process of dialysis is
driven by the difference in concentration of the solutes on the two
sides of the membrane. New buffer solution is introduced until
buffer composition on each side equalises.
[0135] Buffers
3 I: 4 M Urea, 0.5 M Arginine, 0.15 M NaCl, 10 mM PO4 (NaH.sub.2PO4
(2H.sub.2O)/K.sub.2HPO4 (3H.sub.2O)) pH 6.8 J: 2 M Urea, 0.5 M
Arginine, 0.15 M NaCl, 10 mM PO4 (NaH.sub.2PO4
(2H.sub.2O)/K.sub.2HPO4 (3H.sub.2O)) pH 6.8 K: 0 M Urea, 0.5 M
Arginine, 0.15 M NaCl, 10 mM PO4 (NaH.sub.2PO4
(2H.sub.2O)/K.sub.2HPO4 (3H.sub.2O)) pH 6.8
[0136] 1) The Sample (15 ml) is introduced into a dialysis tubing
(20.4 mm diameter and 6 cm height).
[0137] 2) Dialysis tubing is placed in a 2 liters cylinder
containing Buffer I under stirring at 4.degree. C. for 2 hours.
[0138] 3) Dialysis tubing is placed in a 2 liters cylinder (under
stirring) containing Buffer J; at 4.degree. C. for 2 hours.
[0139] 4) Dialysis tubing is placed in a 2 liters cylinder
containing Buffer K (under stirring) at 4.degree. C. O/N. Buffer is
changed and dialysis is pursued 2 more hours at 4.degree. C.
[0140] Millipore Sterile Millex-GV 0.22.mu., 13 mm. Catalogue
number: SLGV0130S.
[0141] All steps are performed at room temperature
(RT.congruent.22.degree- . C.), the antigen appears stable.
[0142] Antigen solution is filtered through a 0.2 .mu.m filter to
prevent any bacterial growth. Antigen is kept at -20.degree. C. in
Nunc vials.
[0143] Characterisation:
[0144] Protein D1/3 E6 His is characterised as follows:
[0145] ProteinD1/3-E6-His is a 273 amino acids long peptide with
112 amino acids coming from Protein D part. ProteinD1/3-E6-His has
a theoretical Molecular Weight of 32 kD and migrates on SDS-PAGE as
a 33 kD protein. ProteinD1/3-E6-His theoretical isoelectric point
is 8.17.
[0146] The viral Protein E6 is a basic protein containing 14
cystein residues, eight of them (Cys 30,33,63,66 and Cys
103,106,136,139) are involved in two C-terminal zinc binding
motifs.
[0147] Protein D 1/3-E6-His is expressed as insoluble protein, in
E. coli-AR 58 strain, with Thioredoxin in Trans, a folding partner.
Cell culture is produced in 400 ml flask.
[0148] 5.4 mg of 95% pure protein is obtained per liter of
culture.
EXAMPLE VI
Construction of an E. coli Strain Expressing Fusion
Protein-D1/3-E6E7-his/HPV16
[0149] 1. Construction of Expression Plasmid
[0150] a) Plasmid pMG MCS prot D1/3 (=pRIT14589) is a derivative of
pMG81 (described Supra) in which the codons 4-81 of NS 1 coding
region from Influenza were replaced by the codons corresponding to
residues Ser 20.fwdarw.Thr 127 of mature protein D of Haemophilus
Influenzae strain 772, biotype 2 (H. Janson et al., 1991, Infection
and Immunity, Jan. p.119-125). The sequence of Prot-D1/3 is
followed by a multiple cloning site (11 residues) and a coding
region for a C-terminal histidine tail (6 His). This plasmid is
used to express the fusion protein D1/3-E6E7-his.
[0151] b) HPV genomic E6 and E7 sequences type HPV16 (Seedorf et
al., Virology 1985, 145, p.181-185) were amplified from HPV16 full
length genome cloned in pBR322 (obtained from Deutsches
Krebsforschungszentrum (DKFZ), Referenzzentrum fur human pathogen
Papillomaviruses--D 69120--Heidelberg) and were subcloned into
pUC19 to give TCA 301 (=pRIT14462).
[0152] c) The coding sequences for E6 and E7 in TCA301 (=pRIT
14462) were modified with a synthetic oligonucleotides adaptor
(inserted between Afl III and Nsi I sites) introducing a deletion
of 5 nucleotides between E6 and E7 genes to remove the stop codon
of E6 and create fused E6 and E7 coding sequences in the plasmid
TCA309(=pRIT 14556) see FIG. 4.
[0153] Construction of Plasmid TCA 311(=pRIT14512): a Plasmid
Expressing the Fusion Protein-D1/3-E6E7-His/HPV16
[0154] The nucleotides sequences corresponding to amino acids
1.fwdarw.249 of fused E6E7 protein were amplified from pRIT14556.
During the polymerase chain reaction, NcoI and Spel restriction
sites were generated at the 5' and 3' ends of the E6E7 fused
sequences allowing insertion into the same sites of plasmid pMGMCS
Prot D1/3 to give plasmid TCA311 (=pRIT14512) (see FIG. 5). The
insert was sequenced to verify that no modification had been
generated during the polymerase chain reaction. The coding sequence
for the fusion protein-D1/3-His is described FIG. 6.
[0155] 2. Transformation of AR58 Strain
[0156] Plasmid pRIT14512 was introduced into E. coli AR58 (Mott et
al., 1985, Proc. Natl. Acad. Sci., 82:88) a defective .lambda.
lysogen containing a thermosensitive repressor of the .lambda. pL
promoter.
[0157] 3. Growth and Induction of Bacterial Strain--Expression of
Prot-D1/3-E6E7-His
[0158] Cells of AR58 transformed with plasmid pRIT14512 were grown
in 100 ml of LB medium supplemented with 50 .mu.gr/ml of Kanamycin
at 30.degree. C. During the logarithmic phase of growth bacteria
were shifted to 39.degree. C. to inactivate the .lambda. repressor
and turn on the synthesis of protein D1/3-E6E7-his. The incubation
at 39.degree. C. was continued for 4 hours. Bacteria were pelleted
and stored at -20C.
[0159] 4. Characterization of Fusion Protein D1/3-E6E7-his
[0160] Frozen cells are thawed and resuspended in 10 ml of PBS
buffer. Cells are broken in a French pressure cell press SLM Aminco
at 20.000 psi (three passages). The extract is centrifuged at
16.000 g for 30 minutes at 4.degree. C.
[0161] After centrifugation of extracts described above, aliquots
of supernatant and pellet were analysed by SDS-polyacrylamide gel
electrophoresis and Western blotting.
[0162] A major band of about 48 kDa, localized in the pellet
fraction, was visualised by Coomassie stained gels and identified
in Western blots by rabbit polyclonal anti-protein-D and by Ni-NTA
conjugate coupled to calf intestinal alkaline phosphatase (Qiagen
cat. n.degree. 34510) which detects accessible histidine tail. The
level of expression represents about 1% of total protein.
EXAMPLE: VIb
[0163] In an analagous fashion the fusion protein of Lipo D 1/3 and
E6-E7 from HPV 16 was expressed in E. coli in the presence of
thioredoxin. The N-terminal of the pre-protein (388 aa) contains
MDP residues followed by 16 amino acids of signal peptide of
lipoprotein D (from Haemophilus Influenzae) which is cleaved in
vivo to give the mature protein (370 aa). Lipoprotein portion (aa 1
to 127) is followed by the proteins E6 and E7 in fusion. The C
terminal of the protein is elongated by TSGHHHHHH.
[0164] The protein was purified by the following protocol:
EXAMPLE VII
Lipoprotein D1/3-E6-E7-HIs (TIT) Purification
[0165] A) Solubilisation.
[0166] Cell paste is suspended to 60 OD.sub.600 in 2 M NaCl, 20 mM
Phosphate (NaH.sub.2PO4/K.sub.2HPO4) pH 7.5 in presence of 1 mM
Pefabloc as protease inhibitor prior cell lysis by three passes
through a French press disruptor (20,000 psi). Lysed cells are
pelleted 30 min at 15,000 g at 4.degree. C. In order to reduce
endotoxin level, bacterial cell pellet containing the recombinant
protein is washed twice in 4 M urea, 2 M NaCl, 20 mM Phosphate pH
7.5, once in 2% Empigen BB, 20 mM Phosphate pH 7.5 and finally
twice in 20 mM Phosphate buffer pH 7.0 to eliminate trace of
detergent (each wash is performed in the same volume used for cell
suspension). LipoProt.D1/3-E6-E7-His (TIT) is solubilised (in the
same volume used for cell suspension) by 8 M urea in 0.2 M
.beta.MercaptoEthanol (=.beta.MeOH), 20 mM PO4 pH 12 overnight at
4.degree. C. followed by a two hours incubation at RT.degree.
versus the same buffer. Cell debris are pelleted 30 min at 15,000 g
at 4.degree. C. Supernatant is kept at -20.degree. C.
[0167] B) Purification
[0168] 1) Anion Exchange Chromatography on Q-Sepharose Fast
Flow.
[0169] 225 ml of frozen sample is thawed at room temperature in a
cold water bath and is applied onto a Q-Sepharose fast flow column
(Pharmacia, XK 26/20) preequilibrated in 8 M urea, 0.2 M
.beta.MEOH, 20 mM PO4 pH 12 (30 ml resin/225 ml supernatant) at 45
cm/h. Column is washed by 8 M urea, 0.2 M MEOH, 20 mM PO4 pH 12,
until OD 280 nm reaches the baseline, followed by a second wash in
8 M urea, 20 mM Phosphate pH 12 (in 2 column volumes) Elution is
performed by NaCl steps (0.1 M, 0.25 M, 0.5 M NaCl, each step in
about 2 column volumes) in 8 M urea, 20 mM Phosphate pH 12, at 45
cm/h. 0.5 M NaCl-eluted fractions are pooled.
[0170] 2) Ion Metal Affinity Chromatography (IMAC).
[0171] 0.5 M NaCl-eluted fractions from Q Sepharose step are pooled
and dialyzed versus 0.2 M NaCl, 8 M urea, 20 mM Phosphate pH 10
before loading onto a Ni2+-NTA (Qiagen) column (XK 26/20,
Pharmacia) preequilibrated in 8 M urea, 20 mM PO4 pH 12 (30 ml
resin/61 ml sample) at 5.6 cm/h. Column is washed in 8 M urea, 20
mM PO4 pH 12 until the base line is reached then by 8 M urea, 20 mM
PO4 pH 10. Antigen is eluted by Imidazole steps (0.025 M, 0.05 M,
0.1 M, 0.15 M, 0.2 M, 0.5 M Imidazole, each step in two column
volumes) in 8 M urea, 20 mM PO4 pH 10, at 45 cm/h. 0.05 M
Imidazole-eluted fractions are pooled.
[0172] C) Concentration.
[0173] Imac sample is concentrated about 5 times (to 0.407 mg/ml)
on a 5 kDa Filtron Omega membrane in a stirred cell from AMICON at
RT.degree..
[0174] D) Dialysis
[0175] Concentrated sample is dialyzed at RT versus
decreasing-urea-concentration steps (4 M, 2 M urea) in 0.5 M
Arginine, 150 mM NaCl, 10 mM PO4 pH 6.8. Last dialysis against 0.5
M Arginine, 150 mM NaCl, 10 mM PO4 pH 6.8 is achieved at 4.degree.
C.
[0176] Results:
[0177] IMAC step is able to eliminate a 32 kD contaminant at 0.025
M Imidazole which eluted also some antigen. 0.05 M Imidazole-eluted
Antigen is estimated pure at 90% by Coomassie blue staining of
SDS-PAGE. After these two purification steps, sample is free of E.
coli contaminants. Western blotting analysis using specific
antigen-N and/or C terminus antibodies shows a heterogeneous
pattern of bands with higher and lower MW than the full length
protein. This pattern suggests the presence of aggregates and
incompletely processed protein and/or degraded one, copurified with
the full length protein.
EXAMPLE VIII
Construction of E.coli Strain B1002 Expressing Fusion
ProtD1/3-E7
[0178] Mutated (cys24.fwdarw.gly,glu26.fwdarw.gln) type HPV16
[0179] 1)-Construction of Expression Plasmid
[0180] Starting Material:
[0181] a)--Plasmid pRIT 14501 (=TCA 308) which codes for fusion
ProtD1/3-E7-His
[0182] b)--Plasmid LITMUS 28 (New England Biolabs cat n.degree.
306-28), a cloning vector pUC-derived
[0183] c)--Plasmid pMG MCS ProtD1/3 (pRIT 14589), a derivative of
pMG81 (described Supra) in which the codons 4-81 of NS1 coding
region from Influenza were replaced by the codons corresponding to
residues Ser 20.fwdarw.Thr 127 of mature protein D of Haemophilus
Influenzae strain 772, biotype 2 (H. Janson et al., 1991, Infection
and Immunity, Jan. p.119-125). The sequence of Prot-D1/3 is
followed by a multiple cloning site (11 residues) and a coding
region for a C-terminal histidine tail (6 His) Construction of
plasmid pRIT 14733(=TCA347): a plasmid expressing the fusion
Protein-D1/3-E7 mutated (cys24.fwdarw.gly, glu26.fwdarw.gln) with
His tail
[0184] The NcoI--XbaI fragment from pRIT 14501 (=TCA 308), bearing
the coding sequence of E7 gene from HPV16, elongated with an His
tail, was subcloned in an intermediate vector Litmus 28 useful for
mutagenesis to give pRIT 14909 (=TCA337) Double mutations cys24gly
(Edmonds and Vousden, J. Virology 63: 2650 (1989) and glu26gln
(Phelps et al, J. Virology 66: 2418-27 (1992) were chosen to impair
the binding to the antioncogene product of Retinoblastome gene
(pRB). The introduction of mutations in E7 gene was realized with
the kit "Quick Change Site directed Mutagenesis (Stratagene cat
n.degree. 200518) to give plasmid pRIT 14681 (=TCA343) After
verification of presence of mutations and integrity of the complete
E7 gene by sequencing, the mutated E7 gene was introduced into
vector pRIT 14589 (=pMG MCS ProtD1/3) to give plasmid pRIT 14733
(=TCA347) (FIG. 7).
[0185] The sequence for the fusion protein-D1/3-E 7 mutated
(cys24.fwdarw.gly, glu26.fwdarw.gln)-His is described in the FIG.
8.
[0186] 2)--Construction of Strain B1002 Expressing ProtD1/3-E7
Mutated (cys 24gly, glu26gln)-His/HPV16
[0187] Plasmid pRIT 14733 was introduced into E.coli AR58 (Mott et
al.,1985, Proc. Natl. Acad. Sci., 82:88) a defective X lysogen
containing a thermosensitive repressor of the .lambda. pL promoter,
to give strain B1002, by selection for transformants resistant to
kanamycine
[0188] 3)--Growth and Induction of Bacterial Strain
B1002--Expression of ProtD1/3-E7 Mutated (cys 24.fwdarw.gly,
glu26.fwdarw.gln)-His/HPV16
[0189] Cells of AR58 transformed with plasmid pRIT 14733 (B1002
strain) were grown at 30.degree. C. in 100 ml of LB medium
supplemented with 50 .mu.gr/ml of Kanamycin. During the logarithmic
phase of growth bacteria were shifted to 39.degree. C. to
inactivate the .lambda. repressor and turn on the synthesis of
ProtD1/3-E7 mutated-His/HPV16. The incubation at 39.degree. C. was
continued for 4 hours. Bacteria were pelleted and stored at
-20.degree. C.
[0190] 4)-Characterization of Fusion ProtD1/3-E7 mut
(cys24.fwdarw.gly, glu26.fwdarw.gln)-His type HPV16.
[0191] Frozen cells were thawed and resuspended in 10 ml of PBS
buffer.Cells were broken in a French Pressure cell press SLM Aminco
at 20 000 psi (three passages). The extract was centrifuged at
16000 g for 30 minutes at 4.degree. C.
[0192] After centrifugation of extracts described above, aliquots
of supernatant and pellet were analysed by SDS-polyacrylamide gel
electrophoresis and Western blotting.
[0193] A major band of about 33 kDa, localized in the pellet
fraction, was visualised by Coomassie stained gels and identified
in Western blots by rabbit polyclonal 22 J 70 anti-protein D, by
monoclonal anti E7/HPV16 from Zymed and by Ni-NTA conjugate coupled
to calf intestinal alkaline phosphatase (Qiagen cat. n.degree.
34510) which detects accessible histidine tail. The level of
expression represents about 3 to5% of total protein.
[0194] Cells of B1002 were separated from the culture broth by
centrifugation. The concentrated cells of B1002 were stored at
-65.degree. C.
EXAMPLE IX
Purification PROTD1/3 E7 (Dmutant) HPV 16
[0195] 1
[0196] a) Preparation of Cell Suspension
[0197] The frozen concentrated cells of B1002 were thawed and
resuspended in a cell disruption buffer at +4.degree. C. (see table
1) to a final optical density OD.sub.650 of 60 (corresponding to a
cell concentration of approximately 25 g DCW L.sup.-1)
[0198] b) Cell Disruption
[0199] The cells were disrupted by two passes at 1000 bar through a
high-pressure homogeniser (Rannie). The broken cell suspension was
collected in a flask maintained at 4.degree. C. CELL DISRUPTION
BUFFER: Na.sub.2 HPO.sub.4 (0.02N), NaCl (2M) pH adjusted to 7.5
with HCl 3N (Merck)
[0200] Purification
[0201] 2a) Dynamic Membrane Filtration (DMF.RTM.-PALL FILTRON)
[0202] 2 Litres of broken cell suspension (OD 60) is loaded on the
DMF.RTM., a dynamic filtration system from PALL, mounted with a 0.2
.mu.m cut-off membrane.
[0203] concentration from 2 Litres to 1L to give sample PCC1
[0204] washing at constant volume with 3 volumes (3L) of
empigen-EDTA buffer (concentration EDTA 1.86 g, Empigen (30%) 3.33
mL, PO.sub.4.sup.3- 0.5M 40.00 mL 40.00 mL) gave sample PD!
[0205] concentration from 1L to 300 mL gave sample PCC2
[0206] washing at constant volume with 10 volumes (3L) of empigen
buffer (concentration L.sup.-1: Empigen 30%, 3.33 mL,
PO.sub.4.sup.3- 0.5M 40 mL) pH 7.5 gave sample PD2
[0207] solubilisation of the protein in by addition of the same
volume (300 mL) of Guanidine hydrochloride 8M buffer (concentration
L.sup.-1 Gu.HCl 764 g;
[0208] Empigen 30% 3.33 mL, PO.sub.4.sup.3- 0.5M 40 mL) pH 7.5
[0209] recovery of the protein: Collection of the permeate--sample
P3 during Concentration to initial volume (300 mL) and
[0210] Diafiltration with 3 volume of Guanidine hydrochloride 4M
buffer (concentration L.sup.-1: Gu.HCl 328.12 g, Empigen (30%) 3.33
mL Po43-0.5M 40.00 mL) pH 7.5.
[0211] All these steps are made in a cold room (2-8.degree. C.), pH
adjusted with 0.5M PO.sub.4.sup.3.sup.-.
[0212] The P3 fraction is store at -20.degree. C. waiting for the
next purification step.
[0213] 2b) Zn-Chelating Sepharose Chromatography
[0214] The P3 fraction is thawed and injected in a packed and
equilibrated Zn-chelating sepharose FF.
[0215] After that, the column is:
[0216] Washed with around 3 volumes of Guanidine hydrochloride 4M
buffer (see above)--sample Zn-FT
[0217] Washed with around 5 volumes of Urea 4M buffer
(concentration L.sup.-1: Urea 240.24 g Empigen 3.33 mL
PO.sub.4.sup.3- 0.5M 40.00 mL)--sample Zn-W
[0218] Eluted with around 3 volumes of Urea 4M-Imidazole 20 mM
buffer (concentration L.sup.-1 Urea 240.24 g, Empigen (30%) 3.33 mL
Imidazole (1.36 g) PO.sub.4.sup.3- 0.5M 40.00 mL pH 7.5) buffer as
above, but concentration L.sup.-1 of Imidazole 34.04 g--sample
Zn-20
[0219] Eluted with Urea 4M-Imidazole 500 mM to the end of the UV
peak--sample Zn-500
[0220] The column is the washed with EDTA 50 mM and NaOH 0.5M. Zn
chelating sepharose eluate (Zn-500) is stored between 2-8.degree.
c. before the next purification step.
[0221] The Zn-chelating sepharose chromatography operations are
carried out at room temperature.
[0222] 2c) Q-sepharose Chromatography
[0223] The Zn-500 fraction is injected in a packed and equilibrated
Q-sepharose FF.
[0224] After that, the column is:
[0225] Washed with around 7 volumes of Urea 4M buffer (see above) -
sample QS-FT
[0226] Washed with around 10 volumes of Urea 4M buffer without
empigen (concentration L.sup.-1 Urea 240.24 g PO.sub.4.sup.3- 0.5M
40.00 mL)--sample QS-W1
[0227] Washed with around 10 volumes of Urea 6M buffer without
empigen (Urea 360.36 g/L)--sample QS-W2
[0228] Eluted with around 5 volumes of Urea 6M-NaCl 200 mM buffer
(conctration L.sup.-1: Urea 360.36 g NaCl 11.69 g, 40.00 MnL
PO.sub.4.sup.3-.
[0229] Eluted with around 3 volumes of Urea 6M-NaCl 500 mM buffer
(as above, but NaCl 29.22 g/L). The exact end of the fraction is
determined by the end of the UV peak.--sample QS-500
[0230] Eluted with 4 volumes of Urea 4M-NaCl 1M buffer (conctration
L.sup.-1 Urea 360.36, NaCl 58.44 g 40.00 mL PO.sub.4.sup.3-
(0.5)--sample QS-1M
[0231] The column is then washed with NaOH 0.5M
[0232] QS-sepharose eluate (QS-500) is stored between 2-8.degree.c
before the next purification step.
[0233] The Q-sepharose chromatography operations are carried out at
room temperature.
[0234] 2d) Ultrafiltration
[0235] The QS-500 fraction is then treated on a 10 kD
utrafiltration unit (Ultrasette--Pall Filtron)
[0236] The product is first concentrated to around 1 mg/mL of
protein and then diafiltrated against 10 volumes of phosphate
buffer.
[0237] The permeate (fraction UF-P) is discarded and the retentate
(fraction UF-R) is stored at 2-8.degree. c. waiting for final
filtration.
[0238] Ultrafiltration operations are carried out at 2-8.degree.
C.
[0239] 2e) Final Filtration
[0240] The final bulk (UF-R fraction) is filtered through a 0.22 cm
sterile filter (Millipak-Millipore) under laminar flow and in an
aseptic class 100 room.
[0241] The final concentration is between 0.5 and 1.0 .mu.g/mL.
[0242] The sterile bulk is stored at -20.degree. C.
EXAMPLE X
Construction of an E. coli Strain Expressing Fusion clyta-E6-his
(HPV 16)
[0243] 1. Construction of Expression Plasmid
[0244] a)--Plasmid pRIT14497 (=TCA307), that codes for fusion
ProtD1/3-E6-His/HPV16
[0245] b)--Plasmid pRIT14661 (=DVA2), an intermediate vector
containing the coding sequence for the 117 C-terminal codons of
LytA of Streptococcus Pneumoniae. Lyta is derived from
Streptococcus pneumoniae which synthesize an N-acetyl-L-alanine
amidase, amidase LYTA, (coded by the lytA gene {Gene, 43 (1986) pag
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.
[0246] 1.b Construction of Plasmid pRIT14634 (=TCA332): a Plasmid
Expressing the Fusion clyta-E6-His/HPV16
[0247] a) The first step was the purification of the large
NcoI-AflII restriction fragment from plasmid pRIT14497 and the
purification of the small AflII-AflIII restriction fragment from
pRIT14661
[0248] b) The second step was linking of clyta sequences to the
E7-His sequences (NcoI and AfilIII are compatible restriction
sites) that gave rise to the plasmid pRIT 14634 (=TCA332), coding
for the fusion protein clyta-E6-His under the control of the pL
promoter. (see FIG. 9) The coding sequence for the fusion protein
clyta-E6-His is decribed in FIG. 10.
[0249] Transformation of AR58 Strain
[0250] Plasmid pRIT14634 was introduced into E. coli AR58 (Mott et
al., 1985, Proc. Natl. Acad. Sci., 82:88) a defective .lambda.
lysogen containing a thermosensitive repressor of the .lambda. pL
promoter.
[0251] Growth and Induction of Bacterial Strain--Expression of
clyta-E6-His
[0252] Cells of AR58 transformed with plasmid pRIT14634 were grown
in 100 ml of LB medium supplemented with 50 .mu.gr/ml of Kanamycin
at 30.degree. C. During the logarithmic phase of growth bacteria
were shifted to 39.degree. C. to inactivate the .lambda. repressor
and turn on the synthesis of protein clyta-E6-his. The incubation
at 39.degree. C. was continued for 4 hours. Bacteria were pelleted
and stored at -20.degree. C.
[0253] 4. Characterization of Fusion clyta-E6-his
[0254] Frozen cells were thawed and resuspended in 10 ml of PBS
buffer. Cells were broken in a French pressure cell press SLM
Aminco at 20.000 psi (three passages). The extract was centrifuged
at 16.000 g for 30 minutes at 4.degree. C. After centrifugation of
extracts described above, aliquots of supernatant and pellet were
analysed by SDS-polyacrylamide gel electrophoresis and Western
blotting.
[0255] A major band of about 33 kDa, localized in the pellet
fraction, was visualised by Coomassie stained gels and identified
in Western blots by rabbit polyclonal anti-clyta antibodies and by
Ni-NTA conjugate coupled to calf intestinal alkaline phosphatase
(Qiagen cat. n.degree. 34510) which detects accessible histidine
tail. The level of expression represents about 3% of total
protein.
EXAMPLE XI
Construction of an E. coli strain expressing fusion clyta-E7-his
(HPV 16)
[0256] 1. Construction of Expression Plasmid
[0257] 1.a Starting Materials
[0258] a)--Plasmid pRIT14501 (=TCA308), that codes for fusion
ProtD1/3-E7-His/HPV16
[0259] b)-Plasmid pRIT14661 (=DVA2), an intermediate vector
containing the coding sequence for the 117 C-terminal codons of
LytA of Streptococcus Pneumoniae.
[0260] 1.b Construction of Plasmid pRIT14626 (=TCA330): a plasmid
expressing the fusion clyta-E7-His/HPV16
[0261] a) The first step was the purification of the large
NcoI-AflIII restriction fragment from plasmid pRIT14501 and the
purification of the small AflII-AflIII restriction fragment from
pRIT14661
[0262] b) The second step was linking of clyta sequences to the
E7-His sequences (NcoI and AflIII are compatible restriction
sites)that gave rise to the plasmid pRIT 14626 (=TCA330), coding
for the fusion protein clyta-E7-His under the control of the pL
promoter.
[0263] (FIG. 11)
[0264] The coding sequence for the fusion protein clyta-E7-His is
decribed in FIG. 12.
[0265] 2. Transformation of AR58 Strain
[0266] Plasmid pRIT14626 was introduced into E. coli AR58 (Mott et
al., 1985, Proc. Natl. Acad. Sci., 82:88) a defective ? lysogen
containing a thermosensitive repressor of the .lambda. pL
promoter.
[0267] 3. Growth and Induction of Bacterial Strain--Expression of
clyta-E7-His
[0268] Cells of AR58 transformed with plasmid pRIT14626 were grown
in 100 ml of LB medium supplemented with 50 .mu.gr/ml of Kanamycin
at 30.degree. C. During the logarithmic phase of growth bacteria
were shifted to 39.degree. C. to inactivate the .lambda. repressor
and turn on the synthesis of protein clyta-E7-his. The incubation
at 39.degree. C. was continued for 4 hours. Bacteria were pelleted
and stored at -20.degree. C.
[0269] 4. Characterization of Fusion clyta-E7-his
[0270] Frozen cells were thawed and resuspended in 10 ml of PBS
buffer. Cells were broken in a French pressure cell press SLM
Aminco at 20.000 psi (three passages). The extract was centrifuged
at 16.000 g for 30 minutes at 4.degree. C. After centrifugation of
extracts described above, aliquots of supernatant and pellet were
analysed by SDS-polyacrylamide gel electrophoresis and Western
blotting.
[0271] A major band of about 35 kDa, localized in the pellet
fraction, was visualised by Coomassie stained gels and identified
in Western blots by rabbit polyclonal anti-clyta antibodies and by
Ni-NTA conjugate coupled to calf intestinal alkaline phosphatase
(Qiagen cat. n.degree. 34510) which detects accessible histidine
tail. The level of expression represents about 5% of total
protein.
EXAMPLE XII
Construction of an E. coli Strain Expressing Fusion clyta-E6E7-his
(HPV 16)
[0272] 1. Construction of Expression Plasmid
[0273] 1.a Starting Materials
[0274] a)--Plasmid pRIT14512 (=TCA311), that codes for fusion
ProtD1/3-E6E7-His/HPV16
[0275] b)--Plasmid pRIT14661 (=DVA2), an intermediate vector
containing the coding sequence for the 117 C-terminal codons of
LytA of Streptococcus Pneumoniae.
[0276] 1.b Construction of Plasmid pRIT14629 (=TCA331): a Plasmid
Expressing the Fusion clyta-E6E7-His/HPV16
[0277] a) The first step was the purification of the large
NcoI-AflII restriction fragment from plasmid pRIT14512 and the
purification of the small AflII-AflIII restriction fragment from
pRIT14661
[0278] b) The second step was linking of clyta sequences to the
E7-His sequences (NcoI and AflIII are compatible restriction
sites)that gave rise to the plasmid pRIT 14629 (=TCA33 1), coding
for the fusion protein clyta-E6E7-His under the control of the pL
promoter. (see FIG. 13)
[0279] The coding sequence for the fusion protein clyta-E6E7-His is
decribed in FIG. 14.
[0280] 2. Transformation of AR58 Strain
[0281] Plasmid pRIT14629 was introduced into E. coli AR58 (Mott et
al., 1985, Proc. Natl. Acad. Sci., 82:88) a defective .lambda.
lysogen containing a thermosensitive repressor of the .lambda. pL
promoter.
[0282] 3. Growth and Induction of Bacterial Strain--Expression of
clyta-E6E7-His
[0283] Cells of AR58 transformed with plasmid pRIT14629 were grown
in 100 ml of LB medium supplemented with 50 .mu.gr/ml of Kanamycin
at 30.degree. C. During the logarithmic phase of growth bacteria
were shifted to 39.degree. C. to inactivate the .lambda. repressor
and turn on the synthesis of protein clyta-E6E7-his. The incubation
at 39.degree. C. was continued for 4 hours. Bacteria were pelleted
and stored at -20.degree. C.
[0284] 4. Characterization of Fusion clyta-E6E7-his
[0285] Frozen cells were thawed and resuspended in 10 ml of PBS
buffer. Cells were broken in a French pressure cell press SLM
Aminco at 20.000 psi (three passages). The extract was centrifuged
at 16.000 g for 30 minutes at 4.degree. C.
[0286] After centrifugation of extracts described above, aliquots
of supernatant and pellet were analysed by SDS-polyacrylamide gel
electrophoresis and Western blotting.
[0287] A major band of about 48 kDa, localized in the pellet
fraction, was visualised by Coomassie stained gels and identified
in Western blots by rabbit polyclonal anti-clyta antibodies and by
Ni-NTA conjugate coupled to calf intestinal alkaline phosphatase
(Qiagen cat. n.degree. 34510) which detects accessible histidine
tail. The level of expression represents about 1% of total
protein.
EXAMPLE XIII
Prot D1/3 E7 his (HPV 18) (E.Coli B1011) Protein D1/3 E7 his HPV
expressed with Thioredoxin in Trans (E.Coli B1012)
[0288] 1)--Construction of Expression Plasmids
[0289] 1).a. Construction of Plasmid TCA316(=pRIT 14532) a Plasmid
Expressing the Fusion Protein-D1/3-E7-His IHPV18
[0290] Starting Materials
[0291] a)--Plasmid pMG MCS prot D1/3 (=pRIT14589) is a derivative
of pMG81 (described in UK patent application n.degree. 951 3261.9
published as WO97/01640 in which the codons 4-81 of NS1 coding
region from Influenza were replaced by the codons corresponding to
residues Ser 20.fwdarw.Thr 127 of mature protein D of Haemophilus
Influenzae strain 772, biotype 2 (H. Janson et al., 1991, Infection
and Immunity, Jan. p.119-125). The sequence of Prot-D1/3 is
followed by a multiple cloning site (11 residues) and a coding
region for a C-terminal histidine tail (6 His) (see FIG. 15). This
plasmid is used to express the fusion protein D1/3-E7-his.
[0292] b)--HPV genomic E6 and E7 sequences of prototype HPV18 (Cole
et al, J. Mol. Biol. (1987) 193,599-608) were amplified from HPV16
full length genome cloned in pBR322 (obtained from Deutsche
Krebsforschungszentrum (DKFZ), Referenzzentrum fur human pathogen
Papillomaviruses--D 69120--Heidelberg) and were subcloned into
pUC19 to give TCA 302 (=pRIT14467).
[0293] Construction of Plasmid TCA 316(=pRIT14532)
[0294] The nucleotides sequences corresponding to amino acids
1.fwdarw.105 of E7 protein were amplified from pRIT14467. During
the polymerase chain reaction, NcoI and SpeI restriction sites were
generated at the 5' and 3' ends of the E7 sequences allowing
insertion into the same sites of plasmid pMGMCS Prot D1/3 to give
plasmid TCA316 (=pRIT14532). The insert was sequenced and a
modification versus E7/HPV18 prototype sequence was identified in
E7 gene (nucleotide 128 G.fwdarw.A) generating a substitution of a
glycine by a glutamic acid (aa 43 in E7, position 156 in fusion
protein). The sequence for the fusion protein-D1/3-E7-His/HPV18 is
described in FIG. 16.
[0295] 1).b. Construction of Plasmid TCA313 (=pRIT14523): a Plasmid
Expressing Thioredoxin
[0296] Starting Materials
[0297] a)--Plasmid pBBR1MCS4(Antoine R. and C. Locht, Mol.
Microbiol. 1992, 6, 1785-1799 ; M. E. Kovach et al. Biotechniques
16, (5), 800-802)which is compatible with plasmids containing ColE1
or P15a origins of replication.
[0298] b)--Plasmid pMG42 (described in WO93/04175) containing the
sequence of promoter pL of Lambda phage
[0299] c)--Plasmid pTRX (Invitrogen, kit Thiofusion K350-01)
bearing the coding sequence for thioredoxin followed by AspA
transcription terminator.
[0300] Construction of Plasmid TCA313(=pRIT14523)
[0301] The fragment EcoRI-NdeI fragment from pMG42, bearing pL
promoter and the NdeI-HindIII fragment from pTRX, bearing the
coding sequence for thioredoxin followed by AspA terminator, were
purified and ligated into the EcoRI and HindIII sites of plasmid
vector pBBR 1MCS4 to give plasmid TCA313(=pRIT14523) (see FIG.
17).
[0302] The sequence for thioredoxin is described in FIG. 18.
[0303] 2)--Transformation of AR58 Strain
[0304] 2).a. To Obtain Strain B1011 Expressing
ProtD1/3-E7-His/HPV18
[0305] Plasmid pRIT14532 was introduced into E. coli AR58 (Mott et
al., 1985, Proc. Natl. Acad. Sci., 82:88) a defective .lambda.
lysogen containing a thermosensitive repressor of the .lambda. pL
promoter, by selection for transformants resistant to
kanamycine.
[0306] 2).b. Construction of Strain B1012 Expressing
ProtD1/3-E7-His/HPV18 and Thioredoxin
[0307] Plasmid pRIT14532 and pRIT14523 were introduced into E. coli
AR58 (Mott et al., 1985, Proc. Natl. Acad. Sci., 82:88) a defective
.lambda. lysogen containing a thermosensitive repressor of the
.lambda. pL promoter, by double selection for transformants
resistant to kanamycin and ampicillin.
[0308] 3)--Growth and Induction of Bacterial Strains B1011 and
B1012--Expression of Prot-D1/3-E7-His/HPV18 Without and With
Thioredoxin in Trans
[0309] Cells of AR58 transformed with plasmids pRIT14532 (B1011
strain) and Cells of AR58 transformed with plasmids pRIT14532 and
pRIT14523 (B1012 strain) were grown at 30.degree. c. in 100 ml of
LB medium supplemented with 50 .mu.gr/ml of Kanamycin for B1011
strain and supplemented 50 .mu.gr/ml of Kanamycin and 100 .mu.gr/ml
of Ampicillin for B1012 strain. During the logarithmic phase of
growth bacteria were shifted to 39.degree. C. to inactivate the
.lambda. repressor and turn on the synthesis of protein
D1/3-E7-his/HPV18 and thioredoxin. The incubation at 39.degree. C.
was continued for 4 hours. Bacteria were pelleted and stored at
-20.degree. C.
[0310] Characterization of Fusion Protein D1/3-E7-his/HPV18
[0311] Preparation of Extracts
[0312] Frozen cells are thawed and resuspended in 10 ml of PBS
buffer. Cells are broken in a French pressure cell press SLM Aminco
at 20.000 psi (three passages). The extract is centrifuged at
16.000 g for 30 minutes at 4.degree. C.
[0313] Analysis on Coomassie-Stained SDS-Polyacrylamide Gels and
Western Blots
[0314] After centrifugation of extracts described above, aliquots
of supernatant and pellet were analysed by SDS-polyacrylamide gel
electrophoresis and Western blotting.
[0315] The fusion protD1/3-E7-His (about 31 kDa) was visualised by
Coomassie stained gels in the pellet fraction for strain B1011 and
partially localized (30%) in the supernatant fraction for strain
1012 and was identified in Western blots by rabbit polyclonal
anti-protein-D and by Ni-NTA conjugate coupled to calf intestinal
alkaline phosphatase (Qiagen cat. n.degree. 34510) which detects
accessible histidine tail. The level of expression represents about
1-3% of total protein as shown on a Coomassie-stained
SDS-polyacrylamide gel.
[0316] For the extract of strain B1012 the thioredoxin (about 12
KDa) was visualised by coomassie stained gel in the supernatant and
identified in western blots by monoclonal anti thioredoxin
(Invitrogen R920-25)
[0317] Purification of Prot D1/3 E7-his/HPV18
[0318] Recombinant HPV 18-ProtD1/3-E7-His is expressed in E. coli
(as described above) AR58 strain. All steps are performed at room
temperature (RT.congruent.22.degree. C.). Proteins are followed by
monitoring OD 280 nm. Between steps, antigens positive fractions
are kept at -20.degree. C.
[0319] Purified antigen is stable one week at -20.degree. C. and
4.degree. C. (no degradation) but appears more susceptible to
oxidation after incubation at 37.degree. C.
[0320] d)--Solubility
[0321] Protein solubility is pH dependent (see below) with decrease
of solubility for pH<7.4:
4 PBS pH 7.4 686 .mu.g/ml 100% PBS pH 7.2 560 .mu.g/ml 81% PBS pH
7.0 498 .mu.g/ml 72% PBS pH 6.8 327 .mu.g/ml 48%
[0322] e)--The HPV 18 Prot D1/3 E7 proteiin is composed of 227
amino acids. Its theoretical molecular weight is 25.9 kDa, and a
theoretical isoelectric point of 5.83. It migrates at about 31.5
kDa in reducing SDS PAGE.
EXAMPLE XIV
Purification of HPV 18 Protein D1/3 E7
[0323] a)--Solubilisation
[0324] Cell paste is suspended to 60 OD.sub.600 in 2 M NaCl, 20 mM
Phosphate. (NaH.sub.2PO4/K.sub.2HPO4) pH 7.6 prior cell lysis by
two passes through a Rannie disruptor. Lysed cells are pelleted 30
min at 9,000 rpm in a JA 10 rotor at 4.degree. C. In order to
reduce endotoxin level, bacterial cell pellet containing the
recombinant protein is washed once in 5 mM EDTA, 2 M NaCl, PBS pH
7.4; once in 4 M urea, 20 mM Phosphate pH 7.4 and finally once in
PBS pH 7.4 to eliminate trace of EDTA (each wash is performed in
twice volume used for cell suspension). HPV18Prot.D1/3-E7-His (TIT
for Thioredoxin In Trans) is solubilised (in the same volume used
for cell suspension) by 6 M Guanidine-Chloride, 50 mM PO4 pH 7.6
overnight at 4.degree. C. Cell debris are pelleted 30 min at 9,000
rpm in a JA 10 rotor at 4.degree. C. Supernatant is supplemented
with 0.5% Empigen BB and incubated 30 min at RT.
[0325] b)--Purification
[0326] 1).a. Immobilized Metal Affinity Chromatography
[0327] 125 .ml of sample are loaded onto a Zn.sup.2+-Chelating
Sepharose FF column (XK 26/20, Pharmacia; 50 ml gel/125 ml
solubilisation) preequilibrated in 0.5% Empigen BB, 6 M
Guanidine-Chloride, 50 mM PO4 pH 7.6 at 4 ml/min. Column is washed
by Guanidine Chloride 6M, PO4 50 mM pH 7.6 until the base line is
reached then by 6 M urea, 0.5 M NaCl, 50 mM PO4 pH 7.6. Antigen is
eluted by 0.25 M-Imidazole in 6 M urea, 0.5 M NaCl, 50 mM PO4 pH
7.6, at 2 ml/min (FIG. 1B). IMAC-eluted sample is dialyzed at
4.degree. C. versus PBS pH 7.4
[0328] 1).b. Affi-Prep Polymixin (Bio-Rad)
[0329] To reduce endotoxin level, 28 mg (37 ml) of antigen are
incubated in batch mode with 2 ml of Affiprep Polymyxin resin
prequilibrated in PBS pH 7.4, over night at room temperature.
Protein recovery is estimate at 60% and endotoxin content is
reduced 6.5 times.
[0330] 1).c. Analysis
[0331] Purified antigen analyzed on reducing-SDS-PAGE presents a
major 30 kDa band with a second one at 55 kDa, after Coomassie Blue
or Silver Staining. In a non reducing SDS-PAGE,
HPV-18-ProtD1/3-E7-His appears mainly like a smear with Molecular
Weight.gtoreq.175 kDa. However this oxidation can be reversed by
addition of 5 mM of .beta.-Mercapto-Ethanol. This pattern is
confirmed by anti ProtD or by anti His Western Blotting
analysis.
[0332] c)--Stability
[0333] Purified antigen is stable one week at -20.degree. C. and
4.degree. C. (no degradation) but appears more susceptible to
oxidation after incubation at 37.degree. C.
[0334] d)--Solubility
[0335] Protein solubility is pH dependent (see below) with decrease
of solubility for pH <7.4:
5 PBS pH 7.4 686 .mu.g/ml 100% PBS pH 7.2 560 .mu.g/ml 81% PBS pH
7.0 498 .mu.g/ml 72% PBS pH 6.8 327 .mu.g/ml 48%
[0336] HPV18-ProtD1/3-E7-His protein is composed of 227 amino
acids. Its theoretical molecular weight is 25.9 kDa. It migrates at
about 31.5 kDa in reducing SDS-PAGE. Theoretical isoelectric point
is 5.83.
EXAMPLE XV
Construction of E.coli strain B1098 expressing fusion
ProtD1/3-E7
[0337] Mutated (cys27.fwdarw.gly,glu29.fwdarw.gln) type HPV18
[0338] 1)-Construction of Expression Plasmid
[0339] Starting Material:
[0340] a)--Plasmid pRIT 14532 (=TCA 316) which codes for fusion
ProtD 1/3-E7-His
[0341] b)--Plasmid LITMUS 28 (New England Biolabs cat n.degree.
306-28), a cloning vector pUC-derived
[0342] c)--Plasmid pMG MCS ProtD1/3 (pRIT 14589), a derivative of
pMG81 (described supra) in which the codons 4-81 of NS1 coding
region from Influenza were replaced by the codons corresponding to
residues Ser 20.fwdarw.Thr 127 of mature protein D of Haemophilus
Influenzae strain 772, biotype 2 (H. Janson et al., 1991, Infection
and Immunity, Jan. p. 119-125). The sequence of Prot-D1/3 is
followed by a multiple cloning site (11 residues) and a coding
region for a C-terminal histidine tail (6 His)
[0343] Construction of Plasmid pRIT 14831(=TCA355): a Plasmid
Expressing the Fusion Protein-D1/3-E7 Mutated (cys27.fwdarw.gly,
glu29.fwdarw.gln) With His Tail
[0344] The NcoI-XbaI fragment from pRIT 14532 (--TCA 316), bearing
the coding sequence of E7 gene from HPV18, elongated with an His
tail, was subcloned in an intermediate vector Litmus 28 useful for
mutagenesis to give pRIT 14910 (=TCA348) By analogy with E7/HPV 16
mutagenesis, double mutations cys27gly and glu29gln were chosen to
impair the binding to the antioncogene product of Retinoblastome
gene (pRB).
[0345] The introduction of mutations in E7 gene was realized with
the kit "Quick Change Site directed Mutagenesis (Stratagene cat
n.degree. 200518). As the sequencing of pRIT14532 had pointed out
the presence of a glutamic acid in position 43 of E7 instead of a
glycine in the prototype sequence of HPV 18, a second cycle of
mutagenesis was realized to introduce a glycine in position 43. We
obtained plasmid pRIT 14829 (=TCA353). After verification of
presence of mutations and integrity of the complete E7 gene by
sequencing, the mutated E7 gene was introduced into vector pRIT
14589 (=pMG MCS ProtD1/3) to give plasmid pRIT 14831 (=TCA355) (see
FIG. 17).
[0346] The sequence for the fusion protein-D1/3-E 7 mutated
(cys27.fwdarw.gly, glu29.fwdarw.gln)-His is described in the FIG.
18.
[0347] 2) Construction of Strain B1098 Expressing ProtD1/3-E7
Mutated (cys 27gly, glu29gln)-His/HPV18
[0348] Plasmid pRIT 14831 was introduced into E.coli AR58 (Mott et
al., 1985, Proc. Natl. Acad. Sci., 82:88) a defective .lambda.
lysogen containing a thermosensitive repressor of the .lambda. pL
promoter, to give strain B1098, by selection for transformants
resistant to kanamycin.
[0349] 3)--Growth and Induction of Bacterial Strain
B1098--Expression of ProtD1/3-E7 Mutated (cys 27.fwdarw.gly,
glu29.fwdarw.gln)-His/HPV18
[0350] Cells of AR58 transformed with plasmid pRIT 14831 (B1098
strain) were grown at 30.degree. C. in 100 ml of LB medium
supplemented with 50 .mu.gr/ml of Kanamycin. During the logarithmic
phase of growth bacteria were shifted to 39.degree. C. to
inactivate the .lambda. repressor and turn on the synthesis of
ProtD1/3-E7 mutated -His/HPV18. The incubation at 39.degree. C. was
continued for 4 hours. Bacteria were pelleted and stored at
-20.degree. C.
[0351] 4)--Characterization of Fusion ProtD1/3-E7 mut
(cys24.fwdarw.gly, glu26.fwdarw.gln)-His Type HPV16
[0352] Frozen cells were thawed and resuspended in 10 ml of PBS
buffer. Cells were broken in a French Pressure cell press SLM
Aminco at 20 000 psi (three passages). The extract was centrifuged
at 16000 g for 30 minutes at 4.degree. C.
[0353] Analysis on Coomassie Stained SDS-Polyacrylamide Gels and
Western Blots
[0354] After centrifugation of extracts described above, aliquots
of supernatant and pellet were analysed by SDS-polyacrylamide gel
electrophoresis and Western blotting. A major band of about 31 kDa,
localized in the pellet fraction, was visualised by Coomassie
stained gels and identified in Western blots by rabbit polyclonal
22 J 70 anti-protein D and by monoclonal Penta-His (Qiagen cat.
n.degree. 34660) which detects accessible histidine tail. The level
of expression represents about 3 to 5% of total protein.
EXAMPLE XVI
Construction of an E. coli Strain Expressing Fusion Protein-D
1/3-E6-his/HPV18
[0355] 1. Construction of Expression Plasmid
[0356] a) Plasmid pMG MCS prot D1/3 (=pRIT14589) is a derivative of
pMG81 (described supra) in which the codons 4-81 of NS1 coding
region from Influenza were replaced by the codons corresponding to
residues Ser 20.fwdarw.Thr 127 of mature protein D of Haemophilus
Influenzae strain 772, biotype 2 (H. Janson et al., 1991, Infection
and Immunity, Jan. p.119-125). The sequence of Prot-D 1/3 is
followed by a multiple cloning site (11 residues) and a coding
region for a C-terminal histidine tail (6 His). This plasmid is
used to express the fusion protein D1/3-E6-his.
[0357] HPV genomic E6 and E7 sequences type HPV18 (Cole et al., J.
Mol. Biol. 1987, 193, p.599-608.) were amplified from HPV18 full
length genome cloned in pBR322 (obtained from Deutsches
Krebsforschungszentrum (DKFZ), Referenzzentrum fur human pathogen
Papillomaviruses--D 69120--Heidelberg) and were subcloned into
pUC19 to give TCA 302 (=pRIT14467).
[0358] Construction of Plasmid TCA 314(=pRIT14526): a Plasmid
Expressing the Fusion Protein-D1/3-E6-His/HPV18
[0359] The nucleotides sequences corresponding to amino acids
[0360] 1.fwdarw.158 of E6 protein were amplified from pRIT14467.
During the polymerase chain reaction, NcoI and Spel restriction
sites were generated at the 5' and 3' ends of the E6 sequences
allowing insertion into the same sites of plasmid pMGMCS Prot D1/3
to give plasmid TCA314 (=pRIT14526) (see FIG. 21). The insert was
sequenced to verify that no modification had been generated during
the polymerase chain reaction. The coding sequence for the fusion
protein-D1/3-E6-His is described in FIG. 22.
[0361] Transformation of AR58 Strain
[0362] Plasmid pRIT14526 was introduced into E. coli AR58 (Mott et
al., 1985, Proc. Natl. Acad. Sci., 82:88) a defective .lambda.
lysogen containing a thermosensitive repressor of the .lambda. pL
promoter.
[0363] 3. Growth and Induction of Bacterial Strain--Expression of
Prot-D1/3-E6-His
[0364] Cells of AR58 transformed with plasmid pRIT14526 were grown
in 100 ml of LB medium supplemented with 50 .mu.gr/ml of Kanamycin
at 30.degree. C. During the logarithmic phase of growth bacteria
were shifted to 39.degree. C. to inactivate the .lambda. repressor
and turn on the synthesis of protein D1/3-E6-his. The incubation at
39.degree. C. was continued for 4 hours. Bacteria were pelleted and
stored at -20C.
[0365] 4. Characterization of Fusion Protein D1/3-E6-his
[0366] Frozen cells are thawed and resuspended in 10 ml of PBS
buffer. Cells are broken in a French pressure cell press SLM Aminco
at 20.000 psi (three passages). The extract is centrifuged at
16.000 g for 30 minutes at 4.degree. C. After centrifugation of
extracts described above, aliquots of supernatant and pellet were
analysed by SDS-polyacrylamide gel electrophoresis and Western
blotting.
[0367] A major band of about 32 kDa, localized in the pellet
fraction, was visualised by Coomassie stained gels and identified
in Western blots by rabbit polyclonal anti-protein-D and by Ni-NTA
conjugate coupled to calf intestinal alkaline phosphatase (Qiagen
cat. n.degree. 34510) which detects accessible histidine tail. The
level of expression represents about 3-5% of total protein.
EXAMPLE XVII
Construction of an E. coli strain expressing fusion
Protein-D1/3-E6E7-his/HPV18
[0368] 1. Construction of Expression Plasmid
[0369] a) Plasmid pMG MCS prot D1/3 (=pRIT14589) is a derivative of
pMG81 (described supra) in which the codons 4-81 of NS 1 coding
region from Influenza were replaced by the codons corresponding to
residues Ser 20.fwdarw.Thr 127 of mature protein D of Haemophilus
Influenzae strain 772, biotype 2 (H. Janson et al., 1991, Infection
and Immunity, Jan. p.119-125). The sequence of Prot-D1/3 is
followed by a multiple cloning site (11 residues) and a coding
region for a C-terminal histidine tail (6 His). This plasmid is
used to express the fusion protein D1/3-E6E7-his.
[0370] b) HPV genomic E6 and E7 sequences type HPV18 (Cole et al.,
J. Mol. Biol. 1987, 193, 599-608) were amplified from HPV18 full
length genome cloned in pBR322 (obtained from Deutsches
Krebsforschungszentrum (DKFZ), Referenzzentrum fur human pathogen
Papillomaviruses--D 69120--Heidelberg) and were subcloned into
pUC19 to give TCA 302 (=pRIT14467).
[0371] c) The coding sequences for E6 and E7 in TCA302 (=pRIT
14467) were modified with a synthetic oligonucleotides adaptor
(inserted between Hga I and Nsi I sites) introducing a deletion of
11 nucleotides between E6 and E7 genes, removing the stop codon of
E6 and creating fused E6 and E7 coding sequences in the plasmid
TCA320(=pRIT 14618) see FIG. 23.
[0372] Construction of Plasmid TCA 328(=pRIT14567): a Plasmid
Expressing the Fusion Protein-D1/3-E6E7-His/HPV18
[0373] The nucleotides sequences corresponding to amino acids
[0374] 1.fwdarw.263 of fused E6E7 protein were amplified from
pRIT14618. During the polymerase chain reaction, NcoI and SpeI
restriction sites were generated at the 5' and 3' ends of the E6E7
fused sequences allowing insertion into the same sites of plasmid
pMGMCS Prot D1/3 to give plasmid TCA328 (=pRIT14567) (see FIG. 24).
The insert was sequenced to verify that no modification had been
generated during the polymerase chain reaction. The coding sequence
for the fusion protein-D1/3-E6E7-His is described in FIG. 25.
[0375] 2. Transformation of AR58 Strain
[0376] Plasmid pRIT14567 was introduced into E. coli AR58 (Mott et
al., 1985, Proc. Natl. Acad. Sci., 82:88) a defective .lambda.
lysogen containing a thermosensitive repressor of the .lambda. pL
promoter.
[0377] 3. Growth and Induction of Bacterial Strain--Expression of
Prot-D1/3-E6E7-His
[0378] Cells of AR58 transformed with plasmid pRIT14512 were grown
in 100 ml of LB medium supplemented with 50 .mu.gr/ml of Kanamycin
at 30.degree. C. During the logarithmic phase of growth bacteria
were shifted to 39.degree. C. to inactivate the .lambda. repressor
and turn on the synthesis of protein D1/3-E6E7-his. The incubation
at 39.degree. C. was continued for 4 hours. Bacteria were pelleted
and stored at -20C.
[0379] 4. Characterization of Fusion Protein D1/3-E6E7-his
[0380] Frozen cells are thawed and resuspended in 10 ml of PBS
buffer. Cells are broken in a French pressure cell press SLM Aminco
at 20.000 psi (three passages). The extract is centrifuged at
16.000 g for 30 minutes at 4.degree. C.
[0381] After centrifugation of extracts described above, aliquots
of supernatant and pellet were analysed by SDS-polyacrylamide gel
electrophoresis and Western blotting. A major band of about 48 kDa,
localized in the pellet fraction, was visualised by Coomassie
stained gels and identified in Western blots by rabbit polyclonal
anti-protein-D and by Ni-NTA conjugate coupled to calf intestinal
alkaline phosphatase (Qiagen cat. n.degree. 34510) which detects
accessible histidine tail. The level of expression represents about
1% of total protein.
EXAMPLE XVIII
Vaccine Formulations
[0382] Vaccines are formulated with a Protein from the above
examples expressed in E. coli from the strain AR58, and as
adjuvant, the formulation comprising a mixture of 3 de -O-acylated
monophosphoryl lipid A (3D-MPL) and aluminium hydroxide or 3D-MPL
and/or QS21 optionally in an oil/water emulsion, and optionally
formulated with cholesterol.
[0383] 3D-MPL: is a chemically detoxified form of the
lipopolysaccharide (LPS) of the Gram-negative bacteria Salmonella
minnesota.
[0384] Experiments performed at Smith Kline Beecham Biologicals
have shown that 3D-MPL combined with various vehicles strongly
enhances both the humoral and a THI type of cellular immunity.
[0385] QS21: is one saponin purified from a crude extract of the
bark of the Quillaja Saponaria Molina tree, which has a strong
adjuvant activity: it activates both antigen-specific
lymphoproliferation and CTLs to several antigens.
[0386] Vaccine containing an antigen of the invention containing
3D-MPL and alum may be prepared in analogous manner to that
described in WO93/19780 or 92/16231.
[0387] Experiments performed at Smith Kline Beecham Biologicals
have demonstrated a clear synergistic effect of combinations of
3D-MPL and QS21 in the induction of both humoral and TH1 type
cellular immune responses. Vaccines containing an antigen such
antigens are described in U.S. Pat. No. 5,750,110.
[0388] The oil/water emulsion is composed of 2 oils (a tocopherol
and squalene), and 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).
[0389] Experiments performed at Smith Kline Beecham Biologicals
have proven that the adjunction of this O/W emulsion to MPL/QS21
further increases their immunostimulant properties.
[0390] Preparation of Emulsion SB62 (2 Fold Concentrate)
[0391] 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 5 g of DL alpha tocopherol and 5 ml of
squalene are vortexed to mix thoroughly. 90 ml 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.
[0392] Preparation of Prot.D1/3 E7 QS21/3D MPL oil in Water
Formulation
[0393] ProtD1/3-E7 (5 .mu.g) was diluted in 10 fold concentrated
PBS pH 6.8 and H.sub.2O before consecutive addition of SB62, 3 D
MPL (5 .mu.g), QS21 (5 .mu.g) and 50 .mu.g/ml thiomersal as
preservative at 5 min interval. The emulsion volume is equal to 50%
of the total volume (50 .mu.l for a dose of 100 .mu.l). All
incubations were carried out at room temperature with agitation.
The adjuvants controls without antigen were prepared by replacing
the protein by PBS.
[0394] Tumour Regression Experiments (HPV 16) with PROT D E7
[0395] Vaccine Antigen: Fusion Protein ProtD E7
[0396] Protein D is a lipoprotein exposed on the surface of the
Gram-negative bacteria Haemophilus influenzae.
[0397] The inclusion of the 109 first residues of the protein D as
fusion partner is incorporated to provide the vaccine antigen with
bystander help properties. The antigen was formulated with QS21
3D-MPL and SB62 as described supra.
EXAMPLE XIX
In vivo Tumour Regression Experiments Tumour cell line TC1:
[0398] Primary lung epithelial cells from C57BL.6 mice were
immortalised by HPV 16 E6 and E7 and then transformed with an
activated ras oncogene, producing a tumourigenic cell line
expressing E6 and E7 (Lin KY et al. 1996). The E7 expression has
been verified by FACS analysis of fixed and permeabilised TC1 cells
using the mouse anti-HPV 16 E7 Mab (Triton Corp. Alameda,
Calif.)
[0399] Tumour Growth:
[0400] TC 1 cells growing in vitro culture were trypsinised, washed
two times in serum-free medium and were injected S.C. in the right
flank of the mice. To assess treatment of established tumours, TC1
cells were injected at a dose of 3.times.10e4 cells/mouse. One and
two weeks after the tumour cell injection, mice were vaccinated
with 5 .mu.g in 100 .mu.l of protD 1/3 E7 His intra foot pad (50
.mu.l/foot pad) in PBS or in the 3D-MPL, QS21 and SB62 or with PBS
or with the adjuvant alone. Five C57BL/6 mice (Iffa Credo) were
used in each group. Mice were monitored twice a week for tumour
growth. The mean tumour mass/group is shown in FIG. 26, the mice
vaccinated with protD 1/3 E7 His in PBS or with PBS or the adjuvant
alone developed progressively growing tumours (0-1 tumour-free
animal/group). On the contrary, four out of five mice vaccinated
with protD1/3 E7 His in adjuvant did not develop a tumour, one
animal developed a very small and stable tumour at day 40. This
results indicate that the protein protD1/3 E7 His from HPV 16
formulated in adjuvant is able to induce the regression of small
established tumours expressing this antigen.
[0401] Immunological Read Out
[0402] Proliferation Assay:
[0403] For in vitro assay, Lymphocytes were prepared by crushing
the spleen or the popliteal lymph nodes from the vaccinated mice at
day 69.
[0404] An aliquot of 2.times.10e5 cells was plated in triplicate in
96 well plates with decreasing concentrations (10, 1, 0.1 .mu.g/ml)
of protD 1/3 E7 His coated or not onto latex microbeads (Sigma) to
restimulate the cells in vitro (72 Hrs). T cell proliferation was
measured by 3H thymidine incorporation.
[0405] FIGS. 27 and 28 compares the ability of protD E7 to
stimulate the proliferation of splenocytes and lymph node cells
primed in vivo either by PBS, 3D-MPL, QS21 SB62, ProtD1/3 E7 His
and ProtD1/3 E7 His+the adjuvant of 3D-MPL, QS21, SB62 and shows
that high proliferative responses in spleen were detected only in
mice immunised with protD1/3 E7 His in adjuvant compared to the
other groups.
[0406] Antibody Response
[0407] Individual serum were taken at the same time as the organs
were taken and submitted to indirect ELISAs.
[0408] 5 .mu.g/ml of purified E7 protein was used as coated
antigen. After saturation in PBS+1% newborn calf serum 1 Hr at
37.degree. C., the sera were serially diluted (starting at
{fraction (1/100)}) in the saturation buffer and incubated O/N at
4.degree. C. or 90 min at 37.degree. C. After washing in PBS Tween
20 0.1%, biotinylated goat Anti mouse Ig ({fraction (1/1000)}) or
goat anti mouse Ig subclass (total IgG, IgGl, IgG2a, IgG2b)
antisera (1/5000) were used as second antibodies, after an
incubation of 90 min at 37.degree. C., streptavidin coupled to
peroxydase was added and TMB (tetra-methyl-benzidine/peroxide) was
used as substrate, after 10 min. the reaction was stopped with
H2SO4 0.5 M and the O.D.450 was determined.
[0409] The subclass-specific anti E7 titers elicited by the
vaccinations in the different groups of mice are shown in FIG. 29
as a comparison of the relative mean midpoint dilution of the
serum.
[0410] These results show that a weak antibody response is
triggered with 2 injections of ProtD 1/3 E7 HPV16 alone.
[0411] Much more anti-E7 antibodies are generated when ProtD1/3 E7
was injected in the presence of the adjuvant SB62, QS21+3D-MPL.
[0412] No IgA nor IgM were detected in any of the serum samples
even in the serum of the mice that received ProtD 1/3 E7 in the
adjuvant SB62, QS21+3D-MPL (data not shown) On the contrary, the
total IgG level was slightly increased by the vaccination of the
mice with ProtD 1/3 E7 alone and was greatly increased by the
addition of the adjuvant SB62, QS21+3D-MPL to the protein. The
analysis of the concentrations of the different IgG subclass show
that a mixed antibody response has been induced as the
concentration of all types of IgG subclass analyzed (IgG1, IgG2a,
and IgG2b) were increased in the serum of the mice that received
the adjuvanted antigen, compared to the concentration observed in
the serum of mice that received the antigen or the adjuvant alone.
The predominant isotype found was IgG2b which represented more than
80% of the total of IgG), this isotype is generally said to be
associated with the induction of a TH1 type immune response.
EXAMPLE XX
In vivo Tumour Protection Experiments
[0413] Mice were immunised 2 times at 14 days interval with either
PBS, adjuvant of example 1, 5 .mu.g of protD1/3 E7 His or 5 .mu.g
of protD1/3 E7 His in the adjuvant of example 1 intra foot pad in a
volume of 100 .mu.l (50 .mu.l/foot pad).
[0414] Tumour Growth:
[0415] Four weeks after the latest vaccination mice were challenged
with 2.times.10e5 TC1 cells/mouse S.C. in the flank. TC1 cells
growing in vitro culture, were trypsinised and washed two times in
serum-free medium and injected. 5 mice used in each group were
monitored twice a week for tumour growth.
[0416] FIG. 30 shows that vaccination with the E7 protein in the
SB62 QS21, 3D-MPL adjuvant protect the mice against the development
of tumour (only one animal/5 has a very small and stable tumour) in
all the other groups, that received the E7 protein without the
adjuvant or the adjuvant alone developed growing tumours.
[0417] Immunological Read Out
[0418] Three weeks after the latest vaccination, before the tumour
challenge 5 mice in each group were sacrified for immunological
read out.
[0419] Proliferation Assay
[0420] For in vitro assay, Lymphocytes were prepared as described
above from the spleen and from the popliteal draining lymph nodes.
An aliquot of 2.times.10e5 cells was plated in triplicate in 96
well plates with decreasing concentrations (10, 1, 0.1 .mu.g/ml) of
protD 1/3 E7 His coated or not onto latex microbeads (Sigma) to
restimulate the cells in vitro (72 Hrs). T cell proliferation was
measured by 3H thymidine incorporation.
[0421] FIGS. 31 and 32 show repectively that, both with splenocytes
or popliteal lymph node cells, as it was observed in the
therapeutic settings, a better lymphoproliferative activity was
obtained for the mice that received the E7 protein in the SB62
QS21, 3D-MPL adjuvant antibody response.
[0422] FIG. 33 shows that as in the therapeutic settings, a better
antibody response was observed in the serum of mice vaccinated with
the ProtD1/3 E7 protein formulated in the 3D-MPL, QS21 O/W
adjuvant. A mixed antibody response was triggered, as all the IgG
subclass tested (IgG 2a, IgG2b, IgG1), in this case also, IgG2b was
the predominant isotype found, representing 75% of the total
IgG.
EXAMPLE XXI
Vaccination Experiments with Prot D1/3 E7 (HPV 18)
[0423] Mice were vaccinated twice, 2 weeks apart, with 5 .mu.g in
100 .mu.l of protD 1/3 18 E7 His intra foot pad (50 .mu.l/foot pad)
in PBS or QS21, 3D-MPL and SB62, DQ MPL as described in WO96/33739
or DQ alum MPL as described in WO98/15827. Eight 6-8 weeks old
Balb/c mice (Iffa Credo) were used in each group.
[0424] 14 days post II, the spleen and lymph nodes were taken for
immunological read out and blood sampling for serology.
[0425] Immunological Read Out:
[0426] Proliferation Assay:
[0427] For in vitro assay, lymphocytes were prepared by crushing
the spleen or the popliteal lymph nodes from the vaccinated mice at
day 28
[0428] An aliquot of 2.times.10e5 cells was plated in triplicate in
96 well plates with decreasing concentrations (10, 1, 0.1, 0.01
.mu.g/ml) of protD 1/3 18 E7 His to restimulate the cells in vitro
(72 Hrs). T cell proliferation was measured by 3H thymidine
incorporation. The results are expressed as stimulation index (cpm
sample/cpm baseline)
[0429] FIGS. 34 and 35 compares the ability of protD 1/3 18 E7 to
stimulate the proliferation of splenocytes or lymph node cells
primed in vivo either by ProtD1/3 18 E7 His or Prot D1/3 18 E7
His+adjuvant and shows that only a basal lymphoproliferation is
seen in mice that received the protein alone, on the contrary, high
proliferative responses in spleen and very high responses in lymph
nodes were detected in mice immunised with protD1/3 18 E7 His in
adjuvant.
[0430] Cytokine Production
[0431] The cytokines (IL-5 and IFNg) produced in the culture
supernatant after a 96 Hrs period of in vitro re-stimulation of
spleen or lymph node cells, with medium or with the ProtD1/3 18E7
(1 or 3 .mu.g/ml) was measured by ELISA as described:
[0432] IFNg (Genzyme)
[0433] Quantitation of IFN.gamma. was performed by Elisa using
reagents from Genzyme. Samples and antibody solutions were used at
50 .mu.l per well. 96-well microtiter plates (Maxisorb
Immuno-plate, Nunc, Denmark) were coated overnight at 4.degree. c.
with 50 .mu.l of hamster anti-mouse IFNg diluted at 1.5 .mu.g/ml in
carbonate buffer pH 9.5. Plates were then incubated for lhr at
37.degree. c. with 100 .mu.l of PBS containing 1% bovine serum
albumin and 0.1% Tween 20 (saturation buffer). Two-fold dilutions
of supernatant from in vitro stimulation (starting at {fraction
(1/2)}) in saturation buffer were added to the anti-IFNg-coated
plates and incubated for 1 hr 30 at 37.degree. c. The plates were
washed 4 times with PBS Tween 0.1% (wash buffer) and
biotin-conjugated goat anti-mouse IFNg diluted in saturation buffer
at a final concentration of 0.5 g/ml was added to each well and
incubated for 1 hr at 37.degree. c. After a washing step, AMDEX
conjugate (Amersham) diluted {fraction (1/10000)} in saturation
buffer was added for 30 min at 37.degree. c. Plates were washed as
above and incubated with 50 .mu.l of TMB (Biorad) for 15 min. The
reaction was stopped with H.sub.2SO.sub.4 0.4N and read at 450 nm.
Concentrations were calculated using a standard curve (mouse
IFN.gamma. standard) by SoftmaxPro (four parameters equation) and
expressed in pg/ml.
[0434] IL5 (Pharmingen)
[0435] Quantitation of IL5 was performed by Elisa using reagents
from Pharmingen. Samples and antibody solutions were used at 50
.mu.l per well. 96-well microtiter plates (Maxisorb Immuno-plate,
Nunc, Denmark) were coated overnight at 4.degree. c. with 50 .mu.l
of rat anti-mouse IL5 diluted at 1 .mu.g/ml in carbonate buffer pH
9.5. Plates were then incubated for lhr at 37.degree. c. with 100
.mu.l PBS containing 1% bovine serum albumin and 0.1% Tween 20
(saturation buffer). Two-fold dilutions of supernatant from in
vitro stimulation (starting at {fraction (1/2)}) in saturation
buffer were added to the anti-IFNg-coated plates and incubated for
1 hr 30 at 37.degree. c. The plates were washed 4 times with PBS
Tween 0.1% (wash buffer) and biotin-conjugated rat anti-mouse IL5
diluted in saturation buffer at a final concentration of 1 .mu.g/ml
was added to each well and incubated for 1 hr at 37.degree. c.
After a washing step, AMDEX conjugate (Amersham) diluted {fraction
(1/10000)} in saturation buffer was added for 30 min at 37.degree.
c. Plates were washed as above and incubated with 50 .mu.l of TMB
(Biorad) for 15 min. The reaction was stopped with H.sub.2SO.sub.4
0.4N and read at 450 nm. Concentrations were calculated using a
standard curve (recombinant mouse IL5) by SoftmaxPro (four
parameters equation) and expressed in pg/ml.
[0436] Starting with spleen cells, no IL-5 could be detected
whatever the group tested, on the contrary, a very high production
of IFNg production was observed in all groups, with only a slight
increase in the group of mice that received the SBAS1c adjuvanted
protein compared to the other groups. This suggest the induction of
a TH1 type of immune response.
[0437] Regarding lymph node cells, a very weak IFNg production was
obtained in the group of mice that received the protein alone and a
5-10 fold increase is observed with the adjuvanted protein. IL5
could only be detected in the group of mice receiving the SBAS2
adjuvanted protein.
[0438] FIGS. 36 and 37 compares the ability of ProtD1/3 18 E7 His
to stimulate the production of cytokines 'IFNg and IL5) after in
vitro re-stimulation of spleen or lymph node cells
respectively.
[0439] Antibody Response
[0440] Individual serum were taken at the same time as the organs
and submitted to indirect ELISAs.
[0441] 2.5 .mu.g/ml of purified of protD1/3 18E7 protein HPV 18 was
used as coated antigen. After saturation in PBS+1% newborn calf
serum 1 Hr at 37.degree. C., the sera were serially diluted
(starting at {fraction (1/100)}) in the saturation buffer and
incubated O/N at 4.degree. C. or 90 min at 37.degree. C. After
washing in PBS Tween 20 0.1%, biotinylated goat Anti mouse Ig
({fraction (1/1000)}) or goat anti mouse Ig subclass (total IgG,
IgGI, IgG2a, IgG2b) antisera ({fraction (1/5000)}) were used as
second antibodies, after an incubation of 90 min at 37.degree. C.,
streptavidin coupled to peroxydase was added and TMB
(tetra-methyl-benzidine/peroxide) was used as substrate, after 10
min. the reaction was stopped with H2SO4 0.5 M and the O.D.450 was
determined.
[0442] A very weak antibody response is triggered with 2 injections
of ProtD 1/3 18 E7 alone. The total IgG level was greatly increased
by the addition of adjuvants to the protein vaccine.
[0443] The analysis of the concentrations of the different IgG
subclass show that when the protein was injected in the presence of
adjuvants, DQS21 3D-MPL or SB62, QS21/3D-MPL, a slight increase of
the IgG2a subtype percentage was obtained: 28% IgG1, 48% IgG2a and
43% IgG1, 44% IgG2a respectively, compared to 46% of IgG1, 32% of
IgG2a with the non adjuvanted protein. The strongest antibody
response is obtained with the protein formulated in DQ alum with a
clear shift in the isotype concentration (80% IgG1, 8% IgG2a). As
the IgG2a isotype in Balb/c mice is generally conidered to be
associated with the induction of a TH1 type of immune response,
these results suggested that the DQS21, 3D-MPL and SB62 QS21/3D-MPL
adjuvants tend toincrease the THI type profile of the humoral
response while SBAS5 induce a clear TH2 type of response.
[0444] FIG. 38. The comparison of the midpoint dilution of the
serum and relative percentage of the different isotypes elicited by
the vaccinations in the different groups of mice are shown.
[0445] Conclusion:
[0446] We have demonstrated that the fused protein: {fraction
(1/3)} Prot D and early protein E7 of HPV 16 induced a potent
systemic antitumour immunity and the fusion protein ProtD1/3 and E7
of HPV18 has also been showed to be immunogenic in mice Vaccination
with the prot D1/3 E7 HPV16 fusion protein protected the mice from
a tumour challenge with E7 expressing tumour cells and eliminated
small pre-established tumours expressing the E7 of HPV16 injected
at a distant site from the vaccination site.
[0447] We have demonstrated that the ProtD1/3 E7 HPV16 protein in
adjuvant is capable of enhancing helper T cell proliferation
suggesting that the antitumour immune response induced by this
vaccine is at least in part associated with a CD4+ T cell
response.
[0448] We have also demonstrated that a better antibody response
was triggered by the vaccination with the ProtD1/3 E7 in the
presence of the 3D-MPL containing adjuvant. The predominant isotype
found in the serum of C57BL/6 mice being IgG2b suggesting that a
THI type immune response was raised.
Sequence CWU 1
1
23 1 220 PRT Homo sapien 1 Met Asp Pro Ser Ser His Ser Ser Asn Met
Ala Asn Thr Gln Met Lys 1 5 10 15 Ser Asp Lys Ile Ile Ile Ala His
Arg Gly Ala Ser Gly Tyr Leu Pro 20 25 30 Glu His Thr Leu Glu Ser
Lys Ala Leu Ala Phe Ala Gln Gln Ala Asp 35 40 45 Tyr Leu Glu Gln
Asp Leu Ala Met Thr Lys Asp Gly Arg Leu Val Val 50 55 60 Ile His
Asp His Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe 65 70 75 80
Pro His Arg His Arg Lys Asp Gly Arg Tyr Tyr Val Ile Asp Phe Thr 85
90 95 Leu Lys Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr
Met 100 105 110 Ala Met His Gly Asp Thr Pro Thr Leu His Glu Tyr Met
Leu Asp Leu 115 120 125 Gln Pro Glu Thr Thr Asp Leu Tyr Cys Tyr Glu
Gln Leu Asn Asp Ser 130 135 140 Ser Glu Glu Glu Asp Glu Ile Asp Gly
Pro Ala Gly Gln Ala Glu Pro 145 150 155 160 Asp Arg Ala His Tyr Asn
Ile Val Thr Phe Cys Cys Lys Cys Asp Ser 165 170 175 Thr Leu Arg Leu
Cys Val Gln Ser Thr His Val Asp Ile Arg Thr Leu 180 185 190 Glu Asp
Leu Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys Ser 195 200 205
Gln Lys Pro Thr Ser Gly His His His His His His 210 215 220 2 663
DNA Homo sapien 2 atggatccaa gcagccattc atcaaatatg gcgaataccc
aaatgaaatc agacaaaatc 60 attattgctc accgtggtgc tagcggttat
ttaccagagc atacgttaga atctaaagca 120 cttgcgtttg cacaacaggc
tgattattta gagcaagatt tagcaatgac taaggatggt 180 cgtttagtgg
ttattcacga tcacttttta gatggcttga ctgatgttgc gaaaaaattc 240
ccacatcgtc atcgtaaaga tggccgttac tatgtcatcg actttacctt aaaagaaatt
300 caaagtttag aaatgacaga aaactttgaa accatggcca tgcatggaga
tacacctaca 360 ttgcatgaat atatgttaga tttgcaacca gagacaactg
atctctactg ttatgagcaa 420 ttaaatgaca gctcagagga ggaggatgaa
atagatggtc cagctggaca agcagaaccg 480 gacagagccc attacaatat
tgtaaccttt tgttgcaagt gtgactctac gcttcggttg 540 tgcgtacaaa
gcacacacgt agacattcgt actttggaag acctgttaat gggcacacta 600
ggaattgtgt gccccatctg ttctcagaaa ccaactagtg gccaccatca ccatcaccat
660 taa 663 3 822 DNA Homo sapien 3 atggatccaa gcagccattc
atcaaatatg gcgaataccc aaatgaaatc agacaaaatc 60 attattgctc
accgtggtgc tagcggttat ttaccagagc atacgttaga atctaaagca 120
cttgcgtttg cacaacaggc tgattattta gagcaagatt tagcaatgac taaggatggt
180 cgtttagtgg ttattcacga tcacttttta gatggcttga ctgatgttgc
gaaaaaattc 240 ccacatcgtc atcgtaaaga tggccgttac tatgtcatcg
actttacctt aaaagaaatt 300 caaagtttag aaatgacaga aaactttgaa
accatggcca tgtttcagga cccacaggag 360 cgacccagaa agttaccaca
gttatgcaca gagctgcaaa caactataca tgatataata 420 ttagaatgtg
tgtactgcaa gcaacagtta ctgcgacgtg aggtatatga ctttgctttt 480
cgggatttat gcatagtata tagagatggg aatccatatg ctgtatgtga taaatgttta
540 aagttttatt ctaaaattag tgagtataga cattattgtt atagtttgta
tggaacaaca 600 ttagaacagc aatacaacaa accgttgtgt gatttgttaa
ttaggtgtat taactgtcaa 660 aagccactgt gtcctgaaga aaagcaaaga
catctggaca aaaagcaaag attccataat 720 ataaggggtc ggtggaccgg
tcgatgtatg tcttgttgca gatcatcaag aacacgtaga 780 gaaacccagc
tgactagtgg ccaccatcac catcaccatt aa 822 4 273 PRT Homo sapien 4 Met
Asp Pro Ser Ser His Ser Ser Asn Met Ala Asn Thr Gln Met Lys 1 5 10
15 Ser Asp Lys Ile Ile Ile Ala His Arg Gly Ala Ser Gly Tyr Leu Pro
20 25 30 Glu His Thr Leu Glu Ser Lys Ala Leu Ala Phe Ala Gln Gln
Ala Asp 35 40 45 Tyr Leu Glu Gln Asp Leu Ala Met Thr Lys Asp Gly
Arg Leu Val Val 50 55 60 Ile His Asp His Phe Leu Asp Gly Leu Thr
Asp Val Ala Lys Lys Phe 65 70 75 80 Pro His Arg His Arg Lys Asp Gly
Arg Tyr Tyr Val Ile Asp Phe Thr 85 90 95 Leu Lys Glu Ile Gln Ser
Leu Glu Met Thr Glu Asn Phe Glu Thr Met 100 105 110 Ala Met Phe Gln
Asp Pro Gln Glu Arg Pro Arg Lys Leu Pro Gln Leu 115 120 125 Cys Thr
Glu Leu Gln Thr Thr Ile His Asp Ile Ile Leu Glu Cys Val 130 135 140
Tyr Cys Lys Gln Gln Leu Leu Arg Arg Glu Val Tyr Asp Phe Ala Phe 145
150 155 160 Arg Asp Leu Cys Ile Val Tyr Arg Asp Gly Asn Pro Tyr Ala
Val Cys 165 170 175 Asp Lys Cys Leu Lys Phe Tyr Ser Lys Ile Ser Glu
Tyr Arg His Tyr 180 185 190 Cys Tyr Ser Leu Tyr Gly Thr Thr Leu Glu
Gln Gln Tyr Asn Lys Pro 195 200 205 Leu Cys Asp Leu Leu Ile Arg Cys
Ile Asn Cys Gln Lys Pro Leu Cys 210 215 220 Pro Glu Glu Lys Gln Arg
His Leu Asp Lys Lys Gln Arg Phe His Asn 225 230 235 240 Ile Arg Gly
Arg Trp Thr Gly Arg Cys Met Ser Cys Cys Arg Ser Ser 245 250 255 Arg
Thr Arg Arg Glu Thr Gln Leu Thr Ser Gly His His His His His 260 265
270 His 5 1116 DNA Homo sapien 5 atggatccaa gcagccattc atcaaatatg
gcgaataccc aaatgaaatc agacaaaatc 60 attattgctc accgtggtgc
tagcggttat ttaccagagc atacgttaga atctaaagca 120 cttgcgtttg
cacaacaggc tgattattta gagcaagatt tagcaatgac taaggatggt 180
cgtttagtgg ttattcacga tcacttttta gatggcttga ctgatgttgc gaaaaaattc
240 ccacatcgtc atcgtaaaga tggccgttac tatgtcatcg actttacctt
aaaagaaatt 300 caaagtttag aaatgacaga aaactttgaa accatggcca
tgtttcagga cccacaggag 360 cgacccagaa agttaccaca gttatgcaca
gagctgcaaa caactataca tgatataata 420 ttagaatgtg tgtactgcaa
gcaacagtta ctgcgacgtg aggtatatga ctttgctttt 480 cgggatttat
gcatagtata tagagatggg aatccatatg ctgtatgtga taaatgttta 540
aagttttatt ctaaaattag tgagtataga cattattgtt atagtttgta tggaacaaca
600 ttagaacagc aatacaacaa accgttgtgt gatttgttaa ttaggtgtat
taactgtcaa 660 aagccactgt gtcctgaaga aaagcaaaga catctggaca
aaaagcaaag attccataat 720 ataaggggtc ggtggaccgg tcgatgtatg
tcttgttgca gatcatcaag aacacgtaga 780 gaaacccagc tgatgcatgg
agatacacct acattgcatg aatatatgtt agatttgcaa 840 ccagagacaa
ctgatctcta ctgttatgag caattaaatg acagctcaga ggaggaggat 900
gaaatagatg gtccagctgg acaagcagaa ccggacagag cccattacaa tattgtaacc
960 ttttgttgca agtgtgactc tacgcttcgg ttgtgcgtac aaagcacaca
cgtagacatt 1020 cgtactttgg aagacctgtt aatgggcaca ctaggaattg
tgtgccccat ctgttctcag 1080 aaaccaacta gtggccacca tcaccatcac cattaa
1116 6 371 PRT Homo sapien 6 Met Asp Pro Ser Ser His Ser Ser Asn
Met Ala Asn Thr Gln Met Lys 1 5 10 15 Ser Asp Lys Ile Ile Ile Ala
His Arg Gly Ala Ser Gly Tyr Leu Pro 20 25 30 Glu His Thr Leu Glu
Ser Lys Ala Leu Ala Phe Ala Gln Gln Ala Asp 35 40 45 Tyr Leu Glu
Gln Asp Leu Ala Met Thr Lys Asp Gly Arg Leu Val Val 50 55 60 Ile
His Asp His Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe 65 70
75 80 Pro His Arg His Arg Lys Asp Gly Arg Tyr Tyr Val Ile Asp Phe
Thr 85 90 95 Leu Lys Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe
Glu Thr Met 100 105 110 Ala Met Phe Gln Asp Pro Gln Glu Arg Pro Arg
Lys Leu Pro Gln Leu 115 120 125 Cys Thr Glu Leu Gln Thr Thr Ile His
Asp Ile Ile Leu Glu Cys Val 130 135 140 Tyr Cys Lys Gln Gln Leu Leu
Arg Arg Glu Val Tyr Asp Phe Ala Phe 145 150 155 160 Arg Asp Leu Cys
Ile Val Tyr Arg Asp Gly Asn Pro Tyr Ala Val Cys 165 170 175 Asp Lys
Cys Leu Lys Phe Tyr Ser Lys Ile Ser Glu Tyr Arg His Tyr 180 185 190
Cys Tyr Ser Leu Tyr Gly Thr Thr Leu Glu Gln Gln Tyr Asn Lys Pro 195
200 205 Leu Cys Asp Leu Leu Ile Arg Cys Ile Asn Cys Gln Lys Pro Leu
Cys 210 215 220 Pro Glu Glu Lys Gln Arg His Leu Asp Lys Lys Gln Arg
Phe His Asn 225 230 235 240 Ile Arg Gly Arg Trp Thr Gly Arg Cys Met
Ser Cys Cys Arg Ser Ser 245 250 255 Arg Thr Arg Arg Glu Thr Gln Leu
Met His Gly Asp Thr Pro Thr Leu 260 265 270 His Glu Tyr Met Leu Asp
Leu Gln Pro Glu Thr Thr Asp Leu Tyr Cys 275 280 285 Tyr Glu Gln Leu
Asn Asp Ser Ser Glu Glu Glu Asp Glu Ile Asp Gly 290 295 300 Pro Ala
Gly Gln Ala Glu Pro Asp Arg Ala His Tyr Asn Ile Val Thr 305 310 315
320 Phe Cys Cys Lys Cys Asp Ser Thr Leu Arg Leu Cys Val Gln Ser Thr
325 330 335 His Val Asp Ile Arg Thr Leu Glu Asp Leu Leu Met Gly Thr
Leu Gly 340 345 350 Ile Val Cys Pro Ile Cys Ser Gln Lys Pro Thr Ser
Gly His His His 355 360 365 His His His 370 7 663 DNA Homo sapien 7
atggatccaa gcagccattc atcaaatatg gcgaataccc aaatgaaatc agacaaaatc
60 attattgctc accgtggtgc tagcggttat ttaccagagc atacgttaga
atctaaagca 120 cttgcgtttg cacaacaggc tgattattta gagcaagatt
tagcaatgac taaggatggt 180 cgtttagtgg ttattcacga tcacttttta
gatggcttga ctgatgttgc gaaaaaattc 240 ccacatcgtc atcgtaaaga
tggccgttac tatgtcatcg actttacctt aaaagaaatt 300 caaagtttag
aaatgacaga aaactttgaa accatggcca tgcatggaga tacacctaca 360
ttgcatgaat atatgttaga tttgcaacca gagacaactg atctctacgg ttatcagcaa
420 ttaaatgaca gctcagagga ggaggatgaa atagatggtc cagctggaca
agcagaaccg 480 gacagagccc attacaatat tgtaaccttt tgttgcaagt
gtgactctac gcttcggttg 540 tgcgtacaaa gcacacacgt agacattcgt
actttggaag acctgttaat gggcacacta 600 ggaattgtgt gccccatctg
ttctcagaaa ccaactagtg gccaccatca ccatcaccat 660 taa 663 8 220 PRT
Homo sapien 8 Met Asp Pro Ser Ser His Ser Ser Asn Met Ala Asn Thr
Gln Met Lys 1 5 10 15 Ser Asp Lys Ile Ile Ile Ala His Arg Gly Ala
Ser Gly Tyr Leu Pro 20 25 30 Glu His Thr Leu Glu Ser Lys Ala Leu
Ala Phe Ala Gln Gln Ala Asp 35 40 45 Tyr Leu Glu Gln Asp Leu Ala
Met Thr Lys Asp Gly Arg Leu Val Val 50 55 60 Ile His Asp His Phe
Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe 65 70 75 80 Pro His Arg
His Arg Lys Asp Gly Arg Tyr Tyr Val Ile Asp Phe Thr 85 90 95 Leu
Lys Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr Met 100 105
110 Ala Met His Gly Asp Thr Pro Thr Leu His Glu Tyr Met Leu Asp Leu
115 120 125 Gln Pro Glu Thr Thr Asp Leu Tyr Gly Tyr Gln Gln Leu Asn
Asp Ser 130 135 140 Ser Glu Glu Glu Asp Glu Ile Asp Gly Pro Ala Gly
Gln Ala Glu Pro 145 150 155 160 Asp Arg Ala His Tyr Asn Ile Val Thr
Phe Cys Cys Lys Cys Asp Ser 165 170 175 Thr Leu Arg Leu Cys Val Gln
Ser Thr His Val Asp Ile Arg Thr Leu 180 185 190 Glu Asp Leu Leu Met
Gly Thr Leu Gly Ile Val Cys Pro Ile Cys Ser 195 200 205 Gln Lys Pro
Thr Ser Gly His His His His His His 210 215 220 9 879 DNA Homo
sapien 9 atgaaagggg gaattgtaca ttcagacggc tcttatccaa aagacaagtt
tgagaaaatc 60 aatggcactt ggtactactt tgacagttca ggctatatgc
ttgcagaccg ctggaggaag 120 cacacagacg gcaactggta ctggttcgac
aactcaggcg aaatggctac aggctggaag 180 aaaatcgctg ataagtggta
ctatttcaac gaagaaggtg ccatgaagac aggctgggtc 240 aagtacaagg
acacttggta ctacttagac gctaaagaag gcgccatggt atcaaatgcc 300
tttatccagt cagcggacgg aacaggctgg tactacctca aaccagacgg aacactggca
360 gacaggccag aattggccag catgctggac atggccatgt ttcaggaccc
acaggagcga 420 cccagaaagt taccacagtt atgcacagag ctgcaaacaa
ctatacatga tataatatta 480 gaatgtgtgt actgcaagca acagttactg
cgacgtgagg tatatgactt tgcttttcgg 540 gatttatgca tagtatatag
agatgggaat ccatatgctg tatgtgataa atgtttaaag 600 ttttattcta
aaattagtga gtatagacat tattgttata gtttgtatgg aacaacatta 660
gaacagcaat acaacaaacc gttgtgtgat ttgttaatta ggtgtattaa ctgtcaaaag
720 ccactgtgtc ctgaagaaaa gcaaagacat ctggacaaaa agcaaagatt
ccataatata 780 aggggtcggt ggaccggtcg atgtatgtct tgttgcagat
catcaagaac acgtagagaa 840 acccagctga ctagtggcca ccatcaccat
caccattaa 879 10 292 PRT Homo sapien 10 Met Lys Gly Gly Ile Val His
Ser Asp Gly Ser Tyr Pro Lys Asp Lys 1 5 10 15 Phe Glu Lys Ile Asn
Gly Thr Trp Tyr Tyr Phe Asp Ser Ser Gly Tyr 20 25 30 Met Leu Ala
Asp Arg Trp Arg Lys His Thr Asp Gly Asn Trp Tyr Trp 35 40 45 Phe
Asp Asn Ser Gly Glu Met Ala Thr Gly Trp Lys Lys Ile Ala Asp 50 55
60 Lys Trp Tyr Tyr Phe Asn Glu Glu Gly Ala Met Lys Thr Gly Trp Val
65 70 75 80 Lys Tyr Lys Asp Thr Trp Tyr Tyr Leu Asp Ala Lys Glu Gly
Ala Met 85 90 95 Val Ser Asn Ala Phe Ile Gln Ser Ala Asp Gly Thr
Gly Trp Tyr Tyr 100 105 110 Leu Lys Pro Asp Gly Thr Leu Ala Asp Arg
Pro Glu Leu Ala Ser Met 115 120 125 Leu Asp Met Ala Met Phe Gln Asp
Pro Gln Glu Arg Pro Arg Lys Leu 130 135 140 Pro Gln Leu Cys Thr Glu
Leu Gln Thr Thr Ile His Asp Ile Ile Leu 145 150 155 160 Glu Cys Val
Tyr Cys Lys Gln Gln Leu Leu Arg Arg Glu Val Tyr Asp 165 170 175 Phe
Ala Phe Arg Asp Leu Cys Ile Val Tyr Arg Asp Gly Asn Pro Tyr 180 185
190 Ala Val Cys Asp Lys Cys Leu Lys Phe Tyr Ser Lys Ile Ser Glu Tyr
195 200 205 Arg His Tyr Cys Tyr Ser Leu Tyr Gly Thr Thr Leu Glu Gln
Gln Tyr 210 215 220 Asn Lys Pro Leu Cys Asp Leu Leu Ile Arg Cys Ile
Asn Cys Gln Lys 225 230 235 240 Pro Leu Cys Pro Glu Glu Lys Gln Arg
His Leu Asp Lys Lys Gln Arg 245 250 255 Phe His Asn Ile Arg Gly Arg
Trp Thr Gly Arg Cys Met Ser Cys Cys 260 265 270 Arg Ser Ser Arg Thr
Arg Arg Glu Thr Gln Leu Thr Ser Gly His His 275 280 285 His His His
His 290 11 720 DNA Homo sapien 11 atgaaagggg gaattgtaca ttcagacggc
tcttatccaa aagacaagtt tgagaaaatc 60 aatggcactt ggtactactt
tgacagttca ggctatatgc ttgcagaccg ctggaggaag 120 cacacagacg
gcaactggta ctggttcgac aactcaggcg aaatggctac aggctggaag 180
aaaatcgctg ataagtggta ctatttcaac gaagaaggtg ccatgaagac aggctgggtc
240 aagtacaagg acacttggta ctacttagac gctaaagaag gcgccatggt
atcaaatgcc 300 tttatccagt cagcggacgg aacaggctgg tactacctca
aaccagacgg aacactggca 360 gacaggccag aattggccag catgctggac
atggccatgc atggagatac acctacattg 420 catgaatata tgttagattt
gcaaccagag acaactgatc tctactgtta tgagcaatta 480 aatgacagct
cagaggagga ggatgaaata gatggtccag ctggacaagc agaaccggac 540
agagcccatt acaatattgt aaccttttgt tgcaagtgtg actctacgct tcggttgtgc
600 gtacaaagca cacacgtaga cattcgtact ttggaagacc tgttaatggg
cacactagga 660 attgtgtgcc ccatctgttc tcagaaacca actagtggcc
accatcacca tcaccattaa 720 12 239 PRT Homo sapien 12 Met Lys Gly Gly
Ile Val His Ser Asp Gly Ser Tyr Pro Lys Asp Lys 1 5 10 15 Phe Glu
Lys Ile Asn Gly Thr Trp Tyr Tyr Phe Asp Ser Ser Gly Tyr 20 25 30
Met Leu Ala Asp Arg Trp Arg Lys His Thr Asp Gly Asn Trp Tyr Trp 35
40 45 Phe Asp Asn Ser Gly Glu Met Ala Thr Gly Trp Lys Lys Ile Ala
Asp 50 55 60 Lys Trp Tyr Tyr Phe Asn Glu Glu Gly Ala Met Lys Thr
Gly Trp Val 65 70 75 80 Lys Tyr Lys Asp Thr Trp Tyr Tyr Leu Asp Ala
Lys Glu Gly Ala Met 85 90 95 Val Ser Asn Ala Phe Ile Gln Ser Ala
Asp Gly Thr Gly Trp Tyr Tyr 100 105 110 Leu Lys Pro Asp Gly Thr Leu
Ala Asp Arg Pro Glu Leu Ala Ser Met 115 120 125 Leu Asp Met Ala Met
His Gly Asp Thr Pro Thr Leu His Glu Tyr Met 130 135 140 Leu Asp Leu
Gln Pro Glu Thr Thr Asp Leu Tyr Cys Tyr Glu Gln Leu 145 150 155 160
Asn Asp Ser Ser Glu Glu Glu Asp Glu Ile Asp Gly Pro Ala Gly Gln 165
170 175 Ala Glu Pro Asp Arg Ala His Tyr Asn Ile Val Thr Phe Cys Cys
Lys 180 185 190 Cys Asp Ser Thr Leu Arg Leu Cys Val Gln Ser Thr His
Val Asp Ile 195 200 205 Arg Thr Leu Glu Asp Leu Leu
Met Gly Thr Leu Gly Ile Val Cys Pro 210 215 220 Ile Cys Ser Gln Lys
Pro Thr Ser Gly His His His His His His 225 230 235 13 1173 DNA
Homo sapien 13 atgaaagggg gaattgtaca ttcagacggc tcttatccaa
aagacaagtt tgagaaaatc 60 aatggcactt ggtactactt tgacagttca
ggctatatgc ttgcagaccg ctggaggaag 120 cacacagacg gcaactggta
ctggttcgac aactcaggcg aaatggctac aggctggaag 180 aaaatcgctg
ataagtggta ctatttcaac gaagaaggtg ccatgaagac aggctgggtc 240
aagtacaagg acacttggta ctacttagac gctaaagaag gcgccatggt atcaaatgcc
300 tttatccagt cagcggacgg aacaggctgg tactacctca aaccagacgg
aacactggca 360 gacaggccag aattggccag catgctggac atggccatgt
ttcaggaccc acaggagcga 420 cccagaaagt taccacagtt atgcacagag
ctgcaaacaa ctatacatga tataatatta 480 gaatgtgtgt actgcaagca
acagttactg cgacgtgagg tatatgactt tgcttttcgg 540 gatttatgca
tagtatatag agatgggaat ccatatgctg tatgtgataa atgtttaaag 600
ttttattcta aaattagtga gtatagacat tattgttata gtttgtatgg aacaacatta
660 gaacagcaat acaacaaacc gttgtgtgat ttgttaatta ggtgtattaa
ctgtcaaaag 720 ccactgtgtc ctgaagaaaa gcaaagacat ctggacaaaa
agcaaagatt ccataatata 780 aggggtcggt ggaccggtcg atgtatgtct
tgttgcagat catcaagaac acgtagagaa 840 acccagctga tgcatggaga
tacacctaca ttgcatgaat atatgttaga tttgcaacca 900 gagacaactg
atctctactg ttatgagcaa ttaaatgaca gctcagagga ggaggatgaa 960
atagatggtc cagctggaca agcagaaccg gacagagccc attacaatat tgtaaccttt
1020 tgttgcaagt gtgactctac gcttcggttg tgcgtacaaa gcacacacgt
agacattcgt 1080 actttggaag acctgttaat gggcacacta ggaattgtgt
gccccatctg ttctcagaaa 1140 ccaactagtg gccaccatca ccatcaccat taa
1173 14 390 PRT Homo sapien 14 Met Lys Gly Gly Ile Val His Ser Asp
Gly Ser Tyr Pro Lys Asp Lys 1 5 10 15 Phe Glu Lys Ile Asn Gly Thr
Trp Tyr Tyr Phe Asp Ser Ser Gly Tyr 20 25 30 Met Leu Ala Asp Arg
Trp Arg Lys His Thr Asp Gly Asn Trp Tyr Trp 35 40 45 Phe Asp Asn
Ser Gly Glu Met Ala Thr Gly Trp Lys Lys Ile Ala Asp 50 55 60 Lys
Trp Tyr Tyr Phe Asn Glu Glu Gly Ala Met Lys Thr Gly Trp Val 65 70
75 80 Lys Tyr Lys Asp Thr Trp Tyr Tyr Leu Asp Ala Lys Glu Gly Ala
Met 85 90 95 Val Ser Asn Ala Phe Ile Gln Ser Ala Asp Gly Thr Gly
Trp Tyr Tyr 100 105 110 Leu Lys Pro Asp Gly Thr Leu Ala Asp Arg Pro
Glu Leu Ala Ser Met 115 120 125 Leu Asp Met Ala Met Phe Gln Asp Pro
Gln Glu Arg Pro Arg Lys Leu 130 135 140 Pro Gln Leu Cys Thr Glu Leu
Gln Thr Thr Ile His Asp Ile Ile Leu 145 150 155 160 Glu Cys Val Tyr
Cys Lys Gln Gln Leu Leu Arg Arg Glu Val Tyr Asp 165 170 175 Phe Ala
Phe Arg Asp Leu Cys Ile Val Tyr Arg Asp Gly Asn Pro Tyr 180 185 190
Ala Val Cys Asp Lys Cys Leu Lys Phe Tyr Ser Lys Ile Ser Glu Tyr 195
200 205 Arg His Tyr Cys Tyr Ser Leu Tyr Gly Thr Thr Leu Glu Gln Gln
Tyr 210 215 220 Asn Lys Pro Leu Cys Asp Leu Leu Ile Arg Cys Ile Asn
Cys Gln Lys 225 230 235 240 Pro Leu Cys Pro Glu Glu Lys Gln Arg His
Leu Asp Lys Lys Gln Arg 245 250 255 Phe His Asn Ile Arg Gly Arg Trp
Thr Gly Arg Cys Met Ser Cys Cys 260 265 270 Arg Ser Ser Arg Thr Arg
Arg Glu Thr Gln Leu Met His Gly Asp Thr 275 280 285 Pro Thr Leu His
Glu Tyr Met Leu Asp Leu Gln Pro Glu Thr Thr Asp 290 295 300 Leu Tyr
Cys Tyr Glu Gln Leu Asn Asp Ser Ser Glu Glu Glu Asp Glu 305 310 315
320 Ile Asp Gly Pro Ala Gly Gln Ala Glu Pro Asp Arg Ala His Tyr Asn
325 330 335 Ile Val Thr Phe Cys Cys Lys Cys Asp Ser Thr Leu Arg Leu
Cys Val 340 345 350 Gln Ser Thr His Val Asp Ile Arg Thr Leu Glu Asp
Leu Leu Met Gly 355 360 365 Thr Leu Gly Ile Val Cys Pro Ile Cys Ser
Gln Lys Pro Thr Ser Gly 370 375 380 His His His His His His 385 390
15 684 DNA Homo sapien 15 atggatccaa gcagccattc atcaaatatg
gcgaataccc aaatgaaatc agacaaaatc 60 attattgctc accgtggtgc
tagcggttat ttaccagagc atacgttaga atctaaagca 120 cttgcgtttg
cacaacaggc tgattattta gagcaagatt tagcaatgac taaggatggt 180
cgtttagtgg ttattcacga tcacttttta gatggcttga ctgatgttgc gaaaaaattc
240 ccacatcgtc atcgtaaaga tggccgttac tatgtcatcg actttacctt
aaaagaaatt 300 caaagtttag aaatgacaga aaactttgaa accatggcca
tgcatggacc taaggcaaca 360 ttgcaagaca ttgtattgca tttagagccc
caaaatgaaa ttccggttga ccttctatgt 420 cacgagcaat taagcgactc
agaggaagaa aacgatgaaa tagatgaagt taatcatcaa 480 catttaccag
cccgacgagc cgaaccacaa cgtcacacaa tgttgtgtat gtgttgtaag 540
tgtgaagcca gaattgagct agtagtagaa agctcagcag acgaccttcg agcattccag
600 cagctgtttc tgaacaccct gtcctttgtg tgtccgtggt gtgcatccca
gcagactagt 660 ggccaccatc accatcacca ttaa 684 16 227 PRT Homo
sapien 16 Met Asp Pro Ser Ser His Ser Ser Asn Met Ala Asn Thr Gln
Met Lys 1 5 10 15 Ser Asp Lys Ile Ile Ile Ala His Arg Gly Ala Ser
Gly Tyr Leu Pro 20 25 30 Glu His Thr Leu Glu Ser Lys Ala Leu Ala
Phe Ala Gln Gln Ala Asp 35 40 45 Tyr Leu Glu Gln Asp Leu Ala Met
Thr Lys Asp Gly Arg Leu Val Val 50 55 60 Ile His Asp His Phe Leu
Asp Gly Leu Thr Asp Val Ala Lys Lys Phe 65 70 75 80 Pro His Arg His
Arg Lys Asp Gly Arg Tyr Tyr Val Ile Asp Phe Thr 85 90 95 Leu Lys
Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr Met 100 105 110
Ala Met His Gly Pro Lys Ala Thr Leu Gln Asp Ile Val Leu His Leu 115
120 125 Glu Pro Gln Asn Glu Ile Pro Val Asp Leu Leu Cys His Glu Gln
Leu 130 135 140 Ser Asp Ser Glu Glu Glu Asn Asp Glu Ile Asp Glu Val
Asn His Gln 145 150 155 160 His Leu Pro Ala Arg Arg Ala Glu Pro Gln
Arg His Thr Met Leu Cys 165 170 175 Met Cys Cys Lys Cys Glu Ala Arg
Ile Glu Leu Val Val Glu Ser Ser 180 185 190 Ala Asp Asp Leu Arg Ala
Phe Gln Gln Leu Phe Leu Asn Thr Leu Ser 195 200 205 Phe Val Cys Pro
Trp Cys Ala Ser Gln Gln Thr Ser Gly His His His 210 215 220 His His
His 225 17 109 PRT Homo sapien 17 Met Ser Asp Lys Ile Ile His Leu
Thr Asp Asp Ser Phe Asp Thr Asp 1 5 10 15 Val Leu Lys Ala Asp Gly
Ala Ile Leu Val Asp Phe Trp Ala Glu Trp 20 25 30 Cys Gly Pro Cys
Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp 35 40 45 Glu Tyr
Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn 50 55 60
Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu 65
70 75 80 Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala
Leu Ser 85 90 95 Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu
Ala 100 105 18 684 DNA Homo sapien 18 atggatccaa gcagccattc
atcaaatatg gcgaataccc aaatgaaatc agacaaaatc 60 attattgctc
accgtggtgc tagcggttat ttaccagagc atacgttaga atctaaagca 120
cttgcgtttg cacaacaggc tgattattta gagcaagatt tagcaatgac taaggatggt
180 cgtttagtgg ttattcacga tcacttttta gatggcttga ctgatgttgc
gaaaaaattc 240 ccacatcgtc atcgtaaaga tggccgttac tatgtcatcg
actttacctt aaaagaaatt 300 caaagtttag aaatgacaga aaactttgaa
accatggcca tgcatggacc taaggcaaca 360 ttgcaagaca ttgtattgca
tttagagccc caaaatgaaa ttccggttga ccttctaggt 420 caccagcaat
taagcgactc agaggaagaa aacgatgaaa tagatggagt taatcatcaa 480
catttaccag cccgacgagc cgaaccacaa cgtcacacaa tgttgtgtat gtgttgtaag
540 tgtgaagcca gaattgagct agtagtagaa agctcagcag acgaccttcg
agcattccag 600 cagctgtttc tgaacaccct gtcctttgtg tgtccgtggt
gtgcatccca gcagactagt 660 ggccaccatc accatcacca ttaa 684 19 227 PRT
Homo sapien 19 Met Asp Pro Ser Ser His Ser Ser Asn Met Ala Asn Thr
Gln Met Lys 1 5 10 15 Ser Asp Lys Ile Ile Ile Ala His Arg Gly Ala
Ser Gly Tyr Leu Pro 20 25 30 Glu His Thr Leu Glu Ser Lys Ala Leu
Ala Phe Ala Gln Gln Ala Asp 35 40 45 Tyr Leu Glu Gln Asp Leu Ala
Met Thr Lys Asp Gly Arg Leu Val Val 50 55 60 Ile His Asp His Phe
Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe 65 70 75 80 Pro His Arg
His Arg Lys Asp Gly Arg Tyr Tyr Val Ile Asp Phe Thr 85 90 95 Leu
Lys Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr Met 100 105
110 Ala Met His Gly Pro Lys Ala Thr Leu Gln Asp Ile Val Leu His Leu
115 120 125 Glu Pro Gln Asn Glu Ile Pro Val Asp Leu Leu Gly His Gln
Gln Leu 130 135 140 Ser Asp Ser Glu Glu Glu Asn Asp Glu Ile Asp Gly
Val Asn His Gln 145 150 155 160 His Leu Pro Ala Arg Arg Ala Glu Pro
Gln Arg His Thr Met Leu Cys 165 170 175 Met Cys Cys Lys Cys Glu Ala
Arg Ile Glu Leu Val Val Glu Ser Ser 180 185 190 Ala Asp Asp Leu Arg
Ala Phe Gln Gln Leu Phe Leu Asn Thr Leu Ser 195 200 205 Phe Val Cys
Pro Trp Cys Ala Ser Gln Gln Thr Ser Gly His His His 210 215 220 His
His His 225 20 837 DNA Homo sapien 20 atggatccaa gcagccattc
atcaaatatg gcgaataccc aaatgaaatc agacaaaatc 60 attattgctc
accgtggtgc tagcggttat ttaccagagc atacgttaga atctaaagca 120
cttgcgtttg cacaacaggc tgattattta gagcaagatt tagcaatgac taaggatggt
180 cgtttagtgg ttattcacga tcacttttta gatggcttga ctgatgttgc
gaaaaaattc 240 ccacatcgtc atcgtaaaga tggccgttac tatgtcatcg
actttacctt aaaagaaatt 300 caaagtttag aaatgacaga aaactttgaa
accatggcgc gctttgagga tccaacacgg 360 cgaccctaca agctacctga
tctgtgcacg gaactgaaca cttcactgca agacatagaa 420 ataacctgtg
tatattgcaa gacagtattg gaacttacag aggtatttga atttgcattt 480
aaagatttat ttgtggtgta tagagacagt ataccgcatg ctgcatgcca taaatgtata
540 gatttttatt ctagaattag agaattaaga cattattcag actctgtgta
tggagacaca 600 ttggaaaaac taactaacac tgggttatac aatttattaa
taaggtgcct gcggtgccag 660 aaaccgttga atccagcaga aaaacttaga
caccttaatg aaaaacgacg atttcacaac 720 atagctgggc actatagagg
ccagtgccat tcgtgctgca accgagcacg acaggaacga 780 ctccaacgac
gcagagaaac acaagtaact agtggccacc atcaccatca ccattaa 837 21 278 PRT
Homo sapien 21 Met Asp Pro Ser Ser His Ser Ser Asn Met Ala Asn Thr
Gln Met Lys 1 5 10 15 Ser Asp Lys Ile Ile Ile Ala His Arg Gly Ala
Ser Gly Tyr Leu Pro 20 25 30 Glu His Thr Leu Glu Ser Lys Ala Leu
Ala Phe Ala Gln Gln Ala Asp 35 40 45 Tyr Leu Glu Gln Asp Leu Ala
Met Thr Lys Asp Gly Arg Leu Val Val 50 55 60 Ile His Asp His Phe
Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe 65 70 75 80 Pro His Arg
His Arg Lys Asp Gly Arg Tyr Tyr Val Ile Asp Phe Thr 85 90 95 Leu
Lys Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr Met 100 105
110 Ala Arg Phe Glu Asp Pro Thr Arg Arg Pro Tyr Lys Leu Pro Asp Leu
115 120 125 Cys Thr Glu Leu Asn Thr Ser Leu Gln Asp Ile Glu Ile Thr
Cys Val 130 135 140 Tyr Cys Lys Thr Val Leu Glu Leu Thr Glu Val Phe
Glu Phe Ala Phe 145 150 155 160 Lys Asp Leu Phe Val Val Tyr Arg Asp
Ser Ile Pro His Ala Ala Cys 165 170 175 His Lys Cys Ile Asp Phe Tyr
Ser Arg Ile Arg Glu Leu Arg His Tyr 180 185 190 Ser Asp Ser Val Tyr
Gly Asp Thr Leu Glu Lys Leu Thr Asn Thr Gly 195 200 205 Leu Tyr Asn
Leu Leu Ile Arg Cys Leu Arg Cys Gln Lys Pro Leu Asn 210 215 220 Pro
Ala Glu Lys Leu Arg His Leu Asn Glu Lys Arg Arg Phe His Asn 225 230
235 240 Ile Ala Gly His Tyr Arg Gly Gln Cys His Ser Cys Cys Asn Arg
Ala 245 250 255 Arg Gln Glu Arg Leu Gln Arg Arg Arg Glu Thr Gln Val
Thr Ser Gly 260 265 270 His His His His His His 275 22 1152 DNA
Homo sapien 22 atggatccaa gcagccattc atcaaatatg gcgaataccc
aaatgaaatc agacaaaatc 60 attattgctc accgtggtgc tagcggttat
ttaccagagc atacgttaga atctaaagca 120 cttgcgtttg cacaacaggc
tgattattta gagcaagatt tagcaatgac taaggatggt 180 cgtttagtgg
ttattcacga tcacttttta gatggcttga ctgatgttgc gaaaaaattc 240
ccacatcgtc atcgtaaaga tggccgttac tatgtcatcg actttacctt aaaagaaatt
300 caaagtttag aaatgacaga aaactttgaa accatggcgc gctttgagga
tccaacacgg 360 cgaccctaca agctacctga tctgtgcacg gaactgaaca
cttcactgca agacatagaa 420 ataacctgtg tatattgcaa gacagtattg
gaacttacag aggtatttga atttgcattt 480 aaagatttat ttgtggtgta
tagagacagt ataccgcatg ctgcatgcca taaatgtata 540 gatttttatt
ctagaattag agaattaaga cattattcag actctgtgta tggagacaca 600
ttggaaaaac taactaacac tgggttatac aatttattaa taaggtgcct gcggtgccag
660 aaaccgttga atccagcaga aaaacttaga caccttaatg aaaaacgacg
atttcacaac 720 atagctgggc actatagagg ccagtgccat tcgtgctgca
accgagcacg acaggaacga 780 ctccaacgac gcagagaaac acaagtaatg
catggaccta aggcaacatt gcaagacatt 840 gtattgcatt tagagcccca
aaatgaaatt ccggttgacc ttctatgtca cgagcaatta 900 agcgactcag
aggaagaaaa cgatgaaata gatggagtta atcatcaaca tttaccagcc 960
cgacgagccg aaccacaacg tcacacaatg ttgtgtatgt gttgtaagtg tgaagccaga
1020 attgagctag tagtagaaag ctcagcagac gaccttcgag cattccagca
gctgtttctg 1080 aacaccctgt cctttgtgtg tccgtggtgt gcatcccagc
agactagtgg ccaccatcac 1140 catcaccatt aa 1152 23 383 PRT Homo
sapien 23 Met Asp Pro Ser Ser His Ser Ser Asn Met Ala Asn Thr Gln
Met Lys 1 5 10 15 Ser Asp Lys Ile Ile Ile Ala His Arg Gly Ala Ser
Gly Tyr Leu Pro 20 25 30 Glu His Thr Leu Glu Ser Lys Ala Leu Ala
Phe Ala Gln Gln Ala Asp 35 40 45 Tyr Leu Glu Gln Asp Leu Ala Met
Thr Lys Asp Gly Arg Leu Val Val 50 55 60 Ile His Asp His Phe Leu
Asp Gly Leu Thr Asp Val Ala Lys Lys Phe 65 70 75 80 Pro His Arg His
Arg Lys Asp Gly Arg Tyr Tyr Val Ile Asp Phe Thr 85 90 95 Leu Lys
Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr Met 100 105 110
Ala Arg Phe Glu Asp Pro Thr Arg Arg Pro Tyr Lys Leu Pro Asp Leu 115
120 125 Cys Thr Glu Leu Asn Thr Ser Leu Gln Asp Ile Glu Ile Thr Cys
Val 130 135 140 Tyr Cys Lys Thr Val Leu Glu Leu Thr Glu Val Phe Glu
Phe Ala Phe 145 150 155 160 Lys Asp Leu Phe Val Val Tyr Arg Asp Ser
Ile Pro His Ala Ala Cys 165 170 175 His Lys Cys Ile Asp Phe Tyr Ser
Arg Ile Arg Glu Leu Arg His Tyr 180 185 190 Ser Asp Ser Val Tyr Gly
Asp Thr Leu Glu Lys Leu Thr Asn Thr Gly 195 200 205 Leu Tyr Asn Leu
Leu Ile Arg Cys Leu Arg Cys Gln Lys Pro Leu Asn 210 215 220 Pro Ala
Glu Lys Leu Arg His Leu Asn Glu Lys Arg Arg Phe His Asn 225 230 235
240 Ile Ala Gly His Tyr Arg Gly Gln Cys His Ser Cys Cys Asn Arg Ala
245 250 255 Arg Gln Glu Arg Leu Gln Arg Arg Arg Glu Thr Gln Val Met
His Gly 260 265 270 Pro Lys Ala Thr Leu Gln Asp Ile Val Leu His Leu
Glu Pro Gln Asn 275 280 285 Glu Ile Pro Val Asp Leu Leu Cys His Glu
Gln Leu Ser Asp Ser Glu 290 295 300 Glu Glu Asn Asp Glu Ile Asp Gly
Val Asn His Gln His Leu Pro Ala 305 310 315 320 Arg Arg Ala Glu Pro
Gln Arg His Thr Met Leu Cys Met Cys Cys Lys 325 330 335 Cys Glu Ala
Arg Ile Glu Leu Val Val Glu Ser Ser Ala Asp Asp Leu 340 345 350 Arg
Ala Phe Gln Gln Leu Phe Leu Asn Thr Leu Ser Phe Val Cys Pro 355 360
365 Trp Cys Ala Ser Gln Gln Thr Ser Gly His His His His His His 370
375 380
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